Στην παρούσα Ειδική Ερευνητική Εργασία μελετόνται οι φωτοφυσικές ιδιότητες γνωστών χρωμοφόρων μορίων. Συγκεκριμένα μελετάται η Coumarin 30, η Fluorescein Isothiocyanate και η Dichlorofluorescein, τόσο με φασματοσκοπία σταθερής κατάστασης όσο και με φασματοσκοπία χρονικής ανάλυσης μέσω της τεχνικής Time Correlated Single Photon Counting. Η διερεύνηση γίνεται με τα μόρια αυτά να βρίσκονται σε μορφή διαλυμάτων με στόχο τον προσδιορισμό της συμπεριφοράς τους όταν αυτά βρισκονται σε διαφορετικά περιβάλλοντα (διαλύτες). Επιπλέον ερευνώνται οι αλληλεπιδράσεις των μορίων αυτών μέσω της φασματοσκοπίας σταθερής κατάστασης και χρονικής ανάλυσης όταν βρίσκονται σε μορφή μειγμάτων. / In this Master Thesis, the phophysical properties of chromophrores are investigated. Specifically, Coumarin 30, Fluorescein Isothiocyanate and Dichlorofluorescein are studied with steady – state spectroscopy as well as with time – resolved spectroscopy via the technique of Time Correlated Single Photon Counting. The study of these molecules focuses also on solvent – solute effects. Finally, interactions between them are also studied through steady – state spectroscopy and time resolved spectroscopy.

Un dispositif permettant le confinement d'ions calcium pendant des heures a été réalisé. Dans une première étape les ions ont été détectés utilisant deux méthodes électroniques: l'éjection résonnante à l'aide d'un multiplicateur d'électrons, et la détection par un circuit résonnant. L'influence d'un gaz tampon sur les durées de confinement et l'évolution de la charge d'espace a été mise en évidence par cette dernière méthode. les ions ont ensuite été détectés par la fluorescence des raies de résonance du CaII soit en utilisant un gaz tampon, soit par double-résonance optique-optique. l'excitation directe des transitions interdites 4S-3D des ions a permis d'aborder des mesures de la durée de vie naturelle des niveaux métastables par une méthode originale. des résultats très précis pour ces durées de vie ont été obtenus. Nous avons déterminé les taux de réaction pour la désexcitation des états métastables vers le niveau fondamental (quenching) en mesurant la variation de la durée de vie effective des niveaux en présence de différents gaz tampon. une étude en équilibre (fluorescence sensibilisée) ainsi qu'une étude dynamique des populations des deux états du doublet ont fourni des taux de réaction pour le mélange des niveaux de structure fine.

Le but de cette thèse est de sonder la flexibilité de NCp7 et de Δ(-)PBS, deux bio-molécules impliquées dans le second saut de brin de la transcription inverse du VIH. Deux stratégies expérimentales ont été mises en place. Un nouveau montage de spectroscopie ultra-rapide de fluorescence par down-conversion a été construit. Les dynamiques de quenching de la 2-aminopurine (2Ap), insérée en position 6, 8 et 10 de la boucle Δ(-)PBS ont pu être entièrement résolues à une résolution sub-ps. Pour chaque position, 4 temps de vie ont été révélés. Des mesures d'anisotropie confirment que les deux composantes < 5 ps sont liées à un empilement de la 2Ap avec les Guanines avoisinantes. Cet empilement est site-spécifique, prouvé par l'augmentation significative de leurs amplitudes lorsque la 2Ap est située près de la tige (position 10). La faible proportion de conformations reliées à un quenching collisionnel est significative de la faible exposition des 2Ap au solvant et de l'encombrement général de la boucle. La seconde approche avait pour but d'étudier l'effet du repliement du squelette protéique de [35-50] NCp7 autour de son atome de zinc par CID et par LID. Les spectres CID de la protéine nue sont expliqués par le modèle du proton mobile et une description détaillée d'un schéma de fragmentation spécifique autour du Tryptophane (Trp) a été soulignée, attribué une Lysine voisine. Un seul fragment issu de l'excitation à 266 nm a été identifié, son apparition entre en compétition avec les fragments CID du Trp. L'effet général du repliement autour du Zinc se traduit par une augmentation du taux de fragmentation autour du Trp et par une perte de spécificité pour le reste du spectre.Les flexibilités de Δ(-)PBS et NCp7 ont été respectivement évaluées par spectroscopie ultra-rapide de type down-conversion et par spectrométrie en phase gazeuse. La première méthode nécessite l'utilisation d'une sonde fluorescente non invasive, la 2-aminopurine (2Ap), placée en position 6, 8 et 10 de la boucle Δ(-)PBS. Notre résolution temporelle permet de résoudre entièrement les dynamiques locales de quenching et d'anisotropie de la 2Ap. Les composantes liées au quenching statique et quenching collisionnel ont été discriminées et révèlent les degrés d'empilement / encombrement locaux de la boucle. L'effet du repliement de [35-50] NCp7 autour de son atome de zinc a été étudié par CID et par LID à 266 nm. La protéine nue présente un interessant shéma de fragmentation autour du Tryptophane (Trp), exalté par la complexation avec le zinc, au prix une perte de spécificité pour le reste du spectre. Un seul fragment LID a été identifié, un mécanisme de sa formation est proposé.

