Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space

Specific In Vivo Staining of Astrocytes in the Whole Brain after Intravenous Injection of Sulforhodamine Dyes

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders

Joint Observation of the Galactic Center with MAGIC and CTA-LST-1

MAGIC is a system of two Imaging Atmospheric Cherenkov Telescopes (IACTs), designed to detect very-high-energy gamma rays, and is operating in stereoscopic mode since 2009 at the Observatorio del Roque de Los Muchachos in La Palma, Spain. In 2018, the prototype IACT of the Large-Sized Telescope (LST-1) for the Cherenkov Telescope Array, a next-generation ground-based gamma-ray observatory, was inaugurated at the same site, at a distance of approximately 100 meters from the MAGIC telescopes. Using joint observations between MAGIC and LST-1, we developed a dedicated analysis pipeline and established the threefold telescope system via software, achieving the highest sensitivity in the northern hemisphere. Based on this enhanced performance, MAGIC and LST-1 have been jointly and regularly observing the Galactic Center, a region of paramount importance and complexity for IACTs. In particular, the gamma-ray emission from the dynamical center of the Milky Way is under debate. Although previous measurements suggested that a supermassive black hole Sagittarius A* plays a primary role, its radiation mechanism remains unclear, mainly due to limited angular resolution and sensitivity. The enhanced sensitivity in our novel approach is thus expected to provide new insights into the question. We here present the current status of the data analysis for the Galactic Center joint MAGIC and LST-1 observations

Measurement of the B0s→μ+μ− Branching Fraction and Effective Lifetime and Search for B0→μ+μ− Decays

A search for the rare decays Bs0→μ+μ- and B0→μ+μ- is performed at the LHCb experiment using data collected in pp collisions corresponding to a total integrated luminosity of 4.4  fb-1. An excess of Bs0→μ+μ- decays is observed with a significance of 7.8 standard deviations, representing the first observation of this decay in a single experiment. The branching fraction is measured to be B(Bs0→μ+μ-)=(3.0±0.6-0.2+0.3)×10-9, where the first uncertainty is statistical and the second systematic. The first measurement of the Bs0→μ+μ- effective lifetime, τ(Bs0→μ+μ-)=2.04±0.44±0.05  ps, is reported. No significant excess of B0→μ+μ- decays is found, and a 95% confidence level upper limit, B(B0→μ+μ-)<3.4×10-10, is determined. All results are in agreement with the standard model expectations.A search for the rare decays $B^0_s\to\mu^+\mu^-$ and $B^0\to\mu^+\mu^-$ is performed at the LHCb experiment using data collected in $pp$ collisions corresponding to a total integrated luminosity of 4.4 fb$^{-1}$. An excess of $B^0_s\to\mu^+\mu^-$ decays is observed with a significance of 7.8 standard deviations, representing the first observation of this decay in a single experiment. The branching fraction is measured to be ${\cal B}(B^0_s\to\mu^+\mu^-)=\left(3.0\pm 0.6^{+0.3}_{-0.2}\right)\times 10^{-9}$, where the first uncertainty is statistical and the second systematic. The first measurement of the $B^0_s\to\mu^+\mu^-$ effective lifetime, $\tau(B^0_s\to\mu^+\mu^-)=2.04\pm 0.44\pm 0.05$ ps, is reported. No significant excess of $B^0\to\mu^+\mu^-$ decays is found and a 95 % confidence level upper limit, ${\cal B}(B^0\to\mu^+\mu^-)<3.4\times 10^{-10}$, is determined. All results are in agreement with the Standard Model expectations

Nanoparticules fluorescentes à base de Pluronic : application à l'imagerie intravitale de la vascularisation par microscopie à deux photons et au transport de molécules

Classic fluorescent dyes are not necessary efficient in two-photon absorption. Their low two-photon absorption efficiency often requires high laser power and important dye concentrations. Therefore, new dyes and other administration strategies need to be developed specifically for intravital two-photon microscopy. In collaboration with chemists, specialized in the synthesis of molecules with a high two-photon absorption cross-section, different dyes have been synthesized. Most of these dyes are hydrophobic and not directly suitable for biological applications. The work presented in this thesis consisted of encapsulating hydrophobic molecules in biocompatible Pluronic block copolymers. In water, Pluronic unimers with hydrophobic and hydrophilic blocks self-assembled in hydrophilic micelles forming a hydrophobic core around the molecules. This strategy has been used already for the transport and delivery of different hydrophobic molecules in living organism. In the present study, this strategy has been transposed to transport ultra sensitive two-photon dyes in the blood plasma for deep vascular imaging in vivo.Les chromophores classiques ne sont pas toujours efficaces en absorption à deux photons. Leur faible efficacité nécessite l'utilisation de fortes puissances laser et de grandes concentrations en colorants. Dans ce sens, la microscopie à deux photons in vivo requière le développement de nouvelles stratégies de marquage utilisant des chromophores spécialement dédiés à la microscopie à deux photons. Dans le cadre de collaborations avec des chimistes spécialisés dans la synthèse de molécules à forte section efficace d'absorption à deux photons, différents chromophores ont été synthétisés. Ces molécules organiques sont souvent hydrophobes et ne sont pas utilisables directement pour les applications en biologie. Le travail effectuer ici a consisté à encapsuler ces molécules dans des micelles de copolymères biocompatibles, les Pluronic. Les Pluronic sont des matériaux pouvant s'auto assembler en milieu aqueux sous forme de micelles et permettent de solubiliser des composés hydrophobes. Cette stratégie est déjà utilisé pour permettre de transporter différents composés hydrophobes dans les organismes vivants et a été utilisée ici pour transporter des chromophores ultrasensibles à deux photons dans le sang de manière à imager la vascularisation in vivo

