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    Protein Engineering of Enzymes for Plastic Biodegradation
    (2024) Joho, Yvonne
    Plastics are increasingly prevalent in our daily lives, and the management of plastic waste has developed into a significant environmental concern. Conventional recycling methods fall short of achieving a genuinely circular plastic economy. Enzymatic depolymerisation represents an intriguing opportunity for addressing the limitations of chemical and mechanical plastic recycling. Employing enzymatic hydrolysis to depolymerize post-consumer plastic waste holds the potential to establish a true circular economy. Nevertheless, it is essential to note that natural enzymes, in their current state, are not yet suitable for industrial applications; significant enhancements are required to make them viable for large-scale purposes. The discovery of new plastic-degrading enzymes opens new possibilities for enzyme engineering. A breakthrough occurred in 2016 with the discovery of Ideonella sakaiensis, the first bacteria known to be capable of using PET as its primary carbon source and completely degrading it into its monomers. The initial catalytic step in this process involves IsPETase, an enzyme with great potential for industrial applications. The doctoral thesis aims to improve IsPETase limited thermal stability and enzymatic activity. To achieve this, a combinatory approach of engineering strategies was employed, including ancestral sequence reconstruction (ASR), structure-based rational design, and directed evolution (DE). ASR is a method to understand the evolutionary history of a particular enzyme and is increasingly used for protein engineering due to the beneficial properties of ancestral enzymes. Our study resulted in a better understanding of the path of evolution of IsPETase and resulted in ancestral enzymes with enhanced properties. Simultaneously, a structure-based approach using iterative rounds of engineering and rational design was performed, resulting in the most significant improvement of IsPETase. Finally, to further improve IsPETase this thesis aimed to employ directed evolution. First, this thesis reviewed the successful approaches in improving plastic-degrading enzymes via directed evolution and highlighted the limitations of the research such as other properties including the protein solubility that has not been explored yet. Second, to address this research gap, a directed evolution was employed using a high throughput approach with FACS sorting and a Split-GFP system to screen for active and more soluble variants. Overall, we combined the beneficial mutations from each approach resulting in the Combi-PETase, a variant that has a significantly improved activity, thermal stability of 27 degrees and up to 25-fold in protein yields.
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    Elucidating the variability and transcriptional properties of human rDNA repeats using long-read sequencing technologies.
    (2024) Weiss, Emiliana
    Background The sequencing and assembly of the human genome have been instrumental in unravelling the regulation of gene expression and understanding the role of genome organisation. Yet, significant gaps remain, particularly concerning ribosomal RNA (rRNA) and genes (rDNA), which are organized into extensive repeat arrays and play dual roles: encoding rRNAs essential for ribosomal assembly and function as well as maintaining genome stability. Additionally, exploration of rDNA arrays within the human genome has long been hampered by the inherent limitations of short-read sequencing technologies, which fail to capture the full complexity and linear continuity of these regions. This thesis confronts challenges posed by the repetitive and variable nature of rDNA regions, especially within the intergenic spacer (IGS) regions, which have been historically difficult to analyse due to their sequence diversity. Methods To better understand rDNA structure, we implemented advanced techniques such as head-to-tail distance analysis on the HG002 sample, revealing significant unit length variability and genetic diversity, including INDELs. Additionally, our use of a polishing strategy on Nanopore ultra-long reads with PacBio HiFi reads aimed to enhance base accuracy, but also highlighted the challenges of maintaining crucial genetic variants for accurate epigenetic analysis. Results and Conclusions The study presented here represents an important advance in the genomic analysis of the rDNA by employing long-read sequencing technologies to examine the rDNA arrays in their entirety, from promoter regions to IGS areas. Our findings emphasize the need for meticulous rDNA annotation and balance in sequencing to preserve essential variations crucial for downstream applications. This includes understanding the implications of copy number variation (CNV) and sequence diversity, where sequencing technology and coverage significantly influence accuracy. We also demonstrated that low sequencing coverage and adaptive sampling can lead to underrepresentation of rDNA regions, affecting CNV estimates. Moreover, our study extends to the analysis of CpG methylation patterns within rDNA, uncovering distinct methylation states-methylated, unmethylated, and semi-methylated-each with unique regulatory roles. Machine learning models have further shown that sequence content is predictive of methylation status, underscoring the intricate relationship between genetic sequence and epigenetic regulation. Our study highlights a non-random nature of methylation within the rDNA locus by unveiling distinct methylation and association patterns between neighbouring rDNA units sharing the same methylation type. Significance and Future Directions Overall, our study provides a deeper understanding of rDNA. This study establishes a foundation for further research by showcasing the capability of long-read sequencing to advance genomic studies, particularly in analysing complex and repetitive regions. This advancement is a crucial step toward addressing the knowledge gap in understanding specific, poorly characterized regions of the human genome. Our study will be relevant to researchers studying diseases such as cancer and ribosomopathies - both of which result from abnormalities in ribosome function. Future studies could build on the work presented in this thesis to facilitate the identification of specialized ribosomes, which may differ in their role in protein synthesis under normal and disease states.
