Marqueurs pathologiques et pistes thérapeutiques dans la Sclérose Latérale Amyotrophique Sporadique

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting both upper and lower motor neurons.The disease is responsible for a progressive paralysis leading to death within 5 years after the symptom onset, with no curative treatment available so far. Studying pathophysiological processes, identifying new biomarkers of the disease, and developing new therapeutic prospects are currently the three main fields of ALS research. In this work, we first aimed to develop a new microscopy technique allowing super-resolutive imaging of the human brain, with the aim of studying tissue samples at the nanoscale level with specific immunostaining. Our results on brain samples from the Neuropathology Department of the University Hospital of Angers allowed us to characterize precisely the architecture of pathological protein aggregates observed in neurodegenerative disorders. Extending this approach to brain and spinal cord samples from patients affected with ALS will contribute to characterize the composition and organization of intra-neuronal lesions, and reveal underlying mechanisms involved in the diseases. Concomitantly, we aimed to identify new ALS biomarkers on the basis of pathophysiological mechanisms involved in the disease. Thanks to the expertise of the research unit MitoLab (Angers), we analyzed the ultrastructural organization, mitochondrial metabolism, stress response and collagen synthesis of controls- and patients derived skin fibroblasts. Our results highlighted alterations of purine and pyrimidine metabolisms, and an increase of the collagen synthesis in fibroblasts derived from ALS patients. These parameters could be used as markers for ALS diagnosis and prognosis, and may turn out to be valuable tools in clinical practice and therapeutic research. Finally, joining the Canadian research team of Pr Jean-Pierre JULIEN (CERVO Institute, Qc), we participated in the development of a new therapeutic approach in ALS based on the use of monoclonal antibodies targeting pathological TDP-43 aggregates in neurons. The treatment decreased TDP-43 proteinopathy in vitro in cultured neuronal cells, and in vivo in TDP-43A315T transgenic mice models. On the basis of these results, a new research project has started with the aim of humanizing the antibodies to consider immunotherapy for future therapeutic trialsLa sclérose latérale amyotrophique (SLA) sporadique est une pathologie neurodégénérative affectant les motoneurones, responsable d’une paralysie diffuse d’aggravation rapidement progressive aboutissant au décès des patients dans les 5 années suivant le diagnostic. Il n’existe à ce jour aucun traitement de la maladie. L’étude des lésions neuronales à l’origine de la maladie, l’identification de biomarqueurs de la SLA, et la mise au point de nouvelles approches thérapeutiques sont les domaines de recherche sur lesquels les efforts sont actuellement concentrés. Le premier objectif de ce travail de thèse a été d’optimiser l’analyse histologique des lésions neuronales observées chez les patients atteints de maladies neurodégénératives grâce à l’utilisation des nouvelles techniques d’imagerie super-résolutive, qui permettent un immunomarquage spécifique à l’échelle nanoscopique. Nos premiers résultats obtenus à partir de la banque de cerveaux du département de Neuropathologie du CHU d’Angers ont permis de caractériser avec précision les agrégats protéiques observés dans les processus de neurodégénérescence, ouvrant un nouveau champ pour l’exploration du tissu cérébral et médullaire des patients atteints de SLA. Le second travail de cette thèse a porté sur l’identification de biomarqueurs de la SLA à partir des hypothèses physiopathologiques impliquant la protéine TDP-43, des déficits mitochondriaux et les anomalies du cytosquelette. Grâce à l’expertise de l’équipe MitoLab (Angers), nous avons exploré l’ultrastructure cellulaire, le métabolisme énergétique et protéique, l’architecture du réseau mitochondrial, et la réponse au stress d’une biocollection de fibroblastes issue de patients atteints de SLA sporadique constituée prospectivement. Nos données ont permis d’objectiver une altération de certaines voies métaboliques, ainsi qu’une augmentation de la synthèse du collagène. Ces résultats font de ces deux paramètres de potentiels marqueurs diagnostique et pronostique de la maladie. La 3ème approche expérimentale de cette thèse a été menée au sein de l’équipe du Professeur J.-P. JULIEN (Institut CERVO Canada). Nous avons étudié l’adressage et l’action d’anticorps ciblant les agrégats de protéine TDP-43 au niveau du système nerveux central. Nos résultats obtenus par immunomarquages fluorescents et études histologiques ont montré l’efficacité de l’adressage du traitement ainsi qu’une diminution des lésions chez des souris transgéniques porteuses de la mutation TDP-43A315T. Sur la base de ces résultats, des projets complémentaires ont été initiés dans la perspective de tester l’efficacité cette nouvelle approche chez l’homme

