Hfq est une protéine bactérienne qui a un rôle pleiotropique. La principale fonction de la protéine Hfq bactérienne consiste à répondre aux stress que peut rencontrer la bactérie lors d’un changement environnemental, en utilisant essentiellement un contrôle post-transcriptionnel. La protéine, par sa capacité à interagir avec les ARN et notamment les petits ARN non codant, permet ainsi une régulation rapide de l’expression génétique. En outre la protéine interagit aussi avec l’ADN qu’elle aide à se structurer. Les mutations dans le gène qui code pour Hfq ont des effets pleïotropes (déterminant plusieurs caractères phénotypiques).D’un point de vue structural, la protéine adopte un repliement de type Sm, caractérisé par un oligomère toroïdal reposant sur la formation d’un feuillet β continu à 30 brins. Cependant, outre cette région Sm N-terminale, Hfq possède également une région C-terminale (CTR) de taille et de séquence variables selon les bactéries. Mon travail de thèse a porté sur l’analyse de cette région CTR chez la bactérie Escherichia coli. Cette région a en effet la capacité de former une structure de type amyloïde : structures auto-assemblées in vivo, à proximité de la membrane interne et dans le nucléoïde.Par l’utilisation de diverses techniques physico-chimiques (microscopie moléculaire, spectroscopie et microscopie infrarouge, dichroïsme circulaire et diffusion aux petits angles), mon travail a consisté à caractériser l’assemblage de cette région de Hfq ainsi que les facteurs l’influençant en particulier la présence d’acide nucléique. Une partie de mon travail de thèse a aussi consisté à mettre en place une méthode d’imagerie corrélative innovante permettant d’analyser la signature chimique et morphologique d’une fibre amyloïde unique. Mon travail a enfin porté sur l’analyse de l’effet de composés inhibant l’agrégation de la structure amyloïde, ce qui pourrait constituer une piste pour développer une nouvelle classe d’antibiotiques. / Hfq is a pleiotropic bacterial protein that determines several phenotypic characteristics. Its main function is to facilitate responses to stresses that bacteria may encounter during environmental changes, mainly by using post-transcriptional genetic control. The protein, by its capacity to interact with RNA, in particular small non-coding RNA, enables a rapid regulation of gene expression. In addition, the protein also interacts with DNA and compacts it. From a structural point of view, the protein adopts an Sm-like fold, characterized by a toroidal oligomer formed by a continuous 30-stranded β-sheet. Besides its conserved N-terminal Sm domain, Hfq also possesses a C-terminal region (CTR) that can vary in size and sequence between bacteria. My PhD work focused on the analysis of this CTR region in Escherichia coli bacteria. Indeed, this region has the capacity to form an amyloid structure. This structural dynamic is related to the formation of self-assembled structures in vivo, in the proximity of the inner membrane and in the nucleoid.Using various physicochemical techniques (molecular microscopy, spectroscopy and infrared microscopy, circular dichroism and small angle X-ray scattering), my work consisted in characterizing the assembly of this region of Hfq, as well as the factors influencing its assembly (in particular, the presence of nucleic acids). A part of my work consisted in setting up an innovative correlative–imaging method to analyze the chemical and morphological signature of a single amyloid fibre. Finally, my work focused on the analysis of the effect of compounds that inhibit the aggregation of the amyloid structure, which could constitute a new way to develop a novel class of antibiotics.

Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop. In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.

No description available.

No description available.

