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Increasing Zn content of cereals, particularly rice, is an approach for combating global Zn malnutrition. This project aimed to investigate the use of CNPs as a Zn carrier to increase rice Zn content. CDs bound with Zn were used as a foliar spray in the study to determine their effectiveness in delivering Zn to rice plants. The CNPs utilized in the study neither had detrimental effect on the growth nor showed deleterious effect on physiology of the rice plants tested. The possible physiological and molecular mechanism of a Zn efficient rice variety (IR36) and a Zn inefficient variety (IR26) were also studied under CNP. ZnCD application significantly increased shoot and seed Zn content of the IR36 variety, and this response was not seen in the IR26 variety. In contrast, the Fe content of IR 36 seeds increased significantly compared to the bulk Zn application. This validates prior research that found a positive relationship between Zn and Fe uptake in rice. Nonetheless, the responses of the two rice varieties revealed differences in Zn uptake and grain loading demonstrating varying Zn utilization efficiency. Comparative transcriptome and proteome analysis were used to determine the molecular mechanism by which rice responds to CNP-under contrast Zn utilization genetics. RNA-Seq-based transcriptomic analysis found 251 upregulated genes in IR36 in response to ZnCDs that were not upregulated in IR26. These set of activated IR36-specific genes implicated in Zn uptake, grain filling, auxin signalling, and abiotic stress signalling pathways. Our finding clearly demonstrates that the genetics of Zn use efficiency is further amplified by ZnCDs. The comparative proteomics analysis showed ZnCD application to IR36 upregulated 38 proteins, while IR26 upregulated 210 proteins, with just 2 similarities between the two groups. These IR36-specific proteins were heavily implicated in pathways like glucose metabolism, and photosynthesis. Findings of this study could be successfully used in formulation of a CNP based nano-fertilizer which could be used to address the global Zn malnutrition. In addition, greater understanding of the genetic mechanism of Zn utilization efficiency will open up new avenues for breeding and engineering cereals to increase their grain Zn content.

The opium poppy, Papaver somniferum L., is a pharmaceutically important species that is of commercial significance in the state of Tasmania, Australia. Poppies are grown for seed and notably for their alkaloids, including morphine, codeine and thebaine. Alkaloids accumulate in the capsule during plant development, sequestered to vesicles within the latex of the reticulated network of laticifer cells. Laticifers, as the repository for alkaloids in poppies, are potentially an important component in the yield of alkaloids, and there are aspects of these cells that have not been fully investigated. The studies herein examined the volumetric capacity of capsule laticifers; the allocation of biomass to poppy capsules; and the capability of laticifers to deliver alkaloids to the capsule through the bulk movement of latex. Capsule laticifer capacity is of relevance in the cropping system used in Australia, where capsules are not lanced and remain intact until harvesting, which occurs after plant senescence. Variations in the volumetric capacity may represent a component of the alkaloid yield from capsules, and the first investigation assessed the quantity and variability of this parameter in field grown poppies. The latex volume was calculated for capsules that were harvested at one week, and at two weeks after flowering. It was demonstrated that the volume of laticifers per unit area of tissue was established early in capsule development (before the first week of flowering) and did not increase thereafter, yet capsule morphine content continued to rise. The capacity determined at either stage did not predict the end-yield of morphine, but there was a positive linear relationship with capsule mass, and capsule mass was the most important factor in capsule morphine yield. It was concluded that the volumetric capacity of capsule laticifers was established early in capsule development, and therefore has limited utility as a component that may be manipulated to increase capsule alkaloid yields. Factors influencing capsule mass were further investigated. The within-plant allocation of biomass to capsules was investigated in a field study of P. somniferum plant morphology. Greater than 80% of the capsule biomass was allocated to the upper three capsules, with the senescence of reproductive structures more prevalent on lower branches. The findings indicated that in the development of this commercial line of poppies there was sequential regulation of the sinks (fruit), and that the majority of reproductive biomass was allocated to the early developing capsules in terms of both fruit and seed. An argument was presented to suggest that developmental processes and within-plant resource limitation may influence the allocation of biomass to reproductive structures in poppies. Due to the complex interplay of sink strength and feedback mechanisms, and with so little modelling of within-plant biomass allocation in poppies, additional studies were suggested. The capability of laticifers to deliver alkaloids to the capsule through the bulk movement of latex was conducted through an investigation of the vesicle populations and osmotic potential of both capsule latex and stem latex in P. somniferum. Vesicle populations were examined during plant development, at localised positions and in terms of their volume. Capsule latex contained significantly greater vesicle numbers, packed vesicle volume (PVV), and osmotic potential than stem latex, and these significant differences occurred as a steep gradient across the thalamus of the poppy capsule. Latex from three sampled positions on plants of P. somniferum also differed in the development of vesicle populations, with capsule latex exhibiting a steep increase in vesicle number after flowering. The findings indicated that the development of latex (within laticifers) was localised to separate organs, and that in toto latex movement to the capsule was unlikely. In summary, the research in this thesis reveals that laticifers represent a consistent volume in capsule tissue; that capsule mass is important for alkaloid yield and that the upper capsules have an advantage in the development of that mass; and that the in toto movement of latex through laticifers into the capsule is improbable.

