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: Automating test scripts for Android UI testing

authors: Turner, Jessica Dawn

subjects: capture/replay testing

abstract: The Espresso capture/replay testing tool for Android applications creates tests that are prone to test fragility, in that when small changes occur to the user interface, tests break and are unable to be re-run. To reduce the fragility and its impact inherent in Espresso tests, we take a modeldriven development approach to test generation. Using interaction sequence models as the basis for generation, we are able to create test scripts that can be run in Android Studio identically to manually recorded tests. This process simplifies scripts when compared with those generated by recording and reduces the time required by developers to create and maintain the test suite, resulting in higher quality testing and validation of Android user interfaces.

text: Automating test scripts for Android UI testing The Espresso capture/replay testing tool for Android applications creates tests that are prone to test fragility, in that when small changes occur to the user interface, tests break and are unable to be re-run. To reduce the fragility and its impact inherent in Espresso tests, we take a modeldriven development approach to test generation. Using interaction sequence models as the basis for generation, we are able to create test scripts that can be run in Android Studio identically to manually recorded tests. This process simplifies scripts when compared with those generated by recording and reduces the time required by developers to create and maintain the test suite, resulting in higher quality testing and validation of Android user interfaces.

year: 2024

title: Visitors' satisfaction with heritage sites in New Zealand: Causes and complexities, clusters and causes

authors: Trinh, Thi Thu

subjects: Heritage Tourism

abstract: The thesis presents findings from a survey of over 1,000 visitors at three New Zealand heritage sites. These sites were Te Puia, the Rotorua Bathhouse Museum and the Rangiriri Battlefield Interpretation Centre. All three represent a key period in New Zealand’s history from the period of approximately 1840 to 1900, but in the case of Te Puia there is also a continuing contemporary cultural importance. This last site, located in Rotorua, was founded as the Maori Arts and Crafts Institute and was established to perpetuate Maori tradition skills in areas such as carving and weaving. Its location was in part determined by the volcanic nature of the valley, long inhabited by members of Te Arawa tribal people. The site has a strong connection with tourism as Te Arawa have entertained tourists from the mid-nineteenth century in the volcanic area. The site therefore represents a tourism site from the perspective of history, culture and natural heritage. The Bathhouse Museum represents a period of late colonial architecture while the third site, the Rangiriri Battlefield is based on the remnants of the Pa (Maori fortifications) that was the site of a battle between the colonial government forces and the Maori Kingi movement on November 23rd 1840.

The motive for the research was to provide a profile of visitors for the respective sites and their management, and then to assess to what degree socio-demographics might be explanatory variables in determining future visitation. The core theories being employed revolved around concepts of levels of interest in heritage and historic sites, the intellectual search for knowledge, and the degree to which people became involved in the activity of heritage site visitation. The work was driven by the finding that only about 11 per cent of visits to cultural tourism sites were ‘purposeful’ tourists as defined by McKercher and Du Cros (2002). Being purposeful implies having specific degrees of interest, of becoming involved and possibly seeking meanings that implied senses of identity. That is, self-awareness accrued from having a better understanding of the past as a means of knowing about the present. This conceptualisation implies use of the theories of involvement, benefits and self-awareness, and the managerial aspects of interpretation.  Normally such an approach has been seen by many researchers as a determinant of satisfaction, but in this thesis satisfaction is not seen as simply an end to a process. Rather, this thesis argues that to be satisfied entails not only cognitive and affective components, but also the conative. That conative component can include making recommendations to others, making visits to other heritage sites, or joining organisations associated with heritage sites such as the New Zealand Historic Place Trust. These form key themes in the literature review.

Unfortunately, while these premises emerged from the literature review and informed the hypotheses that are later described in the thesis, they were not wholly supported by the data. It is suggested that one reason for this, from a statistical perspective, was that measures used were subject to multi-collinearity and auto-correlation – put simply, many of the variables are not independent from each other. For example, it is suggested that satisfaction is actually enhanced by subsequently being able to make recommendations to friends and others; that the act of making a recommendation enhances one’s own self in both the eyes of that friend or through an enhanced self- perception of being helpful, and thus auto-correlation may exist between these variables.

