We investigated the reactivity and bonding of perfluoroethyl groups (C2F5) on Cu(111) under ultra high vacuum conditions. Perfluoroethyl moieties bonded to the surface were generated by the dissociative adsorption of perfluoroethyl iodide. Temperature-programmed reaction/desorption (TPR/D) and reflection- adsorption infrared spectroscopy (RAIRS) revealed abounding reaction pathways, and a variety of intermediates were either identified or inferred. The major desorption products, hexafluoro-2-butyne and hexafluorocyclobutene, were detected at 360K and 440K, and some octafluorobutene was observed at 320K at higher coverages, implicating that two fluorine atoms were abstracted step-by-step from the C2F5 on Cu(111). Two sets of signature IR bands were recognized. One set (2054cm-1, 1409cm-1, 1210cm-1) was found to correlate with the surface-bound trifluorovinyl moieties which were also confirmed by directly generating this species from trifluorovinyl iodide. The other set of vibrational features (1322cm-1, 1224cm-1, 950cm-1) presumably implied the trifluoro- ethylidyne intermediate on the surface. Hence, C2F5(ad) underwent the £\-F and £]-F elimination reactions in sequence to yield trifluorovinyl which eventually led to hexafluoro-2-butyne. The alternative route was that C2F5(ad) proceeded via the £\-F elimination reaction twice to render trifluoroethylidyne which ultimately resulted in hexafluorocyclobutene. To our knowledge, the occurrence of the sequential £\-F and£]-F elimination pathway, or the double £\-F elimination reaction has never been observed in any single system.

Yes / Rpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation. / BBSRC [grant number: BB/L006510/1] and the Wellcome Trust [grant number: 092107/Z/10/Z]. K.K. was supported by the UPStream network [EU, FP7, ITN project 290257]

No description available.

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No description available.

High surface area tungsten oxynitride samples have been prepared by the temperature programmed reaction (TPR) of W0₃ with NH₃. All samples were characterized by X-ray diffraction (XRD), nitrogen physisorption, and CO chemisorption. In addition, some sample compositions were determined by elemental analysis. Samples were prepared at various heating rates (β), allowing a Redhead analysis to be carried out giving an activation energy of nitridation of 109 kJ mol⁻¹. A heating rate of 0.016 K S⁻¹ gave optimum synthesis conditions. Solid state intermediates were studied by interruption of the temperature program at various stages. No distinct suboxide phases formed along the synthesis path were found using XRD. An increase in surface area, CO uptake and nitrogen weight content, were found to occur as the reaction progressed. Reactivity experiments showed reasonable hydro deoxygenation (HDO) and hydrodenitrogenation (HDN) activities, but little hydrogenation (HYD) or hydrodesulfurization (HDS) activities. / Master of Science

Le complexe de pores nucléaires est le seul point d'entrée et de sortie du transport nucléocytoplasmique. Le panier nucléaire, l'un de ses principaux composants, s'est avéré impliqué dans la régulation des gènes et pourrait jouer un rôle majeur dans le contrôle de la qualité de l'export d'ARNm. Cependant, on sait peu de choses sur le fonctionnement du panier dans l'export nucléaire et la régulation des gènes. La principale composante structurelle du panier, la TPR (Translocated Promoter Region), est considérée comme l'acteur principal de la fonction de contrôle de la qualité du panier. Il reste à établir par quel mécanisme cette protéine assure la sélection des mRNP compétentes pour l'exportation. Malgré son implication connue dans le contrôle de la qualité des mRNP, l'exportation et la maturation, des questions demeurent: que fait vraiment le panier, qu'est-ce qui définit le contrôle qualité, comment le panier nucléaire est-il capable d'identifier l'ARN qui n'est pas compétent pour l'exportation et quels sont les rôles de différentes protéines composant le panier nucléaire. Récemment, il a été montré que la protéine TPR est présente dans deux populations, l'une dans le nucléoplasme et l'autre liée au NPC. Nos études préliminaires utilisant FRAP (Fluorescence Recorvery After Photobleaching) et la microscopie à molécule unique montrent que les molécules nucléoplasmiques de TPR ne sont pas impliquées dans un échange rapide avec les