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Cryptococcosis is a global opportunistic infection that can result in a fatal meningitis. Despite improved diagnostics, current treatments have issues including resistance, limited availability and toxicity, highlighting a need for new therapies. Interferon-γ has shown promise as an adjunctive treatment for cryptococcosis, but the mechanism of protection is not yet understood – a role of interleukin-1ß has been proposed, but not validated. Therefore, I sought to determine the significance of interferon-γ and interleukin-1ß in cryptococcosis. Firstly, I examined their effect on murine J774 cell interactions with C. neoformans, as immune responses in pulmonary cryptococcosis are macrophage-centric. Controversially, interferon-γ worsened J774 control of intracellular C. neoformans. I then established an inhalation infection mouse model of pulmonary cryptococcosis to confirm these findings in vivo. Using this model, I show that a single dose of interferon-γ at the time of infection failed to improve host outcomes while infection with an interferon-γ producing strain of C. neoformans, H99a IFNγ, significantly accelerated mouse mortality. However, infections of transgenic mice lacking interleukin1ß showed no differences in survival or fungal burden. However, as mice are not readily compatible with intravital imaging, the effect these cytokines on pulmonary hostpathogen interactions could not be assayed. Therefore, I also established a precision cut lung slice model of cryptococcosis so that live pulmonary immune responses to C. neoformans could be visualised ex vivo. Using this model to examine the first 24 hours of infection, I confirm that macrophages are the main cells to initially respond to inhaled C. neoformans, although do not appear to mediate early clearance – phagocytosis was limited and did not appear to inhibit fungal replication. In conclusion, I have provided evidence that interferon-γ may be dangerous if given to immunocompetent cryptococcosis patients, and present an ex vivo infection model that enables live pulmonary immune responses to be visualised.

Brassicas are important crops susceptible to significant losses caused by disease. Breeding resistant lines can mitigate the effects of pathogens. The first layer of active defence in plants is based on the perception of pathogen- (or microbe-) associated molecular patterns (PAMPs/MAMPs) leading to PAMP-triggered immunity (PTI). In this study I studied the response to various PAMPs including Necrosis & Ethylene-inducing peptide 1-like proteins (NLPs) in 3 different plant species from Brassicaceae. This PhD research investigates the immune system of Brassicas in different aspects, and how it is contributing to quantitative disease resistance (QDR). I developed a segregating population from a cross between two NLP responsive and non-responsive Brassica napus accessions and revealed that recognition of NLP induces the resistance against Botrytis cinerea. In silico mapping of the region associated with NLP-recognition on B. napus genome was accomplished with an improved BSA pipeline and the most significant peak was identified on chromosome A04 spread over a 2.5Mbp region. KASP markers were designed and tested on F2 individuals of the BSA population to narrow down the region and to reduce the number of the candidate genes. I identified 4 KASP markers tightly linked to the phenotype. I also genetically mapped the locus responsible for high NLP-induced ROS burst, and show that BnaBSK1.A01 is involved in modulating the NLP response, and further supported by functional tests of the gene in A. thaliana. The genetic tolerance under drought stress conditions is another agronomically important trait for the changing climate conditions in the world. The effect of environmental conditions, including drought stress, on the PTI responses of the plants observed quite a lot during the study. To investigate the effect of abiotic stress conditions, I optimised reproducible drought stress conditions to enable further mapping of induced QTL on Brassica oleracea genome and design and RNA-Seq experiment to reveal underlying genes. As a result, I showed that drought stress induces both PTI and disease resistance against B. cinerea in B. oleracea. The work will provide new insight into crop improvement, enabling more reliable QDR for controlling Brassica diseases to be developed.

Autophagy is the process by which a cell sends cytosolic material to the lysosome for digestion. In this work, I investigated the importance of autophagy to lipid droplet formation and protein turnover in the neural stem cells, neurons, and glia of the larval Drosophila brain. The role of the endomembrane system in autophagy in neurons and neural stem cells was also considered, as was the movement of acid-resistant protein from neural stem cell lineage to the surrounding cortex glia. The location of various proteins involved in nutrient sensing and autophagic induction was also determined, and the reproducibility of manual image classification attempts was assessed using deep learning.

Disease transmitted by mosquitoes present some of the most pressing challenges facing human health today. Land-use change is a key driver of disease emergence, however the mechanisms linking environmental covariates of change, such as temperature, to the transmission potential of mosquitoes is poorly understood. Studies exploring these relationships have largely been correlative in nature, and thus have limited capacity to predict dynamics through space and time. Mechanistic approaches provide a valuable framework for understanding the processes underlying transmission, however they suffer from a dearth of field data on fundamental mosquito ecology. In both approaches, the environmental data used is typically coarse in scale and interpolated from weather stations located in open areas. In reality, local climatic conditions can vary considerably over fine spatial and temporal scales, particularly in dynamic working landscapes. Wild mosquitoes experience and respond to this highly dynamic environment, and failing to account for this variation may have significant implications for the accuracy of epidemiological models. This thesis uses an established epidemiological framework to explore the effects of tropical forest conversion to oil palm plantation on the potential for Ae. albopictus mosquitoes to transmit disease. Using field-derived microclimate data and published thermal responses of mosquito traits, I first examine how the scale of environmental data affects predictions of mosquito demography under land-use change. Next, I conduct field experiments to investigate whether microclimate heterogeneity across a land-use gradient drives variation in the rates of larval development. By pairing fine-scale microclimate data with temperature-dependent trait estimates, I find that forest conversion significantly increases the potential of Ae. albopictus to transmit disease. Together, these findings advance our understanding of Ae. albopictus ecology, and highlight the importance of incorporating fine-scale environmental and mosquito data into epidemiological frameworks to better understand disease risk in changing landscapes.

