This dissertation comprises identifying the structural determinants of binding selectivity as demonstrated in three systems. The first system involves the structure determination of Keap1-small molecule fragment complexes to locate binding surfaces. The second system involves the structural determination of a NEMO/IKKbeta complex to serve as a platform for future fragment binding validation studies. The third system involves the structural investigation of a bacterial phosphoglycosyltransferase found in Campylobacter concisus to find the active site. Keap1 binding of Nrf2 is a regulatory mechanism to inhibit the transcription factor activity of Nrf2 to upregulate Nucleoporin p62 (p62). Nucleoporin p62 is a regulator of tau protein aggregates in Alzheimer's disease. The determination of binding hot spots in the Keap1 active site could serve as a starting point for the development of inhibitors as a treatment method for Alzheimer’s disease. To achieve this, I have developed a crystal form of Keap1 that allows for fragment-based study of binding in the active site via small molecule fragment screening and X-ray crystallography. Analysis of collected data has resulted in the solution of four structures, one containing a peptide fragment and three containing small molecule fragments that occupy a region of binding within the Keap1 active site, demonstrating the utility of the crystal form and affording information on binding hot spots. Nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) is a transcription factor and has been linked to cancer, inflammation, and immune dysfunction. The enzyme complex IκB kinase (IKK) is a regulator of NF-κB and consists of three subunits: IKK-α, IKK-β, and NEMO. If NEMO activity is abrogated, IKK is unable to activate NF-κB, making it a promising therapeutic target. My research has found crystallization conditions and performed trials of phase determination on an N terminal IKKβ-binding construct of NEMO containing previously uncharacterized regions of this protein. Glycosylation is a commonly occurring post-translational modification that affects a number of processes including protein folding, trafficking, cell-cell interactions and host immune response. The phosphoglycosyl transferase PglC is an essential part of the Campylobacter glycosylation pathway and a possible antibacterial target. My research determined the crystallization conditions and has developed complexes and protein constructs for phase determination of this single-pass transmembrane protein and will in the future provide a platform for structure-based inhibition of this protein.

Macrocycles (MCs) have become an increasing area of interest for drug design efforts, especially for classically “difficult” targets like protein-protein interactions (PPIs). And although there are many examples of successful MC drugs derived from natural products, there is little information about the characteristics of compounds with effective pharmacological and physicochemical properties. In this dissertation, I describe the development of design guidelines for new MC drugs based on a representative set of known inhibitor MCs and their target proteins. Analysis of both the individual MC structures and their interactions in the protein complex resulted in identification of several structural and physicochemical features likely to promote favorable binding and bioavailability. Additionally, investigation of the binding sites on the proteins suggest that MCs can bind targets conventionally considered “non-druggable,” strengthening the argument for exploring MC compounds to increase the druggable target space. Furthermore, this work includes the application of the proposed design guidelines to the development of synthetic MC libraries for a PPI target, the NEMO/IKKβ complex.

ACQUISITION OF PROPERTY FROM UNLAWFUL POSSESSOR In my diploma thesis I deal with the issue of the acquisition of property from unlawful possessor as an exception to the traditional civil law principle nemo plus iuris ad alium transferre potest quam ipse habet (also called as nemo dat rule), the principle, which lays down that no one can transfer more rights than he has himself. The topic is extremely actual since as of 1st of January 2014 the Act No. 89/2012 Coll., The Civil Code, which - in comparison to the previous legislation - provides diametric changes in the understanding and in the legislation of the property acquisition from unlawful possessor, became effective. Until the adoption of the new Civil Code, the legislation in this area was inadequate and due to dichotomy between Civil and Commercial Code it was also fragmented. The thesis is composed of an introduction, five main chapters (which are further divided into sub-chapters) and a conclusion. Each chapter deals with different aspects of acquisition of property from unlawful possessor. In the first introductory part I give my thesis some theoretical fundamentals and define basic terminology used in this paper: property, good faith. I'm also trying to analyze nemo plus iuris principle and conflict of protection of property rights with the...

