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This thesis describes the examination of novel block copolymers that contain Ru(bpy)32+ complexes incorporated into one block of diblock copolymers made by ROMP. With the intent of exploring the potential usefulness of these interesting materials in applications such as light-harvesting and sensing, a systematic study of the solution self-assembly, luminescence properties, and the ability of the metal complex to engage in electron and energy transfer reactions has been conducted. / The solution self-assembly of block copolymers that contain Ru(bpy) 32+ complexes was examined first. Using a series of these block copolymers, a detailed study of the effects of block length, block ratio, polymer concentration and solution conditions on the copolymer self-assembly is presented. Using TEM, a number of morphologies were reproducibly observed including star micelles, large compound micelles, tubules, and interestingly, vesicles. These structures all contain the metal complex Ru(bpy)3 2+ within their core domains. / The luminescence properties of two block copolymers containing Ru(bpy) 32+ were examined: one polymer self-assembled into star micelles, the other into vesicles. Comparison of the unassembled polymer chains and the self-assembled polymers indicated that self-assembly, and confinement of the Ru(bpy)32+ complexes into the core domains of the aggregates, did not seriously adversely affect the luminescence properties of the metal complex. Measurement of the luminescence lifetime decay of the polymers suggested that energy migration occurred among the metal complexes along the polymer chain. The ability of the metal complexes within self-assembled structures to participate in electron transfer reactions with small molecules was also explored. It was found that from within the core domains of self-assembled structures, the Ru(bpy)32+ complexes could still engage in electron transfer reactions with molecules on the outsides or the insides of the aggregates, likely a result of energy migration. / The ability of Ru(bpy)32+ complexes within the cores of micelles to participate in energy transfer was explored. Micelles were formed in aqueous solutions using polymers that possessed both the metal complex and a water-soluble block. Several methods were attempted to encapsulate two molecules, a derivative of coumarin 2 and an Os(bpy)3 2+-based molecule, inside these micelles. It was observed that Ru(bpy) 32+ could act as an energy acceptor from the coumarin derivative, and could act as an energy donor to the osmium-based complex. Encapsulation of the small molecules greatly enhanced the efficiency of energy transfer, by non-covalently bringing the small molecules in close proximity to the Ru(bpy)32+ complexes. / Polymers were synthesized that contained a Ru(bpy)3 2+-based block and were terminated with the molecular recognition unit biotin. These polymers, upon self-assembly, formed micelles with biotin groups on their periphery. The addition of the protein streptavidin, which has a strong binding affinity for biotin, resulted in the aggregation of the self-assembled structures. This established the potential for self-assembled metal-containing aggregates to form higher-order structures. / Early work is presented in Appendix A involving block copolymers that contain hydrogen-bonding groups. Several methods were attempted to elucidate the solution morphologies of these polymers, namely IR, 1H NMR, DLS, and pyrene fluorescence. The transition of this initial work to polymers that contain the Ru(bpy)32+ complex is also described.

The oligomerisation of protein subunits is an area of much research interest, in particular the relationship to protein function. In the last decade, the potential to control the interactions involved in order to design constructs with tuneable oligomeric properties in vitro has been pursued. The subject of this thesis is the quaternary structure of members of the peroxiredoxin family, which have been seen to assume an intriguing array of organisations. Human Peroxiredoxin 3 (HsPrx3) and Mycobacterium tuberculosis alkyl hydroperoxide reductase (MtAhpE) catalyse the detoxification of reactive species, preferentially hydrogen peroxide and peroxynitrite respectively, and form an essential part of the antioxidant defence system. As well as their biomedical interest, the ability of these proteins to form organised supramolecular assemblies makes them of interest in protein nanotechnology. The work described focusses on the elucidation of the quaternary structure of both proteins, resolving previous debates about their oligomeric state. The factors influencing oligomerisation were examined through biophysical characterisation in different conditions, using solution techniques including chromatography, light and X-ray scattering, and electron microscopy. The insight gained, along with analysis of the protein-protein interfaces, was used to alter the quaternary structure through site-directed mutagenesis. This resulted in a level of control over the protein’s oligomeric state to be achieved, and novel structures with potential applications in nanotechnology to be generated. The activity of the non-native structures was also assessed, to begin to unravel the relationship between peroxiredoxin quaternary structure to enzyme activity. The formation and structure of very high molecular weight complexes of HsPrx3 were explored using electron microscopy. The first high resolution structural data for such a complex is presented, analysis of which allowed the theory of an assembly mechanism to be proposed.

