NMR spectroscopy in the presence of ultrasound and other perturbations

The work described in this thesis has been concerned with exploring the potential uses of ultrasound in Nuclear Magnetic Resonance (NMR) spectroscopy, The NMR spectra of liquids provide detailed structural information that may be deduced from the chemical shifts and spin-spin coupling, that are evident in the narrow resonances, arising from some of the nuclear broadening interactions being reduced to zero. In the solid state, all of the nuclear broadening interactions are present and broad lines in the NMR spectrum are observed. Current techniques employed to reduce the line widths in solids are based on coherent averaging techniques such as MAS NMR1,2 which can remove first order interactions. Recently DOR3 and DAS4 have become available to remove higher order interactions. SINNMR (Sonically Induced Narrowing of the NMR spectra of solids) has been reported by Homer et al5 and developed by Homer and Howard6 to reduce the line widths of solids. The basis of their work is the proposal that a colloidal suspension of solid particles can be made to move like large molecules by using ultrasonic agitation. The advantage of the technique is that the particles move incoherently removing all of the nuclear interactions responsible for broad lines. This thesis describes work on the extension of SINNMR by showing that the line width of 27AI and 11B for the glass Na20/B203/AI203 can be reduced by placing solid particles in a colloidal suspension. Further line width reduction is possible by applying ultrasound, at 2 MHz, of sufficient intensity. It is proposed that a cavitation field is responsible for imparting sufficient rotational motion to the solid particles to partially average the nuclear interactions responsible for broad lines. Rapid stirring of the colloidal suspension generates turbulent flow, however, the motion is insufficient to narrow the line widths for 27AI in the glass. Investigations of sonochemical reactions for in situ rate measurements by NMR have been made. 8y using the Weissler reaction7, it has been shown that ultrasonic cavitation is possible up to 10MHz. Preliminary studies have been carried out into the rate of ultrasonic polymerisation of methylmethacrylate by NMR. Long range order in liquid crystals can imposed when they are aligned in the presence a magnetic field. The degree of alignment can be monitored by NMR using, for example a deuterated solute added to the liquid crystal8. Ultrasonic streaming can then be employed to deflect the directors of the liquid crystal from their equilibrium position, resulting in a change In the NMR spectrum. The angle of deflection has been found for the thermotropic liquid crystal (I35) to be ca, 35° and for the lyotropic (ZLI-1167) to be ca, 20°, Mechanical stirring can used to re- orientate the liquid crystal but was found to give a smaller deflection, In a separate study, that did not use ultrasound, it has been found that the signal to noise ratio of 13C NMR signals can be enhanced by rapidly stirring a Iiquid. Accelerating the diffusion of nuclei out of the coil region enables M0 to be re-established more rapidly than the normal relaxation process. This allows the pulse repetition rate to be reduced without saturating the spin system. The influence of varying the relaxation delay, acquisition time and inter-pulse delay have been studied and parameters optimised. By studying cholesterol the technique was found to be most effective for nuclei with long relaxation times, such as quaternary carbon sites. Key Worde: NMR, Ulf.rasciund, 1,.lqi.fi!:l cryllltalt!h SCll1C1otlemlstryl I!r1hano~d algnflllf