Incontinentia pigmenti (IP, OMIM # 308300) est une maladie génétique liée au chromosome X qui est létale chez les garçons. Chez les filles, une dermatose débute peu après la naissance et évolue selon une séquence complexe d’événements associant de l’inflammation, de l’hyperprolifération cellulaire et de l’apoptose. En dehors de ce problème cutané, les patientes IP peuvent aussi souffrir d’anomalies oculaires, dentaires et cérébrales. Les défauts cérébraux affectent environ 30% des patientes et se caractérisent par de l’épilepsie et des troubles cognitifs et/ou moteurs. Le gène causant IP code pour la protéine NEMO, un composant essentiel de la voie de signalisation NF-kB, qui régule les processus immunitaires, inflammatoires, ainsi que la prolifération et la mort cellulaire. Chez approximativement 70% des patientes IP, le même réarrangement chromosomique élimine presque intégralement le gène NEMO, générant une perte d’activation de la voie NF-kB. L’invalidation du gène Nemo chez la souris (femelles Nemo +/-) fournit un modèle pour l’étude de la dermatose associée à IP en récapitulant les événements cutanés observés chez les patientes.Dans le but de mieux définir les anomalies cérébrales des patientes IP ainsi que leur origine qui reste obscure, nous avons analysé le cerveau des souris Nemo +/-. Nous montrons ici, en utilisant un protocole d’IRM adapté à des cerveaux de sept jours (P7), que des anomalies sont détectées dans une fraction des échantillons. Ces anomalies sont hétérogènes et localisées de manière aspécifique, comme chez l’Homme. Elles incluent des zones hémorragiques diffuses, des cavités et de l’atrophie du corps calleux. Cela démontre l’utilité du système murin Nemo +/- pour étudier également les défauts cérébraux des patientes IP. De manière intéressante, ces lésions cérébrales peuvent être détectées avant la naissance, au jour 18 du développement, et ne sont pas causées par un dysfonctionnement neuronal, astrocytaire ou des oligodendrocytes. Utilisant une approche immunitaire couplée à une analyse in toto de cerveaux P7 après mise en transparence (Technique 3DISCO), nous avons détecté des anomalies vasculaires qui suggèrent que ce compartiment serait à l’origine des défauts cérébraux des patientes IP. / Incontinentia pigmenti (IP, OMIM # 308300) is an X-linked genetic disease which is lethal in boys. In girls, it causes a skin disease that begins soon after birth and evolves along a complex sequence of events involving inflammation, cell hyperproliferation and apoptosis. IP patients can also suffer from ocular, dental and brain anomalies. Brain defects affect about 30% of patients and are characterized by epilepsy and/or cognitive/motor disorders. The IP-causing gene encodes the protein NEMO, an essential component of the signaling pathway NF-kB that regulates immunity, inflammation, proliferation and cell death process. In approximately 70% of IP patients, the same chromosomal rearrangement eliminates almost completely the NEMO gene, generating a loss of activation of the NF-kB pathway. Invalidation of the gene in Nemo mice (Nemo +/- females) provides a model for the study of the cutaneous events observed in IP patients.In order to better define the IP brain anomalies of patients and their origin, which remains obscure, we analyzed the brains of Nemo +/- mice. We show here, using a MRI protocol adapted for brain of seven days (P7), that anomalies are detected in a fraction of the samples. These anomalies are heterogeneous and localized in a non-specific manner, as in humans. They include diffuse hemorrhagic areas, cavities and atrophy of the corpus callosum. This demonstrates the usefulness of the murine system Nemo +/- to also study the IP patient's brain defects. Interestingly, these brain lesions can be detected before birth, at day 18 of development, and are not caused by a neuron, astrocyte or oligodendrocyte dysfunction. Using an immune approach coupled to an in toto analysis of P7 brain, after treating them by chemicals to induce their transparency (iDISCO technique), we detected vascular anomalies, suggesting that this compartment causes the brain defects in IP patients.

