Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16875号 / 工博第3596号 / 新制||工||1543(附属図書館) / 29550 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 佐藤 啓文, 教授 田中 一義, 教授 梶 弘典 / 学位規則第4条第1項該当

Les protéines à domaine FIC (Filamentation induced by cAMP) sont très répandues chez les bactéries où elles catalysent l’ajout d’une modification post-traductionnelle contenant un phosphate à une protéine cible, en utilisant différents co-substrats comme l’ATP. Certaines de ces protéines sont des toxines sécrétées par des pathogènes humains, mais la fonction de la plupart d’entre elles reste mystérieuse. Plus d’une dizaine de structures de protéines FIC ont été déterminées récemment, qui ont permis d’élucider leur mécanisme catalytique. L’une des sous-familles de protéines FIC possède un glutamate dans leur site catalytique, dont il a été proposé qu’il aurait une fonction auto-inhibitrice pour la fixation de l’ATP. Durant ma thèse, j’ai étudié la structure et les mécanismes de régulation de deux familles de protéines FIC : les protéines FIC à glutamate inhibiteur, et la toxine AnkX de la bactérie pathogène Legionella pneumophila.La première étude s’est intéressée à la protéine FIC de la bactérie pathogène Enterococcus faecalis (EfFIC), qui fait partie de la sous-famille des protéines FIC possédant un glutamate inhibiteur. J’ai résolu plusieurs structures cristallographique d’EfFIC, qui ont permis de caractériser son site catalytique et comment elle fixe l’AMP et l’ADP. En utilisant une propriété fréquemment observée d’auto-AMPylation (modification par l’AMP), j’ai montré au moyen d’ATP radioactif qu’EfFIC possède une activité basale d’auto-AMPylation, et j’ai identifié une nouvelle activité de dé-AMPylation. En m’inspirant des métaux observés dans mes structures cristallographiques, j’ai montré que l’alternance entre les activités d’AMPylation et de de-AMPylation dépend de la nature du métal fixé dans le site actif et de la présence du glutamate. Ce glutamate régulateur est également présent chez une protéine humaine, HYPE, qui possède une double activité d’AMPylation et dé-AMPylation d’ une chaperone du réticulum endoplasmique. Par un test de fluorescence, j’ai enfin montré que l’activité de HYPE était elle aussi régulée par les métaux comme celle de EfFIC. Ces résultats suggèrent un nouveau modèle de régulation partagé par des protéines FIC de la bactérie à l’homme.La seconde étude a porté sur la toxine AnkX de Legionella pneumophila, qui modifie les petites protéines G de la famille de Rab Rab1 et Rab35 (régulatrices du trafic cellulaire) par une molécule de phosphocholine (PC). En utilisant des liposomes de composition contrôlée, j’ai montré qu’AnkX interagit avec les membranes, et j’ai identifié par mutagenèse son domaine d’interaction avec les membranes. Au moyen de petites GTPases Rab ancrées artificiellement à la surface de liposomes par une queue 6his remplaçant le lipide naturel, j’ai montré que l’activité d’AnkX est stimulée par la présence de membranes. Des résultats préliminaires suggérent que Rab35 est un meilleur substrat que Rab1a, ce qui pourrait renseigner sur la fonction et le compartiment cellulaire où se trouve la toxine. J’ai ensuite mené une étude structurale d’AnkX par diffusion des rayons X aux petits angles (SAXS), qui permet d’obtenir des informations structurales en solution. AnkX est constitué d’un domaine FIC, de répétitions ankyrine et d’un domaine C-terminal. L’analyse en SAXS montre que ces domaines s’organisent en forme de fer à cheval, suggérant un modèle d’association bi-partite du complexe AnkX/Rab aux membranes. L’ensemble de ces résultats conduit à un modèle dans lequel l’activité d’AnkX est régulée spatialement par les membranes, ce qui pourrait lui permettre de cibler à la fois les petites GTPases Rab cellulaires et le compartiment membranaire. / FIC (Filamentation induced by cAMP ) domain containing proteins are widespread in bacteria where they use different substrate such ATP to modify a target protein with a phosphate containing post-translational modification. Some of those proteins are secreted toxins from pathogens but the function of the majority stay unknown. Some recently resolved structures explain the catalytic mechanism. A subfamily of FIC proteins was proposed to be auto-inhibited for ATP binding by a glutamate in their active site. In my thesis, I lead a structural and biochemical study of two FIC proteins family: the auto-inhibited by a glutamate FIC proteins and the Legionella pneumophila toxin AnkX.For the first study, I focused on the pathogenic bacteria Enterococcus faecalis protein EfFIC that is an auto-inhibited FIC protein. I solved several crystallographic structures to characterize the active site and the AMP and ADP binding. Using the classic auto-AMPylation (modification with an AMP molecule) mechanism and radioactive ATP, I showed that EfFIC is active and I identified a new de-AMPylation activity. Using metals found in my crystallographic structures, I showed that the AMPylation and de-AMPylation switch is controlled by the nature of the metal bound in the active site and that this switch is inhibitory glutamate-dependent. This glutamate is found in human HYPE that shows a double AMPyaltion and de-AMPylation activity of the ER chaperone BIP. Using fluorescence assays, I showed that those two activities are alors regulated by metals as in EfFIC. Those results point on a new regulation model shared between FIC proteins from bacteria to human.The second study focused on Legionella pneumophila toxin AnkX that modifies small GTPases Rab1 and Rab35 with a phosphocholine (PC) molecule. Using controlled composition liposomes, I showed that AnkX interact with membranes and mapped the interaction domain by mutagenesis. With artificially anchored to nickel containing liposomes surface Rab GTPases, I demonstrated the stimulation of AnkX activity by the membranes. Preliminary results also suggest that Rab35 is a better substrate than Rab1a, giving information on AnkX function and localization during infection. I lead a small angle X-ray scattering (SAXS) study on AnkX that gave low-resolution structural information on AnkX in solution. The analyses of SAXS results show that AnkX is horseshoe shaped, suggesting an association with the membrane and Rab of AnkX model. In this model, membranes spatially regulate AnkX, allowing a targeting of Rab and cellular compartment targeting.