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    Limiting Hyperinsulinaemia To Prevent Type 2 Diabetes
    (2024) Shamoon, Muhammad
    The rising prevalence of type 2 diabetes (T2D) is a major concern globally. This thesis focuses on severe insulin resistant subtype of T2D (SIR-T2D) in which hyperinsulinaemia and obesity, accompanied by insulin resistance, occur prior to onset of diabetes. We hypothesised that interventions aimed at curtailing the development of hyperinsulinaemia would prevent SIR-T2D. Main aims were to determine in a mouse model of SIR-T2D if (i) intermittent fasting (IF) and (ii) deficiency of the system B(0) neutral amino acid transporter AT1 (B0AT1, Slc6a19 gene) could prevent hyperinsulinaemia and T2D. NODk mice, derived from non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D) but protected from T1D due to congenic replacement of Idd1-H2g7 MHC II locus with an autoimmune diabetes low risk Idd1-H2k MHC II locus, were used. Male NODk mice fed a Western diet (WD) develop a SIR-T2D like phenotype characterised by excessive weight gain, hyperinsulinaemia, dyslipidaemia, and severe hyperglycaemia. To investigate IF, male NODk mice were fed chow diet (CD), WD, or WD with 15 h of fasting twice per week (WD+IF) from 6 to10 weeks (short-term (ST) protocol) and 6 to 30 weeks (long-term (LT) protocol) of age. Metabolic characteristics of mice were serially assessed, including i.p.GTT after 3 and 17 weeks on diet, histological examination of pancreases and livers, and the incidence of diabetes by 30 weeks of age. To investigate B0AT1 deficiency, male NODk.Slc6a19 wild type (WT) and KO mice were fed CD or WD from 8 weeks of age for 5 days-WD challenge (WDC protocol), and CD or WD in ST and LT protocols as for the IF studies. Additional readouts included urine amino acid profiles, and on day 4 of WDC protocol, i.p.GTT and oral GTT testing. IF attenuated WD-induced whole body and fat depot weight gains, hyperinsulinaemia and hypertriglyceridaemia, improved glucose tolerance, and prevented SIR-T2D. WD resulted in increased pancreatic fat deposits, enlarged islet size, frequent pseudo-rosette structured islets, presence of intracytoplasmic inclusion bodies in endocrine cells, with some apoptotic endocrine cells. IF lessened islet enlargement and prevented pseudo-rosette formation. Peri-islet and peri-ductal inflammation were present in both WD and WD-IF mice pancreases. Although WD feeding did not elevate serum transaminases, marked hepatic steatosis, hepatocyte ballooning and mild to moderate hepatic lobular inflammation were found, partially mitigated by IF. Finally, with WD-feeding there was not much evidence of renal function impairment. The phenotype of the CD-fed NODk.Slc6a19 KO compared to WT mice included marked aminoaciduria (confirming B0AT1 deficiency), normal body weight, behaviour and appearance at 6 weeks of age, mildly reduced body and fat depot weights at 30 weeks of age, normal glucose tolerance, reduced basal and glucose stimulated insulinaemia, higher basal and glucose-stimulated GLP-1 concentrations and markedly increased plasma FGF-21 concentrations. As for IF, B0AT1 deficiency attenuated WD-induced body and fat depot weight gains, hyperinsulinaemia and hypertriglyceridaemia, improved glucose tolerance, and prevented SIR-T2D. GLP-1 and FGF-21 concentrations remained higher in the KO-WD compared to WT-WD mice, and curtailed WD-induced falls in serum adiponectin. CD-fed KO mice had normal pancreas and liver histological appearances. In WD-fed KO compared to WT mice pancreases, fat deposition, inflammation, pseudo-rosette islet structures and endocrine cell apoptosis were less evident. WD-induced changes in liver were not different between two genotypes. Finally, KO mice had heavier kidneys, but renal function was normal in both genotypes and not adversely affected by WD-feeding. In conclusion, both IF and B0AT1 deficiency attenuated WD-induced hyperinsulinaemia and completely prevented diabetes in WD-fed male NODk mice. IF and pharmacological targeting of B0AT1 are promising interventions for the prevention of SIR-T2D.
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    Saproxylic Insights: Unveiling Gut Microbial Dynamics in Forest Floor Invertebrates and Their Frass in the Tallaganda Region, NSW
    (2024) Forteza, Imelda
    Host-microbe associations have been, and continue to be, an area of great interest. Even within a well-defined and well-studied gut microbial community, little attention has been paid to how the dynamics of the host's gut environment may undergo shifts, potentially exhibiting concurrent variations along the spectrum of microbial independence within that particular microenvironment, ranging from loose and opportunistic host-microbe associations at one end to intimate and specific associations at the other. This study explored factors influencing gut microbial assembly and diversity among forest floor invertebrate hosts: saproxylic velvet worms, saprophagous wood cockroaches, funnel web spiders, and their immediate environment, the frass. All individuals used in this study were found in the Tallaganda Region of New South Wales, Australia. This study utilized the V4-V5 region of the 16S rRNA gene sequences to characterize the gut microbial structure and composition of the hosts in the wild, and in velvet worms along a gradient of semi-isolated refugia. A six-week feed experiment on velvet worms, wood cockroaches, huntsman and funnel web spiders showed higher gut microbial diversity in wood cockroaches than in velvet worms and spiders combined. Moreover, the microbiome of the fed groups seemed to be unaffected by feeding regimes, which included feeding or fasting. Low microbial diversity and highly variable gut microbial communities were observed across all velvet worms, funnel web, and huntsman spiders' gut samples. The core bacterial phyla across all hosts' gut samples were Proteobacteria, Actinobacteria and Firmicutes, with prevalence ranging from 40 -95 %. Interestingly, all these three taxa were also found in frass samples. In summary, combined with the results of the feeding experiments, while the core microbiome has been observed in at least 40% of the samples of interest, the likelihood of a persistent core microbial population in these species is rather low, in contrast to the wood cockroach, which appears to have a well-established community of gut microbes. These findings suggest additional research is essential to understanding the complexities of gut microbial communities and highlight the hosts' ability to dynamically engage with transient microbial communities within their direct surroundings, dead wood microhabitats. This nuanced understanding provides an ecological insight into the host-gut microbial associations and the dead wood microenvironment. It contributes to the ongoing discourse on the ecological dynamics of host-microbe associations, emphasizing the contextual significance of microbial independence along a spectrum of host-microbe interactions. These findings illuminate the complexities inherent in the interplay between hosts and their microbial communities, offering valuable insights into dead wood microhabitats.
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    Engineering the cis and trans DNA cleavage of CRISPR-Cas12a orthologues
    (2024) Newman , Anthony
    CRISPR-Cas (Clusters of Regularly Interspaced Short Palindromic Repeats - CRISPR associated) systems are host defences of prokaryotes that have been harnessed for biotechnology applications, due to their programmable nuclease activities. CRISPR-Cas12a is the signature effector protein of type V-A CRISPR-Cas systems and is widely used for both gene editing and molecular detection. Cas12a orthologues have high structural similarity and low sequence similarity, and a conserved mechanism of CRISPR-RNA (crRNA)-guided double-stranded DNA cleavage with a single RuvC nuclease domain. Cas12a has a bi-lobed structure, where recognition (REC) and nuclease (NUC) lobes respectively recognise and cleave target DNA. Cas12a has highly specific target DNA cleavage (in cis), which is harnessed for genome engineering. After target DNA cleavage, Cas12a unleashes non-specific degradation of single stranded nucleic acids (in trans), which has been repurposed for molecular detection technologies. Three natural Cas12a orthologues (As - Acidaminococcus sp. BV3L6, Lb - Lachnospiraceae bacterium ND2006, Fn - Francisella tularensis subsp. novicida U112) have been well characterised, and widely used for genome editing and molecular detection. AsCas12a and LbCas12a are efficient genome editors of mammalian cells, LbCas12a has the most robust trans cleavage activity for molecular detection, while FnCas12a has both low trans cleavage and editing efficiency. This raises the question: what protein structure and sequence features drive these differences? Stable RNA and DNA interactions are crucial to the crRNA-programmable DNA cleavage activity of Cas12a, and unknown interactions drive non-specific trans cleavage. In this thesis, I used structural alignments of three Cas12a orthologues (AsCas12a, LbCas12a, FnCas12a) to inform mutagenesis of divergent structural motifs and key residues, to determine what drives their different cis and trans cleavage activities. Thus, I characterised in vitro and in vivo the role of crRNA binding moieties, of REC lobe-crRNA:DNA heteroduplex stacking interactions, and of protein-DNA electrostatic interactions near the RuvC nuclease domain. Although crRNA binding is required for gene editing activity, engineering stronger crRNA binding was not sufficient to rescue the low editing activity of FnCas12a. Furthermore, although relaxation of REC-heteroduplex stacking interactions quickened the rate-limiting step of target DNA cleavage, this caused weaker crRNA binding and decreased activity in vivo for the three Cas12a orthologues. A hitherto uncharacterised 'NUC loop' was identified as a divergent structural element between Cas12a orthologues. This anti-parallel beta sheet structure extends from the NUC lobe towards the REC, potentially interacting with the crRNA:DNA target heteroduplex. Disruption of this loop was deleterious to the function of FnCas12a and LbCas12a, while modestly enhancing the gene editing of AsCas12a. Finally, key residues in the NUC were found drive trans cleavage through electrostatic interactions, which were neutralised by increasing ionic strength. I exploited this observation to rationally design Cas12a orthologues and optimal buffer conditions with enhanced trans cleavage activity, decreasing DNA detection times by 2 to 3-fold. In addition, these engineered Cas12a orthologues had enhanced editing efficiencies in human cell lines. Overall, this inter-orthologue survey highlights the structural and sequence features that drive the different properties of Cas12a orthologues. This work details how DNA cleavage mechanisms subtly differ between orthologues and provides a blueprint for rational engineering of cis and trans cleavage.
