No description available.

L’ouverture à la concurrence du transport de fret ferroviaire en Europe entraîne des évolutions de structures significatives. La SNCF - opérateur historique – doit engager une mutation sans précédent. De nouveaux opérateurs s’implantent en France et doivent recruter et former du personnel. Dans un premier temps, le fonctionnement de ces nouveaux acteurs s’effectue principalement grâce à du personnel retraité de la SNCF. On assiste également à la création d’Opérateurs Ferroviaires de Proximité (OFP). Ces entités, généralement de petite taille, viennent compléter le travail effectué par les entreprises ferroviaires sur des dessertes terminales. Les OFP ont également besoin de compétences ferroviaires. Notre recherche porte donc sur les moyens nécessaires pour construire des compétences ferroviaires dans le respect des principes de sécurité et en intégrant le fonctionnement spécifique des structures OFP. La création d’une structure virtuelle peut contribuer à l’identification et la construction de compétences ferroviaires. / The opening to compétition of rail freight in Europe leads to significant changes in structures. The SNCF, incumbent, must commit unprecedented change. New operators setting up in France and need to recruit and train staff. At first, these new operators mainly use SNCF retired staff. Furthermore, Short Lines establishing are observed. These entities, generally small, complement the work of railway undertakings for the last kilometres. Short Lines also need skills rail. Our research focuses on necessary means to acquire skills rail in accordance with safety principles and by working according to Short Lines specific operating. Virtual structure creation can help skills rail identification and définition.

abstract: Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps. / Dissertation/Thesis / Ph.D. Chemistry 2013

No description available.

DNA replication is one of the most fundamental processes in all living organisms. Its semi-discontinuous nature dictates that the lagging strand is synthesized in short fragments called Okazaki fragments. In eukaryotes, each Okazaki fragment is initiated by an ~ 30-40 nucleotide-long RNA-DNA hybrid primer that is synthesized by Pol α-primase complex. To ensure genomic stability, the RNA primer has to be excised, any misincorporations by Pol α have to be corrected for and finally the resulting nick has to be sealed generating a contiguous strand. This feat is accomplished by a highly coordinated and regulated process called Okazaki fragment maturation. At the center of this process are 5’ nucleases, which are structure-specific nucleases that catalyze the incision of phosphodiester bonds one nucleotide into the 5’ end of ssDNA/dsDNA junctions. Previous structural and biochemical studies have shed some light on the mechanism of FEN1 substrate recognition, its catalysis and regulation. However, many gaps in our understanding of this remarkable nuclease still persist. Moreover, the choice between the short- and long-flap pathways is still elusive. Finally, the mechanism of the coordination among the different enzymatic activities of the polymerase, the nuclease and the ligase during Okazaki fragment maturation is still debatable. In this work, we set out to study FEN1 substrate recognition, catalysis and regulation using single molecule techniques. We show that FEN1 employs a sophisticated substrate recognition mechanism through which it actively distorts the DNA to ~100˚ bent angle. It also displays a remarkable selectivity towards its cognate substrate and avoids off-target substrate by a lock-down mechanism that commits the enzyme for catalysis on cognate substrates while promoting the dissociation of non-cognate substrates. We further characterized FEN1 reaction from substrate binding/bending to product handoff and built a comprehensive kinetic scheme that shows FEN1 releasing its product in two steps. Finally, we uncovered an unprecedented role of FEN1 in the choice between short- and long-flap pathways.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22301号 / 医博第4542号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 椛島 健治, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13401号 / 論医博第2225号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 渡邊 直樹, 教授 江藤 浩之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23066号 / 医博第4693号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 溝脇 尚志, 教授 藤田 恭之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Fluorescence-based assays have gained an ever-increasing popularity in life sciences. One of these rapidly emerging techniques is Protein Induced Fluorescence Enhancement (PIFE). Traditional explanations of PIFE focused exclusively on the role of the protein and largely neglected the role of the mediator DNA. In the same time, the existing models of PIFE were denying its exactly opposite effect. In the first part of the current dissertation we focus on a better understanding of PIFE, stimulated by the direct observation of its opposite effect, Induced Fluorescence Enhancement Quenching (PIFQ). This study offered us the leverage for obtaining on-demand fluorescence modulation in cyanine dyes. The following two chapters harvest this control over fluorescence modulation to generate two biotechnology applications: a sensitive potassium sensor with embedded fluorescent transducer, and a simple protocol for the fluorescent detection of His-tagged proteins. In the last part, a variety of fluorescence tools including Förster resonance energy transfer, fluorescence enhancement, and fluorescence quenching are employed for a much more complex task; to demystify the behavior of the human Maturation of Okazaki Fragments (MOF) machinery. First, we reconstituted the human MOF reaction and showed that it behaves considerably different than its well-established yeast homolog. Subsequently, our toolbox of fluorescence-based assays was used to pinpoint the kinetics and dynamics that lead to this unexpected MOF behavior.