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Multiple sclerosis is a progressive autoimmune disease where myelin is gradually stripped from axons. Axon degeneration inevitably follows protracted myelin loss ultimately leading to irreversible neurological decline. To better understand the cellular mechanisms associated with the axon loss phase of the disease, spinal cord axons from the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis were examined using correlated in vivo time-lapse microscopy and serial section transmission electron microscopic (ssTEM) reconstruction. A novel technique, termed near infrared burning (NIRB), was developed that took advantage of a femtosecond-pulsed mode locked laser’s ability to create photoconvertable fiducial markers for routine identification of previously imaged axons for ssTEM reconstruction. This combination of imaging techniques revealed the subcellular milieu that underlies axon degeneration at both the light and electron microscopic level. In particular, paranodal regions of axons in EAE animals contained a significantly higher population of mitochondria with large rounded, electron lucid, vesiculated mitochondria with unorganized cristae compared to controls. This effect was largely restricted to the paranodal region and was not always associated with direct immune cell interaction or myelin loss. Together, these results suggest a novel mechanism for axon degeneration that is not only focal in nature, but decoupled with myelin loss in the EAE animal model of multiple sclerosis. / Department of Biology

Viruses and their morphology have been detected and studied with electron microscopy (EM) since the end of the 1930s. The technique has been vital for the discovery of new viruses and in establishing the virus taxonomy. Today, electron microscopy is an important technique in clinical diagnostics. It both serves as a routine diagnostic technique as well as an essential tool for detecting infectious agents in new and unusual disease outbreaks. The technique does not depend on virus specific targets and can therefore detect any virus present in the sample. New or reemerging viruses can be detected in EM images while being unrecognizable by molecular methods. One problem with diagnostic EM is its high dependency on experts performing the analysis. Another problematic circumstance is that the EM facilities capable of handling the most dangerous pathogens are few, and decreasing in number. This thesis addresses these shortcomings with diagnostic EM by proposing image analysis methods mimicking the actions of an expert operating the microscope. The methods cover strategies for automatic image acquisition, segmentation of possible virus particles, as well as methods for extracting characteristic properties from the particles enabling virus identification. One discriminative property of viruses is their surface morphology or texture in the EM images. Describing texture in digital images is an important part of this thesis. Viruses show up in an arbitrary orientation in the TEM images, making rotation invariant texture description important. Rotation invariance and noise robustness are evaluated for several texture descriptors in the thesis. Three new texture datasets are introduced to facilitate these evaluations. Invariant features and generalization performance in texture recognition are also addressed in a more general context. The work presented in this thesis has been part of the project Panvirshield, aiming for an automatic diagnostic system for viral pathogens using EM. The work is also part of the miniTEM project where a new desktop low-voltage electron microscope is developed with the aspiration to become an easy to use system reaching high levels of automation for clinical tissue sections, viruses and other nano-sized particles.

Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates. / Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013

Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates. / Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013

In order to improve understanding of diesel soot oxidation, an experimental rig was designed and set up, in which the soot oxidation conditions, such as temperature, oxygen partial pressure, and CO2 partial pressure, could be varied independently of each other. The oxidizing gas flow in the oxidizer was under laminar condition. This test rig comprised a naturally-aspirated single cylinder engine which acted as the soot generator, and a separate premixed oxidation burner system in which soot extracted from the engine was oxidized under controlled conditions. Diesel soot was extracted from the engine exhaust pipe and from the engine pre-combustion chamber, and the soot-laden gas was then conveyed to the burner where it was oxidized. The burner was positioned vertically and it had a flat flame whose thickness was only a few millimetres. The hot gases from the flame flew upwards through a quartz transparent tube which acted as the soot oxidation duct. The soot-laden gas from the engine was premixed with the feedgas (itself a premixed mixture of methane, air, oxygen, and nitrogen) to the burner. The soot particles passed vertically through the flame front and continued burning in the post-flame gas flowing through the quartz tube oxidation duct. The oxygen concentration and temperature of the post-flame soot oxidation gas were controllable by adjusting the flowrate and composition of the burner feedgas. Diesel soot particles were sampled at different heights along the centreline of the quartz tube above the burner. Profiles of oxygen concentration, temperature, and soot particle velocity in the oxidation zone were thus measured. Morphology and size distributions of the sampled diesel soot particles were analyzed by means of Transmission Electron Microscopy (TEM) and a computer software called ImagePro Plus. Subsequently, the specific surface oxidation rates of the soot particles were worked out based on soot particle size distributions. The TEM micrographs obtained in this study showed that the diesel soot agglomerates existed in forms of clusters and chains, each containing between a small number and thousands of individual, mostly spherical tiny particles. Of order 97% of the individual spherical particles (spherules) had a size range from 10 to 80 nm. Occasionally, individual spherules of about 150 nm in diameter could be observed. The diesel soot particles sampled from the pre-chamber of the engine had different size distributions from those sampled from the exhaust of the engine, indicating that the soot underwent an oxidation process in the combustion chamber. Soot oxidation experiments were performed in the burner post-flame gas under oxygen partial pressures ranging from 0.010 to 0.050 atm and temperatures from 1520 to 1820 K. The test results showed that the oxidation rates of the diesel soot extracted from the diesel engine were generally lower than those predicted by the well-known Nagle and Strickland-Constable formula; however, the measured oxidation rates were higher than the predictions made with another well-known formula - the Lee formula. The soot extracted from the engine pre-chamber appeared not to oxidize as fast as the soot extracted from the exhaust of the engine. CO2 gas injection to the post-flame oxidation gas at constant oxygen partial pressure and oxidation temperature seemed to have accelerated the diesel soot oxidation rate. Based on the experimental results of this study and the results of other researchers, modifications to the Nagle and Strickland-Constable formula and to the Lee formula were accomplished. Also, an empirical expression, as an alternative to semi-empirical formulae, was worked out and presented in the thesis.