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    Flame Synthesized Nanoparticles and Miniaturized Opto-electronic Devices for Advanced Gas Sensing
    (2024) Abideen, Zain Ul
    Advancements in gas sensing technology are crucial for enhancing environmental safety, industrial monitoring, and health diagnostics. This research explores the transformative potential of flame-synthesized metal oxide nanoparticles and miniaturized opto-electronic devices in creating highly sensitive and selective gas sensors. The study delves into optimizing flame spray pyrolysis for developing nanostructured materials, with a focus on manipulating process parameters to refine the physical properties of nanoparticles for enhanced sensing capabilities. A pivotal advancement is the promising strategy of engineering oxygen vacancies in thick semiconductor films using deep ultraviolet photoactivation. This method significantly enhances the room-temperature detection capabilities for volatile organic compounds, exemplified by the enhanced sensitivity and decreased response times of ZnO sensors to ethanol. Specifically, the introduction of oxygen vacancies by low temperature deep ultraviolet photoactivation leads to about a 58% increase in ZnO sensitivity, coupled with a 51% and 64% reduction in response and recovery times, respectively. The approach demonstrates a broader potential for tuning electronic structures and surface activities of semiconductor sensors, achieving lower detection limits (as low as 2 ppb) and improved selectivity at relatively low operating temperatures. Further innovations are realized in the engineering of three dimensional nano-heterojunction networks. By incorporating oxygen vacancies into NixOy-ZnO nanoscale heterojunctions through deep ultraviolet photoactivation, the sensing performance is significantly boosted. This results in an 88% increase in sensitivity to ethanol and a 30-fold enhancement in selectivity against a range of volatile organic compounds at room temperature. The heterojunctions, characterized by their high porosity and efficient charge separation, facilitate deeper penetration and interaction with target gas molecules, leading to unprecedented sensitivity and selectivity levels. Theoretical analyses corroborate these findings, showing a substantial increase in analyte adsorption energy due to the presence of oxygen vacancies. The research further culminates in the development of an innovative dual-sensing approach through the integration of plasmonic and electrical sensing in a single metamaterial sensing device. This multifunctional sensing platform, combining chemiresistive and plasmonic techniques, is a significant leap forward in gas detection technology. It leverages the unique properties of a zinc oxide and gold nanoparticle-based metamaterial, achieving simultaneous detection and discrimination of various volatile organic compounds. The dual-sensing system is further augmented by machine learning models capable of accurately predicting gas types with 32% accuracy and concentrations with a coefficient of determination (R2) of 0.96. This approach not only demonstrates exceptional sensitivity and selectivity but also opens new avenues for smart sensor applications in environmental, industrial, and health sectors. Overall, this research represents a substantial contribution to the field of chemical and gas sensing technology, offering innovative approaches for the development of highly sensitive, selective, and multifunctional sensors. The findings underscore the potential of nano-engineered materials in advancing smart sensor technologies, with significant implications across various domains including environmental monitoring, industrial safety, and health-related applications.
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    Packing materialities: marketising trans prosthetics
    This thesis is concerned with the practice of packing undertaken by trans men and transmasculine people, and with the commercialisation of products designed for use in this practice. To pack is to wear an object in your underwear to emulate in some way or shape a penis, and to provide the feeling or impression of genitals that the wearer does not possess. This thesis charts the development of a market for products to be used in this practice: from simple foam padding to hand-crafted silicone packers, to multifunction prosthetics that can be used for sex and 'standing to pee'. I describe these products collectively as 'packsthetics', a portmanteau of packer and prosthetic, because, as I demonstrate, it is difficult to make clear distinctions between these two terms as either product descriptions or in their use in practice. Employing Michel Callon's (2021) framework of 'market agencement' to understand the development of a packsthetics market, as well as concepts from Actor-Network Theory and queer theory, I demonstrate how packsthetics are enacted through their marketisation. This is crucial to the ability of packsthetic products to become someone's genitals. Attending to how packsthetic products are commercialised, I trace a heterogeneous web of relationships, with both human and non-human elements, that cohere to enact the packsthetic. These include packsthetic histories, websites, silicone and pigments, YouTube videos and associated reviews, an imaginary of the trans 'community', and the normative penis. Understanding packing not as a discreet and individualised practice, but as enacted through such an assemblage, provides an opening for deeper consideration of packsthetics' materiality, particularly how the packsthetics market tries to 'realistically' represent an idealised version of the penis. I illustrate how this 'realism' and its mimetic origin in the penis are materially constructed and culturally constituted. Understanding both the packsthetic and the penis as enacted provides an opening to challenge the 'naturalness' of genitals and highlights how embodied sex and gender for all of us-trans and non-trans both-is always already interwoven with the material world.
