School of BioSciences - Theses

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    Investigating the function of the LEUNIG regulatory complex in the basal land plant Marchantia polymorpha
    Li, Qiwei ( 2022)
    Abstract Some 500 million years ago plants colonized the land, which had a major transformative effect on the plant’s biosphere. The evolutionary leap from an aquatic to a land-based existence involved changes in life history, physiology and the body plan of the plant. Some key innovations associated with land plant evolution included the development of waterproof cuticles and production of desiccation tolerant spores. Underlying the adaption to a terrestrial environment were changes in gene regulation and increased sophistication in shaping transcriptional networks. However, it is not clear how transcriptional networks have evolved in land plants. Work described in this thesis focuses on the LEUNIG (LUG) regulatory complex and its role in the basal land plant Marchantia polymorpha. Since LUG lacks DNA binding domains, the recruitment to DNA targets relies on interactions with the coregulator SEUSS (SEU) and SEU-LIKE (SLK) proteins as well as associated transcription factors (TFs). Previous studies have shown that the LUG complex regulates various developmental processes in flowering plants ranging from embryo patterning to leaf and flower development. For example, LUG/SEU interact with the MADS-box transcription factors SEP3 and AP1 to control the floral organ identity. To investigate whether the LUG complex regulation is conserved in basal land plant, single MpLUG and two MpSEU (MpSEU1, MpSEU2) were identified in Marchantia genome and subsequent yeast and in planta assays revealed that MpLUG interacts with both MpSEU1 and MpSEU2, while strong protein interactions were observed between MpMADS1 and MpLUG/MpSEU1. These results suggest that the components of the LUG complex might be conserved in Marchantia. Use of promoter-reporter constructs established that the MpLUG expression overlaps with MpSEU1 in the growing regions of the thallus as well as in the gametangiophores. Overlap in promoter activity was also apparent between MpLUG and MpSEU2, although in this case it was limited to rhizoids arising from the vegetative thallus. Differences between MpSEU1 and MpSEU2 promoter activities suggested that these genes have diversified and that separate MpLUG-MpSEU complexes might exist in Marchantia. Reducing MpLUG activity through knockdown generated small, slow growing plants with an unbranching thallus composed of prothallus-like cells. In addition, CRISPR/Cas9-induced mutant analysis identified chimeric Mplug and Mpseu1 lines suggesting that these genes perform essential functions in the plant, whereas a non-chimeric Mpseu2 mutant did not display a noticeable phenotype. Furthermore, this study also found that transgenic plants over-expressing MpMADS1Res, which are resistant to endogenous miRNA degradation, showed a similar phenotype to the transgenic lines with reduced MpLUG activity. In summary, this study identifies the main constituents of the LUG regulatory complex in Marchantia. Furthermore, it establishes that interactions occur between this complex and MpMADS1 and that this interaction has functional significance. Based on these findings, it is proposed that MpLUG and MpSEU1 limit activity of MpMADS1 within the vegetative apical notch and in doing so, enable cells within this region to begin differentiation.
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    Evolution of epigenetic control of reproduction and development in therian mammals, marsupials and eutherians
    Ishihara, Teruhito ( 2022)
    Genomic imprinting in mammals is a complex phenomenon that is essential for normal development in therian mammals (eutherians and marsupials). However, it is currently unclear how and why imprinting evolved in the common ancestors of therian mammals. There are several differences in the imprinting mechanisms identified to date between marsupials and eutherians, but the ancestral condition is unknown. Therefore, the similarities and differences in imprinting mechanisms between marsupials and eutherians allow comparative studies to examine how imprinting evolved and is maintained in therian mammals. This thesis used the tammar wallaby, Macropus eugenii, as the marsupial model to examine the extent to which epigenetic regulation of mammalian germline development is conserved. It then examined similarities and differences in mechanisms of genomic imprinting in therian mammals, focussing on