Science

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  • (2021) Al-Nakashli, Russul
    Thesis
    Cellular uptake of nanoparticles plays a crucial role in their biomedical applications. Despite abundant studies trying to understand the interaction between nanoparticles and cells, the influence of cell geometry and the drug loading efficiency on the uptake of nanoparticles remains unclear. In this study, poly(vinyl alcohol) (PVA) is micropatterned on polystyrene cell culture plates using UV photolithography to control the spreading area and shape of cancerous and normal cell lines. The effects of these factors on the cellular uptake of HPMA-based micelles were investigated. Cancer cells with larger sizes and circular shapes, had higher total micelle uptake, On the other hand, normal cells showed opposite behaviours. Moreover, the effect on drug loading efficiency on the cellular uptake of polymeric micelles together with cellulose nanoparticles was also investigated. Both nanoparticles used as nanocarrier of the potent anticancer agent, ellipticine (EPT) at varying amounts. In the self- assembled P(HPMA-co-MAA)-b-PMMA micelles, the drug loading efficiency of EPT was observed to range between 50.0% to 62.6% depending on the ratio between drug and polymer. The IC50 value of U87MG cells were 0.67, 0.64 and 0.92 μg/mL. for EPT-200, EPT-100 and EPT-50 respectively, showing a higher toxicity in micelles with higher drug loading content. However, the cytotoxicity values measured from U87MG spheroids were lower than those measured in the 2D model. Cellular uptake was studied by confocal microscopy and flow cytometry, showing that the higher EPT loaded micelles were taken up more efficiently by the cells. Additionally, self-fluorescent cellulose nanofibers (PHEA-g-CNFs) were also used to investigate the effect of loading efficiency on the cellular uptake. Additional negatively charged carboxylic groups of CNFs allowed for protonated EPT loading by electrostatic interactions at various drug loading capacities from 1.68 to 8.07 wt %. In vitro cytotoxicity of blank and EPT-nanocellulose was also investigated on 2D and 3D cellular models. Lower drug loading was favorable for cellular uptake of cancer and healthy cells. The results indicate that the geometry of cells and the drug loading efficiency influence the nanoparticle uptake and may shed light on the design of functional nanoparticles for anticancer drug delivery.

  • (2020) Hu, Shangfeng
    Thesis
    The neural cell adhesion molecule 2 (NCAM2) is a cell adhesion molecule (CAM) of the immunoglobulin superfamily (IgCAMs). NCAM2 plays an important role in the developing and mature nervous system by regulating neurite outgrowth and synapse formation and maintenance. NCAM2 is also involved in neurodevelopmental and neurodegenerative brain disorders, such as autism spectrum disorder (ASD) and Alzheimer’s disease (AD), respectively. β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartic protease, which plays a key role in AD by cleaving the amyloid precursor protein (APP) to produce the toxic Aβ peptide accumulating in brains of AD patients. Synaptic levels of NCAM2 are reduced in the hippocampus of AD patients and Aβ treated cultured hippocampal neurons. Since BACE1 activity is increased in brains of AD patients, we investigated whether there is a functional interaction between NCAM2 and BACE1. Our study shows that NCAM2 can be cleaved by BACE1 and BACE2, a homolog of BACE1. The cleavage site of NCAM2 by BACE1 and BACE2 is within the extracellular domain region, which is close to the transmembrane domain of NCAM2. The intracellular domain of NCAM2 is not required for the cleavage. Our study also shows that BACE-dependent cleavage of NCAM2 is not enhanced by Aβ. The BACE-dependent turnover of NCAM2 is higher in hippocampal than in cortical neurons possibly due to higher exo- and endocytosis of BACE1 in hippocampal neurons when compared to cortical neurons. We also found that the cell surface levels of BACE1 and the shedding of BACE1 are increased in NCAM2 knock-out neurons, whereas the total levels and endocytosis of BACE1 are not affected by NCAM2 deficiency. Taken together, our study demonstrates that NCAM2 is cleaved by BACE1 and BACE2 and regulates cell surface levels of BACE1 most likely by regulating the delivery of BACE1 to the cell surface.

