No description available.

The Application of Magnetic Resonance (MRI) and Computed Tomography Imaging (CT) in the Diagnosis and Management of Maxillofacial Tumours. For decades maxillofacial surgeons over the world have been frustrated by the high and often fatal recurrence of certain advanced jaw tumours. This study conclusively proves that Computed Tomography and especially Magnetic Resonance Imaging significantly decreases recurrence of Odontogenic Keratocyst and Ameloblastoma and allows surgical planning to avoid these recurrences. / Doctor Scientiae (Odontology) - DSc(Odont)

Amyotrophic lateral sclerosis (ALS) is a devastating and progressive disease that results in selective death of motor neurons in the cortex, brain stem and spinal cord. ALS is the most common adult onset motor neuron disease resulting in paralysis and death, commonly within 2 – 5 years of symptom onset, yet there remains no effective treatment for the disease. The majority of ALS cases show no hereditary link (referred to as sporadic ALS or sALS); however, ~10% of cases show a dominant pattern of inheritance (referred to as familial ALS or fALS). Over 170 different mutations in human Cu, Zn superoxide dismutase (SOD1) have been identified to account for ~20% of fALS. SOD1 is a ubiquitously expressed homodimeric antioxidant enzyme. It is widely accepted that mutations in SOD1 result in a gain of toxic function, rather than a loss of native function. A prominent hypothesis for the gain of function is the formation of protein aggregates, which have been shown to be toxic to motor neurons. Protein aggregation is observed in a number of neurodegenerative disorders, including Alzheimer’s, Huntington’s and Parkinson’s disease. Each β-rich monomer of SOD1 binds one catalytic Cu ion and one structural Zn ion. The metallation state of SOD1 significantly influences the structure, dynamics, activity, stability, and aggregation propensity. A similar trend has been observed in a number of metalloenzymes and as such a method to rapidly and accurately quantitate metal ions in proteins is of great importance. Here a review of previous methods using the chromogenic chelator PAR to quantitate metal ions in proteins is presented. Three methods are assessed for their accuracy, precision and ease of use. The methods vary in accuracy, which is highest only under the specific conditions it was designed for. A robust new method is presented here that uses spectral decomposition software to accurately resolve the absorption bands of Cu and Zn with high precision. This method may be successful as a more general method for metal analysis of proteins allowing for the quantitation of additional metal combinations (e.g. Zn/Co, Ni/Cu, Ni/Co). Thermodynamic stability has widely been implicated as playing a major role in the aggregation of globular proteins. Metal loss significantly decreases the global stability of SOD1 and as such metal-depleted (apo) forms of SOD1 have largely been the focus of SOD1 investigations. Recent studies, however, suggest that complete global unfolding is not required for protein aggregation. Local unfolding has been investigated and proposed to be sufficient to induce irreversible protein aggregation in the absence of global destabilization. Enhanced local unfolding has been observed in a number of disease-related proteins. Since SOD1 aggregation may occur from partially unfolded forms, NMR temperature dependence studies have been carried out on the most abundant form of SOD1 in vivo, the fully metallated (holo) dimer, to provide a residue specific picture of subglobal structural changes in SOD1 upon heating. Amide proton (N1H) temperature coefficients report on the hydrogen bonding status of a protein. A curved N1H temperature dependence indicates that the proton populates an alternative conformation generally within 5 kcal/mol of the ground state. NMR temperature dependence studies of pseudoWT indicate that the thermal unfolding process of holo pWT begins with “fraying” of the structure at its periphery. In particular, increased disorder is observed in edge strands β5 and β6, as well as surrounding the zinc binding site. The local stability and conformational heterogeneity of ALS-associated mutants G93A, E100G and V148I was also assessed. All mutants display similar local unfolding patterns to pseudoWT, but also show distinct differences in the hydrogen bonding network surrounding the mutation site. Interestingly, each mutation regardless of its structural context results in altered dynamics at the β-barrel plug, a key stabilizing element in SOD1. A significant proportion of residues (~30%) access alternative states in both pseudoWT and mutants, however, overall mutants appear to be able to access higher free energy alternative states compared to pseudoWT. The implications of these results for the mechanism of protein aggregation and disease are discussed.