The sibling species Anopheles gambiae s.s. and Anopheles coluzzii are the most important vectors of human malaria in sub-Saharan Africa. They are thought to be undergoing speciation with gene flow with rare viable hybrids but are reproductively isolated by assortative mating and ecological divergence. As such important vectors, they are the focus of novel control strategies based on mosquito releases. One of the known drivers of their ecological speciation is their divergent larval ecological adaptation that is possibly linked to rice domestication in Africa. The potential impact of such divergence has never been integrated into mosquito rearing to improve alternative vector control methods such as Sterile Insect Technique (SIT) and Genetically Modified Mosquitoes (GMM), that are needed to accelerate the progress towards malaria elimination. These innovative vector control methods depend on mass rearing of millions of mosquitoes in a manner that is both efficient and economic, to produce mosquitoes that are of adequate quality, able to favourably compete with wild populations. In this thesis, we investigated the phenotypic plasticity of these sibling species to typical stressors in the rice field ecosystem with a focus on ammonia in their larval habitat. Experiments were conducted in small containers and in contrasted microcosms to test the direct effects of mineral water and increasing ammonia concentrations on larval development and to highlight divergent reaction norms between the sibling species. We also evaluated the use of zeolite to improve larval water quality management in An. gambiae s.l. insectary. To further understand the dynamics of the nitrogen cycle in larval rearing trays that led to larval mortality, we characterised their bacteria communities using 16S rRNA gene sequencing. Functional filters were applied to identify candidate bacteria species beneficial and detrimental to larval development and these were validated by qPCR. Our results suggest that genotype-by-environment interactions associated with rice domestication event in Africa are indeed an important driver of the eco-speciation between the sibling species. An. coluzzii was more tolerant to ammonia and rice-field like conditions supporting the idea that this may have driven its speciation from the ancestral An. gambiae s.s. We show that mineral water is beneficial for improved mosquito yield and phenotypic quality of adult mosquitoes in the insectary and this can be used to improve rearing protocols for these species. For the first time, we demonstrated that zeolite can be used to improve rearing results for An. gambiae s.l., providing a water conserving alternative for rearing mosquitoes for mass release programmes, especially in arid regions. Furthermore, the ensuing analyses of bacterial communities larval trays is also a novel endeavour which led to the identification of 1031 bacteria species and of several key species with various opportunities for further improvement of larval rearing towards mass release purposes and/or for novel direct vector control. In conclusion, we have made modest contributions towards the control of these malaria vectors and the fight to eliminate this multifaceted disease. It is therefore important that policy makers in malaria endemic countries ensure that policy reformations in irrigational agriculture and urbanization consider the impact of policy on these disease vectors that are of immense public health importance.

Defective in cullin neddylation-like (DCNL) proteins are known to coordinate the addition of NEDD8 to the cullin subunit of the largest family of ubiquitin E3 ligases, the Cullin-RING ligases (CRLs), in a process known as neddylation. The human genome encodes five DCNL proteins which are thought to exhibit a large degree of overlap in function, with only a few neddylation processes having been definitively ascribed to a single DCNL homologue. It currently remains unclear whether these DCNL proteins have functions that extend beyond their roles in cullin neddylation. In the present study we now describe a novel role for one of the family members, DCNL5, in the programmed cell death response known as apoptosis. We have shown that cells lacking DCNL5 function fail to promote caspase 8 cleavage – an important early activation step - in response to various inducers of the extrinsic apoptosis pathway. Caspase 8 cleavage and activation requires polyubiquitination which is known to be mediated by cullin 3 in coordination with the dual ubiquitin and NEDD8 ligase RBX1. This process is thought to occur in lipid rafts at the plasma membrane and in the cytosol. In the present work, we provide the first indication that DCNL5 is able to translocate out of the nucleus where it was previously thought to be exclusively located, and this occurs in response to TNFα-related apoptosis-inducing ligand (TRAIL) stimulation. In addition, we present evidence for the first known interaction between DCNL5 and cullin 3 in U2OS cells under endogenous conditions. The DCNL5 KO cells demonstrated a lack of a polyubiquitination event that occurs in WT cells; unmodified caspase 8 was shown to associate with a polyubiquitinated protein (the identity of which we were unable to determine) in response to TRAIL and this interaction was absent in KO cells, perhaps representing the key mechanism underlying DCNL5 involvement. This emerging function for DCNL5 in promoting caspase 8 cleavage was confirmed in multiple cancer cell lines including U2OS, H460 and HeLa cells. Importantly, we demonstrated that siRNA-mediated silencing of DCNL5 prevented CASP8 cleavage. A lot of our work suggested that DCNL5’s role in CASP8 activation is mediated by the cullin CUL3. However, treatment with the neddylation inhibitor MLN4924 caused a reduction, but not a total loss of caspase 8 cleavage, suggesting that if cullin 3 is involved, it may be independent of its neddylation status. This hints at the surprising possibility that a CRL complex exists that that does not require neddylation for some of its function. Furthermore, while our data suggests that DCNL5 may regulate apoptosis via cullin 3, we were unable to exclude a cullin neddylation-independent role for DCNL5 in this process. Future work will need to answer this question by identifying and characterising the molecular target of DCNL5 in apoptosis signalling to ascertain the precise mechanism underlying DCNL5 regulation of this clinically important signalling event.

B lymphocytes play a central role in humoral immunity but also have antibody-independent functions. Studies to date have focused on B cells within blood and secondary lymphoid organs. This thesis sought to address the question of whether B cells reside in non-lymphoid organs (NLOs), and to determine their phenotype and function. Using intravenous labelling and parabiosis, we identified a population of bona-fide self-renewing, tissue-resident B cells, represented mainly by innate-like CD5+ B-1 cells, across murine NLOs, including lung, liver, kidney and bladder. The size and phenotype of this tissue-resident B-cell subset was influenced by genetic background, age and the microbiome, with an expanded population evident in pet-store mice. Extravascular B cells had less diverse Igh repertoire with fewer N-additions compared to blood, suggesting their prenatal origin. Seeding of these B cells into NLOs was independent of their antigen specificity. Using strains of genetically modified mice with higher (PI3Kδ E1020K-B, Siglec-G-/-) or lower (μMT-) numbers of tissue-resident B cells in NLOs, we tested the function of these cells in the context of urinary tract infection. The number of tissue-resident B cells inversely correlated with bacterial clearance suggesting that B cells negatively regulate anti-microbial responses. Tissue-resident B cells were spatially co-localised with macrophages and had a profound effect on macrophage polarisation, promoting an anti-inflammatory M2 phenotype, an effect at least partially driven via interleukin (IL)-10. Finally, in human NLOs we found a similar enrichment for non-naïve less diverse B cells when compared to blood and spleen, with indices for innate-like and regulatory phenotype. In conclusion, these data identify a critical role for tissue-resident B cells in modulating organ immunity, determining inflammatory 'set-point' of resident and recruited myeloid cells, with important clinical implications.