  • (2020) Pereyra, Joao
    Thesis
    Evidence is mounting that the incidence and intensity of harmful algal blooms is increasing, most likely as a result of climate change and its consequential environmental effects. Bloomforming cyanobacteria can produce an array of natural products including toxic secondary metabolites known as cyanotoxins. Among the most significant cyanotoxins from a water management as well as a biomedical perspective are saxitoxin and its chemical analogues, known collectively as the paralytic shellfish toxins (PSTs). This thesis expands our understanding of PST production in cyanobacteria, with a focus on the genetic and molecular elements responsible for the formation of specific PSTs that collectively give rise to unique toxin profiles. Toxic cyanobacteria strains from around Australia were investigated to determine correlations between phenotype, genotype and toxicity. Examination of the toxic strains, belonging to the species Dolichospermum circinale, revealed strain-specific PST profiles. A comparative analysis of these strains revealed that morphology, phylogeny or geography did not appear to be responsible for the variation in toxin quotas. Next generation sequencing was used to analyse and compare the genomes of a subset of these toxic strains to identify genetic elements responsible for PST profile variation. The presence of genes putatively involved in the formation and regulation of PSTs was consistent in all four genomes, as well as a toxic D. circinale reference genome. However, analysis of the gene cluster responsible for the biosynthesis of PSTs (sxt) uncovered tailoring genes previously not reported in any other homologous cluster, and essential for the production of specific PSTs making up the toxin profile of each strain. An expression system was developed for the characterisation of cryptic tailoring enzymes from the sxt gene cluster. Using this system, the sxtX gene, putatively responsible for the conversion of saxitoxin to neosaxitoxin, was expressed as a series of truncated SxtX polypeptides, providing insight into the likely cofactor required for this enzymatic step. The results of this thesis present novel insights into the genetic and molecular factors governing PST production and regulation, expanding our knowledge of these cyanobacterial neurotoxins.

  • (2020) von Lupin, Carolin
    Thesis
    Lanthipeptides belong to the group of ribosomally synthesised and post-translationally modified peptide natural products. The steps of lanthipeptide biosynthesis include the dehydration and cyclisation of specific amino acids to form the characteristic lanthionine residues. Lanthipeptides are divided into four different classes depending on the enzymes catalysing these post-translational modifications. The final step of their biosynthesis involves a proteolytic cleavage that generates the biologically active compound. Lanthipeptides are best known for their antibacterial activity but have also been reported to exhibit antifungal, antiviral and antinociceptive bioactivities, which makes them of commercial interest. This thesis describes the discovery of the first lanthipeptide biosynthesis gene cluster from a marine proteobacterium, the endosymbiont Pseudoalteromonas sp. HM-SA03. In silico analyses of the gene cluster revealed characteristics of class I as well as class II lanthipeptides suggesting the formation of a new (sub-)class that may be specific to Gram-negative bacteria. Multiple sequence alignments of precursor peptides showed that the leader peptides lacked the conserved FNLD/FDLD motif, which was reported to be essential for enzyme recognition. The identified class I modification enzymes were observed to incorporate dehydrated residues and lanthionines in vivo in Escherichia coli. Additionally, three potential proteolytic cleavage sites were identified in the precursor peptides. Mass spectrometric analyses of crude extracts from Pseudoalteromonas sp. HM-SA03 confirmed a double-glycine motif to present the preferred cleavage site which is characteristic for the type of class II bifunctional transporter/protease that was encoded within the lanthipeptide gene cluster. Furthermore, the activity of putative regulators of the gene cluster was analysed in luciferase reporter assays. A two-component system was found to be a positive regulator for the transcription of the structural and self-immunity genes, whereas an unknown protein was demonstrated to function as a repressor for the transcription of the entire gene cluster. The findings of this study identified a novel lanthipeptide biosynthetic pathway with unusual characteristics that will enhance the current knowledge and contribute to the expansion of the current classification system. Future studies of the described lanthipeptides are anticipated to reveal potentially valuable bioactivities.

