The intestinal protozoan parasite, Giardia duodena/is, is a major cause of diarrhoeal disease world wide and is one of the most common parasites of man. Resistance to antigiardial drugs have been reported both clinically and in vitro, thereby making the treatment of this medically significant pathogen an area of continuing research. G. duodenalis is the best characterised example of the archezoa, the earliest eukaryotes, which predate fusion with purple non-sulphur bacteria in the genesis of mitochondria. Giardia is dependent on fermentative catabolism and substrate level phosphorylation for ATP generation. While lacking a cytochrome mediated electron transport system in the reduction of 02 to water by cytochrome oxidase, this microaerophilic protozoan has been shown to actively consume oxygen at rates comparable with the aerobic protozoa. This observation raises issues concerning electron transport, xenobiotic detoxification and the maintenance of an optimum intracellular thiol redox ratio in G. duodena/is. In this context drug activation and resistance can then be investigated.G. duodenalis contains cysteine as its major low molecular weight thiol instead of the nearly ubiquitous glutathione (GSH) and its analogues (Brown et al., 1993). Trypanothione, a GSH conjugate predominating in the trypanosomatids, was also absent from Giardia. Apart from cysteine, significant levels of sulphite, thioglycolic acid and coenzyme A were also detected. In Giardia intracellular cysteine is maintained in its reduced state by a broad-range thioredoxin-like disulphide reductase, thereby assuming a role analogous to the GSH system of most other eukaryotes in the maintenance of an optimum intracellular thiol redox ratio (Brown et al., 1995c). Pure G. duodenalis disulphide reductase is a dimeric flavoprotein containing 1 mol FAD per subunit (Mr- 35 kDa) which catalyses the NADPH (Km = 8µM) -dependent reduction of 5,5'-dithio­ bis(2-nitrobenzoic acid) to thionitrobenzoate without the addition of an intermediate electron transport protein. However, a candidate electron transport protein, which enhances disulphide reductase activity 6 fold, was partially purified from Giardia suggesting that G. duodenalis contains a thioredoxin-reductase like system consisting of a disulphide reductase and an intermediate electron transport protein (analogous to thioredoxin). Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are consistent with its being a member of the bacterial thioredoxin reductase class of disulphide reductases (Brown et al., 1995c) and explains the so called "glutathione-induced thiol-reduct.ase activity" previously observed in G. duodenalis (Smith et al., 1988). This is the first report of a thioredoxin-like disulphide reductase to be purified from the anaerobic protozoa, and it appears to be the major disulphide reducing system in Giardia (Brown et al., 1995c).Spectrophotometric and in situ nondenaturing polyacrylamide gel analysis of superoxide dismut.ase (SOD), catalase, and peroxidase revealed that G. duodenalis does not contain any of the conventional mechanisms of reactive oxygen species (ROS) detoxification (Brown et al., 1995b). Furthermore, SOD, catalase and peroxidase were not detected in drug-(furazolidone and metronidazole) and peroxide- (H2O2 and t­ butylhydroperoxide) resistant Giardia lines, nor in parasites cultured under conditions favouring their expression. The same technique readily detected at least one of the conventional enzymes of oxidative stress in the bacterium Escherichia coli and in the protozoans Trichomonas vaginalis and Tritrichomonas foetus.Instead of the conventional mechanisms of oxidative stress management, Giardia contains an NADH oxidase, which tetravalently reduces oxygen to water, thereby abrogating the requirement of SOD catalase and peroxidase. The oxidase is complemented by a membrane-associated NADH peroxidase (Brown et al., 1995a; Brown et al., 1995b). Pure G. duodenalis NADH oxidase is a monomeric flavoprotein (M1- 46 kDa) containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide and was able to utilise both NADH (Km = 4µM) and NADPH (Km = 16µM) as electron donors (Brown et a1., 1995b). Inhibitor studies suggested that NADH oxidase contained a thiol group as part of the active site. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors, utilising both NADH and NADPH, which suggests that NADH oxidase may play a role as a mixed-function oxidase depending upon environmental oxygen tension. NADH oxidase also catalysed the reduction of the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR studies. This is the first report of furazo1idone reduction in the anaerobic protozoa (Brown et al., 1995b). Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)+ oxidoreduct.ase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H.The N-terminal sequence of G. duodenalis NADH oxidase showed high homology with other sequenced NADH oxidases from Streptococcus faecalis and Serpulina hyodysenteriae which are both intestinal anaerobic bacteria. Indeed, N­ tenninal sequence data taken together with the enzymatic, physical and spectral properties showed that G. duodenalis NADH oxidase has striking similarities with the NADH oxidases from the anaerobic bacteria (Brown et al., 1995b). The 02 scavenging NADH oxidase may function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, in the maintenance of an optimum intracellular redox ratio, and explains the previously observed futile "respiration" in Giardia. A candidate NADH oxidase gene was located on a 4.8 kb Bam HI cleaved genomic DNA fragment in hybridisation studies using an oligonucleotide designed around the NADH oxidase N­ tenninal sequence. To our knowledge NADH oxidase and disulphide reductase are the first flavoenzymes to be purified from Giardia.Giardia demonstrated a capacity to readily develop resistance to rare earth oxyanions. A conventional mechanism of oxyanion resistance in E. coli involving an arsenite activated ATPase efflux pump was examined in oxyanion-resistant Giardia. However, preliminary data could not find evidence for an ATPase-driven efflux mechanism of oxyanion resistance which suggests that alternative mechanisms of oxyanion resistance are present in Giardia.Resistance to peroxide and the rare earth oxyanions, and the presence of an H2O-producing NADH oxidase, NADH peroxidase, broad range disulphide reductase, cysteine and thioglycolic acid (a free radical trap) accounts for the apparent lack of conventional detoxification mechanisms in G. duodenalis. This shows that G. duodenalis has novel mechanisms of oxidative stress management and xenobiotic detoxification. Considering these novel pathways of detoxification, which are different from those of the higher eukaryotes, a broader perspective, outside the previously characterised detoxification mechanisms, must be considered before the mechanisms by which Giardia develops drug resistance are understood.The bacterial-like mechanisms described in this thesis (Brown et al., 1993; Brown et al., 1995a; Brown et al., 1995b; Brown et al., 1995c) add further support to proposal by Cavalier-Smith (1993) that Giardia is representative of the most ancient eukaryotes, bridging the gap between the bacteria and eukaryotes which was based upon 16S rDNA sequence analysis, ultrastructural and limited metabolic studies (Cavalier­ Smith, 1987; Edwards et al., 1992; Sogin et al., 1989; Cavalier-Smith, 1993).