Experimental and kinetic studies of the escherichia coli glucuronylsynthase : an engineered enzyme for the synthesis of glucuronides

The E. coli glucuronylsynthase is a glycosynthase enzyme derived from E. coli {u03B2}-glucuronidase. It catalyses the conjugation of a glucopyranuronic acid unit to an alcohol acceptor. These glucuronide conjugates are important markers for doping in sports drug testing and drug metabolism in pharmaceutical trials. Up till now, the optimal glucuronylsynthase reaction conditions were unknown which may have reduced the previously reported yields. This thesis covers the development and optimisation of the glucuronylsynthase-mediated glucuronylation reaction. This was achieved by determining the enzyme kinetics associated with the glucuronylsynthase enzyme and devising a synthetic strategy to overcome the water solubility issues associated with steroidal substrates. HPLC-UV was used to monitor and quantitate the components of the glucuronylsynthase reaction required for enzyme kinetic analysis. This HPLC assay was used to determine the kinetic parameters (Km, kcat) of the {u03B1}-D-glucuronyl fluoride 51 donor and a range of acceptor substrates. Substrate inhibition and product inhibition were observed during these kinetic investigations and inhibition constants are determined in each case. The optimised glucuronylsynthase-mediated reaction conditions were also determined using HPLC-UV. Different variations (pH, temperature, enzyme concentration, substrate concentration/equivalents) were altered and their effect on enzyme activity and product yield determined by HPLC-UV. Improved reaction rates and yield were observed in the glucuronylsynthase reactions involving phenol 52 and DHEA O-(carboxymethyl)oxime 116 when applying optimised conditions. To overcome the poor water solubility of steroidal substrates, carboxymethoxylamine 115 was condensed to the ketone of DHEA 55 and testosterone 7 to synthesise ionisable oxime-analogues with greatly enhanced water solubility. This provided greater yields of the steroid glucuronide conjugates compared to the direct glucuronylsynthesis of the parent steroids. Greater enzyme efficiency was also achieved due to more concentrated reaction volumes being used. The oxime moiety was cleaved under mild titanium(III) hydrolysis to complete the synthesis of the steroid glucuronide conjugate in high overall yields in three steps. Application of this steroid solubilisation strategy on a half gram scale was demonstrated. A solid-phase extraction procedure was adapted to a small library of steroids to isolate a variety of purified steroid glucuronides. This screen illustrated the broad application of the enzyme and served as a trial for a larger screen of substrates. The combination of the optimised methodology and screening procedures sets the foundation to expand the substrate repertoire for the glucuronylsynthase reaction. Upon reviewing the glucuronylsynthase procedure and alternate means of glucuronylation, the glucuronylsynthase procedure compares well, and is better than these alternative procedures in many ways. This includes an experimental procedure that is easy to set-up, mild and applicable to a broad range of substrates. Other attributes include a short and simple donor synthesis and the absence of by-products (to date) which allows high isolated yields and purity

The use of augmented reality apps to make laboratories more accessible to people with colour vision deficiencies

Colour is an important part of how we understand the world around us, from choosing fresh fruit and vegetables to reading traffic lights. Learning and research in science also has a large focus on colour, using colour and colour-based observations to inform experiments and interpret results. For people with a colour blindness, better described as colour vision deficiency (CVD), the colour focus of science can be an implicit barrier to learning or science-based careers. This study first explores the ways in which chemistry experiments can be inaccessible to people with CVD and next assesses tools that could be used to improve the accessibility of laboratories in STEM. Recognising components of experiments that may prove challenging to CVD participants is a proactive approach to improving CVD accessibility, but they can be difficult to identify. There are several augmented reality (AR) apps available for mobile devices which can simulate CVD vision but no scientific evidence has been shown on their accuracy. During this study, four of these AR CVD simulating apps were evaluated using the Farnsworth D15 hue-based colour vision test results from eleven participants. The best performing CVD simulating app, based on scientific evidence, was then used to evaluate current undergraduate chemistry experiments at The University of Sydney. Of the 24 experiments identified as being potentially challenging for students with CVD, we applied CVD simulation to five chemistry experiments experienced by First Year students. In the final part of this study, we evaluated the potential of AR apps assisting CVD participants in a laboratory environment. Farnsworth D15 colour test were used again to evaluate colour naming, colour filtering and colour shifting functions of AR CVD assistive apps. These CVD assistive apps were then applied during a live chemistry experiment to evaluate their real-life laboratory applicability. The results of this study will be presented along with recommendations of which apps and practices are best applied to improve the accessibility of STEM laboratories to CVD students