The technique of ¹H spin echo n.m.r. has been used for the non-invasive study of enzyme catalysed ¹H/²H equilibrium isotope exchange at the C-2 position of lactate in suspensions of human erythroeytes. The intracellular environment of the enzymes involved in this exchange has been investigated by comparing the exchange properties of the enzymes in the intact cell with the properties they display in vitro. A study of the exchange of the lactate C-2 substituent with solvent, which is catalysed by a coupled system of four glycolytic enzymes, has teen used to examine the kinetic properties of the individual enzymes in vitro. Measurements of the exchange in the intact cell have been used to investigate the in situ kinetic properties of one of these enzymes, glyceraldehydephosphate dehydrogenase. Contrary to the conclusions of previous studies with the isolated enzyme in vitro, these measurements have shown that the enzyme is not rate determining for glycolytic flux in the human erythrocyte and that it is unlikely that it is bound to the cell membrane in situ. A study of ¹H/²H exchange between the C-2 positions of methyl labelled lactate molecules, catalysed by lactate dehydrogenase, has been used to investigate the in situ kinetic properties of this enzyme. Comparison of these properties with those it displays in vitro indicate that the free intracellular NAD(H) concentration in the erythrocyte is only approximately 10% of the total extractable concentration. A considerable fraction of the coenzyme must be bound, therefore, in the intact cell. This type of experiment should be widely applicable to a variety of tissues and possibly to different dehydrogenases. Theoretical aspects of bulk isotope exchange kinetics in multi-enzyme systems are examined and the effects of chemical flux, and of isotope effects, on the measurement of isotopic flux are considered. The advantages of the n.m.r. method over conventional radioactive tracer techniques are described. It is concluded that ¹H n.m.r. studies of ¹H/²H isotope exchange may be used to obtain information about the kinetic properties of enzymes in intact cellular systems. The technique should be a useful complement, therefore, to the currently more widely used n.m.r. methods employing the ³¹P and ¹³C nuclei and to other methods used for the non-invasive study of metabolism.

A quantitative understanding of metabolic control depends on a knowledge of the enzymes involved. The extrapolation of studies in vitro to the intact cell is controversial because the intracellular environment is relatively poorly characterised, particularly with respect to the interactions between weakly-associated enzymes. There is a clear need to study enzymes directly in the cell, yet there are few suitable techniques. Metabolites have been very successfully studied in cells by the non-invasive technique of nuclear magnetic resonance (NMR). NMR studies of enzymes in the cell have, however, been prevented by difficulties in assigning the resonances from the many proteins within the cell. A method for studying a specific enzyme in the cell has been developed, using Saccharomyces cerevisiae and phosphoglycerate kinase (PGK) as a model system. Using an inducible expression system, PGK was synthesised in the cell without significant synthesis of other proteins. With 5-fluorotryptophan in the growth medium, fluorine-labelled PGK was formed in situ. Fluorine is an excellent label for NMR since it is absent from most cells and has a high receptivity to NMR detection. ¹⁹ F NMR was used to study PGK in the intact cell. Comparisons with measurements in vitro showed that PGK was exposed to only a small fraction of the total intracellular [ADP], implying some form of compartmentalisation. The NMR relaxation properties observed in vivo and in vitro were compared with theoretical predictions. This showed that PGK was not part of a complex in the cell and that the viscosity of the cytoplasm, relative to water, was c. 4 at 30 °C. Fluorine-labelled pyruvate kinase and hexokinase have also been prepared; the spectra of these proteins in vitro are responsive to their ligands, and further work will study these proteins in vivo. NMR techniques were also applied to study the kinetics of PGK in the cell. PGK and GAPDH catalyse an ATP↔P_i exchange which is near-equilibrium in wild-type cells. ³¹P magnetisation transfer experiments in genetically manipulated cells showed that the reaction becomes unidirectional if the PGK activity is reduced by 95 %. Net flux is reduced by less than 30 %. In low-PGK cells, the ATP↔P_i exchange from oxidative phosphorylation can be isolated from that of glycolysis, facilitating direct measurements of the P:O ratio. In the cells studied, the P:O ratio was 2 to 3.

The effect of short, strong radiofrequency pulses on the nuclear spin system is examined. Providing the durations of the pulses are short with respect to coupling constants within the spin system, they may be described by simple rotation operators which are exponential functions of the angular momentum operators. Operator algebra can be used to define the interaction of such pulses with the spin system, and the mutual interaction of a sequence of pulses. The case of a simple coupled spin system is examined in detail and it is found that a vector model can be used to describe the motion of the expectation values of the observables. This model also allows treatment of such 'non-classical 1 effects as coherence transfer and multiple-quantum coherence. The proposal is also made that certain types of pulse imperfection may be compensated by using specially constructed sequences of small numbers of pulses, which are termed 'composite pulses'. Their compensatory action is illustrated by computer simulation, and by experimental results. In the case of certain symmetrical composite pulses, operator algebra can be used to understand their overall effect in the presence of pulse imperfections, suggesting their use in such critical applications as multiple spin echo trains. Another class of symmetrical composite pulses provides rotations by arbitrary angles around the z-axis of the rotating reference frame, and is expected to be of use in multiple-quantum spectroscopy.