Celiac disease is triggered by ingestion of gluten from wheat, rye and barley in genetically susceptible patients. In the western population, only 1% of individuals develop celiac disease despite 35% being genetically predisposed. This suggests that environmental exposure plays a role in aetiology of disease. This thesis links bacterial exposure with celiac disease as patients could recognise molecular mimics of gluten derived from Pseudomonas species. We uncovered the mechanism behind immune cell recognition of wheat-gluten peptides and bacterially-derived molecular mimics of these peptides. Future efforts to target key interactions identified in this study may offer therapeutic benefit.

Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.

The plant cytoskeleton is commonly considered a vital component of cell growth and development; however, it also plays a critical role in plant immunity. During plant immunity, the cytoskeleton orchestrates rapid and precise immune-associated processes. For instance, the cytoskeleton mobilizes and orients the movement of organelles, proteins, and chemical signaling. To counter plant immunity, bacterial pathogens deliver virulence proteins, known as T3Es (type III effectors), into plant cells through a needle-like apparatus called the type III secretion system (T3SS). A novel T3E, called RipU, interacts with the cytoskeleton. Data has shown that RipU co-localizes with cytoskeletal markers in tobacco leaves. Ectopic expression of RipU can suppress PTI responses like ROS bursts or seedling growth inhibition. Tomato plants inoculated with Rs K60 lacking RipU showed less wilting and root colonization, suggesting that RipU plays a role in pathogenesis and virulence. Furthermore, inducible expression of RipU in Arabidopsis dramatically alters plant development. These plants have wavy roots, branching root hairs, and underdeveloped true leaves. Our results suggest that by targeting the cytoskeleton, RipU contributes to Rs K60s pathogenicity and virulence.

Interleukin-10 (IL-10) is a pleotropic cytokine implicated in infection-associated immunopathology, autoimmunity, and allergy. It was tested as a potential therapeutic agent against several inflammatory and autoimmune disorders. However, systemic IL-10 administration proved to be of limited value, which indicated that its production needed to be carefully targeted in order to be efficacious therapeutically. Hence, research focus shifted on understanding the regulation of IL-10’s downstream targets, so they could be utilised instead in a more cell-specific approach to therapy.Macrophages are regulated by the modulation of metabolic events, and can shift their functional status in response to their changing microenvironmental stimuli. The focus of this project was to investigate how IL-10 impacted on inflammatory macrophage metabolism and whether manipulating targets of IL-10 could result in a reduced inflammatory burden overall. Here, I uncover two targets of IL-10, both part of the arginine-polyamine pathway, as being attractive candidates for modulating macrophage polarisation. The first target, Spermine oxidase (Smox), is a catabolic enzyme of the polyamine pathway, which was upregulated in lipolysaccharide (LPS)-stimulated inflammatory macrophages, but was significantly inhibited in the presence of IL-10. Chemical inhibition of Smox in LPS-treated cells significantly reduced cytotoxicity, pro-inflammatory cytokine secretion, and reactive oxygen species (ROS) production.The second target that is more extensively characterised in this thesis is the mitochondrial isoform of arginase, Arginase-2 (Arg2), which was significantly enhanced by the presence of IL-10 in LPS-stimulated murine and human macrophages. I demonstrated that Arg2 was essential for enhancing mitochondrial respiration in both quiescent and inflammatory macrophage cells. I further delineated that Arg2 enhanced macrophage mitochondrial respiration by enhancing the activity and assembly of a particular node of the electron transport chain at Complex II/Succinate dehydrogenase (Sdh). The product of Sdh, the metabolite fumarate, was found to be downregulated in Arg2–/– macrophages, while the substrate of Sdh, succinate was upregulated. This finding correlated with a significant increase in the expression of the transcription factor Hypoxia Inducible Factor-1α (HIF-1α) and the pro-inflammatory cytokine Interleukin-1β (IL-1β) in the Arg2–/– cells. Collectively, this is the first report of the Arg2 isoform affecting mitochondrial oxidative output in an immune cell type, which in turn modulates the overall inflammatory output of the cell.