Strong light can cause damage and be lethal for photosynthetic organisms. An increase of thermal dissipation of excess absorbed energy at the level of photosynthetic antenna is one of the processes protecting against deleterious effects of light. In cyanobacteria, a soluble photoactive carotenoid binding protein, Orange Carotenoid Protein (OCP) mediates this process. The photoactivated OCP by interacting with the core of phycobilisome (PB; the major photosynthetic antenna of cyanobacteria) triggers the photoprotective mechanism, which decreases the energy arriving at the reaction centres and PSII fluorescence. The excess energy is dissipated as harmless heat. To regain full PB capacity in low light intensities, theFluorescence Recovery Protein (FRP) is required. FRP accelerates the deactivation of OCP.In this work, I present my input in the understanding of the mechanism underlying the OCPrelated photoprotection. I further characterized the FRP of Synechocystis PCC6803, the model organism in our studies. I established that the Synechocystis FRP is shorter than what it was proposed in Cyanobase and it begins at Met26. Our results also revealed the great importance of a high OCP to FRP ratio for existence of photoprotection. The most remarkable achievement of this thesis is the in vitro reconstitution of the OCPrelated mechanism using isolated OCP, PB and FRP. I demonstrated that light is only needed for OCP photoactivation but OCP binding to PB is light independent. Only the photoactivated OCP is able to bind the PB and quench all its fluorescence. Based on our in vitro experiments we proposed a molecular model of OCP-related photoprotection. The in vitro reconstituted system was applied to examine the importance of a conserved salt bridge (Arg155-Glu244) between the two domains of OCP and showed that this salt bridge stabilises the inactive form of OCP. During photoactivation this salt bridge is broken and Arg155 is involved in the interaction between the OCP and the PB. The site of OCP binding in the core of a PB wasalso investigated with the in vitro reconstituted system. Our results demonstrated that the terminal energy emitters of the PB are not needed and that the first site of fluorescence quenching is an APC trimer emitting at 660 nm. Finally, we characterised the properties of excited states of the carotenoid in the photoactivated OCP showing that one of these states presents a very pronounced charge transfer character that likely has a principal role in energy dissipation. Our results strongly suggested that the OCP not only induces thermal energy dissipation but also acts as the energy dissipator.

Notre génome, véritable mode d'emploi de chaque cellule et organisme, est constamment menacé par de multiples agents endogènes ou exogènes qui endommagent la biomolécule d'ADN. Ces lésions résultantes, de nature diverse, sont notamment impliquées dans les processus de vieillissement cellulaire, de cancérogénèse et de mort cellulaire. Afin de contrer ces effets néfastes, les organismes ont développé différents systèmes de réparation de l'ADN capables de prendre en charge spécifiquement chaque type de dommages. Parmi ces voies métaboliques, la réparation par excision de base (BER) répare chaque jour des dizaines de milliers de dommages, incluant les bases alkylées, oxydées ou désaminées, les sites abasiques ou encore certaines cassures de brin. Dans le présent travail, nous exposons la mise au point d'un nouveau biocapteur pour la détection des activités enzymatiques du BER. L'outil se caractérise par un set de sondes nucléiques autocomplémentaires, fluorescentes ou pro-fluorescentes, immobilisées sur microbilles paramagnétiques. Chaque sonde est modifiée par l'introduction sélective d'une lésion, substrat d'une activité enzymatique ciblée (ADN N-glycosylase, AP-endonucléase). L'activité d'excision/incision de la lésion, conduit à la coupure de la sonde et à la déshybridation de la structure. L'analyse et la quantification du clivage spécifique est réalisée en fluorescence, soit à partir du surnageant par spectrofluorimétrie, soit des billes par cytométrie en flux. Ce dispositif permet la détection multiplexée des activités enzymatiques de protéines purifiées ou au sein d'extraits nucléaires. Egalement, des applications dans le criblage d'inhibiteurs de la réparation de l'ADN sont envisageables dans le cadre de recherches pré-cliniques. L'adaptation de ces tests in vitro à la détection de la réparation de l'ADN in cellulo a fait l'objet de développements préliminaires.