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    Functional characterisation of Nudix hydrolase effectors from phytopathogenic fungi
    (2024) McCombe, Carl
    Crop production is constrained by plant-pathogenic fungi, threatening the ability of society to feed a growing global population. Central to the intricate molecular interactions occurring between plants and pathogenic fungi are small pathogen-secreted proteins (effectors). Understanding the functions of effectors offers a potential pathway towards unravelling the sophisticated interplay that occurs between cropping plant species and their devastating fungal pathogens. In this thesis, I have identified and characterised effectors from pathogenic fungi that are functional Nudix hydrolase enzymes. AvrM14 is a Nudix hydrolase effector from Melampsora lini (flax rust). To elucidate AvrM14's enzymatic activity, an exhaustive substrate screen was conducted, revealing AvrM14's remarkable substrate selectivity, the effector only hydrolysed m7Gp5G (P1-(5'-7-methyl-guanosyl)-P5-(5'-guanosyl)-pentaphosphate). While m7Gp5G does not occur naturally, 7-methyl guanosine (m7G) is a distinctive molecular structure from the 5' cap of eukaryotic mRNA. I demonstrate that AvrM14 can remove the protective 5' cap from mRNA, and this activity requires a glutamate amino acid within the putative active site. Notably, homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage and mRNA decapping activity is conserved in related Nudix hydrolase effectors across the Melampsora genus. When expressed in planta, AvrM14 inhibits the immune-related reactive oxygen species burst and hypersensitive cell-death response. Significantly, the same pivotal glutamate amino acid is indispensable both for the mRNA decapping and the immune-suppressive activities. The findings support a model whereby the Nudix hydrolase effectors from Melampsora spp. decap plant mRNA to suppress immune responses and ultimately promote the infection process. Nudix hydrolase effectors from Magnaporthe and Colletotrichum spp. are more closely related to each other than the mRNA decapping effectors of Melampsora spp. Substrate screening with purified effector proteins revealed that the Magnaporthe and Colletotrichum effectors hydrolyse inositol pyrophosphate signalling molecules. I determined the crystal structure of a M. oryzae Nudix effector (MoNudix), identifying remarkable similarity to Homo sapiens diphosphoinositol polyphosphate phosphohydrolase 1 (HsDIPP1). HsDIPP1 also hydrolyses inositol pyrophosphates, and using targeted mutagenesis I demonstrate that like HsDIPP1, MoNudix utilises basic amino acids to facilitate inositol pyrophosphate binding and hydrolysis. Inositol pyrophosphates serve as plant messengers for shifts in phosphate availability: diminished inositol pyrophosphate levels activate phosphate starvation response transcription factors. Consistent with this, the production of Nudix hydrolase effectors from M. oryzae and Colletotrichum spp. in Nicotiana benthamiana elevated the expression of phosphate starvation responsive genes as measured by qPCR. Phosphate starvation induction requires amino acids involved in the hydrolysis of inositol pyrophosphates in vitro, and all qPCR data was corroborated using an innovative promoter/reporter system developed during the study. When the expression of one inositol pyrophosphate hydrolysing effector from M. oryzae was reduced using RNAi, there was a significant decline in the pathogen's virulence on rice. Collectively, the results indicate that Magnaporthe and Colletotrichum Nudix hydrolase effectors target inositol pyrophosphate signalling molecules to initiate plant phosphate starvation responses. To date, the virulence functions of fungal effectors have remained largely unidentified. Through this study, I have established that the predicted Nudix hydrolase effectors from pathogenic fungi act as enzymes, likely targeting mRNA caps and inositol pyrophosphates. All together, these data advance our understanding effector function and the complex interplay occurring between pathogenic fungi and their plant hosts.
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    Rights under the 1997, 2006 and 2007 Thai Constitutions: Influences on, and Uses by, the Advantaged
    (2024) Bishop , Sarah
    This thesis draws on analysis of Thai-language constitution drafting records and court decisions to explore the Thai experience with rights recognition and litigation under the 1997, 2006 and 2007 Thai constitutions. In doing so, it focuses especially on the rights to equality, non-discrimination, human dignity and life and body; and the influence on the recognition of those rights and uses of those rights made by those who were advantaged. The motivation for this exploration came from a sense that existing portrayals of the Thai experience with constitutional rights recognition and litigation in that period were limited in their scope and often inaccurate, and that the lack of breadth and the inaccuracies in the portrayals could reflect broader distortions in the study of law in contexts like that in Thailand that have been observed by others and could have undesirable real-world consequences. Within the Thai context this thesis is significant primarily for four reasons. The first is because it shows the presence of dynamics behind the constitutional recognition of rights that suggest a much greater significance for that recognition than do most existing analyses. The second is because it shows an extent of engagement with the rights that is much greater than is reflected in the existing literature, and so contributes to challenging ideas of a particular Thai aversion to engaging rights. The third is because it shows a variety of engagement with the rights and constitutions, and impacts of that engagement, that are more diverse than those reflected in the existing literature. And the fourth is because it shows the limiting of the potential of and for rights litigation in Thailand in that period to have not been inevitable or necessarily a product of court failure, but likely a product of the choices made by constitution drafters and the pressures and incentives created by how the rights were used. Beyond the Thai context, the thesis is significant primarily for five reasons. The first is for the response it provides to distortions in the study of law in contexts like that in Thailand. The second is for the ways it draws on analysis of constitution drafting records to explore influences of the advantaged on the recognition of rights. The third is for the attention it brings to factors that contribute to, and to the effects of, court non-interventions. The fourth is for the exposure it brings to types and effects of uses of rights by the advantaged that are not emphasised in the existing literature on uses of rights by that group. And, the fifth, especially relevant for the study of less active courts and dejudicialisation, is for the attention it brings to reasons why and ways that courts might choose to limit their own roles.
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    Memories of Repatriates from Japan to North Korea: Stories of Separated Families and Narratives of Reunification
    (2024) Lee, Joowhee
    This dissertation examines the lives and family histories of Zainichi Koreans who boarded repatriation ships from Japan to North Korea about 60 years ago in the context of the postcolonial reshaping of East Asia and the Cold War. The first repatriation ship departed from Japan to North Korea on 14 December 1959. In total, 93,340 people--including 6,730 Japanese people (about 1,800 Japanese spouses married to Korean men and their children)--were repatriated from Japan to North Korea between 1959 and 1984. The thesis focuses on the experiences of people who moved to North Korea as teenagers (referred to here as the "1.5 generation" of returnees--as distinct from the first generation, who chose to move as adults, and the second generation, who were born in North Korea). The vast majority of the first generation originated from the southern part of Korea, but nonetheless decided to move to North Korea with their families. Existing research commonly views the repatriation as a symbolic and historical event in the post-war political order of Northeast Asia: a region which was at the intersection of Cold War and Hot War conflicts and postcolonial processes. Existing studies on escaped Zainichi Korean returnees (those who have left North Korea as refugees) also explore how they were influenced by the perspectives and ideologies of postcolonial nation-states (South Korea, North Korea or Japan) and often examine this history within the frame of the Cold War inter-state competition. Returnees are an example of an excluded group who do not fit into the frame of dominant memories of the post-war postcolonial states. Drawing on interviews conducted with members of the "1.5 generation" who have escaped from North Korea and are now living in Japan and South Korea, my research approaches returnees' life histories and family histories as historical (re)constructions that are shaped by their interactions with the evolving structures of inclusion and exclusion in Japan, South Korea and North Korea. Returnee memories are testimonies about repatriation and life in North Korea, but the ways in which the returnees create meaning within those memories are also configured by the political and social contexts in which they were (and are) living. By creating space to listen attentively to the contradictions within the lives and family histories of individuals--rather than framing these individuals and their stories through national, ethnic, or ideological lenses--it is possible to shed new light on the structures that supress crucial aspects of these memories. This thesis thus treats the Cold War/Hot War, not as a past event, but rather as a structural force that influences the microlevel and private spheres of individuals and families in Northeast Asia today, and proposes that documenting the memories of forgotten people from a holistic point of view is a practice which may help to develop a post-Cold War imagination.