molécules assemblant avec les paniers ancrés au NPC et présentent différentes sous-populations basées sur la diffusion. L'analyse de études protéomiques préliminaires de notre laboratoire a révélé que l’interactome de TPR présente un enrichissement inattendu en protéines impliquées dans la maturation de l'ARNm, notamment l'épissage et les facteurs de traitement de l'extrémité 3'. Ces résultats pourraient suggérer des interactions complexes des nouvelles fractions nucléoplasmiques de TPR avec la machinerie de maturation des ARNms et nous amènent à poser les questions suivantes : Quelle est la fonction de la protéine du panier TPR lorsqu'elle n'est pas associée au NPC, et la TPR nucléoplasmique participe-t-elle au métabolisme de l'ARN nucléaire, reliant potentiellement les processus nucléaires au contrôle de la qualité au NPC? Mon projet s'est concentré sur l'étude des fonctions et de la dynamique de la protéine du panier nucléaire TPR à l'aide de techniques d'imagerie fluorescente en cellule vivante et de suivi de protéine unique. Nous avons pu identifier la dynamique et la localisation des différentes populations de TPR à partir des profils de diffusion de leurs trajectoires, qui peuvent être réparties en 5 catégories : Dirigée, Brownienne, Restreinte, Confinée et Butterfly. Nos données suggèrent que les trajectoires confinées pourraient être liée à l’association de TPR à la chromatine tandis que les browniennes représenteraient les molécules de TPR diffusant librement dans le noyau. De plus, nous avons constaté que les trajectoires dirigées et restreintes pourraient être liées à la maturation de l'ARN vu que ces deux sous-populations de TPR sont les plus affectées lorsque la transcription est inhibée. Également, en absence de la transcription par l’ARN polymérase II, TPR forme des granules dans le nucléoplasme, suggérant son implication durant la transcription active. Ainsi, notre étude montre que la fraction nucléoplasmique du TPR est subdivisée en fractions non associées aux pores hétérogènes qui pourraient jouer plusieurs rôles dans le métabolisme de l'ARN et la qualité de l'export. / The nuclear pore complex is the only entry and exit point for the nucleocytoplasmic transport. The nuclear basket, one of its main components, was shown to be involved in gene regulation and could play a major role in quality control of mRNA export. However, little is known on how the basket functions in nuclear export and gene regulation. The main structural component of the basket, TPR (Translocated Promoter Region), is thought to be the main actor in the quality control function of the basket. It is yet to be establish by which mechanism this protein ensures the selection of competent mRNPs for export. With all these involvement of the basket in quality control, export, and maturation, one question remains: What is the basket really doing, what defines quality control, how the nuclear basket can identify RNAs that aren’t competent for export, and what are the roles of the different proteins that make up the basket. Recently it was shown that TPR is present in two populations, one in the nucleoplasm and another bound at the NPC. Our preliminary studies using FRAP (Fluorescence Recovery After Photobleaching) and single molecule microscopy shows that the nucleoplasmic TPR molecules aren’t exchanging with the baskets anchored at the NPC and present different subpopulations based on diffusion. Analysis of preliminary proteomics studies from our laboratory revealed an interactome with an unexpected enrichment of proteins involved in mRNA maturation notably splicing and 3’ end processing factors. These results imply complex interactions of the new fractions of TPR and lead us to ask these following questions: What is the function of the basket protein TPR when it is not associated with the NPC, and does nucleoplasmic TPR participate in nuclear RNA metabolism, potentially linking nuclear processes to quality control at the NPC? My project focused on investigating the functions and dynamics of the nuclear basket protein TPR using fluorescent live-cell and single-protein imaging techniques. We were able to identify the dynamics and localization of the different populations of TPR based on the diffusion profiles of their trajectories, which can be divided in 5 categories: Directed, Brownian, Restricted, Confined and Butterfly. Our data suggest that the confined population might be linked to chromatin association of TPR, whereas the Brownian would represent the free diffusing TPR molecules in the nucleus. We further found that the Directed and Restricted trajectories could be linked to RNA maturation as these two subpopulations of TPR are most affected when transcription is inhibited. Moreover, in absence of transcription, TPR forms granules in the nucleus, suggesting its implication during active transcription. Altogether, our study shows that the nucleoplasmic fraction of TPR is subdivided in heterogenous diffusive fractions that could play several roles in the metabolism of RNA and quality of export

