Title: Fast Dynamic Processes in Solution Studied by NMR Spectroscopy Author: Mária Šoltésová Department: Department of low temperature physics, Charles University in Prague, and Department of Materials and Environmental Chemistry, Stockholm University Supervisor: doc. RNDr. Jan Lang, Ph.D., Department of low temperature physics, Charles University in Prague, and Prof. Jozef Kowalewski, Department of Materials and Environmental Chemistry, Stockholm University Abstract: Nuclear magnetic resonance (NMR) spectroscopy is capable to deliver a detailed information about the dynamics on molecular level in a wide range of time scales, especially if accompanied by suitably chosen theoretical tools. In this work, we employed a set of high-resolution NMR techniques to investigate dynamics processes in several weakly interacting molecular systems in solution. Van der Waals interactions play an important role in inclusion complexes of crypto- phane-C with chloroform or dichloromethane. The complex formation was thoroughly investigated by means of 1H and 13C NMR experiments along with the quantum- chemical density functional theory (DFT) calculations. We characterized kinetics, thermodynamics, as well as fine details of structural rearrangements of the complex formation. Internal dynamics of oligo- and...

Nuclear magnetic resonance (NMR) spectroscopy has been extensively used to investigate numerous systems of interest, ranging from collections of molecules to living organisms. However, NMR suffers from one key drawback: an inherent lack of detection sensitivity, as compared to other common forms of spectroscopy. This is due to the minute nuclear magnetic moments and low nuclear spin polarization levels at thermal equilibrium (~10-5 to 10-6), and thus necessitates the use of relatively large sample volumes. One way to overcome this low detection sensitivity is to introduce a species with highly non-equilibrium nuclear spin polarization, such as `hyperpolarized' xenon-129. Hyperpolarized xenon can either be used as its own chemical sensor (due to its exquisitely sensitive chemical shift range), or the non-equilibrium polarization may be transferred from xenon to another molecule of interest (such as a protein or inclusion complex). Hyperpolarized xenon is produced through a process known as spin-exchange optical pumping (SEOP), where the angular momentum from resonant, circularly-polarized light is transferred to the electronic spins of an alkali-metal, and is subsequently transferred to the xenon nuclei through gas-phase collisions. While SEOP has been extensively characterized throughout the years, new experimental techniques and emerging technologies have considerably advanced the field in recent years, and may enable a new understanding of the underlying physics of the system. The first five chapters in this dissertation review background information and the principal motivations for this work. Chapter one reviews the basics of NMR, from the various components of the nuclear spin Hamiltonian and different spin-relaxation pathways to the reasons behind the low polarization of nuclear spins at thermal equilibrium and a few alternative methods to `boost' the NMR signal. Chapter two discusses the fundamental aspects of SEOP, including the electronic spin polarization of the alkali-metal, polarization transfer to the xenon nuclei, and different avenues for the spin polarization to be depleted. The third chapter covers the practical considerations of SEOP from the viewpoint of an experimentalist; namely, the experimental differences when using a variety of alkali metals and noble gases, as well as different SEOP apparatuses and experimental parameters. Chapter four details a variety of different light sources that may be used for SEOP; specifically, the use of laser diode arrays (LDAs) are reviewed, including LDAs that have been frequency-narrowed for more efficient light absorption by the alkali metal. The fifth background chapter covers a variety of magnetic resonance applications of hyperpolarized xenon, including molecular biosensors, specific and non-specific binding with proteins, materials studies, and in vivo applications. The sixth chapter is used as an overview of the dissertation research, which is presented in chapters seven through eleven. Chapter seven details the arrangement of the particular SEOP apparatus used in this research, as well as the experimental protocol for producing hyperpolarized xenon. The eighth chapter accounts the implementation and characterization of the first frequency-narrowed LDA used in this research, as well as an equal comparison to a traditional broadband LDA. Chapter nine introduces the use of in situ low-field NMR polarimetry, which was used to distinguish an anomalous dependence of the optimal OP cell temperature on the in-cell xenon density; the low-field set-up is also used to examine the build-up of nuclear spin polarization in the OP cell as it occurs. The tenth chapter covers the use of high power, frequency-narrowed light sources that are spectrally tunable independent of laser power; this allows for the study of changes to the optimal spectral offset as a function of in-cell xenon density, OP cell temperature, and laser power. Xenon polarization build-up curves are also studied to determine if the spectral offset of the laser affects the nuclear spin polarization dynamics within the OP cell. Finally, chapter eleven accounts the use of high power, broadband LDAs to perform SEOP in which cesium is used as the alkali metal; these results demonstrate (for the first time) that the xenon polarization generated by cesium optical pumping can surpass that of rubidium OP under conditions of high laser flux and elevated in-cell xenon densities.