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    Last Letters Home: The Forgotten Voices of Iranian Volunteer Soldiers During the Iran-Iraq War (1980 - 1988)
    (2024) Nooraninejad, Setayesh
    Last Letters Home: The Forgotten Voices of Iranian Volunteer Soldiers During the Iran-Iraq War (1980 - 1988) Abstract This study focuses on the writings of Iranian volunteer soldiers during the Iran-Iraq War of 1980-88, particularly their will letters. The research explores the motivations and objectives of these soldiers by analyzing their final correspondences, which served as a means to convey their last wishes and messages to their families and loved ones. The act of will-writing allowed them to make sense of their experiences in the war and address various aspects such as debts, fears, hopes, and the desire to be remembered. Writing held significant importance for these soldiers as they confronted the possibility of death. The study highlights the shift from oral wills to written wills during the war, even by semi-literate and illiterate soldiers. It argues that the war played a decisive role in popularizing the culture of will-writing among Iran's lower-middle class. The emergence of this new form of writing offers insights into individual and national identities at a grassroots level. It explores the impact of the state's policy of 'Exporting the Revolution' on soldiers' nationalist motivations and their relationship with the concept of martyrdom. The thesis divides the war into different phases and examines the soldiers' national and individual identities within each phase. It discusses the soldiers' secular anti-imperialist nationalism, their use of Islam as a practical ideology to achieve independence, and their alignment with the discourse of exporting revolution. The soldiers' letters also reflect their regional attachments and friendships, strengthening their sense of national loyalty. Furthermore, the research delves into the increasing emphasis on martyrdom in soldiers' letters, particularly in the later stages of the war. It explores how martyrdom became a means of achieving spiritual victory and compensating for military defeats. The study also analyzes the soldiers' anxieties about death and the challenges associated with pursuing martyrdom, including the need for self-purification and restitution for past sins. Overall, the study highlights the significance of will-writing during the Iran-Iraq War, shedding light on the motivations, identities, and experiences of ordinary volunteer soldiers. It emphasizes the transformative power of the war in shaping the culture of will-writing and the emergence of lower-class writers during this period. The research fills a gap in the scholarly focus on popular writing and provides insights into the experiences of soldiers and the socio-political context of the war.
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    Executives' Goal Orientation and Characteristics: Influences on Firm Decision-Making, Search, and Performance
    (2024) Orbes Cervantes, Irina
    Upper echelons theory (UET; Hambrick 2007; Hambrick & Mason 1984) proposes that a firm is a reflection of its top managers (Lin & Lin 2019; Richard, Wu & Markoczy 2019). Specifically, executive leaders shape firm strategy and develop strategic responses to enhance search, knowledge acquisition and performance (Hambrick 2007). Building on this logic, I explore the roles of chief executive officers (CEOs) and top management teams (TMTs) as strategic decision-makers and the architects of firms' strategies (Calori, Johnson & Sarnin 1994). I seek to understand the characteristics and attributes that influence their choices and decisions. These attributes likely influence their behaviour, preferences and motivations and the lenses through which they assess information. In turn, these factors play an important role in predicting firms' overall strategic directions and performance consequences (Bromiley & Rau 2016; Hambrick, Humphrey & Gupta 2015). I analyse this complex decision-making process by studying two important executive attributes: (1) CEO goal orientation, which concerns the types of goals that individuals set in achievement situations, their areas of focus and how these goals influence self-regulation (Dweck 1986) and (2) TMT functional diversity, which refers to the degree of different career experiences among TMT members (Carpenter, Geletkanycz & Sanders 2004). This thesis comprises three main chapters, which consider large publicly traded US firms (S&P 500) from 2006 to 2019. CEO and TMT measures are used as proxies for firm-level analysis. In the first study, I describe the process of developing and validating a content analysis dictionary of the goal orientations of CEOs. This process encompasses two dictionaries, each based on a distinct construct of goal orientation: learning orientation and performance orientation. Their formulation was grounded in theoretical foundations and an analysis of the frequency of words in surveys as well as shareholder letters. Following a validation process, these dictionaries demonstrated their efficacy in accurately predicting CEO goal orientations. The second study examines the impact of CEO goal orientation on firm search behaviour in response to performance shortfalls. This study reveals that learning oriented CEOs who face historical performance shortfalls and performance oriented CEOs who experience social performance shortfalls tend to increase their search. This underscores the notion that individual motivations affect leaders' attention, leading to the initiation of search when experiencing performance shortfalls. The third study investigates how CEO learning orientation and TMT functional diversity interplay in firms' decision-making processes, focusing on knowledge acquisition strategies and firm performance. The findings suggest that CEO learning orientation and TMT functional diversity strengthen the relationship between knowledge acquisition and firm performance. However, when the two are at a high level, the strengthening effect is not as pronounced. Further, when the two are at a low level, the relationship weakens. In conclusion, this study highlights the ways that CEOs' psychological attributes and TMTs' characteristics influence firm outcomes, advancing UET. By integrating psychological and sociological theories, this work paves the way for a more comprehensive understanding of CEOs' motivations, TMTs' diversity, leadership behaviours and their collective impact on strategic outcomes. The data for this study were gathered from shareholder letters, annual firm reports, industry-level data and executive profiles from various databases.