Fluorescent Pluronic micelles for in vivo two-photon imaging of the vasculature and active molecule delivery

Les chromophores classiques ne sont pas toujours efficaces en absorption à deux photons. Leur faible efficacité nécessite l'utilisation de fortes puissances laser et de grandes concentrations en colorants. Dans ce sens, la microscopie à deux photons in vivo requière le développement de nouvelles stratégies de marquage utilisant des chromophores spécialement dédiés à la microscopie à deux photons. Dans le cadre de collaborations avec des chimistes spécialisés dans la synthèse de molécules à forte section efficace d'absorption à deux photons, différents chromophores ont été synthétisés. Ces molécules organiques sont souvent hydrophobes et ne sont pas utilisables directement pour les applications en biologie. Le travail effectuer ici a consisté à encapsuler ces molécules dans des micelles de copolymères biocompatibles, les Pluronic. Les Pluronic sont des matériaux pouvant s'auto assembler en milieu aqueux sous forme de micelles et permettent de solubiliser des composés hydrophobes. Cette stratégie est déjà utilisé pour permettre de transporter différents composés hydrophobes dans les organismes vivants et a été utilisée ici pour transporter des chromophores ultrasensibles à deux photons dans le sang de manière à imager la vascularisation in vivo.Classic fluorescent dyes are not necessary efficient in two-photon absorption. Their low two-photon absorption efficiency often requires high laser power and important dye concentrations. Therefore, new dyes and other administration strategies need to be developed specifically for intravital two-photon microscopy. In collaboration with chemists, specialized in the synthesis of molecules with a high two-photon absorption cross-section, different dyes have been synthesized. Most of these dyes are hydrophobic and not directly suitable for biological applications. The work presented in this thesis consisted of encapsulating hydrophobic molecules in biocompatible Pluronic block copolymers. In water, Pluronic unimers with hydrophobic and hydrophilic blocks self-assembled in hydrophilic micelles forming a hydrophobic core around the molecules. This strategy has been used already for the transport and delivery of different hydrophobic molecules in living organism. In the present study, this strategy has been transposed to transport ultra sensitive two-photon dyes in the blood plasma for deep vascular imaging in vivo

Robotized telesurgery in contact with moving organs

Le geste médical assisté par ordinateur en salle d'opération n'a pas cessé de progresser lors de ladernière décennie. Ce phénomène est lié à l'évolution des techniques de chirurgie et notamment àl'apparition des techniques dites mini-invasives. Cette pratique bien que révolutionnaire car ellepermet de diminuer les risques d'infection a introduit de nombreux problèmes. En effet, lechirurgien est limité dans ses gestes à cause de la restriction de mouvement imposée par les trocarts.De ce fait, la communauté robotique s'est assez rapidement intéressée aux problématiques deschirurgiens afin de développer des dispositifs robotisés adaptés. Un des grands thèmes étudié estcelui de la téléopération. Cette dernière consiste à utiliser deux dispositifs robotisés communémentappelés: maître et esclave. Dans le contexte médical, le robot maître est manoeuvré par le chirurgienet le robot esclave est au contact de l'organe à opérer. La première génération de ces systèmes nepermettait pas au chirurgien de ressentir les efforts qu'il exerçait sur les organes du patient. Unedeuxième génération de dispositifs est apparue en introduisant le retour d'efforts. Au delà desproblèmes de stabilité et de commande rencontrés, de tels systèmes permettent de réduire lesimperfections du geste humain ou d'en augmenter les capacités. Une des gênes constatées par leschirurgiens lors de l'utilisation de tels systèmes est le mouvement physiologique des organes àopérer.Cette thèse s'est principalement focalisée sur le thème de la compensation des mouvementsphysiologiques dans le cadre de la téléopération avec retour d'efforts. L'objectif étant de démontrerqu'il est possible pour le chirurgien de téléopérer un organe soumis à des mouvementsphysiologiques, et notamment le mouvement respiratoire, sans qu'il ne ressente cette perturbation.[...]Computer aided medical gesture kept increasing during the last decade. This phenomenon is linkedto the evolution of surgical technique and specially the use of minimally invasive surgery. Thisrevolutionary technique allows reducing infection risks but introduces new problems. Indeed, thesurgeon has limited motion capability because the use of trocars. Therefore, robotics communityinterest has kept growing in order to develop specific robotized devices. One of the most currentstudied topics is teleoperation in which two robots are used: the master and the slave. In a medicalcontext, the master robot is manipulated by the surgeon and the slave robot is in contact with theoperated organ. The first generation of those systems did not allow the surgeon to feel how muchforce he applies on the patient. Then with the second generation, force-feedback was introduced.Even if this technique introduces new problems such as control and stability, it also allows medicalgesture improvements. One commonly cited problem is the physiological motion of organs forexample.This thesis mainly focuses on the subject of physiological motion compensation in the context ofmedical force feedback teleoperation. The aim is to prove that the surgeon is able to teleoperate anorgan submitted to a physiological motion, and especially respiratory motion, without feeling thisdisturbance.First of all, the choice of the teleoperation scheme is important. In the literature, many can be found.To simplify this study, a two communication channel scheme has been chosen. This means that onlyone information is sent from the master to the slave and another one from the slave to the master. Inorder to fulfill a task in the free space and in contact with the environment, and to compensate aforce disturbance, the force-position scheme is selected. In this case, the slave sends forceinformation and the master sends back position information.[...