Autophagy involves the formation of an endoplasmic reticulum-derived membrane termed a phagophore which expands to engulf cytoplasmic cargo before sealing to form an autophagosome. Amino acid starvation is amongst the most potent autophagic stimuli, however whilst the key signalling complexes involved in starvation-induced autophagy are known, the precise regulatory mechanisms remain poorly understood. The serine/threonine kinase ULK1 and close homolog ULK2 assume the most upstream position in the autophagic signalling cascade and play a crucial yet enigmatic role in coordinating the autophagic machinery. To further understand the mechanisms of starvation-induced autophagy, I performed a number of unbiased phosphoproteomic screens to identify ULK substrates before classifying their roles in starvation-induced autophagy. Analysis of these datasets has revealed that loss of ULK results in significant changes to the phosphoproteome and has yielded a high confidence list of potential substrates whilst also offering interesting insights into the veracity of the published ULK consensus signature. Amongst the novel phosphorylation targets are components of the retromer and AMPK complexes along with multiple components of the class III PI3K VPS34 complex. The pseudokinase p150, scaffolding component of the VPS34 complex, is phosphorylated by ULK1 in vitro and in vivo at serine 861. CRISPR-based knockout of p150 results in inhibition of autophagy and endosomal trafficking, whilst mutating the phosphorylated residue in p150 alters both omegasome establishment and autophagic flux. Furthermore, incorporation of phosphomutant p150 into the VPS34 complex modulates its lipid kinase activity in vitro. These data identify a novel ULK-dependent signalling axis and help illuminate the complexities of signal transduction in autophagy.

Macroautophagy (hereby autophagy) is a catabolic pathway whereby a double membrane organelle called an autophagosome, engulfs cytoplasmic components in order to degrade them via the lysosome. While autophagy is induced in response to various stresses to promote homeostasis and cell survival, a key mechanistic step in initiation of autophagosome formation is thought to be regulated by the serine/threonine kinases ULK1/2. ULK1/2 kinase activity is required for amino acid starvation-induced and other types of autophagy. Our lab recently showed that inhibition of ULK1 with the potent compound MRT68921, results in formation of stalled autophagosomes that are positive for early and late autophagy markers. These data suggested that ULK1 is not only regulating initiation of autophagosome formation but also later stages, such as elongation and fusion with lysosomes. Here, I compared the most selective and potent ULK1 inhibitors available to date as to what autophagosomal defects they cause. From this work it is concluded that ULK1 inhibition may block autophagosome flux by affecting dissociation of autophagy-initiating protein complexes from the forming autophagosome and influencing its closure. In order to understand the underlying mechanisms regulating this process I performed a proximity ligation assay (BioID) using MRT68921 as a tool to enrich for autophagy regulators trapped on stalled autophagosomes. With this approach ATG2B was identified, among others, as a protein that is enriched on autophagosomal structures upon ULK1 inhibition. I here show that ULK1 phosphorylates ATG2B upon autophagy induction in response to amino acid starvation. It is known that loss of ATG2A/B results in accumulation of arrested autophagosomes, consistent with that seen by ULK1 kinase inhibition. Thus, ATG2B and its homologue ATG2A might be key substrates of ULK1, the phosphorylation of which is required for proper autophagosome formation.

Cilia and flagella are highly conserved organelles required for eukaryotic cells to perform sensory and motility functions. They are found in many cells of the human body and are essential for the survival of single celled protozoans. These organelles are nucleated from a basal body after docking to the plasma membrane and are structurally similar to centrioles. Docking of the basal body requires the presence of transitional fibres, which mature from distal appendages on the centriole. These structures rely on the presence of the Cep164 protein to carry out docking and ciliogenesis. Understanding of basal body docking and flagellum length regulation in the Trypanosoma brucei cell is limited and is thought to involve the transitional fibres and surrounding structures. Three diverse Cep164 orthologues in the T. brucei parasite have been identified, but knowledge on their function is limited as functional analysis of these proteins has not been carried out. This project, in collaboration with the TrypTag project used a bioinformatic approach to identify basal body proteins in the T. brucei cell. Candidate proteins were confirmed as basal body components through endogenous tagging and co-localisation studies. The Cep164 orthologues (Cep164A, Cep164B and Cep164C) localised to the distal section of the mature basal body only, with Cep164C showing a cell cycle dependent localisation. Functional analysis of the Cep164 proteins was performed through the generation of inducible RNAi cell lines. Ablation of these proteins showed a functional role of flagellum length regulation, or formation of the transitional fibres and correct basal body docking. This work provides further understanding on the three Cep164 proteins in the T. brucei cell and proposes how the cell regulates its flagellum length.

Complex communities are formed by association of plant roots with microbes in soil, and significantly determine plant nutrition, stress tolerance, and pathogen resistance. These communities are potentially shaped by differential microbial colonisation of roots, competition between colonizing strains, and selective recruitment of microbes by plants - all these processes are poorly understood. To better understand community assembly in the root and how it can affect plant growth and health, this thesis explored experimentally the ecological factors shaping microbial communities. I found that plant host species rather than soils defined microbial communities, although community assembly appears to differ between plant species, as both relatively invariant and dynamic assembly patterns are observed. Phenotypic characterization of rhizobacteria isolates indicated that bacterial functions define root-associated bacteria beyond phylogenetic identities, so that differential colonization and selection are plausible mechanisms for assembly. Nevertheless, isolate host origin was not an important predictor of colonization ability in a gnotobiotic in planta experimental system, although competitive or mutualistic interactions could be very important for colonization and plant growth outcomes. An additional chapter exploring the microbial ecology of a 'replant' disease in a commercial orchard confirmed that disease risk associated with soil legacy was associated with changes in diversity and abundance of unculturable phyla. This project highlights the potential of culture-independent approaches, but also their limitations. Microbial function and microbe-microbe interactions are clearly important for rhizobacterial community assembly, but their elucidation may require more data-intensive approaches. In terms of predicting which microbes might colonize particular plants and whether colonization is likely to be beneficial, this thesis only scratched the surface of the problem. Perhaps not surprisingly, environmental context is very important for understanding when rhizobacteria are likely to be beneficial, while the complexities of bacterial competition and mutualism suggest that stochasticity is important in determining which microbes are successful on which plant.

