title: Characterisation of the ATP-dependent ligase, Lig E:  An investigation into its significance in Neisseria gonorrhoeae

authors: Pan, Jolyn

abstract: The rapid rise in multidrug-resistant Neisseria gonorrhoeae isolates has caused major public concern for the treatment of its sexually transmitted disease, gonorrhoea. Previously easily treatable, gonorrhoea is now spreading at alarming rates due to the bacterium’s ability to take up DNA from its environment without regulation. With approximately 106 million new cases annually, thorough investigations into novel gonococcal pathways that can be targeted to limit its survival and spread are crucial. One possible target is the disruption of its DNA repair systems, which include ligases that seal breaks in DNA, and would greatly discourage bacterial growth. Interestingly, in addition to its replicative ligase, N. gonorrhoeae expresses a minimal ATP-dependent ligase, Ligase E (Lig E), which contains a signal peptide that indicates a likely periplasmic location as opposed to the cytosolic location of genomic DNA. Hence, I hypothesise that Lig E is working on repairing fragmented extracellular DNA which has a role in both biofilm formation and bacterial competence, both of which would be greatly beneficial for the persistence and acquisition of antibiotic resistance genes in N. gonorrhoeae.

In this thesis, I focussed on characterising Lig E from N. gonorrhoeae (Ngo-Lig E) to determine its contribution to gonococcal phenotype, as well as its structure, function and cellular location. To analyse its biological role, several ngo-lig E mutants were created in vivo and their growth, stress response, biofilm formation as well as infection and adhesion to a human cell line were studied. I showed that Ngo-Lig E is not essential for normal planktonic N. gonorrhoeae growth and survival; however, it is important for its biofilm formation and subsequent adhesion onto human cells. Following this, I further investigated the potential role of Ngo-Lig E on biofilm formation by looking at its morphology via scanning electron microscopy and confocal laser scanning microscopy after growing them under constant shear forces with a continual supply of fresh media. Data from this supported earlier indications that Ngo-Lig E is important for N. gonorrhoeae in biofilm formation, as well as the extent of damage the bacterium can cause to human tissue. Taking its natural competence into account, I also investigated the potential role of Ngo-Lig E in the uptake of fragmented DNA during transformation and found a decreased ability of N. gonorrhoeae to repair and take up nicked DNA when ngo-lig E was absent. Investigations into the potential location of Ngo-Lig E showed that it is likely transported into both the periplasmic and extracellular space in N. gonorrhoeae, where it may perform the aforementioned dual-role in biofilm formation and DNA uptake. Finally, I solved the three-dimensional structure of Ngo-Lig E, highlighting its minimal architecture and lack of complete encirclement around its substrate DNA. 

The work presented in this thesis provides the first report of a solved structure of Lig E from a human pathogen, the first report of in vivo characterisation of this enzyme, as well as the first report of attempting to characterise its role and location. Collectively, this thesis has allowed for the establishment of important characteristics of Ngo-Lig E that were previously unknown. Although further research into this enzyme is necessary, the results presented here bring to light a potential novel pathway that may be targeted by future drug developments to tackle the emerging threat of multidrug-resistant N. gonorrhoeae in our community.

text: Characterisation of the ATP-dependent ligase, Lig E:  An investigation into its significance in Neisseria gonorrhoeae The rapid rise in multidrug-resistant Neisseria gonorrhoeae isolates has caused major public concern for the treatment of its sexually transmitted disease, gonorrhoea. Previously easily treatable, gonorrhoea is now spreading at alarming rates due to the bacterium’s ability to take up DNA from its environment without regulation. With approximately 106 million new cases annually, thorough investigations into novel gonococcal pathways that can be targeted to limit its survival and spread are crucial. One possible target is the disruption of its DNA repair systems, which include ligases that seal breaks in DNA, and would greatly discourage bacterial growth. Interestingly, in addition to its replicative ligase, N. gonorrhoeae expresses a minimal ATP-dependent ligase, Ligase E (Lig E), which contains a signal peptide that indicates a likely periplasmic location as opposed to the cytosolic location of genomic DNA. Hence, I hypothesise that Lig E is working on repairing fragmented extracellular DNA which has a role in both biofilm formation and bacterial competence, both of which would be greatly beneficial for the persistence and acquisition of antibiotic resistance genes in N. gonorrhoeae.

In this thesis, I focussed on characterising Lig E from N. gonorrhoeae (Ngo-Lig E) to determine its contribution to gonococcal phenotype, as well as its structure, function and cellular location. To analyse its biological role, several ngo-lig E mutants were created in vivo and their growth, stress response, biofilm formation as well as infection and adhesion to a human cell line were studied. I showed that Ngo-Lig E is not essential for normal planktonic N. gonorrhoeae growth and survival; however, it is important for its biofilm formation and subsequent adhesion onto human cells. Following this, I further investigated the potential role of Ngo-Lig E on biofilm formation by looking at its morphology via scanning electron microscopy and confocal laser scanning microscopy after growing them under constant shear forces with a continual supply of fresh media. Data from this supported earlier indications that Ngo-Lig E is important for N. gonorrhoeae in biofilm formation, as well as the extent of damage the bacterium can cause to human tissue. Taking its natural competence into account, I also investigated the potential role of Ngo-Lig E in the uptake of fragmented DNA during transformation and found a decreased ability of N. gonorrhoeae to repair and take up nicked DNA when ngo-lig E was absent. Investigations into the potential location of Ngo-Lig E showed that it is likely transported into both the periplasmic and extracellular space in N. gonorrhoeae, where it may perform the aforementioned dual-role in biofilm formation and DNA uptake. Finally, I solved the three-dimensional structure of Ngo-Lig E, highlighting its minimal architecture and lack of complete encirclement around its substrate DNA. 

