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Improving the differentiation of human pluripotent stem cells into functional haematopoietic stem cells, called 'iHSCs'
No full text© 2025 Ritika SaxenaThe generation of therapeutically useful haematopoietic stem cells (HSCs) from in vitro differentiated pluripotent stem cells (PSCs) has been challenging, due to our incomplete understanding of and ability to replicate the developmental processes that give rise to HSCs in the human embryo. HSCs emerge from the aortic floor in the aorta-gonad-mesonephros (AGM) region of the embryo between weeks 4-6 of human gestation. HSCs from the AGM can be distinguished from haematopoietic progenitors arising in the extra embryonic yolk sac during earlier waves of haematopoiesis by their selective expression of HOXA cluster genes.
Our laboratory has recently established a method for the generation of haematopoietic cells from PSCs in vitro that are capable of multi-lineage reconstitution and long-term engraftment in immune deficient mice. Using our established swirling embryoid body platform, work in this thesis has characterized intermediate differentiation stages during development of HSC-like cells in vitro, termed ‘iHSCs’, and further validated their function through transplantation experiments in immune deficient mice.
A HOXA5::tdTomato PSC reporter line generated in our laboratory was used to identify the development of a dominant HOXA+CXCR4+ALDH1A2+ mesoderm population after 3 days of differentiation that was analogous to a population found in a gastrulating human embryo. This work indicated that our in vitro derived mesoderm has a counterpart in the human embryo. HOXA5::tdTomato+ mesoderm subsequently generated HOXA5::tdTomato+ vasculature and haematopoietic cells, confirming the maintenance of the developmental ‘switch’ to HOXA-expressing trajectory regulated by early mesoderm patterning, and recapitulating the sequence of events during AGM haematopoiesis.
Similarly, we generated PSC reporter lines for RUNX1, the master regulator of haematopoiesis and the most reliable marker for haemogenic potential. HSCs emerge from haemogenic endothelial precursors via an endothelial to haematopoietic transition (EHT) and express RUNX1 throughout this stage. Using the RUNX1::mCherry reporter line, we benchmarked the emergence of key cell surface markers during AGM haematopoiesis in vitro. We observed that CD34+CD45-RUNX1::mCherry+ cells first appeared as a subset of CXCR4+ arterial endothelium, clearly indicating emergence of haemogenic cells from arterial precursors. We complemented cell surface marker expression with single cell resolved transcriptomic profiles, compared it to human embryo reference datasets and established a detailed roadmap for the generation of iHSCs in vitro. We also improved an existing EHT assay, increasing its haematopoietic efficiency, thereby generating a screening platform for future validation of conditions that enhance iHSC generation.
Finally, functional evaluation of iHSCs by transplantation into immune deficient mice showed high level multilineage engraftment in both adult and neonatal recipients. Recipient mice also contained a donor HSC compartment in the bone marrow that was enriched in RUNX1::mCherry+ cells, recapitulating key aspects of normal haematopoietic development and validating the RUNX1 reporter in vivo. By incorporating protocol modifications and using iHSCs generated later in the differentiation, we improved the frequency of engrafting HSCs in adult mice. We also found that neonatal mice provided a more sensitive transplantation model to detect engrafting HSCs.
Overall, we can now more faithfully recapitulate the developmental events during AGM haematopoiesis in vitro, allowing us to generate iHSCs that are capable of long term, multilineage reconstitution at a higher frequency. These findings bring us closer to our goal of generating a clinical source of HSCs for patients in the future
From Outbreaks to Evolution: Understanding Infectious Diseases Through Genomics
No full text© 2025 Mona Lisa TaoukUnderstanding the evolution and transmission of pathogens is vital for addressing the growing burden of infectious diseases in an interconnected world. This thesis applies pathogen whole genome sequencing (WGS) to explore the dynamics of disease transmission, demonstrate the utility of genomic surveillance during outbreaks, and refine phylogenetic methods to improve evolutionary analyses. Together, these studies bridge the gap between genomic research and actionable public health strategies, providing insights that are both scientifically novel and practically impactful.
In Chapter 2, a detailed investigation of Neisseria gonorrhoeae transmission across Victoria, Australia, identified 233 genomic clusters over five years, including several persistent clusters disproportionately involving heterosexual transmission networks. By integrating WGS with epidemiological data, the study quantified the impact of COVID-19 public health measures, revealing a 40% reduction in the effective reproductive number (Re) and a concurrent decrease in genomic diversity due to border closures. These findings highlight the resilience of certain transmission networks and provide a framework for targeted interventions in close-contact pathogens.
Chapter 3 showcases the pivotal role of genomic surveillance during the 2022 global mpox outbreak. Analysis of viral genomes revealed limited intra-host diversity and identified three non synonymous mutations shared among early Australian cases, suggesting common transmission chains. By characterising the virus’s evolutionary rate and minor variant patterns, this work provided actionable data that informed local outbreak response strategies. The findings demonstrate how timely, high-resolution genomic data can track viral adaptation and guide public health decisions during emerging infectious disease threats.
