title: Characterisation of the transcriptional regulator CysB in Neisseria gonorrhoeae

authors: Klaus, Hannah Rose

subjects: Neisseria gonorrhoeae

abstract: Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted infection gonorrhoea. Rapid emergence of antibiotic resistant strains of N. gonorrhoeae has given rise to multi-drug resistant gonorrhoea. Combined with increasing incidence and the most recent resistance event in 2018, there is an urgent need for new antimicrobial treatments for gonorrhoeal infection. N. gonorrhoeae has a limited number of transcriptional regulators, one of which, CysB, is essential and regulates genes required for de novo cysteine biosynthesis. The cysteine biosynthetic pathway is a promising target for the development of novel antimicrobials. Cysteine plays an important role in protein folding and function, and is the precursor for many thiol-containing molecules within N. gonorrhoeae including effective oxidative stress defences such as glutathione. N. gonorrhoeae displays unique differences in assimilation of sulfur and cysteine biosynthesis, yet little is known about how this altered pathway is regulated in N. gonorrhoeae.
The cysteine regulon consists of the genes required for sulfur transport, assimilation and cysteine biosynthesis. The transcriptional regulator CysB regulates this regulon in other organisms and is essential in N. gonorrhoeae. Understanding how N. gonorrhoeae regulates gene expression will improve our understanding of the pathogenicity of N. gonorrhoeae. In order to understand how CysB mediates gene expression, we need to characterise DNA-binding activity and elucidate the three-dimensional structure. In this thesis, we determine the DNA-binding activity of CysB, its three-dimensional structure and investigate conformational changes of CysB. Our results indicate that CysB regulates genes in the altered cysteine regulon, but that CysB does not respond to the inducer N-acetylserine. The three-dimensional structure of CysB is the first reported full-length CysB structure and was solved to 2.73 Å. Analysis of this structure, coupled with small angle scattering data suggest that CysB from N. gonorrhoeae lacks the ability to undergo the hypothesised large scale conformational changes upon inducer binding. This indicates that CysB may have an alternate mechanism in N. gonorrhoeae.
Collectively, the data presented in this thesis represents key advances in our understanding of how N. gonorrhoeae regulates gene expression by characterisation of the transcriptional regulator CysB. Data presented here is the basis for future work to identify other targets within N. gonorrhoeae that may be investigated for the development of new antimicrobials for treatment of extensively drug resistant gonorrhoea.

text: Characterisation of the transcriptional regulator CysB in Neisseria gonorrhoeae Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted infection gonorrhoea. Rapid emergence of antibiotic resistant strains of N. gonorrhoeae has given rise to multi-drug resistant gonorrhoea. Combined with increasing incidence and the most recent resistance event in 2018, there is an urgent need for new antimicrobial treatments for gonorrhoeal infection. N. gonorrhoeae has a limited number of transcriptional regulators, one of which, CysB, is essential and regulates genes required for de novo cysteine biosynthesis. The cysteine biosynthetic pathway is a promising target for the development of novel antimicrobials. Cysteine plays an important role in protein folding and function, and is the precursor for many thiol-containing molecules within N. gonorrhoeae including effective oxidative stress defences such as glutathione. N. gonorrhoeae displays unique differences in assimilation of sulfur and cysteine biosynthesis, yet little is known about how this altered pathway is regulated in N. gonorrhoeae.
The cysteine regulon consists of the genes required for sulfur transport, assimilation and cysteine biosynthesis. The transcriptional regulator CysB regulates this regulon in other organisms and is essential in N. gonorrhoeae. Understanding how N. gonorrhoeae regulates gene expression will improve our understanding of the pathogenicity of N. gonorrhoeae. In order to understand how CysB mediates gene expression, we need to characterise DNA-binding activity and elucidate the three-dimensional structure. In this thesis, we determine the DNA-binding activity of CysB, its three-dimensional structure and investigate conformational changes of CysB. Our results indicate that CysB regulates genes in the altered cysteine regulon, but that CysB does not respond to the inducer N-acetylserine. The three-dimensional structure of CysB is the first reported full-length CysB structure and was solved to 2.73 Å. Analysis of this structure, coupled with small angle scattering data suggest that CysB from N. gonorrhoeae lacks the ability to undergo the hypothesised large scale conformational changes upon inducer binding. This indicates that CysB may have an alternate mechanism in N. gonorrhoeae.
Collectively, the data presented in this thesis represents key advances in our understanding of how N. gonorrhoeae regulates gene expression by characterisation of the transcriptional regulator CysB. Data presented here is the basis for future work to identify other targets within N. gonorrhoeae that may be investigated for the development of new antimicrobials for treatment of extensively drug resistant gonorrhoea.

