No description available.

@font-face { font-family: "Arial"; }@font-face { font-family: "ＭＳ 明朝"; }@font-face { font-family: "Calibri"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0in 0in 0.0001pt; text-indent: 0.5in; line-height: 200%; font-size: 11pt; font-family: "Times New Roman"; }p.MsoBodyText, li.MsoBodyText, div.MsoBodyText { margin: 0in 0in 6pt; text-indent: 0.5in; line-height: 200%; font-size: 11pt; font-family: "Times New Roman"; }span.BodyTextChar { font-family: Calibri; }div.Section1 { page: Section1; } A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica has broad antimicrobial activity in tryptone peptone rich medium. This protein, which we call ‘escapin’, belongs to an L-amino acid oxidase family. The goals of my project were 1) to determine the products of escapin’s oxidation of its main substrate L-lysine, 2) to characterize the antimicrobial effects of escapin’s products, and 3) determine bactericidal mechanisms of action of these products. Escapin is a powerful bactericidal agent against several bacteria species including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio harveyi. Escapin operates through a two-step process: 1) deamination of L-amino acids (especially L-lysine) by enzymatic activity to produce escapin intermediate products of L-lysine (EIP-K), hydrogen peroxide, and ammonia; and 2) EIP-K simultaneously reacts with hydrogen peroxide to generate escapin end products (EEP-K). EIP exists as an equilibrium mixture of the linear a-keto analogue of lysine and its cyclic forms, and the relative amount of the linear form increases with pH decreases. The powerful bactericidal effect of escapin requires the simultaneous presence of hydrogen peroxide and EIP-K in weak acidic conditions, which suggests that linear form of EIP-K with hydrogen peroxide is responsible for the bactericidal effect of escapin. Using E. coli MC4100 as a model, the mechanism of action of escapin was examined. Brief treatment with EIP-K + H2O2, but not EIP-K or H2O2 alone, causes irreversible DNA condensation with a time course similar to the bactericidal effect. A mutant strain resistant to EIP-K + H2O2 was isolated, and a single point mutation was found in the oxidative stress regulator gene (oxyR). Through a complementary assay, it was shown that wild type E. coli is conferred resistance to EIP-K + H2O2 by carrying mutated oxyR plasmid. Furthermore, in this bactericidal effect, heat or cold shock does not substitute for hydrogen peroxide induced oxidative stress. Thus, escapin’s powerful bactericidal effect may be through irreversible DNA condensation mediated through hydrogen peroxide generating an oxidative stress response, but the pathway mediating EIP-K’s synergistic effect is still unclear.

Donald Judd, Dan Flavin, Carl Andre et Sol LeWitt sont parmi les principaux représentants de l'art minimal aux Etats-Unis. Les oeuvres analysées dans cette thèse sont majoritairement tirées de leur pratique des années soixante et soixante-dix, et se rattachent à la sculpture. À partir de ces oeuvres, notre recherche se propose de montrer les rapports entre l'esthétique et le politique, en regard d'un questionnement sur la rationalité que ces oeuvres induisent. Dans les premier et second chapitres, nous présentons une analyse initiale des oeuvres en exergue, et une mise en situation contextuelle. Celle-ci s'établit en regard d'un corpus critique et d'un commentaire sur le climat épistémologique de l'époque étudiée. Le troisième chapitre renvoie aux sources formelles et théoriques, en maintenant l'idée de filiation -et d'infléchissement de ces mêmes filiations -qui se dégagent des chapitres précédents. Les deux derniers chapitres reprennent les principaux éléments théoriques que nous avons retenus pour les besoins de notre analyse. Nous y étudions, à l'aune des pensées de Deleuze et d'Adorno, l'articulation particulière de l'esthétique et du politique dans l'art minimal. Le politique s'y appuie sur une interrogation de la rationalité qui travaille l'idée de totalité et d'immédiateté de la perception. Avec la prémisse de la reconduction dans l'oeuvre d'instances paradoxales basées sur une synthèse connective ou une répétition profonde qui sous-tend la répétition sérielle première. En somme, il s'agit dans le commentaire critique des oeuvres et des artistes choisis, d'étudier des notions qui se posent comme paradigmes de l'ambiguïté de l'oeuvre minimale. L'ancrage théorique privilégié en découle. L'investigation de concepts prégnants dans l'oeuvre minimale, tels la différence et la répétition, le matériau et les rapports forme/contenu, et la mimésis qui renvoie aux problématiques du simulacre ou du reflet, qualifient la représentation dans l'oeuvre minimale. Une analyse renouvelée de ces termes récurrents demandait un champ théorique inusité. Ce dernier ouvre à une qualification de l'oeuvre minimale basée, non pas uniquement sur les certitudes de ce que l'on voit, mais également sur ce qui échappe à la perception immédiate. Cette conjonction spécifique reconduit une oeuvre déterminée par ce qui l'indétermine. Et une interrogation de l'esthétique et du politique à travers celle d'une rationalité où perce le sensible. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Art minimal, Esthétique, Politique, Rationalité, Éléments.

