Das Verständnis der Mechanismen, nach denen biologische Systeme ablaufen, ist ein wichtiger Fokus der aktuellen Strukturbiologie. Kernmagnetische Resonanzspektroskopie (NMR) ist eine geeignete Technik, um solche Ziele anzustreben sowie Struktur und Dynamik von Biomolekülen zu erforschen, um komplementäre Informationen zum Verständnis von Proteinfunktionalität zu erhalten. Rasante Fortschritte sind vor nicht langer Zeit auf dem Gebiet biologischer Festkörper-NMR (ssNMR) erzielt worden, was zu vollständiger Strukturaufklärung zahlreicher Peptide und kleiner Proteine, der Beschreibung von Protein-Komplexbildung sowie der der dynamischen Eigenschaften kleiner Proteine geführt hat. Festkörper-NMR ist die Methode der Wahl bei struktureller und dynamischer Charakterisierung von Membranproteinen und aggregierten amyloidogenen Systemen, die schwer löslich und kaum mit Lösungs-NMR oder Röntgenkristallographie zugänglich sind. Moderne Festkörper-NMR ist noch immer limitiert, was Auflösung und Empfindlichkeit betrifft, und macht weitere Entwicklungen auf den Gebieten der Probenpräparation und des Pulssequenz-Designs erforderlich. In meiner Arbeit untersuche ich die potenzielle Verwendung von Deuterierung in der Protein Festkörper-NMR zur Erhöhung von Empfindlichkeit und Auflösung in 15N-1H Korrelationsexperimenten. Der erzielte Fortschritt auf diesen Gebieten erlaubt die Verfolgung von Proteinrückgratbewegungen mit hoher Genauigkeit, die vorher nicht verfügbar war. Wir zeigen zum ersten Mal, dass TROSY Experimente für Festkörper-NMR gewinnbringend sind. Außerdem wurde eine Pulssequenz für 13C-13C J Kopplung zur Erhöhung der Auflösung in der Kohlenstoff-Dimension entwickelt. / Understanding the mechanisms how biological systems work is an important objective of current structural biology. Nuclear magnetic resonance (NMR) spectroscopy is a well suited technique to approach these goals and to study structure and dynamics of biomolecules in order to obtain complimentary information for understanding functionality of proteins. Recently, rapid progress has been made in the field of biological solid state NMR (ssNMR), which resulted in complete structure elucidation of several peptides and small proteins, the characterization of protein complex formation and the characterization of dynamic properties of small proteins. Solid state NMR is the method of choice for structural and dynamic characterization of membrane proteins and aggregated amyloidogenic systems, which are poorly soluble and can not be easily studied by solution state NMR and X-ray spectroscopy. Modern solid state NMR is still limited in resolution and sensitivity, and requires developments in sample preparation and pulse sequence design. In my thesis, I study the potential use of deuteration in protein solid state NMR for sensitivity, as well as for resolution enhancement in 15N-1H correlation experiments. Achieved progress in these fields allows to monitor backbone motion with high accuracy, which has not been available before. We show for the first time that TROSY type experiments can be beneficial for solid state NMR. In addition, a pulse sequence for 13C-13C J decoupling was developed to increase resolution in the carbon dimension.