It has long been recognized that our understanding of how RNA adapts its complex three-dimensional structure and undergoes conformational fluctuations has played a central role in our understanding of the biological functions of RNA. Our current understanding of the vast and diverse set of RNA conformational dynamics is the culmination of several decades of biophysical research applying several ensemble and single-molecule techniques. In this journey, each of the biophysical techniques have provided a unique perspective into the dynamic processes of RNA and revealed information about distinct RNA dynamics occurring over a broad range of timescales. In recent years, a new, promising single-molecule biophysical technique called single-molecule field effect transistors (smFETs) has been developed. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that enables single-molecule studies of RNA conformational dynamics observed at microsecond temporal resolution for a long period of time. The broad range of timescales opens immediate prospects for smFETs to provide a unique perspective into understanding RNA conformational dynamics that are presently inaccessible in other single-molecule approaches. The primary focus of this thesis is to understand how RNA stem-loops undergo folding and unfolding. Stem-loops are one of the most common secondary structural motifs in RNA and act as a fundamental building block for complex RNA structures. Despite their fundamental importance, a complete unifying picture of the folding mechanism of RNA stem-loops has been difficult to achieve, primarily due to the rugged nature of their folding energy landscapes. In Chapter 2, experimental methods that were developed to enable smFET studies of RNA conformational dynamics are described. This includes the development of a high-throughput fabrication process that generates high signal to noise ratio (SNR) smFET devices and the development and validation of nucleic acid tethering strategies that enables controlled tethering of biomolecules onto smFET devices. Utilizing these methods, Chapter 3 establishes smFET as a general single-molecule approach to characterize the folding dynamics of RNA stem-loops. Finally, Chapter 4 explores the use of smFETs to investigate the molecular mechanism in which a model RNA stem-loop undergoes folding and unfolding. Collectively, this thesis demonstrates how smFETs can be applied to uniquely capture and describe the folding energy landscapes of RNA and reveal new insights to how RNAs fold and unfold.

Traveling to space requires a great deal of energy. This then limits the size of spacecraft accessible to transport to space. An optimization of a flexible tube that could be used as a satellite deployable structure was conducted by varying the cross section of the tube and its composite material properties. The material properties manipulated include the selection of a fiber, matrix, filler volume ratio, and orientation. HEEDS, a commercially available software, conducts the optimization process using the SHERPA algorithm. In the optimization, the finite element code, ABAQUS, iteratively performs two simulations. First, ABAQUS determines the stress distribution along the tube when wrapping the tube in its stored configuration. Second, ABAQUS finds the first natural frequency of the deployed structure. The objective function driving the optimization process is minimizing the weight and strain energy of the tube to create a light but highly flexible tube. This provides benefits of avoiding a violent deployment and lowering the dynamic response of the spacecraft during deployment. Three optimizations were performed with 1000 iterations each, using different initial geometries. While all three produce very similar results, one design converges to a clear best result. Using the best design, a series of deployment simulations are performed, using different boundary conditions to represent various scenarios. These boundary conditions include a free body dynamic response to deployment, a restricted response to only allow for rotation about the direction of deployment, and an increased damping deployment. Energy is dissipated differently comparing the results, showing that the most realistic case, being a free body deployment, has the lowest effect on the system. The spacecraft can dissipate energy by oscillating in the other axis. While damping does reduce the settling time for the deployed tube, there is notable oscillation in the middle of the tube seen in the transient state. / Master of Science / The size and weight of a spacecraft is important when considering its feasibility to launch to space. By creating a spacecraft that can be stowed in a small configuration and deploy, new parameters arise, thus new designs can be created. This paper observes using different shapes and materials to create an expandable tube, providing a support structure for a satellite or spacecraft. HEEDS, an optimization software, uses the SHERPA code to select the shape of the cross section and create a composite material. Composite material selection is comprised of a fiber, a matrix, a filler volume ratio, and an angle for the fibers to lay at. After selecting these parameters, HEEDS calls a finite element software, ABAQUS, to perform two simulations. The first simulation wraps the tube around a central hub and observes stress at each timestep. The second simulation finds the first mode of natural frequency of the deployed model. Using user defined constraints that revolve around the safety factor of the stress and minimum frequency, each iteration is marked as feasible or infeasible. An objective function is used to evaluate the best design. This paper focuses on minimizing the weight of the tube and the strain energy inside of the objective function. By minimizing the strain energy, the tube will deploy less violently and cause less rotation due to deployment. HEEDS performs 1000 iterations on three different initial geometry. While there are similar defining factors of each final design, there is one design that is better than the other two. Using the best design, ABAQUS runs three different deployment simulations to observe the deployment behavior. These scenarios encompass different dynamic simulations and show that a realistic deployment where the spacecraft is free to rotate on all axis is safe.