two important conserved imprinted genes, IGF2R and MEST. Sex-specific DNA methylation reprogramming in developing germ cells is essential for establishing sex-specific imprints in gametes. In the tammar, reprogramming of DNA methylation in male germ cells was highly correlated with male germ cell development, as seen in mice. This suggests that global changes in DNA methylation are associated with male germ cell differentiation in therian mammals. In contrast to males, meiotic arrest, which is initiated specifically in female germ cells during germline sexual differentiation, was associated with H3K27me3-associated chromatin remodelling on the promoter of a meiotic initiation factor, STRA8 but not on a second meiosis initiation factor, MEIOSIN. These results suggest that global epigenetic reprogramming, which is important for the establishment of sex-specific imprinting in gametes, also plays an essential role in normal germ cell differentiation in marsupials as it does in eutherians. In marsupials, both IGF2R and MEST lack a promoter DMR whereas eutherian IGF2R and MEST each have a promoter differentially methylated region (DMR). Despite the differences in possession of promoter DMRs between marsupials and eutherians, all the key enzymes, DNMT3A, DNMT3A2 and DNMT3L were present in therian mammals. This suggests that marsupials may lack the protein-protein interactions required to recruit DNA methylation at certain genes, such as the IGF2R promoter. In the mouse, Igf2r has a DMR in the promoter and a second DMR (ICR) in intron 2. Eutherian IGF2R is regulated by an antisense long non-coding RNA, Airn, which is expressed from the intronic DMR in mice. Airn silences neighbouring genes, Solute carrier family 22 member 2 (Slc22a2) and Slc22a3 that are important for placental function. Marsupials also have an antisense non-coding RNA, ALID, expressed from a DMR, although the exact function of ALID is currently unknown. While the eutherian IGF2R DMR is located in intron 2, the marsupial IGF2R DMR is located in intron 12. In the tammar, only SLC22A3 and not SLC22A2 was imprinted in the placenta. This suggests that SLC22A3 imprinting has been strongly selected in both groups of therian mammals, despite the differences in the DMR location between eutherian and marsupials. Tammar SLC22A3 protein was localised in the endodermal cell layer of the placenta where nutrient transport takes place, suggesting that the control of nutrient transport from placenta to fetus is a critical evolutionary function of genomic imprinting in both marsupials and eutherians. This thesis also confirmed the presence of active histone modification at paternally expressed genes (PEGs), including Mest, in both mouse sperm and mouse zygote paternal pronuclei. This suggests that the histone modification in sperm might be a mechanism to define the epigenetic status of the paternal genome to establish imprinting patterns in zygotes. Furthermore, genes that initiate paternal expression upon zygotic genome activation and lose imprinted expression as embryogenesis proceeds were identified. The presence of such transient PEGs suggests that imprinting may have evolved to ensure a paternal contribution to early development during zygotic genome activation. In eutherians MEST has a distinct DMR, while the tammar MEST has none. This suggested that MEST imprinting may be regulated by differential histone modifications. Detailed characterisation of the tammar MEST gene locus led to the discovery of a conserved lncRNA, MESTIT1, which, like human MESTIT1, is present in sperm. Although the actual role of this conserved lncRNA is currently unknown, it is possible that this transcript may help to retain the active histone modification in therian sperm as the lncRNA Kcnq1ot1 does in mouse sperm. This raises the question as to whether histone modifications rather than DNA methylation may represent a more ancestral form of imprinting control. The results from this research using this alternative model species to examine the evolution of imprinting has provided new information, insights and many questions into how imprinting may have evolved in the common ancestor of therian mammals. Since no imprinted genes have been identified in monotremes as yet, it is thought that mammalian genomic imprinting evolved after the therian mammals diverged from the monotremes. This study supports that conclusion, but further work is needed on the question of the evolution of imprinting as well as on the unique and diverse marsupial fauna, only one of which was studied in this thesis.
