Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de récepteurs transmembranaires. Ils sont impliqués dans une large variété de processus physiologiques et par conséquent ils représentent une cible thérapeutique d'intérêt pour le développement de médicaments. Plusieurs études ont démontré que les RCPGs sont capables d'interagir entre eux pour former des complexes oligomériques. Cependant, leur existence in vivo et leur rôle fonctionnel reste sujet à débats. Afin de mieux appréhender ce phénomène, nous avons utilisé un RCPG de classe C comme modèle d'étude, le récepteur de l'acide γ-aminobutyrique (GABAB), qui est impliqué dans une grande variété de désordres neurologiques et psychiatriques. Son originalité réside dans le fait qu'il est un hétérodimère obligatoire composé de deux sous-unités : GABAB1 et GABAB2 (GB1 et GB2). La liaison de l'agoniste sur GB1 conduit à l'activation de GB2. Au cours de ma thèse, nous avons montré en utilisant une nouvelle approche biophysique basée sur un marquage fluorescent enzymatique appelé Snap-tag que, contrairement aux récepteurs métabotropiques du glutamate, le récepteur GABAB forme des dimères de dimères (tétramères). Cette organisation hétéro-oligomérique est assurée par des contacts stables entre les domaines extracellulaires des sous-unités GB1. De plus, nous avons apporté des données en faveur de l'existence physiologique de cet assemblage en utilisant des membranes de cerveau de rat et de souris. Dans une seconde partie, nous avons souhaité déterminer les conséquences fonctionnelles de cette organisation. Nos résultats suggèrent une efficacité de couplage à la protéine G réduite du récepteur GABAB lorsqu'il est associé en dimères de dimères. Collectivement, nos données rapportent pour la première fois, l'existence de larges complexes allostériques de RCPGs dans le cerveau. / The G-protein coupled receptors (GPCR) constitute the main family of transmembrane receptors. They are involved in many physiological processes and, as a consequence, they represent a therapeutic target of interest for the development of new drugs. Few studies have demonstrated that GPCRs are able to interact with each other to form oligomeric complexes. However, the existence in vivo and the functional interest of these oligomers remain a subject of intense debates. To address this issue, we have used a class C GPCR as a model, the γ-aminobutyrate B receptor (GABAB), which is involved in a wide variety of neurological and psychiatric disorders. This receptor has the particularity to be an obligatory heterodimer composed of two subunits GABAB1 and GABAB2 (GB1 and GB2). Agonist binding on GB1 leads to G-protein activation by GB2. During my thesis, we developed a new biophysical approach based on an enzyme-mediated fluorescent labeling calle d Snap-Tag and showed that, unlike metabotropic glutamate receptors, GABAB forms dimers of dimers (tetramers). This oligo-heterodimers organization is mediated via stable contacts between extracellular domains of GB1 subunits. Furthermore, we brought evidence of the physiological reality of this assembly using rat and mouse brain membranes. Then, we aimed at assessing what would be the functional rational of the GABAB dimer of heterodimers. Our results suggest that the GABAB receptor has a lower G protein-coupling efficacy when associated into dimers of dimers. Altogether, our data report for the first time, the existence of large allosteric GPCR complexes in the brain.

