The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.

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Nitrification, a key step in biological nitrogen removal processes, is the oxidation of ammonia into nitrate performed by ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) under aerobic condition. Researchers have focused on factors affecting the performance of nitrification for decades, but the inorganic carbon limitation on nitrification had been neglected. However, the increase in nitrogen in wastewater has increased the need to evaluate and improve our understanding of this limitation. In a previous research, the hypothesis that different inorganic carbon concentrations would enrich different AOB populations has been examined. In this study, the focus was on the effect of inorganic carbon concentration on NOB, which has a close relationship with AOB. Two 5L lab–scale continuous–flow stirred tank reactors (CSTR) were operated to evaluate the nitrification performance and microbial ecology of nitrifier populations acclimated under inorganic carbon sufficient (high–IC) and limited (low–IC) conditions for approximately 700 days. During the operation period, both bioreactors were able to maintain satisfactory nitrification efficiency higher than 95% at an influent ammonium concentration of 250 mg–N/L. Nitrate was the major end product and no significant nitrite accumulation was observed. To evaluate the effects of inorganic carbon on NOB community structures, cloning/sequencing and real–time PCR were applied to target and quantify the two common NOB genera, Nitrospira and Nitrobacter, as no molecular probe targeting all known NOB is available presently. The results showed that these two genera were both found in the two reactors. Nitrospira was the dominant NOB population in the high–IC bioreactor, while Nitrobacter was dominant in the low–IC one after one year acclimation. Kinetic analysis revealed that NOB enriched in the two reactors have different kinetic performances. However, IC concentration did not show a significant impact on the nitrite oxidizing kinetics of NOB in the batch tests.