Chapter 4 advances phylogenetic practice by evaluating how single nucleotide polymorphism (SNP) filtering strategies influence phylogenetic accuracy. The comparative analysis of strict- versus soft-core alignments in bacterial datasets showed that stricter filtering can substantially alter tree topology and cluster definition, impacting downstream epidemiological interpretations. These results refine methodological approaches in genomic epidemiology, ensuring that phylogenetic analyses more accurately reflect pathogen evolution and spread.
Collectively, these chapters demonstrate how pathogen WGS can be applied to address distinct challenges across infectious disease research and public health: identifying and quantifying transmission networks, informing outbreak responses through real-time genomic surveillance, and refining phylogenetic methods to improve the accuracy of evolutionary inferences. By integrating genomic data with epidemiological context, this thesis provides a practical framework for using genomics to understand and respond to the spread of infectious diseases, contributing to more targeted and effective control strategies
Persecution of non-binary people in the Bangladesh/ Myanmar investigation
No full textIn this rewritten judgment, Claerwen O’Hara authorises the investigation into the situation in Bangladesh/ Myanmar in a manner which brings the experience of Hijra, transgender and intersex peoples to the forefront of the decision. Whilst the Chamber’s original decision only briefly references sexual violence against Hijra individuals, O’Hara discusses in depth the persecution of gender-diverse Rohingya people on the intersecting grounds of gender, ethnicity and religion. In doing so, O’Hara offers an interpretation of gender under Article 7(3) of the Statute which encompasses gender-diverse people and the socially constructed nature of gender
Fatty acids and fatty alcohol esters as novel markers of authenticity and extraction method of commercial avocado oil
No full textThe lack of regulations on avocado oil authenticity enabled producers to blend it with cheaper oils without declaring it on the label. This study explored fatty acids (FA) and fatty alcohol esters (FAE) as innovative markers of avocado oil purity and extraction method. Only 40 % of the samples met the FA standards. Regarding FAE, extra virgin samples showed a maximum concentration of 2674.12 ± 570.98 mg total FAE·kg−1 oil, significantly higher than the 640.98 ± 220.06 mg total FAE·kg−1 oil observed in refined samples, making FAE novel markers of extraction method. Furthermore, a canonical discriminant analysis using Wilk's statistic (Λ = 0.008, F126, 1028.2 = 8.87, p < 0.0001), based on fault concentrations (high, medium, low) of the allegedly pure samples, revealed that the high- and medium-fault clusters aligned closely with declared blends containing canola or safflower oils. FA and FAE are promising molecules to detect adulteration in avocado oil
A pilot multicenter randomized controlled trial on individualized blood pressure targets versus standard care among critically ill patients with shock
No full textBACKGROUND: Minimizing relative hypotension, or mean arterial pressure (MAP) deficit, by targeting patients' own pre-illness MAP (individualized MAP) during vasopressor therapy is a potential strategy to improve outcomes among ICU patients with shock. We conducted a prospective, open label, parallel-group, pilot RCT to assess feasibility and safety of this intervention compared to standard care. METHODS: Thirty-seven eligible patients, aged 40 years or older and receiving vasopressor support for shock, were randomly allocated to individualized MAP target (N = 17) or standard MAP target (N = 20) at two multidisciplinary ICUs in Australia and Ireland. Pre-specified endpoints were time-weighted average MAP-deficit (i.e., percentage difference between patients' pre-illness MAP and achieved-MAP), percentage time spent with > 20% MAP-deficit, major adverse kidney events (MAKE-14), 14-day and 90-day all-cause mortality, and cardiovascular adverse events within 28 days of randomization. All comparisons of efficacy outcomes were exploratory. RESULTS: The median MAP-deficit and percentage time with > 20% MAP-deficit with individualized MAP vs. standard MAP were 7% [interquartile range: 2-16] vs. 18% [9-23] (p = 0.048), and 8% [0-43] vs. 53% [14-75] (p = 0.03), respectively. MAKE-14 (2/17 (12%) vs. 4/20 (20%), p = 0.67), 14-day mortality (1/17 (6%) vs. 3/20 (15%), p = 0.61), 90-day mortality (2/17 (12%) vs. 4/20 (20%), p = 0.67) and cardiovascular adverse events were similar for both groups. CONCLUSIONS: This pilot RCT demonstrated that an individualized MAP target strategy was feasible to implement. No adverse safety signals were evident. These data and study procedures helped inform the design of a definitive RCT on the question of individualized MAP targets among critically ill patients with shock. STUDY REGISTRATION: ACTRN12618000571279
Hazardous gambling behavior is associated with amplified emotional reactivity to gambling outcomes