Mediators of monocyte activity in inflammation

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. CRP is currently one of the best markers of inflammatory disease and disease activity. One of the keys cells involved in inflammation within chronic inflammatory diseases is the monocyte. Monocytes are able to modulate inflammation through cytokine expression, cytosolic peroxide formation, adhesion molecule expression and subsequent adhesion/migration to sites of inflammation. CRP has been previously shown to bind directly to monocytes through Fc receptors. However this observation is not conclusive and requires further investigation. The effects of incubation of CRP with human primary and monocytic cell lines were examined using monocytic cytokine expression, adhesion molecule expression and adhesion to endothelial cells and intracellular peroxide formation, as end points. Monocytic intracellular signalling events were investigated after interaction of CRP with specific CRP receptors on monocytes. These initial signalling events were examined for their role in modulating monocytic adhesion molecule and cytokine expression. Monocyte recruitment and retention in the vasculature is also influenced by oxidative stress. Therefore the effect of 6 weeks of antioxidant intervention in vivo was examined on monocytic adhesion molecule expression, adhesion to endothelial cells ex vivo and on serum CRP concentrations, pre- and post- supplementation with the antioxidants vitamin C and vitaInin E. In summary, CRP is able to bind FcγRIIa. CRP binding FcγR initiates an intracellular signalling cascade that phosphorylates the non-receptor tyrosine kinase, Syk, associated with intracellular tyrosine activating motifs on the cytoplasmic tail of Fcγ receptors. CRP incubations increased phosphatidyl inositol turnover and Syk phosphorylation ultimately lead to Ca2+ mobilisation in monocytes. CRP mediated Syk phosphorylation in monocytes leads to an increase in CD 11b and IL-6 expression. CRP engagement with monocytes also leads to an increase in peroxide production, which can be inhibited in vitro using the antioxidants α-tocopherol and ascorbic acid. CRP mediated CD 11b expression is not redox regulated by CRP mediated changes in cytosolic peroxides. The FcyRIla polymorphism at codon 131 effects the phenotypic driven changes described in monocytes by CRP, where R/R allotypes have a greater increase in CD11b, in response to CRP, which may be involved in promoting the monocytic inflammatory response. CRP leads to an increase in the expression of pro-inflammatory cytokines, which alters the immune phenotype of circulating monocytes. Vitamin C supplementation reduced monocytic adhesion to endothelial cells, but had no effect on serum levels of CRP. Where long-term antioxidant intervention may provide benefit from the risk of developing vascular inflammatory disease, by reducing monocytic adhesion to the vasculature. In conclusion CRP appears to be much more than just a marker of ongoing inflammation or associated inflammatory disease and disease activity. This data suggests that at pathophysiological concentrations, CRP may be able to directly modulate inflammation through interacting with monocytes and thereby alter the inflammatory response associated with vascular inflammatory diseases.

Intermediate pyrolysis and product identification by TGA and Py-GC/MS of green microalgae and their extracted protein and lipid components

The thermo-chemical conversion of green microalgae Chlamydomonas reinhardtii wild type (CCAP 11/32C), its cell wall deficient mutant C. reinhardtii CW15 (CCAP 11/32CW15) and Chlorella vulgaris (CCAP 211/11B) as well as their proteins and lipids was studied under conditions of intermediate pyrolysis. The microalgae were characterised for ultimate and gross chemical composition, lipid composition and extracted products were analysed by Thermogravimetric analysis (TG/DTG) and Pyrolysis-gaschromatography/mass-spectrometry (Py-GC/MS). Proteins accounted for almost 50% and lipids 16-22 % of dry weight of cells with little difference in the lipid compositions between the C. reinhardtii wild type and the cell wall mutant. During TGA analysis, each biomass exhibited three stages of decomposition, namely dehydration, devolatilization and decomposition of carbonaceous solids. Py-GC/MS analysis revealed significant protein derived compounds from all algae including toluene, phenol, 4-methylphenol, 1H-indole, 1H-indole-3methyl. Lipid pyrolysis products derived from C. reinhardtii wild type and C. reinhardtii CW15 were almost identical and reflected the close similarity of the fatty acid profiles of both strains. Major products identified were phytol and phytol derivatives formed from the terpenoid chain of chlorophyll, benzoic acid alkyl ester derivative, benzenedicarboxylic acid alkyl ester derivative and squalene. In addition, octadecanoic acid octyl ester, hexadecanoic acid methyl ester and hydrocarbons including heptadecane, 1-nonadecene and heneicosane were detected from C. vulgaris pyrolysed lipids. These results contrast sharply with the types of pyrolytic products obtained from terrestrial lignocellulosic feedstocks and reveal that intermediate pyrolysis of algal biomass generates a range of useful products with wide ranging applications including bio fuels.