title: Structural and biochemical characterisation of O-acetylserine  sulphydrylase (CysK) from Neisseria gonorrhoeae

authors: McGarvie, Jack

subjects: Biochemistry

abstract: Neisseria gonorrhoeae is the causative bacteria of the sexually transmitted infection (STI), gonorrhoea. Rapid emergence of antibiotic resistant strains of N. gonorrhoeae has given rise to almost untreatable gonorrhoea over the last few decades. There is an urgent need for development of new antimicrobial treatments for gonorrhoea infection. Synthesis of the amino acid cysteine is a promising new target for the development of new antimicrobials. Cysteine plays a vital role in protein folding and function, and synthesis of glutathione for protection against oxidative stress during infection. 
The final step of the two-step cysteine biosynthesis pathway is catalysed by the enzyme O-acetylserine sulphydrylase (OASS). Most bacteria have two isoforms of OASS, OASS-A/CysK that utilises sulphide and O-acetylserine for the synthesis of cysteine and OASS-B/CysM that utilises thiosulphate. N. gonorrhoeae has just one OASS isoform (annotated as CysK) in its genome. N. gonorrhoeae displays unique differences in its sulphate acquisition pathway for cysteine biosynthesis, yet little is known about these pathways in N. gonorrhoeae. We propose CysK is a potential antimicrobial target. This 
thesis biochemically characterises the structure and function of CysK to determine its role in cysteine biosynthesis. Kinetic characterisation demonstrates that CysK has O-acetylserine sulphydrylase activity and displays positive cooperativity with respect to substrates, O-acetylserine and sulphide. Sulphide shows partial allosteric inhibition, and the enzyme does not use thiosulphate as a substrate. The structure of CysK was solved to 2.49 Å by X-ray crystallography and shows CysK belongs to the tryptophan synthase β superfamily and adopts a homodimeric structure consisting of two monomers. The structure supports positive cooperativity as co-factor binding residues are in inactive and active conformations in each monomer of the dimer, respectively. Many CysK enzymes form complexes with the first enzyme in the two-step cysteine synthesis pathway, CysE. It appears CysE and CysK from N. gonorrhoeae are unable to form a complex, indicating an alternate pathway for regulation of sulphur flux and cysteine production. Attempts to investigate the in vivo role of CysK were inconclusive.
The research presented in this thesis represents a major leap in our understanding of the  uncharacterised cysteine biosynthesis pathway in N. gonorrhoeae. Data presented here is the basis for future work using the kinetics and structure of CysK to guide computational inhibitor design, to identify lead compounds for CysK inhibition, and thereby development of new antimicrobials for treatment of extensively drug resistant gonorrhoea.

text: Structural and biochemical characterisation of O-acetylserine  sulphydrylase (CysK) from Neisseria gonorrhoeae Neisseria gonorrhoeae is the causative bacteria of the sexually transmitted infection (STI), gonorrhoea. Rapid emergence of antibiotic resistant strains of N. gonorrhoeae has given rise to almost untreatable gonorrhoea over the last few decades. There is an urgent need for development of new antimicrobial treatments for gonorrhoea infection. Synthesis of the amino acid cysteine is a promising new target for the development of new antimicrobials. Cysteine plays a vital role in protein folding and function, and synthesis of glutathione for protection against oxidative stress during infection. 
The final step of the two-step cysteine biosynthesis pathway is catalysed by the enzyme O-acetylserine sulphydrylase (OASS). Most bacteria have two isoforms of OASS, OASS-A/CysK that utilises sulphide and O-acetylserine for the synthesis of cysteine and OASS-B/CysM that utilises thiosulphate. N. gonorrhoeae has just one OASS isoform (annotated as CysK) in its genome. N. gonorrhoeae displays unique differences in its sulphate acquisition pathway for cysteine biosynthesis, yet little is known about these pathways in N. gonorrhoeae. We propose CysK is a potential antimicrobial target. This 
thesis biochemically characterises the structure and function of CysK to determine its role in cysteine biosynthesis. Kinetic characterisation demonstrates that CysK has O-acetylserine sulphydrylase activity and displays positive cooperativity with respect to substrates, O-acetylserine and sulphide. Sulphide shows partial allosteric inhibition, and the enzyme does not use thiosulphate as a substrate. The structure of CysK was solved to 2.49 Å by X-ray crystallography and shows CysK belongs to the tryptophan synthase β superfamily and adopts a homodimeric structure consisting of two monomers. The structure supports positive cooperativity as co-factor binding residues are in inactive and active conformations in each monomer of the dimer, respectively. Many CysK enzymes form complexes with the first enzyme in the two-step cysteine synthesis pathway, CysE. It appears CysE and CysK from N. gonorrhoeae are unable to form a complex, indicating an alternate pathway for regulation of sulphur flux and cysteine production. Attempts to investigate the in vivo role of CysK were inconclusive.
The research presented in this thesis represents a major leap in our understanding of the  uncharacterised cysteine biosynthesis pathway in N. gonorrhoeae. Data presented here is the basis for future work using the kinetics and structure of CysK to guide computational inhibitor design, to identify lead compounds for CysK inhibition, and thereby development of new antimicrobials for treatment of extensively drug resistant gonorrhoea.