Many human diseases including cancer have been associated with altered cellular metabolism and a changed oxygen consumption in cells. Fluorophores are sensitive to their local environment due to their long life times in transient dark states. A recent study successfully utilized this sensitivity to image differences in oxygen concentrations in cells using transient state (TRAST) microscopy together with fluorescent labels [1]. A natural continuation of this study is to investigate the possibilities of using this method with natural fluorophores already present in cells and thereby avoid articial labeling. Flavin adenine dinucleotide (FAD) is an auto fluorescent coenzyme that is naturally present in cells and involved in cellular metabolism. This project is an exploratory pilot study for cellular measurements with the aim to investigate if FAD can be used to probe oxygen concentrations in aqueous solution using transient state monitoring and fluorescence correlation spectroscopy (FCS). This thesis includes the results from FCS and TRAST experiments on FAD in aqueous solutions with different oxygen concentrations as well as different ascorbic acid concentrations. The performed experiments showed that FAD monitored with TRAST is sensitive to differences in oxygen concentrations for the aqueous solutions used in this study.

No description available.

Die lichtaktivierte Kinase Phototropin aus Chlamydomonas reinhardtii, die photoaktivierte Adenylatcyclase (PAC) aus Euglena gracilis und das BLUF-Protein Slr1694 aus Synechocystis sp. PCC 6803 wurden in Hinblick auf die molekularen Details der primären photochemischen Prozesse sowie der Signalweiterleitung untersucht. Phototropin wurde mit Hilfe von Arginin aus Escherichia coli in Milligramm Mengen isoliert. Ohne Arginin wurde E. coli cAMP Rezeptorprotein assoziiert aufgefunden, welches eine hohe Homologie zu einer cAMP aktivierten Kinase aus C. reinhardtii besitzt. Volllängen Phototropin bildet wie einzelne LOV-Domänenkonstrukte ohne Kinasedomäne den Flavin-Triplettzustand und das kovalente Cysteinyl-Addukt. Der Zerfall des Signalzustandes ist in Anwesenheit von ATP beschleunigt und deutet auf Photorezeptor-Kinase Interaktion hin. Strukturelle Änderungen in der Kinasedomäne wurden durch FTIR-Differenzspektroskopie gezeigt. Über ELDOR-Spektroskopie wurde der Abstand der Photorezeptordomänen auf etwa 25 Angstrom bestimmt. Mutationen in Slr1694 an S28, N31 und W91 zeigten keine konservierten Einfluss auf die Dynamik des Signalzustands. Die Entfernung der Seitenkette von S28 führte zu einer 15 nm Rotverschiebung des Absorptionsspektrums aufgrund veränderter Wasserstoffbrückenkoordination des Kofaktors. Die Einführung von positiv geladenen Seitenketten an Stelle von N31 erhöhte die Kofaktorbindung von phosphorylierten Flavinen. Künstliche Kofaktoren wie Roseoflavin konnten in Slr1694 durch Koexpression eines prokaryotischen Flavintransporters erreicht werden. Die Rolle von M152 in PAC für die Signalweiterleitung wurde anhand der lichtaktivierten cAMP Synthese-Aktivität gezeigt. Durch ultraschnelle IR-Spektroskopie wurde die Beteiligung der Seitenketten von Y8 sowie Q50 bestätigt und eine genauere Beschreibung der Wasserstoffbrücken im langlebigen Signalzustand ermöglicht. / The light activated kinase Phototropin from Chlamydomonas reinhardtii, the photoactivated adenylylcyclase (PAC) from Euglena gracilis and the BLUF protein Slr1694 from Synechocystis sp. PCC 6803 were investigated concerning the molecular details of the primary photochemistry as well as signal transduction. Phototropin was isolated from Escherichia coli in mg amounts after solubilization with arginine. Without arginine E. coli cAMP receptor protein, which shows high homology to a cAMP activated kinase from C. reinhardtii, was copurified. Full length Phototropin shows similar photochemistry to LOV-domain containing proteins without the kinase including triplet and covalent cysteinyl adduct formation. Signaling state decay is accelerated in the presence of ATP and suggests photoreceptor-kinase interaction. FTIR spectroscopy showed light induced structural changes in the