La pseudouridylation, l'isomérisation du résidu urine (U) en pseudouridine ([PSI]) est la modification post-transcriptionnelle la plus fréquemment retrouvée dans les ARN. Elle est catalysée par une enzyme ARN:PSI-synthase. Chez les archées et les eucaryotes, cette activité est également portée par des particules ribonucléoprotéiques à boîtes H/ACA (RNP H/ACA). Chez les archées, le complexe comprend quatre protéines invariables dont l'ARN:PSI-synthase aCBF5 et trois protéines partenaires L7Ae, aNOP10 et aGAR1, ainsi qu'un ARN guide qui cible par appariement de bases la position de l'uridine à modifier de l'ARN substrat. Le rôle des partenaires a pu être identifié par des analyses structure-fonction basées sur des approches biochimiques, biophysiques et radiocristallographiques. Au cours de ce travail, nous avons démontré l'existence de disparités fonctionnelles entre les ARN guides d'un même organisme, et l'importance de l'interaction entre L7Ae et aNOP10 pour le positionnement correct de l'ARN substrat. Nous avons testé in vitro l'assemblage et l'activité de particules reconstituées en présence d'ARN guides non conventionnels. L'étude sur la dynamique de l'ARN substrat lors de la pseudouridylation a également été abordée et a permis de déterminer que aGAR1 n'était pas nécessaire pour le mécanisme de turnover de la particule, que la température jouait un rôle crucial pour cette activité, et que la nature du nucléotide cible ainsi que la longueur de l'ARN substrat étaient des éléments importants pour la sélection de cet ARN. Nous avons également mis au point une nouvelle technique basée sur le phénomène de FRET permettant de suivre l'association de l'ARN substrat à la RNP H/ACA / Pseudouridylation reaction that consists in the isomerization of uridines (U) into pseudouridines (PSI) is the most frequent post-transcriptional modification found in RNAs. It is catalyzed by enzymes with RNA:PSI-synthase activity. In Archaea and Eukarya, ribonucleoprotein particles, the so-called box H/ACA RNPs, possess such activity. In Archaea, the box H/ACA complex comprises four invariable proteins namely the RNA:PSI-synthase aCBF5 and three protein partners L7Ae, aNOP10 and aGAR1, and specific to each RNP, an RNA acting as a guide to secure by base pairing the RNA substrate and define the position to be modify. During these last years, several crystal structures of components of archaeal H/ACA RNP and fully assembled RNP have been resolved. Complementary biochemical and biophysical studies allowed detailed structure-function analyses to identify the role of the different components. During this work we identified functional differences between two RNA guides expressed in the same archaea, and demonstrated that the interaction between L7Ae and aNOP10 is important for a correct positioning of substrate RNA. We also tested in vitro the assembly and activity of RNP reconstituted on H/ACA-like guide RNAs. We investigated dynamics of substrate RNA during the pseudouridylation. We found that aGAR1 was not necessary for the turnover of the particle, that the temperature was crucial for such activity, and that the chemical structure of the targeted residue and length of the substrate RNA were important determinants for substrate selectivity. Finally, we have also developed a new technic based on FRET adapted to monitor binding of the susbtrate RNA to the box H/ACA RNP enzyme

