School of BioSciences - Theses
Permanent URI for this collection
Search Results
1 - 10 of 41
-
ItemThe diversity and behaviour of Dermaptera (earwigs) in the southern Australian grain belt( 2018)Dermaptera (earwigs) are a cosmopolitan order of insects that have recently come to the attention of the Australian grain industry. Researchers have been sampling Dermapteran diversity and abundance across the southern Australian grain belt, but resolution of the sample is stifled by taxonomic impediment, particularly for specimens of the morphologically uniform Anisolabididae family. Molecular methods can provide much needed resolution to the study of Australian Dermaptera. I barcoded known Dermaptera species from across the southern Australian states to assess the utility of barcoding for Dermapteran biodiversity research. I also assessed the diversity of the Anisolabididae by combining morphological identification and DNA barcodes. I then estimated their phylogeny with a larger molecular dataset comprised of two mitochondrial and two nuclear gene fragments under a maximum-likelihood framework. Anisolabididae males were divided into seven morphospecies based on the shape of the forceps and parameres (a male genital structure), and these morphospecies were corroborated by barcodes (low within versus between-species genetic distance). Paramere shape distinguished two putative genera, Anisolabis Fieber and Gonolabis Burr. The molecular phylogeny did not support the monophyly of the genera, rather forming clades distinguished by the shape of the forceps. The evolutionary significance of paramere versus forceps morphology in Dermapteran taxonomy is discussed. Anisolabididae were only found in Western and South Australia and showed apparent endemism. Extending the study of Australian Anisolabididae beyond grains-producing regions may reveal a diverse endemic fauna, almost entirely unexplored heretofore.
-
ItemGroup culture of preimplantation embryos creates a microenvironment that determines development, viability, and response to external stressors( 2018)Over 400,000 babies worldwide are born following in vitro fertilisation (IVF) treatment every year and millions of babies have been born from IVF over the last 40 years (Adamson et al. 2018, Niederberger et al. 2018). Despite this, approximately half of couples undergoing IVF treatment do not achieve a successful pregnancy, even after multiple treatment cycles (Chambers et al. 2017, Fitzgerald et al. 2017). The period of in vitro culture is a crucial stage in determining not only the success of IVF treatment but also the long-term health of these babies (Feuer and Rinaudo 2016). Due to new embryo selection and tracking technologies, clinical embryologists increasingly culture embryos individually rather than in groups. However, individual culture can cause stress to the embryo (Reed 2012)because embryos are deprived of paracrine signals, which promote the development of embryos cultured in groups (Wydooghe et al. 2017). The identities and actions of most of these embryo-secreted molecules remain poorly understood. Using a mouse model, this study compares the development of embryos cultured individually or in groups, and how other culture conditions influence the development of individually cultured embryos. Firstly, it was found that any period of individual culture was detrimental to development rates, cell numbers and percentage of inner cell mass (ICM), and the precompaction stage was no more sensitive to this stress than the post-compaction embryo. Reducing the volume of culture media was beneficial to individually cultured embryos, but only under 5% oxygen, not atmospheric oxygen, indicating that there is an interaction between optimal embryo density and oxygen concentration. Reducing the media volume further by culturing embryos in microwells increased the % ICM and influenced the speed of cleavage and hatching. Addition of embryo-conditioned media to individually cultured embryos also increased hatching rate and cell numbers. The interaction between individual culture and atmospheric oxygen was investigated in more detail. Compared to group culture under 5% oxygen, either individual culture (at 5% oxygen) or atmospheric oxygen (group culture) caused slower cleavages, lower development rates, and fewer cells, plus changes to glucose and amino acid metabolism. Embryos cultured individually under 20% oxygen were affected further, so that combining the two detrimental conditions resulted in earlier and longer delays to development, fewer and smaller blastocysts, and more exaggerated changes to metabolism compared to those cultured under 5% oxygen in groups. There was very little effect of individual culture under 5% oxygen on fetal and placental development compared to group cultured embryos. However, individual culture under 20% oxygen resulted in smaller and skinnier fetuses, and lighter placentas compared to embryos cultured in groups. This may be because the further the culture conditions are from optimal, the more resources an embryo uses to adapt and survive, or there is an accumulation of trauma, and the manifestations of stress become more exaggerated. Embryos in culture secrete high levels of IL-6, and supplementing culture media with IL-6 increased hatching rate and cell numbers, showing IL-6 to be an embryotrophic cytokine. However, it is just one of hundreds of embryo-secreted molecules that may be involved in paracrine signalling during group culture. This study demonstrates that individual culture is detrimental to mouse embryos, especially in combination with atmospheric oxygen. This raises concerns regarding the increasing prevalence of single embryo culture in human IVF. Furthermore, these data demonstrate the cumulative nature of stress during embryo culture and highlight the importance of optimising each element of the culture system.