X-ray diffraction has been used to investigate the structure and structural behaviour of several inorganic solids under variable pressure and temperature conditions. A brief description of the diamond anvil cell (d.a.c.) and its applications to the high- pressure investigations is given. A spectroscopic system built for pressure calibration is discussed, plus an account of powder diffraction data analysis including a detailed description of a software package engineered for the specific reduction of powder data obtained under variable pressure. Potassium nitrate (KNO3) has seven polymorphs in the pressure range 0.0-4.0 GPa. This material has been studied with energy dispersive powder diffraction (EDXRD) to 9.3 GPa. The structure of the non-ambient phase IV refined by neutron diffraction at 0.36 GPa has been confirmed, the compressibility of the high-pressure phase measured and found to be anisotropic with axial compression ratios a:b:c = 1.0:0.64:0.50. The valence induced structural transition at 0.8 GPa in EuPd2Si2 has been studied using EDXRD and uni-axial compressibilities calculated. A basic explanation of the electronic theory which accompanies the subtle shortening of the a lattice parameter in the tetragonal asymmetric unit is also given. The structures of two compounds CH3HgX (X = I, Cl) have been investigated using single crystal and angle-dispersive powder techniques respectively. The crystal structure of methyl mercuric iodide is reported. CH3HgCl has been observed above and below the temperature induced I/II phase boundary at 162.5 K. A hypothesis detailing the iso-structural nature of CH3HgCl low temperature phase II and the ambient phase I of the analogous methyl mercuric bromide is also tested.

The structure and solvation of ions are studied from different aspects using various kinetic, spectroscopic and thermodynamic techniques. The solvation of a wide range of ions, both simple and complex, is investigated by calculation of sets of single-ion transfer chemical potentials for a series of binary aqueous solvent mixtures. Observed trends are discussed in terms of the effects on ion solvation of solute and solvent structure and solute-solvent interactions. Structure, solvation and also reactivity of sane low- spin iron (II) di-imine complexes containing unsymmetrical Schiff base ligands are investigated using a variety of techniques. The occurrence of diastereoisomerism in complexes of this type is probed using complementary techniques which highlight different aspects of this phenomenon. Thus, the differing reactivities of, and the differing spatial orientations in, possible diastereoisomeric forms are utilized in detecting diastereoisomers by kinetic and 1H nmr spectroscopic methods respectively. In addition, for complexes of this type, links between structure, solvation and reactivity are investigated using kinetic data, solubility data, ultraviolet/visible absorption spectra and 1H nmr spectra obtained for the complex species.

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Fixed jacket offshore structures. which react environmental wave loading. are generally constructed using tubular steel members. When subject to load. severe surface and through-thickness stress gradients occur due to local bending of the tubular wall. The cyclic nature of the environmental wave loading results in high stress concentration at the joints. which can lead to fatigue cracking. British Gas currently operates twelve fixed offshore structures. two of which. the Rough A-Complex structures. are now ageing and showing signs of fatigue cracking. The objective of the work described in this thesis is the development of a fracture mechanics-based methodology for the assessment of fatigue cracking in these structures. The fracture mechanics approach uses the stress intensity factor (SIl) to characterise crack-tip conditions. and provides a means of analysing the behaviour of cracks. The SIF is defined in terms of the crack site stress distribution and the change in structural compliance with crack size. Difficulties in the application of fracture mechanics lie in the derivation of accurate solutions for the SIF. The tubular joints of offshore jacket structures present particular difficulties due to their complex loading and geometry. The current work starts with a review and assessment of tubular joint fracture mechanics models. followed by a numerical study of cracked tubular joints using shell finite element (FE) models incorporating line spring crack representation. Based on the results of this study. a general fracture mechanics model for the prediction of SIF solutions for tubular joints. is derived and assessed. The general fracture mechanics model is incorporated into a crack growth model. which is best implemented using sophisticated commercial software. Crack growth and fatigue life predictions obtained are validated against full scale tubular joint fatigue data. Finally. a complete methodology for the assessment of fatigue cracks in any tubular joint is proposed, and applied to the assessment of fatigue cracking in the Rough A-Complex structures.

No description available.