year: 2022

title: A novel machine learning model to predict the moment capacity of cold-formed steel channel beams with edge-stiffened and un-stiffened web holes

authors: Dai, Yecheng

subjects: cold-formed steel

abstract: A novel machine learning model, eXtreme Gradient Boosting (XGBoost), was used for the purpose of evaluating the moment capacity of cold-formed steel (CFS) channel beams with edge-stiffened web holes subject to bending. A total of 1620 data points were generated for training the XGBoost model, using an elasto-plastic finite element model which was validated against 12 sets of test data taken from the existing literature. The R2 score of XGBoost predictions for the moment capacity was around 99%. The performance of current design equations was evaluated through the comparison of their results against those obtained from the XGBoost model. The moment capacities obtained from the XGBoost testing dataset were also compared with that determined from the existing design equations for un-stiffened holes (USH) and edge-stiffened holes (ESH). The moment capacities determined from the current design equations for USH and ESH were found to be excessively conservative by 38.3%, and unconservative by 36.2% on average, respectively. Therefore, new design equations were proposed based on the results of parametric study using the XGBoost model. In the detailed parametric analysis, the effects of web depth, section thickness, and beam length on the moment capacity of channel beams (CFSCB) with ESH were considered. From the results of XGBoost outputs, the absolute percentage error of new design equations for that based on the strengths of unperforated CFSCB was 8.78%, and for that based on the strengths of CFSCB with USH, the absolute percentage error was 13.7%. Additionally, a reliability analysis was performed to evaluate the accuracy of the proposed equations that were used to predict the moment capacity of CFS channel beams with ESH subject to bending. The reliability indices of all the proposed equations were greater than 2.5 which can be reliable as per the guidelines of AISI.

text: A novel machine learning model to predict the moment capacity of cold-formed steel channel beams with edge-stiffened and un-stiffened web holes A novel machine learning model, eXtreme Gradient Boosting (XGBoost), was used for the purpose of evaluating the moment capacity of cold-formed steel (CFS) channel beams with edge-stiffened web holes subject to bending. A total of 1620 data points were generated for training the XGBoost model, using an elasto-plastic finite element model which was validated against 12 sets of test data taken from the existing literature. The R2 score of XGBoost predictions for the moment capacity was around 99%. The performance of current design equations was evaluated through the comparison of their results against those obtained from the XGBoost model. The moment capacities obtained from the XGBoost testing dataset were also compared with that determined from the existing design equations for un-stiffened holes (USH) and edge-stiffened holes (ESH). The moment capacities determined from the current design equations for USH and ESH were found to be excessively conservative by 38.3%, and unconservative by 36.2% on average, respectively. Therefore, new design equations were proposed based on the results of parametric study using the XGBoost model. In the detailed parametric analysis, the effects of web depth, section thickness, and beam length on the moment capacity of channel beams (CFSCB) with ESH were considered. From the results of XGBoost outputs, the absolute percentage error of new design equations for that based on the strengths of unperforated CFSCB was 8.78%, and for that based on the strengths of CFSCB with USH, the absolute percentage error was 13.7%. Additionally, a reliability analysis was performed to evaluate the accuracy of the proposed equations that were used to predict the moment capacity of CFS channel beams with ESH subject to bending. The reliability indices of all the proposed equations were greater than 2.5 which can be reliable as per the guidelines of AISI.

year: 2022

title: Fanning the divine spark: Gaining understandings of micro-interactions in New Zealand classrooms

authors: Eley, Elizabeth Martha Ann

subjects: micro-interactions

abstract: New Zealand’s Treaty of Waitangi was signed by representatives of Iwi Māori (Indigenous tribal groups) and the British Crown.  Inherent in the heritages of both signatory groups is the ontological positioning that children are born of greatness; carriers of the divine spark from Tama-nui-te-rā (the central sun) and created in the image of God.  Almost all New Zealand children attend school.  For some, their ongoing classroom experiences with educators fans their divine spark and works towards the realisation of their innate potential.  For others, schooling is not a positive experience and their divine spark may be neither recognised nor celebrated.  