Giving students the choice of authentic assessments in the Chemistry laboratory

Incorporating authentic assessments into a laboratory program can improve students’ engagement and satisfaction whilst developing highly valuable employment skills (Schultz et al., 2022; Sokhanvar et al., 2021). It can promote a deeper understanding by having students translate their data and findings to a real-world audience of stakeholders – occasionally not from the scientific community (Jopp, 2020). Another assessment strategy is to empower students with a choice of assessments to complete. A choice of assessments can increase student motivation, confidence and engagement whilst reducing the anxiety around assessments (Patall et al., 2010; Garside et al., 2009). This promotes self-regulated learning, self-efficacy and can lead to improved academic performance (Jopp & Cohen, 2020). At The University of Sydney, students complete four experiments in their laboratory program during their first semester of first year chemistry. Each experiment has a different style of authentic assessment associated with it. Students are provided with the opportunity to select one of these four experiments (and its associated authentic assessment) to submit as their major laboratory assessment for the semester. At the end of semester, students (n = ~750) were questioned about why they had selected the assessment they submitted. In this presentation, we analyse how popular the different authentic assessments were amongst students and look at some of the motives behind why these assessments were selected. REFERENCES Garside, J., Nhemachena, J. Williams, J., & Topping, A. (2009). Repositioning assessment: Giving students the ‘choice’ of assessment methods. Nurse Education in Practice, 9(2), 141-148. Jopp, R. (2020). A case study of a technology enhanced learning initiative that supports authentic assessment. Teaching in Higher Education, 25(8), 942-958. https://doi.org/10.1080/13562517.2019.1613637 Jopp, R. & Cohen, J. (2020). Choose your own assessment – assessment choice for students in online higher education. Teaching in Higher Education 27(6), 738-755. https://doi.org/10.1080/13562517.2020.1742680 Patall, E. A., Cooper, H., & Wynn, S.R. (2010). The effectiveness and relative importance of choice in the classroom. Journal of Educational Psychology, 102(4), 896. Schultz, M., Young, K., Gunning, T., & Harvey, M. (2022). Defining and measuring authentic assessment: a case study in the context of tertiary science. Assessment and Evaluation in Higher Education, 47(1), 77-94. Sokhanvar, Z. Salehi, K., & Sokhanvar, F. (2021) Advantages of authentic assessment for the improving the learning experience and employability skills of higher education students: A systematic literature review. Studies in Educational Evaluation, 70, 101030

