High-resolution magic angle spinning (HRMAS) NMR spectroscopy is used to study solid samples that are normally difficult to analyze due to broadening of peaks. Solid-phase peptide synthesis can bind peptides to an insoluble resin that can be analyzed with HRMAS NMR spectroscopy. A combination of HRMAS NMR and IRMPD spectroscopy, along with computational chemistry, was applied to analyze and evaluate the structure of resin-bound glutathione. Two-dimensional 1H-1H NMR experiments such as COSY, TOCSY, and ROESY were employed to assign and predict the structure of the resin-bound peptide. IRMPD results were used along with calculated protonated structures and spectra to evaluate the conformation of the peptide. The experimental spectrum was compared to the spectra and structures of the protonated species to hypothesize the most favoured structure. Molecular mechanics, molecular dynamics and DFT calculations were implemented to collect structures that best resembled the free and resin-bound glutathione peptide. The results from these methods were compared to determine the structure that is most probable for the glutathione peptide. A semi-folded conformation is the structure the resin-bound GSH most preferred as concluded from the NMR and DFT results. The IRMPD results were analyzed as separate from the resin-bound experiments and suggested protonated GSH had a folded conformation. FK-13 was another peptide synthesized using the solid-phase peptide synthesis technique. The peptide was synthesized using a modified technique different from conventional methodology used in the past. The peptide was also analyzed using COSY, TOCSY, and ROESY to confirm that the synthesis was done correctly and hypothesize a structure. The low substitution of the peptide on the resin gave rise to minimal NOE interactions, but there was some evidence suggesting that the synthesis was successful and the peptide adopted a cyclic conformation. These initial results are useful for future analyses and conformational studies of this resin-bound peptide. Further work needs to be done for both peptides to explore the structures in more detail. The explicit model of solvation should be used to explore the effect of solvent molecules on the conformation of the glutathione peptide as opposed to the implicit model that PCM provides. FK-13 could be synthesized better so that a higher substitution is achieved and better NMR results are obtained. The IRMPD results obtained by the McMahon group can then be compared to the NMR results and computational calculations can be performed to obtain realistic structures of the peptide.

To unravel the mysteries of complex biological processes carried out by biomolecules it is necessary to adopt a multifaceted approach, which involves employing a wide variety of tools both computational and experimental. In order to gain a clear understanding of the function of biomolecules their three dimensional structure is required. X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy are the only two methods capable of providing high-resolution three-dimensional structure of biomolecules. NMR has the advantage of allowing the study of structure of biomolecules in solution and is better equipped to characterize the dynamics of the protein. Protein structure determination by NMR spectroscopy consists of recombinant expression of isotopically labeled proteins, purification, data collection, data processing, resonance assignment, distance restraint and angular restraint generation, structure calculation and structure validation. Apart from 3D structure determination of biomolecules NMR has become the method of choice for studying transient protein-protein interactions, which are notoriously difficult to study at higher resolution by other methods. Mass spectrometry plays an important role in enabling rapid identification of biomolecules and their modifications. The high sensitivity and resolution mass spectrometry offers makes it the method of choice for studying post-transitional modification of proteins. Use of computers in biology has played an essential role in elucidating those structure function relationships in biomolecules that are not possible to study by experimental techniques. The first chapter of this thesis deals with the introduction of methods used in this study. A brief introduction about the theory of Nuclear Magnetic Resonance (NMR) spectroscopy is given. Protein NMR methods used for structure determination of medium sized proteins are discussed. A part of this chapter discusses about the application of mass spectrometry in biochemistry and the use of tandem MS/MS experiments in identification of proteins and peptide fragments. Finally, the last part of this chapter gives an introduction about the theory of molecular dynamics and techniques used in the post processing