year: 2021

title: Djadjiling Rockshelter: 35,000 ¹⁴C Years of Aboriginal Occupation in the Pilbara, Western Australia

authors: Law, W. Boone

subjects: Djadjiling Rockshelter

abstract: The Pleistocene settlement of the arid zone is a prominent research theme in Australian archaeology (Hiscock 2008:45- 62; Hiscock and Wallis 2004; Marwick 2002a, 2002b; O’Connor et al. 1998; Smith 1987, 2005; Thorley 1998; Veth 1993, 1995, 2005). Of particular interest is the inland Pilbara region of the western arid zone, which until recently was reported to have been first occupied between c.20,000 BP and c.26,000 BP (Brown 1987:27; Edwards and Murphy 2003:45; Maynard 1980:7). The recent test excavations at Juukan-1 rockshelter suggest the region was occupied before 32,920±270 BP (Slack et al. 2009:34). Our research at Djadjiling rockshelter supports this result by demonstrating an Aboriginal presence at the site c.35,000 years ago. Not only is the site unique for its antiquity, but excavations have recovered a large flaked stone assemblage from the earliest occupational phase. The evidence demonstrates repeated early site use, and a sequence of intermittent occupation throughout the late Pleistocene and Holocene. The preliminary findings are presented below.

text: Djadjiling Rockshelter: 35,000 ¹⁴C Years of Aboriginal Occupation in the Pilbara, Western Australia The Pleistocene settlement of the arid zone is a prominent research theme in Australian archaeology (Hiscock 2008:45- 62; Hiscock and Wallis 2004; Marwick 2002a, 2002b; O’Connor et al. 1998; Smith 1987, 2005; Thorley 1998; Veth 1993, 1995, 2005). Of particular interest is the inland Pilbara region of the western arid zone, which until recently was reported to have been first occupied between c.20,000 BP and c.26,000 BP (Brown 1987:27; Edwards and Murphy 2003:45; Maynard 1980:7). The recent test excavations at Juukan-1 rockshelter suggest the region was occupied before 32,920±270 BP (Slack et al. 2009:34). Our research at Djadjiling rockshelter supports this result by demonstrating an Aboriginal presence at the site c.35,000 years ago. Not only is the site unique for its antiquity, but excavations have recovered a large flaked stone assemblage from the earliest occupational phase. The evidence demonstrates repeated early site use, and a sequence of intermittent occupation throughout the late Pleistocene and Holocene. The preliminary findings are presented below.

year: 2010

title: The general cognitive ability of Maori and European subjects: a comparative study of Queensland test performances and psychometric properties