The zoonotic tapeworm, Echinococcus granulosus, mainly cycles between domestic dogs and sheep, but presents a significant risk to human health and is a source of economic loss for livestock industries. In the UK, E. granulosus is endemic and historically restricted to mid-Wales, surrounding English border regions and the Hebridean Scottish Islands. E. equinus, a non-zoonotic species that mainly cycles between hunting hounds and horses is also endemic. Following a fall in E. granulosus prevalence after a hydatid control programme in mid-Wales in 1979-89, recent surveys report a re-emergence of infection in Wales and case reports suggest a wider distribution beyond known prevalence hotspots. Information on the molecular epidemiology of Echinococcus spp., in particular E. granulosus, to support this emergent and re-emergent picture is needed. This thesis uses questionnaires, coproantigen ELISA, PCR, DNA sequencing and histopathology to study Echinococcus spp. in UK farm dogs, hunting hounds, zoo canids and livestock, collectively called the HyData project. The thesis finds evidence that E. granulosus distribution extends far beyond known hotspots in the UK and shows that E. granulosusis involved in the re-emergence of infection in Wales. In a study of 46 UK sheep farms, 17.4% housed dogs positive for Echinococcus spp. coproantigen, with a widespread national distribution; 10.9% had dogs positive for E. granulosus coproDNA in Wales, reporting for the first time in the North of England, Scotland and Northern Ireland. Positive results were significantly associated with location in Wales (p<0.05), supporting a picture of re-emergent Echinococcus spp. transmission in that region. In a study of 32 UK hunting hound packs, 9.4% hunts tested positive for Echinococcus spp. coproantigen, reported for the first time in the North West and South West of England and the Scottish Borders. A further pack in the North West of England is the first reported positive for E. granulosus coproDNA. In a study of canids and hyaenids in 22 UK zoos, 22.7% of collections (all in England) housed species testing positive for Echinococcus spp. coproantigen, with the first reported UK cases in African hunting dog, European grey wolf, Iberian wolf, Arctic fox, Black-backed jackal collections and the first reported UK case of E. equinus by coproPCR in the European Wolf. A survey of 87 hydatid cases (and 261 controls) in cattle and sheep slaughtered at 15 abattoirs in England and Wales reported 7.1% of samples positive for Echinococcus spp. on PCR; Of samples submitted as hydatid, 32.9% were confirmed as Echinococcus spp. by PCR and 26.9% as E. granulosus by DNA sequencing. Matching of cattle movement records from 23 PCR-confirmed E. granulosus cases reported cattle travelling in Wales or adjacent counties as significantly more likely to have hydatid disease (OR=15.47, p<0.0001). Four cases had never entered Wales: two adjacent in Gloucestershire and Herefordshire and two further afield in Staffordshire and North Yorkshire/Humber. A proof-of-concept exercise to evaluate meat inspection for hydatidosis estimated a diagnostic sensitivity of 30.68%, (95%CI: 11.91-49.44) and specificity of 99.48%, (95%CI: 99.48-100.0) suggesting likely underreporting of disease at meat inspection, although more data are needed to optimise this calculation. Factors associated with increased risk of Echinococcus spp. infection were common to all canine study groups. Over a third of participating farms (36.2%), 79.1% of hunts and 80% of zoos reported feeding raw meat and offal from fallen stock to canines; 44.7% of farms reported witnessing farm dogs scavenge carcases of fallen stock on-farm. Only 57.5% of farms, 29.6% of hunts and 23.5% of zoos were administering a suitable wormer at a minimum dose for E. granulosus control. In farm dogs, sub-optimal worming was significantly associated with a positive E. granulosus PCR result (p<0.05). Routine faeces collection was reported by all hunts, all zoos and 55% of farms; however, 44.4% of zoos, 50% of hunts and 83% of farms then reported disposing of faeces by means, such as muck heaps and farm fields, which could potentially contaminate agricultural land with Echinococcus spp. eggs if used as unprocessed fertilizer, posing an under-researched risk to public and animal health. The thesis findings call for effective and targeted E. granulosus control in dogs and livestock and for nationwide prevalence studies to further explore a renewed public health risk. As an exercise in baseline data gathering, the approach of the HyData project and the thesis findings would inform the planning phase of a UK hydatid control programme.

Background: Programmed Cell Death (PCD) is the genetically controlled death of specific cells following developmental or environmental stimuli. There is potential for PCD to be manipulated through genetic engineering to increase productivity in commercially relevant plant and algae species. KISS OF DEATH (KOD) has been identified as a short peptide involved in the regulation of PCD in Arabidopsis thaliana. Increasing our knowledge of KOD and PCD in general could lead to important innovations in biotechnology. Methods: We investigated the potential of Cell Penetrating Peptides (CPPs) as a tool to assay potential short peptides in microalgae and protoplasts, in the model organisms Chlamydomonas reinhardtii and A. thaliana. We also heterologously expressed KOD in C. reinhardtii to gain further insights into microalgal PCD. Finally, we screened for and tested KOD homologues in A. thaliana. Results: Data from study of CPPs indicates that CPPs may not be suitable for the study of PCD in microalgae as they induce cell death even without a cargo. The expression of KOD in C. reinhardtii led to unexpected results. It was found that liquid cultures, which expressed KOD, maintained cell density in stationary phase for longer periods than controls. This suggests that the heterologous expression of KOD in C. reinhardtii prevented cell death. A KOD homologue was identified which had a PCD sensitive phenotype, providing more evidence for the role of small peptides in PCD. Conclusion: This thesis has provided further insights into the mechanisms of PCD in plants and algae; however, more work is required to develop our knowledge further before PCD can be manipulated to increase the productivity of plants and algae through biotechnology.