Cystic fibrosis (CF) is an inherited disorder caused by a mutation in the CFTR gene and characterised by progressive lung damage due to cycles of infection and inflammation. Gene-based therapies have been proposed as a potential treatment approach for CF. MicroRNA (miRNA) are short 21-24 nucleotide duplex RNAs responsible for post-transcriptionally regulating protein expression. We have identified a miRNA of interest, miR-146a which has previously been shown to target IRAK1 and TRAF6 signalling, thereby reducing inflammation. Herein we investigate the potential role of miR-146a in CF and its therapeutic potential. Delivery of exogenous miRNA therapeutics is a growing area of interest. However, their clinical translation is limited by the availability of safe and effective delivery systems. Novel star-polypeptide/peptoids are promising gene vectors capable of forming polyplexes with nucleic acids. In this work, we evaluate for the first time the star-polypeptides as miRNA carriers to prepare innovative miRNA-nanomedicines for treatment of CF via nebulisation.The levels of IRAK1 and TRAF6 gene and protein expression are significantly higher in CF versus non-CF bronchial epithelial cells while miR-146a expression is significantly lower in CF versus non-CF bronchial epithelial cells. IRAK1 and TRAF6 levels in CFBE41o-cells decreased by ~50% when miR-146a was delivered to these cells using Ribojuice™ while pro-inflammatory IL-8 production in response to LPS was attenuated by 56%. Stable nano-sized, star-miR-146a polyplexes can be formed which are capable of being nebulised and significantly reduce IRAK1 (~50%) protein and IL-8 production (~23%) in CFBEs. Overall a device-integrated, star-miRNA nanoparticle platform has been developed.

Defective haemostasis can contribute to development of a bleeding diathesis, whereas dysregulated coagulation increases the risk of thrombotic events. Acute or chronic inflammation contributes to haemostatic imbalance by promotion of pathological activation of blood coagulation, although the molecular basis for inflammation-driven thrombosis remains poorly defined. In this thesis, I sought to identify novel biological parameters underlying phenotypic heterogeneity in people with severe haemophilia (PWH). Plasma from PWH was found to be more likely to have increased plasma fibrinolytic activity compared to plasma from a healthy age-matched cohort. This was found to arise due to significantly increased plasmin generation activity and/or increased resistance to thrombin-activatable fibrinolysis inhibitor (TAFI) activity in PWH plasma. In addition, a novel positive correlation was identified between increased plasma plasmin generation capacity and development of joint damage in PWH, whereas PWH body weight was inversely correlated with their plasmin generation capacity. In the second part of this study, I showed that macrophage polarisation resulted in divergent effects on macrophage-mediated procoagulant and fibrinolytic activity. Macrophage procoagulant activity induced by exposure to bacterial (lipopolysaccharide, LPS) or viral (single-stranded RNA) mimetics was dependent upon a glycolytic shift in macrophages that could be reversed by chemical inhibitors of glycolysis, thus identifying a novel link between haemostatic macrophage activity and cellular metabolism. Finally, I identified that b-glucan-induced myeloid cell trained immunity in vitro and in vivo lowered the threshold for induction of procoagulant and antifibrinolytic activity by secondary proinflammatory stimuli, and that this phenomenon was dependent upon b-glucan-induced metabolic and epigenetic modifications. Furthermore, I found that the protoporphyrin-IX component of free haem was a novel inducer of trained immunity in myeloid cells, which also promoted exaggerated procoagulant and antifibrinolytic activity upon exposure to subsequent pro-inflammatory stimuli.Collectively, this study used novel assays to define new components of fibrinolytic pathway activity as mediators of phenotypic heterogeneity in PWH. In addition, I identified new molecular mechanisms and cellular pathways underlying myeloid cell procoagulant activity, that may contribute to the pathophysiology of thrombo-inflammatory disease.

