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The work described in this thesis is concerned with the development of new applications of the photo-CIDNP (photochemically induced dynamic nuclear polarization) technique to aspects of protein structure and folding. Chapters 1 and 2 are introductory chapters; Chapter 1 describes the theoretical basis of the CIDNP phenomenon in terms of the underlying spin chemistry of the radical pair mechanism, while Chapter 2 presents the apparatus, photosensitizer and pulse sequences used, along with some important experimental considerations. Chapter 3 describes how ¹⁵N CIDNP can be used to probe the accessibility of tryptophan side-chains in both native and denatured states of proteins. The polarization of indole nitrogens in uniformly ¹⁵N labeled protein is detected in a two-dimensional ¹⁵N-¹ H NMR heteronuclear correlation experiment. Chapter 4 describes two new techniques offering considerable improvements in the quality of photo-CIDNP spectra of proteins. Both focus on the problem of progressive photo-degradation of the flavin dye and in both cases a larger number of scans can be accumulated before the flavin is exhausted than would otherwise be possible. In Chapter 5, the potential of stopped-flow photo-CIDNP spectroscopy for the study of protein folding is explored. Rapid dilution of denatured protein into a buffer solution is used to initiate a refolding process which is followed using short laser pulses to generate ¹H CIDNP in the side-chains of exposed aromatic residues. In Chapter 6, the field dependence of amino acid photo-CIDNP intensities is investigated using a stopped-flow CIDNP device that allows sample irradiation over a range of magnetic fields (0.1-7 T) within the bore of a 9.4 T NMR magnet and rapid transfer into the NMR tube for detection. Finally, in Chapter 7 two photo-CIDNP techniques that probe the exposure of aromatic residues in partially folded states are described. Both involve transfer of polarization to the native state for detection. One approach achieves this kinetically by rapid refolding, and the other involves monitoring exchange cross peaks in a two-dimensional CIDNP spectrum under conditions where the two states are interconverting.

Particulate adsorption by Mucor flavus and Neurospora crassa is a physical property of the cell wall, independent of both cellular metabolism and the production of extracellular polymers. Initial attractive forces responsible for particulate adsorption by N. crassa are mainly electrostatic in nature and this mechanism probably holds for M. flavus also. The outer glucan layer of the cell wall of N. crassa, although able to adsorb particulates, was not as efficient as the underlying protein layer at particle adsorption. Young, growing mycelium generally adsorbs the , -, largest amount of particles, due to the continued production of adsorption sites and the entrapment of particulates by hyphae. Factors increasing adsorption include nutrient starvation of mycelium and incubation with low concentrations of magnesium ions. Relatively high concentrations of mercury and copper ions decrease adsorption perhaps due to the precipitation and adsorption of the corresponding metal sulphides on the mycelial surface thereby effectively physically interfering with further particulate adsorption. Optimum conditions for adsorption are a temperature of 250C and a slightly acid pH value. Also, small particles are more readily adsorbed than large particles. Mucor flavus can adsorb clays and this ability may be used to treat industrial effluents which contain large amounts of clay minerals. An acid pH and a temperature of 250C are optimum conditions for clay adsorption by the fungus. Low concentrations of montmorillonite and kaolinite increase biomass production by Aspergillus niger in submerged culture, due to the inhibition of pellet formation by the fungus. The clays cause A. niger to grow in a more filamentous form and presumably would affect other fungi in a similar way. The use of clays to control fungal morphology may be important in several industrial fermentations. Low concentrations of the fungicide thiram stimulated the growth of Aspergillus niger in the presence of montmorillonite. Immobilization of fungi by magnetic means is possible due to their ability to adsorb magnetite. This method could also be used to remove fungi from fermentation media as an alternative to filtration or centrifugation. Even though older hyphae of Penicillium chrysogenum are unable to adsorb magnetite, this fungus can still be magnetically immobilized if it is grown from a spore suspension in the presence of magnetite. Either the spores or young hyphae of this fungus adsorb magnetite, producing pellets with magnetic properties. Magnetite adsorption occurs optimally at a temperature of 250C and is constant over a wide range of pH values. Waste mycelium of A. niger from the surface fermentation method of citric acid production can be magnetically removed from solution after adsorbing magnetite. Dilute solutions of sodium hydroxide and sodium bicarbonate desorbed magnetite attached to mycelial surfaces. Silver is accumulated selectively by A. niger waste mycelium produced by the surface fermentation method of citric acid production. The process is rapid, maximum uptake occurring twenty minutes after initial exposure of the mycelium to a silver solution. Silver accumulation by the mycelium is relatively insensitive to changes in pH and temperature, a slight decrease in uptake only occurring at a temperature of 800C. Dilute solutions of H2S04 and HNO3 desorb silver from the mycelial surface. However, this process is relatively inefficient and more effective desorbents need to be found to make the silver accumulation process economically viable.

Thesis (PhD)--University of South Australia, 2004.

The surface stoichiometry of five samples, two semiconductors (GaAs and InP) and three minerals (galena (PbS), pyrite (FeS2) and sphalerite (Zn, Fe) S), has been determined by a range of surface analysis techniques: ToF-SIMS, XPS, RBS, NRA and PIXE. The strengths and limitations of the five surface and interface analytical techniques have also been assessed by challenging each technique with the above mentioned samples. As a result, a strategy tree for the quantitative surface analysis of real mixed mineral systems has been developed for novice investigators as an operational guide. ToF-SIMS is a technique able to detect species from hydrogen to very complex organic modules with very high sensitivity. SIMS is generally not a quantitative technique due primarily to the matrix effect, i.e. the strong dependence of secondary ion yields on the chemical composition of the sample. However, for well defined systema, quantitative analyses are possible if suitable calibration standards are accessible. / In this study, a preliminary RSF (Relative Sensitivity Factor) study has been conducted on five samples to give RSFs of the major constituents of each sample. XPS is broadly recognised as a surface analysis technique with the ability of determining the elemental composition near the surface region as well as chemical information, but the depth resolution of XPS is poor. RBS is capable of revealing the concentration depth profiles within the sample without destroying the solid surface. RBS is also considered a good quantitative analytical technique. The main disadvantages of RBS are the poor surface sensitivity and the low efficiency in detecting light elements in a high Z substrate. PIXE is a non-destructive multi-element analytical technique based on X-ray spectroscopy and good at detecting medium Z elements (Z~/= 20~30). By using L X-ray lines instead of K X-ray lines, PIXE is also able to detect high Z (>40) elements. However, due to the very low detection efficiency of Si (Li) detectors (typical PIXE detectors) at low X-ray energies, elements lighter than sodium can not be analysed. Another major shortcoming of PIXE is the lack of depth information. Various surface textures of pyrite have been studied in order to investigate the influence of surface conditions on the quantitative results for the individual techniques utilised in this study and the activation induced chemical alteration on pyrite surfaces has also been studied in detail. By comparing the results from these surface analysis techniques, a good understanding of the elemental composition of the mixed minerals has been achieved. / Thesis (PhD)--University of South Australia, 2004.