The actin cytoskeleton is a complex and dynamic network of protein filaments involved in a great number of cellular processes essential for cellular homeostasis and survival. The dynamic nature of the cytoskeleton requires careful control of actin stabilisation and severing. Cofilin is an important promoter of actin turnover, promoting the severing and disassembly of actin filaments, resulting in increased availability of actin and driving assembly of new actin structures . A major reported negative regulator of cofilin is p21 activated kinase 1 (PAK1). These dynamic structures and the importance of actin turnover result in targets for manipulation by bacterial pathogens, such as Salmonella Typhimurium’s ability to enter non-phagocytic cells, and Shigella flexneri to assemble actin ‘comet tails’ for intracellular propulsion and bacterial dissemination. This work sought to investigate the role that cofilin and PAK1, as well as its upstream and downstream regulators has in actin remodelling, and how this may be usurped by bacterial enteric pathogens, with a particular focus on Shigella flexneri comet tail formation. Shigella LPS O-antigen null mutants have been previously shown to have poor comet tail formation, however the mechanism behind this defect remains controversial. Previous studies have focused on the relationship between LPS length and the functionality of IcsA, the protein primarily responsible for intracellular motility. Here we demonstrate that LPS length does not affect IcsA’s ability to bind the vital actin related proteins necessary to produce actin comet tails, leading to investigation of the wider actin dynamics and the role they play in Shigella infection post-invasion. This work establishes key roles for both PAK1 and cofilin in bacterial manipulation of the actin cytoskeleton, demonstrating that the activation of both cofilin and PAK is required for Salmonella invasion and Shigella comet tail invasion. I provide evidence that the artificial activation of PAK and cofilin via ATP and calcium signalling results in the restoration of several attenuated bacterial mutant strains ability to manipulate the actin cytoskeleton. I provide evidence that Salmonella is able to limit its invasion, by the inhibition of cofilin post-invasion via the bacterial effector SopB, preventing the overloading of host cells and prolonging infection. I propose that Shigella activates cofilin post-invasion in response to caspase-1 activation by the bacterial effector protein IpaB, maintaining the dynamic cytoskeleton required for comet tail formation. This work also provides a potential mechanism for the failure of R-LPS Shigella mutants to form actin comet tails by indicating that the loss of O-antigen results in the secretion impairment of IpaB, resulting in its association to the bacterial membrane. Together these finding outline a novel function for IpaB’s activation of caspase-1 in the promotion of comet tail formation and provides further insight into the relationship between inflammation and actin reorganisation.

Mycolic acids are essential components of the unique, lipid rich cell wall of M. tuberculosis. However, enzymes involved in the biosynthesis of mycolic acids remain under exploited as drug targets despite one of the early and hallmark anti-TB drug isoniazid which inhibits mycolate biosynthesis. Previous studies from our laboratory identified mycolate processing enzymes and transporters. A mycolyl reductase was identified to play a role in the final reduction step in mycolic acid biosynthesis in Mycobacterium smegmatis and Corynebacterium glutamicum. Using gene knockdowns I have now extended these studies to slow growing mycobacteria like Mycobacterium tuberculosis and Mycobacterium bovis BCG. The results in this thesis clearly indicated that the mycolyl reductase Rv2509 involved in the final stages of mycolic acid biosynthesis is essential in M.tuberculosis and M.bovis BCG. Furthermore using BLAST-P alignments and predictions of a 3D structure we identified unique domains and residues in the mycolate processing enzymes, and present functional studies on the same. This unique domain and other residues identified from in silico analysis of the structure of Rv2509 were important for the functionality of the protein following complementation studies. Purification of the protein of Rv2509 led to the identification of the possible substrate.

African trypanosome infections are characterised by antigenic variation to avoid host immunity and by the production of transmission stages to promote disease spread. Laboratory-adapted ('monomorphic') lines of Trypanosoma brucei are reported to switch their expressed VSG antigenic coat at a much lower frequency than 'pleomorphic' populations recently transmitted through tsetse flies. These laboratory-adapted parasites also lose the capacity to differentiate into transmission competent 'stumpy forms'. It is unclear if the reduced rate of antigenic variation is directly coupled to the loss of pleomorphism or whether the processes, although co-selected by multiple passage, are independent. To address whether monomorphism caused a concomitant change in the frequency of antigenic variation, an 'inducible monomorphism' model was exploited. This exploited pleomorphic RNAi cell lines that would inducibly silence genes required for stumpy formation. This provided a tool to switch pleomorphism 'on' or 'off' inducibly, without long term passage. Thereafter, two approaches were used to ask if the induction of monomorphism directly influenced antigenic variation: in vitro flow cytometry-based VSG switch assays, and VSGseq, a targeted sequencing approach. These assays demonstrated that the induction of monomorphism did not reduce VSG switch rate nor generate a reduction in expressed VSG diversity, or change the expressed VSG subset. To extend this analysis further, the prolonged in vitro passage of a pleomorphic cell line was used to select isogenic monomorphic populations with reduced capacity to generate stumpy forms. These selected monomorphs cells did not exhibit a reduction in VSG switch rate compared to the parental pleomorphic population, thus corroborating the observations with the induced monomorphic cells. To understand the loss of pleomorphism in the selected cells, an 'evolve and resequence' approach and RNAseq analysis was adopted. Interestingly, the 'selected monomorphs' were depleted of transcripts whose expression was associated with the stumpy and insect forms of the parasite. Particularly, a number of CCCH zinc finger proteins, such as ZC3H20, were significantly downregulated. These RNA-binding proteins could represent novel regulators of slender to stumpy differentiation. Overall our results demonstrate that T. brucei antigen switch frequency and pleomorphism can be uncoupled, and provide new insight into the molecular control of stumpy formation.

Fc receptor-like 6 (FCRL6) is an immunoreceptor tyrosine-based inhibitory motif-bearing transmembrane receptor upregulated on human cytotoxic T and NK cells during chronic immune activation. It has been suggested to act as an inhibitory receptor by possibly interacting with human leukocyte antigen-DR (HLA-DR), but its function remains largely unknown. We initially investigated the role of FCRL6 in T cells using its murine counterpart. However, Fcrl6 expression was absent in developing, mature or activated mouse T cells. Therefore, we generated a human FCRL6 (hFCRL6) expressing transgenic mouse to investigate the function of this receptor. The expression of HLA-DR on antigen presenting cells resulted in reduced in vitro proliferation of hFCRL6+ CD4+ T cells. However, we were unable to recapitulate this inhibitory effect of the hFCRL6:HLA-DR axis in an in vivo environment. Further characterisation of mouse immune populations revealed Fcrl6 expression in natural killer (NK) and developing B cells. Analysis of Fcrl6-/- mice showed that the development of these cells as well as T cells and the splenic proportion of most major immune populations remained unaffected by FCRL6 deletion. We observed a reduction in the proportion of splenic macrophages and NK cells in Fcrl6-/- and NK conditional Fcrl6-/- mice, respectively. However, NK cell responses in a chronic retroviral infection model as well as a tumour model remained unaffected by FCRL6 deletion. Similarly, B cell antibody production in response to retroviral infection was also unaffected by FCRL6 deletion. Overall, data obtained here suggested that the mouse ortholog of FCRL6 might not possess the potential immunoregulatory capacity of its human counterpart and thus may have evolved to mediate non-immunoregulatory functions. Further studies will be required to define the role of mouse FCRL6 in NK cells and macrophages in addition to hFCRL6 in NK cells and T cells.