Neutrophils are the first cells recruited to an immune stimulus stemming from infection or sterile injuries via a mixture of chemoattractant cues. In addition to eliminating pathogens, neutrophils coordinate the overall inflammation by activating and producing inflammatory signals in the tissue while modulating the activation of other immune cells which in some cases leads to adverse tissue damage. Over amplified or chronic neutrophil recruitment directly leads to autoimmune diseases including rheumatic arthritis, diabetes, neurodegenerative diseases, and cancer. Dampening neutrophil recruitment is a strategy to intervene in neutrophil-orchestrated chronic inflammation. Despite intensive research over the past several decades, clinical studies targeting neutrophil migration have been largely unsuccessful, possibly due to the prominent redundancy of adhesion receptors and chemokines. Additional challenges lie in the balance of dampening detrimental inflammation while preserving immunity. Neutrophils are terminally differentiated cells that are hard to study in cell culture. Mouse models are often used to study hematopoiesis, migration, and chemotaxis of neutrophils but is very labor intensive. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified eight miRNAs as potent suppressors of neutrophil migration. We have generated transgenic zebrafish lines that overexpresses these candidate miRNAs where we recapitulated the mitigation in neutrophil motility and chemotaxis to tissue injury or infection. Among those we further characterized two miRNAs which have not been reported to regulate neutrophil migration, namely miR-722 and miR-199. MiR-722 downregulates the transcript level of rac2 through binding to the rac2 3'UTR. Furthermore, miR-722-overexpressing larvae display improved outcomes in both sterile and bacterial systemic models, which correlates with a robust upregulation of the anti-inflammatory cytokines in the whole larvae and isolated neutrophils. miR-722 protects zebrafish from lethal lipopolysaccharide challenge. In addition, overexpression of mir-722 reduced chemotaxis of human neutrophil like cells, indicating that miR-722 is a potential agent to reduce inflammation in humans. MiR-199, decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. In summary, our results reveal previously unknown functions of these miRNAs, and provide potential avenues to modulate neutrophil migration as well as lead to discoveries of novel factors which can regulate this process. We have also discovered a non-classical role of CDK2 in regulating neutrophil migration which provides directions for alleviating systemic inflammation and a better understanding of neutrophil biology.

Phosphorus (P) is an essential macronutrient for plant growth and development. P deficiency is becoming one of the most limiting factors for crop productivity. It has been discovered that vascular tissue-mediated systemic signaling plays important roles in plant responses to P deficient growth conditions. In order to understand vascular tissue-specific molecular alterations in response to P deficiency, I used Plantago major as a model species to study the transcriptomic alterations in vascular tissues because it is fast and easy to dissect pure vascular tissues from this plant. I identified 237 differentially expressed genes involved in various roles to P deficiency, such as “phosphate metabolism and remobilization”, “sucrose metabolism, loading and synthesis” and “plant hormone metabolism and signal transduction”. In addition, translating ribosome affinity purification (TRAP) was used to identify 547 differentially expressed genes from the Arabidopsis vascular tissues. AtERF, one of the downregulated genes, was chosen for further functional characterization. My results demonstrated that AtERF is specifically expressed in vascular tissues and it encodes a transcription factor. Over-expression of AtERF led to a purple vein phenotype, decreased growth of shoots and roots, and reduced Pi concentrations in shoots and roots. The erf mutant plants displayed larger shoots and roots, and increased Pi concentration in shoots and roots. Molecular analysis in the over-expression and mutant plants showed that genes related to hormone metabolisms and root architecture establishment might be the major players enabling plants to cope with low P. The discoveries from this study may be used to implement strategies for the production of crops with increased P uptake efficiency.

