Cette thèse traite du recouplage et du découplage des interactions dipolaires homonucléaire et hétéronucléaire en RMN des solides. Premièrement, deux types de techniques permettant d’établir des corrélations à travers l’espace entre des noyaux quadripolaires et de spin-1/2 sont présentés : Multiple-Pulse Cross-Polarization (MPCP) et Dipolar - Multiple Heteronuclear Quantum Coherence (D-HMQC). Nous comparons différentes séquences de recouplage dipolaire telles que SFAM, SR41² pouvant être utilisées avec D-HMQC. Secundo, nous proposons un nouvel exemple de technique de découplage de l’interaction diplolaire homonucléaire basée sur les principes de symétrie. Il fonctionne à de hautes vitesses de rotations à l'angle magique (MAS) (vR > 25 kHz) et sa version lissée appelée SAM (Smoothed Amplitude Modulation) est complémentaire d’autres méthodes antérieures, telles que DUMBO ou FSLG/PMLG. Troisièmement, une nouvelle méthode de recouplage homonucléaire appelée SPIP, permet la création de cohérences double quanta (DQ) entre noyaux de spin 1/2, tel que 1H. Comparée aux techniques DQ antérieures, cette séquence n’exige que des champs rf modérés, même à vitesses MAS extrêmement rapides. En outre, il affiche plus de robustesse à l’anisotropie de déplacement chimique et à l’off-résonnance. Quatrièmement, une méthode de traitement, appelée covariance, est employée en RMN des solides pour produire des spectres de corrélation homonucléaire (HOMCOR) 2D. Elle permet de s’affranchir des problèmes de troncature du signal, sans aucune connaissance préalable des positions et des largeurs des résonances, d'une manière plus performante que le traitement par transformée de Fourier habituel. / This thesis deals with the recoupling and decoupling of dipolar homonuclear and heteronuclear interactions in solid-state NMR.First, two kinds of techniques for establishing the through-space correlations between quadrupolar nuclei and spin 1/2 nuclei are presented. One is MPCP (Multiple-pulse Cross-Polarization) recoupling sequence and the other one is D-HMQC (Dipolar assisted- Heteronuclear Multiple Quantum Coherence) sequence. We evaluate different kinds of dipolar recoupling techniques such as SFAM, SR41², that are used in our D-HMQC. Second, we propose a new 1H homonuclear dipolar decoupling technique labeled SAM (Smooth Amplitude Modulation). This method that is a symmetry-based sequences, works mainly at fast or ultra-fast Magic Angle Spinning (MAS) rates (vR > 25 kHz) and is complementary to previous methods, such as DUMBO, FSLG/PMLG. Third, a novel symmetry-based homonuclear recoupling method (SPIP) that excites double-quantum (DQ) coherences between spin-1/2 nuclei, such as 1H, is presented. Compared to previous 1H DQ-recoupling techniques, this pulse sequence requires moderate rf field, even at ultra-fast MAS speed. Furthermore, it displays higher robustness to both chemical shift anisotropy and spreads in resonance frequencies.Fourth, a new processing scheme, called covariance, is employed in solid state NMR to produce homo-nuclear correlation (HOMCOR) 2D spectra. It can accommodate signal truncation much better than the usual 2D-FT data treatment, without any previous knowledge of the positions and line-widths of the resonances. Besides, covariance methods can be applied to HETCOR NMR data to generate two HOMCOR indirect-covariance spectra.