-
ItemInterspecific hybridization as a tool for enhancing climate resilience of reef-building corals( 2018)The world’s coral reefs are facing unprecedented changes in temperature and carbonate chemistry caused by the increasing concentration of atmospheric CO2. Recent massive loss of corals across the world suggests that their rate of adaptation and/or acclimatization is unlikely fast enough to keep pace with climate change. This thesis examines interspecific hybridization as a conservation management tool to develop coral stock with enhanced climate resilience and adaptive potential. I start this thesis by discussing the potential benefits and risks of hybridization, and exploring the legal framework associated with hybrids and hybridization (chapter 1). Next, I present the results of interspecific fertilization trials, as well as stress experiments on coral larvae (chapter 2) and recruits (chapter 3) conducted to compare fitness of purebred and hybrid offspring. To understand mechanisms that may have contributed to the observed holobiont fitness differences, bacterial and algal endosymbiont communities associated with these corals were examined using 16S rRNA gene and ITS2 metabarcoding (chapter 4), and coral host gene expression patterns were assessed using RNA sequencing (chapter 5). The following findings and key conclusions have emerged from this thesis. Firstly, all four tested pairs of Acropora species from the Great Barrier Reef were cross-fertile, but the degree of prezygotic barriers varied (chapters 2, 3). In both years in which hybridization was attempted (2015, 2016), the majority of the target species pairs had no or limited temporal isolation (i.e., similar spawning dates and times). The only clear temporal isolation was between the ‘early spawner’ A. tenuis and the ‘late spawner’, A. loripes, although their gametes were still compatible. Gametic incompatibility varied between species pairs and the year of hybridization tests (which involved the same coral species collected from different locations). Levels of cross fertility ranged from no prezygotic barriers in both directions (chapter 3), to successful fertilization in one direction only, and in once case, unsuccessful fertilization in both directions (chapter 2). The observed variations in gametic incompatibility may be a consequence of differences in gamete-gamete recognition molecules. Secondly, hybrid corals were generally as fit as or more fit than parental purebred species (chapters 2, 3). At the embryonic stage, hybrid embryos developed normally and at similar rates as purebred embryos (chapter 3). At the larval stage, survival and settlement of hybrid larvae under 10 days exposure to ambient and elevated temperatures were mostly similar to that of purebreds, but higher than purebreds in a small number of cases (chapter 2). Hybrid recruits also had similar algal endosymbiont uptake rates and photochemical efficiency as that of purebred recruits (chapter 3). Under seven months exposure to ambient and elevated temperature and pCO2 conditions, however, some hybrids showed higher survival and grew larger than parental purebred species under both conditions (chapter 3). Overall, maternal effects were observed in hybrids of the A. tenuis x A. loripes cross (i.e., hybrids had similar fitness to the maternal parent species), and over-dominance in hybrids of the A. sarmentosa x A. florida cross (i.e., hybrids had higher fitness than both parental species), with some variations between traits and treatment conditions. While fitness of these hybrids in the field and their reproductive potential are yet to be investigated, these findings provide proof-of concept that interspecific hybridization may enhance coral resilience and this approach may therefore increase the success of coral reef restoration programs. Thirdly, the observed holobiont fitness differences between offspring groups were likely due to host-related factors (chapter 5), but not the microbial communities associated with these corals (chapter 4). No differences in the bacterial and microalgal endosymbiont community composition were found between hybrid and purebred corals (chapter 4). Microbial communities of these seven months old recruits were highly diverse and lacked host specificity. Winnowing of the communities occurred over time, resulting in less diverse microbial communities that differed between the two species pair crosses by two years of age. Transcriptome-wide gene expression analysis for the A. tenuis x A. loripes cross showed clear maternal patterns (chapter 5), consistent with the observed fitness results. Hybrids had similar gene expression patterns to their material parents, and