Téléchirurgie robotisée au contact d'organes mobiles

Computer aided medical gesture kept increasing during the last decade. This phenomenon is linkedto the evolution of surgical technique and specially the use of minimally invasive surgery. Thisrevolutionary technique allows reducing infection risks but introduces new problems. Indeed, thesurgeon has limited motion capability because the use of trocars. Therefore, robotics communityinterest has kept growing in order to develop specific robotized devices. One of the most currentstudied topics is teleoperation in which two robots are used: the master and the slave. In a medicalcontext, the master robot is manipulated by the surgeon and the slave robot is in contact with theoperated organ. The first generation of those systems did not allow the surgeon to feel how muchforce he applies on the patient. Then with the second generation, force-feedback was introduced.Even if this technique introduces new problems such as control and stability, it also allows medicalgesture improvements. One commonly cited problem is the physiological motion of organs forexample.This thesis mainly focuses on the subject of physiological motion compensation in the context ofmedical force feedback teleoperation. The aim is to prove that the surgeon is able to teleoperate anorgan submitted to a physiological motion, and especially respiratory motion, without feeling thisdisturbance.First of all, the choice of the teleoperation scheme is important. In the literature, many can be found.To simplify this study, a two communication channel scheme has been chosen. This means that onlyone information is sent from the master to the slave and another one from the slave to the master. Inorder to fulfill a task in the free space and in contact with the environment, and to compensate aforce disturbance, the force-position scheme is selected. In this case, the slave sends forceinformation and the master sends back position information.[...]Le geste médical assisté par ordinateur en salle d'opération n'a pas cessé de progresser lors de ladernière décennie. Ce phénomène est lié à l'évolution des techniques de chirurgie et notamment àl'apparition des techniques dites mini-invasives. Cette pratique bien que révolutionnaire car ellepermet de diminuer les risques d'infection a introduit de nombreux problèmes. En effet, lechirurgien est limité dans ses gestes à cause de la restriction de mouvement imposée par les trocarts.De ce fait, la communauté robotique s'est assez rapidement intéressée aux problématiques deschirurgiens afin de développer des dispositifs robotisés adaptés. Un des grands thèmes étudié estcelui de la téléopération. Cette dernière consiste à utiliser deux dispositifs robotisés communémentappelés: maître et esclave. Dans le contexte médical, le robot maître est manoeuvré par le chirurgienet le robot esclave est au contact de l'organe à opérer. La première génération de ces systèmes nepermettait pas au chirurgien de ressentir les efforts qu'il exerçait sur les organes du patient. Unedeuxième génération de dispositifs est apparue en introduisant le retour d'efforts. Au delà desproblèmes de stabilité et de commande rencontrés, de tels systèmes permettent de réduire lesimperfections du geste humain ou d'en augmenter les capacités. Une des gênes constatées par leschirurgiens lors de l'utilisation de tels systèmes est le mouvement physiologique des organes àopérer.Cette thèse s'est principalement focalisée sur le thème de la compensation des mouvementsphysiologiques dans le cadre de la téléopération avec retour d'efforts. L'objectif étant de démontrerqu'il est possible pour le chirurgien de téléopérer un organe soumis à des mouvementsphysiologiques, et notamment le mouvement respiratoire, sans qu'il ne ressente cette perturbation.[...