of MD trajectories to elucidate the dynamics of proteins. The second chapter of this thesis is concerned with NMR characterization of a novel protein-protein interaction between the glycolytic enzyme Triosephosphate isomerase and the redox protein Thioredoxin. Chemical shift perturbation studies have been done to map the binding interfaces of these proteins. The structure of the complex was then modeled using NMR restraints based docking using the known 3D structure of these proteins. The docked complex reveals crucial insights into the glutathione mediated redox regulation of Triosephosphate isomerase and the role of thioredoxin as a deglutathionylating agent. Enzyme activity assays of Triosephosphate isomerase were done to show the inhibitory effects of s-glutathionylation of Cys217 and the role of thioredoxin as a deglutathionylating agent. The third chapter of the thesis is aimed to address some important issues related to the inhibition of Plasmodium falciparum Triosephosphate isomerase by S-glutathionylation. Oxidative stress induces protein glutathionylation which is a reversible post translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione. Mass spectrometric analysis of the kilnetics of glutathionylation along with enzyme activity assays clearly show that gluthionylation of either Cys-13 (situated in the dimmer interface) or Cys-217 (situated in Helix G) can render the enzyme inactive. Molecular dynamics simulations provide a mechanistic basis of inhibition and predict that glutathionylation at Cys217 allosterically induces loop 6 disorder. The fourth chapter of this thesis addresses the stabilizing effect of introduction of a cross-strand disulfide bond across a non-hydrogen bonded position of an antiparallel beta sheet. Multidimensional heteronuclear NMR experiments have been used to get the backbone and side-chain resonance assignments, distance and angular restraints. In addition RDC based restraints have been used to calculate the structure of oxidixed form of L79C, T89C thiroedoxin. The observation of predominantly –RH staple conformation among the NMR ensemble in typical of cross-strand disulfides. The fifth chapter of this thesis deals with the dynamics of thioredoxin using computational methods.In this chapter analysis of known complexes of thiroedoxin was done to determine binding hot spot residues using free energy calculations. The physicochemical basis for the multispecificity of thioredoxin is probed using molecular dynamics simulations. In this chapter it has been shown that conformational selection plays a very important role in thioredoxin target recognition.

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (leaves 149-161). Also available via World Wide Web.

This thesis combines synthetic studies for isotopic enrichment with solid-state characterisation techniques to investigate two classes of microporous materials: zeolites and metal-organic frameworks (MOFs). These materials have a wide range of successful applications, from industrial catalysis to medicine, resulting in the increasing need for both a complete understanding of their unique structural features and synthetic methods to target new frameworks. Nuclear magnetic resonance (NMR) spectroscopy, thanks to its sensitivity to the local, atomic-scale, environment and its element specificity, is applied, in combination with powder X-ray diffraction (PXRD), electron microscopy, N2 adsorption and mass spectrometry, to the study of these materials. Oxygen atoms play a crucial role in the structure and chemistry of zeolites and MOFs, making 17O NMR an excellent tool for chemical and structural investigations. However, the low natural abundance of this isotope (0.037%) and the cost of 17O-enriched reactants require the development of atom-efficient synthetic processes for isotopic enrichment. In the first part of this work, the unconventional assembly-disassembly-organisation-reassembly (ADOR) method is applied to the Ge-doped UTL framework and optimised in reduced-volume conditions for economic enrichment to obtain 17O- and 29Si-enriched UTL-derived zeolites. In situ and ex situ solid-state characterisation studies show that isotopic enrichment not only enables a more detailed spectroscopic investigation, but also provides new insights into the mechanism of the ADOR process and its sensitivity to experimental conditions. In the second part of this work, dry gel conversion synthesis and a novel steaming procedure are studied as cost-effective 17O-enrichment pathways for Al, Ga and Sc mixed-metal terephthalate MOFs. 17O solid-state NMR spectroscopy, in combination with PXRD and electron microscopy, is employed to investigate cation disorder and 17O NMR spectra are shown to be sensitive to substitution of metal centers and conformational changes upon interaction with guest molecules.