USING LIVE STUDENT PEER ASSESSMENT WITH AUTOMATED INSTANT FEEDBACK

Peer assessment and feedback enables students to develop objectivity in relation to standards which can then be transferred to their own work (Liu & Carless, 2006). However, providing feedback, particularly in large classes, can be labour intensive (eg. collating scores and comments). As such, it can be challenging to provide effective feedback in a timely manner which has been shown to promote retention and the correction of inaccurate responses (Epstein et al., 2002). We have recently utilised the online student data and engagement system (SRES, Liu et al., 2017) to run our peer assessments of our student oral presentations within our undergraduate chemistry laboratories. Students are able to grade their peers’ presentations in real time via mobile devices which is captured by SRES, alongside the Academic(s) grading. The system automatically collates both student and academics scores and immediately posts this grade and feedback to the Learning Management System (LMS) of the presenting student(s). Students have immediate access to this feedback to construct self-reflections or to discuss their performance with their teacher whilst the experience is still “fresh”. We will discuss its implementation and how it addresses topics such as mitigating academic misconduct, improving student engagement and reducing the academic burden in running these assessments. REFERENCES Epstein, M.L., Lazarus, A.D., Calvano, T.B. et al. (2002). Immediate Feedback Assessment Technique Promotes Learning and Corrects Inaccurate first Responses. The Psychological Record, 52, 187–201. Liu, D. Y. T., Bartimote-Aufflick, K., Pardo, A., & Bridgeman, A. J. (2017). Data-Driven Personalization of Student Learning Support in Higher Education. In Learning Analytics: Fundaments, Applications, and Trends, Peña-Ayala, A., Ed. Springer International Publishing. Liu, N. & Carless, D. (2006). Peer feedback: the learning element of peer assessment. Teaching in Higher Education, 11(3), 279-290

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin.

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites

What does empathy look like to you? Investigating student and staff opinions

Empathy is a key factor in successful human interaction. Many contemporary issues can be linked to a lack of ability for individuals to truly understand the perspectives of those that they interact with. In the context of teaching and learning, if teaching staff cannot truly understand the complex lives of their students, it is likely that students will be unable to reach their full potential and proceed into society as fully realised members of their respective communities (Levin et al., 2012; Robertson et al., 2015; Tudor, 1993). As such, any intervention that increases the ability of teaching staff to connect to the students, benefits not only the student but also the workforce they go on to contribute to (Haertel et al., 1981). What is unknown, however, is how teaching staff perceive their role in this empathic relationship, especially in the sciences (chemistry, biology, physics, etc.). It is additionally unclear how these perceptions are affected by either subject area or the teaching staff’s previous teaching and life experience. This project would seek to interview teaching staff across a range of disciplines in order to unpack their views around empathy and how best to employ it in their teaching practices. Largescale questionaries undertaken with undergraduate students would allow a comparison between the perceptions of students with the teaching staff. Ideally, the results of this project would allow for a better understanding of how empathy can best be supported and embedded into the practices of teaching staff both within a university context but also into all teaching practices across society. REFERENCES Haertel, G. D., Walberg, H. J., & Haertel, E. H. (1981). Socio-psychological environments and learning: A quantitative synthesis. British Educational Research Journal, 7(1), 27-36. Levin, D., Hammer, D., Elby, A., & Coffey, J. (2012). Becoming a responsive science teacher: Focusing on student thinking in secondary science. National Science Teachers Association Arlington, VA. Robertson, A. D., Scherr, R., & Hammer, D. (2015). Responsive teaching in science and mathematics. Routledge. Tudor, I. (1993). Teacher roles in the learner-centred classroom. ELT Journal, 47(1), 22-31.

Student Relationship Engagement System Community of Practice (SRES-CoP)

NOTE: It is recommended you bring a laptop to this workshop but not essential. GOAL To deliver a hands-on workshop that will expand knowledge on SRES, derive inspiration through demonstrated practices and develop networks with SRES users across the institutions. BACKGROUND The Student Relationship Engagement System (SRES) is a bespoke student engagement system that allows you collect, filter and output student data. Data can be collected from direct input, learning management system (LMS) synchronisation or directly from the student themselves. The information output can be as simple as a filtered spreadsheet through to automated emails and webpages. This output can be tailored to teachers (eg listing at-risk students) or students (eg providing personalised messages or feedback). The multitude of functions and options that SRES provides has resulted in unique and innovative applications of SRES which deserve recognition. AIMS In this session, we will share our collective experiences and practices of SRES across a range of disciplines and Australian institutions. Participants from all science disciplines and SRES experience (or lack of) are invited. For new or non-SRES users, it’s an opportunity to see what SRES is and what applications are possible. For experienced users, the workshop can facilitate the implementation of more advanced practices or inspire new ways to utilise SRES. Overall, the objective of the workshop is to capture how educators use SRES and to establish a network of users that can facilitate support beyond this workshop. DELIVERABLES Through this workshop we aim to facilitate the following deliverable outcomes: Introduce SRES and hear some SRES experiences and practices from a panel of SRES users; Collect a list of SRES practices and their creators (to share with the community); Demonstration: Escape room-style Lab Safety Activity Dedicated time for participants to interact with SRES mentors to troubleshoot their current SRES issues or build a practical solution to ideas they wish to implement in SRES. PROPOSED WORKSHOP SCHEDULE Introduction (10 mins) – What is SRES? How does it work? Panel examples (40 mins) – How have the workshop panelist used SRES in their practices? Workshop task (20 mins) – How do you use SRES in your practices? Networking Session (20 mins) –Talk ideas or troubleshoot SRES challenges with colleagues and workshop panelists.