Toxoplasma gondii is capable of infecting almost any nucleated cell of warm-blooded animals and it is estimated that one-third of the world population is infected with latent toxoplasmosis. To date, there are no effective treatments available to treat and control the spread of this disease, and the survival pathways of this organism remain unclear. This thesis investigates the role of sirtuin in modulating T. gondii survival using sirtuin activators and inhibitors in tandem with gene identification, molecular cloning, protein expression, enzymatic characterization and in silico analysis of this novel parasite protein. Taken together, the findings in the present study suggest that T. gondii Sir2A may not play a role in the survival of RH strain T. gondii.

The GENIE Project Team at HHMI Janelia Research Campus have developed a multiple genetically-encoded fluorescent indicators including the jGCaMP8 Calcium indicator family, the Voltron and Positron voltage indicators, and animal models incorporating these indicators. This poster provides updates on developments in each tool and how to obtain them. Chemigenetic voltage indicator improvement: Voltron2 showed a 65% increase in 1AP sensitivity and 3x better subthreshold membrane potential detection. Mutations in the voltage-sensing domain (Ace2N) were swapped between various types of voltage indicators and compared with patch-clamp. A new positive-going variant, Positron2, showed a 300% improvement in sensitivity. jGCaMP8s and 8m transgenic mice: TetO-jGCaMP8s mouse has ~5X increased SNR compared to TetO-GCaMP6s. Sensitivity and fast kinetics match AAV performance. TIGRE jGCaMP8s/m knock-in mice stably expressed in targeted neuronal subpopulations with reduced tetTA toxicity effects. Mice available soon from JAX.

Supplementary data for a manuscript submitted to the American Journal of Physiology-Endocrinology and Metabolism. This data includes the organ weights for experimental mice at 47-weeks of age, the location of the knockout locus, and the results from two different preptin mouse serum kit experiments.

This study addresses some outstanding issues in the epigenetics (DNA and histone modifications) and epitranscriptomics (RNA modifications) fields for gene regulation and embryonic stem cell (ESC) state maintenance that have not been resolved yet. These comprise: i) Investigate combinatorial gene regulatory functions of all histone-demethylases (HDMs, a class of epigenetic regulators) in murine ESCs. ii) Identify the novel m6A RNA methylation-reader/binding proteins and dissect their roles in maintaining the murine ESC state. iii) Examine underlying mechanisms that link epigenetic and epitranscriptomic pathways for gene regulation to maintain the ESC state.

Geraniin is a natural compound present abundantly in the rambutan rind and has the potential to ameliorate diet-induced metabolic syndrome. This study was carried out to identify whether geraniin's interaction with gut microbiota is the main mechanism for its efficacy against metabolic syndrome. Male Sprague Dawley rats were treated with various doses of geraniin and an enriched extract of geraniin (GEE). We observed that 5mg geraniin and 115mg GEE supplementation significantly improved diet-induced metabolic aberrations while increasing the relative abundance of a few butyrate-producing bacteria and plasma butyrate. These findings suggest that geraniin and its enriched extract are promising therapeutic agents against diet-induced metabolic syndrome.

Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear. Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative in vitro model is critical to elucidate the signaling pathways orchestrating CSF production in the CP. This research aims to characterize an in vitro culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.

Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer-related deaths worldwide. Women consistently show lower CRC incidence and mortality rates than men.In addition, pre-menopausal female patients have better 5-year survival rates than age-matched male patientsand post-menopausal patients, supporting a pivotal role for oestrogen in protecting women against CRC. Oestrogen protective effects in CRC havebeen mainly attributedto the oestrogen receptor beta(ERbeta)but its expression is lost during CRC progression.The role of the G-protein coupled membrane oestrogen receptor GPER, which remains expressed after ERbeta loss,is currently under debate. Wehypothesised that oestrogen can protect against CRC progressionviaGPER,bymodulating the Wnt/beta-catenin proliferative pathway which is commonly hyperactivated in CRC. In support of a sex-specific regulation of the Wnt/beta-catenin pathway, we found that expression levels of several Wnt/beta-catenin signalling pathway genes associated differentially with survival in each female:malecohort from 3 independent CRC patient datasets. We investigated the ability of GPER modulation to impact Wnt/beta-catenin signalling inCRC cell lines with its natural ligand 17beta-oestradiol, the specific agonist G1 and antagonist G15. We detected limited impact of these GPER modulators on Wnt/beta-catenin signalling under basal conditions. However, we have shown in HT29 cellsexposed to pharmacological overactivation of the Wnt pathway, G1 significantly prevented the Wnt-induced increase inexpression of the oncogene JUN.Other Wnt targets such as AXIN2and KCNN4were not impacted by G1 treatment, nor was there a detectable change in beta-catenin nuclear accumulation. Thus, our resultssuggest that GPER signalling might selectively reduce the tumorigenic effects of the Wnt/beta-catenin pathway hyperactivationin CRC by limiting JUNexpression. This novel finding provides a mechanism by which GPER may protect against CRC progression.Furthermore, we have validated,in an independent cohort, apreviously reported female-specificand late-stage-dependent negative survival association of GPERexpression in CRC patients, supporting a potentialnegative shift of GPER actions in late-stage CRC.Our results highlight the importance of sexual dimorphism, tumour stage and location in order to truly understand the role of oestrogen in colorectal cancer.

A major aim in evolutionary biology is to understand the repeatability of adaptation and the role of hybridization during invasion. The thesis used two alien plant species, Cakile edentula and C. maritima, to investigate phenotypic and genotypic signals of adaptation and the contribution of hybridization on two continents. Parallel latitudinal clines in phenology and size evolved in the alien ranges in only 100-150 generations, mirroring the native ranges in both species. Moreover, I found hybridization occurred in both invaded ranges and evidence that introgression of the same regions has aided rapid parallel climate adaptation in two separate invasions of C. maritima.

Atypical teratoid rhabdoid tumours (ATRTs) are high-grade malignant paediatric brain tumours with a less than one-year survival rate after diagnosis. Current treatment for ATRT results in long-term side effects on ATRT patients. Here is an urgent need to discover targeted therapies that could be used to treat patients with ATRT. As part of the Hudson Monash Paediatric Precision Medicine Program, this project provides a framework for applying an integrative multi-omics approach in paediatric cancer precision medicine. This thesis demonstrates that ATRT cells preserved clinically relevant genetic architecture, and informed on novel therapeutic strategies for ATRT.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and is a leading cause of mortality worldwide. The pathophysiology of sepsis is characterised by endothelial dysfunction, hypercoagulation, and sustained hyperinflammation and are key events that lead to multi-organ failure and death. A growing body of literature now suggests that the vascular endothelium plays a critical role in driving early events of sepsis progression. Upon bacterial infection, the endothelium releases a specific class of extracellular vesicle called exosomes. Exosomes are plasma membrane-derived lipid nanovesicles (30 – 120nm) capable of transporting biological material from donor to recipient cells. Biological material including DNA, protein, mRNA, and microRNA (miRNA). While the nature of the signals that drive the sustained hyperinflammatory response observed in sepsis are unknown, there are suggestions that genetic elements may be playing a role. miRNAs are short ~22 nucleotide posttranscriptional and translational repressors of gene expression. They bind mRNA with partial or complete complementarity, which results in translational inhibition of mRNA or its’s catalytic cleavage. Therefore, miRNAs can alter cell signalling pathways, gene expression and physiological responses including inflammation. An increased exosomal load containing a dysregulated miRNome released into the bloodstream by an infected endothelium may be driving the sustained an excessive inflammatory response observed in sepsis. There were three primary objectives of the research presented in this thesis. First, to demonstrate whether Staphylococcus aureus (S. aureus) infection of endothelial cells affected exosome production and exosomal miRNA packaging and expression. Secondly, to deliver S. aureus infected endothelial exosomes to monocytes and observe alterations to the inflammatory phenotype of these cells. Thirdly, to identify the specific miRNA pathways involved in driving the proinflammatory phenotype in monocytes. In this study, we demonstrated that S. aureus infection does not alter exosome production in endothelial cells. However, the small RNA (Delivery of S. aureus infected endothelial exosomes to monocytes increased CD11b and MHCII expression. Both CD11b and MHCII are upregulated in monocytes in response to proinflammatory stimuli and are, therefore, markers of monocyte activation. Increased proinflammatory marker expression correlated with heightened production of the proinflammatory cytokine IL-6 and dampened production of the antinflammatory cytokine IL-10. Serum levels of both IL-6 and IL-10 in sepsis have the highest correlation with mortality ¬in-vivo (3). Interestingly, mTOR protein expression was elevated following delivery of S. aureus infected endothelial exosomes. We demonstrated that inhibition of mTOR using miR-99a and miR-99b mimetics heightened IL-6 and dampened IL-10 production following S. aureus infection. Whereas inhibition of miR-99a and miR-99b using antagomirs facilitated increased mTOR activity which had the opposing effects on IL6 and IL-10 production. Collectively, we have demonstrated that endothelial cells release exosomes containing dysregulated miRNA expression profile following S. aureus infection. These exosomes shift monocytes into a proinflammatory phenotype resulting in heightened IL-6 production, a key proinflammatory cytokine that mediates the initial events leading to sepsis. We demonstrated that this increase in IL-6 production was directly correlated with mTOR activity. Overall, we have identified a novel pathway by which the endothelium drives the proinflammatory phenotype of monocytes in response to infection. Our data suggest that blocking miR-99a and miR-99b inhibition of mTOR following infection may be appropriate treatment strategies for critically ill sepsis patients. Further work is required to assess the effects endothelial exosomes have on other innate immune cells such as macrophages, neutrophils, dendritic cells and to identify the exact mTOR signalling cascade involved.