  • (2020) Inman, Victoria
    Thesis
    There is limited biological and ecological data on hippos, most of which originates from riverine/lacustrine populations, with none on the Okavango Delta population in its unique wetland habitat. This thesis aimed to investigate the Delta’s hippo population (size/distribution) and examine small-scale seasonal variations in hippo pod dynamics, habitat selection, and behaviour. The research provides baseline ecological and behavioural data on hippos in wetland habitats and gives insight into their adaptability to changes in water availability. This is particularly important as the Delta’s waters are threatened by climate change and human pressure, which will reduce habitat for hippos. I utilised thirteen years of aerial survey data to examine temporal and spatial patterns of hippo populations in the Delta. Hippos preferentially occurred in large lagoons within seasonal swamps, avoiding the dense aquatic vegetation and deep water of the permanent swamp/main channels. Since the mid-1990s, Botswana’s hippo population has grown significantly, likely due to long-term increases in rainfall and inflow. The most recent survey (2018) emphasises Botswana as a stronghold for this species in Africa, however, the exceptionally small flood event of 2019 will likely have led to substantially reduced hippo numbers. This work demonstrates the value in long-term monitoring programs. I developed a method to monitor (count/age) hippos using a drone, achieving more accurate counts than ground and aerial surveys. Using this method, I examined seasonal changes in hippo pods (size/density/demographic composition/distribution) related to varying surface water availability. In the low flood season, hippos occurred in larger pods in higher densities, aggregating on the minimal water still available. All seasons were characterised by near-constant changes in pods, emphasising their dynamic nature and challenging the notion of stable hippo groupings. I conducted 24-hour activity budgets within the Delta and Chobe River to increase the currently limited knowledge of hippo behaviour and to investigate how behaviour changed temporally, spatially, and seasonally in variable landscapes. Large differences in behaviour between pods (even those within close proximity) indicate hippos are highly flexible, taking advantage of available resources, but also emphasising the effects of humans on hippo populations.  

  • (2020) Pang, Clara
    Thesis
    In this thesis, I utilise the vast amount of competition literature that has accumulated to conduct four literature surveys testing the predictions of traditional strategy theory on the role of competition in the evolution of plant strategies and life histories. Traditional theory predicts that seedlings emerging from large seeds have the advantage over those emerging from small seeds in environments of dense vegetation. Through my synthesis I show that large seed size is associated with high competitive ability but only when plants are competing against other seedlings. My findings offer a new interpretation of seed size strategies suggesting that seedlings have little chance of establishing in dense communities but rather, mainly emerge in open spaces. Next I tested whether shade avoidance plasticity was an adaptive response under competition. Shade avoidance responses have often been thought of as a strategy plants use to outcompete their neighbours for light resources. I found that shade avoidance plasticity was not associated with an increase in competitive performance as has been predicted by theory but that instead the adaptive value of shade avoidance lies in gathering resources to allow earlier reproduction under competition. I tested if these findings extended to plasticity in general which theory predicts gives plants a competitive advantage by allowing them to adjust growth to acquire more of the available resources than their competitors. I found that plasticity was not associated with an increase in competitive ability in either short or long-lived species. Instead plasticity was correlated with increased reproductive efficiency in short-lived species whilst this did not occur in long-lived species. Finally, I tested whether functional traits specific leaf area (SLA) and maximum height were good predictors of competitive ability. I found that neither maximum height nor SLA were good predictors of competitive performance. My findings highlighted that the theoretical basis of our understanding of competition needs to be improved before we can effectively use functional traits to predict competitive outcomes. Overall, this thesis challenges some of the key foundational assumptions of strategy theory and illustrates the need for a change in the way we measure competitive ability.