Einleitung: Mit der vorliegenden Studie wurden zum ersten Mal beim Menschen die Auswirkungen von Dehydration (Dehy) und anschließender Rehydratation (Rehy) auf cerebrale volumenregulatorische Metabolite (myo-Inosit (mI), N-Azetylaspartat+N-Azetylaspartylglutamat (tNAA), Kreatin (Cr), Glyzerophosphocholin+Phosphocholin (Cho) und Glutamat+Glutamin (Glx)) sowie auf Flüssigkeitsverschiebungen untersucht. Methoden: 14 Radsportler (26.6 (22.7/29.8) Jahre, Median und 25./75. Perzentile) wurden mittels 1H-Spektroskopie (1H-MRS) in der okzipitoparietalen grauen Substanz (GM) und parietalen weißen Substanz rechts (WMR) und links (WML) untersucht (GE Signa Horizon 3T94; PRESS: TE 30ms, TR 6000ms, VOI 8ml). Die Messungen erfolgten vor, direkt nach Dehy und nach Rehy (180min, Zufuhr von 150\% der verlorenen Körpermasse (KM)). Zusätzlich wurde durch T2-Relaxationsmessungen der Atrophieindex alpha (Verhältnis cerebrales Gewebewasser (HW) zu Liquor (CSF)) bestimmt. Resultate: Die KM der Probanden reduzierte sich durch Dehy um 3.7 (3.4/4.1)% und stieg durch Rehy wieder um 4.5 (3.7/5.3)% an (Wilcoxon: p / Introduction: In the present study the influence of Dehy on cerebral volume regulatory metabolites (myo-Inositol (mI), N-Acetyl-aspartata+N-Acetyl-aspartyl-glutamate (tNAA), Creatine (Cr), Glycerophosphocholine+Phosphocholine (Cho) and Glutamate+Glutamine (Glx)) and fluid shifts has been investigated for the first time in humans. Methods: 14 cyclists (26.6 (22.7/29.8) y, median and 25./75. percentile) have been examined with proton NMR spectroscopy in the occipito-parietal gray matter (GM) and the right (WMR) and left (WML) parietal with matter (GE Signa Horizon 3T94; PRESS: TE 30ms, TR 6000ms, VOI 8ml). Spectra were acquired before, immediately after Dehy and after rehydration (Rehy). Rehy took place during 180min and 150% of lost body weight (BW) was substituted. Additionally the atrophy index alpha (ratio between cerebral water and liquor) was assessed (T2 signal decay as a function of echo time). Results: BW of volunteers has been decreased 3.7 (3.4/4.1)% after Dehy and increased 4.5 (3.7/5.3)% after Rehy (Wilcoxon: p

This research advances the current understanding of the carbonation reaction in porous materials by investigating pH changes during the hardening process of lime, the role of pore-water in the dissolution process of calcium hydroxide and the effects of pore size on precipitation of calcium carbonate solid phases. To achieve this, carbonation is studied within a thin film of an aqueous solution of calcium hydroxide, that simulates the conditions existing in porous media once most of the liquid water has evaporated. The research introduces novel approaches such as the use of specially manufactured micro-electrodes used to measure pH variations during the carbonation process. The effect of pore size on the solid phases precipitated by carbonation is investigated using a novel lime based material called nano-lime. Influence of pore-water on the hardening process of lime is studied in formulated lime using impedance spectroscopy: an electrochemical technique which is new in the study of lime based materials. Overall, results demonstrate that the micro-electrodes can operate reliably in very alkaline environments such as those produced by the dissolution of lime. Their potentiometric response, in fact, was found to be Nernstian up to pH 14. Furthermore, the electrode response proved to be sufficiently sensitive and reproducible to differentiate, on the basis of pH, between the formation of calcite and vaterite. It is likely that these micro-electrodes are currently the only analytical tools capable of monitoring high pHs in confined places and, for this reason, they can be considered highly valuable for the study of chemical processes involving very alkaline waters. The study on the role of pore-water in the hardening process of formulated lime has, instead, demonstrated the potential of impedance spectroscopy as a non-destructive technique for real time in situ monitoring of the reaction between lime and hydraulic additives.

