Graduation date: 1980

Bacterial infection is a leading cause of morbidity and mortality arising from platelet transfusions (1, 2). Storage of platelet products at room temperature (20 to 24ºC) provides ideal conditions for bacterial proliferation (1, 3-6). Furthermore, platelets are stored in plasma containing bioavailable iron that bacteria require to survive (7). Thus we hypothesize that the inclusion of iron chelators will bind and remove iron, thereby inhibiting bacterial growth in both culture medium and platelet concentrates. Additionally, we hypothesize that residual red blood cells (RBCs) in platelet units may contribute bioavailable iron that promotes bacterial growth. To test these hypotheses, we first assessed growth of Staphylococcus epidermidis in culture medium after treatment with the iron chelators deferoxamine (DFO) or phytic acid. DFO significantly inhibited bacterial growth in a dose dependent manner (p < 0.009). Conversely, phytate only inhibited bacterial growth at concentrations ≥ 100 mM (p < 0.001); at ≤ 5 mM, phytate supplied S. epidermidis with additional nutrients and significantly promoted growth (p < 0.001). Subsequently, we monitored the change in RBCs over time. Hemolysis, methemoglobin, and iron levels all significantly increased over the 7-day storage period (p < 0.001) releasing bioavailable iron. Indeed, we found that S. epidermidis growth in iron-poor medium drastically increased with the addition of RBCs, thus supporting our second hypothesis. Surprisingly, the inclusion of DFO in minimal medium did not demonstrate a bacteriostatic effect in the presence of RBCs. The inhibitory effect of DFO was likely overcome by iron released from the elevated methemoglobin levels arising from the direct interaction of DFO with hemoglobin. Previous studies demonstrate that methemoglobin releases iron more quickly than normal hemoglobin (8). Lastly, we evaluated the effect of DFO on microbial growth in platelet concentrates using the BacT/ALERT system. The presence of DFO significantly inhibited S. epidermidis growth in buffy coat platelets in a dose dependent manner (p < 0.001). With these findings, the inclusion of iron chelators is a promising approach to preventing transfusion-transmitted bacterial infection and providing patients with a safer platelet product.

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of harmful hydroxyl-free radicals. Ceruloplasmin is the major multicopper ferroxidase in blood; however, hephaestin, a membrane-bound ceruloplasmin homolog, has been implicated in the export of iron from duodenal enterocytes into blood. These ferroxidases supply transferrin, the iron-carrier protein in plasma, with Fe(III). Transferrin circulates through blood and delivers iron to cells via the transferrin receptor pathway. Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between the ferroxidase and transferrin. In Chapter 3, the putative interaction of transferrin with ceruloplasmin and a soluble form of recombinant hephaestin was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance, a stable interaction between the two proteins was not detected. The lack of interaction between hephaestin and transferrin prompted the investigation into the localization of hephaestin in the human small intestine. Hephaestin has been reported to have both intracellular and extracellular locations in murine tissue. In the Appendix, the location of hephaestin in human tissue was investigated using a novel polyclonal antibody. Hephaestin was localized to the basolateral membrane and an intracellular location of the enterocyte, as well as a novel location in the myenteric plexus of the duodenum. The delivery of iron to cells via the transferrin receptor pathway is well established; however, little is known about the interaction of transferrin with the transferrin receptor at the molecular level. In Chapters 4 and 5, surface plasmon resonance was employed to further characterize the binding event between transferrin and the transferrin receptor. It was found that mutations affecting iron release in transferrin did not impact receptor binding. However, when N-lobe residues predicted to form contacts with the transferrin receptor were targeted, significant changes in the transferrin receptor binding kinetics and affinity were observed.

The subject of this thesis is the gas sensing properties of sensors. / Thesis (PhD)--University of South Australia, 2001.