  • (2020) Liu, Angela
    Thesis
    Yellowtail kingfish (Seriola lalandi, Valenciennes 1833; YTK) farming is considered the greatest opportunity for aquaculture expansion and diversification in Australia. Consequently, the demand for pelleted feed is expected to increase significantly for this species. Nutritional research on YTK has focused on macronutrient requirements and testing alternative protein and lipid sources to fishmeal and fish oil. However, there is a lack of information on micronutrient requirements and their effects on the growth and health of YTK. Therefore, it is important to acquire more knowledge on this topic to optimise the feed formulation for YTK. Choline is an essential nutrient typically supplemented in aquafeed and is abundant in raw materials such as fishmeal and soybean meal. Substituting raw materials for alternative food sources without knowing the choline requirement or its role in YTK performance might hinder YTK production. Accordingly, the main aim of this thesis was to improve our understanding of dietary choline on the growth performance and liver health of juvenile YTK at sub-optimal water temperatures. Liver was selected as a target organ because it is one of the major sites that facilitates various metabolic processes, including choline metabolism, and is susceptible to choline-deficiency induced liver disease. This thesis further explored the efficacy of using naturally-occurring stable isotopes to better understand the interactive effects of dietary choline and water temperature on nutrient assimilation of YTK. The aim was achieved and addressed by conducting two independent experiments with a series of interlinked studies. The research first determined the digestible choline requirement of YTK using a dose-response approach. The choline supplementation of a practical diet formulation at two temperatures (16 °Cand 24 °C) that bracket the culturing water temperatures commonly experienced by farmed YTK in Australia was also conducted. The digestible choline requirement of juvenile YTK was estimated to be 27.3 mg kgBW−1 d−1 or 1.94 g kg−1 diet (choline chloride supplementation: 0.0−10.0 g kg−1 diet), which is higher than for most finfish species. Choline and lipid apparent digestibility improved with increasing dietary choline. In contrast to many animal models, higher liver lipid deposition (in the form of free fatty acids and triacylglycerol) was associated with increasing dietary choline in YTK. However, this species did not exhibit signs of fatty liver disease related lesions which indicates that, if not beneficial, this amount of lipid is not detrimental to juvenile YTK. Upon further examination, dietary choline does not significantly alter the liver lipid composition but might protect liver health of YTK. This research confirmed that supplementing choline in fishmeal-based practical diets was necessary for juvenile YTK reared between 16 °C and 24 °C. Water temperature was largely responsible for the change in liver lipid composition and morphology; however, there was no indication of disease in fish fed practical diets. Importantly, this research demonstrated that the current industry practice of supplementing 3.0 g CC kg−1 diet to a fishmeal-based formula would ensure that the production performance and liver health of juvenile YTK are not compromised when cultured between 16 °C and 24 °C. Furthermore, using naturally-occurring stable isotopes, this research showed that fish had an affinity for assimilating certain raw materials such as sodium caseinate, poultry meal, meat meal, lupin, and wheat flour, irrespective of their inclusion levels. However, dietary choline content and water temperature did not significantly alter the assimilation of raw materials in the practical diet. The results indicate that higher inclusion of poultry meal, meat meal, lupin, and wheat flour in YTK feed is possible and might offer complementary information to digestibility studies. This thesis is the first to determine the choline requirement of YTK and established a scientific knowledge platform for further research on choline metabolism and its interaction with metabolically-linked nutrients. The research also provides scientifically-validated information that will help guide aquafeed formulation to reduce production costs and improve the growth performance and quality of YTK.

  • (2021) Jilani, Sidra
    Thesis
    Solvent rich BMGs (>77% solvent element) are unique among bulk glass-formers (of which most are rich in solute element content), in that they are often quite ductile and exhibit physical, chemical and functional traits more akin to the solvent element. A major fundamental aim of this project was to be explore the effects of topology and composition on the glass-forming ability of solvent rich glasses based on geometric packing efficiency/ cluster models that focus on structural aspects of glass-formation and electro-chemical aspects of the constituents based on electronic contributions and magic number theory. The specific rules that dictate glass-formation were analysed with respect to small atom solvent, medium atom solvent and large atom solvent subgroups of ternary alloys with respect to the atomic size of all constituents. (This was also extended to pseudo-binary type topologies). A comprehensive analysis of existing solvent-rich glasses along with a complimenting experimental program were undertaken to decipher more specific criteria for solvent-rich glass-formation. The approach intends to focus on solvent-rich compositions which shall ideally introduce predominantly metallic bonding among the atomic constituents by maximising the like-like atomic bonds in the amorphous structure. Potentially leading to extended ductility and increases the likeliness that the resultant material may exhibit many of the characteristic properties of the solvent metal. A number of new metallic glasses and in some cases bulk metallic glasses were discovered though the course of this study. It was found that topology was playing a significant role in glass-formation for all the three atomic size categories, and that different glass-forming criteria not only existed for compositions in the three solvent size categories but also for the different atomic size and coordination families within each category, encompassing solute and solvent centred clusters, shared solute atom topological arrangements and varying degrees of medium range order all contributed in various ways for different topologies, which dictate and limit maximum solvent/solute contents for each case. Relationships between atomic bonding bridges and magic number theory were also observed in relation to glass forming ability and thermal stability.