Ce travail porte sur la synthèse et la caractérisation d’oxydes de manganèse de formule généraleCaxMnyOz avec y/x > 1. Par une approche de chimie du solide, les propriétés redox de ces matériauxseront corrélées à leur composition chimique et leur structure cristalline ce qui constitue une étude enamont de l’application pour ces systèmes pouvant jouer le rôle de tampon à oxygène dans desprocessus catalytiques. Les composés ont été synthétisés par autocombustion en voie aqueuse etcaractérisés d’un point de vue structural. Les propriétés redox ont été évaluées par ATG et lespropriétés de réductibilité par H2-TPR pour certains composés. Quelles que soient la structure, ladimensionnalité ou la valence du manganèse dans les composés oxydés (Mn4+/Mn3+), le manganèsese réduit totalement en Mn2+ sous atmosphère réductrice (Ar/H2). Les composés réduits cristallisentdans une solution solide de type NaCl de formule Ca1-xMnxO. Les mécanismes de réduction dumanganèse au sein de ces structures ont été appréhendés sur la base des analysesthermogravimétriques. Afin de faire varier les températures de réduction, l’iono-covalence de la liaisonMn-O a été modifiée via des substitutions cationiques (i) sur les sites du calcium principalement ausein de réseaux 2D ou (ii) sur les sites du manganèse essentiellement dans les réseaux 3D avec laprésence d’Al3+ ou de Fe3+ de rayons ioniques comparables respectivement à Mn4+ et Mn3+. Lessolutions solides ont été caractérisées d’un point de vue structural mais aussi pour leurs propriétésredox. Il convient de souligner que les phases substituées au Fer présentent des propriétés redox toutà fait remarquables dans la mesure où les ions Fe3+ tout comme Mn4+/3+ se réduisent d’abord en Fe2+puis en Fe° qui est dès lors expulsé hors de la matrice oxyde. Lors de la réoxydation, le fer réintègrele réseau 3D et les matériaux sont cyclables dans des conditions réductrices puis oxydantes à l’air àdes températures inférieures à 1000°C. / This work deals with the synthesis and characterization of manganese oxides with CaxMnyOz formulaand y/x >1. According to a solid state chemistry scope, the redox properties of these materials will becorrelated to their chemical composition and crystallographic structure which constitutes a forehandexploratory study of compounds that are intended to be used for the automotive catalysis exhaustbased on the three-way catalysis principle. The materials were synthesized by aqueous selfcombustionroute and structurally characterized. Redox properties and reducibility properties wereevaluated by TGA and H2-TPR, respectively. Whatever the structure, the dimensionality of the networkor the manganese valence in the starting oxidized material, all of the manganese ions are completelyreduced (Mn2+) in a Ar/H2 atmosphere. The reduced compounds crystallize in a rock-salt type solidsolution with the formula Ca1-xMnxO. The mechanisms of manganese reduction within these structureswere explored on the basis of TGA analysis. In order to tune the reduction temperatures, the ionocovalenceof the Mn-O bond has been modified by either cationic substitution of calcium in the 2Dnetworks or either substitution of manganese in the 3D networks. In this last case, Substituting ionswere Al3+ and Fe3+ which ionic radii comparable to Mn4+ and Mn3+, respectively. Solid solutions havebeen characterized from a structural point of view but also for their redox properties. One shouldnotice that iron substituted compounds exhibit remarkable redox properties because Fe3+ ions firstreduce in Fe2+ iron before a final reduction in Fe° that is consequently expulsed from the matrix. Atreoxidation, iron returns into the 3D network and cycling can be observed when reducing and oxidizingat temperatures lower than 1000°C.