abstract: The behaviors of various amorphous materials are characterized at high pressures to deduce phase transitions, coordination changes, densification, and other structural or electronic alterations in the system. Alongside, improvements on high pressure techniques are presented to measure equations of state of glassy materials and probe liquids using in-situ high resolution nuclear magnetic resonance (NMR) spectroscopy. 27Al NMR is used to quantify coordination changes in CaAl2O4 glass pressure cycled to 16 GPa. The structure and coordination environments remain unchanged up to 8 GPa at which 93% of the recovered glass exists as 4-fold Al, whereas the remaining population exists as [5,6]Al. Upon densification, [5,6]Al comprise nearly 30% of observed Al, most likely through the generation of 3-coordinated oxygen. A method to determine the volumetric equation of state of amorphous solids using optical microscopy in a diamond anvil cell is also described. The method relies on two dimensional image acquisition and analysis to quantify changes in the projected image area with compression. The area analysis method is used to determine the compression of cubic crystals, yielding results in good agreement with diffraction and volumetric measurements. A NMR probe capable of reaching 3 GPa is built to understand the nature of magnetic field gradients and improve upon the resolution of high pressure studies conducted in a diamond anvil cell. Field gradients in strength up to 6 G/cm are caused largely by mismatches in the magnetic susceptibility between the sample and gasket, which is proven to shift the chemical shift distribution by use of several different metallic gaskets. Polyamorphic behavior in triphenyl phosphite is studied at pressures up to 0.7 GPa to elucidate the formation of the glacial phase at high pressures. A perceived liquid-liquid phase transition is shown to follow a positive Clapeyron slope, and closely follows the predicted glass transition line up to 0.4 GPa and temperatures below 270 K. A drastic change in morphology is indicative of a transformation from liquid I to liquid II and followed by optical microscopy. / Dissertation/Thesis / Ph.D. Chemistry 2012

O objetivo do presente estudo foi avaliar a efusão e o posicionamento do disco articular através de imagens por ressonância magnética nuclear (RMN) pré e pós-operatórios de duas técnicas de artrocentese da articulação temporomandibular. Foram incluídos 26 pacientes com deslocamento de o disco articular sem redução (DDSR), divididos aleatoriamente em dois grupos: artrocentese com 1 agulha com distensão do compartimento superior da ATM (A1) e artrocentese convencional com 2 agulhas (A2). Para comparação dos valores da efusão articular entre as intervenções nos diferentes momentos (antes e após um ano das artrocenteses), foi utilizado o teste qui-quadrado. Todas as análises foram realizadas com nível de significância de 5%. Em relação à efusão, após a realização dos tratamentos, foi observada uma diferença estatisticamente significativa entre as diferentes categorias de efusão (p=0,009), sendo essa diferença evidente no grupo de artrocentese convencional. Do total da amostra, apenas um caso não houve qualquer modificação do posicionamento do disco articular. Pode-se concluir que a artrocentese convencional foi capaz de alterar a variável efusão de maneira estatisticamente significativa, enquanto a artrocentese de agulha única e distensão do compartimento superior não. Ambas as técnicas foram responsáveis por alterar o posicionamento da cabeça mandibular, ou do complexo cabeça-disco, projetando-os nessa última situação para uma posição mais anterior o que pode ser verificado no exame de RMN final, com um aumento da distância interincisal máxima. O uso de artrocentese com emprego de uma única agulha é uma técnica mais simples, do que a artrocentese convencional o que possibilita um maior conforto para o paciente com menor tempo de procedimento e com resultados satisfatórios. / The aim of the present study was to evaluate the effusion and positioning of the articular disc through nuclear magnetic resonance imaging (NMRI) before and after two different arthrocentesis techniques for the temporomandibular joint. 