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    Modern human evolution: insights from primate comparative genomics and archaic hominins
    Vespasiani, Davide Maria ( 2022)
    A fine characterisation of the genetic basis underlying human-specific phenotypic traits is of primary importance both for biomedical and evolutionary studies. Computational predictions based on variant functional annotations coupled with in vitro experimental validations can shed light into the processes that have shaped modern humans phenotypic diversity and predisposition to diseases. In this thesis, by using a combination of both computational analyses and experimental assays, I investigate the potential functional and evolutionary significance of genomic diversity within modern human populations as well as between humans and chimpanzees. I begin by studying the consequences of admixture between modern humans and archaic hominins. Specifically, from a set of Denisovan and Neanderthal archaic variants, previously identified segregating within present-day Papuans, I study whether archaic introgression might concur in shaping phenotypic diversity within this human group, by looking at the patterns of introgression across chromatin functional states and human cell types. I show that a substantial proportion of these archaic variants lie within cis-regulatory elements, particularly those active within immune-related cells. I further show how Denisovan introgression might have larger consequences on immune phenotypic traits compared to Neanderthals', potentially highlighting archaic variants of putative evolutionary relevance. I proceed by designing and performing a MPRA to quantify the molecular phenotype associated with the presence of a small subset of Denisovan cis-regulatory alleles. This study informed about the feasibility of using this high-throughput approach and revealed that at least 3 of the Denisovan alleles I have shortlisted are associated with a significant reduction in the expression levels of the reporter gene compared to their modern human counterparts in immune-related cells. Finally, I compare the regulatory landscape during early development between primates by analysing a dataset of chromatin accessibility previously generated from 6 humans and 6 chimpanzees iPSCs. By comparing the patterns of accessibility for a set of 151,266 orthologous regions I identify 52,352 peaks with inter-species significant differences in accessibility. Functional annotation of these peaks revealed that they show signatures associated with the potential presence of developmental regulatory elements. I then investigate the set of DNA motifs likely contained within the chromatin accessible peaks and integrate my data with gene expression and higher-order chromatin organisation information obtained from the same cell lines to study the existence of coordinated epigenetic changes that are also associated with gene expression variation between species. Together, the findings from my thesis highlight the role of variation at cis-regulatory elements as an important source of phenotypic diversity both between and within species. Moreover, they also demonstrate how integrating computational predictions with laboratory-based experimental validation can favour a deeper understanding about the consequences of any genetic and epigenetic variation.
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    Exploring the roles of bromodomain protein 2 (PfBDP2) and acetylated histone variant PfH2A.Z and PfH2B.Z in Plasmodium falciparum chromatin biology
    Azizan, Mohd Suffian ( 2022)
    The eukaryotic Plasmodium falciparum parasite employs multiple levels of gene regulation to alter its morphology throughout its complex life cycle to survive different environments it encounters in mosquitoes and humans. Thus, epigenetic mechanisms that control gene regulation are integral to the parasite’s survival. Histone lysine acetylation is the post-translational modification of histones, generally associated with eukaryotic transcriptional activation. This epigenetic modification is recognised by bromodomain, and bromodomain-containing proteins (BDPs) can recruit transcription factors to promote gene expression. In the P. falciparum genome, eight bromodomain-containing proteins were identified but only two have been functionally characterised. This thesis detailed the works done on the uncharacterised P. falciparum bromodomain protein 2 (PfBDP2). I explored cellular and epigenomic localisations of PfBDP2 in the asexual schizont-stage P. falciparum parasites via biochemical assays and native chromatin immunoprecipitation, which revealed that PfBDP2 is a nuclear protein expressed throughout the asexual cycle and is enriched within the 5' intergenic region of invasion genes in schizonts. PfBDP2 is also enriched within heterochromatin, particularly across the promoters of silent multigene families and across var gene introns. This points to PfBDP2 being a chromatin protein with dual epigenetic roles, associated with both gene expression and silencing. This thesis also reports a growth delay upon the conditional disruption of PfBDP2 expression in vitro, indicating its importance for normal growth during the asexual intraerythrocytic cycle. The inability to recover stable PfBDP2 knockouts following knockout induction suggests that PfBDP2 may be essential to blood-stage growth and could be druggable by novel antimalarials. Finally, this thesis also detailed the epigenomic localisation of two unique Apicomplexan histone variants, PfH2A.Z and PfH2B.Z, and their acetylated cognates, PfH2A.Zac and PfH2B.Zac, previously shown to be the binding targets of PfBDP2. PfSir2A is a histone deacetylase shown to anti-correlate with PfH2A.Z deposition at heterochromatin in the P. falciparum genome, fulfilling a critical role in the maintenance of chromatin structure. Using crosslinked chromatin immunoprecipitation, the genome-wide localisation of the histone variants was mapped in both wild-type and PfSir2A-KO parasites. There was an enrichment of both PfH2A.Zac and PfH2B.Zac across all euchromatic intergenic regions of P. falciparum genome in the wild-type parasites. This pattern was similar in PfSir2AKO parasites but differed in the aberrant, increased enrichment of the acetylated histone variants within heterochromatin, signalling a breakdown of heterochromatin-euchromatin boundaries. This indicates an important structural function of the two histone variants in maintaining chromatin boundaries in P. falciparum. Additionally, PfH2A.Zac and PfH2B.Zac appeared to be enriched at specific loci around var genes, most importantly var introns, suggesting a role in var gene regulation and lending further evidence to the acetylated histone variants being in vivo binding targets of PfBDP2.