Orientador: Carlos Henrique Inácio Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T13:20:36Z (GMT). No. of bitstreams: 1 Murakami_LeticiaMariaZanphorlin_D.pdf: 5383539 bytes, checksum: 1a45d203e6e3c5a992791b8ce893aa36 (MD5) Previous issue date: 2014 / Resumo: Chaperonas moleculares e proteínas de choque térmico (Heat shock protein, Hsp) atuam contra a agregação e o enovelamento incorreto de proteínas, que são os agentes causais de doenças neurodegenerativas, como por exemplo, Alzheimer e Parkinson. A Hsp90 é uma das mais importantes chaperonas moleculares, considerada essencial para a viabilidade celular em eucariotos, pois está associada com a maturação de proteínas atuantes na sinalização e ciclo celular. Além disso, foi demonstrado que a Hsp90 está envolvida na estabilização do fenótipo tumoral de diversos tipos de câncer, destacando a sua importância biomédica. A interação com co-chaperonas, proteínas auxiliares das chaperonas, permite que a Hsp90 atue como uma proteína "hub", ou seja, um ponto central de regulação de diversas proteínas. Muitas dessas co-chaperonas possuem um ou mais domínios do tipo TPR (do inglês, tetratricopeptide repeat) que interagem com o C-terminal da Hsp90. No presente projeto de doutorado, investigamos as características estruturais e termodinâmicas da interação entre o domínio C-terminal da Hsp90 (C-Hsp90) e a co-chaperona TPR Tom70 humana, utilizando técnicas de reação-cruzada acoplada à espectrometria de massas (LC-MS/MS), calorimetria de titulação isotérmica (ITC), espalhamento de raios-X à baixos ângulos (SAXS) e modelagem molecular. Os resultados de LC-MS/MS e ITC evidenciaram novas regiões na interação do complexo C-Hsp90/Tom70 que envolve a hélice A7 presente na Tom70 e experimentos de SAXS revelaram a estrutura em baixa resolução das proteínas C-Hsp90, Tom70 e do complexo C-Hsp90/Tom70. Além disso, investigamos o efeito do celastrol, um composto com potencial atividade anti-câncer, na conformação e na função da Hsp90. Na presença do composto, a Hsp90 sofre um processo de oligomerização e a natureza dos oligômeros foi determinada por ferramentas bioquímicas e biofísicas, tais como espalhamento dinâmico de luz (DLS), cromatografia de exclusão molecular analítica acoplada a espalhamento de luz em multiângulos (SEC-MALS) e eletroforese em gel nativo. Interessantemente, a oligomerização induzida pelo celastrol não afetou a atividade de proteção da Hsp90 contra a agregação protéica e a capacidade de ligação as co-chaperonas com enovelamento tipo TPR. Este é o primeiro trabalho a apontar um possível mecanismo para a ação do celastrol sobre a Hsp90. Coletivamente, nossos resultados e descobertas contribuem para uma melhor compreensão dos mecanismos moleculares relacionados à interação entre chaperonas e co-chaperonas, bem como, chaperonas e potenciais ligantes. / Abstract: Molecular chaperones and heat shock proteins (Hsp) act against protein aggregation and misfolding, which are the causal agents of neurodegenerative diseases such as Alzheimer and Parkinson. Hsp90 is one of the most important molecular chaperones, considered essential for cell viability