La spectroscopie RMN alliée à l’utilisation de solvants cristal-liquide fortement et faiblement orientants est une stratégie efficace pour élucider les structures et distributions conformationnelles de petites molécules organiques rigides et flexibles en solution, et déterminer les ordres orientationnel et positionnel des solutés comme des solvants orientés. Dans une première partie, afin d’explorer les différentes contributions aux couplages dipolaires d’un soluté donné, la très faible amplitude de l’ordre orientationnel d’une molécule quasi-sphérique, le tetramethylallène, dissoute dans un nématique thermotrope est exploitée. Dans cette situation limite, le caractère prédominant des mécanismes de réorientation et de vibration moléculaire est mis en évidence, et estimé. Dans une seconde partie, les données RMN obtenues à partir de solutés de petites tailles dissous dans des solvants smectiques sont combinées aux résultats de calculs reposant sur des concepts de thermodynamique statistique et de la théorie de la fonctionnelle de densité. L’efficacité de cette méthode dans la détermination des paramètres d’ordres positionnel du solvant et orientationnel des molécules-sondes est démontrée aussi bien dans le cas de phases conventionnelles smectiques A que celui plus délicat de smectiques interdigitées Ad. La stratégie d’analyse proposée est ensuite étendue à l’investigation des structures tridimensionnelles et équilibres conformationnels de molécules flexibles bioactives ou biomimétiques. Dans une perspective méthodologique, à l’aide d’études expérimentale et théorique portant sur le biphényle, molécule symétrique constituée d’un unique rotor, il est tout d’abord démontré l’intérêt des méthodes de simulations par dynamique moléculaire pour évaluer l’ensemble des couplages dipolaires d’un soluté donné dans une phase thermotrope, ultérieurement utilisés comme paramètres initiaux dans une analyse spectrale itérative, et in fine déterminées précisément. L’analyse spectrale chronophage et dont l’aboutissement est incertain si les paramètres initiaux sont difficiles à estimer, en est ainsi facilitée. Puis, les distributions conformationnelles d’anti-inflammatoires non stéroïdiens de dérivés salicylés et profènes, fluorés ou non, constitués d’un ou deux rotors indépendants sont présentées. Via l’utilisation inédite du modèle AP-DPD dans les solvants nématiques (chiraux) lyotropes faiblement orientants, et à partir des couplages dipolaires homo- et hétéronucléaires notamment obtenus grâce à l’expérience RMN GET-SERF, créée à propos pour permettre l’extraction simple et rapide des couplages 1H-19F, les surfaces d’énergie potentielle de ces biomolécules sont décrites de façon satisfaisante. Enfin, les équilibres conformationnels de deux stilbénoïdes constitués de deux rotors coopératifs sont déterminés dans deux solvants cristal-liquide, l’un fortement, l’autre faiblement orientant. Ces études comparatives permettent de discuter la fiabilité, la précision et l’accessibilité des observables RMN extraites dans les phases, et d’établir la complémentarité des analyses RMN réalisées dans ces solvants. / NMR spectroscopy in weakly and highly orienting media is used as a route for dealing with orientational, positional, structural and conformational problems of a variety of small rigid and flexible organic molecules in solution. First, the very weak orientational order of a quasi-spherical molecule dissolved in a nematic phase is exploited for exploring the role of the different contributions to the observed dipolar coupling. In such a limit condition, a predominant effect of the non-rigid reorientation-vibration coupling term emerges. Then, NMR data obtained from small rigid probes dissolved in smectic solvents are combined with a statistical thermodynamic density functional theory, in order to measure the positional order parameters of both solutes and solvent. The methodology gives good results when applied to a conventional smectic A liquid crystal and to the more delicate case of an interdigitated smectic Ad phase. The strategy is subsequently extended to the investigation of structure, order and conformational equilibrium of flexible bioactive or biomimetic molecules dissolved in various partially ordered NMR solvents. A first experimental and theoretical study is presented on the symmetric single-rotor molecule of biphenyl dissolved in a thermotropic liquid crystal. This test-case indicates molecular dynamics simulations are a promising tool for estimating a set of dipolar couplings of a solute in a thermotropic solvent, to be used as starting set of parameters in a standard operator-mediated NMR spectral analysis. Then, we report the conformational study of some single- and two-rotor nonsteroidal anti-inflammatory drugs, belonging to the families of salicylates and profens, dissolved in weakly orienting chiral nematic PBLG phases. A new pulse sequence, the Gradient Encoded heTeronuclear 1H-19F SElective ReFocusing NMR experiment (GET-SERF), is proposed here for the trivial edition of all 1H-19F couplings in one single NMR experiment, for a given fluorine atom. Starting from homo- and heteronuclear dipolar couplings, difficult to extract in thermotropic solvents because of a too complex spectral analysis, the torsional distributions of such molecules can be satisfactory described by the Additive Potential model combined with the Direct Probability Description of the torsional distribution in terms of Gaussian functions (AP-DPD approach). Finally, the conformational and orientational study of two stilbenoids displaying cooperative torsions is discussed in both a highly and weakly ordering liquid crystal phase. This comparative study allows to draw some conclusions on reliability, accuracy and accessibility of desired data in the two phases. Overall, this work proves NMR in liquid crystals is a flexible and meaningful tool for studying order, structure and conformation and it can greatly benefit from the availability of several aligning media inducing a different degree of order.