Pathological markers and therapeutic prospects in Sporadic Amyotrophic Lateral Sclerosis

La sclérose latérale amyotrophique (SLA) sporadique est une pathologie neurodégénérative affectant les motoneurones, responsable d’une paralysie diffuse d’aggravation rapidement progressive aboutissant au décès des patients dans les 5 années suivant le diagnostic. Il n’existe à ce jour aucun traitement de la maladie. L’étude des lésions neuronales à l’origine de la maladie, l’identification de biomarqueurs de la SLA, et la mise au point de nouvelles approches thérapeutiques sont les domaines de recherche sur lesquels les efforts sont actuellement concentrés. Le premier objectif de ce travail de thèse a été d’optimiser l’analyse histologique des lésions neuronales observées chez les patients atteints de maladies neurodégénératives grâce à l’utilisation des nouvelles techniques d’imagerie super-résolutive, qui permettent un immunomarquage spécifique à l’échelle nanoscopique. Nos premiers résultats obtenus à partir de la banque de cerveaux du département de Neuropathologie du CHU d’Angers ont permis de caractériser avec précision les agrégats protéiques observés dans les processus de neurodégénérescence, ouvrant un nouveau champ pour l’exploration du tissu cérébral et médullaire des patients atteints de SLA. Le second travail de cette thèse a porté sur l’identification de biomarqueurs de la SLA à partir des hypothèses physiopathologiques impliquant la protéine TDP-43, des déficits mitochondriaux et les anomalies du cytosquelette. Grâce à l’expertise de l’équipe MitoLab (Angers), nous avons exploré l’ultrastructure cellulaire, le métabolisme énergétique et protéique, l’architecture du réseau mitochondrial, et la réponse au stress d’une biocollection de fibroblastes issue de patients atteints de SLA sporadique constituée prospectivement. Nos données ont permis d’objectiver une altération de certaines voies métaboliques, ainsi qu’une augmentation de la synthèse du collagène. Ces résultats font de ces deux paramètres de potentiels marqueurs diagnostique et pronostique de la maladie. La 3ème approche expérimentale de cette thèse a été menée au sein de l’équipe du Professeur J.-P. JULIEN (Institut CERVO Canada). Nous avons étudié l’adressage et l’action d’anticorps ciblant les agrégats de protéine TDP-43 au niveau du système nerveux central. Nos résultats obtenus par immunomarquages fluorescents et études histologiques ont montré l’efficacité de l’adressage du traitement ainsi qu’une diminution des lésions chez des souris transgéniques porteuses de la mutation TDP-43A315T. Sur la base de ces résultats, des projets complémentaires ont été initiés dans la perspective de tester l’efficacité cette nouvelle approche chez l’homme.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting both upper and lower motor neurons.The disease is responsible for a progressive paralysis leading to death within 5 years after the symptom onset, with no curative treatment available so far. Studying pathophysiological processes, identifying new biomarkers of the disease, and developing new therapeutic prospects are currently the three main fields of ALS research. In this work, we first aimed to develop a new microscopy technique allowing super-resolutive imaging of the human brain, with the aim of studying tissue samples at the nanoscale level with specific immunostaining. Our results on brain samples from the Neuropathology Department of the University Hospital of Angers allowed us to characterize precisely the architecture of pathological protein aggregates observed in neurodegenerative disorders. Extending this approach to brain and spinal cord samples from patients affected with ALS will contribute to characterize the composition and organization of intra-neuronal lesions, and reveal underlying mechanisms involved in the diseases. Concomitantly, we aimed to identify new ALS biomarkers on the basis of pathophysiological mechanisms involved in the disease. Thanks to the expertise of the research unit MitoLab (Angers), we analyzed the ultrastructural organization, mitochondrial metabolism, stress response and collagen synthesis of controls- and patients derived skin fibroblasts. Our results highlighted alterations of purine and pyrimidine metabolisms, and an increase of the collagen synthesis in fibroblasts derived from ALS patients. These parameters could be used as markers for ALS diagnosis and prognosis, and may turn out to be valuable tools in clinical practice and therapeutic research. Finally, joining the Canadian research team of Pr Jean-Pierre JULIEN (CERVO Institute, Qc), we participated in the development of a new therapeutic approach in ALS based on the use of monoclonal antibodies targeting pathological TDP-43 aggregates in neurons. The treatment decreased TDP-43 proteinopathy in vitro in cultured neuronal cells, and in vivo in TDP-43A315T transgenic mice models. On the basis of these results, a new research project has started with the aim of humanizing the antibodies to consider immunotherapy for future therapeutic trial