only up to 10 differentially expressed genes were observed between them. In contrast, hundreds of genes were found differentially expressed between purebred A. tenuis and A. loripes, as well as between hybrids that had different maternal parents. Due to insufficient material available for the A. sarmentosa x A. florida cross at the end of the seven months aquarium experiment, transcriptome analysis was not conducted for this cross. Findings from this thesis support the notion that interspecific hybridization may improve coral resilience and facilitate adaptation to climate change. Further, as genetic diversity within species is predicted to decline as a consequence of high mortality disturbances such as mass bleaching events, interspecific hybridization can be used to restore losses in genetic diversity. If future studies can demonstrate high fitness of hybrid corals in the field and in advanced generations, hybrid corals may serve as a stock for reef managers for reseeding degraded reefs and/or enhancing resilience of healthy reefs.
-
ItemHormone-responsive networks influence urethral closure and phallus growth( 2018)Phallus development in mammals is androgen-dependent but can be affected by oestrogen. Many genes that are important in regulating phallus development in mice are also androgen and/or oestrogen responsive. The balance between androgen receptor (AR) and oestrogen receptor α (ERα) activity is important to maintain normal penile development. Interfering this balance either by treating with sex steroids or AR inhibitors, such as flutamide, induces abnormal phallus phenotype in mice and rat. However, very few studies have looked into the global regulation of gene networks on phallus development under the direct effects of sex steroids. Different from eutherian mammals, tammar wallaby has a unique phallus developmental process, where the phallus differentiation occurs postnatally. Such unique postnatal development allows us to observe the direct effects of sex hormones on phallus development without considering maternal physiology and placental transfer. In this thesis, male tammar wallabies were treated with oestrogen and females were treated with the adiol from day 20-40 postpartum (pp) during the established androgen imprinting window (day 25-30 pp). Another group of male tammars were castrated at day 25 pp. Phalluses were collected for morphological comparison at day 150 pp, when urethral closure was almost completed, and for transcriptomic analysis at day 50 pp when they first become sexually dimorphic to examine changes in multiple signalling pathways, including SHH/WNT signalling pathways, IGF1 signalling pathway and AP-1 signalling pathway, as well as to identify novel hormonal responsive lncRNAs, such as, lnc-BMP5, lnc- ZBTB16 and lnc-RSPO4, during phallus development in the tammar. Administration of oestrogen to males inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment during the imprinting window in females did not promote phallus elongation but did initiate urethral closure. Along with multiple stage hormonal treatments conducted by Leihy, et al., (2004) in the tammar phallus, the data suggests that short-term exposure of androgen during the window of sensitivity is necessary for urethral closure, but a constant level of androgen is required to maintain later phallus elongation. Hormonal manipulation altered gene expression in multiple signalling pathways during tammar phallus development. Adiol treatment suppressed SHH expression and its associated genes, including MAFB, WNT5A and RSPO4, but upregulated IGF1 and AP 1 genes in male phalluses at day 50 pp. After day 50 pp, SHH was transiently increased in male phalluses, which might be regulated by the increase level of IGF2 in the liver. The androgen-SHH switch might be the signalling constraint for the timing of phallus differentiation. While, the later activation of both endocrine IGF2-SHH and androgen- IGF1 signalling may be required to trigger and maintain male urethral closure. More interestingly, the activation of androgen dependent window (day 40-120 pp) altered IGF1 signalling, which may not only maintain phallus elongation by regulating cell proliferation, but also contribute to urethral closure by increasing the proliferation in urorectal septum. During the androgen dependent window, IGFBP3 expression was negatively correlated with that of IGF1 in phalluses, suggesting that IGFBP3 may negatively regulate IGF1 signalling in the tammar phalluses. Hormonal manipulation also altered non-coding gene expression profile of phalluses. I identified several predicted trans-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive coding genes IGF1, a