Les encéphalites limbiques à autoanticorps anti-CASPR2 sont des atteintes du système nerveux central caractérisées par des troubles de la mémoire et des crises d’épilepsie. La protéine CASPR2 (Contactin-associated protein-like 2), avec son partenaire TAG-1, est connue pour son rôle dans le rassemblement des canaux potassiques voltage-dépendants (Kv1.1 et Kv1.2) dans la région juxtaparanodale des nœuds de Ranvier ; régions essentielles pour la conduction rapide des messages nerveux. Par ailleurs, de plus en plus d’études suggèrent un rôle de CASPR2 dans la plasticité synaptique et l’excitabilité neuronale, en lien avec les symptômes observés chez les patients présentant des anticorps anti-CASPR2. Cependant, le rôle pathogénique des anticorps anti-CASPR2 dans les encéphalites limbiques reste loin d’être compris. Au cours de ma thèse, j’ai souhaité améliorer la connaissance des mécanismes pathologiques des anticorps anti-CASPR2 de patient dans l’encéphalite limbique auto-immune. Pour cela, j’ai déterminé les caractéristiques biologiques des anticorps anti-CASPR2, suggérant un rôle direct des anticorps sur la fonction de CASPR2 en ciblant les domaines N-terminaux de la protéine. De plus, j’ai identifié deux mécanismes d’action potentiels des anticorps anti-CASPR2 sur l’interaction entre CASPR2 et TAG-1 et sur l’expression des canaux Kv1.2 en surface. Ces travaux impliquent d’avantage les anticorps anti-CASPR2 dans la pathogénicité des encéphalites limbiques auto-immunes / Anti-CASPR2 autoimmune limbic encephalitis is a central nervous system disorder characterized by memory disorders and epilepsy. CASPR2 (Contactin-associated protein-like 2) with its partner TAG-1, is known for its role in the clusterisation of voltage-dependent potassium channels (Kv1.1 and Kv1.2) in the juxtaparanodal region of node of Ranvier; which are essential for the rapid conduction of nerve signals. In addition, an increasing number of studies suggest a role of CASPR2 in synaptic plasticity and neuronal excitability, in relation with the symptoms observed in patients with anti-CASPR2 antibodies. However, the pathogenic role of anti-CASPR2 antibodies in limbic encephalitis remains far from clear. During my thesis I wished to improve our understanding of the mechanisms mediated by anti-CASPR2 antibodies in limbic encephalitis. To this end, I determined the biological characteristics of anti-CASPR2 antibodies, suggesting a direct role of antibodies on CASPR2 function by targeting its N-terminal domains. Furthermore, I identified two potential mechanisms of anti-CASPR2 antibodies on CASPR2/TAG-1 interaction and on Kv1.2 cell surface expression. These works further implicate anti-CASPR2 antibodies in the pathogenicity of autoimmune limbic encephalitis

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

Covalent modification of histones is an important factor in the regulation of the chromatin structure implicated in DNA replication, repair, recombination, and transcription, as well as in RNA processing. In recent years, histone methylation has emerged as one of the key modifications regulating chromatin function. However, the mechanisms involved are complex and not well understood. Histone 3 lysine 4 (H3K4) methylation is deposited by a family of histone H3K4 methyltransferases (HMTs) that share a conserved SET domain. In mammalian cells, six family members have been characterized: Setd1a and Setd1b (the mammalian orthologs of yeast Set1) and four Mixed lineage leukemia (Mll) family HMTs, which share limited similarity with yeast Set1 beyond the SET domain. Several studies demonstrated that the H3K4 methyltransferases exist as multiprotein complexes. To functionally dissect H3K4 methyltransferase complexes, GFP tagging of the core subunit Ash2l and the complex-specific subunits Cxxc1 and Wdr82 (Setd1a/b complexes) Men1 (Mll1/2 complexes), and Ptip (Mll3/Mll4 complexes), was used. The fusion proteins were successfully expressed in mouse embryonic stem cells (ES cells), analyzed by confocal microscopy, Mass Spectrometry (MS) and ChIP-seq. Ptip was the only subunit able to bind mitotic chromatin. Additionally, both Ptip and Wdr82 were found to associate with cell cycle regulators, suggesting a possible role of the two proteins or respective complexes in cell cycle regulation. Mass Spectrometry revealed that Wdr82 and Ptip interact with members of he PAF complex, and ChIP-seq showed that Wdr82, Cxxc1 and Ptip positively modulate pluripotency genes. Thus, Setd1a/b and Mll3/4 complexes might act together in the regulation of embryonic stem cells identity. Protein pull downs identified at least one new Setd1a/b interactor, Bod1l that is orthologous to the yeast protein Sgh1, a component of the Set1C complex. Furthermore, our MS and ChIP-seq data suggested that only Mll2 complex binds to bivalent promoters, wheras Mll2 and Setd1a complexes might function together in a set of promoters.