The reniform nematode, Rotylenchulus reniformis Linford & Oliveira, is a sedentary species of plant parasitic nematode that is widely distributed in tropical and subtropical regions and causes significant economic loss. There has been little molecular characterisation of R. reniformis, particularly in relation to the function of its effectors. Recent genomic and transcriptomic resources have become available that provide evidence of the complex suite of effector genes in R. reniformis. Expanded families of putative effector genes have been described for other plant parasitic nematodes. In particular it was noted that the Globodera pallida genome encoded a large number (30) of complete glutathione synthetase-like genes in comparison to the free-living nematode C. elegans which has a solitary glutathione synthetase (gs) gene. In this study, we have identified a profusion of 73 complete glutathione synthetase-like genes from the R. reniformis genome and transcriptomes. The phylogeny of R. reniformis GS-like genes divides this family into three major clades: Clade 1 contains only one sequence that is the likely ancestor of the R. reniformis GS gene family; Clades 2 and 3 represent two independent expansions that acquire their unique functions during evolution. In addition, most Clade 3 GS do carry a signal peptide for secretion while Clade 1 & 2 GS do not. Furthermore, most Clade 3 gs are most highly expressed in the parasitic female stage whereas Clade 1 & 2 gs are up-regulated in the non-parasitic stages. In situ analysis showed Clade 3 gs are expressed in the gland cell of R. reniformis which is a common site of nematode effector synthesis. In contrast, Clade 1 & 2 gs are expressed in the intestine tissues. Glutathione synthetase is a key enzyme in the second step of glutathione biosynthesis. Biochemical analysis of GS from R. reniformis confirmed the functional diversity between each clade. Clade 1 GS exhibited the canonical GS enzyme activity which was all-but lacking in Clade 2 & 3 GSs. Crystallography was then exploited to investigate the structural differences between canonical and non-canonical GSs, indicating that an alternative substrate may be accepted by non-canonical GS. This project also set out to investigate the functions of R. reniformis GS. None of the R. reniformis GS, including canonical GS could complement the Arabidopsis GS mutant gsh2. In addition, Arabidopsis overexpressing Clade 3 GS showed enhanced susceptibility to the cyst nematode Heterodera schachtii. In conclusion, this study revealed evolved functional diversity of this expanded large GS family by phylogenetic, biochemical, structural and functional evidence.

Using a novel library of natural products isolated from temperate zone plants, the antiparasitic activity of 643 Phytopure library compounds were determined against intraerytrocytic P. falciparum, the blood-stream form of T. b brucei and axenic amastigotes of L. mexicana. Twelve compounds with a 50% inhibitory effect (EC50) values of less than 6 μM were detected against P. falciparum, 25 compounds with an EC50 values of less than 2.8 μM against T. b brucei, and 23 compounds with an EC50 values of less than 2.8 μM against L. mexicana. The cytotoxicity effects, and thus their selectivity of action against each parasite, of these selected compounds were determined against a human liver cell line (HepG2) to establish priorities for further work. Here, four structurally-related triterpene compounds (700022, 700107, 700136 and 700240) were shown to have activity against axenic and intramacrophage amastigote stages with reasonable selectivity when compared to the THP-1 and HepG2 human cells. By exposing promastigote L. mexicana to increasing concentrations over 28 weeks, a 700022 resistant line was generated in vitro. Promastigotes of this resistant cell line were 7.5-fold more resistant to 700022 than compared to the parental wild type line, with axenic promastigotes having a 40-fold increase in resistance. Interestingly, the 700022 resistant promastigotes had a 25% smaller cell surface area and a 85% reduction in flagellum length. The 700022-resistant line was cross resistant to the related triterpenes 700107, 700136 and 700240 and miltefosine (11.8-fold compared to wild type strain). The potential for mutations within genes (LmMT/LmRos3) that encode subunits of the miltefosine transporter complex were investigated. No mutations were associated with LmMT, with three nonsynonymous mutations found in LmRos3. This thesis also reports the evaluation of transgenic L. mexicana expressing a novel NanoLuc luciferase, and a PEST-tagged variant, as a tractable, rapid and sensitive system for antileishmanial compound screening. The validity of this approach is demonstrated by a screen of the MMV Pathogen Box. The opportunity afforded by the transgenic L. mexicana expressing NanoLuc-PEST in an in vitro infected macrophage model is also demonstrated. These transgenic L. mexicana offer an opportunity for high-throughput screening programmes that assess the more clinically-relevant activity against intracellular amastigote parasite without the time, specialist and post-assay processing burdens associated with current high-content imaging techniques.

Salmonella enterica serovar Typhi is an intracellular bacterial pathogen that only infects humans. Remarkably, a BLOC-3 and Rab32-dependent anti-microbial pathway was identified to control the killing of S. Typhi in mouse macrophages and restrict S. Typhi infection in humans. This pathway can be counteracted by a broad-host Salmonella enterica serovar Typhimurium with its 2 effectors GtgE and SopD2. Yet very little is known about this novel pathway. Here, I used an explorative quantitative proteomics approach in combination with Rab32 pulldown assay and identified 3 proteins – RabGDIa, RabGDIß and Vps13C that specifically bind Rab32. With preliminary evidences achieved indicating a possible role of Vps13C and RabGDI in Salmonella infection, this study provides some clues for future investigation on discovering other components of this novel pathway. In addition to that, this study determined that a post-translational modification of GtgE leads to an increased virulence of S. Typhimurium during infection, which is likely via its increased ability to cleave Rab32.