candidate gene regulating urethral closure and phallus growth, AR and ESR1. Interestingly, more than 50% of ARcorrelated coding genes and lncRNAs were also co-expressed with ESR1. In addition, I identified and validated three novel cis-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium in day 20 and day 90 post-partum (pp) male phalluses, and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 pp. LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. In addition, another lncRNA, LEAT1, which has a short but highly conserved region in mammals, was also co-expressed with its neighbouring regulating gene, EFNB2, in the tammar phalluses at day 30 and day 50 pp. Thus, exogenous steroid hormones appear to have significant effects on the complex network of both coding genes and lncRNAs controlling phallus development. Phallus development is under strictly programmed molecular regulation of SHH and IGF1 signalling as well as lncRNAs, which are susceptible to exogenous sex steroids. This study offered new insights to understand the complex gene networks between coding genes and lncRNAs during phallus development under the regulation of a balanced endocrine environment.
-
ItemThe bryophyte flora of Lord Howe Island: taxonomy, diversity and biogeography( 2018)Before this study the known Lord Howe Island bryophyte flora (mosses, liverworts and hornworts) totalled 173 species, consisting of 131 mosses, 40 liverworts and 2 hornworts. For this study I conducted one month of field studies on the island, during which I collected more than 650 specimens, and also studied collections from the island held in Australian herbaria and other collections available in overseas herbaria. Fourteen moss, 32 liverwort and 1 hornwort species are newly reported from Lord Howe Island, including one liverwort new to science. A further 2 moss and 2 liverwort varieties are also new to the island. Twenty-eight moss and 11 liverwort species are discounted from the island’s flora, as well as 3 moss varieties and 1 liverwort variety. As a result, the known bryophyte flora now totals 178 species, consisting of 117 moss species (122 taxa), 58 liverwort species (60 taxa) and 3 hornwort species. These totals exclude 5 moss and 2 liverwort species whose taxonomic status or presence on the island is considered uncertain. One liverwort variety, Heteroscyphus echinellus var. echinellus, is new to Australia. Fourteen bryophyte species and one variety are endemic to the island. Spiridens muelleri, previously thought to be the same as S. vieillardii from New Caledonia, is shown to be a separate species endemic to Lord Howe Island. Chiloscyphus howeanus is also shown to be a legitimate species endemic to the island. Cololejeunea elizabethae is described as a new species, also endemic to the island. Trachyloma wattsii, considered to be endemic to Lord Howe Island, is supported by a molecular analysis as a legitimate species most closely allied to T. planifolium. Confusion about the correct identities of the two Ptychomitrium species on the island is resolved through a revision of the genus for Australia. A previously unrecorded morphological character of Atrichum androgynum is described from a study of Lord Howe Island plants, and a molecular analysis shows that South American plants previously ascribed to A. androgynum do not belong to that species. Hypnodendron vitiense is shown to be paraphyletic, but not as circumscribed by Touw (1971). The Lord Howe Island plants appear to belong to a morphologically cryptic species distinct from H. vitiense s.str, and substantial genetic variation within H. vitiense subsp. australe as currently circumscribed suggests that it might include more than one taxon. Other molecular investigations clarify the relationship between Lord Howe Island populations and mainland Australian populations of a number of moss species. An original and novel investigation of the potential modes of transport of bryophyte propagules to and from the island is made, and a hypothesis is formed about the origins of its bryophyte flora and the biogeographic relationships to the Australian land mass and other western Pacific islands, including New Zealand and New Caledonia. The nearest region of the Australian mainland is shown to be the most likely origin of most of the island’s bryoflora, with the injection of propagules into the high-level jet stream by storms the most likely dispersal mechanism. The presence of numerous otherwise tropical species on the island is probably a result of dispersal by tropical cyclones moving into the Pacific from north-eastern Australia. Migratory birds are shown to be another potential vector for bryophyte dispersal to the island.