kinase domain. The distance of the photoreceptor domains of 25 Angstrom was determined by ELDOR spectroscopy. Mutation of the side chains of S28, N31 and W91 in Slr1694 showed no conserved influence on the dynamic of the signaling state. Removal of the hydroxyl group of S28 lead to a 15 nm red shift of the absorption spectrum as a result of altered hydrogen bond coordination of the cofactor. Introduction of positively charged side chains at the position of N31 strengthened the binding of phosphorylated flavins. An artificial flavin like roseoflavin was introduced in Slr1694 by coexpression of a bacterial flavin transporter. The essential role of M152 in PAC for signal transduction was shown by determination of light activated cAMP synthesis activity. Ultrafast IR spectroscopy confirmed the contribution of Y8 and Q50 in the photocycle and gave a more detailed description of the hydrogen bonding situation in the signaling state.

Ces dernières années, l’OMS a émis un signal d’alarme à propos de l’occurrence majeure de résistance bactérienne qui constitue un problème de santé publique global. A ce titre, la recherche de nouvelles cibles enzymatiques et le développement de nouveaux antibactériens ciblant ces dernières de manière sélective constitue alors un enjeu actuel impératif. La mise en évidence d’une nouvelle enzyme de la famille des thymidylates synthases par l’équipe de Myllykallio en 2002 et son étude a permis de faire de cette dernière une cible de choix pour la conception de nouveaux antibactériens par sa présence exclusive chez des bactéries pathogènes pour l’Homme, sa non-similarité structurelle avec l’enzyme thymidylate synthase classique et son mécanisme particulier mettant en jeu un couple de cofacteurs oxydo-réducteurs(NADPH/FAD). Ce manuscrit, divisé en trois grandes parties, s’intéresse dans un premier temps à la synthèse métallo-catalysé de nouveaux analogues nucléotidiques du FAD substitué sur l’azote centrale par une chaîne acyclique de type alkényle phosphonate. Dans un second temps, le manuscrit traite de la translation de cette même chaîne latérale sur l’azote N1 couplée à un large panel de base hétéroaromatiquebicyliques ou tricycliques par deux réactions clés : une alkylation régiosélective en conditions deVorbrüggen et une étape de métathèse croisée. Enfin, la troisième partie porte sur la préparation d’acyclonucléosides comportant un motif d’intérêt de type gem-difluoromethylphosphonate connu comme étant un mime isostérique et isoélectronique du groupement phosphate. L’incorporation de ce motif a permis la synthèse de petite librairie d’ANPs inédits à visée anti-FDTS et anti-virales. / In recent years, WHO has warned against the major occurrence of bacterial resistance as a global public health problem. As such, the search for new enzymatic targets and the development of new antibacterials targeting them selectively is therefore an imperative challenge. The discovery of a new enzyme among the family of thymidylate synthases by Myllykallio’s team in 2002 and its study has made it a prime target for the design of new antibacterials by its sole presence in pathogenic bacteria, its structural dissimilarity with the classical thymidylate synthase enzyme and its singular mechanism involving a pair of oxido-reducingcofactors (NADPH / FAD).This manuscript, divided into three main parts, is initially interested in the metallocatalytic synthesis of new nucleotide analogues of FAD substituted on the central nitrogen by analkenyl phosphonate acyclic chain. In the second part, the manuscript deals with the translation of this same side chain on nitrogen N1 on a large panel of bicylic or tricyclic heteroaromatic base through two keyreactions: a regioselective alkylation under Vorbrüggen conditions and a cross metathesis step. Finally, th ethird part relates to the preparation of acyclonucleosides comprising a gem-difluoromethylphosphonatefunctional group which is known to be an isosteric and isoelectronic mimic of the phosphate group. The incorporation of this moeitie has allowed the synthesis of a small library of novel ANPs for anti-FDTS andanti-viral purposes.