L’objectif de cette thèse est d’identifier les politiques qui permettraient de réduire les émissions de GES du transport de fret routier au moindre coût pour la collectivité. Pour cela, nous estimons, sur la période 2030-2050, les quantités évitées ainsi que le coût économique dans 6 scénarios correspondant à différentes politiques de réductions des émissions. Le coût économique comprend la somme des coûts pour les utilisateurs de transport et pour les pouvoirs publics ainsi que les coûts des externalités. Nous utilisons, quand ils sont disponibles, des jeux d’hypothèses définis par le ministère des transports, en particulier pour l’évolution des trafics et les valeurs unitaires des externalités. Pour chaque scénario, le coût économique par tonne de GES évitée est très sensible aux hypothèses, en particulier aux valeurs unitaires retenues pour les externalités (valorisation des co-bénéfices). Dans les hypothèses centrales, plusieurs scénarios présentent un coût négatif, c'est-à-dire un bénéfice pour la collectivité en plus de la réduction des émissions. C’est le cas de notamment de la taxe carbone, aussi longtemps qu’elle reste inférieure aux coûts externes engendrés par les poids lourds. Pourtant, quand cette taxe augmente, la hausse correspondante du prix de transport conduit les chargeurs à renoncer à une partie de leur activité, ce qui limite l’augmentation souhaitable. L’augmentation de 40 à 60 tonnes du poids maximum autorisé des poids lourds est une mesure très favorable aux utilisateurs de transport car elle permet de réduire significativement le coût du transport. Prenant en compte les gains d’externalités, la perte fiscale des pouvoirs publics, ainsi que les coûts d’infrastructure (investissement pour renforcer les ponts et les routes et entretien), ce scénario présente également un bénéfice pour la collectivité. Le scénario d’installation de caténaires sur le réseau autoroutier pour permettre la circulation de poids lourds hybrides est efficace en matière de quantité de GES évitée et le coût d’évitement par CO2e reste limité. En revanche, dans le scénario d’électrification du fret urbain (remplacement des camions diesels par des camions électriques dans les villes) le coût de la tonne évitée est élevé si les camions électriques n’ont que deux tonnes de charge utile alors qu’il devient un bénéfice économique si, grâce au progrès technologique, les camions électriques ont 6 tonnes de charge utile en 2030 (pour une autonomie de 100 km).Le remplacement des poids lourds diesels par des poids lourds au Gaz Naturels Véhicules réduit le coût du transport et bénéficie donc aux transporteurs et chargeurs tandis que les pouvoirs publics supportent une perte de recette fiscale. En tenant en compte les gains d‘externalités, cette mesure permettrait un coût économique limité. Enfin, le chargeur a la possibilité de réduire ses émissions de GES, avec un coût raisonnable pour l’entreprise et un bénéfice pour la collectivité, en diminuant la fréquence de ses envois. Pourtant, aucune de ces mesures n’est susceptible d’offrir seule une réduction suffisante des émissions. Pour atteindre l’objectif de facteur 4 à un coût raisonnable pour la collectivité, différentes mesures doivent être envisagées en complément / The purpose of this thesis is to identify policies that would allow to mitigate road freight GHG emissions at the lowest cost for the community. With this in mind, for six scenarios corresponding to different mitigation policies we estimate the mitigated emissions along with the economic cost, over the 2030-2050 period. The overall economic cost includes the cost for transport users, for public authorities, and an economic valuation of transport externalities. When available, we used sets of assumptions retained by the French Ministry of Transports for official traffic projections and for the unitary valuation of external costs. For every scenario, the economic cost by avoided ton of CO2 is highly sensitive to these assumptions, especially to the unitary value of external costs (valuation of co-benefits). In the central set of assumptions, several scenarios result in a negative economic cost of GHG mitigation, id est a benefit to the community in addition to GHG cuts.This is notably the case for the carbon tax, as long as it remains lower than the external costs of freight road transportation. However, when the tax rate is rising, the resulting increase in transport prices leads shippers to reduce their activity, which limits the desirable tax rate increase. Increasing the maximum authorized weight of heavy trucks from 40 to 60 tons would be a very beneficial measure towards transport users by allowing a substantial transport costs reduction. This scenario is also beneficial for the community, when accounting for the avoided external costs, the financial loss for public authorities and infrastructure costs (the required reinforcement of roads and bridges and increased maintenance costs). The installation of catenaries on motorways to supply hybrid heavy trucks is efficient to reduce CO2 emissions, and the economic cost per avoided ton of CO2 remains moderate. Quite the opposite, the economic cost is high in the scenario of electrification of urban freight (where diesel trucks are replaced by electric trucks for urban goods delivery), if electric trucks only have a maximum payload of 2 tons. On the contrary, it turns into an economic benefit if, thanks to technological progress, electric trucks have a payload of 6 tons in 2030, for a range of 100 km.The replacement of diesel trucks by natural gas vehicles reduces transport costs and is therefore beneficial towards carriers and loaders, but public authorities support a loss of taxation revenues. However, when accounting for the avoided external costs, the overall economic cost happens to be limited.Finally, shippers can reduce GHG emissions for a reasonable economic cost by reducing the frequency of shipments, this measure also being beneficial for the community.However, none of these measures is capable to achieve by itself the official goal of dividing by 4 the level of GHG emissions at a reasonable cost for the community, implying that additional measures have to be considered jointly