Iron oxide is a crucial hydrophilic mineral in the minerals, metals and materials industries. Colloidal iron oxide dispersions have proven difficult to dewater to high solid-loading during routine hydometallurgical processes. Despite this, there are a limited number of studies integrating interfacial chemistry, flocculation and dewatering behaviour of iron oxide in the literature. / In this study, fundamental investigations of the principles underpinning hematite pulp dewatering behaviour were examined to determine the effectiveness of four polyacrylamide ploymers (home-polymer, PAM N, anionic carboxylate substituted copolymer, PAM A, anionic sulphonate substituted copolymer, PAM S, and cationic trimethyl amino ethyl substituted copolymer, PAM C) as flocculants and four metal salts, Mn(NO), MnSO, and MnC1and KSO) as coagulants. Effects of flocculant structure type and concentration, metal salt type and pH on the surface chemistry, pulp rheology and the dewaterability of dispersions were considered. Experiments were conducted at the isoelectric point (iep-pH 8.5), below (pH 6) and above (pH 11) the iep of the hematite to investigate particle charge effect in the presence and absence of the coagulants and/or flocculants. / Investigations into the polymer adsorption mechanisms conducted using infrared spectroscopy found PAM A to chemically adsorb onto hematite particles via bidentate chelation on either side of the iep, and via monodentate chelation at the iep. These bonding mechanisms were found to influence the pulp particle interactions (rheology) and hence dewatering behaviour. Hydrogen bonding also occurred between PAM A and PAM N and the hematite particles. Spectroscopic evidence was also provided, for the first time, of the partial hydrolysis of non-ionic polyacrylamide at high pH. / Flocculation in general was found to be most effective when the polymer was of similar charge to the hematite particles. Settling rates of over 100 m/h were achieved at and below the iep after flocculation with the charged polymers, whilst consolidation was generally unaffected by flocculation at any pH. Bridging mechanisms between polymers of similar charge to the particles was most effective. Electrostatic and charge patch flocculation mechanisms prevailing between oppositely charged polymer and hematite particles provided less-improved dewatering. / Surface chemistry was greatly influenced by the anionic polymers at and below the iep of hematite. Addition of sulphate ions caused pronounced particle surface charge suppression with significant zeta potential reduction below the iep. The sulphate ion also facilitated PAM A adsorption above the iep which led to a significant improvement in settling rate. Dewatering of flocculated pulps was more efficient at and below the iep of hematite, with the negatively charged dispersion at high pH generally comprising a turbid supernatant. Manganese salts in synergy with PAM A significantly enhanced settling rates at and below the iep, however, the effect was subtle when compared to the addition of the sulphate ion. Application of mechanical shear to the pre-sedimented pulps produced outstanding improvement in particle consolidation, with up to 20 wt% increase in solid loading observed. / Thesis (PhDApSc(MineralsandMaterials))--University of South Australia, 2008.

Thesis (PhD)--University of South Australia, 1997

Superoxide, a reduced form of dioxygen is produced in natural waters from abiotic thermal processes such as iron(II) oxygenation photochemically, and biologically in natural waters. Superoxide is highly redox-active due to the presence of an unpaired electron and plays an important role in the geochemical cycling of trace metals and degradation of organic pollutants in natural waters. It also has a significant effect on the physiology of aquatic microorganisms and has been implicated in the growth of toxic microalga in coastal areas of Japan, Australia and Canada. The generation of superoxide by both biotic and photochemical pathways is described in this thesis and attention given to selected reactions of superoxide in simulated natural waters. Particular attention in this work has been given to the interaction of superoxide and iron and the impact of this interaction on iron availability to Chattonella marina, a red-tide phytoplankton frequently associated with fish-mortalities in Australia and Japan. Superoxide production from both phototchemical and biological sources is measured using a highly sensitive chemiluminescence technique and the effect of superoxide production on iron transformation under various environmental conditions is investigated by employing spectrophotometric techniques with high sensitivity and temporal resolution. The intermediacy of superoxide in iron acquisition mechanism by C. marina is also investigated using a radioisotope labeling technique. Our experimental results show that superoxide in natural waters is produced via reduction of dioxygen. The reduction of dioxygen takes place by transfer of electrons by the photo-excited quinone moieties present in natural organic matter or by reductases located on the outer cell membrane of organisms' surface. The experimental results suggest that the fate of iron in marine waters is closely related to the superoxidedioxygen redox couple. Superoxide can reduce a wide range of organically complexed iron(III) species to the more soluble iron(II) redox state, thus affecting its bioavailability. A simple kinetic model for redox-cycling of iron in the presence of superoxide is developed. In addition, by coupling the model for redox-cycling of iron with the forms of iron acquired by C. marina, a generalized mathematical model for iron acquisition is presented which satisfactorily describes all results obtained.

Integral membrane proteins perform a wide range of biological processes, including respiration, signal transduction and molecular transport. Structural information is necessary for a full understanding of the mechanisms by which integral membrane proteins work. Ferrous iron transporter protein B (FeoB) is an integral membrane protein of Escherichia coli which is considered to transport ferrous iron in to bacteria. But there are no definite proofs or clear indications of the precise mechanism of ferrous transport. By expressing and crystallizing the G-protein domain (FeoGP) and FeoB, it will be helpful to know about the iron transport system. In order to express FeoB and FeoGP, expression vector pFeoB (FeoB in pGEX-4T-1) and pFeoGP (FeoB in pGEX-4T-1) were made. FeoB and FeoGP proteins were expressed and purified. Using vapour diffusion method crystallization trials of FeoB and FeoGP were done. Crystals of FeoGP are observed and no crystal formation for FeoB. Native crystals of FeoGP diffracted to 2.2 ?? resolution, and mant-GMPPNP crystals to 2.6 ??. Preliminary data processing indicate space group P212121 for native crystals, with cell dimensions 46 x 119 x 146 ??. The data set is 100% complete, Rmerge 0.08, and I/ ?? 3.2.