L'objectif général de cette thèse est de contribuer à l'avancement de la connaissance de l'impact de l'activité àméso-échelle du Courant Nord (CN) au large du Var sur sa circulation en aval et des interactions de ce courantde bord avec la dynamique côtière, particulièrement dans une baie semi-fermée peu profonde : la baie deHyères. Ces travaux se sont appuyés sur deux configurations numériques réalistes à haute résolution basées surle code NEMO et emboîtées avec AGRIF : une première de la façade méditerranéenne française à unerésolution spatiale de 1,2 km et une seconde le long des côtes varoises à 400 m de résolution.La comparaison des simulations obtenues avec des observations (radar HF, ADCP, glider, SST satellite) apermis de confirmer le réalisme des configurations, et de montrer l'apport d'une résolution de l'ordre de 400 msur la dynamique dans une baie mais également sur le CN et son écoulement en aval.Enfin, une étude de paramétrisation de l'advection horizontale et du mélange vertical a permis d'améliorerl'impact d'un downscaling dans la région d'étude, et particulièrement concernant la représentation de lacirculation au sein de la baie semi-fermée. / The main objective of this work is to improve our knowledge on the impact of the Northern Current (NC)mesoscale activity off the Var coast on its downstream flow and on the links between this boundary current andthe coastal dynamics, particularly in a semi-enclosed bay and shallow: the bay of Hyères. To do so, twonumerical realistic configurations at high-resolution were used. Based on the NEMO code and nested withAGRIF, the first one covers the French Mediterranean coasts at 1,2 km and the second one covers the Varcoasts with a spatial resolution of 400 m.Simulations comparisons with ocean observations (HF radar, ADCP, glider, satellite SST) confirm therealism of the configurations, and show the contribution of a 400 m spatial resolution on the simulateddynamics in the bay but also on the NC and its downstream flow.Finally, a parametrization study on the horizontal advection terms and vertical mixing provide an improvementof the impact of a downscaling in the studied area, and particularly for the simulated dynamics in the semienclosedbay.

Erwerb vom Nichtberechtigten Diese Arbeit beschäftigt sich mit der Problematik des Erwerbs des Eigentums vom Nichtberechtigten, das ein Durchbrechen des Prinzips, dass niemand mehr Recht übertragen kann, als er selbst hat, vorstellt. Das Problem wird sowohl aus der juristisch-theoretischen Sicht als auch aus der juristisch-philosophischen behandelt, wenn der Konflikt zwischen dem Schutz des guten Glaubens, des Verkehrsschutzes und dem Schutz des Eigentums, als ein das in der Charta verankerten Grundrechte, gelöst wurde. Das gegenwärtige Bürgerliche Gesetzbuch führt das Institut des gutgläubigen Erwerbs wieder in die tschechische Rechtsordnung ein und das sowohl für die Rechtsgeschäfte zwischen Kaufleuten als auch zwischen Zivil-Personen. Zu dieser Problematik wurde schon vieles verfasst. Nichts desto trotz gibt es immer noch Streitfragen in diesem Bereich. Im Zusammenhang mit der neuen Regelung entstand auch eine Reihe von Teilproblemen, auf die hinzudeuten ist. Einige von ihnen wurden unter anderem durch einige Mängel in der tschechischen Regelung verursacht, die in den Kontrast zu der besser durgeführten deutschen Regelung gestellt wurden. Die folgende Arbeit ist in vier Hauptkapitel gegliedert, die weiter in Unterkapiteln geteilt wurden. Das erste Kapitel widmet sich den theoretischen Grundbegriffen, wie...