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    Investigating factors influencing low fertility in Asia
    (2024) Tan, Jolene
    In many high-income Asian societies, low fertility and closely related trends, such as increasing rates of singlehood, delayed marriage and rising age at first birth, have been widely observed and studied. However, many significant contributing factors in the domains of socioeconomic status, attitudes and culture remain that have not been comprehensively and empirically studied. These contextual factors are important because they provide insights into the environment in which family processes occur and help us understand the motivations behind diverse family-formation behaviours. The theoretical and empirical objectives of this thesis focus on the interplay of individual, social and contextual factors that influence reproductive behaviours. This approach allows us to look beyond mere fertility numbers to gain a deeper understanding of the underlying dynamics that shape family-formation behaviours. By considering how different factors operate in specific contexts to influence marriage and fertility patterns, the findings that arise from this thesis may inform the development of family support policies tailored to an array of family-planning needs. This thesis comprises five published papers that present evidence regarding the impact of socioeconomic, attitudinal and cultural factors on marriage and fertility, with specific emphasis on South Korea, Singapore and Taiwan. These papers draw on a wide range of cross-sectional, retrospective and longitudinal data. Conceptually, the various factors are situated at different levels - the micro level (individual or couple), the meso level (family) and the macro level (institutional) - to provide a holistic perspective on fertility. The papers employ a combination of analytical methods, including latent class analysis, survival analysis, multilevel regression analysis, and dominance analysis, to investigate the factors associated with marriage desires, childbearing behaviour and population policy perceptions. Paper 1 examines the influence of education on women's transition to first birth in South Korea. Paper 2 analyses variation in family-related attitudes among never-married individuals in Singapore. Paper 3 explores the impact of couples' division of labour on their fertility behaviour in Taiwan. Paper 4 presents a cross-national analysis of the relationship between intergenerational support and fertility in East Asia. Paper 5 evaluates attitudes towards pronatalist policies in Singapore. The findings demonstrate that declining birth rates stem from an amalgamation of socioeconomic and cultural factors that contribute to the increasing prevalence of singlehood, a growing preference for smaller families and rising tension between women's work and family roles. Efforts that prioritise individuals' family aspirations, reduce barriers to an equitable division of labour between partners, and improve equity in family formation are important in providing support for reproductive choices.
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    The Evolution of Chinese Maritime Law Enforcement Agencies, 1949-2018: Domestic and External Interactions
    (2024) Yang, Fang
    Why and how have China's maritime law enforcement agencies (MLEAs) evolved from being instruments primarily focused on maintaining economic, social and environmental order at sea to becoming active frontline players in maritime territorial disputes? Existing literature on MLEAs offers isolated and somewhat contradictory views about the structure, capability, role and function of China's MLEAs, leading to different conclusions on Chinese maritime policy-making. By examining three path-dependent phases and one transitional period in chronological order, this thesis argues that the interactions between domestic and external factors throughout different periods contributed to the Chinese central government's shift from a maritime economy-centric agenda to a rights protection-oriented maritime territorial agenda. Consequently, this shift resulted in changes in the structure, capability, role and function of the MLEAs. During Phase One (1949-1980s), the Chinese central government's preoccupation with land-based threats, both domestically and externally, resulted in the dominance of the "land dominates the sea" model. This model determined the early fragmented structure of China's maritime administration and law enforcement, as the central government relied on land-based sectors managing limited maritime activities. Subsequently, from the early 1980s onwards, the continued involvement of pre-existing land-based sectors in maritime administration and law enforcement further reinforced the fragmented structure of the MLEAs through a path-dependent process. During Phase Two (1980s-early 2000s), the Chinese central government's Reform and Opening policy served as a domestic factor which incentivised its participation in and accession to the United Nations Convention on the Law of the Sea in 1982, while the latter served as a favourable external factor by granting China with extended maritime space, jurisdiction, legal framework and experience for ocean governance. This two-level interaction facilitated the Chinese central government's development of a marine economy-centric maritime agenda. This new agenda further led to the establishment of multiple MLEAs under various ministries which were land-based actors previously involved in maritime affairs. The transitional period in the mid-2000s highlighted some impacts created by the structural and jurisdictional problems of the MLEAs. While these problems constrained the MLEAs' ability in conducting law enforcement, including rights protection operations in disputed waters, some MLEAs actively pursued their own agenda and interests within the limit of central authority, as exemplified in the SOA/CMS' development of the Regular Rights Protection Patrol Scheme. Phase Three (late 2000s-2018) witnessed the emergence of the MLEAs as a unique set of domestic factors contributing to China's evolving maritime territorial policies and agenda. Their increased interactions with active foreign claimants and agencies resulted in heightened tension in the region, which, in turn, prompted the Chinese central government's reflection and adjustment in its maritime territorial policies and adoption of a series of more proactive, rights protection-focused maritime policies, including the restructuring of the MLEAs in 2013. Nevertheless, the restructuring process before 2018 was slow and incomplete, as the central government prioritised boosting the MLEAs' rights protection capability to address external challenges rather than resolving the structural and jurisdictional problems of the MLEAs thoroughly. The study on the evolution of the MLEAs provides empirical insights into Chinese domestic maritime actors and enhances our understanding of Chinese maritime policymaking. Moreover, the application of the two-level interaction framework bridges gaps in the existing literature and contributes to the scholarship on the domestic-external nexus in China studies and International Relations.
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    Buddhist Logic and the Liar Paradox
    (2024) Bogacz, Szymon
    In this thesis, I present and defend the Dharmakirtian approach to the liar paradox. I argue that the natural way of understanding the liar paradox is epistemological (related to seemings, acceptances, inference, and knowledge) and that most current approaches to this paradox miss something important by analysing it as a logical phenomenon (related to arguments, proofs, truth, validity, and derivability). According to the Dharmakirtian approach, the liar paradox is a piece of inference (a transition between cognitions about the liar sentence's truth value) and this piece of inference cannot produce knowledge that the liar sentence is true or not true. The Dharmakirtian approach that I develop and my arguments in its support extrapolate from philosophical works of Indian Buddhist epistemologists and logicians, Dignaga and Dharmakirti. I present and defend the Dharmakirtian approach in the context of contemporary studies of the liar paradox, philosophy of logic, epistemology, and philosophy of language. By doing so, I employ Buddhist philosophical material to analyse the liar paradox and propose an epistemological approach to this paradox for the first time.