  • (2021) Soares, Georgia
    Thesis
    The Great Oxidation Event (GOE: c. 2.45-2.3 Ga) was accompanied by a series of global glaciations that placed significant environmental pressure on extant microbial life and, perhaps, led to the evolution of eukaryotes. However, the direct impact of the GOE on life remains unclear due to a lack of well-preserved, fossiliferous strata from this time. Here, a novel fossil form is described from the well-preserved shallow water part of a microbialite reef complex that lies between the 1st and 2nd of three global glaciations of the GOE, within the c. 2.4 Ga Turee Creek Group, Western Australia. Referred to as branching organic siliceous structures (BOSS), these novel fossils are organic-rich structures, up to 3 cm high, that are attached to the sides and tops of microbialites in up to 4 separate beds that have intercolumn sediment composed of phosphatic peloidal grainstone to pebble conglomerate. The unique occurrence of BOSS with phosphorite shows that they thrived in an oxygenated, nutrient-rich environment. BOSS display a range of morphology, from simple buds, through cylindrical or simple branching structures, to taller multi-level branching structures. The best-preserved BOSS are composed of kerogen-rich micrite with a faint texture of fine fibres that show a weakly radiating texture from the base to the outer, upper margins of BOSS. The interior parts of BOSS are commonly partially to wholly replaced by fine-grained silica (chalcedony to microquartz), which formed early in diagenesis, or possibly even during growth. Silica replacement of BOSS commenced as point-source growth of individual scattered spherulites and progressed to whole-scale replacement of the interior. BOSS interiors also contain two forms of pyrite: as necklaces of fine aggregates distributed in clusters; and as euhedral, sometimes zoned, grains in the exterior rinds of BOSS with silicified core zones. Sulfur isotopic data from the fine pyrite necklaces provides evidence of a sulfate reducing microbial community within BOSS. Petrographic analysis and morphological comparison show that BOSS are biological and dissimilar to any known type of abiological structure or microbialite. Rather, BOSS are most similar to eukaryotic algal-grade organisms based on their size, morphology and carbon isotope data. BOSS potentially illustrate that eukaryotes evolved up to 750 Ma earlier than previously recognised, coinciding with significant environmental change during the GOE.

  • (2021) Watt, Tavis
    Thesis
    The aim of this dissertation was to determine the benefit and underlying mechanisms of artistic and physical activities, when used as adjuncts to established clinical therapies. Using both categorical questions and standardised ratings, the mechanisms of behavioural activation, belonging, common factors, and flow states were measured in relation to perceived benefit and psychological distress. Six empirical studies used samples from the Australian Defence Force (ADF), a civilian art therapy program, and a population of university students. In the first three studies, participants from the ADF’s Arts for Recovery, Resilience, Teamwork and Skills (ARRTS) Program reported on their experience in the program. The first study retrospectively surveyed former participants (N = 31) and found the perceived benefit and positive experience of mechanisms lasted up to 24 months. The second study (N = 92) surveyed participants during and after the program using standardised ratings. The ratings for all mechanisms plus reductions in psychological distress increased during the one-month program but thereafter largely disappeared within six months. The third study (N = 35) surveyed ADF respondents using categorical questions. Their responses confirmed the benefits and experience of the mechanisms. The fourth study (N = 20), using outpatients in an art therapy program, confirmed the benefit and experience of the mechanisms seen in the ADF studies. The fifth and sixth studies used a nonclinical sample of university students (N=394) to analyse the potential mediating role of the four mechanisms and their factor structure, respectively. In the fifth study, each mechanism individually mediated the relationship between higher levels of physical activity and lower levels of psychological distress. In an integrated model, the mechanisms of behavioural activation and belonging remained as significant mediators. In the sixth study, an exploratory factor analysis revealed that the four mechanisms were distinct. Studies one to three found reductions in anxiety and depressive symptoms, with all underlying mechanisms being experienced by respondents. Study four found the mechanisms were also experienced by art therapy participants. Study five determined that in the context of sport belonging and behavioural activation were the most prominent of the mechanisms. Study six developed a shorter preliminary questionnaire to measure the mechanisms potentially reducing participant burden. The results of the studies are discussed with respect to the role of the four mechanisms in delivering benefit to participants in adjunct activities.