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    A population genetics investigation of the New Zealand endemic Lophomyrtus bullata (Myrtaceae), a species of conservation concern due to the threat of myrtle rust : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2023) Macdonald, Amy Leigh
    Lophomyrtus bullata Burret (Myrtaceae) is a shrub or small tree species endemic to New Zealand. The species is a member of the Myrtaceae family and is one of two species in the Lophomyrtus genus. The conservation status of L. bullata was raised to “Threatened – Nationally Critical” following the establishment of the fungal pathogen Austropuccinia psidii (G. Winter) Beenken 2017 in New Zealand in 2017. Repeated infections of the fungal disease can lead to extensive loss of foliage, premature dropping of fruits and plant death. Infected L. bullata exhibit all these symptoms, and rapidly declining regional populations indicate a high potential for localised extinction. The regional disappearance of L. bullata could isolate remaining populations, decreasing gene flow and connectivity. Additionally, rapidly decreasing population sizes could increase the potentially harmful effects of inbreeding and genetic drift, reducing the species’ fitness and increasing its risk of extinction. An understanding of the genetic variation and structure of L. bullata populations could help determine its vulnerability to such genetic effects. To this end, Illumina sequencing was used to develop microsatellite markers for L. bullata. From the 1,351,112 successfully paired and merged sequences, 55 microsatellite loci were isolated, and primer pairs were designed. After an initial screening of the 55 primer pairs, 12 were identified as polymorphic and amplified consistently. These 12 microsatellite markers were genotyped across 452 samples representing 18 populations of L. bullata, one population of L. obcordata, and two populations of L. bullata x L. obcordata putative hybrids. The 18 populations of L. bullata showed low genetic differentiation, low expected heterozygosity and very few private alleles. Lophomyrtus bullata populations in the upper North Island region had the highest expected heterozygosity, and there is evidence of a correlation between expected heterozygosity and decreasing latitudinal distance. High genetic differentiation was observed between the populations of L. bullata and L. obcordata and between the two hybrid populations and the L. bullata populations. The population structure results suggest the presence of two to five genetic clusters within L. bullata. The clusters reflect the geographic location of the populations, in addition to a potential North versus South cline. These patterns could have formed due to past climatic events such as glacial cycling and volcanic activity, isolation by distance, or some combination of these. These results suggest that L. bullata is potentially vulnerable to the negative effects of genetic stochasticity. The continued spread and infection of Austropuccinia psidii could significantly exacerbate these effects. However, the higher genetic diversity and differentiation of the putative hybrid populations suggest a possible avenue for the species to acquire new adaptive variation, though this would heavily depend on the hybridisation mechanics of the species, for which there is little information. Additionally, the significant population structure of L. bullata identified in this study can be used as a guide for conservation practices, including seed banking, population management and re-vegetation projects.
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    Characterisation of pseudogene-like EP400NL in chromatin remodelling and transcriptional regulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Li, Zidong
    EP400 is an ATP-dependent chromatin remodelling enzyme that has been implicated in DNA double-strand break repair and transcription regulation including Myc-dependent gene expression. It was previously shown that the ectopic expression of the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. This prompted the question of whether the EP400 N-terminal-Like (EP400NL) gene, which resides next to the EP400 gene locus, also plays a similar role in epigenetic transcriptional regulation to the full-length EP400 protein. To characterize the function of the EP400NL nuclear complex, a stable cell line expressing TAP-tagged EP400NL was established, and the EP400NL complex was affinity purified and analyzed by mass spectrometry. EP400NL was found to form a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, despite no histone acetyltransferase activity being detected, the EP400NL complex displayed H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. In addition to a role in H2A.Z deposition, it was also determined that the transcriptional coactivator function of EP400NL is required for serum and IFNγ- mediated transcriptional activation of the immune checkpoint gene PD-L1. EP400NL, cMyc and multiple identified ATPases such as BRG1, RuvBL1/RuvBL2 were shown to be recruited to the promoter region of PD-L1. To further demonstrate the importance of EP400NL in regulating Myc and IFNγ-mediated PD-L1 expression, CRISPR/Cas9 mediated EP400NL indels were introduced in H1299, a human non-small cell lung carcinoma cell line. These EP400NL indel cell lines show compromised gene induction profiles with significantly decreased PD-L1 expression from both Myc and IFNγ stimulation experiments. In contrast to full-length EP400NL, two deletion mutants (Δ246- 260 and Δ360-419) lacked the ability to enhance the expression level of PD-L1 mRNA or protein, indicating that these regions are important for coactivator activity. Collectively, these data show that EP400NL plays a role as a transcription coactivator for cMyc-mediated gene expression and provides a potential target to modulate PD-L1 expression in cancer immunotherapy.