Thermal stability is an important and interesting physical property of proteins. A common method to study it by is circular dichroism (CD) spectroscopy. The aim of this study was to test methods to improve thermal stability analysis by CD spectroscopy. Experiments were performed using the Abp1p SH3 domain from yeast as a model protein. Thermal denaturation was monitored at multiple wavelengths. It was concluded that for data sets of reasonable quality the choice of wavelength does not affect the results. An approach to estimate stability of thermophilic proteins was tested where thermal stability was measured at different concentrations of the denaturant GuHCl. The thermochemical data was used to estimate the stability in absence of GuHCl by extrapolation. The results were compared to those obtained from CD spectroscopy and differential scanning calorimetry. It was found that a stabilizing effect from low concentrations of GuHCl complicated the extrapolation. It is likely that this method is more successful if there is no stabilizing effect. The effect of ΔCp in stability parameter calculations was investigated with an experimentally and theoretically determined ΔCp. This was further investigated with synthetic data sets. The ΔCp used in calculations had no notable effect, as long as there was no cold denaturation. Although ΔCp is not necessary in calculations, it is an interesting parameter itself. ΔCp can be calculated from the thermochemical data used for extrapolation. The results in this study demonstrate robustness in thermal stability analysis by CD spectroscopy and a potential for development.

O metaboloma é definido como a coleção qualitativa e quantitativa de todos os metabólitos de baixa massa molecular presentes nas células, os quais participam de reações bioquímicas necessárias para a manutenção, crescimento e fisiologia celular. A avaliação metabolômica permite o delineamento do processo bioquímico de sistemas a fim de ampliar o entendimento de como as patologias se manifestam. A Espectroscopia de Ressonância Magnética Nuclear (RMN) é usada para investigar uma variedade de processos biológicos em diversos sistemas. A magnética aplicada a células e biópsias de tecido intacto de melanoma murino B16F10 contribuiu para a caracterização bioquímica de biomarcadores das diferentes fases de progressão tumoral do melanoma murino B16F10. Os resultados obtidos neste estudo permitiram a identificação de 33 metabólitos possíveis, envolvidos na carga lipídica e no metabolismo secundário da via glicolítica, que favorecem o crescimento e a progressão tumoral. A presença da taurina, prolina, serina, fenilalanina, que aumentaram quantitativamente, são possíveis marcadores da invasão, progressão e metastatização. A análise quantitativa desses metabólitos mostrou diferença significativa em 11 compostos, dos quais 9 estão diretamente envolvidos na expressão das respostas proliferativas, de morte celular e angiogênese, nos diferentes períodos de crescimento do melanoma B16F10, avaliados neste estudo. Desta forma, os achados obtidos neste estudo, quando associados no futuro a outros fatores, poderão ser úteis no diagnóstico e auxiliar na escolha terapêutica alvo com maior especificidade e menores efeitos colaterais. RMN pode ter um importante impacto na monitorização de metabólitos em células e tecidos tumorais, possibilitando a detecção mais precoce de tumores malignos, em suma, através da combinação de métodos de ressonância magnética. / The metabolome is defined as the qualitative and quantitative collection of all low weight molecular metabolites in cells that participate in biochemical reactions necessary for the maintenance, growth and physiology of cell. The metabolomic evaluation allows the design of systems of biochemical process in order to broaden the understanding of how diseases manifest. The Nuclear Magnetic Resonance Spectroscopy (NMR) is used for investigating a variety of biological processes in several systems. The magnetics applied to cell and tissue biopsies intact murine melanoma B16F10 contributed to the biochemical characterization of biomarkers of different stages of tumor progression of murine melanoma B16F10. The results obtained in this study allowed the identification of 33 potential metabolites involved in lipid content in the secondary metabolism of the glycolytic pathway, which promote growth and tumor progression. The presence of taurine, proline, serine, phenylalanine, which quantitatively increased, is possibly markers of invasion and metastasis progression. Quantitative analysis of these metabolites showed a significant difference in 11 compounds, of which 9 are directly involved in the expression of proliferative responses, cell death and angiogenesis in different periods of growth of B16F10 melanoma, evaluated in this study. Thus, the findings from this study, when associated in the future with other factors, may be useful in the diagnosis and may assist in choosing therapeutic target with greater specificity and fewer side effects. NMR may have a significant impact on monitoring metabolites in tumor cells and tissues, allowing for earlier detection of malignant tumors; in short, by combining MRI methods.