Characterization of Cells Interactions with Patterned Azopolymer-Based Materials using SEM, AFM and Video Microscopy

International audienc

Metabo-lipidomics of Fibroblasts and Mitochondrial-Endoplasmic Reticulum Extracts from ALS Patients Shows Alterations in Purine, Pyrimidine, Energetic, and Phospholipid Metabolisms

International audienceAmyotrophic lateral sclerosis (ALS) is characterized by a wide metabolic remodeling, as shown by recent metabolomics and lipidomics studies performed in samples from patient cohorts and experimental animal models. Here, we explored the metabolome and lipidome of fibroblasts from sporadic ALS patients (n = 13) comparatively to age- and sex-matched controls (n = 11), and the subcellular fraction containing the mitochondria and endoplasmic reticulum (mito-ER), given that mitochondrial dysfunctions and ER stress are important features of ALS patho-mechanisms. We also assessed the mitochondrial oxidative respiration and the mitochondrial genomic (mtDNA) sequence, although without yielding significant differences. Compared to controls, ALS fibroblasts did not exhibit a mitochondrial respiration defect nor an increased proportion of mitochondrial DNA mutations. In addition, non-targeted metabolomics and lipidomics analyses identified 124 and 127 metabolites, and 328 and 220 lipids in whole cells and the mito-ER fractions, respectively, along with partial least-squares-discriminant analysis (PLS-DA) models being systematically highly predictive of the disease. The most discriminant metabolomic features were the alteration of purine, pyrimidine, and energetic metabolisms, suggestive of oxidative stress and of pro-inflammatory status. The most important lipidomic feature in the mito-ER fraction was the disturbance of phosphatidylcholine PC (36:4p) levels, which we had previously reported in the cerebrospinal fluid of ALS patients and in the brain from an ALS mouse model. Thus, our results reveal that fibroblasts from sporadic ALS patients share common metabolic remodeling, consistent with other metabolic studies performed in ALS, opening perspectives for further exploration in this cellular model in ALS