The NF-kB signalling pathway is a critical mediator of the cellular responses to inflammatory cytokines. The IκB kinase (IKK) complex, which is composed of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ/NEMO) acts as the master regulator of NF-κB transcription factor activity. Seminal genetic studies in knockout (KO) mouse embryonic fibroblasts (MEFs) have defined two pathways of NF-κB activation; a canonical pathway, activated in response to cytokines such as TNFα/IL-1β, that requires NEMO and predominantly IKKβ catalytic activity; and a non-canonical pathway, activated in response to a subset of TNF-family cytokines, which requires IKKα and NIK kinase. We have generated and validated CRISPR-Cas9 IKKα, IKKβ and IKKα/β DKO HCT116 colorectal cancer cell lines to interrogate novel functions of the I kappa B kinases in colorectal cancer, including the relative contributions of these kinases to the activation of NF-κB signalling pathways downstream of TNFα induction. Contrary to the seminal studies in KO MEFs, IKKα appeared to make a more significant contribution to canonical NF-κB induction in these cells than IKKβ. Western blot studies demonstrated that both IKKs contributed to the phosphorylation and degradation of IκB and the phosphorylation of the NF-κB subunit, p65 at Serine 536. However, high-content immunofluorescence studies demonstrated that IKKα KO cells were defective in TNFα-induced nuclear translocation of p65 compared to WT and IKKβ KO cells. Additionally, NF-κB-driven luciferase reporter assays showed that IKKα, but not IKKβ, KO cells exhibited significantly reduced NF-κB-dependent gene expression following TNFα stimulation. We also have evidence to suggest that the phosphorylation site at Serine 468 on p65, previously defined as an IKKβ-dependent site, is in-fact an IKKα-dependent site in these cells. Furthermore, IKKα knockout revealed a potentially important role for IKKα activity in preventing the stabilisation of NIK protein following prolonged TNFα stimulation. RNA sequencing analysis of wild-type, IKKα KO, IKKβ KO and IKKα/β DKO cells stimulated with TNFα was performed to identify genes whose expression were differentially deregulated by IKK KO. These analyses confirmed the importance of IKKα for canonical NF-κB gene expression. Furthermore, IKKβ knockout had unexpected effects on the expression of a broad range of genes involved in chromatin organisation, cytoskeletal organisation, mitotic cell cycle control and the DNA damage response. During the characterisation of IKK KO cells it was discovered that the expression of NEMO was downregulated at the protein, but not mRNA level by approximately 50% in IKKα KO cells and 90% in IKKα/β DKO cells. IKKβ KO cells, meanwhile, exhibited wild-type NEMO expression. Emetine-chase and radioactive pulse chase labelling experiments demonstrated that the half-life of NEMO in IKKα and IKKα/β DKO cells was significantly shortened due to enhanced proteasomal turnover. Bioinformatics analyses predicted significant regions of intrinsic structural disorder within NEMO, particularly at the N- and C-termini, the former of which overlapped with the IKK binding domain. On this basis, the susceptibility of NEMO to in vitro degradation by the 20S proteasome was examined, with NEMO proving be a highly effective substrate of the 20S proteasome. Importantly, IKKα and IKKβ were both shown to protect NEMO from proteasomal degradation, leading us to propose a model whereby interaction with IKK kinase subunits sequesters/masks intrinsically disordered regions in NEMO that would otherwise make NEMO a highly effective substrate for ubiquitin-dependent and/or ubiquitin-independent proteasomal degradation. BMS-345541 is a commercially available allosteric inhibitor of IKKβ that has been used extensively in numerous studies, including a report that proposed novel functions for IKKβ in mitotic cell cycle progression (Blazkova et al., 2007). Similar antiproliferative effects to those reported by Blazkova et al., were observed during the characterisation of a novel ATP-competitive inhibitor of IKKβ, AZD2230. In depth characterisation of the selectivity of AZD2230 and BMS-345541, however, revealed that the antiproliferative effects of AZD2230 and BMS-345541 are, in fact, due to off-target inhibition, potentially at the level of RNA Polymerase II C-terminal domain phosphorylation, and hence general transcription. Collectively, these studies reveal novel functions of the IKK kinases in NF-κB signalling and inform therapeutic strategies for targeting chronic canonical NF-κB activation in colorectal cancer.

Protein-protein interactions (PPI) mediate numerous biological processes, but inhibiting these interactions with small molecules has been difficult to achieve in drug discovery. A small number of successes have shown that some PPIs are amenable to inhibition. Computational algorithms designed to measure the druggability of PPIs have been developed based on these successes. These algorithms have identified the interaction between the NF-κB essential modulator (NEMO) and I𝜅B kinase β (IKKβ) as a candidate for inhibition. Furthermore, in vivo peptide-based inhibition of the NEMO-IKKβ interface has shown benefits in attenuating the NF-𝜅B response in cellular and animal models. In addition to its intrinsic interest as a drug target, developing inhibitors against the NEMO/IKKβ interaction may help in the development of improved methods for PPI inhibition. In this thesis, the production of full-length, recombinant forms of soluble NEMO is described. This protein was used in a variety of biochemical assays to advance our understanding of NEMO structure and function. Furthermore, a fluorescence anisotropy (FA) assay was developed to screen for compounds inhibiting the NEMO/IKKβ PPI. Hits from the FA assay were tested by several methods to confirm true inhibition. Additionally, the FA assay was used to accurately measure the affinity of NEMO for IKKβ and to assess the degree of cooperativity in IKKβ binding. The oligomeric state of NEMO has been characterized through the development of a panel of NEMO cysteine to alanine mutants, using polyacrylamide gel electrophoresis analysis, analytical ultracentrifugation, and fluorescence anisotropy. These data represent the first comprehensive characterization of full-length human NEMO, and may provide a path toward development of drug-like inhibitors of the NEMO/IKKβ interaction.