La Spectroscopie RMN (SRMN) du 1H et du 31P permet de détecter et de mesurer in vivo de façon non-invasive la concentration de composés biologiques qui sont pertinents à l’étude des aspects variés du métabolisme cérébral comme la neurotransmission (glutamate, GABA), la densité neuronale (N-acetyl-aspartate) et gliale (myo-inositol) ou le métabolisme énergétique (phosphocreatine, ATP), entre autres. Ainsi, l’analyse des profils biochimiques permet d’étudier longitudinalement l’évolution de la physiologie cérébrale en conditions pathologiques ou normales. Par ailleurs, à ultra-haut champ magnétique la SRMN bénéficie d’une sensibilité et d’une résolution spectrale accrues, maximisant l’information métabolique exploitable. Au cours de cette thèse, nous nous sommes surtout intéressés à l’étude du vieillissement cérébral normal. Une étude longitudinale en 1H et 31P a été menée in vivo à 17.2 Tesla afin de suivre les altérations métaboliques pendant 14 mois chez deux cohortes de rats Dark Agouti âgés d’un mois et 8 mois au départ de l’étude. Les concentrations ainsi que les temps de relaxation T1 et T2 de plus de 20 métabolites ont été mesurés jusqu’à l’âge de 22 mois. Nous avons notamment observé une augmentation des concentrations de myo-inositol et des macromolécules dans les 4 volumes d’intérêt (VOI) étudiés. Dans le VOI Main, comprenant principalement du cortex mais aussi du corps calleux et de l’hippocampe, ces changements métaboliques ont été accompagnés par une augmentation des niveaux de glutamine et de composés contenant de la choline (tCho). Ces observations sont cohérentes avec une possible neuro-inflammation modéré au cours du vieillissement. Aucun changement du NAA a été observé sur le Main VOI, thalamus et putamen caudé (striatum). Additionnement, une réduction des temps T2 pour le NAA total, la tCho et les macromolécules a été observée, en accord avec une altération du milieu cellulaire et une accumulation de fer dans les tissus avec l’âge. Etonnamment, nous avons observé un effet corrélé avec le nombre d’examens RMN, qui a été fortement manifesté par une augmentation significative des temps T1 de nombreux métabolites.Un deuxième axe de travail pendant cette thèse a été la mise en place des outils méthodologiques nécessaires à la réalisation des études par SRMN du 1H et du 31P à 7 Tesla chez l’homme. Des séquences d’imagerie spectroscopique 2D ont été développées pour obtenir des cartes de concentration des métabolites 31P et 1H respectivement par la sélection d’une coupe ou bien d’un voxel par écho-stimulé. Un schéma de suppression d’eau WET a été optimisé pour son application à 7 T. Des modes d’excitation et de saturation du signal extérieur (OVS) en « anneau » ont été implémentés avec la méthode de transmission parallèle pour son application en imagerie spectroscopique 1H par l’optimisation des configurations statiques d’excitation ou « shimming-B1 ». Cette approche a permis d’appliquer des champs d’excitation plus homogènes et de réduire le dépôt d’énergie chez le sujet par rapport à l’utilisation des bandes OVS classiques. Des expériences in vitro ont été menées pour démontrer leur faisabilité. Enfin, un module de saturation BISTRO a été implémenté pour l’acquisition in vivo de cartes métaboliques en 31P. L’efficacité du module BISTRO a été démontrée et ce module peut être adapté pour des expériences 31P de transfert d’aimantation, ouvrant la voie de l’étude du métabolisme énergétique cérébral chez l’homme à très haut champ magnétique. / 1H and 31P nuclear magnetic resonance spectroscopy allows to detect and to measure in vivo and non-invasively the concentrations of biologically relevant compounds associated to metabolic processes such as neurotransmission (glutamate, GABA), neuronal and glial density (N-acetyl-aspartate, myo-inositol) and energetic metabolism (phosphocreatine, ATP) among others. Knowledge of the biochemical profile provides a mean to evaluate the metabolic state of the brain in pathological cases or in evolving physiological conditions, such as aging. Yet, the neural basis of age-related cognitive dysfunction in normal brain aging remains