The central aim of this study is to combine theorist Roland Barthes' ideas on visual and textual thinking with leading contemporary approaches to Western advertising design with a view to their possible application in China. The result is a newly coined concept the present author refers to as 'Advertising Thinking'. This new term puts forward a fresh method that goes beyond the conventional way of dealing with advertising design. At present, there are very few in-depth studies of advertising in China, making it crucial for Chinese designers, creative directors, design tutors, entrepreneurs and business people to gain a better understanding of the subject. The intention is for this understanding developed in the current study to enable China's advertising industry and its discourse to go beyond the superficial stage it is presently at. In order to achieve this an in-depth analysis of the status quo of China's story creative advertising history is needed. From this it can be seen that imitation, celebrity endorsement and the lack of aesthetic appeal in Chinese advertising design are issues that urgently need to be addressed. Through an in-depth study of advertising theory key concepts in advertising need to be explored. Following an introduction that outlines the key concepts in Barthes' writing - hidden meaning, narrative, and emotion - numerous case studies are explored in the chapters that follow to demonstrate different approaches to Advertising Thinking. These case studies include: Sandy Hook 'Even' (USA) 2016; Stay in School (Australia) 2014; Coca Cola 'Pool Boy' (USA) 2017; and Black Currant Tango 'St George' (UK) 1996. Following this, the research analyses five Chinese advertisements. From the analysis of these case studies it can be seen that Chinese advertisers and advertising professionals need to critically explore Chinese traditional culture and apply key concepts in contemporary Western advertising theory - a number of points are made that are crucial to moving Chinese advertising design forwards so that it can realise its potential.

The consequences to wildlife of living in contaminated areas with chronic low-dose rates of radiation are still relatively unknown. Laboratory studies using acute radiation have demonstrated that invertebrates are relatively radioresistant compared to other taxa. However, there is little scientific evidence to show how chronic low dose rates affect invertebrates. This is problematic for understanding the consequences to wildlife living in highly contaminated areas and also testing assumptions made for invertebrates by the International Commission on Radiological Protection (ICRP). This thesis was designed to address a number of recommendations have been suggested to improve radioecological studies and help reduce the uncertainty as to effects at low dose rates. These include environmentally relevant laboratory studies (Chapters 2 and 4), improved dosimetry and dose assessments (Chapter 3), investgating confounding factors (Chapter 4) and continuity between laboratory experiments and field work conducted in the Chernobyl Exclusion Zone (CEZ) (Chapter4). Chapter 2 presents an environmentally-relevant experiment testing how bumblebee reproduction and life history is affected by chronic low-dose rates. Unexpectedly, at dose rates equivalent to the CEZ, queen production declined and reproductive timing was altered. The estimation of dose rates to establish a dose-effect relationship for wild animals is difficult and a common criticism of radioecological studies, therefore, Chapter 3 tests whether the common approach to measuring only external ambient dose rates is suitable and whether the inclusion of life-history traits significantly alters the dose rate. The findings from this chapter reiterate the necessity to use dose-assessment tools to test different parameters to estimate dose rate in different scenarios to account for unknown variation. Chapter 4 demonstrates that in areas of elevated dose rates in the CEZ parasite burden was higher and bumblebees did not live as long. These results were reinforced by a laboratory study, which determined bumblebees exposed to increased radiation doses had high parasite burdens and were infected quicker, resulting in reduced longevity. The data in this thesis detected effects below the current dose bands used in international radioprotection and therefore advocate these dose bands be re-evaluated. However, the data do not support studies which have measured adverse effects at dose rates similar to background and suggest that confounding factors such as habitat quality and co-stressors need to be included in field and laboratory studies.

The recent identification of two novel influenza-like viruses in bats, H17N10 and H18N11 virus, and the discovery of serologically positive Eidolon helvum bats in Ghana for avian H9 virus prompted my hypothesis that, in addition to the large repertoire of zoonotic viruses hosted, bats may serve as asymptomatic reservoir species to conventional influenza A viruses found in birds and mammals. To begin to test this hypothesis, the susceptibility of three bat cell lines, derived from lung epithelial cells of Eidolon helvum, Carollia perspicillata and Tadarida brasiliensis (TB1-Lu), to low pathogenicity avian viruses (H2N3 [A/mallard duck/England/7277/06] virus and H6N1 virus [A/turkey/England/198/09] virus), and human viruses (USSR H1N1 virus [A/USSR/77] and pandemic H1N1 2009 virus [A/California/07/2009]) was determined. All three species of bat epithelial cells were found to be more robust and resistant to influenza virus infections than permissive MDCK cells. Infected bat cells produced lower levels of viral RNA and viral progeny, and were more viable than correspondingly treated MDCK cells. Interestingly, bat cells were more susceptible and replication permissive to avian than human influenza viruses. Among the bat cells, TB1-Lu cells were the least susceptible to influenza virus infection and that appears to be related to a lack of sialic acid α2,6-Gal receptors, mammalian virus-preferred host receptors, which were present in the other two bat species. The innate mechanisms underlying resistance to influenza virus infection in bats remains to be determined, however, inhibition of the JAK/STAT pathway was found not to affect virus production from infected bat cells suggesting that JAK/STAT signalling may not have a major role in influenza virus resistance in bat cells. Modulation of NF-κB signalling was found to affect virus production suggesting that tight regulation of NF-κB may be key in controlling the pro-inflammatory response to influenza infection in bat cells and warrants further investigation.