This thesis applies the ab initio techniques of computational chemistry to studies of molecular clusters containing covalent (strong) or van der Waals (weak) bonds formed in chemistry and biochemistry in the temperature range 10-300 K. Van derWaals complexes with an enthalpy of formation from reactants of less than 25 kJ mol-1 and covalent clusters are described in this thesis. The first group of van der Waals complexes involved the molecule carbon monoxide that possesses a small permanent dipole that could lead to dipole - induced dipole interaction and dipole - dipole interaction with another reactant in addition to dispersion. The substrates investigated were methanimine and cyanogen where endergonic unstable molecules were formed, and the clustering of carbon monoxideon a porphin surface leading to the formation of carbon - carbon fragments. TheFaraday effect was invoked to suggest that this was the original method by which thechirality of the D-sugars was selected. Coordination of imino-compounds on thesame surface involving induction and electrostatic interactions could lead to the preferential formation of L-aziridones, hydrolysable to L-amino-acids.The preferred formation of D-ribose, and the more stable D-2-deoxyribose, andnucleotides polymerisable to deoxyribonucleic acids was described. The second group of van der Waals complexes involved the polymerisation of acetylene molecules, to di- and tri-acetylene complexes where the exchange interaction involved the quadrupole moment of the acetylene radical reacting with acetylene or diacetylene. The reaction of carbon monoxide was extended to include its interaction with diacetylene. The entire potential energy surface for the interaction with diacetylene was investigated. The reaction was shown to be endergonic to produce a reactive species, here postulated to rearrange with a reasonable activation energy toform an aldehyde. The energetics of the formation of diacetylene, triacetylene andhigher polymers was briefly investigated. The reactivity of the acetylene polymeraldehydes with other substrates was briefly investigated. This work has apparently laid a firm basis both, qualitative and quantitative, tounderstand some of the weakest interactions in nature involving the simplest ofreactions that have been important in atmospheric chemistry.