Dans ce travail, nous nous sommes concentrés sur la création de nanostructures chirales optiquement actives en fabriquant des nanohélices de silice fluorescente afin d’obtenir des matériaux souple, nanométriques, optiquement actifs pour des applications en tant que matériaux nanophotoniques. Dans cette optique, des nanohélices de silice chirales ont été utilisées pour greffer et organiser des nanocristaux inorganiques fluorescents achiraux tels que des quantums dots, des chromophores, des molécules et des polymères fluorescents selon différentes approches. Ces hélices inorganiques ont été formées par procédé sol-gel en utilisant des auto-assemblages hélicoïdaux organiques de molécules amphiphiles (amphiphile gemini cationique, avec un contre-ion chiral le tartrate) en tant que modèles. Tout d'abord, la surface de la silice hélicoïdale a été fonctionnalisée par l’APTES afin de greffer des quantum dots inorganiques ZnS-AgInS2 possédant divers ligands. Dans la deuxième partie, le polymère de dérivé anthracénique fluorescent a été organisé par dépôt et adsorption à la surface de silice hélicoïdale. Afin d’étudier les propriétés chiroptiques, différentes caractérisations ont été réalisées telle que la spectroscopie du dichroïsme circulaire (CD) et celle de la luminescence circulairement polarisée (CPL).Le premier chapitre présente l’étude bibliographique sur différents systèmes d’auto-assemblage organiques chiraux et leurs propriétés chiroptiques. Les études sur la formation de systèmes auto-assemblés chiraux dans différentes conditions, leur morphologie structurale, les techniques de fabrication et leurs applications sont discutées suivies de l'utilisation de nanocristaux fluorescents, à savoir, les quantums dots (QD) et les polymères fluorescents achiraux sur lesquels les propriétés chiroptiques peuvent être obtenues et leurs applications dans les nanodispositifs optiques, les capteurs et la nano-photonique.Dans la première partie du deuxième chapitre, différentes techniques de caractérisation telles que le microscope électronique en transmission (TEM), le microscope électronique en transmission haute résolution (HRTEM), la microscopie confocale, la spectroscopie UV-Vis, celle de la fluorescence, du dichroïsme circulaire (CD) et de la luminescence circulairement polarisée (CPL) sont décrites. Dans la deuxième partie, la synthèse du gemini 16-2-16 ainsi que son mécanisme d'auto-assemblage, et sa transformation en réplica de silice par l'intermédiaire de la chimie sol-gel sont décrits. Ces nanohélices de silice sont fonctionnalisées par le 3-aminopropyltriéthoxysilane (APTES). Leur analyse est effectuée par analyse thermogravimétrique (TGA) et analyse élémentaire (EA).Dans le troisième chapitre, nous nous sommes concentrés sur la synthèse de QDs inorganiques ((ZnS)x-1(AgInS2)x) avec différentes compositions rapport molaire et leurs caractérisations par TEM, TGA, EA, spectroscopie infrarouge à transformée de Fourier (FTIR), mesures de potentiel zêta, spectroscopie d'absorption et d'émission. Quatre types de ligands ont été utilisés, par échange de ligand, pour recouvrir les QDs : sulfure d'ammonium (AS), acide 3-mercaptopropionique (MPA), l-cystéine (L-Cys) et l'oleylamine (OLA). Ces QDs sont greffés à la surface des hélices de silice modifiée par de l’amine suite à des interactions ioniques. Diverses techniques ont été utilisées pour confirmer leur greffage à la surface des hélices de silice, et les propriétés optiques ont été étudiées par spectroscopie d'absorption et d'émission. Après le greffage, différents résultats ont été observés selon le ligand utilisé : la caractérisation par TEM montre que les QDs sont greffés à la surface des hélices de silice. [...] / In this work, we focused on the creation of optically active chiral nanostructures by fabricating fluorescent silica nanohelices in order to obtain optically active nanoscale soft materials for applications as nanophotonics materials. For this purpose, silica chiral nanohelices were used for grafting and organizing achiral fluorescent inorganic nanocrystals, dyes, molecules, and fluorescent polymers through different approaches. These inorganic helices were formed via sol-gel method using organic helical self–assemblies of surfactant molecules (achiral and cationic gemini surfactant, with chiral counterion, tartrate) as templates. First, the surface of helical silica was functionalized by APTES in order to graft inorganic quantum dots ZnS-AgInS2 with different capping ligands. In the second part, fluorescent anthracene derivative polymer was organized via deposition and absorption on the surface of helical silica. To investigate the chiroptical properties, circular dichroism and circularly polarised luminescence characterization were performed.In the first chapter, the bibliographic study on different chiral organic self-assembling systems and their chiroptical properties are shown. The studies on the formation of chiral self-assembled systems in different conditions, structural morphology, fabrication techniques and their applications are discussed followed by the use of fluorescent nanocrystals, i.e., quantum dots (QDs) and achiral fluorescent polymers on which chiroptical properties can be obtained and their applications in optical nanodevices, sensors, and nano-photonics.In the first part of the second chapter, different characterisation techniques such as transmission electron microscope (TEM) , high resolution transmission electron microscope (HRTEM), and confocal microscopy, UV-Vis spectroscopy and fluorescence spectroscopies, as well as circular dichroism (CD) and circularly polarised luminescence (CPL) spectroscopies are described. In the second part, the synthesis of Gemini 16-2-16 as well as their self-assemblies mechanism, and their transformation to silica replica via sol-gel chemistry are described. These silica nanohelices are functionalized by 3-aminopropyltriethoxysilane (APTES). Their analysis is performed by Thermogravimetric analysis (TGA) and elementary analysis (EA).In the third Chapter, we focused on the synthesis of inorganic ((ZnS)x-1(AgInS2)x) QDs with different compositions molar ratio and its characterizations by TEM, TGA, EA, Fourier-transform infrared spectroscopy (FTIR), zeta potential measurements, absorption, and emission spectroscopy. Four types of ligands were used to cap the QDs via phase ligand exchange as follows: ammonium sulphide (AS), 3-mercaptopropionic acid (MPA), l-cysteine (L-Cys) and the fourth one is oleylamine (OLA). These QDs are grafted on the surface of amine-modified silica helices through ionic interaction. Various techniques were used to show the grafting of QDs on the surface of silica helix, and their optical properties were studied using absorption and emission spectroscopy. After grafting, in each case of ligands, different results were observed as follows: The TEM characterization shows that QDs are grafted on the surface of silica helices. In the case of AS-capped QDs, the helical morphology of silica helices after grafting is destroyed; therefore the further ananlysis was not possible. While, in the cases of QDs with three other ligands MPA, OLA and L-cys, dense and homogeneous grafting of the QDs were observed by TEM and the helical morphology was preserved after their grafting. The HRTEM images were taken on the MPA-QDs@silica helices and energy-dispersive x-ray (EDX) analysis was performed in STEM mode, confirming the QDs elements present on the silica surfaces. [...]

