Les matrices vitreuses aluminosilicatées de lanthanides {SiO2 –Al2O3 – (CaO) – Y2O3 – La2O3} constituent, une matrice potentielle d’intérêt nucléaire, dans le cadre du stockage des déchets des actinides. Alors que de nombreuses études sont encore menées pour établir leurs propriétés macroscopiques (durabilité, mécanismes de vitrification, etc…), nous nous sommes attachés à décrire l’environnement proche des noyaux qui composent le réseau, c'est-à-dire leur structure à une échelle locale. Les verres aluminosilicatés sont constitués de tétraèdres d’aluminium et de silicium. Plus précisément, ils sont constitués d’entités Qn(mAl) silicium et d’entités qn(mSi) aluminium. Cependant, il est rare d’aboutir à une description en ces termes. La Résonance Magnétique Nucléaire haute résolution solide (RMN MAS 27Al et 29Si) est un des moyens d’y parvenir. Nous proposons ici le développement de séquences d’impulsions permettant l’identification de ces unités puis la description de leurs connectivités via leurs liaisons chimiques, basées sur le filtrage des cohérences MultiQuanta associées aux couplages scalaires J2 (Si-O-Si) et J2 (Si-O-Al). Cette approche RMN permet donc d’affiner la compréhension des réseaux aluminosilicatés quels qu’ils soient. Couplé à la spectroscopie RAMAN, elle a pu ensuite nous servir comme référence pour établir la structure et le comportement à long terme de ces matrices, suite à des expériences de lixiviation statique et d’irradiation a pratiquées au cyclotron du CEMHTI. / Aluminosilicate glasses of rare earth {SiO2 –Al2O3 – (CaO) – Y2O3 – La2O3} can be considered like a potential matrix of nuclear waste. This type of glass appears to be suitable matrix for the specific immobilization of trivalent actinides. Whereas many studies dealt with the macroscopic properties of these matrix (durability, mechanism, etc…), we investigate here the local environment surrounding the nucleus constituting the vitreous network. Thus, we can say that we investigate the local structure of the glass. Aluminosilicate glasses are constituted with silicon and aluminium tetrahedra. They are more especially based on a mixture of silicon Qn(mAl) and aluminium qn(mSi) units. Up to now, few experiments allow to describe the vitreous network with this terminology. A solution is the High Resolution Solid State NMR : 27Al and 29Si 1D or 2D MAS NMR experiments. We proposed here to provide and improve NMR pulse sequence to evidence NMR signatures of chemically bounded Al-O-Si and Si-O-Si molecular motifs and to establish an approximate picture of medium range order, thanks to 27Al/29Si heteronuclear and 29Si/29Si homonuclear NMR experiments associated to MultiQuantum filtering based on indirect scalar coupling J2 Al-O-Si/J2 (Si-O-Si) and. Combining RAMAN spectroscopy, Spin counting and MultiQuantum filtering allow establishing an approximate and new picture of medium range order in aluminosilicate compounds. Thus, they offer a reference to describe the changes in the structure and the long term behavior of our aluminosilicate glasses of rare earth, induced by static lixiviation and a irradiation practiced to cyclotron in CEMHTI laboratory .

The beta-decay interaction contains two terms which consist of invariant products of two scalars and two tensors respectively. The relative absolute magnitude of these two terms is fairly well established but there has been some controversy over their relative sign. In this thesis the form of the interaction is investigated by means of an analysis of the second-forbidden decay spectrum of Cs137 and it is concluded that the relative sign of the scalar and tensor terms is negative. / Thesis / Master of Science (MS)

Sophisticated electromagnetic pulse sequences that control spin dynamics have been developed in Nuclear Magnetic Resonance (NMR) over the last few decades. However, due to more and more demanding criteria, such as unknown parameters, larger bandwidths, higher signal to noise ratio (SNR), less power consumption, etc., new pulse sequences are constantly needed. This thesis presents new pulse sequences for several important applications of NMR. / Engineering and Applied Sciences

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Our understanding of the folding of membrane proteins lags behind that of soluble proteins due to the challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding landscapes, Chapter 2 investigates the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 $\mathit{\Chi}_{SDS}$), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal binding pocket is disrupted, with transmembrane residues becoming more solvent-exposed. Folding of pSRII from an SDS-denatured state harbouring a covalently-bound retinal chromophore shows deviations from an apparent two-state behaviour. SDS denaturation to form the sensory opsin apo-protein is reversible. This chapter establishes pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin. In Chapter 3, SDS-denatured pSRII, acid-denatured pSRII and sensory opsin obtained by hydroxylamine-mediated bleaching of pSRII were characterised by solution state NMR. 1D $^1$H and $^{19}$F NMR were first used to characterise global changes in backbone amide protons and tryptophan side-chains. Residue-specific changes in backbone amide chemical shifts and peak intensities in 2D [$^1$H,$^{15}$N]-correlation spectra were analysed. While only small changes in the chemical environment of backbone amides were detected, changes in backbone amide dynamics were identified as an important feature of SDS- and acid-denatured pSRII and sensory opsin. $^{15}$N relaxation experiments were performed to study the backbone amide dynamics of SDS-denatured pSRII, reflecting motions on different timescales, including fast fluctuations of NH bond vectors on the ps-ns timescale and the lack of exchange contributions on the µs timescale. These studies shed insight on differences in the unfolding pathways under different denaturing conditions and the crucial role of the retinal chromophore in governing the structural integrity and dynamics of the pSRII helical bundle. Hydrogen bonds play fundamental roles in stabilising protein secondary and tertiary structure, and regulating protein function. Successful detection of hydrogen bonds in denatured states and during protein folding would contribute towards our understanding on the unfolding and folding pathways of the protein. Previous studies have demonstrated residue-specific detection of stable and transient hydrogen bonds in small globular proteins by measuring $^1{\it J}_{NH}$ scalar coupling constants using NMR. In Chapter 4, different methods for measuring $^1{\it J}_{NH}$ scalar coupling were explored using RalA, a small GTPase with a mixed alpha/beta fold, as proof-of-concept. Detection of hydrogen bonds was then attempted with OmpX, a beta-barrel membrane protein, both in its folded state in DPC micelles and in the urea-denatured state. While $^1{\it J}_{NH}$ measurement holds promise for studying hydrogen bond formation, further optimisation of NMR experiments and utilisation of perdeuterated samples are required to improve the precision of such measurements in large detergent-membrane protein complexes. Naturally occurring split inteins can mediate spontaneous trans-splicing both in vivo and in vitro. Previous studies have demonstrated successful assembly of proteorhodopsin from two separate fragments consisting of helices A-B and helices C-G via a splicing site in the BC loop. To complement the in vitro unfolding/folding studies, pSRII assembly in vivo was attempted by introducing a splicing site in the loop region of the beta-hairpin constituting the BC loop of pSRII. The expression conditions for the N- and C-terminal pSRII-intein segments were optimised, and the two segments co-expressed. However, the native chromophore was not observed. Further optimisation is required for successful in vivo trans-splicing of pSRII and application of this approach towards understanding the roles of helices and loops in the folding of pSRII.