La spectroscopie de Résonance Magnétique Nucléaire (RMN) Haute Résolution à l’angle magique (HRMAS) joue un rôle de plus en plus prépondérant pour le diagnostic médical. Cette technique permet d’établir les empreintes ex vivo des métabolites de tissus sains et pathologiques. Cependant, pour certains métabolites, les valeurs des déplacements chimiques des groupes de protons peuvent légèrement varier en fonction de l’environnement des tissus ou cellules, particulièrement de son acidité. Cet effet gêne l’estimation correcte des concentrations des métabolites lorsqu’on utilise des algorithmes fondés sur des bases de métabolites. Ce travail est dévolu aux méthodes d’optimisation des bases de métabolites, notamment aux algorithmes de correction des changements de déplacements chimiques. Deux méthodes de traitement du signal ont été développées pour l’optimisation simple et rapide des signaux / spectres : contraction/expansion du signal moyennant ré-échantillonnage et fractionnement du spectre. Une autre méthode, QM-QUEST, conjuguant la simulation par Mécanique Quantique et la quantification, a été mise en œuvre. Cette dernière permet l’ajustement plus robuste des spectres en limitant l’implication de l’utilisateur et préserve les empreintes correctes des métabolites. Son efficacité est démontrée pour la quantification de spectres RMN de biopsies cérébrales humaines d’oligodendroglioma, obtenues à 11.7 Tesla et de spectres de cellules acquis à 9.4 T par la technique RMN-HRMAS. Etant donné la nécessité de simulation rapide des signaux RMN basée sur la Mécanique Quantique, une partie du travail est vouée à une méthode approchée accélérant la simulation. L’algorithme fondé sur la fragmentation du système de spins pourrait devenir une partie importante de la méthode d’optimisation QM-QUEST et sema mis en œuvre en tant qu’option de simulation de la méthode NMR-SCOPE, module du logiciel jMRUI. / From day to day, the role of HRMAS (High-Resolution Magic Angle Sinning) Nuclear Magnetic Resonance Spectroscopy (NMRS) in medical diagnosis is increasing. This technique enables setting up metabolite profiles of ex vivo pathological and healthy tissue. Automatic spectrum quantitation enables monitoring of diseases. However for several metabolites, the values of chemical shifts of proton groups may slightly differ according to the micro-environment in the tissue or cells, in particular to its pH. This hampers accurate estimation of the metabolite concentrations mainly when using quantitation algorithms based on a metabolite basis-set. The present word is devoted to the optimization of NMR metabolite basis set signals, particularly to the algorithms of chemical shift mismatch correction. Two sighal processing (“warping”) methods were developed for simple and fast spectrum optimization : signal stretching/shrinking (resampling) and spectrum splitting. Then, another optimization method, QM-QUEST, coupling Quantrum Mechanical simulation and quantitation algorithms was implemented. The latter provides more robust fitting while limiting user involvement and respects the correct fingerprints of metabolites. Its efficiency is demonstrated by accurately quantitating signals from tissue samples of human brains with oligodendroglioma, obtained at 11.7 Tesla and spectra of cells acquired at 9.4T by HRMAS-NMR. As the necessity of fast NMR signal simulation based on quantum Mechanics is raised in the thesis, a part of the word is dedicated to an approximate method speeding-up the calculations. The algorithm based on spin-system fragmentation could become an important part of the QM-QUEST optimization method and will be implemented as an option of simulation in NMR-SCOPE, module of the jMRUI software package.