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Thesis advisor: Mary F. Roberts / Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis, often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids such as phosphatidylcholine (PC), which is enriched in the outer leaflet of eukaryotic cell membranes, are not well understood. Molecular dynamics (MD) simulation and mutagenesis results strongly indicate that PI-PLC interacts with PC head groups via cation-π interactions with aromatic tyrosine residues, and suggest that cation-π interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins. Aromatic amino acids can not only form cation-π interactions at the interface but also insert into membranes and have hydrophobic interactions with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side-chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring while their enhanced hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis PI-PLC identifies those involved in cation-π interactions with PC. Cation-π interactions provide a likely molecular mechanism for BtPI-PLC PC specificity but do not account for its preference for bilayers containing a small fraction of anionic lipids. MD simulations and fluorescence correlation spectroscopy (FCS) vesicle binding measurements of positively charged amino acids as well as surface tyrosine residues are used to formulate a complete model of BtPI-PLC specific binding to mixed anionic phospholipid/PC membrane. DNF2, a new plant protein with homology to bacterial PI-PLC, is confirmed to be the first plant small PI-PLC enzyme that can cleave both PI and glycosylphosphatidylinositol (GPI) anchored proteins. GPI-anchored protein cleavage also confirms that DNF2 plays an important role in symbiosome, the intracellular compartment formed by the plant that contains nitrogen fixing bacteria. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Le terme de sulfoprotéomique est utilisé pour désigner l’étude de la sulfatation des protéines. Bien que la sulfatation soit depuis peu considérée comme une MPT d’une importance majeure, il y a toujours peu de travaux scientifiques qui y sont consacrés en comparaison avec ce qui se fait sur la phosphorylation notamment. Ce retard s’explique notamment par la difficulté à analyser les espèces protéiques sulfatées dans les conditions classiques utilisées en protéomique, notamment par spectrométrie de masse. Ces travaux de thèse visent justement à développer des méthodes d’analyses par spectrométrie de masse dédiées à l’étude de la sulfatation des protéines, afin d’augmenter le champ des connaissances de cette MPT. Pour cela, nous avons largement utilisé le mode d’ionisation négatif, très peu, voire jamais utilisé en protéomique, avec deux techniques de fragmentation pour réaliser des spectres MS/MS, à savoir les fragmentations CID et HCD. Les résultats obtenus nous ont permis de mettre en évidence une méthode d’analyse permettant la formation d’ions spécifiques de la sulfatation et de la phosphorylation (qui sont isobariques), permettant ainsi une identification certaine de chacune des deux MPTs. Nous avons également entrepris d’étudier le rôle de la sulfatation d’un récepteur cellulaire, CXCR4, dans son interaction avec son ligand naturel, la chimiokine SDF-1/CXCL12. Cette étude a été menée par électrophorèse capillaire, et pourra constituer une base de travail solide pour des futures analyses mettant en œuvre le couplage entre l’électrophorèse capillaire et la spectrométrie de masse pour une meilleure caractérisation des complexes formés entre les partenaires protéiques. / Sulfoproteomics term designs protein sulfation studies. It appears during the 2000’s, when the interest for others Post-Translational Modifications (PTMs) than phosphorylation and glycosylation was growing up. Even though sulfation is thought to be an important PTM, a weak number of publications has emerged about it, notably if we compare with the huge quantity of phosphorylation papers. This difference is mainly due to the difficulty to correctly analyze sulfated proteins and peptides in the classical ways of proteomics, as in mass spectrometry for example. The goal of this thesis is to develop mass spectrometry methods dedicated to the characterization of sulfated species, in order to improve the knowledge of this PTM. To do that, we have mainly used negative ion mode, which is almost never used, with two fragmentations techniques for the MS/MS spectra, which are CID and HCD. Results obtained allow us to pinpoint an analytical method allowing the differentiation between sulfation and phosphorylation (they are isobaric), based on the presence of specific ion for each PTM in MS/MS. In another part of the project, we have investigated the role of sulfation in the interaction between a cellular receptor, CXCR4, and its in vivo ligand, the chemokine SDF-1/CXCL12. We used capillary electrophoresis for this work, and it could be a good basis for future analyses using capillary electrophoresis coupled with mass spectrometry, in order to have a better characterization of the observed complexes.

This thesis presents results on the study of electromagnetic (EM) activity in the MicroBooNE Liquid Argon Time Projection Chamber (LArTPC) neutrino detector. The LArTPC detector technology provides bubble-chamber like information on neutrino interaction final states, necessary to perform precision measurements of neutrino oscillation parameters. Accelerator-based oscillation experiments heavily rely on the appearance channel νµ → νe to make such measurements. Identifying and reconstructing the energy of the outgoing electrons from such interactions is therefore crucial for their success. This work focuses on two sources of EM activity: Michel electrons in the 10-50 MeV energy range, and photons from π^0 decay in the ~30-300 MeV range. Studies of biases in the energy reconstruction measurement, and energy resolution are performed. The impact of shower topology at different energies is discussed, and the importance of thresholding and other reconstruction effects on producing an asymmetric and biased energy measurement are highlighted. This work further presents a study of the calorimetric separation of electrons and photons with a focus on the shower energy dependence of the separation power.