The work presented in this thesis provides the first report of a solved structure of Lig E from a human pathogen, the first report of in vivo characterisation of this enzyme, as well as the first report of attempting to characterise its role and location. Collectively, this thesis has allowed for the establishment of important characteristics of Ngo-Lig E that were previously unknown. Although further research into this enzyme is necessary, the results presented here bring to light a potential novel pathway that may be targeted by future drug developments to tackle the emerging threat of multidrug-resistant N. gonorrhoeae in our community.

year: 2025

title: Conventional, remote, virtual, and simulated work-integrated learning: A meta-analysis of existing practice

authors: Wood, Yvonne I.

subjects: COVID-19

abstract: As a result of the COVID-19 pandemic, many organizations offering work-integrated learning (WIL) opportunities were no longer able to support student placements, resulting in many institutions searching for a range of innovative solutions. Many have redesigned their conventional placement-based WIL activities or programs to enable students to meet graduate profile criteria through alternative means such as virtual or simulated WIL experiences. The literature shows there are many models of WIL beyond conventional work placements. Therefore, a meta-analysis was conducted of these WIL models, to provide a complied synthesis of existing practice from within the WIL field specifically focused on two books, the International Journal of Work-Integrated Learning, and three national WIL association conference publications. The trends and qualities that emerge from the meta-analysis of existing practice can inform the redevelopment across the different modes of conventional, remote, virtual, and simulated WIL experiences.

text: Conventional, remote, virtual, and simulated work-integrated learning: A meta-analysis of existing practice As a result of the COVID-19 pandemic, many organizations offering work-integrated learning (WIL) opportunities were no longer able to support student placements, resulting in many institutions searching for a range of innovative solutions. Many have redesigned their conventional placement-based WIL activities or programs to enable students to meet graduate profile criteria through alternative means such as virtual or simulated WIL experiences. The literature shows there are many models of WIL beyond conventional work placements. Therefore, a meta-analysis was conducted of these WIL models, to provide a complied synthesis of existing practice from within the WIL field specifically focused on two books, the International Journal of Work-Integrated Learning, and three national WIL association conference publications. The trends and qualities that emerge from the meta-analysis of existing practice can inform the redevelopment across the different modes of conventional, remote, virtual, and simulated WIL experiences.

year: 2020

title: Integrating education for sustainability into secondary science classes: Practices and perceptions of New Zealand teachers

authors: Gundersen, Mandy

abstract: Global challenges like climate change and biodiversity loss necessitate a collective shift towards sustainable living. Education for Sustainability aims to equip students with the essential knowledge, skills, attitudes and agency to meet sustainability goals and forge a better future. It is a holistic approach, yet single-subject school structures at the secondary level mean it must be integrated into subjects such as science. However, there is limited research, particularly in New Zealand, on how this integration is practiced in secondary science classes.

Grounded in an interpretivist paradigm, this study interviewed five New Zealand secondary science teachers to understand their conceptualisations of sustainability and sustainability education. It explored how their understanding was translated into practice through detailed descriptions of lessons and units. The study provides insights into how these teachers integrate sustainability across junior science, the new National Certificate of Educational Achievement Level One science standards, and senior science subjects, while also elaborating on the benefits they believed students received and the challenges they faced in this integration.

This research found that these New Zealand teachers had a broad understanding of sustainability and recognised key components of sustainability education. They saw sustainability as a way to make science engaging and to develop scientific literacy among their students. To incorporate sustainability into science, they had all designed units which included place-based approaches, integrated topics, considered multiple perspectives, and interwove mātauranga Māori, or New Zealand indigenous knowledge. The boundaries of science as a subject, senior-level assessment requirements, and the time needed for some sustainability-focused activities were noted as challenges to sustainability’s integration.

The research recommends that for the effective integration of sustainability, support is required, particularly regarding teacher education and the provision of resources that exemplify sustainability in science. Furthermore, curriculum and assessment changes designed to incorporate sustainability must remain clear about the required science content to ensure students are robustly prepared for senior science studies.

text: Integrating education for sustainability into secondary science classes: Practices and perceptions of New Zealand teachers Global challenges like climate change and biodiversity loss necessitate a collective shift towards sustainable living. Education for Sustainability aims to equip students with the essential knowledge, skills, attitudes and agency to meet sustainability goals and forge a better future. It is a holistic approach, yet single-subject school structures at the secondary level mean it must be integrated into subjects such as science. However, there is limited research, particularly in New Zealand, on how this integration is practiced in secondary science classes.

Grounded in an interpretivist paradigm, this study interviewed five New Zealand secondary science teachers to understand their conceptualisations of sustainability and sustainability education. It explored how their understanding was translated into practice through detailed descriptions of lessons and units. The study provides insights into how these teachers integrate sustainability across junior science, the new National Certificate of Educational Achievement Level One science standards, and senior science subjects, while also elaborating on the benefits they believed students received and the challenges they faced in this integration.

This research found that these New Zealand teachers had a broad understanding of sustainability and recognised key components of sustainability education. They saw sustainability as a way to make science engaging and to develop scientific literacy among their students. To incorporate sustainability into science, they had all designed units which included place-based approaches, integrated topics, considered multiple perspectives, and interwove mātauranga Māori, or New Zealand indigenous knowledge. The boundaries of science as a subject, senior-level assessment requirements, and the time needed for some sustainability-focused activities were noted as challenges to sustainability’s integration.

The research recommends that for the effective integration of sustainability, support is required, particularly regarding teacher education and the provision of resources that exemplify sustainability in science. Furthermore, curriculum and assessment changes designed to incorporate sustainability must remain clear about the required science content to ensure students are robustly prepared for senior science studies.

year: 2025