Abstract The present thesis describes a method for characterization of liquid crystals in confined spaces by means of NMR of probe atoms and molecules. 129Xe isotope enriched xenon gas and 13C isotope enriched methyl iodide and methane were used as probes. Behavior of solutes and liquid crystals confined to porous materials was investigated using 129Xe and 13C NMR spectroscopy. Uniaxial nematic liquid crystals Phase 4 and ZLI 1115 were used as a medium. Controlled pore glass with well defined and known properties was used as a porous material. The behavior of liquid crystals and solutes in various different size pores, temperatures and magnetic fields at different solute concentrations was explained. The average pore diameter of the material varied from mesopores to macropores. The studied temperature range covered solid, nematic and isotropic phases of bulk liquid crystals, and the highest magnetic field was 2.5 times stronger than the lowest one used (4.70 T). The chemical shifts, intensities, and line shapes of the resonance signals from the solutes were observed to contain lots of information about the effect of confinement on the state of the liquid crystals. Especially the line shape of the 13C resonances of methyl iodide was observed to be very sensitive to the liquid crystal orientation distribution in the pores. By varying experimental conditions the relative contribution of field and the surface forces of pore walls to the orientation of liquid crystal molecules inside the pores was seen to change quite drastically. In addition, it was also observed that when the sample is cooled very rapidly, xenon atoms do not squeeze out from the freezing medium but they are occluded inside the solid lattice, and their chemical shift is very sensitive to crystal structure. Furthermore, because solutes experienced on average isotropic environment inside the smallest pores, isotropic value of the shielding tensor could be determined at exactly the same condition and temperature as anisotropic counterpart between the pore particles. Thus, for the first time in the solution state, shielding anisotropies could be determined as a function of temperature.

Dans ce travail, nous nous concentrons sur la caractérisation de l'environnement physico-chimique des éléments présents dans des multicouches à base de cobalt qui travaillent dans les domaines des rayons X mous et extrême ultra-violet (EUV). L'observation des modifications des interface des deux systèmes Co/Mo2C et Co/Mo2C/Y lors du recuit est important pour l'amélioration de leurs performance optique. Ils ont été étudiés en combinant des méthodes non destructives, spectroscopie d'émission des rayons X, résonance magnétique nucléaire, réflectométrie de rayons X et des méthodes destructrices, spectrrométrie de masse d'ions secondaires par temps de vol et la Microscopie électronique en transmission pour étudier leurs propriétés interfaciales. Ce travail vise non seulement à la conception et la fabrication de nouveaux éléments optiques pour faire face au développement des sources et des applications dans les demaines des rayons X mous et l'EUV, mais aussi à développer une méthodologie combinant des simulations et des expérimentations consacrée à l'analyse des interfaces dans structures multicouches afin d'améliorer leur propriétés optiques. / In this work, we focus on the characterization of physico-chemical environment of the element present in Co-based multilayers working in the soft x-ray and EUV ranges. The observation of interface changes of both systems Co/Mo2C and Co/Mo2C/Y upon annealing is important for improving their optical performance. They were studied by combining non-destructive methods, x-ray emission spectroscopy, nuclear magnetic resonance spectroscopy, x-ray reflectometry and destructive methods, time-of-flight secondary ions mass spectroscopy and transmission electron microscopy to investigate their interface properties. This work aims not only at designing and fabricating new optical elements to face the development of sources and applications in the EUV and soft x-ray ranges, but also at developing a methodology combining simulations and experiments devoted to the interface analysis in these multilayer structures in order to improve their optical properties.