This thesis describes the application of a wide variety of data processing methods, in particular the Maximum Entropy Method (MEM), to data from Nuclear Magnetic Resonance (NMR) experiments. Chapter 1 provides a brief introduction to NMR and to data processing, which is developed in chapter 2. NMR is described in terms of the classical model due to Bloch, and the principles of conventional (Fourier transform) data processing developed. This is followed by a description of less conventional techniques. The MEM is derived on several grounds, and related to both Bayesian reasoning and Shannon information theory. Chapter 3 describes several methods of evaluating the quality of NMR spectra obtained by a variety of data processing techniques; the simple criterion of spectral appearance is shown to be completely unsatisfactory. A Monte Carlo method is described which allows several different techniques to be compared, and the relative advantages of Fourier transformation and the MEM are assessed. Chapter 4 describes in vivo NMR, particularly the application of the MEM to data from Phase Modulated Rotating Frame Imaging (PMRFI) experiments. In this case the conventional data processing is highly unsatisfactory, and MEM processing results in much clearer spectra. Chapter 5 describes the application of a range of techniques to the estimation and removal of splittings from NMR spectra. The various techniques are discussed using simple examples, and then applied to data from the amino acid iso-leucine. The thesis ends with five appendices which contain historical and philosophical notes, detailed calculations pertaining to PMRFI spectra, and a listing of the MEM computer program.

No description available.

The application of Nuclear Magnetic Resonance (NMR) spectroscopy in the determination of nutrition labeling component data (NLCD) was investigated, with the intent of using this methodology as a primary method to calibrate FTIR instrumentation for NLCD confirmation or screening on a routine basis. Unlike previous NMR studies, this work used three strategies to attain accuracy and reproducibility of NLCD through: (i) appropriate setting of operational parameters for spectral acquisition; (ii) resonance selection by optimizing the signal in proportion to the nuclei population and (iii) integration of resonances by pre-defined fixed chemical shift ranges. Both of 13C NMR spectra and 1H NMR spectra were shown to provide robust and acceptable results on the condition of appropriate acquisition of spectra for quantization purposes and the adoption of standard procedures for spectral processing, integration and calculation purposes. A quantitative approach of NLCD including trans content was determined by the interpretation resonance signals of 13C's and 1H's from methylene groups presented in triglyceride complex of fats and oils. An alternative method based on partial-least-squares (PLS) calibrations was provided as well, the latter proved to be especially useful in dealing with overlapping bands frequently found in 1H spectra. With the diagnostic provided by PLS, the trans and cis signals were shown to be separated in 1H spectra. It is the premise for the trans fat determination based on 1H spectra. Unit conversion from mole to weight % was addressed and a solution was developed based on NMR data per se, without significant assumptions. Validation involving the analysis of three different lipid types (model triacylglycerols, refined and hydrogenated oils) demonstrated that NMR predictions of NLCD were in good agreement with those results either from samples' actual values as well as those obtained using GC and FTIR predictions. Thus with appropriate integration of instrumentation, software and spectral processing accessories, both 13C and 1H NMR can determine NLCD, but with the capability to determine trans, 1H NMR is more practical than 13C NMR due to its much shorter spectral acquisition time. Thus NMR can serve as a primary method for the calibration of FTIR instrumentation, a practical instrumental method for routine NLCD determination and screening.

In this dissertation, the intermolecular interactions between soil organic matter (SOM) and organofluorine compounds have been studied at the molecular-level using Nuclear Magnetic Resonance (NMR) spectroscopy. NMR probes the local magnetic environment surrounding atomic nuclei, and is uniquely capable as an analytical tool to probe molecular environments in complex disordered materials, such as soils. Several NMR techniques were employed in this work, including Pulse Field Gradient (PFG)-NMR based diffusion measurements, solid-state cross-polarization (CP), saturation transfer difference (STD) spectroscopy, and reverse-heteronuclear saturation transfer difference (RHSTD) spectroscopy. Using organofluorine compounds as molecular probes, xenobiotic interactions with SOM were studied. Using 1H{19F} RHSTD, the interaction sites in humic acid for organofluorine compounds were identified by direct molecular-level methods. Protein and lignin were identified as major binding sites, with different preferences exhibited for these sites by dissimilar organofluorine compounds: aromatic organofluorine compounds display varied preference for aromatic humic acid sites while perfluorooctanoic acid exhibits near total selectivity for protein-derived binding sites. The mechanisms underlying these preferences were probed in the solution state. Using crucial knowledge from the humic acid studies, a detailed molecular-level investigation of xenobiotic interactions in an intact and unmodified whole soil was made possible. A direct and in situ elucidation of the components in soil organic matter that interact with small organofluorine xenobiotic molecules has been presented, allowing, for the first time, resolution of multiple interactions occurring for xenobiotics simultaneously at different sites within a whole soil.