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    Behavioural Response of Marine and Estuarine Invertebrates to Contaminated Sediment
    Woods, Josephine Lenore Dorothea ( 2022)
    Behavioural ecotoxicology is important for understanding real world responses to toxicity. Alterations in behaviour may not elicit immediate lethal consequences but can have implications for the overall fitness of individuals. Research on behavioural responses of marine and estuarine invertebrates is increasing, but the direction of responses and how they vary between toxicants is largely unknown. In a systematic literature review of this information in Chapter 1, I determined there were seven major categories of behavioural change in response to toxicity: locomotion, movement, feeding, burrowing, courtship, response to stimulus and other behaviours. The overall trend across all species was for a reduction in locomotion and feeding with exposure to a toxicant. Burrowing behaviour tended toward avoidance of sediment with exposure. Although some work explored complex ecological responses such as competition and changes in ecological functions, most work was focussed on simple behaviours, such as decreases in feeding or closure of valves. There was a lack of studies incorporating more complex biological interactions such as predation and competition. There was also a lack of research into the impact on functional processes, such as sediment reworking or nutrient removal. This review also highlighted the absence of research on behavioural responses in some taxonomic groups, especially annelids. In addressing this gap, I exposed the annelid (Simpliseita aequisetis) to copper contaminated sediment in a behavioural avoidance test. The behavioural avoidance response of two gastropods with different feeding regimes was also examined in Chapter 2. Although tested under varying concentrations of copper for different durations, none of the three species responded to the contaminant. None were able to select an uncontaminated environment in the scenarios tested. The lack of response of the gastropods could be interpreted as tolerance of toxicity. The annelid may also be tolerant of copper, or perhaps be utilising other behavioural strategies to increase tolerance. To determine if S. aequisetis used other behaviour to decrease toxic impact, I undertook a standard ‘toxicity test’ with behavioural responses as endpoints in Chapter 3. The annelid buried at a shallower depth, and bioturbation (measured as depth of oxic sediment and percentage of reworked area) decreased with increasing copper concentration. Exposure of the amphipod Gammaropsis sp. to copper also showed a decline in sediment reworking. This decrease in bioturbation showed toxic exposure can cause alterations in functional processes, as bioturbation is important for many chemical cycles in the sediment. Analysis of copper in the sediment, water, burrow, annelid and porewater showed body burden did not increase with increasing sediment concentration. The alteration in sediment reworking behaviour appears to have allowed tissue concentrations of S. aequisetis to stay below toxic concentrations. Finally, I undertook a manipulative field experiment in Chapter 4 to determine if behaviours measured in the laboratory were similar under field conditions and in the context of the macroinvertebrate community. Sediments were dosed with copper and zinc and community responses measured after 4 weeks. The low level of copper was likely to have caused a hormetic response, with the abundance of the opportunistic amphipod species Paracorphium sp. increasing with increasing copper. There was no observable decline in any species for both copper and zinc manipulations. The thesis highlights the knowledge gaps in behavioural ecotoxicology and provides insight into the complexities of behavioural response in the laboratory and the field. This thesis also demonstrates that behavioural responses to pollution can reduce the impact of toxicity and can have secondary impacts on ecosystem function.
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    Uncovering the interplay between nutrient availability and cellulose biosynthesis inhibitor activity
    Ogden, Michael ( 2022)
    All plant cells are surrounded by a dynamic, carbohydrate-rich extracellular matrix known as the cell wall. Nutrient availability affects cell wall composition via uncharacterized regulatory mechanisms, and cellulose deficient mutants develop a hypersensitive root response to growth on high concentrations of nitrate. Since cell walls account for the bulk of plant biomass, it is important to understand how nutrients regulate cell walls. This could provide important knowledge for directing fertilizer treatments and engineering plants with higher nutrient use efficiency. The direct effect of nitrate on cell wall synthesis was investigated through growth assays on varying concentrations of nitrate, measuring cellulose content of roots and shoots, and assessing cellulose synthase activity (CESA) using live cell imaging with spinning disk confocal microscopy. A forward genetic screen was developed to isolate mutants impaired in nutrient-mediated cell wall regulation, revealing that cellulose biosynthesis inhibitor (CBI) activity is modulated by nutrient availability. Various non-CESA mutants were isolated that displayed CBI resistance, with the majority of mutations causing perturbation of mitochondria-localized proteins. To investigate mitochondrial involvement, the CBI mechanism of action was investigated using a reverse genetic screen, a targeted pharmacological screen, and -omics approaches. The results generated suggest that CBI-induced cellulose inhibition is due to off-target effects. This provides the groundwork to investigate uncharacterized processes of CESA regulation and adds valuable knowledge to the understanding of CBI activity, which could be harnessed to develop new and improved herbicides.