to be elucidated and it has been shown to develop at different rates depending on the structural region.At ultra-high magnetic fields, magnetic resonance spectroscopy (MRS) benefits from an increased signal-to-noise ratio and a higher chemical shift dispersion, resulting in an increased sensitivity and spectral resolution. To exploit these advantages, 1H and 31P longitudinal studies were carried out in vivo at 17.2 Tesla in the aging rat brain to evaluate the progressive metabolic changes within the same individuals from the ages of 1 to up to 22 months of age using two rat cohorts with 1 and 8 months of age at the beginning of the study. For the 1H MRS studies, T1 and T2 metabolite relaxation times were measured at each exam in order to control age-related variations and to calculate absolute metabolite concentrations. 1H neurochemical profiles from four volumes of interest (VOI) in the brain were studied, revealing a progressive increase in myo-inositol and macromolecule content throughout the brain. In our main VOI composed mostly of cortex but also of corpus callosum and hippocampus, increased levels of choline-containing compounds (tCho) and glutamine were also observed, suggesting a mild neuroinflammation. No changes in NAA were observed in our main VOI, the thalamus or the caudate putamen (striatum). T2 decreases were observed with age for total NAA, tCho and macromolecules. Notably, unexpected effects correlated with the number of NMR exams were observed, the most prominent effect being an increase of the T1 relaxation times of the majority of metabolites.The second axis of the work done during this thesis was to set up an experimental framework for MR spectroscopic imaging (MRSI) studies at 7 Tesla in the human brain. 2D MRSI pulse sequences were developed for the acquisition of 31P and 1H metabolite maps using either slab selection or STEAM localization, respectively. A WET water suppression scheme was numerically optimized for its application at 7 T. Static B1-shimming configurations were implemented to reduce the inhomogeneity of the excitation field in the volume of interest and to generate outer-volume suppression (OVS) “ring” modes to saturate the signal in the periphery of the head. This approach allows to reduce the energy deposition in comparison to conventional OVS bands. Experiments were done in vitro showing their feasibility. The performance of standard OVS bands was also compared to a B1-insensitive train to obliterate signal (BISTRO) scheme in vivo using a double-tuned 1H/31P phased-array coil in a single-channel configuration for transmission. The demonstrated suppression efficacy of BISTRO opens the way for its use as a frequency-selective pre-saturation module for future 31P magnetization transfer experiments for the study of brain energy metabolism at very high magnetic field.

Copper is one of the essential metal ions for aerobic organisms. Two well known functions of copper in the biological systems are electron transfer and molecular oxygen interaction. Thus, this metal can be found in haemocyanin, an oxygen carrier protein, and superoxide dismutase, an enzyme that involves in electron transfer. In addition, having a positive redox potential allows copper to be involved in redox chemistry. It is the redox properties of copper that are responsible for many important biochemical processes. Although the copper-containing oxidases have been well studied over the years, certain mechanistic details such as reaction intermediates remain to be elucidated. Several research groups have been trying to study this by trying to mimic the native systems, synthesizing bulky organic molecules with copper-binding and oxidative capabilities. However, these model systems are only applicable in organic solvents at low temperatures. In this study, three naturally occurring peptides, amyloid-ß, bacitracin, and histatin 5, have been shown to display the oxidative chemistry when complexed with CuII. A combination of spectroscopic (UV-Vis and NMR) and reactivity was used in studying their metal-binding properties as well as in elucidating their catalytic mechanism.