It has long been observed that salts affect proteins in a variety of ways, yet comprehensive explanations for different salt effects are still lacking. In the work presented here, the effect of salts on proteins has been investigated through three different effects: the hydrophobic effect; their conformational stability; the hydrogen bonding network of water in a protein’s hydration shell. UV-vis absorbance and fluorescence spectroscopy were used to monitor changes in two model systems, the phenol-acetate contact pair and the model enzyme ribonuclease t1. It was shown that salts affect the hydrophobicity of the contact pair according to their charge density, induced image charges play an important role in the observed salt-induced increase of ribonuclease t1 stability, and that salts affect ribonuclease t1 activity through modulation of the hydrogen bonds of water in the enzyme’s hydration shell. This work contributes a greater understanding of the effect of salts on proteins.

It has long been observed that salts affect proteins in a variety of ways, yet comprehensive explanations for different salt effects are still lacking. In the work presented here, the effect of salts on proteins has been investigated through three different effects: the hydrophobic effect; their conformational stability; the hydrogen bonding network of water in a protein’s hydration shell. UV-vis absorbance and fluorescence spectroscopy were used to monitor changes in two model systems, the phenol-acetate contact pair and the model enzyme ribonuclease t1. It was shown that salts affect the hydrophobicity of the contact pair according to their charge density, induced image charges play an important role in the observed salt-induced increase of ribonuclease t1 stability, and that salts affect ribonuclease t1 activity through modulation of the hydrogen bonds of water in the enzyme’s hydration shell. This work contributes a greater understanding of the effect of salts on proteins.

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-08-20T13:53:22Z No. of bitstreams: 1 WELLINGTON CORDEIRO OLIVEIRA - DISSERTAÇÃO (PPGEM) 2018.pdf: 8813868 bytes, checksum: a538ae4b12c154e17d31068945d4febb (MD5) / Made available in DSpace on 2018-08-20T13:53:22Z (GMT). No. of bitstreams: 1 WELLINGTON CORDEIRO OLIVEIRA - DISSERTAÇÃO (PPGEM) 2018.pdf: 8813868 bytes, checksum: a538ae4b12c154e17d31068945d4febb (MD5) Previous issue date: 2018-04-06 / Os aços ferramenta têm uma importante participação no mercado mundial no que se refere a engenharia. São usados na indústria metal-mecânica como ferramentas de corte, moldes, punções, entre outras aplicações. Devido a sua grande importância, é imprescindível discutir a relação entre a microestrutura e as propriedades mecânicas, devido ser uma prática comum na indústria especificar os tratamentos térmicos com base principalmente na dureza final do material, sem considerar que para um mesmo valor de dureza um aço ferramenta pode apresentar diferentes propriedades mecânicas, isto dependendo do ciclo de tratamento térmico aplicado. Logo, a seleção do tratamento térmico é um aspecto tecnológico relevante no desempenho destes aços. Tendo em vista a vasta possibilidade de propriedades mecânicas obtidas (tenacidade a fratura) com diferentes tratamentos térmicos, esse trabalho teve como objetivo especificar um aço ferramenta nacional e um conjunto de parâmetros de tratamento térmico que possibilite a utilização deste material como forma de substituição de um outro aço importado utilizado na confecção de lâminas de corte usadas no processamento de placas de chumbo. O aço selecionado para o desenvolvimento deste trabalho foi o aço AISI D6, a seleção desse aço foi feita tomando-se como base as características obtidas através de análises realizadas em campo e de microdureza e microestrutura das lâminas de corte já existentes no processo confeccionadas com aço importado. Com o aço selecionado em mãos, aplicou-se dois tratamentos térmicos diferentes de têmpera e revenido (um proposto pelo fabricante e outro proposto pelo trabalho em execução), onde avaliou-se o efeito dos tratamentos térmicos sobre as propriedades mecânicas e microestrutural do material. Através da análise dos resultados da relação entre dureza e tenacidade, constatou-se que para os dois tratamentos térmicos aplicados, não houve variações significativas das propriedades mecânicas do material, e que os dois tratamentos apresentaram resultados satisfatórios. O aço ferramenta nacional especificado apresentou-se como alternativa para substituição do aço importado utilizado atualmente. Testes obtidos em campo com a lâmina fabricada com o aço nacional especificado nesse estudo e que recebeu o tratamento térmico proposto pelo fabricante (FAB), corroboraram com a análise das propriedades, visto que se constatou aumento do tempo de vida de corte em mais de 30%. / The tool steels have an important participation in the world market with regard to engineering. They are used in the metal-mechanic industry as cutting tools, molds, punches, among other applications. Because of its great importance, it is essential to discuss the relationship between the microstructure and the mechanical properties, since it is a common practice in industry to specify thermal treatments based mainly on the final hardness of the material, without considering that for a same hardness value a steel tool may have different mechanical properties, depending on the heat treatment cycle applied. Therefore, the selection of heat treatment is a relevant technological aspect in the performance of these steels. Considering the wide possibility of mechanical properties obtained (fracture toughness) with different thermal treatments, this work had the objective of specifying a national tool steel and a set of thermal treatment parameters that allow the use of this material as a substitute for a another imported steel used in the manufacture of cutting blades used in the processing of lead plates. The steel selected for the development of this work was AISI D6 steel, the selection of this steel was made taking as a base the characteristics obtained through field analysis and microhardness and microstructure of the cutting blades already in the process made with steel imported. With the selected steel in hand, two different tempering and tempering treatments were applied (one proposed by the manufacturer and another proposed by the work in progress), where the effect of the thermal treatments on the mechanical and microstructural properties of the material was evaluated. By analyzing the results of the relationship between hardness and toughness, it was found that for the two thermal treatments applied, there were no significant variations of the mechanical properties of the material, and that both treatments presented satisfactory results. The specified national steel tool was presented as an alternative to replace the currently used imported steel. Field tests with the blade manufactured with the national steel specified in this study and that received the thermal treatment proposed by the manufacturer (FAB), corroborated with the analysis of the properties, as it was observed an increase in the cutting life time in more than 30 %.