-
ItemTargeted gene flow for conservation: northern quolls and the invasive cane toad( 2018)Global biodiversity is declining at an unprecedented rate. Within declining populations, however, there are some individuals who are able to survive the threat. Unfortunately in many cases these adaptive traits are not common enough to prevent extinction, particularly when threats are rapid and severe. But by understanding how species respond to certain threats conservationists may be able to boost adaptive potential in threatened populations. Targeted gene flow is a novel conservation tool that involves moving individuals with relevant traits to areas where they could be beneficial for conservation. Although the implications are wide reaching, this idea is yet to be attempted on a wild population. In this thesis, I set out to test the feasibility of targeted gene flow as a conservation tool, using the endangered northern quoll (Dasyurus hallucatus) as a model species. Northern quolls have experienced dramatic declines since the introduction of the invasive cane toad (Rhinella marina) because the quolls unsuspectingly attack the toxic toads. There are, however, a small number of remnant quoll populations that have survived the toad invasion, seemingly because they do not attack toads. It is this potential “toad-smart” behaviour I hoped to harness using targeted gene flow. If it was possible to breed toad-smarts into still threatened areas of the northern quoll’s range, managers could boost adaptive potential and population survival. The first step was to understand how some individuals could survive alongside toads. In the preliminary chapters of this thesis, I examine toad-exposed northern quolls to see how they react to cane toads. I found that quolls from areas invaded by cane toads were indeed toad-smart – they didn’t attack toads. Using a common garden experiment, I then demonstrated this toad-smart behaviour had a heritable basis, meaning I could potentially breed the trait into threatened populations. The next step was to explore how best to implement targeted gene flow for quolls, including investigating any potential negative impacts. I used population modelling to explore the optimal timing and number of individuals introduced to maximise population survival whilst maintaining species-level genetic diversity. I then set up an experimental field trail, releasing both toad-smart and toad-naïve northern quolls onto a toad-infested island. Despite unforeseen circumstances that resulted in a dramatic reduction in population size, I was able to demonstrate no negative implications of targeted gene flow from the first stage of the experiment. This thesis shares the process of exploring a new conservation strategy, from initial conception to field trials. I provide evidence that targeted gene flow could reverse declines of threatened northern quoll populations – demonstrating a genetic basis for toad-smart behaviour, showing little evidence of outbreeding depression, and presenting the ideal management approach for implementing the tool in threatened populations. The resulting strategy is not limited to northern quolls, but instead has widespread applications for other threatened populations. Even the most endangered populations often have some individuals who are resistant to a threat. If conservations can understand and harness these adaptive traits, targeted gene flow could prove an invaluable tool for conserving threatened species.