A thesis concerning the effects of water deficit on stomatal traits in plants. The relationships between different traits and their influence on overall stomatal anatomy is discussed. Genetic work to investigate molecular regulation of stomatal development is also presented and discussed.

Nitrogen (N), an essential plant macronutrient, is among the most limiting factors of crop yield. To sustain modern agriculture, N is often amended in soil in the form of chemical N fertilizer, a major anthropogenic contributor to nutrient pollution that affects climate, biodiversity and human health. To achieve agricultural sustainability, a comprehensive understanding of the regulation of N response in plants is required, in order to engineer crops with higher N use efficiency. Recently, epigenetic mechanisms, such as histone modifications, have gained increasing importance as a new layer of regulation of biological processes. However, our understanding of how epigenetic processes regulate N uptake and assimilation is still in its infancy. To fill this knowledge gap, we first performed a meta-analysis that combined functional genomics and network inference approaches to identify a set of N-responsive epigenetic regulators and predict their effects in regulating epigenome and transcriptome during plant N response. Our analysis suggested that histone modifications could serve as a regulatory mechanism underlying the global transcriptomic reprogramming during plant N response. To test this hypothesis, I applied chromatin immunoprecipitation-sequencing (ChIP-Seq) to monitor the genome-wide changes of four histone marks (H3K27ac, H3K4me3, H3K36me3 and H3K27me3) in response to N supply in tomato plants, followed by RNA-Seq to profile the transcriptomic changes. To investigate the organ specificity of histone modifications, I assayed shoots and roots separately. My results suggest that up to two-thirds of differentially expressed genes (DEGs) are modified in at least one of the four histone marks, supporting an integral role of histone modification in regulating N response. I observed a synergistic modification of active histone marks (H3K27ac, H3K4me3 and H3K36me3) at gene loci functionally relevant to N uptake and assimilation. Surprisingly, I uncovered a non-canonical role of H3K27me3, which is conventionally associated with repressed genes, in modulating active gene expression. Interestingly, such regulatory role of H3K27me3 is specifically associated with highly expressed genes or low expressed genes, depending on the organ context. Overall, I revealed the multi-faceted role of histone marks in mediating the plant N response, which will guide breeding and engineering of better crops with higher N use efficiency