This realisation thus leads, in the conclusions of Chapter Eight, to new suggestions for potential future researchers concerning ways of looking at the nature of involvement that draw on distinctions between situational and enduring involvement. Finally, it also needs to be noted that tourists are not lay historians, but are the makers of their holidays, and hence the debate is contextualised within the act of being on holiday, which itself is a period of escape and relaxation for many. Hence the relationships being examined in this thesis are complex, interactive and yet rewarding to untangle.

text: Visitors' satisfaction with heritage sites in New Zealand: Causes and complexities, clusters and causes The thesis presents findings from a survey of over 1,000 visitors at three New Zealand heritage sites. These sites were Te Puia, the Rotorua Bathhouse Museum and the Rangiriri Battlefield Interpretation Centre. All three represent a key period in New Zealand’s history from the period of approximately 1840 to 1900, but in the case of Te Puia there is also a continuing contemporary cultural importance. This last site, located in Rotorua, was founded as the Maori Arts and Crafts Institute and was established to perpetuate Maori tradition skills in areas such as carving and weaving. Its location was in part determined by the volcanic nature of the valley, long inhabited by members of Te Arawa tribal people. The site has a strong connection with tourism as Te Arawa have entertained tourists from the mid-nineteenth century in the volcanic area. The site therefore represents a tourism site from the perspective of history, culture and natural heritage. The Bathhouse Museum represents a period of late colonial architecture while the third site, the Rangiriri Battlefield is based on the remnants of the Pa (Maori fortifications) that was the site of a battle between the colonial government forces and the Maori Kingi movement on November 23rd 1840.

The motive for the research was to provide a profile of visitors for the respective sites and their management, and then to assess to what degree socio-demographics might be explanatory variables in determining future visitation. The core theories being employed revolved around concepts of levels of interest in heritage and historic sites, the intellectual search for knowledge, and the degree to which people became involved in the activity of heritage site visitation. The work was driven by the finding that only about 11 per cent of visits to cultural tourism sites were ‘purposeful’ tourists as defined by McKercher and Du Cros (2002). Being purposeful implies having specific degrees of interest, of becoming involved and possibly seeking meanings that implied senses of identity. That is, self-awareness accrued from having a better understanding of the past as a means of knowing about the present. This conceptualisation implies use of the theories of involvement, benefits and self-awareness, and the managerial aspects of interpretation.  Normally such an approach has been seen by many researchers as a determinant of satisfaction, but in this thesis satisfaction is not seen as simply an end to a process. Rather, this thesis argues that to be satisfied entails not only cognitive and affective components, but also the conative. That conative component can include making recommendations to others, making visits to other heritage sites, or joining organisations associated with heritage sites such as the New Zealand Historic Place Trust. These form key themes in the literature review.

Unfortunately, while these premises emerged from the literature review and informed the hypotheses that are later described in the thesis, they were not wholly supported by the data. It is suggested that one reason for this, from a statistical perspective, was that measures used were subject to multi-collinearity and auto-correlation – put simply, many of the variables are not independent from each other. For example, it is suggested that satisfaction is actually enhanced by subsequently being able to make recommendations to friends and others; that the act of making a recommendation enhances one’s own self in both the eyes of that friend or through an enhanced self- perception of being helpful, and thus auto-correlation may exist between these variables.

This realisation thus leads, in the conclusions of Chapter Eight, to new suggestions for potential future researchers concerning ways of looking at the nature of involvement that draw on distinctions between situational and enduring involvement. Finally, it also needs to be noted that tourists are not lay historians, but are the makers of their holidays, and hence the debate is contextualised within the act of being on holiday, which itself is a period of escape and relaxation for many. Hence the relationships being examined in this thesis are complex, interactive and yet rewarding to untangle.

year: 2013