Caspr2 est une molécule d'adhérence impliquée dans diverses pathologies neurologiques telles que l'autisme et l'encéphalite limbique (EL). Les mécanismes pathogéniques restent inconnus. Caspr2 est associé aux canaux potassiques Kv1.1/1.2 aux juxtaparanoeuds et au segment initial (SI). Dans un premier article publié dans Front. Cell. Neurosci. (2015), nous avons mis en évidence que les autoanticorps anti-Caspr2 issus de patients atteints d'EL ciblent majoritairement les neurones GABAergiques. Caspr2 est localisé au niveau des axones et des terminaisons présynaptiques inhibitrices dans les neurones d'hippocampe en culture. De plus, nous avons généré une chimère Caspr2-Fc soluble qui a permis d’identifier TAG-1 comme récepteur de Caspr2 localisé au niveau du compartiment somato-dendritique postsynaptique. Les neurones incubés avec des IgGs de patients, présentent une densité diminuée des clusters de Géphyrine marqueur des post-synapses inhibitrices. Ces anticorps sont d'isotype IgG4 et reconnaissent le plus communément des épitopes de la région Discoïdine-LaminineG1. Un blocage fonctionnel de Caspr2 au niveau synaptique permettrait de comprendre l'hyperexcitabilité associée à l'EL. Dans un second article en préparation, nous avons étudié la régulation de l’expression de Caspr2 au SI. Nous avons utilisé différentes constructions et identifié les domaines LamineG2-EGF1 extracellulaires de Caspr2 requis pour son expression axonale. De plus, les domaines cytoplasmiques de liaison aux protéines 4.1B et PDZ sont impliqués dans la rétention de Caspr2 et MPP2 au SI. Notablement, l'expression de TAG-1 ou ADAM22 induit des effets opposés sur l'expression de Caspr2 au SI. / Caspr2 is a cell adhesion molecule associated with neurologic diseases, such as autism spectrum disorders and limbic encephalitis. The underlying pathogenic mechanisms are still unknown. Caspr2 is associated with the voltage-gated potassium channels Kv1.1/1.2 localized at the axon initial segment (AIS) and the juxtaparanodes in myelinated axons. In a first paper published in Front. Cell. Neurosci. (2015), we characterized anti-Caspr2 autoantibodies from limbic encephalitis (LE) patients and showed that these autoantibodies preferentially targeted GABAergic neurons. Caspr2 was localized along axons and at the presynaptic terminals of inhibitory neurons in hippocampal cultures. Next, we generated a soluble Caspr2-Fc chimera to identify TAG-1 as a receptor for Caspr2 localized at the somato-dendritic compartment and post-synapses. We determined that neurons displayed decreased synaptic gephyrin clusters when incubated with anti-Caspr2 IgGs from LE patients. The autoantibodies mainly bound the N-terminal Discoidin-LamininG1 domains and were of the IgG4 isotype. They may exert functional blocking activity on inhibitory connections underlying the hyperexcitability linked with LE. In a second article in preparation, we examined the regulated expression of Caspr2 at the AIS using deletion and reporter constructs. We mapped the LamininG2 and EGF1 modules in the ectodomain as implicated in the axonal distribution of Caspr2 and the cytoplasmic motifs for binding to 4.1B and PDZ proteins as implicated in Caspr2 AIS retention together with MPP2. Strikingly, co-expression with TAG-1 and ADAM22 induced opposite effects on AIS Caspr2 distribution.