Ever since the discovery of exotic phenomena like high temperature (Tc) superconductivity in the cuprates and colossal magnetoresistance in the manganites, strongly correlated electron systems have become the center of attention in the field of condensed matter physics research. This renewed interest has been further kindled by the rapid development of sophisticated experimental techniques and tremendous computational power. Computation plays a pivotal role in the theoretical investigation of these systems, because one cannot explain their complicated phase diagrams by simple, exactly solvable models. Among the plethora of experimental techniques, various kinds of high energy electron spectroscopies are fast gaining importance due to the multitude of physical properties and phenomena which they can access. However the physical processes involved and the interpretation of the spectra obtained from these spectroscopies are extremely complex and require extensive theoretical modelling. This thesis is concerned with the theoretical modelling of a certain class of high energy electron spectroscopies, viz. the core-level electron spectroscopies, for strongly correlated systems of various kinds. The spectroscopies covered are Auger electron spectroscopy (AES), core-level photoemission spectroscopy (core-level PES) and X-ray absorption spec- troscopy (XAS), which provide non-magnetic information, and also X-ray magnetic circular and linear dichroism (XMCD and XMLD), which provide magnetic information. .

Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.

No description available.

Das Schalten und das Auslesen der magnetischen Ausrichtung einzelner winziger magnetischer Informationsspeicher müssen zu wirklich nanoskopischer Dimension entwickelt werden, um mit der Miniaturisierung von modernen, nanoelektronischen Bauteilen Schritt zu halten. Daher sind neue Konzepte, den magnetischen Zustand von Nanostrukturen elektronisch gezielt zu beeinflussen, derzeitig im Mittelpunkt wissenschaftlicher Untersuchungen. Diese Arbeit befasst sich mit dem zuverlässigen Einstellen der Magnetisierung eines rein horizontal kontaktierten, nanoskopischen Magneten, in zwei stabile Zustände. Ein spinpolarisierter Strom wird bei Raumtemperatur in eine Leiterbahn unterhalb des magnetischen Nanopillars injiziert. Spindiffusion durch den Kontakt zwischen der Leiterbahn (Cu) und dem Pillar (CoFe) ruft eine Spin-Akkumulation im Nanopillar hervor, der durch den Spin Transfer Torque-Effekt (STT) vermittelt wird. Bei diesem Prozess verursachen die akkumulierten Elektronenspins ein auftretendes Netto-Moment, das senkrecht auf die Magnetisierungsorientierung des Nanopillars wirkt und so das Schalten ermöglicht. In den STT-induzierten Schaltexperimenten wird der magnetische Zustand des Nanopillars durch eine bildgebendes Messverfahren mittels Rasterröntgentransmissionsmikroskopie (STXM) erfasst. So konnte gezeigt werden, dass sich die Magnetisierung des Pillars auch gegen das Oersted-Feld des Schaltstroms reversibel schalten lässt. / “Changing and detecting the orientation of nanomagnetic structures, which can be used for durable information storage, needs to be developed towards true nanoscale dimensions for keeping up the miniaturization speed of modern nano electronic components. Therefore, new concepts for controlling the state of nano magnets are currently in the focus of research in the field of nanoelectronics. Here, we demonstrate reproducible switching of a purely metallic nanopillar placed on a lead that conducts a spin-polarized current at room temperature. Spin diffusion across the metal-metal (Cu to CoFe) interface between the pillar and the lead causes spin accumulation in the pillar, which may then be used to set the magnetic orientation of the pillar by means of Spin Transfer Torque (STT). In our experiments, the detection of the magnetic state of the nanopillar is performed by direct imaging via scanning transmission x-ray microscopy (STXM)” [1]. Therefore it could be demonstrated, to reversibly switch the nanopillar’s magnetic state even against the Oersted field which is induced by the switching current. Furthermore we could show, that magnetization switching is possible by a pure spin current that is diffusively transported beneath the nanopillar.