STUDENT ENGAGEMENT THROUGH DATA MAPPING IN AN UNDERGRADUATE ENVIRONMENTAL CHEMISTRY LABORATORY

We are all too familiar with the map visualisations in media depicting the spread and severity of COVID-19 across the world. The representation of statistical data on a map is a powerful tool that can effectively convey factors such as magnitude, density and spatial variations. Analysing data in this format can help identify trends (eg “hotspots”, “patient zero”) from large datasets. Whilst students outside the discipline of geosciences may be familiar with analysing a data map; constructing one would be a rare experience. In our undergraduate environmental chemistry laboratory, students analyse the metal ion content and hardness of water samples collected on campus. We have used Google Maps Application Programming Interface (API)1 to allow students to geotag their results on a Google Map. The resulting bubble map is live and continually updated as students complete the lab and submit their results.2 This map is shared with the cohort so students can view the evolution of data, their contribution to the “project” and generate their own hypotheses as to why certain concentrations may be linked to certain locales (eg. age of building). This approach offers rich context-based learning that could be modified to address other datasets/contexts, locations, and disciplines

CHEMISTRY IN THE TIME OF COVID-19: REFLECTIONS ON A VERY UNUSUAL SEMESTER

Educators around the world have been challenged to adapt their teaching and pastoral care rapidly in response to the COVID-19 pandemic. The academic members of the Chemistry Education and Communication Theme (CECR) from the School of Chemistry, The University of Sydney have reflected on the challenges and successes during this most unusual semester. As many would no doubt agree, the transition was rapid, and overwhelming with the sheer volume of shared resources and recommendations from our colleagues around the world. In this talk, we will discuss some of the specific tools and techniques employed across a range of teaching modes including lectures, tutorials, and the laboratory. Of particular note, we will explore the importance of communication and pastoral care that we found central to many of our teaching successes. While we recognise each classroom holds a unique context, we believe that the sharing and discussion of our communities’ journeys in navigating this time will hold valuable lessons to inform both our immediate practice and the long-term impact on our teaching

Benznidazole-Resistance in Trypanosoma cruzi Is a Readily Acquired Trait That Can Arise Independently in a Single Population

Benznidazole is the frontline drug used against Trypanosoma cruzi, the causative agent of Chagas disease. However, treatment failures are often reported. Here, we demonstrate that independently acquired mutations in the gene encoding a mitochondrial nitroreductase (TcNTR) can give rise to distinct drug-resistant clones within a single population. Following selection of benznidazole-resistant parasites, all clones examined had lost one of the chromosomes containing the TcNTR gene. Sequence analysis of the remaining TcNTR allele revealed 3 distinct mutant genes in different resistant clones. Expression studies showed that these mutant proteins were unable to activate benznidazole. This correlated with loss of flavin mononucleotide binding. The drug-resistant phenotype could be reversed by transfection with wild-type TcNTR. These results identify TcNTR as a central player in acquired resistance to benznidazole. They also demonstrate that T. cruzi has a propensity to undergo genetic changes that can lead to drug resistance, a finding that has implications for future therapeutic strategies