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    Breeding a diverse and resilient reef
    Lamb, Annika Mae ( 2022)
    Coral reef ecosystems are biologically, culturally, and economically valuable but are under threat from a suite of stressors, the most pressing of which are rising sea surface temperatures and the associated increased frequency and severity of summer heat waves. High water temperatures cause corals to lose their algal symbionts (bleach) and may result in mass bleaching events, mass coral mortality, and the degradation of reefs. Managed breeding is an active intervention that involves generating stock for deployment into degraded systems; it has the potential to combat population declines and boost population fitness. Many reef-building corals lend themselves to managed breeding because they produce large numbers of gametes through mass-spawning that can be collected and crossed in controlled ways. The aim of my PhD was to conduct crosses within and amongst mass-spawning coral species to test intervention approaches and their ability to generate genetically diverse and resilient corals for reef restoration. Firstly, I conducted parentage analyses of intraspecific crosses and found that the genetic diversity of coral stock can be optimised by minimising the handling of gametes and combining the egg-sperm bundles of multiple colonies in bulk reactions. Secondly, I conducted interspecific crosses between coral species and tested the performance of interspecific hybrids relative to their purebred counterparts. Under simulated heat waves in aquaria and a natural heat wave in the ocean, coral hybrids performed as well as one of the purebred parental species or intermediately to both parental species in terms of their growth, survivorship, and bleaching resistance. Finally, I demonstrated that F1 interspecific hybrids can reproduce to generate viable F2 hybrid and backcrossed offspring and have greater reproductive fitness than purebreds. The viability of coral hybrids in the field and lack of outbreeding depression in F1 hybrids or the early life stages of F2 hybrids is indicative that interspecific hybridisation could rapidly boost genetic diversity in degraded populations. The reproductive viability of F1 hybrids further demonstrates that introgression of beneficial alleles that boost resilience could occur between purebred species via interspecific hybridisation and that outplanted hybrids could propagate on reefs. Bulk coral crosses and interspecific hybridisation may therefore be useful tools for reef restoration initiatives.
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    Comparative Genetics of Invertebrate Moulting
    Perry, Caitlyn Louise ( 2022)
    The development of molecular phylogenetics in the 1990s led, among many other discoveries, to the finding of a monophyletic clade of moulting invertebrates dubbed Ecdysozoa. Including arthropods and nematodes (alongside numerous smaller groups) Ecdysozoa is unrivalled among living groups for the number of species it contains and for the diversity of these species; moulting is the only non–molecular trait known to be common to all modern ecdysozoans. However, our understanding of how moulting is regulated draws on only a very small number of species, with the vast majority of these being insects of economic importance. Naturally, the sheer size of Ecdysozoa precludes a comprehensive investigation of moulting regulation across this clade; furthermore, many ecdysozoan groups (for instance, aquatic taxa) would be far less suited to laboratory culture and manipulation than the average insect. However, ecdysozoan genome sequencing, once largely restricted to genetic model organisms, pest species and disease vectors, has begun to extend across a far broader range of taxa. This thesis reports my exploration of molecular data in the context of the evolution of moulting. The Ecdysozoa hypothesis was accepted quickly by most workers in arthropod taxonomics, but this should not lead us to underestimate just how significant a change the embrace of Ecdysozoa represented. The better part of a century of investigations into the molecular basis of moulting had been conducted under the assumption that the non–moulting annelid worms were the sister taxon to arthropods, and thus that moulting had either evolved repeatedly or had been lost repeatedly among invertebrates. In order to explain this historical context, I have produced a review of the history of arthropod taxonomy in the Western tradition. This account runs from Aristotle to the present day, but has a particular focus on the development of the Ecdysozoa hypothesis. My experimental work on the genetics of moulting regulation began with an examination of the cytochrome P450 oxidase family CYP307 in the fruit fly genus Drosophila. Although the precise catalytic role of these enzymes has proved elusive, they are required for the synthesis of ecdysteroids, the master moulting regulators of arthropods. It has been known for around fifteen years that multiple duplications in the CYP307 family have occurred in Drosophila, but the functional significance of these duplications has been unclear. I have attempted to address this question by transgenic substitution of CYP307 paralogs, finding that even enzymes which are closely related at the level of amino acid sequence are not functionally interchangeable. The CYP307 family is only one of many acting in the synthesis of ecdysteroids from dietary sterols; almost all of these were first identified in D. melanogaster, and although comparative analyses have generally suggested good conservation of these enzymes across arthropods, exceptions are known. Building on the CYP307 investigation and its suggestion of rapid functional differentiation following duplications in this family, I sought to identify all duplications and losses of ecdysteroid synthesis genes across all available arthropod genomes (at the time I ceased collecting data, 923 genera were represented by at least one genome assembly). In some cases, I was able to connect observed copy number changes to ecological factors (e.g. whether the predominant dietary sterol was of animal, plant