26 patients with dislocation of the articular disc without reduction (ADDwoR) were included, and randomly divided into two groups: arthrocentesis using 1 needle with distention of the upper compartment of the TMJ (A1), and conventional arthrocentesis with 2 needles (A2). The chi-square test was used to compare the joint effusion values between the interventions at different moments (before and after one year of arthrocentesis). All analyzes were performed with a significance level of 5%. Regarding effusion, after the treatments, a statistically significant difference was observed between the different effusion categories (p = 0.009), which was evident in the conventional arthrocentesis group. Of the total sample, only one case did not have a modification of the position of the articular disc. It can be concluded that conventional arthrocentesis was able to change the effusion variable in a statistically significant way, whereas the single needle arthrocentesis and distention of the upper compartment did not. Both techniques were responsible for altering the position of the mandibular head, or the disc-head complex, projecting them in the latter situation, to a more anterior position with an increase in the maximum interincisal distance verified in the final NMRI examination. Arthrocentesis with a single needle is simpler than the conventional technique, for it allows greater comfort to the patient, a shorter procedure and satisfactory results.

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Made available in DSpace on 2014-06-11T19:35:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-20Bitstream added on 2014-06-13T18:46:15Z : No. of bitstreams: 1 flumignan_dl_dr_araiq.pdf: 7430377 bytes, checksum: 039ba441b6c5a41c8d7804f4d186d79c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Uma das características mais interessantes da instrumentação analítica moderna é o número de variáveis que podem ser medidas simultâneamente em uma única amostra. Dentre as técnicas analíticas, a RMN 1 H e 13 C, destaca se como uma das mais importantes na análise de “fingerprintings” de misturas complexas. Por outro lado, problemas na qualidade de combustíveis comercializados, têm sido constantemente relatados, tornando relevante o desenvolvimento de novos métodos de análise alternativos que complementem os aplicados atualmente. No Brasil a qualidade da gasolina comercial é regulamentada pela Resolução ANP nº 309. A seleção das amostras mensais de gasolinas comerciais representativas com diferentes similaridades foi realizada por HCA, após a determinação de diversos parâmetros físico químicos estabelecidos pela Resolução ANP nº. 309, em 400 amostras coletadas durante abril a setembro de 2005 em postos revendedores da região Centro Oeste de São Paulo. A partir da amostra mais complexa, determinada por CG MS, foram otimizadas as condições de RMN 1 H (volume de amostra: 30 L, volume de solvente: 600 L e tempo de análise: 2 min), e RMN 13 C (volume de amostra: 200 L, volume de solvente: 600 L e tempo de análise: 10 min), que apresentaram razão sinal/ruído e tempo de análise adequados, contribuindo para a construção do banco de dados dos perfis espectrais. O banco de dados foi constituído das intensidades espectrais de cada perfil para cada deslocamento químico, bem como dos parâmetros físico químicos estabelecidos pela Resolução ANP nº. 309 e de suas respectivas características de qualidade. O algoritmo SIMCA RMN 1 H e SIMCA RMN 13 C mostrou que os perfis espectrais podem ser correlacionados de forma satisfatória e confiável com a qualidade das gasolinas comerciais brasileiras (sensibilidade superior a 90%)... / The most interesting features of modern instruments is the number of variables that can be measured in a single sample. The analytical techniques, one dimensional 1 H and 13 C NMR, stand out as the most important