-
ItemUnderstanding how extreme heat events affect the heat budgets of Australian flying-foxes (Pteropus spp.): roles of morphology, physiology and behaviour( 2018)Heatwaves have increased in intensity, duration and frequency in the recent years as a result of climate change. Australian flying-foxes (Pteropus spp.) have been gravely affected by these extreme heat events with mass die-offs resulting in thousands of dead bats and abandoned pups. In order to prevent or control the death toll, it is important to predict when, where, and how these deaths might occur. I first tested the ability of a simple air temperature threshold to predict when and where flying-fox heat stress related mortality might occur. I used the ACCESS-R meteorological forecast from the Bureau of Meteorology and the flying-fox colony location data from the national flying-fox monitoring program to assess the accuracies of using forecast air temperature to predict die-offs. More than 70% predictions were correct when using 48 hour and 24 hour forecasts. In an effort to better understand the mechanistic basis of heat die-offs, I examined fur properties, thermophysiology and behaviours of flying-foxes relevant to their heat budgets, to gain more insight into how these variables and traits may affect different species, sexes and age classes during extreme heat events. Using museum specimens, I explored the impact of fur depth, fur length and fur solar reflectance on flying-fox heat stress. Grey-headed flying-foxes (Pteropus poliocephalus) had longer and deeper fur, while the black flying-fox (P. alecto) had lower fur solar reflectivity compared to other species. Moreover, females and juveniles had deeper fur compared to males and adults, respectively. I conducted thermophysiological experiments to gain insight on the effect of the natural variation in water vapour pressure on the body temperature, metabolic rate, water loss rate and behaviour of the grey-headed flying-fox at high air temperatures. The body temperature did not significantly vary at the different vapour pressure levels. However, the metabolic rate and the intensity of wing-fanning and panting (thermoregulatory behaviours) were significantly higher at higher vapour pressure levels. By means of these results and other parameters affecting the heat budget of a grey-headed flying-fox, I created and validated a biophysical model to better predict flying-fox heat stress and related mortality, using the NicheMapR mechanistic modelling framework. Analyses revealed that flying fox energy expenditure was most sensitive to the variation in fur depth and incoming solar radiation. Furthermore, the biophysical model showed greater accuracy at predicting flying-fox die-offs compared to the forecast air temperature models (88.0% vs. 72.0%). This indicated the importance of incorporating all environmental variables and animal characteristics to predict heat stress, and not only air temperature. Importantly, this mechanistic approach to predicting die-offs helps to identify key driving forces of heat stress conditions in flying-foxes, and provides a framework to understand the effects by habitat and unusual weather combinations. This in turn will allow authorities to use evidence-based strategies to manage and conserve the flying-fox colonies and species as a whole. Moreover, as flying-foxes are regarded as bioindicators of extreme heat events, these results will help us to gauge the impact of extreme heat events and climate change on other more cryptic taxa.
-
ItemBiochemical mechanisms of biomineralization and elemental incorporation in otoliths: implications for fish and fisheries research( 2018)All vertebrates have small bioinorganic “earstones” in their inner ear labyrinth that are essential for hearing and balance. While otoliths play a vital anatomical role in fish, their true value to science is as biochronometers, largely due to their unique pattern of growth. Otoliths first form in embryo and continue to grow throughout the life of an individual, with a double-banded increment composed of a calcium carbonate-rich region and a protein-rich region being deposited daily. In addition to this, they are considered to be metabolically inert, and do not undergo remodelling or resorption. Consequently, otoliths are employed in a variety of ways in fish ecology. Firstly, an individual fish’s age and growth rate can be estimated through counting increments and measuring their widths. Secondly, analysis of increment trace element:calcium ratios, such as by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), can allow for the reconstruction of environmental histories, aiding in the determination of natal origin, movement, habitat use, diet and the impacts of climate change. The utility of specific trace elements as indicators of environmental change, however, is unclear as there is considerable uncertainty as to whether a given trace element is interacting with the mineral or protein components of an increment. This uncertainty is a consequence of otolith research having been largely focussed upon either microstructure or inorganic chemistry, with very few studies on the protein-rich regions of the otolith. As a result, very little is understood about the biochemical mechanisms of biomineralization or trace element incorporation. This