Ulm, Univ. Diss., 2006.

Techn. Hochsch., Diss., 2005--Aachen.

Thesis advisor: Mary F. Roberts / Thesis advisor: Steven D. Bruner / The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from <italic>Bacillus thuringiensis</italic> is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling <super>31</super>P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A set of crude oil samples with different composition and characteristics is studied by means of Fast Field Cycling (FFC) 1H relaxometry, which probes the distribution of longitudinal relaxation times T1 as a function of the Larmor frequency. Investigation of T1 profiles at different temperatures is able to provide an insight into the dynamics and structural changes of oil components, with our particular interest being the high temperature behaviour of asphaltene. It is well-known that asphaltenes tend to form porous clusters in crude oils, which can cause severe problems for the process of oil extraction. Therefore, FFC experiments are conducted on Stelar Spinmaster FFC2000 in the temperature range 203K < T <443K, where the upper limit of 443K is aimed at approximating the typical maximal in-situ well temperatures. FFC relaxometry data of crude oils at such a high temperature are obtained for the first time with the use of a specially modified NMR probe. Inverse Laplace transformation is applied to the longitudinal agnetization decays, yielding T1 distributions at different frequencies. A comparative analysis of these distributions for different Larmor frequencies and temperatures showed that there is a systematic variation of the frequency dependence of T1 correlating with the asphaltene content in the samples, at temperatures similar to the well conditions.

No description available.

No description available.

L’IRM en champ cyclé (FFC-MRI) permet de dissocier deux processus clés de l’IRM qui dépendent chacun du champ magnétique principal B0 : d’une part, la détection du signal RMN et sa localisation et d’autre part, la relaxation du signal RMN, source de contraste d’intérêt biologique et médical. Le système d’IRM en champ cyclé est la combinaison de deux appareils, l’un est un système d’imagerie RMN et l’autre permet de faire varier le champ magnétique B0 rapidement devant les temps de relaxation. Il est ainsi possible de mesurer la dispersion de la relaxation de l’eau, c’est-à-dire sa variation en fonction du champ magnétique et potentiellement de la cartographier de manière non invasive in vivo. La dispersion est une source de contraste complémentaire, étant donné le lien entre relaxation de l’eau et son environnement moléculaire dans les tissus biologiques. L’objectif de la thèse consiste à développer et évaluer le potentiel de l’IRM en champ cyclé entre 1 T et 2 T sur un modèle de cancer. Ce travail a nécessité des développements instrumentaux et méthodologiques originaux pour intégrer le champ cyclé à des séquences IRM. Les solutions proposées portent tout d’abord sur la mesure précise du champ magnétique au cours du temps, la compensation des courants de Foucault et celle des instabilités de l’alimentation du système de variation de l’intensité de B0. De plus, nous proposons des méthodes d’acquisition avec un gain en signal sur bruit, utilisables pour mesurer la relaxation transversale aussi bien que longitudinale. Enfin une exploration sur modèle animal (tumeur du rein sur souris) a été entreprise. / Fast Field Cycling Magnetic Resonance Imaging (FFC-MRI) has the ability to separate two key processes that both depends on the main field intensity B0. On one hand, signal acquisition and localization and on the other hand NMR relaxation, basis of MRI contrast. The equipment thus combines a standard MR scanner with a secondary system to rapidly switch the magnetic field B0 as compared to relaxation times. FFC enables to measure the evolution of NMR relaxation as a function of magnetic field B0, namely the NMR dispersion (NMRD) profile. Combining it with MRI the NMRD profile can be localized in vivo, together with the usual characterization at fixed B0. The NMRD profile of water carries information on molecular mobility in the surrounding biological tissues, and is thus another source of contrast. The objective of this PhD project was to further develop and evaluate the potential of FFC-MRI between 1 T and 2 T in a cancer model. This work required original instrumental and methodological developments to integrate FFC in MRI. First a precise measurement of magnetic field time profile was developed, as well as the compensation of eddy currents and of irreproducible transients in the secondary system. Moreover acquisition sequences with better signal to noise efficiency and applicable for longitudinal as well as transverse relaxation were implemented. Finally a kidney tumor mouse model was explored with FFC MRI.