Les mers Indonésiennes sont le siège de très fort courants de marée qui interagissent avec la topographie pour créer des ondes internes à la fréquence de la marée que l'on appelle marée interne. Certaines d'entres elles, vont se propager et se dissiper dans l'océan intérieur. Le mélange associé provoque la remontée d'eau plus froide et plus riche en nutriments en surface qui influence le climat tropical et toute la chaine des écosystèmes marins. Surveiller les ressources marines est l'objectif du projet INDESO, dont cette thèse fait partie. Prendre en compte le mélange induit par la marée interne n'est pas facile. En effet, le résoudre entièrement n'est pas possible car les échelles concernées par les différents processus des ondes internes varient de plusieurs milliers de kilomètres (propagation) à quelques centimètres/millimètres (dissipation). De plus en plus de scientifiques introduisent le forçage de la marée dans leur modèle mais sans savoir où va l'énergie et comment les ondes sont dissipées. Dans cette thèse nous cherchons à proposer des outils et des débuts de réponses pour participer à cette meilleure compréhension de la dissipation des ondes internes dans le modèle numérique d'océan NEMO. Nous proposons certaines quantifications que nous comparons aux anciennes paramétrisations. J'ai, tout d'abord, contribué à une étude d'INDESO sur la validation de NEMO grâce à de nombreux jeu de données. Ensuite, j'ai cherché à quantifier et à qualifier le mélange induit par l'introduction de la marée explicite dans le modèle, ainsi que son impact sur les masses d'eau. (c'est redit plus loin)Il produit un refroidissement de surface de 0.3°C avec des maxima atteignant 0.8°C au niveau des sites de génération des ondes internes. Le modèle reproduit 75% de l'énergie attendue de génération des ondes internes, en bon accord avec des études précédentes. L'essentiel de la dissipation a lieu horizontalement (19GW) est proche de celle induite par la paramétrisation couramment utilisée (16GW), alors que, dans la réalité, on s'attend principalement à une dissipation réalisée grâce à des processus verticaux. Le modèle, au dessus des zones de génération, est de façon surprenante en très bon accord avec les mesures in situ de dissipation obtenues lors de la campagne INDOMIX. Par contre, dans les régions distantes des sources de génération, le modèle surestime le mélange par rapport aux observations d'INDOMIX. Dans la dernière partie de cette thèse j'ai commencé à apporter des éléments de réponse à la quantification des puits d'énergie dans NEMO. J'ai pour cela travaillé avec le cas test COMODO, qui est une section d'un fluide stratifié constituée d'une plaine abyssale, d'un talus et d'un plateau, forcée par la marée et sans friction de fond. Le modèle T-UGOm, un modèle hydrodynamique de marée, est comparé au modèle NEMO. Dans ce cadre, nous avons développé une méthode originale pour séparer la marée barotrope de la marée barocline. Elle repose sur la projection en modes normaux. Cette méthode donne, à première vue, des résultats similaires à ceux obtenus grâce à la méthode plus classique de soustraction par la moyenne verticale. Cependant, lorsque l'on regarde plus en détail les diagnostiques d'énergie on trouve que la méthode de projection en modes normaux offre une plus grande précision et un plus grand réalisme pour séparer la marée barotrope de la marée barocline. Plus on monte dans des modes élevés plus les longueurs ondes se raccourcissent dans NEMO par rapport à T-UGOm. Par ailleurs, NEMO dissipe la marée barotrope dans la plaine abyssale, alors qu'il n'y a explicitement pas de friction. Ce ne peut pas être la diffusion verticael ou horizontale qui est à l'œuvre ici, car il n'y a pas de raison physique pour une diffusion sur un fond plat. Le meilleur candidat pour expliquer cette diffusion serait le couplage 2D/3D du time splitting de NEMO. Un travail est en cours pour appliquer cette méthode sur l'ensemble de l'archipel Indonésien. / In the Indonesian seas, large tidal currents interact with the rough topography and create strong internal waves at the tidal frequency, called internal tides. Part of them will eventually propagate and dissipate far away from generation sites. Their associated mixing upwells cold and nutrient-rich water that prove to be critical for climate system and for marine resources. This thesis uses the physical ocean general circulation model, NEMO, as part of the INDESO project that aims at monitoring