to fingerprintings analysis of complex mixtures. On the other hand, problems in Brazilian commercial fuel quality, including adulteration episodes, have been consistently reported, making relevant the development of news alternatives analytical methods in laboratories and governmental agencies. In Brazil, the gasoline quality control is regulated by ANP Resolution nº. 309. The samples selection of representative commercial gasoline was performed by HCA, after the determination of several physicochemical parameters, established by ANP Resolution nº. 309, in 400 gasoline samples collected during April to September 2005, in gas stations of the midwest region of Sao Paulo State. The most complex sample, determined by GC MS, promoted the optimization of 1 H NMR conditions (sample volume: 30 mL, solvent volume: 600 mL and analysis time: 2 min) and 13 C NMR conditions (volume Sample: 200 mL, solvent volume: 600 mL and analysis time: 10 min), whose showed adequated signal to noise ratio and time analysis, contributing for the construction of the spectral profile database. Database was composed by the intensities and chemical shift in the spectral profile, as well as by the physicochemical parameters and its respective quality characteristics. SIMCA 1 H NMR and SIMCA 13 C NMR showed satisfactory and confidential correlation between the spectral profiles and the quality of the Brazilian commercial gasoline (sensibility above 90%). Similarly, PLS 1 H NMR and PLS 13 C NMR provided satisfactory correlations in the prediction of several physicochemical parameters, in other words, showed standards erros results RMSEC, RMSEV and RMSEP with proportional intensities... (Complete abstract click electronic access below)

Made available in DSpace on 2015-03-03T11:52:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-10-20Bitstream added on 2015-03-03T12:06:28Z : No. of bitstreams: 1 000806897.pdf: 13230046 bytes, checksum: a6632b14970fa1f287ff7d3b0ee1abdc (MD5) / Este trabalho descreve os estudos químicos dos extratos orgânicos de fezes e de tecidos de lagartas da borboleta Battus polydamas, as quais foram alimentadas com folhas de Aristolochia giberti. As etapas de isolamento dos constituintes químicos foram precedidas por microfracionamentos e/ou estudos comparativos entre as composições químicas de folhas, fezes e lagartas, o que visou a detecção de substâncias, possivelmente, biotransformadas e uma seleção racional das frações a serem estudadas. Do extrato de fezes de lagartas foram isoladas cinco substâncias inéditas: i) ácido (4S,5S,8R,9S,10R,13R)-8,18-dihidroxi-labdan-15-óico (3), ii) ácido (?)-(4S,5- S,8R,9S,10R,13R)-8-hidroxi-labdan-15,18-dióico (4), iii) ácido 7?,8?,18?-trihidroxi-labdan- 15-óico (19), iv) ácido (?)-(8S,8R')-(3,4-metilenodioxi)-(3',4'-dimetoxi)-9'-O-?-glucopiranil- lignan-9-óico (17) e v) ácido (?)-(8S,8R')-[3,4:3',4'-bis(metilenodioxi)]-9'-O-?- -glucopiranil-lignan-9-óico (18), as quais não foram detectadas no extrato de folhas de A. giberti. Foram ainda isolados do extrato fecal a lignana dibenzilbutirolactônica kusunoquinina (2) e o diterpeno labdânico ácido (?)-(5R,8R,9S,10R,13R)-8-hidroxi-labdan- 15-óico (1), cujas ocorrências, em folhas de A. giberti, foram previamente relatadas na literatura. Além disto, neste trabalho, revisitou-se e corrigiu-se a configuração absoluta de 1, e determinaram-se as configurações absolutas das substâncias 3, 4 e 17. Para isso, empregaram-se as seguintes abordagens: i) estudos de dicroísmo circular vibracional associado com cálculos químico-quânticos, ii) estudos teóricos conformacionais, iii) aplicação de uma metodologia de RMN baseada na derivatização comreagentes quirálicos anisotrópicos (metódo PGME), iv) predições de deslocamentos químicos de RMN de 1H e de 13C, em solução, v) medidas polarimétricas e de dicroísmo circular... / This work describes the chemical studies of the extracts of feces and Battus polydamas larvae, which were fed at laboratory with leaves of Aristolochia giberti. The isolation steps of the chemical constituents of these extracts were preceded by microfractionations and/or comparative studies among the chemical compositions of feces, leaves, and larvae, what aimed at the detection of biotransformed compounds. From the fecal extract were isolated five new compounds: i) (4S,5S,8R,9S,10R,13R)-8,18- dihydroxy-labdan-15-oic acid (3), ii) (?)