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    Bridging the (redox) gap: reconciling biomarker and inorganic proxy records in the Middle Cambrian Currant Bush Formation
    (2024) Kinsley, Jordan
    Redox conditions have exerted a strong control upon organic matter (OM) preservation throughout time, determining oxidant availability and heterotrophic activity, and thus controlling OM remineralisation rates across different marine environments. Despite this, redox effects upon ancient OM at a molecular level are comparatively poorly understood. To elucidate redox effects upon biomarkers, this thesis investigates ~103m of the middle Cambrian Currant Bush Formation (CBF), a unit of cyclical outer ramp carbonates, siltstones and black shales, which preserve biomarkers throughout and indicate redox variability. This work undertook high-resolution multiproxy analysis of the CBF (n=109), combining biomarkers with elemental abundances (U, Mo, V, Re), Fe speciation, organic C and bulk N isotopes, mineralogy, sedimentology, and palaeontology. First, this thesis refines the characterisation of CBF euxinia, identifying S limitation from intermediate Fe speciation values (Fepy/FeHR 0.6-0.8) and Fe limitation (FeHR/FeT vs FeT (wt.%) R2=0.9). S limitation in the CBF is additionally indicated by tracers of low sulphide (DBT/Phen xbar=0.2, HHI xbar=3.2) and sulphate (3-MHI xbar=3.0, max.=6.8). These data plug an important Cambrian gap in 3-MHI records. Second, this thesis describes, in addition to evidence for euxinia (delta15N <1.0permil, TOC/N 20-40, Mn <250ppm) contradictory evidence for oxia throughout CBF deposition (AIR xbar=1.6, Pr/Ph mostly >2, TOC/P mostly < 50, UEF xbar=6 and MoEF xbar=10). Together this indicates rapid oscillation of benthic oxic-anoxic sulphidic redox conditions in the CBF, likely driven by frequent storm incursion and relating to oscillating environmental energy levels evident in alternating silty and muddy laminae. Deposition rate estimates gained from laminae counts, along with constraints upon the maximum possible depositional duration of the CBF, require a high frequency, possibly seasonal or decadal, for lamination depositional events, and indicatively for benthic redox oscillations. This work constitutes a highly detailed characterisation of an ancient episodically anoxic system. Third, this thesis investigates sedimentary cyclicity in the CBF, finding that the carbonate-siltstone-black shale cycles observed describe gradual deepening upwards trends with rapid reversions, most likely produced by tectonic subsidence effects. This work investigates 2 particular cycles (92.2-95.6m depth, n=51) identifying parallel cycling of benthic redox conditions driven by increasing up-cycle limitation upon benthic oxygen (U 1-16ppm, Mo 1-13ppm). A wide variety of biomarker parameters demonstrate cyclicity and strong correlations with U and Mo (C30/C31 hopane vs Mo R2=0.7, diastimgastanes/stigmastanes vs U R2=0.7), identifying an overarching control of benthic redox upon molecular distributions. Observed redox responses of biomarkers include systematic covariation in short/long homologue ratios in steranes and aromatic steroids, hopanes, benzohopanoids, and 2,3,6-trimethylarylisoprenoids (C27/C29 steranes vs C27/C33-35 hopanes R2=0.7), and systematic covariation in hopane and diasterane isomer ratios (C27 hopane Ts/Tm vs C30 hopane/moretane R2=0.9). These responses identify distinct redox effects upon biomarkers in the CBF, with homologue relative abundances controlled by oxidation 'clipping' of side-chains determined at the sediment surface and proportional to seafloor residence time, and isomer ratios controlled by double bond preservation determined by redox conditions during early burial. This represents a major advancement in the understanding of redox responses of biomarkers.
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    Ghair-Siyasi Students and the Ideology of 'Dirty Politics' in Pakistan
    (2024) Al Adawy, Heba
    In November 2019, an umbrella of progressive groups organised a Student Solidarity March in Pakistan. Through the March, activists sought to mobilise the wider student community on issues ranging from fee hikes, deteriorating campus infrastructure, sexual harassment scandals and surveillance of ethnic minorities. More fundamentally, they demanded political rights through student unions, which General Zia ul Haq's military dictatorship banned in 1984. For the activists, unions were not just a consultative body for student affairs, but a means to reclaim the notion of politics from its 'dirty' connotations. While the activists' broad concerns struck a chord among university students, their fundamental demand for political rights through unions invoked apprehensions of violence and anarchy within universities. This political ethnography is situated in the period of these events. It asks: why, thirty-five years after the ban, are students apprehensive about politics through unions? And, how have Pakistani public sector universities been depoliticised? To answer these questions, this thesis examines state practices that nurture distrust towards student politics and unions in Pakistan through, among other things, military historiographies; military-backed social media campaigns against progressive student activists; legal and bureaucratic practices within universities; and, military-endorsed youth development activities. The thesis draws upon 11 months of fieldwork in Pakistan, including multi-sited participant observations and 52 ethnographic interviews. The interviewees included political activists associated with progressive student collectives and others who identified in Urdu as ghair-siyasi (non-political), such as students attending extra-curricular activities, members of ethnic councils on campus, university administrators, and NGO officials working on youth engagement programs. Through analysis of the observational and interview data, this thesis theorises 'dirty politics' as a set of ideological practices that structure certain political effects. Decades after the ban, dirty politics represents violence as a timeless fact about student unions and displaces the militarized state as an object of critique. By elucidating dirty politics as a set of ideological practices, this thesis makes three contributions. First, by describing how these practices render student unions violent and anarchic, it explains the depoliticised condition of Pakistani public sector universities. Second, by analysing the affective and atemporal characteristics of these practices, it shows how dirty politics forecloses critique. Third, by showing how ideology generates distrust of politics in Pakistan, it offers a partial account of the militarized state.
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    Cellular reprogramming for brain repair
    (2024) Mahmoudi, Negar
    The inability of the central nervous system (CNS) to regenerate effectively is a barrier to the successful treatment of traumatic injuries and neurodegenerative diseases. As with other cells in the body, neurons are sensitive to injury and degenerative/inflammatory conditions that often result in cell death. While neurons are frequently lost in response to injury or degeneration, astrocytes on the other hand become activated, proliferative, and assemble to enclose a necrotic region and form glial scars. The astrocyte response to injury presents a valuable therapeutic target as they perform both cytotrophic and cytotoxic functions, sometimes concomitantly, after injury. An adeno-associated virus (AAV) vector encoded with specific transcription factors (TFs) that targets astrocytes and potentially converts/reprograms them to functional neurons is of interest in developing novel therapeutic solutions for traumatic brain injury (TBI). Viral vectors such as lentivirus and retrovirus integrate with host-cell genome and pose a series of risks including insertional mutagenesis, transgene integration, and strong immunogenicity[1,2]. However, AAV does not have the problem associated with the integration of host-cell genome as it forms circular concatemers that persist as episomes in the nucleus of transduced cells and does not integrate into host genomes[3]. This project aims to reprogram reactive astrocytes to neurons using TFs both in vitro and in vivo. Furthermore, a bio-inspired self-assembling peptide (SAP) hydrogel is used to provide spatiotemporal release of AAVs in vivo to address multiple difficulties with viral vector delivery by shielding and confining the vectors to the site of therapeutic need. Additionally, these SAP hydrogels have been functionalised to provide relevant biologically active motifs. These motifs can mimic the native cellular microenvironment of the brain. To further drive the survival, differentiation, and maturation of reprogrammed cells, we have developed a hybrid composite biomaterial, incorporating electrospun short fibres (SFs) loaded with valproic acid (VPA) small molecule within our novel SAP hydrogel matrix. We found that NeuroD1/ and SOX2 TFs can trans-differentiate and dedifferentiate reactive astrocytes to neurons in vitro, respectively. More importantly, both SOX2 and NeuroD1-mediated reprogramming resulted in neural replenishment in vivo. Furthermore, NeuroD1 encoded AAV could significantly reduce the glial scar intensity 28 days post-implantation in vivo in a brain injury model. The presentation of SAP+AAV-NeuroD1 also altered the morphology of astrocytes far away from the lesion site due to the alleviation of inflammatory cells (astrocytes and microglia) in the injury site. This demonstrates the ability of NeuroD1 to reprogram reactive astrocytes to functional neurons and potentially alter the phenotype of reactive astrocytes near the injury site. Thus, this SAP hydrogel is promising as a 3D biomimetic cell culture environment (high water content, stiffness within the range of soft tissue, presenting laminin-derived IKVAV peptide as a bio-functional epitope to encourage cell infiltration into the scaffold for neural tissue engineering) and payload delivery platform for future studies of tissue engineering strategies that can manipulate the inflammatory response to improve functional recovery outcomes. To summarise, the research reported here demonstrates i) functionalisation and materials characterisation, ii) the effect of AAV encoded with SOX2 and NeuroD1 TFs on reprogramming reactive astrocytes to functional neurons, iii) the impact of VPA in neural differentiation and maturation, iv) the effect of SAP hydrogels in AAV distribution in brain injury model in vivo.