is important, as the mechanisms that govern otolith formation and growth underpin the assumptions made in traditional increment analyses. In this thesis, I initially undertook a systematic review of all the literature pertaining to otolith biochemistry, revealing the significant gaps that exist in otolith biochemistry as a discipline. Importantly, I determined that fewer than a score of otolith proteins had been identified – a stark contrast to the hundreds or thousands of proteins that have been identified in comparable biomineral systems such as enamel or bone. Working on black bream (Acanthopagrus butcheri), an extensively studied species endemic to southern Australia, I used size exclusion chromatography coupled with ICP-MS to determine the trace element:protein interactions in endolymph, the inner ear fluid that otoliths are submerged in, and the source of all of its constituents. In this study, I assayed 22 elements, and determined that 12 were solely present in a protein-bound form, 6 were present as free ions, and 4 were present in both forms. This allowed me to make recommendations as to their utility in environmental reconstructions. In my next study, I created a unique, multi-disciplinary workflow that combined transcriptomics with proteomics. In this study, I sequenced the transcriptome of the black bream inner ear and used this to identify proteins from the separated organic phase of otoliths and endolymph from wild caught adult black bream. This resulted in the discovery of hundreds of previously unknown proteins, providing new insights into the likely biochemical mechanisms involved in otolith formation and growth. In my final study, I tested the utility of trace element ratios in environmental reconstructions. Specifically, I compared the ability of different cluster analysis approaches to resolve spatial and temporal differences in the likely spawning and larval nursery habitats of juvenile black bream in the Gippsland Lakes, Australia. The results from my thesis have allowed me to make recommendations as to the utility of trace elements in environmental reconstructions and have revealed exciting new avenues of research that fuse ecology and biochemistry.
-
ItemRegulation of mucilage production in the Arabidopsis seed coat through MADS-box TF family members( 2018)During Arabidopsis seed development, the epidermal cells of the seed coat produce large quantities of pectinaceous mucilage polysaccharides. Following imbition, mucilage is released from these cells and forms a thick protective layer around the germinating seed that can be visualised with Ruthenium Red staining. The seed coat epidermis is therefore an attractive system to investigate the production of cell wall polysaccharides, particularly pectin. Previously, mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) have been shown to be associated with a mucilage extrusion defect that is caused by reduced expression of the β-galactosidase MUCILAGE MODIFIED2 (MUM2). To better understand the role of LUH in regulating MUM2, RNA-Seq analysis was performed on whole seeds and identified genes that were differentially expressed in luh mutants. This transcriptomic analysis not only revealed elevated expression of transcription factors with a known role in epidermal cell differentiation, but also several MADS-box transcription factors that perform roles in floral development and silique shattering such SHATTERPROOF1 (SHP1) and SHP2. This thesis presents evidence that SHP1, SHP2 and SEPALLATA3 (SEP3) are a new class of regulators involved in mucilage polysaccharides modification, as loss of their activity result in mucilage extrusion defects. These mucilage defects are enhanced in shp1 shp2 double mutants and correlate with reduced MUM2 expression. While carbohydrate analysis data failed to show an expected increase in galactose residues attached to pectin in shp1 shp2 and sep3 mutants, the decrease of MUM2 in these mutants is shown to contribute to their mucilage defects as partial rescue of mucilage defects in shp1 shp2 and sep3 was achieved with rescue of MUM2 expression. This is further supported by the use of SRDX repressor motif fusions to SHP2 that not only produced mucilage extrusion defects when introduced into Col-0, but also were correlated with the level of reduced MUM2 levels. Finally, the positive regulation of MUM2 by MADS-box TFs was established through a combination of CArG-box motif mutations in the regulatory sequences of MUM2, demonstrating the requirement of MADS-box TFs for proper MUM2 seed coat expression. This was further supported by luciferase-based transactivation assays, which produced increased MUM2 promoter activity on the addition of SHP1, SHP2 and SEP3. In addition, this positive regulation of MUM2 by SHP1, SHP2 and SEP3 was shown to occur in a complex with LUH, mediated by the co-regulator SEUSS (SEU), in protein-protein interaction assays such as yeast 2-hybrid (Y2H), Y3H, and bimolecular fluorescence complementation (BiFC). Overall, this study provides novel, clear evidence that MADS-box TFs can directly bind to MUM2 and mediate its activation with LUH. Future work includes expanding on and integrating MADS-box TFs into a working model that describes the regulatory pathways controlling mucilage polysaccharides production in the Arabidopsis seed coat.