Pour des raisons évidentes d’opération et de sécurité, l’allumage est un problème essentiel dans les moteurs aéronautiques. La conception d’une chambre de combustion de turbine à gaz intègre de multiples objectifs contradictoires, l’un d’entre eux étant un allumage ou ré-allumage efficace des brûleurs. Parmi les paramètres dont disposent les ingénieurs dans la phase d’optimisation du design, le nombre de systèmes d’injection de carburant et leur espacement sont des points cruciaux qui doivent être fixés dès le début. En effet, de tels choix ont non seulement un impact sur le coût de fabrication et la taille de la chambre mais ils affectent aussi l’efficacité d’un moteur ainsi que ses caractéristiques d’allumage. Afin d’améliorer les connaissances relatives au processus l’allumage dans des moteurs réels, la recherche actuelle combine des expériences fondamentales de plus en plus complexes et des simulations numériques de haute fidélité. Ces actions se concentrent d’une part sur les premiers instants où le noyau de flamme apparaît et d’autre part sur la phase de propagation entre les différents brûleurs. Ces deux phases sont capitales mais restent difficiles à étudier simultanément. Le premier objectif de cette thèse vise à évaluer les modèles SGE sur un seul brûleur expérimental situé au CORIA (France) pour mettre en place une méthodologie fiable afin de réaliser numériquement une séquence d’allumage dans des conditions d’opération réelles et équivalentes aux premiers instants. Une telle étude met en jeu plusieurs phénomènes tels que les écoulement swirlés, l’allumage, l’extinction, la propagation de flamme et les interactions flamme/turbulence. Tous ces processus et mécanismes interagissent et augmentent de façon significative le niveau de difficulté, notamment pour modéliser la combustion turbulente d’un tel allumage. Ces modèles requièrent donc d’être évalués précisément. Ensuite, ce travail examine par la simulation numérique la phase de propagation en utilisant les expériences réalisées sur une chambre composée de plusieurs injecteurs. La comparaison des séquences d’allumage obtenues numériquement avec celles des données expérimentales montre que la SGE reproduit les bonnes tendances et s’avère prédictive. D’un point de vue global, les caractéristiques de propagation du front de flamme en direction des injecteurs voisins sont bien capturées par le numériquemontrant desmodes de propagation identiques à ceux obtenus expérimentalement (radial ou axial) et des temps d’allumage similaires. Pour finir, l’analyse détaillée de ces données numériques a permis d’identifier les mécanismes principaux qui sont à l’origine des différents modes de propagation. / For evident operational and safety reasons, ignition is a key feature of aeronautical gas turbine applications. In fact the design of a gas turbine combustion chamber imposes multiple contradicting objectives one of them being efficient ignition or re-ignition. Among all the parameters available to the engineers, the number of fuel injection systems and their spacing are crucial elements, that must be fixed early on in the design phase. Such choices however not only impact the manufacturing cost and size of the combustor but they also affect the operability of the engine as well as its ignition. To improve knowledge of the ignition process occurring in real engines, current research combines fundamental and increasingly complex experiments complemented by high fidelity numerical simulations. These actions focus on the one hand on the initial instants where the first flame kernel appears as well as the follow-on instants corresponding to the light-around phase or burner to burner flame propagation phase. Both phases are clearly important but are difficult to study simultaneously. The first purpose of this thesis aims at assessing LES models on a single experimental burner located at CORIA (France) to provide a reliable numerical methodology to achieve an ignition sequence in real engines. Indeed, various phenomena are involved in such numerical studies dedicated to real aeronautical combustion chambers and all need to be reproduced by numerics: swirling flows, ignition, quenching, flame propagation, flame/turbulence interactions. All of these processes interact and clearly raise the level of difficulty notably in terms of turbulent combustion modeling of an ignition transient. Having assessed the method on a single burner configuration, the work then investigates the second phase, using a multi-injector experiment simulated by LES to study the flame propagation during ignition. The comparison of numerical fully transient ignition sequences with experimental data shows that LES recovers features found in the experiment. Global events such as the propagation of the flame front to neighboring swirlers are well captured and correct propagation modes (radial or axial) as well as correct overall ignition time delay are obtained. Finally the detailed analysis of LES data allows to identify the driving mechanisms governing each of these propagation modes.