This thesis examines E. coli trafficking and improves its gene delivery efficiency into eukaryotic cells. This was achieved by addressing cellular hurdles responsible for its under-functioning. The new strategies developed in this project would enable bacteria vector to be applied more effectively for DNA vaccination and cancer therapy

The aim of this Ph.D. thesis was to investigate and characterise the role of Human Heterogeneous ribonucleoprotein A1 (hnRNPA1) in Influenza A virus life cycle. We found that hnRNPA1 exerts a protective role in Influenza A infection as enhancing hnRNPA1 levels, impaired viral replication and vice versa. Furthermore, it was deciphered that Nucleoprotein of IAV interacts with hnRNPA1 to subvert and co-opt pro-survival and cell death signaling pathways in infection. Thereby, substantiating it's anti-viral function.

Ribosomes are complex molecular machines that synthesise the cell's proteins. Assembling ribosomes is complex and requires input from over 200 different proteins. Many of these proteins also have additional roles within the cell, however, these additional roles are not well understood. This thesis used a microscopic worm organism, C. elegans, to examine the function of a ribosome assembly protein called RIOK-1. Important roles for RIOK-1 in regulating development, metabolism and immunity were identified. Mutations in RIOK-1 commonly associated with various cancers, and therefore these results may help us better understand the role of RIOK-1 in human health and disease.

Dengue fever is a serious mosquito-borne viral disease caused by the dengue virus (DENV) and multiple infections by the mosquitoes could be severe and life-threatening. Currently, there is no remedy against the virus. Accurate and early detection of dengue is vital to reduce the mortality rate due to the severe type of dengue fever. This thesis identifies markers that have the potential to predict the disease. The identification of these biomarkers may aid clinicians in the prediction of patients who may progress to severe dengue so that early treatment could be undertaken to reduce the tendency of fatal dengue.

Fabry disease (FD) is a rare genetic disorder that leads to the accumulation of lipid in cells. This study aimed to develop a more sensitive method to diagnose FD and produce the protein drug for FD management in a cost-effective manner. In this thesis, the method of detecting the Fabry lipid in human cells using antibody and functional studies of recombinant protein drug against FD that was produced by bacteria were described. The development of new alternatives in this study proposes for more accurate throughputs in mass screening and affordable therapies of FD in clinical settings.

The human immune system plays a crucial role in health, safeguarding against disease and infection. Central to this protection is the ability of immune cells to distinguish ‘healthy’ and ‘diseased’ cells. However, our understanding of the mechanisms underpinning such recognition is incomplete. My Thesis sought to understand how a class of T cells (γδ T cells) recognise potential infection. Using high-resolution imaging we revealed the molecular mechanisms T cells use to recognise bacterial infection. Complementary work informed how these molecular recognition events may lead to an immune response. These discoveries could pave the way for immunotherapeutic development of such T cells.

Every cell in the body contains the exact same DNA but each cell only uses a proportion of the genes depending on its function. In adults, PRC2 is a molecule which switches off many genes that are only required during embryonic development. Incorrect control of which genes are turned off by PRC2 can lead to cancer. My thesis investigated how PRC2 is targeted to certain genes by its partner protein PHF19. PHF19 causes PRC2 to bind tightly to DNA. This does not depend on the DNA sequence but may depend on the 3D organisation of the DNA in the cell.

Candida albicans is a highly adaptable human fungal pathogen that can cause superficial to serious infections. Incidences of antifungal resistance are increasing and pose a major concern to our healthcare system. This thesis examines the molecular components and pathways which enables Candida albicans to develop resistance and tolerance following exposure to antifungals.

This thesis describes the structural, kinetic and biophysical characterization of the M1 and M17 enzymes from the two leading causes of malaria in humans, Plasmodium falciparum and Plasmodium vivax, and the murine model species, Plasmodium berghei. We discovered a novel mechanism of M17 enzyme regulation through manipulation of the active conformation, profiled the extended substrate specificity fingerprints of the Plasmodium M1 and M17 enzymes, and demonstrated the ability of small inhibitory molecules to achieve cross-species and dual-enzyme potency. As Plasmodium M1 and M17 enzymes are validated targets for new anti-malarials, this work will assist in designing effective novel therapeutics.