the Indonesian marine living resources. Models not taking into account tidal missing are unable to correctly reproduce the vertical structure of watermasses in Indonesian seas. However, taking into account this mixing is no simple task as the phenomena involved in tidal mixing cover a wide spectrum of spatial scales. Internal tides indeed propagate over thousands of kilometres while dissipation and mixing occurs at centimetric to millimetric scales. A model capable of resolving all these processes at the same time does not exist. Until now scientists either parameterised the tidal mixing or used models which only partly resolve internal tides. More and more scientists introduce explicit tidal forcing in their models but without knowing where the energy is going and how the internal tides are dissipated. This thesis intends to quantify energy dissipation in NEMO forced with explicit tidal forcing and compares it to the dissipation induced by the currently used parameterization. This thesis also provides new results about the quantification of the tidal energy budget in NEMO. I first contributed to an INDESO study that aimed at validating the model against several observation data sets. In a second and third study, I investigated the mixing produced in the model by explicit tidal forcing and its impact on water mass. Explicit tides forcing proves to produce a mixing comparable to the one produced by the parameterization. It also produces a significant cooling of 0.3 °C with maxima reaching 0.8°C in the areas of internal tide generation. The cooling is stronger on austral winter. The spring tides and neap tides modulate this impact by 0.1°C to 0.3°C. The model generates 75% of the expected internal tides energy, in good agreement with other previous studies. In the ocean interior, most of it is dissipated by horizontal momentum dissipation (19 GW), while in reality one would expect dissipation through vertical possesses. This value is close to the dissipation induced by the parameterization (16 GW). The mixing is strong over generation sites, and only 20% remains for far field dissipation mainly in the Banda and Sulawesi Seas. The model and the recent INDOMIX cruise [Koch-Larrouy et al. (2015)], which provided direct estimates of the mixing, are surprisingly in good agreement mainly above straits. However, in regions far away from the energy generation sites where INDOMIX found NO evidence of intensified mixing, the model produces too strong mixing. The bias comes from the lack of specific set up of internal tides in the model. More work is thus needed to improve the modeled dissipation, which is a theme of active research for the scientific community. I dedicated the last part of my thesis to the quantification of tidal energy sinks in NEMO. I first worked on a simple academic case: the COMODO internal tides test case, which analyses the behaviour of a vertically stratified fluid forced by a barotropic flow interacting over an idealized abyssal plain/slope/shelf topography without bottom friction. The results of the finite element T-UGOm hydrodynamic model are compared with those of NEMO. The central issue in calculating tidal energy budget is the separation of barotropic and baroclinic precesses.

The half-life of two-neutrino double beta decay of 150 Nd has been measured with data taken by the NEMO-3 experiment at the Modane Underground Laboratory. Using 1918.5 days of data recorded with 36.55 g of 150 Nd the half-life of this process is measured to be T2ν 1/2 = (8.99 ± 0.18 (stat.) ± 0.71 (syst.)) × 1018 y. SuperNEMO is the successor to NEMO-3 and will be one of the next generation of 0νββ experiments. It aims to measure 82 Se with an half-life sensitivity of 1026 yr corresponding to ⟨mββ⟩ < 50 - 100 meV. In order to achieve this target, a testing apparatus for the scintillator characterization has been developed and 140 scintillator blocks were characterized for the calorimeter of the SuperNEMO. It was determined that none of the blocks violate defined criteria of uniformity (± 3% for the energy resolution at 1 MeV) nor the criteria of energy resolution (< 16% at 1 MeV). The average uniformity of the blocks was found to be 0.7% and the average energy resolution of these blocks is 12% at 1 MeV. However, it was discovered during the visual tests that 23 out of 140 blocks contain more than 20 visible dust specks, thus alarming the possible contamination of these blocks with the natural radioactive isotopes.