authors: St George, Ross

abstract: Research was undertaken into the performances of New Zealand Maori and European children on the Queensland Test of Cognitive Abilities. In addition to studying the comparative performances of the two ethnic groups at six age levels, major psychometric properties of the instrument were investigated. Such data have an important bearing upon the issue of psychological equivalence across ethnic or culture group samples.
Seven hundred children (303 Maori and 397 European) aged between eight and a half and fourteen and a half years were tested with the Queensland Test. A small statistically significant ethnic group difference was found favouring the European children. Subsequent analyses indicated that the small difference was of little practical significance. Queensland Test subtest data were also analysed, This indicated that subtest performances were likely to be influenced by subject variables in a manner not necessarily evident in the overall test results. Gender differences in performance on the Pattern Matching Subtest were found and are consistent with other data. 
Queensland Test reliability, validity and item characteristics were researched for the two ethnic groups. 
To obtain test-retest reliability estimates and data on criterion related validity longitudinal studies were undertaken with four ethnically mixed class groups Standard 3 to Form 2). 
The Queensland Test was found to be equally reliable with both ethnic groups although the levels of reliability obtained were not considered entirely satisfactory. 
Validity data indicated that the same general intelligence construct could be employed in the interpretation of Queensland Test results with both European and Maori subjects. Content validity issues were considered and it was argued that in terms of both test content and measurement procedures the Queensland Test should provide an equally valid measure of general intelligence with both groups. 
The criterion related validity coefficients were of approximately the same order for each ethnic group although doubts were expressed about the adequacy of the criteria. Construct validity was investigated by considering the relationship of test performance to age and through factor analytic evidence. The analyses supported the view that the Queensland Test was primarily a measure of the same underlying construct within each ethnic group. In terms of prevailing psychological theories on human intelligence this construct was interpreted as being of a general intelligence nature.
Comparative data on item characteristics did not indicate any marked ethnic group differences. More apparent was the need to restructure particular subtests where items failed to discriminate adequately within both ethnic groups. It was concluded that the weight of evidence was in favour of the psychological equivalence of this ability measure with Maori and European children. 
Data for combined ethnic group samples have been reported, Pooled data were employed in some factor analyses and these supported a general intelligence interpretation of Queensland Test results. Preliminary norms were also derived from combined ethnic group data. 
Findings are discussed in relation to the concept of intelligence and its cross-cultural measurement, other Queensland Test research and recent cross-cultural studies on cognitive abilities in New Zealand.

text: The general cognitive ability of Maori and European subjects: a comparative study of Queensland test performances and psychometric properties Research was undertaken into the performances of New Zealand Maori and European children on the Queensland Test of Cognitive Abilities. In addition to studying the comparative performances of the two ethnic groups at six age levels, major psychometric properties of the instrument were investigated. Such data have an important bearing upon the issue of psychological equivalence across ethnic or culture group samples.
Seven hundred children (303 Maori and 397 European) aged between eight and a half and fourteen and a half years were tested with the Queensland Test. A small statistically significant ethnic group difference was found favouring the European children. Subsequent analyses indicated that the small difference was of little practical significance. Queensland Test subtest data were also analysed, This indicated that subtest performances were likely to be influenced by subject variables in a manner not necessarily evident in the overall test results. Gender differences in performance on the Pattern Matching Subtest were found and are consistent with other data. 
Queensland Test reliability, validity and item characteristics were researched for the two ethnic groups. 
To obtain test-retest reliability estimates and data on criterion related validity longitudinal studies were undertaken with four ethnically mixed class groups Standard 3 to Form 2). 
The Queensland Test was found to be equally reliable with both ethnic groups although the levels of reliability obtained were not considered entirely satisfactory. 
Validity data indicated that the same general intelligence construct could be employed in the interpretation of Queensland Test results with both European and Maori subjects. Content validity issues were considered and it was argued that in terms of both test content and measurement procedures the Queensland Test should provide an equally valid measure of general intelligence with both groups. 
The criterion related validity coefficients were of approximately the same order for each ethnic group although doubts were expressed about the adequacy of the criteria. Construct validity was investigated by considering the relationship of test performance to age and through factor analytic evidence. The analyses supported the view that the Queensland Test was primarily a measure of the same underlying construct within each ethnic group. In terms of prevailing psychological theories on human intelligence this construct was interpreted as being of a general intelligence nature.
Comparative data on item characteristics did not indicate any marked ethnic group differences. More apparent was the need to restructure particular subtests where items failed to discriminate adequately within both ethnic groups. It was concluded that the weight of evidence was in favour of the psychological equivalence of this ability measure with Maori and European children. 
Data for combined ethnic group samples have been reported, Pooled data were employed in some factor analyses and these supported a general intelligence interpretation of Queensland Test results. Preliminary norms were also derived from combined ethnic group data. 
Findings are discussed in relation to the concept of intelligence and its cross-cultural measurement, other Queensland Test research and recent cross-cultural studies on cognitive abilities in New Zealand.

year: 1977