Zero-valent iron (ZVI) has been successfully used for the degradation of a wide range of organic contaminants in groundwaters in recent years. The rate of degradation of contaminants by ZVI may be enhanced by use of nanoscale zero valent iron (nZVI) particles which possess higher surface area than the more widely used granular materials. However, the most widely used method of producing nZVI involves the reduction of FeIII by sodium borohydride is expensive. Dithionite can be used to reduce Fe(II) and produce cost effective nZVI under conditions of high pH and in the absence of oxygen. The efficiency of trichloroethylene (TCE) degradation using dithionite nZVI particles (nZVIS2O4) is similar to that of the conventional borohydride particles (nZVIBH4). Oxidation of benzoic acid using the nZVIS2O4 particles results in different byproducts than those produced when nZVIBH4 particles are used. The high concentration of phenol compared to hydroxybenzoic acids suggests that OH addition is not the primary oxidation pathway when one is using the nZVIS2O4 particles. It is proposed that sulfate radicals (SO4−) are produced as a result of hydroxyl radical attack on the sulfite matrix surrounding the nZVIS2O4 particles, with these radicals oxidizing benzoic acid via electron transfer reactions rather than addition reactions. Low yields of oxidants limit the application of nZVI. It has recently been demonstrated that nZVI oxidative efficiency can be enhanced in presence of ethlylendiaminetetraacetic acid (EDTA). Additional insight into the nZVI-mediated process has been obtained from comparative studies of degradation of benzoic acid by nZVI particles and Fenton reagents in the absence and presence of EDTA at different pH. The efficiency of nZVI degradation is significantly hindered by the rapid aggregation of the iron nanoparticles, which may result in a decrease in available reactive surface area. These effects of aggregation can be overcome by surface modification through adsorption of capping agents which provide steric and electrosteric repulsive interactions between particles. Several high molecular weight (HMW) organic polymers have been used for preventing agglomeration of nZVI particles, such as water soluble starch, sodium carboxymethyl cellulose (CMC) and alginate. The degradation capabilities of different functionalized nZVIS2O4 particle were investigated. Iron-based bimetallic particles in which metals such as Pd and Ni have been combined with Fe, have been found to both enhance rates of halogenated organic contaminants reduction and generate more fully dehalogenated products relative to unamended iron. The results presented in this thesis demonstrate that formation of bimetallic particles with nZVI formed from the more cost effective dithionite reduction of ferrous salts also results in dramatic enhancement in reducing ability. The oxidising ability of nZVIBH4 particles can be enhanced dramatically by addition of polyoxometallates (POMs), redox catalysts which result in enhanced production of hydrogen peroxide. The extent of enhancement is quantified by examination of the oxidation of formic acid (to CO2) and kinetic modelling of the results obtained used to investigate the mechanism of the POM-mediated oxidation process.

Large summertime proliferations (or blooms) of the noxious filamentous cyanobacterium Lyngbya majuscula have been observed in the coastal marine waters of Moreton Bay, Queensland. This photosynthetic organism is believed to have a high iron requirement and preliminary studies have shown that the presence of organically complexed iron stimulates growth. Since there is no evidence that Lyngbya produces siderophores to aid iron acquisition, it has been hypothesized that this organism may acquire iron via reduction of ferric complexes to the typically more labile, ferrous form. Both the phylogenetic diversity of the genus Lyngbya and the iron metabolism of L. majuscula are examined in this thesis. Software was developed to assist in the design of peR primers that targeted l6S rRNA, rpoB and Highly Iterated Palindrome (HIP) genetic structures and the subsequent phylogenetic analysis. The mechanism of iron acquisition by L. majuscula and the influence of organic complexation of iron were investigated using radioisotope and chemiluminescence-based techniques. Molecular techniques were also used to investigate the genetics of iron metabolism of L. majuscula. Results of the l6S rRNA analysis indicate that the morpho-genus Lyngbya encompasses a large genetic diversity within the cyanobacteria that is consistent with its reported metabolic and ecological diversity. Five discrete lineages comprised of organisms that fit the morophological definition of Lyngbya were discovered in this analysis. L. majuscula utilises endogenously-produced superoxide as a reductant of ferric complexes to produce the more labile ferrous forms. The nature of the organic complexes has been shown to determine the efficacy of this mechanism. A model of iron acquisition by these reductive processes was developed and was shown to generally describe all known methods of reduction-mediated iron acqUIsItIOn. Finally, the genetics of iron metabolism of L. majuscula was found to be consistent with the mechanism we propose, including the discovery of a component of a ferrous iron uptake mechanism.