Résumé confidentiel / Résumé confidentiel

Abstract Most proteins carry out their functions through interactions with other molecules. Thus, proteins taking part in similar interactions are likely to carry out related functions. One way to determine whether two proteins do take part in similar interactions is by quantifying the likeness of their structures. This work focuses on the development of methods for the comparison of protein-protein and protein-ligand interactions, as well as their application to structure-based classification schemes. A method based on the MultiMer-align (or MM-align) program was developed and used to compare all known dimeric protein complexes. The results of the comparison demonstrates that the method improves over MM-align in a significant number of cases. The data was employed to classify the complexes, resulting in 1,761 different protein-protein interaction types. Through a statistical model, the number of existing protein-protein interaction types in nature was estimated at around 4,000. The model allowed the establishment of a relationship between the number of quaternary families (sequence-based groups of protein-protein complexes) and quaternary folds (structure-based groups). The interactions between proteins and small organic ligands were studied using sequence-independent methodologies. A new method was introduced to test three similarity metrics. The best of these metrics was subsequently employed, together with five other existing methodologies, to conduct an all-to-all comparison of all the known protein-FAD (Flavin-Adenine Dinucleotide) complexes. The results demonstrates that the new methodology captures the best the similarities between complexes in terms of protein-ligand contacts. Based on the all-to-all comparison, the protein-FAD complexes were subsequently separated into 237 groups. In the majority of cases, the classification divided the complexes according to their annotated function. Using a graph-based description of the FAD-binding sites, each group could be further characterized and uniquely described. The study demonstrates that the newly developed methods are superior to the existing ones. The results indicate that both the known protein-protein and the protein-FAD interactions can be classified into a reduced number of types and that in general terms these classifications are consistent with the proteins' functions. / Tiivistelmä Suurin osa proteiinien toiminnasta tapahtuu vuorovaikutuksessa muiden molekyylien kanssa. Proteiinit, jotka osallistuvat samanlaisiin vuorovaikutuksiin todennäköisesti toimivat samalla tavalla. Kahden proteiinin todennäköisyys esiintyä samanlaisissa vuorovaikutustilanteissa voidaan määrittää tutkimalla niiden rakenteellista samankaltaisuutta. Tämä väitöskirjatyö käsittelee proteiini-proteiini- ja proteiini-ligandi -vuorovaikutusten vertailuun käytettyjen menetelmien kehitystä, ja niiden soveltamista rakenteeseen perustuvissa luokittelujärjestelmissä. Tunnettuja dimeerisiä proteiinikomplekseja tutkittiin uudella MultiMer-align-ohjelmaan (MM-align) perustuvalla menetelmällä. Vertailun tulokset osoittavat, että uusi menetelmä suoriutui MM-alignia paremmin merkittävässä osassa tapauksista. Tuloksia käytettiin myös kompleksien luokitteluun, jonka tuloksena oli 1761 erilaista proteiinien välistä vuorovaikutustyyppiä. Luonnossa esiintyvien proteiinien välisten vuorovaikutusten määrän arvioitiin tilastollisen mallin avulla olevan noin 4000. Tilastollisen mallin avulla saatiin vertailtua sekä sekvenssin (”quaternary families”) sekä rakenteen (”quaternary folds”) mukaan ryhmiteltyjen proteiinikompleksien määriä. Proteiinien ja pienien orgaanisten ligandien välisiä vuorovaikutuksia tutkittiin sekvenssistä riippumattomilla menetelmillä. Uudella menetelmällä testattiin kolmea eri samankaltaisuutta mittaavaa metriikkaa. Näistä parasta käytettiin viiden muun tunnetun menetelmän kanssa vertailemaan kaikkia tunnettuja proteiini-FAD (Flavin-Adenine-Dinucleotide, flaviiniadeniinidinukleotidi) -komplekseja. Proteiini-ligandikontaktien osalta uusi menetelmä kuvasi kompleksien samankaltaisuutta muita menetelmiä paremmin. Vertailun tuloksia hyödyntäen proteiini-FAD-kompleksit luokiteltiin edelleen 237 ryhmään. Suurimmassa osassa tapauksista luokittelujärjestelmä oli onnistunut jakamaan kompleksit ryhmiin niiden toiminnallisuuden mukaisesti. Ryhmät voitiin määritellä yksikäsitteisesti kuvaamalla FAD:n sitoutumispaikka graafisesti. Väitöskirjatyö osoittaa, että siinä kehitetyt menetelmät ovat parempia kuin aikaisemmin käytetyt menetelmät. Tulokset osoittavat, että sekä proteiinien väliset että proteiini-FAD -vuorovaikutukset voidaan luokitella rajattuun määrään vuorovaikutustyyppejä ja yleisesti luokittelu on yhtenevä proteiinien toiminnan suhteen.