GNSS Timing Receiver Performance in Urban Canyons

Time synchronization is critical for the operation of radio base stations (RBS) in telecommunication companies. Global navigation satellite system (GNSS) is an existing technology to provide precise timing information to distributed RBSs. GNSS timing receiver is used for providing higher timing accuracy than normal GNSS receiver in this synchronization domain.In this thesis, an experiment method for GNSS timing receiver performance in urban canyon has been designed and implemented to evaluate information and the quality of the one pulse per second (1PPS) signal generated by two different GNSS timing receivers. Multi-path signals and the gathered satellite geometry caused by poor sky visibility is identified as the main influential factors to the performance of the GNSS timing receivers. A mathematical model has been built for estimating the multi-path effect. GNSS planning tools are used to simulate the number of line-of-sight (LOS) satellites and Dilution of Precision (DOP) value.Sentinel is a 1PPS signal analyzing equipment from Calnex. Sentinel has an embedded rubidium clock, GNSS antenna, and receiver, and it can produce 1PPS signals to be used as a reference. In this report, we installed our GNSS antenna of Sentinel on the roof and test GNSS antenna in two specified positions representing urban canyon and rooftop. Recorded NMEA messages from GNSS receiver can help us to study the number of visible satellites, PDOP value and multi-path signals in realistic situations.The results show how the noise and time phase of 1PPS signals will be influenced in urban canyons. Since, the geometry of used satellites is similar to the rooftop situation, the multi-path effect of signals is identified as the main reason of this difference.This information is useful when telecommunication companies want to install their radio base station in urban canyons. It will help Ericsson to understand how their GNSS timing receiver is working and how the urban canyon will influence its performance.Tidssynkronisering är kritisk för driften av radiobasstationer (RBS) i telekommunikationsföretag. Global Navigation Satellite System (GNSS) är en befintlig teknik för att ge exakt tidsinformation till distribuerade basstationer. GNSS-baserade tidsmottagare används för att ge högre timing-noggrannhet än vanlig GNSS mottagare i denna synkroniseringsdomän. I denna avhandling har en experimentmetod för GNSS-timingmottagarnas prestanda i urban canyon utformats och implementerats för att utvärdera den genererade informationen och kvaliteten på en puls per sekund-signal (1PPS). Flervägssignaler och den samlade satellitgeometrin som orsakas av dålig himmelsynlighet identifieras som de mest inflytelserika faktorerna för GNSS-tidsmottagarnas prestanda. En matematisk modell har donstruerats för att estimera multi-path-effekten. GNSS-planeringsverktyg används för att simulera antalet LOS-satelliter och DOP-värde (Dilution of Precision). Sentinel är en 1PPS signalanalysutrustning från Calnex. Sentinel har en inbyggd rubidiumklocka, GNSS-antenn och mottagare, och den kan producera 1PPS-signaler som ska användas som referens. I den här rapporten installerade vi vår GNSS-antenn på Sentinel på taket och GNSS-testantennen i två angivna positioner som representerar urban canyon och tak. Inspelade NMEA-meddelanden från GNSS-mottagare kan hjälpa oss att studera antalet synliga satelliter, PDOP-värde och flervägssignaler i realistiska scenarier. Resultatet visar att ljud- och tidsfasen för 1PPS-signaler påverkas i urban canyons. Eftersom satellitgeometrin liknar den för antenner placerade på taket, så är identifieras flervägsutbredningen som huvudorsak för denna skillnad. Denna information är användbar när telekommunikationsföretag vill installera sina radiobasstationer i urban canyons. Det kommer att hjälpa Ericsson att förstå hur deras GNSS-timingmottagare arbetar och hur urban canyon påverkar dess prestanda

Transactive response DNA-binding protein 43 is enriched at the centrosome in human cells

Abstract The centrosome, as the main microtubule organizing centre, plays key roles in cell polarity, genome stability and ciliogenesis. The recent identification of ribosomes, RNA-binding proteins and transcripts at the centrosome suggests local protein synthesis. In this context, we hypothesized that TDP-43, a highly conserved RNA binding protein involved in the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, could be enriched at this organelle. Using dedicated high magnification sub-diffraction microscopy on human cells, we discovered a novel localization of TDP-43 at the centrosome during all phases of the cell cycle. These results were confirmed on purified centrosomes by western blot and immunofluorescence microscopy. In addition, the co-localization of TDP-43 and pericentrin suggested a pericentriolar enrichment of the protein, leading us to hypothesize that TDP-43 might interact with local mRNAs and proteins. Supporting this hypothesis, we found four conserved centrosomal mRNAs and 16 centrosomal proteins identified as direct TDP-43 interactors. More strikingly, all the 16 proteins are implicated in the pathophysiology of TDP-43 proteinopathies, suggesting that TDP-43 dysfunction in this organelle contributes to neurodegeneration. This first description of TDP-43 centrosomal enrichment paves the way for a more comprehensive understanding of TDP-43 physiology and pathology

Efficient water use through environmentally sound hydroponic production of high quality vegetables for domestic and export markets in Mediterranean countries: final report

This project will develop the integrated low cost technology "ECOPONICS" as a hydroponics system with high water use efficiency (WUE) for water savings and reuse of drainage water. ECOPONICS will produce for domestic and export markets quality fruity vegetables with high nutritional and aroma values to be grown under the unique environmental and climatic conditions of the Mediterranean region, suited to the needs of smallholders as well as horticultural enterprises