-(4S,5S,8R,9S,10R,13R)-8-hydroxy-labdan-15, 18-dioic acid (4), iii) 7?,8?,18?-trihydroxy-labdan-15-oic acid (19), iv) (?)-(8S,8R')-3,4- -methylenedioxy)-(3',4'-dimethoxy)-9'-O-?-glucopyranyl-lignan-9-oic acid (17), and v) (?)-(8S,8R')-[3,4:3',4'-bis(methylenedioxy)]-9'-O-?-glucopyranyl-lignan-9-oic acid (18), which were not detected in the extract of A. giberti leaves, in addition to two known compounds, kusunokinin (2) and (5S,8R,9S,10R,13R)-8-hydroxy-labdan-15-oic acid (1), whose occurrences in A. giberti leaves were previously reported. Moreover, the absolute configuration of 1 was revisited and corrected and the absolute configurations of the compounds 3, 4, and 17 were determined using the following aproaches: i) vibrational circular dichroismmeasurements and quantum chemical calculations, ii) conformational analyses, iii) application of NMR methodology based on derivatizations with chiral anysotropic reagents (PGMEmethod), iv) predictions of 1H and 13C NMR chemical shifts in solution, v) optical activity and electronic circular dichroism measurements, and vi) chemical transformations. A total of thirteen semisynthetic derivatives were obtained...

abstract: Glycosaminoglycans (GAGs) are a class of complex biomolecules comprised of linear, sulfated polysaccharides whose presence on cell surfaces and in the extracellular matrix involve them in many physiological phenomena as well as in interactions with pathogenic microbes. Decorin binding protein A (DBPA), a Borrelia surface lipoprotein involved in the infectivity of Lyme disease, is responsible for binding GAGs found on decorin, a small proteoglycan present in the extracellular matrix. Different DBPA strains have notable sequence heterogeneity that results in varying levels of GAG-binding affinity. In this dissertation, the structures and GAG-binding mechanisms for three strains of DBPA (B31 and N40 DBPAs from B. burgdorferi and PBr DBPA from B. garinii) are studied to determine why each strain has a different affinity for GAGs. These three strains have similar topologies consisting of five α-helices held together by a hydrophobic core as well as two long flexible segments: a linker between helices one and two and a C-terminal tail. This structural arrangement facilitates the formation of a basic pocket below the flexible linker which is the primary GAG-binding epitope. However, this GAG-binding site can be occluded by the flexible linker, which makes the linker a negative regulator of GAG-binding. ITC and NMR titrations provide KD values that show PBr DBPA binds GAGs with higher affinity than B31 and N40 DBPAs, while N40 binds with the lowest affinity of the three. Work in this thesis demonstrates that much of the discrepancies seen in GAG affinities of the three DBPAs can be explained by the amino acid composition and conformation of the linker. Mutagenesis studies show that B31 DBPA overcomes the pocket obstruction with the BXBB motif in its linker while PBr DBPA has a retracted linker that exposes the basic pocket as well as a secondary GAG-binding site. N40 DBPA, however, does not have any evolutionary modifications to its structure to enhance GAG binding which explains its lower affinity for GAGs. GMSA and ELISA assays, along with NMR PRE experiments, confirm that structural changes in the linker do affect GAG-binding and, as a result, the linker is responsible for regulating GAG affinity. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