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    Finding a family for an orphan biomarker - searching for modern, biological sources of cheilanthane molecular fossils
    (2024) Liyanage, Tharika
    Molecular fossils are the hydrocarbon skeletons of functionalised biological molecules. Diagenetic processes in sediments alter these precursor biomolecules to form stable hydrocarbon molecules that can be preserved in the geological record for billions of years. Some molecular fossils can be linked to functionalised biological molecules and specific clades of microorganisms based on similarities in the hydrocarbon skeleton. However, there are still many orphan biomarkers, such as cheilanthanes (tricyclic terpanes), with no known microbial sources or precursor biomolecules. Until cheilanthanes are linked to microbial sources, we are left with inaccurate reconstructions of the ecosystems and environments in Earth's history and possible misinterpretations about the co-evolution of life and Earth. The objectives of this doctoral research were to determine whether there were any living microbial sources of cheilanthanes and to characterise the original biomolecule to contextualise the entire record of cheilanthane biomarkers in the history of life on Earth. First, I assembled the first record of C19-C46 cheilanthanes through the Proterozoic and Phanerozoic Eons. I describe the oldest, demonstrably syngenetic cheilanthanes in the 1.641 Ga Teena Dolostone. Based on the relative abundance of cheilanthanes to steranes and hopanes through the geological record, we propose that bacterial or archaeal sources of cheilanthanes are most likely rather than eukaryotic sources such as Tasmanites green alga, which has been previously proposed. I examined the relative abundance of C19-C46 cheilanthane pseudohomologues to discern patterns that may be indicative of cheilanthane precursor biomolecules' chemical nature. The absence of notable relative abundance maxima indicative of parent molecules' carbon number suggests that cleavage of the regular isoprenoid side chain of cheilanthane precursor biomolecules is efficient during diagenesis or that precursors could be part of a large macromolecular structure. These findings provide testable hypotheses in the search for living microbial sources and precursor biomolecules of cheilanthanes. Second, I conducted closed-system pyrolysis experiments to test whether cleaved hopanoid molecules could produce tetracyclic and tricyclic terpanes. Pyrolysis experiments on C30 diplopterol, a widespread bacterial membrane molecule, generated hopanoid breakdown products, which included tricyclic compounds that coelute with C19-C22 cheilanthanes and a tetracyclic compound that coelutes with or is identical to C24 tetracyclic terpane. Examination of geological samples revealed that these hopanoid breakdown products are common in oils and bitumens. Statistical analyses suggest that these hopanoid breakdown products can strongly influence biomarker proxies utilising cheilanthanes and tetracyclic terpane. This underscores the value of understanding why these cheilanthane abundance ratios are indicative of certain depositional environments. Third, to determine whether there were any living microbial sources of cheilanthanes, I developed a rapid screening technique that makes cheilanthanes detectable in modern environmental samples. This new pyrolysis technique simplifies functionalised biological molecules to their GC-amenable hydrocarbon skeletons. I discovered cheilanthane precursor biomolecules and living microbial sources in several aquatic settings, including Lake Cadagno in Switzerland and Ace Lake in Antarctica. My investigations into the chemical structure of the precursor biomolecules have suggested that there are multiple precursor molecules that bear an acid moiety. This discovery indicates that the cheilanthane precursor biomolecules are a new class of biomolecule. This provides a novel opportunity to bridge this gap between the geological record and past microbial ecology and can begin to contextualise the entire record of cheilanthane biomarkers in the molecular fossil record of life on Earth.
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    Roles and Interplay of Flavonoids, Reactive Oxygen Species, and Cytokinin in the Early Infection of Medicago truncatula by Sinorhizobium meliloti
    (2024) Rae, Angus
    Understanding the mechanisms by which legumes are infected by symbiotic, nitrogen-fixing bacteria (rhizobia) may prove crucial in the pursuit of bio-engineering nitrogen-fixation in current food crops, and as a result, reducing reliance on nitrogen fertilisers. In this thesis, I have examined the roles and interplay of key signalling factors required for the establishment of symbiotic infection of rhizobia in the roots of the model legume Medicago truncatula. Infection of Medicago roots is driven by the production of 'infection threads' by the plant. Infection threads are infection structures that are unique to nitrogen-fixing symbioses, and critical to our understanding of the establishment of the symbiosis in legumes. To facilitate the study of infection, I first developed a new method of imaging infection threads via fluorescent labelling and confocal microscopy, that has allowed high resolution imaging of their structure in 3D cell space. One of the earliest signals during the symbiosis is the production of secondary metabolites called flavonoids by the host root, which activate nodulation genes in rhizobia. The first aim of my thesis focussed on a new role for flavonoids in the formation of infection threads, which has so far not been characterised. Based on the observation that genetic elimination of flavonoids from the root prevents infection thread formation, I sought to identify the responsible flavonoid and to define its mechanism of action. Through isolation of purified root hairs of Medicago roots, I identified flavonoids in root hairs that are induced during the early stages of infection using mass spectrometry. These candidates were then tested for their ability to rescue infection thread formation in flavonoid-deficient roots, which led to the identification of two co-required flavonoids for infection. To characterise a possible mechanism of action, I investigated the role of reactive oxygen species (ROS), which are produced in root hairs during infection and are believed to be necessary for infection thread formation. I showed that a specific flavonoid, 4,4'-dihydroxy-2'-methoxychalcone, is required for ROS production in root hairs during infection, and in combination with another flavonoid identified in the same tissue by the mapping above, can rescue infection in roots in which all other flavonoids are silenced. These results suggest that flavonoids act directly or indirectly to regulate ROS formation in root hairs, and that this is necessary for infection thread formation. After infection threads are initiated, they need to grow towards the cortical cell layers inside the root. The mechanism controlling this process is not understood. I investigated an aberrant infection phenotype in the Medicago cytokinin perception mutant cre1-1, where the nodulation-specific cytokinin receptor CRE1 is mutated. Through this, I have developed a new model for cytokinin activity in infection, whereby cytokinin signalling via CRE1 is required to control cell cycle arrest that is necessary for the directed infection thread progression through the cortex. This links the process of infection thread progression with the process of nodule development in the cortex.
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