With the rapid increase in electrical loads, a considerable percentage of global power systems have been stretched to their designed capacity, leading to a sharp rise in the fault current. High fault current levels have severe impact on power systems. They can increase the likelihood of overcurrent damage, which may destroy electrical facilities or even cause system blackout. The resistive-type superconducting fault current limiter (RSFCL) is a device that can help to mitigate the increasing level of fault currents. It can also contribute to the performance, stability and efficiency of electricity grids. In order to promote the RSFCL more effectively, it is essential to study the device itself and conduct simulations regarding the performance and applicability from the system point of view. Chapter 1 and Chapter 2 of this thesis introduced fundamentals of superconductivity and RSFCLs, respectively. In Chapter 3, a power system model was built, and the transient analysis of short-circuit currents was given. Then, the operating principle of RSFCL was explained, and a step-resistance RSFCL model was introduced. To validate the current limiting performance of the SFCL module, wind farm protection schemes were studied under various fault scenarios. After thorough analysis it was concluded that the optimal allocating strategy of SFCLs was the installation of one SFCL at the integrating point of the system model. Chapter 4 presented a comprehensive study on the performance and optimal allocation strategy of RSFCLs. The two power system models used in this chapter were built based on the UK network standard. To assess the impact of incorporating SC material properties on the performance of SFCLs, three different models were compared throughout the study. Although computing time can be reduced when step-resistance and exponential equation models were used, such simplifications led to strong overestimations of the SFCL performance and resulted in wrong conclusion of optimal installing strategies. For both power system models, the simultaneous use of three SFCLs was the best protection strategy in terms of the performance, economic efficiency and reliability of the overall grids. To draw this conclusion, all the potential combinations of two, three, four, and five SFCLs were studied under a wide number of fault scenarios and measuring strategies. In Chapter 5, a series of experiments were performed to study the magnetic field-angular dependence of the critical current of different commercial YBCO samples. We selected ten 2G-HTS tapes with broad differences in width, fabrication process, and laminar structure. The obtained I_c (B,θ) characteristics of HTS samples were applied in the simulation of RSFCLs, showing unneglectable effect on the first peak limiting performance during faults. This study is helpful to the design and simulation of RSFCLs and other HTS applications which require superconducting wires operating inside magnetic fields. In Chapter 6, we presented a High Frequency (HF) AC-assisted quench study of the YBCO conductor. The differences in quench processes triggered by HF AC field were recorded and studied. We found that AC signals of 10 kHz can trigger quench of the HTS tape. Also, the device proved to be effective at guaranteeing uniform quench of the sample. After then, we placed the experimental device in a magnet, to study whether the quench process can be accelerated by external DC magnetic field. It was found that the DC field can reduce the quench time, but its performance was dependent on the amplitude of transport current and the characteristics of HF AC signals. In addition, the angle between the DC magnetic field and the tape surface showed a huge impact on the quench time. Finally, a comprehensive field-angular dependence study about the quench time of the YBCO sample was conducted, revealing the same pattern as J_c (θ,B) dependence of the tape, but with a greater anisotropy. To summarize, this technique showed outstanding performance regarding quench acceleration and tape protection, and therefore has huge potential to be applied in RSFCLs.