or fungal origin), but many duplications and deletions were entirely unexpected and suggestive of additional changes which require further investigation (e.g. replacement of one enzyme with another having similar activity). In some cases, I went on to analyse selective pressures acting on relevant components of the ecdysteroid synthesis and signalling pathways to determine the effects of loss or duplication of ecdysteroidogenic genes. Despite the numerous copy number changes I observed, my findings were generally consistent with the strong conservation of ecdysteroid synthesis genes described by earlier researchers. As a consequence of this conservation, arthropods provide limited evidence concerning the early evolution of ecdysteroid synthesis. This limitation makes the recent release of genome assemblies from lineages closely related to Arthropoda (namely velvet worms and tardigrades) particularly important for understanding ecdysteroid evolution. I used a transgenic approach similar to that previously applied to Drosophila CYP307 enzymes to investigate the function of four cytochrome P450s from the tardigrade Hypsibius exemplaris which showed strong similarity to ecdysteroidogenic enzymes at the amino acid level. I found that H. exemplaris CYP315A1 does not rescue D. melanogaster Cyp315a1 nulls (despite their clear orthology) and that H. exemplaris CYP18K1 overexpression produces a phenotype quite distinct from that associated with D. melanogaster CYP18A1 overexpression. Tardigrades are unlikely to synthesise ecdysteroids, but they, along with representatives of other ecdysozoan taxa such as the priapulids (penis worms), may provide insights into the diverse steroid metabolic pathways of Ecdysozoa. My research has focused on the potential for integration of large–scale molecular analysis with experimentation in tractable model organisms as a means of understanding ancient evolutionary events. While my cross–arthropod screen revealed many changes to the ecdysteroid synthesis pathway which remain to be fully examined, the results of my transgenic experiments demonstrate the use of bioinformatic approaches in identifying promising targets for more direct examination. The moulting process is one of considerable interest both because of its practical importance (disruption of moulting is potentially a potent insecticidal technique) and because of the intrinsic fascination of the morphological changes it enables. However, the combined bioinformatic and transgenic approach I have used could be applied to any process occurring in a taxon from which a model organism has been established.
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    Environmental DNA sampling as a tool for monitoring freshwater vertebrates
    McColl-Gausden, Emily Frances ( 2022)
    To monitor biodiversity effectively, accurate and sensitive detection methods that can be implemented over large spatial scales are required. It has been demonstrated that environmental DNA (eDNA) sampling can be a valid alternative to many traditional sampling methods. This thesis explored the data and conclusions which could be drawn from eDNA sampling regimes across large spatial scales for individual species and communities, answering key questions on species occupancy and detection method sensitivity. Adaptations of site occupancy-detection models (SODM) are used throughout. First, I investigated the relative sensitivity of single- and multi-species detection methods using a consistent methodological framework across multiple datasets with different study designs. I used SODM to evaluate how the molecular method used impacted the probability that a species was detected. I found that qPCR was generally more sensitive at detecting target species but that different methodological decisions impacted its sensitivity. Second, I used eDNA sampling to conduct an extensive platypus occupancy survey across 37% of the species’ estimated distribution. Using a systematic study design, I demonstrated that correlates of platypus occupancy could be revealed using this efficient survey method. I found that platypuses were less likely to occupy sites in areas with a high proportion of surrounding agricultural land or grasslands and were more likely to occupy sites with increased runoff, less zero-flow days, and suitable banks for burrowing. Third, the 2019/2020 mega-fires impacted south-eastern Australia severely. My pre-fire eDNA data provided the opportunity to investigate if platypus occupancy was impacted by this large-scale event. I developed a SODM extension to incorporate a Before-After Control-impact (BACI) design. After surveying 118 sites in three time periods (pre-fire, 2020 and 2021), I found that platypus occupancy was not significantly impacted by the presence of fire. However, I did find a significant interaction between the proportion of a watershed that was burnt at high severity and rainfall post-fire: platypus occupancy was lower in watersheds with a high proportion of high severity fire that had high rainfall post-fire. This finding is consistent with previous work on the impact of fires on aquatic species. Lastly, leading on from my work on large-scale eDNA surveys for an individual species, I used eDNA metabarcoding to investigate occupancy patterns among fish communities. I used a stratified study design across the state of Victoria to investigate how native and introduced fish responded to environmental factors. I found that for the water availability covariates I considered, native and introduced fishes responded similarly. However, for the land use covariates such as the proportion of a contracted catchment covered by agriculture, urban or forested land, different patterns emerged. Native fish responded more positively to these categories, whereas introduced species responded more negatively. This thesis demonstrated that eDNA sampling is an ideal monitoring tool for individual species or communities over large spatial scales. I showed that SODM and eDNA data can be used to estimate correlates of occupancy efficiently and that eDNA sampling over time enables species responses to major disturbances to be determined across large areas.