in eukaryotes, since it is associated with the maturation of proteins involved in cell cycle and signaling. In addition, it was demonstrated that Hsp90 is implicated in the stabilization of the tumor phenotype of various types of cancer, highlighting its biomedical importance. The interaction with co-chaperones, auxiliary proteins of chaperones, allows that Hsp90 acts as a hub, being a central point for regulation of several other proteins. Many of these co-chaperones have one or more TPR domains that interact with the C-terminus of Hsp90. In this PhD project, we investigated structural and thermodynamic characteristics of the interaction between the C-terminus domain of Hsp90 (C-Hsp90) and the TPR co-chaperone human Tom70, using techniques of cross-linking coupled with mass spectrometry (LC-MS/MS), isothermal titration calorimetry (ITC), small angle X-ray scattering (SAXS) and molecular modeling. The results of LC-MS/MS and ITC revealed new regions involved in the interaction of the C-Hsp90 with Tom70, which encompasses the A7 helix from Tom70, and SAXS experiments unveiled the low resolution structure of the proteins C-Hsp90, Tom70 and the C-Hsp90/Tom70 complex. In addition, we investigated the effect of celastrol, a compound with a potential anti-cancer activity, on the conformation and function of Hsp90. In the presence of celastrol, Hsp90 undergoes oligomerization and the nature of the oligomers was determined by biochemical and biophysical tools such as dynamic light scattering (DLS), size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and native gel electrophoresis. Interestingly, the celastrol-induced oligomerization did not affect the protective activities of Hsp90 against protein aggregation or the capacity to bind TPR co-chaperones. This is the first study to point out a possible mechanism for the action of celastrol on Hsp90. Collectively, our findings contribute to a better understanding of the molecular mechanisms associated to the interaction between chaperones and co-chaperones, as well as chaperones and potential ligands / Doutorado / Quimica Organica / Doutora em Ciências

ªí­±¤Æ¾Ç»â°ì¤¤¡A¬ã¨s¹L´çª÷Äݳ洹ªí­±¤W§lªþºA¤A°ò(C2H5)¤§¤ÏÀ³¡A¤w¦³¬Û·í¦hªº³ø¾É¡C¨ä¦@ÃѬ°£]-H®ø¥h(£]-elimination)§Î¦¨¤A²m(C2H4)²æªþ¬O¥D­n¤ÏÀ³¸ô®|¡C¥»¬ã¨s«h¥H¤£¦Pµ{«×¬t¨ú¥Nªº¤A°ò(ethyl)¡GCF3CF2-¡BCHF2CF2-¡BCF3CHF-¡BCF3CH2-¤ÎCH3CH2-§@¬°¹ï·Ó¡A§lªþ¦bCu(100)³æ´¹­±¤W¥Hµ{·Å¤ÏÀ³/²æªþ(TPR/D)¹êÅç±´°Q¤ÏÀ³¾÷ºc¡Aµ²ªGÅã¥Ü«e¨âºØ¼Ë«~¥ý¶i¦æ£\-F®ø¥h§Î¦¨Ethylideneªí­±¤¤¶¡Åé¡A«eªÌ¦A¦Û¨­°¸¦X©ó350K¥Í¦¨CF3-CF=CF-CF3¡A¦Ó«áªÌ°¸¦X©ó300K¥Í¦¨CHF2-CF=CF-CHF2¡C«á¤TºØ¼Ë«~«hµo¥Í£]®ø¥hª½±µ²£¥ÍCHF=CF2(310K)¡BCH2=CF2(225K)©MCH2=CH2(250K)¡C¥Ñ©ó­n¶i¦æ¸û§C·Å¤§£]®ø¥h¤ÏÀ³®É¡A¹L´çºA§e¥­­±¤ÎEclipsedºc«¬(¦p¥k¹Ï)¡CÂǥѦ¹¬Ý¥X«e¨âºØ¼Ë«~¤§£]¸ô®|¹L´çºA¬O¨ã¦³¨â¹ïF-F¤¬¥¸§@¥Î¡A³o¨Ï±o¹L´çºA¸û¤£Ã­©w¡A¯à»Ùª@°ª¾É­P¤ÏÀ³¸ô®|Â੹¬Û¹ï¯à»Ù¥i¯à¸û§C¤§£\®ø¥h¡C«á¤TºØ¼Ë«~¦æ£]®ø¥h¤§¹L´çºA¦]¦³¸û¤ÖF-F±Æ¥¸¡A©Ò¥H¦æ£]®ø¥h¤ÏÀ³¡C¨Ï¥Î¼ÒÀÀ­pºâªºµ²ªG¤]Åã¥Ü¡A·í©Mª÷ÄÝÁäµ²ªº£\ºÒ¤W¨ã¬t¨ú¥N®É°£³y¦¨ªºÁä¯àÅܱj¡A¥ç·|³y¦¨¦b¹L´çºA¦b¶i¦æª÷ÄÝ-ºÒÁäÂ_µõ®É¯à»Ù¤É°ª¦Ó¶}±Ò¬Û¹ï¯à»Ù¥i¯à¸û§C¤§£\®ø¥h³q¹D¡C¬ã¨sµ²ªG©Ò¤Ï¬M¤A°ò¤W¬t¨ú¥N¦ì¸m¤Îµ{«×¤£¦P©Ò³y¦¨ªº¤ÏÀ³¸ô®|ªº¿ï¾Ü©Ê¡A§¡¥i¥Ñ¤W­z¹L´çºA¤©¥H¦X²zªº¸ÑÄÀ¡C