A cascade of metabolomic studies have been developed in the recent decade. The application of metabolomics in the clinical field has been shown to be promising since even subtle physiological changes can be revealed by a metabolomic study which determines variations in the metabolome.Personalized clinical care is currently proposed for almost all the diseases, however, it is still difficultto be executed for the scarce of clinical biomarkers. This thesis work concerns the applications of both ¹H NMR-based and MS-based metabolomics in the determination of potential serumbiomarkers which help to improve personalized diagnosis and prognosis. It is composed by twoprincipal parts. The first part of the work aims to find out metabolite biomarkers predicting themortality of septic shock before clinical intervention and first 12 hours after hospitalization using NMR-based and LC-MS-based metabolomic methods respectively. The goal of the second part of the work is to seek potential biomarkers predicting the recurrence of HCC before and after RFA treatment. Our findings not only show that both the two applied techniques were useful for the discovery of novel clinical biomarkers, but also show that the two techniques are complementary / De nombreuses études métabolomiques a été développée au cours de la dernière dizaine année.L'application de la métabolomique dans le traitement clinique s'est révélée prometteuse puisque même des faibles modifications physiologiques peuvent être révélées par une étude métabolomique. Des soins cliniques personnalisés sont actuellement proposés pour presque toutes les maladies, mais il est encore difficile de les réaliser à cause du manque de biomarqueurs cliniques. Ce travail de thèse a pour le but de la détermination de biomarqueurs cliniques par la spectroscopie RMN et par la spectrométrie de masse. Il est composé de deux parties : la première partie du travail vise à déterminer les biomarqueurs des métabolites prédisant la mortalité du choc septique avant l'intervention clinique et les 12 premières heures après l'hospitalisation en utilisant respectivement des méthodes métaboliques basées sur la RMN et la LC-MS. L'objectif de la deuxième partie du travail est de rechercher des biomarqueurs potentiels prédisant la récidive du CHC avant et après le traitement RFA. Nos résultats montrent non seulement que les deux techniques appliquées ont été utiles pour la découverte de nouveaux biomarqueurs cliniques, maismontrent également que les deux techniques sont complémentaires.

<p>An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase ¹⁹F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. </p><p>The influence of resin structures for seven commercial resins upon resolution of gel-phase ¹⁹F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.</p>

Carbohydrates are abundant in nature and have functions ranging from energy storage to acting as structural components. Analysis of carbohydrate structures is important and can be used for, for instance, clinical diagnosis of diseases as well as in bacterial studies. The complexity of glycans makes it difficult to determine their structures. NMR spectroscopy is an advanced method that can be used to examine carbohydrates at the atomic level, but full assignments of the signals require much work. Reliable automation of this process would be of great help. Herein studies of Escherichia coli O-antigen polysaccharides are presented, both a structure determination by NMR and also research on glycosyltransferases which assemble the polysaccharides. The computer program CASPER has been improved to assist in carbohydrate studies and in the long run make it possible to automatically determine structures based only on NMR data. Detailed computer studies of glycans can shed light on their interactions with proteins and help find inhibitors to prevent unwanted binding. The WaaG glycosyltransferase is important for the formation of E. coli lipopolysaccharides. Molecular docking analyses of structures confirmed to bind this enzyme have provided information on how inhibitors could be composed. Noroviruses cause gastroenteritis, such as the winter vomiting disease, after binding human histo-blood group antigens. In one of the projects, fragment-based docking, followed by molecular dynamics simulations and binding free energy calculations, was used to find competitive binders to the P domain of the capsid of the norovirus VA387. These novel structures have high affinity and are a very good starting point for developing drugs against noroviruses. The protein targets in these two projects are carbohydrate binding, but the techniques are general and can be applied to other research projects. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 5: Manuscript. Paper 6. Manuscript.

An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase 19F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. The influence of resin structures for seven commercial resins upon resolution of gel-phase 19F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.