Cette thèse s’inscrit dans le cadre d’une collaboration entre l’IM2NP et la société Tagsys, dans le cadre du projet PACID-TEXTILE. L'objectif de la thèse est de créer un tag RFID UHF packagé, très robuste et qui puisse facilement s’intégrer de manière discrète dans les produits textiles. L’idée est de concevoir deux antennes couplées et adaptées aux puces choisies pour l’application. Ces deux antennes sont conçues afin de favoriser le couplage entre elles. La première entité est packagée afin de protéger la puce de l’environnement d’utilisation. Dans une première partie, l’antenne primaire est caractérisée. Le packaging fait l’objet d’études particulières pour répondre aux contraintes de robustesses liées aux domaines du textile (blanchisserie, produit chimique). Une seconde partie du travail vise à caractériser le fil conducteur de manière à en établir une modélisation la plus fidèle possible pour pouvoir être utilisé par les outils de simulation et de conception d’antennes. Des essais de résistances du fil sont également réalisés.Enfin, une troisième partie traite de la conception d'antennes. Les tags conçus doivent avoir une portée minimale de 4m, résister à la déformation et à l'empilement. Ces tags sont soumis à différents tests pour établir leurs performances. A l’aide des moyens de caractérisations disponible au laboratoire IM2NP (plate forme de pré certification RFID), un « Benchmarking » est réalisé sur les différents tags en vue de proposer une étiquette répondant au mieux aux exigences du cahier des charges. / This thesis is part of a collaboration between the company Tagsys and IM2NP, under the PACID-TEXTILE project. The aim of the thesis is to create a robust UHF RFID tag antenna that can be easily and discreetly integrated into textile products.The idea is to design two antennas electromagnetically coupled and adapted for selected chips. Both antennas are designed to facilitate the coupling between them. The first antenna is packaged in a robust material in order to protect the chip from laundry cycles. The second antenna is made up by electro-thread. The first part of the work is to characterize the packaged antenna and packaging material. The second part is to measure the conductivity of the used thread, to establish the most accurate model possible. Tests of wire resistance are also made. Toughness of each wire is also tested.The third part deals with the antenna design. The tags are designed to have a minimum range of 4m. They have to resist deformation and stacking. These tags are subjected to various tests to determine their performance. Using resources available in the laboratory IM2NP (RFID platform), a "Benchmarking " is carried on different tags to find the best that meet the requirements of the specifications.

Das Institut für Markscheidewesen und Geodäsie veranstaltete vom 05. bis 07. Mai 2021, in Freiberg, die gemeinsame Tagung Umwelt, Energie Rohstoffe & 21. Geokinematischer Tag 2021, um die neuesten Entwicklungen im Markscheidewesen und den angrenzenden Fachgebieten vorzustellen und zu diskutieren.

Covalent modification of histones is an important factor in the regulation of the chromatin structure implicated in DNA replication, repair, recombination, and transcription, as well as in RNA processing. In recent years, histone methylation has emerged as one of the key modifications regulating chromatin function. However, the mechanisms involved are complex and not well understood. Histone 3 lysine 4 (H3K4) methylation is deposited by a family of histone H3K4 methyltransferases (HMTs) that share a conserved SET domain. In mammalian cells, six family members have been characterized: Setd1a and Setd1b (the mammalian orthologs of yeast Set1) and four Mixed lineage leukemia (Mll) family HMTs, which share limited similarity with yeast Set1 beyond the SET domain. Several studies demonstrated that the H3K4 methyltransferases exist as multiprotein complexes. To functionally dissect H3K4 methyltransferase complexes, GFP tagging of the core subunit Ash2l and the complex-specific subunits Cxxc1 and Wdr82 (Setd1a/b complexes) Men1 (Mll1/2 complexes), and Ptip (Mll3/Mll4 complexes), was used. The fusion proteins were successfully expressed in mouse embryonic stem cells (ES cells), analyzed by confocal microscopy, Mass Spectrometry (MS) and ChIP-seq. Ptip was the only subunit able to bind mitotic chromatin. Additionally, both Ptip and Wdr82 were found to associate with cell cycle regulators, suggesting a possible role of the two proteins or respective complexes in cell cycle regulation. Mass Spectrometry revealed that Wdr82 and Ptip interact with members of he PAF complex, and ChIP-seq showed that Wdr82, Cxxc1 and Ptip positively modulate pluripotency genes. Thus, Setd1a/b and Mll3/4 complexes might act together in the regulation of embryonic stem cells identity. Protein pull downs identified at least one new Setd1a/b interactor, Bod1l that is orthologous to the yeast protein Sgh1, a component of the Set1C complex. Furthermore, our MS and ChIP-seq data suggested that only Mll2 complex binds to bivalent promoters, wheras Mll2 and Setd1a complexes might function together in a set of promoters.

Feierstunde aus Anlass des Tages der Deutschen Einheit und der Wiederbegründung des Freistaates Sachsen im Plenarsaal des Sächsischen Landtages.