This research explores the relationships and pedagogical practices that underpin the classroom micro-interactions experienced by children.  Children’s schooling experience is made up of small interactions in classrooms that are situated in the social, political and legal contexts of schooling.  Therefore, this research applies both a centripetal and a centrifugal examination of the macro and micro contexts of classroom interactions.  

In examining the macro-context, ontological views of the child and the theoretical frameworks that guide pedagogy are considered.  The Education Acts governing New Zealand schooling have successively contained an ontological contradiction that enforces both a factory model as the preferred means of schooling for New Zealand and a requirement to honour the Treaty of Waitangi, including the worldviews of both signatories.  I conclude that educators within the compulsory schooling sector are not provided with specific guidance on how to navigate these contradictory worldviews.  Neither ontological views of how children are perceived within the system, nor theoretical frameworks that could and should inform views of knowledge and how learning occurs, are made explicit for educators.  

At the micro-context level, relationships and pedagogical practices are explored through case studies in three different New Zealand classrooms.  In each case, from video-recordings of classroom interactions, critical incidents were extracted and used to support interviews with participating teachers, students, parents and members of the wider school community.  Employing culturally responsive research methodologies, the interactions with participants were relational and dialogic.  The research found that classroom micro-interactions are pervasive and complex.  All participants, students, teachers and whānau, draw from the knowledge, understandings and positionings of the individual’s own cultural toolkits in classroom interactions.  Their cultural toolkits contain a range of factors to support learning, all of which were necessary and interdependent.  

The thesis calls for all children to receive an education that fans their inherent divine spark.  While this experience is not yet the case for all, there is opportunity in the rhetoric and structures already within New Zealand’s education system for this to occur.  Some schools and communities have begun to successfully challenge and address the prevailing theoretical and pedagogical status quo.  Therefore, there is reason for radical hope for a system-wide fanning of the divine spark in every child.

text: Fanning the divine spark: Gaining understandings of micro-interactions in New Zealand classrooms New Zealand’s Treaty of Waitangi was signed by representatives of Iwi Māori (Indigenous tribal groups) and the British Crown.  Inherent in the heritages of both signatory groups is the ontological positioning that children are born of greatness; carriers of the divine spark from Tama-nui-te-rā (the central sun) and created in the image of God.  Almost all New Zealand children attend school.  For some, their ongoing classroom experiences with educators fans their divine spark and works towards the realisation of their innate potential.  For others, schooling is not a positive experience and their divine spark may be neither recognised nor celebrated.  

This research explores the relationships and pedagogical practices that underpin the classroom micro-interactions experienced by children.  Children’s schooling experience is made up of small interactions in classrooms that are situated in the social, political and legal contexts of schooling.  Therefore, this research applies both a centripetal and a centrifugal examination of the macro and micro contexts of classroom interactions.  

In examining the macro-context, ontological views of the child and the theoretical frameworks that guide pedagogy are considered.  The Education Acts governing New Zealand schooling have successively contained an ontological contradiction that enforces both a factory model as the preferred means of schooling for New Zealand and a requirement to honour the Treaty of Waitangi, including the worldviews of both signatories.  I conclude that educators within the compulsory schooling sector are not provided with specific guidance on how to navigate these contradictory worldviews.  Neither ontological views of how children are perceived within the system, nor theoretical frameworks that could and should inform views of knowledge and how learning occurs, are made explicit for educators.  

At the micro-context level, relationships and pedagogical practices are explored through case studies in three different New Zealand classrooms.  In each case, from video-recordings of classroom interactions, critical incidents were extracted and used to support interviews with participating teachers, students, parents and members of the wider school community.  Employing culturally responsive research methodologies, the interactions with participants were relational and dialogic.  The research found that classroom micro-interactions are pervasive and complex.  All participants, students, teachers and whānau, draw from the knowledge, understandings and positionings of the individual’s own cultural toolkits in classroom interactions.  Their cultural toolkits contain a range of factors to support learning, all of which were necessary and interdependent.  

The thesis calls for all children to receive an education that fans their inherent divine spark.  While this experience is not yet the case for all, there is opportunity in the rhetoric and structures already within New Zealand’s education system for this to occur.  Some schools and communities have begun to successfully challenge and address the prevailing theoretical and pedagogical status quo.  Therefore, there is reason for radical hope for a system-wide fanning of the divine spark in every child.

year: 2020