-
ItemNovel gene therapy for the treatment of diabetes-induced heart failure( 2018)People with diabetes are at risk of developing myocardial abnormalities known as diabetic cardiomyopathy. This is characterised by diabetes-induced left-ventricular (LV) impairment, which develops independent of hypertension, coronary artery or valvular heart disease, leading to an increased risk of heart failure. To date, there is still no effective or specific treatment for diabetic cardiomyopathy. Hence, the overall aim of my thesis was to investigate the therapeutic potential of targeting two distinct novel pathways, the phosphoinositide 3-kinase (PI3K)p110α axis and the hexosamine biosynthesis pathway (HBP)/O-GlcNAcylation, in the setting of diabetic cardiomyopathy. PI3K(p110α) is a lipid kinase that regulates several physiological functions, including membrane trafficking, adhesion, actin rearrangement, cell growth, and survival. Recent findings from our laboratory and others have highlighted that PI3K(p110α) is cardioprotective in a range of different cardiac pathologies. In Chapter 3, I investigated whether cardiac-directed PI3K(p110α) gene therapy ameliorates diabetic cardiomyopathy in a mouse model of type-1 diabetes (T1D) in vivo. I revealed that administration of recombinant adeno-associated virus-6 constitutively-active PI3K(p110α) (rAAV6-caPI3K) attenuated diabetic cardiomyopathy, even when administered after the initial manifestation of LV diastolic dysfunction. I then proceeded to investigate the cardioprotective effects of rAAV6-caPI3K in the more clinically-prevalent type-2 diabetes (T2D) setting. In Chapter 4, I elucidated that restoration of cardiac PI3K(p110α) activity, through the administration of rAAV6-caPI3K gene therapy, attenuates T2D-induced cardiomyopathy, together with limiting ROS generation. In comparison to the cardioprotective nature of PI3K(p110α), the HBP and subsequent protein O-GlcNAcylation have been implicated in the development of diabetic cardiomyopathy. The generation of β-N-acetylglucosamine (O-GlcNAc) from HBP is a substrate for the post-translational protein modification (PTM) called O-GlcNAcylation. Two specific enzymes regulate the addition and removal of O-GlcNAc modification; O-GlcNAc transferase (OGT) catalyses the addition of GlcNAc to proteins and O-GlcNAc-ase (OGA) facilitates its removal. In Chapter 5, I aimed to elucidate the effect of cardiac manipulation of O-GlcNAcylation in the setting of diabetes-induced cardiac dysfunction in vivo. I demonstrated here that a cardiac-selective increase in OGT (via rAAV6-OGT gene delivery), the enzyme responsible for O-GlcNAcylation, is sufficient to drive cardiac dysfunction and remodelling, resembling that seen in diabetic cardiomyopathy. In contrast, increasing cardiac OGA (via rAAV6-OGA gene delivery), the enzyme responsible for the removal of the O-GlcNAc moiety, attenuates several characteristics of diabetic cardiomyopathy, likely at least in part through the improvement of mitochondrial function. Finally, in Chapter 6 I investigated the impact of O-GlcNAcylation on the PI3K(p110α), pathway and the effect of PI3K(p110α) gene therapy on HBP signalling. I elucidated that PI3K(p110α) can negatively regulate consequences of the HBP and O-GlcNAcylation as part of its cardioprotective actions in diabetic cardiomyopathy, while HBP/O-GlcNAcylation inhibits PI3K(p110α)-mediated signalling, likely contributing to the ability for this pathway to exert cardiac impairments. In conclusion, data from this thesis reveal that gene therapies targeting PI3K(p110α) and HBP/O-GlcNAcylation are viable therapeutic targets for diabetic cardiomyopathy. These results hence provide a basis for pursuing gene therapy for the treatment of diabetes-induced heart failure.
- 1 (current)
- 2
- 3
- 4
- 5