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    Role of phosphorylation in regulating secondary cell wall cellulose synthesis in Arabidopsis
    XU, HUIZHEN ( 2022)
    Plant secondary cell walls (SCWs) are important for plant growth and development as the vascular tissues and fibers support plants with water and mineral transport. Cellulose is the major component of SCWs, and its synthesis is a highly complex process regulated by transcription factors as well as post-translational modifications. Cellulose synthase (CESA) 4, 7 and 8 are essential enzymes that catalyze the synthesis of SCW cellulose and form a cellulose synthase complex (CSC) that is active at the plasma membrane. The CSCs move at the plasma membrane; a process driven by the catalytic activity of the CESAs. The behaviour of the CSC is an important character of cellulose synthesis and SCW patterning. Protein phosphorylation is arguably the most common post-translational modification in many cells and affects CESA behaviour during primary wall synthesis. However, how SCW CESA phosphorylation contributes to secondary wall production is not understood well. Chapter 1 provides a brief overview about plant cell wall cellulose synthesis, especially secondary cell wall biosynthesis. There are five main aspects discussed, including secondary cell wall patterns, transcriptional regulation during SCW formation, CESA structures and the function of each domain, the effects of phosphorylation on cellulose synthesis, and environmental effects on SCW production. In Chapter 2, proteomic and phospho-proteomic changes were characterized during the transition from primary to secondary wall synthesis using the VASCULAR-RELATED NAC-DOMAIN7 (VND7)-inducible system. A vast number of phosphorylation sites, especially in SCW-related proteins, were detected. The phosphorylation changes of putative and selected phosphorylation sites in primary and secondary cell wall CESAs were analyzed in detail. This phospho-proteomic dataset provides more insights into phospho-protein changes during the process of SCW biosynthesis. In Chapter 3, phosphorylation sites in each SCW CESA were analyzed and mutated to examine if and how phosphorylation regulates SCW biosynthesis. Most of the selected phospho-mutants, either phospho-null or phospho-mimic versions, restored the phenotype of SCW cesa mutants, and did not show significant differences from wild type control. However, one conserved phosphorylation sites in CESA4, S374, did affect SCW biosynthesis, as single-site phospho-null mutant (CESA4S374A) showed dwarf phenotype with deformed xylem vessels, similar to cesa4 mutant. Sequencing and qRT-PCR confirmed the correct amino acid substitutions and gene expression, respectively. Further, both bioinformatic analysis of protein structure and sequence alignments indicated that S374 in CESA4 was likely to be externally exposed and phosphorylated. Thus, phosphorylation in the position of S374 in CESA4 potentially works to positively regulate SCW cellulose biosynthesis. In Chapter 4, an immunoprecipitation approach of a YFP tagged CESAS7 in the VND7-inducible system was used to pull out potential proteins interacting with SCW CESAs, focusing on protein kinases. Thirteen highly enriched kinases were in this way found to potentially associate with the CESAs. One interesting but unknown receptor-like kinase, AT1G09440, may potentially play a role in SCW formation. Subcellular localization analysis further showed that this protein kinase was secreted from the Golgi to the plasma membrane where it is likely to have its main function. In Chapter 5, the conclusion for this research and some future work directions are proposed.