No full textBACKGROUND AND AIMS: Emotion dysregulation has been suggested to play a role in gambling-related harm, but past gambling research has typically assessed emotion dysregulation via self-report surveys rather than in a gambling context. Here, we sought to investigate how the severity of participants' hazardous gambling behavior was associated with their emotional reactivity and choice behavior within a simulated slot-machine task. METHODS: Participants (N = 100) recruited via Prolific completed a behavioral task involving repeated choices between two simulated slot-machines. When chosen, slot-machines could produce one of five outcome types (win/near-win/neutral/near-loss/loss). After each outcome, participants reported their subjective emotional valence. Emotion data were analysed using a beta-autoregressive computational model, allowing us to extract per-participant estimates of trial-by-trial emotional reactivity to different slot-machine outcomes. RESULTS: Correlation analyses revealed that people who engaged in more hazardous gambling behavior (higher PGSI scores) showed greater emotional reactivity to all slot-machine outcome types (all Spearman ρ > |0.31|, all p < 0.01, corrected for multiple comparisons). There were no significant associations between patterns of choice behavior and PGSI scores. DISCUSSION AND CONCLUSIONS: Within a simulated slot-machine task, individuals who engaged in more hazardous gambling behavior showed greater emotional reactivity in general (more positive emotional reactions to wins and more negative emotional reactions to unpleasant events such as losses and near-wins). These results are consistent with a model in which emotion dysregulation is a risk factor for gambling-related harm, and serve to validate this model in a more naturalistic setting
A thematic analysis of what Australians state would change their minds on climate change
No full textWhat do Australians believe would change their current opinions about climate change? In this study, we used audience segmentation analysis through the Six Americas Short Survey to identify groups of climate opinion holders within a representative sample of Australians. We had 4857 participants tell us what it would take to change their current opinions about climate change and leveraged OpenAI’s Generative Pre-Trained Transformer (GPT) to identify the presence or absence of themes (Nothing, Evidence and Information, Trusted Sources, Action, and Unsure) and subthemes in their responses. GPT performed at near-human levels, proving to be a highly useful tool for thematic analysis. Our analyses revealed that strong climate denialists and believers tended to display greater dogmatism, with increased likelihood of stating that nothing would change their mind and lower likelihood of being unsure. Results also highlighted the need for diverse forms of evidence and information and the importance of trusted sources of information across audience segments. These findings provide support for GPT’s utility in managing large datasets in the social sciences and offer participant-informed insights into climate opinion change
Boys Do Cry: a randomised controlled trial testing the effects of a music video promoting help-seeking for mental health difficulties in Australian men
No full textBACKGROUND: In Australia and internationally, it is men who predominately die by suicide. Men are less likely than women to seek help for their mental health difficulties and this may contribute to their higher suicide rates. We created a 4-minute music video encouraging Australian men to seek help for mental health difficulties (Boys Do Cry). We aimed to assess in a randomised controlled trial (RCT) whether the Boys Do Cry video increased men's intentions to seek help for mental health difficulties from baseline (T1) to post-intervention (1 week = T2). METHODS: We conducted an online single-blind, two-arm RCT comparing the effects of Boys Do Cry against a control video. Analyses were undertaken on an intent-to-treat basis using linear mixed effects models with variables for trial arm, occasion of measurement and their interaction. Intervention effectiveness was assessed by comparing the mean difference between arms in change of the total score on the General Help-Seeking Questionnaire (GHSQ) from T1 to T2. RESULTS: 476 participants were randomised (intervention = 243; control = 233). At T1, GHSQ means were similar (intervention = 45.28; control = 45.70). After viewing the videos, GHSQ means increased in both arms (intervention = 47.33; control = 46.59), with no evidence of a difference in scores at T2 (modelled mean difference = 0.62, 95% CI -1.11 to 2.35, p = 0.485). Similar results were observed for all secondary outcomes. No adverse events were observed. CONCLUSIONS: Boys Do Cry demonstrated some evidence of a positive effect on help-seeking intentions among Australian men; however, so too did the control video, and no significant difference was observed. TRIAL REGISTRATION: ANZCTR No. 2,621,001,008,819. Registered 30 July 2021
Collaborative coding in inductive content analysis: Why, when, and how to do it
No full textInductive content analysis (ICA) is a useful method for analyzing qualitative data in genetic counseling research. It is particularly relevant when the goal is to examine and improve practices or develop recommendations. Although ICA can be undertaken by a single analyst, ideally there is involvement of multiple analysts (or co-coders). Co-coding can bring many benefits to qualitative analysis that sits within a constructivist paradigm, including developing a representation of the data that is not only understandable to more than one individual but also richer and more nuanced. It also provides an opportunity for mentoring more junior researchers and can be an efficient way to analyze large datasets. However, co-coding requires important planning and consideration, and there is currently a paucity of clear guidance. In this paper, we provide an outline of the small body of existing literature on this topic and propose six flexible step-by-step components of our approach to co-coding in ICA, based on our own work. We have utilized it to analyze reporting practices and perspectives for diagnostic genomic sequencing, informed consent for genetic testing, data sharing and storage, and genomic newborn screening, among other topics. To illustrate these components, we present some example vignettes to show how these procedures can be applied in different scenarios and with different analysts