La Diagonalisation Conjointe par Congruence (DCC) d'un ensemble de matrices apparaît dans nombres de problèmes de traitement du signal, tels qu'en Analyse en Composantes Indépendantes (ACI). Les développements récents en ACI sous contrainte de non négativité de la matrice de mélange, nommée ACI semi-non négative, permettent de tirer profit d'une modélisation physique réaliste des phénomènes observés tels qu'en audio, en traitement d'image ou en ingénierie biomédicale. Par conséquent, durant cette thèse, l'objectif principal était non seulement de concevoir et développer des algorithmes d'ACI semi-non négative basés sur de nouvelles méthodes de DCC non négative où la matrice de passage recherchée est non négative, mais également d'illustrer leur intérêt dans le cadre d'applications pratiques de séparation de sources. Les algorithmes de DCC non négative proposés exploitent respectivement deux stratégies fondamentales d'optimisation. La première famille d'algorithmes comprend cinq méthodes semi-algébriques, reposant sur la méthode de Jacobi. Cette famille prend en compte la non négativité par un changement de variable carré, permettant ainsi de se ramener à un problème d'optimisation sans contrainte. L'idée générale de la méthode de Jacobi est de i) factoriser la matrice recherchée comme un produit de matrices élémentaires, chacune n'étant définie que par un seul paramètre, puis ii) d'estimer ces matrices élémentaires l'une après l'autre dans un ordre spécifique. La deuxième famille compte un seul algorithme, qui utilise la méthode des directions alternées. Un tel algorithme est obtenu en minimisant successivement le Lagrangien augmenté par rapport aux variables et aux multiplicateurs. Les résultats expérimentaux sur les matrices simulées montrent un gain en performance des algorithmes proposés par comparaison aux méthodes DCC classiques, qui n'exploitent pas la contrainte de non négativité. Il semble que nos méthodes peuvent fournir une meilleure précision d'estimation en particulier dans des contextes difficiles, par exemple, pour de faibles valeurs de rapport signal sur bruit, pour un petit nombre de matrices à diagonaliser et pour des niveaux élevés de cohérence de la matrice de passage. Nous avons ensuite montré l'intérêt de nos approches pour la résolution de problèmes pratiques de séparation aveugle de sources. Pour n'en citer que quelques-uns, nous sommes intéressés à i) l'analyse de composés chimiques en spectroscopie par résonance magnétique, ii) l'identification des profils spectraux des harmoniques (par exemple, de notes de piano) d'un morceau de musique mono-canal par décomposition du spectrogramme, iii) l'élimination partielle du texte se trouvant au verso d'une feuille de papier fin. Ces applications démontrent la validité et l'intérêt de nos algorithmes en comparaison des méthodes classique de séparation aveugle de source. / The Joint Diagonalization of a set of matrices by Congruence (JDC) appears in a number of signal processing problems, such as in Independent Component Analysis (ICA). Recent developments in ICA under the nonnegativity constraint of the mixing matrix, known as semi-nonnegative ICA, allow us to obtain a more realistic representation of some real-world phenomena, such as audios, images and biomedical signals. Consequently, during this thesis, the main objective was not only to design and develop semi-nonnegative ICA methods based on novel nonnegative JDC algorithms, but also to illustrate their interest in applications involving Blind Source Separation (BSS). The proposed nonnegative JDC algorithms belong to two fundamental strategies of optimization. The first family containing five algorithms is based on the Jacobi-like optimization. The nonnegativity constraint is imposed by means of a square change of variable, leading to an unconstrained problem. The general idea of the Jacobi-like optimization is to factorize the matrix variable as a product of a sequence of elementary matrices which is defined by only one parameter, then to estimate these elementary matrices one by one in a specific order. The second family containing one algorithm is based on the alternating direction method of multipliers. Such an algorithm is derived by successively minimizing the augmented Lagrangian function of the cost function with respect to the variables and the multipliers. Experimental results on simulated matrices show a better performance of the proposed algorithms in comparison with several classical JDC methods, which do not use the nonnegativity as constraint prior. It appears that our methods can achieve a better estimation accuracy particularly in difficult contexts, for example, for a low signal-to-noise ratio, a small number of input matrices and a high coherence level of matrix. Then we show the interest of our approaches in solving real-life problems. To name a few, we are interested in i) the analysis of the chemical compounds in the magnetic resonance spectroscopy, ii) the identification of the harmonically fixed spectral profiles (such as piano notes) of a piece of signal-channel music record by decomposing its spectrogram, iii) the partial removal of the show-through effect of digital images, where the show-through effect were caused by scanning a semi-transparent paper. These applications demonstrate the validity and improvement of our algorithms, comparing with several state-of-the-art BSS methods.