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    Establishing systems to characterise MH pathogenic RyR1 variants : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 2021) Stephens, Jeremy
    Malignant hyperthermia (MH) is a potentially fatal, autosomal dominant, metabolic disorder triggered in susceptible individuals upon exposure to volatile anaesthetics. Following the onset of an MH episode, a patient will enter a hypermetabolic state, displaying the symptoms of intense muscle contraction, metabolic acidosis, increased oxygen consumption. Prolonged episodes can result in rhabdomyolysis. If left untreated, MH can manifest as an increase in body temperature and death by cardiac arrest. MH is diagnosed by the invasive in vitro contracture test, which requires a muscle tissue biopsy. DNA screening has been implemented and is commonly used to diagnose a genetic predisposition to MH; however, the test is currently limited to fifty variants confirmed to be pathogenic out of approximately 350 variants linked to the disorder. DNA-based tests are limited because of the technical difficulties associated with functional analysis. Thus, additional variants must be functionally characterised. The structural implications of MH-linked variants potentially leading to the onset of MH are not yet well defined. Potential structural changes induced by pathogenic variants have been modelled in silico, where variants were mapped to the rabbit RyR1 structure characterised by cryo electron microscopy. However, this does not confirm the role the variants play in the structural and functional alteration of the channel. To address, this a functionally significant region of RyR1 was cloned for recombinant expression in E. coli. The RyR1 region was shown to be soluble and efforts were made to purify the protein. However, the protein could not be purified to an extent acceptable for either biochemical analysis or crystallisation trials or for subsequent X-ray crystallography. A number of pathogenic variants were instead modelled in silico to provide some insights into their potential pathogenic functional role. The viability of a new cell-based system for the functional characterisation of variants was also tested. Patient derived myoblasts were immortalised using lentivirus transduction with the cDNA for human telomerase and cyclin dependent protein kinase 4. The genome editing tool CRISPR Cas 9 was then used to successfully introduce the pathogenic variant c.14497C>T p.his 4833 tyr into the genome of MH negative myoblasts. Functional characterisation of the introduced variant has yet to be performed.
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    Characterising a biologically relevant protein-G4 interaction : HP1α and TERRA : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Roach, Ruby
    Our genetic material is intricately folded and protected through the formation of a compact nucleoprotein complex, termed heterochromatin. In addition to controlling the expression of genes, heterochromatin formation is important for the structural integrity of our genome, specifically for the centromeres, the central attachment point of our chromosomes, and also the telomeres, the ends of our chromosomes. The way in which the heterochromatin in these areas is formed and maintained is though the recruitment and binding of the pivotal chromatin regulator, Heterochromatin Protein 1α (HP1α). The current model that explains how and why HP1α is recruited to, and maintained at, regions of constitutive heterochromatin is simple: HP1α binds post-translational modifications on histones (eg. H3K9me3). However, this binding is not high affinity, therefore may not be the sole determinant in HP1α localisation. At the centromeres, it has been shown that a long non-coding RNA transcribed from the peri-centromeres is responsible for recruiting HP1α to this crucial region. At the telomeres, it is proposed that the long non-coding RNA transcribed from the telomeric DNA is responsible for this same purpose. Because of its guanine-rich sequence, it is able to form a non-canonical nucleic acid structure, the G-quadruplex, which may provide the specificity for heterochromatin formation at telomeres. This TElomeric Repeat-containing RNA (TERRA) has been implicated in telomeric elongation pathways, relating it to the immortalisation of cancer cells. It was found that HP1α can specifically recognise the parallel topology of TERRA, binding with high affinity through HP1α’s unstructured hinge. HP1α was also shown to bind other G-quadruplexes of parallel topology, specifically DNA present in promoter and regulatory regions of many proto-oncogenes, implicating HP1α in potential G-quadruplex-dependent gene expression regulatory mechanisms. The interaction shown here between HP1α and TERRA shows a novel mechanism of telomeric heterochromatin formation, providing crucial insights into telomere maintenance and health in human cells, and furthering our understanding of the role of G-quadruplexes in the epigenome.