abstract: Nanomaterials have attracted considerable attention in recent research due to their wide applications in various fields such as material science, physical science, electrical engineering, and biomedical engineering. Researchers have developed many methods for synthesizing different types of nanostructures and have further applied them in various applications. However, in many cases, a molecular level understanding of nanoparticles and their associated surface chemistry is lacking investigation. Understanding the surface chemistry of nanomaterials is of great significance for obtaining a better understanding of the properties and functions of the nanomaterials. Nuclear magnetic resonance (NMR) spectroscopy can provide a familiar means of looking at the molecular structure of molecules bound to surfaces of nanomaterials as well as a method to determine the size of nanoparticles in solution. Here, a combination of NMR spectroscopic techniques including one- and two-dimensional NMR spectroscopies was used to investigate the surface chemistry and physical properties of some common nanomaterials, including for example, thiol-protected gold nanostructures and biomolecule-capped silica nanoparticles. Silk is a natural protein fiber that features unique properties such as excellent mechanical properties, biocompatibility, and non-linear optical properties. These appealing physical properties originate from the silk structure, and therefore, the structural analysis of silk is of great importance for revealing the mystery of these impressive properties and developing novel silk-based biomaterials as well. Here, solid-state NMR spectroscopy was used to elucidate the secondary structure of silk proteins in N. clavipes spider dragline silk and B. mori silkworm silk. It is found that the Gly-Gly-X (X=Leu, Tyr, Gln) motif in spider dragline silk is not in a β-sheet or α-helix structure and is very likely to be present in a disordered structure with evidence for 31-helix confirmation. In addition, the conformations of the Ala, Ser, and Tyr residues in silk fibroin of B. mori were investigated and it indicates that the Ala, Ser, and Tyr residues are all present in disordered structures in silk I (before spinning), while show different conformations in silk II (after spinning). Specifically, in silk II, the Ala and Tyr residues are present in both disordered structures and β-sheet structures, and the Ser residues are present primarily in β-sheet structures. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2017

Le peptidoglycane est un biopolymère essentiel délimitant la bactérie et dont la synthèse est le produit de réactions catalysées par des complexes protéiques finement régulés spatio-temporellement. Le point culminant de ce processus est l'assemblage et l'insertion des chaînes réticulées de peptidoglycane dans la structure déjà existante par les PBPs, cibles des β-lactames. Les travaux présentées ici portent sur deux importants régulateurs peu caractérisés au niveau atomique, les Lpos (chez Escherichia coli), qui stimulent les activités des PBPs. En utilisant principalement la RMN, les premières structures à haute-résolution de ces protéines ont été obtenues et quelques interactions ont pu être étudiées. Les résultats présentés permettent de mieux comprendre comment ces facteurs, ancrés à la membrane externe, atteignent leur cible et régulent directement la synthèse du peptidoglycane. / Peptidoglycan is an essential biopolymer which surrounds the bacterial cell. Its biosynthesis is the product of large macromolecular complexes tightly regulated in space and time. The peak of this process is the assembly and insertion of peptidoglycan reticulated chains in the existing network by Penicillin-Binding Proteins (PBPs), targets of β-lactam antibiotics. The work presented here focuses on two important regulators of peptidoglycan growth lacking atomic-scale characterization, the Lpo proteins (in Escherichia coli). These proteins are directly stimulating PBPs activities. Using mainly NMR, their structure was obtained, for the first time, and, for one of them, the interaction with its protein partner was investigated. The results provide new insights on how these proteins reach their targets from the outer-membrane and on how their regulatory function is determined.