To date there are no effective treatments for prion diseases, and these diseases are always fatal in both humans and animals. Additionally, the gold standard for diagnosis of these disease remains to be the analysis of biopsied brain tissue obtained post mortem. Consequently, there is a continued demand for therapeutics and ante-mortem diagnostics for prion diseases. This project addresses these demands by investigating candidate therapeutic and diagnostic ligands for prion diseases. This study investigated recombinant prion proteins (rPrPs) as inhibitors in scrapie and bovine spongiform encephalopathy (BSE) in vitro amplification by protein misfolding cyclic amplification (PMCA). Three ovine rPrPs with the polymorphisms VRQ, ARQ and ARR and hamster rPrP were tested against scrapie PMCA in dilution series to calculate IC50 values. The two most potent inhibitors, VRQ and ARQ, were then similarly tested against bovine spongiform encephalopathy (BSE) amplification. The most potent inhibitor of both disease types, the ovine rPrP VRQ, was then observed to inhibit a range of different scrapie and BSE strains at a fixed concentration. It is recommended that further investigation into rPrP inhibitors is performed. Strain characterisation of scrapie was investigated using rPrP inhibitors, following observations that the rPrP inhibitors generate a pattern of inhibition at a set concentration. Although this pattern of inhibition was repeatable in scrapie amplification by PMCA, this was limited to a single round of PMCA. Ultimately, this limited the application of this method to only amplification efficient prion strains and isolates. It is recommended that this method be investigated further in combination with the amplification of different isolates in substrates of different genotypes over multiple rounds of PMCA, as well as the analysis of glycoform ratios by western blotting. Here it was also identified that the imidazole used in the elution buffer for immobilised metal affinity chromatography (IMAC) can inhibit prion amplification in a strain dependent manner. This inhibition could be used in combination with the proposed method as a multi-faceted assay of prion strain characterisation. The use of next generation phage display (NGPD) to map the epitopes of autoantibodies in the sera of scrapie infected sheep was also investigated. This was performed to identify peptides that were immunoreactive to autoantibodies specific to the disease state. The identification of diagnostic peptides would then enable the development of an ante-mortem serological diagnostic test for scrapie. NGPD successfully selected immunoreactive peptides, of which 39 were selected for validation by peptide enzyme-linked immunosorbent assays (ELISAs). Although none of the peptides demonstrated diagnostic specificity by peptide ELISA, an optimised ELISA methodology was developed for future use in the validation of NGPD selected peptides. Further variations in the NGPD method, as well as validation by immunoassay, can be investigated to identify diagnostic peptides immunoreactive to scrapie specific autoantibodies.

Humans are exposed to high levels of amorphous silica on a daily basis, via the diet and the use of cosmetic and pharmaceutical products. Amorphous silica particles (10-200 nm) have also been developed for use in biomedical applications, including as binding agents in tissue repair, drug and gene therapy delivery agents, coatings for medical contrast agents and as vaccine adjuvants. Numerous studies have already been conducted to evaluate the cellular toxicity of these silica particles but still little is known about their effects both in vitro and in vivo, especially of nanosilica particles under 10 nm in diameter. The aim of this thesis was to investigate the cellular and in vivo activity of < 10 nm diameter nanosilica particles with different properties (e.g., size and dissolution rate in dilute conditions) as it may infer upon safety after exposure via the diet and intravenous administration (biomedical applications). First, the cytotoxicity of sub-10 nm nanosilica particles, fully characterized by size, dissolution rate, zeta-potential and by NMR spectroscopy, on immune cell function was assessed using transformed and cancerous cell lines and primary cells. The particles were toxic to the immune cells in a dose dependent manner and impaired certain cellular functions. Primary cells were most susceptible to nanosilica induced death and, of the primary cells, phagocytes were most susceptible to its cytotoxicity. Further investigations were conducted to assess the effect of nanosilica on T cells, as there was evidence suggesting that nanosilica particles were directly interacting with these cells. Nanosilica particles 3.6 nm in diameter were found to have a significant effect on T cell function. The particles induced numerous markers of T cell activation, including CD25 and CD69 on CD4 T cells, CD8 T cells, gamma-delta T cells and NK/NKT cells, CD95 on CD4 and CD8 T cells, CD40L, FoxP3, LAP, GARP on CD4 T cells, and IFN-gamma production, but it did not induce T cell proliferation. The particles were found to activate T cells regardless of their antigenic specificity. Further investigations showed that nanosilica interacts with the T cell receptor complex, the first documented case of a non MHC-coated nanoparticle directly interacting with this receptor complex. The nanoparticulate induced signalling through Zap70, LAT, and, eventually, through NFAT but not through MAPK. Similar signalling in the literature has been shown to induce a hyporesponsive T cell state (anergy) or activation induced cell death. The induction of the CD25 and CD69 T cell activation markers was limited to nanosilica particles below 10 nm in size, while similarly sized iron hydroxide nanoparticles (3-5 nm) only induced low levels of CD69 expression on T helper cells. Finally, it was shown that nanosilica is capable of inducing T cell activation in whole blood, though the T cell responses were greatly attenuated. Although identification of activation pathway in vivo remains elusive, the nanosilica particles were shown to have therapeutic value, decreasing murine subcutaneous tumour growth rate and significantly reducing the formation of lung metastases. Whether these in vivo responses are related to T cell activation identified in vitro remains unclear.

Thymic epithelial cells (TECs) are indispensable for the development of T cells in the thymus. Two subtypes of TECs exist in the thymus, medullary mTECs and cortical cTECs. Both mTECs and cTECs originate from endodermal thymic epithelial progenitor cells (TEPCs) in the embryo, but how the differentiation of TEPCs is regulated is not well understood. The aims of this thesis were to establish the role of Notch signalling in TEPC differentiation, and how it interacts with known regulators such as FOXN1 and the NFκB pathway. Gene expression data showed that Notch is active in TEPCs and exhibits a correlation with the mTEC lineage. Loss of Notch function led to a significant reduction in the number of mTECs in the thymus, and this can be attributed to aberrant mTEC specification. Furthermore, the duration of Notch activity in determining mTEC number appears limited to the early phase of organogenesis, and precedes RANK/NFκB mediated mTEC proliferation. Gain of Notch function resulted in a considerable shift to a primitive, TEPC-like phenotype, and subsequently a latent increase in mTEC frequency. Finally, transcriptomic and functional analyses pointed to a cross-repressive mechanism between Notch and FOXN1 in TEPCs. Taken together, these results identified Notch as a novel regulator of mTEC specification, likely through maintaining the potency of fetal TEPCs, a prerequisite for mTEC lineage commitment.

Parasites have a major impact on host condition and fitness and thereby represent a strong selective force for individuals in wild populations. The main defence against parasite infection and associated morbidity is the host immune response, and consequently it is expected for there to be strong selection eroding genetic variation underlying immune responses in natural populations. However, studies in the wild have found considerable heritable variation underlying immune responses. Few studies have investigated the genetic variants underlying immunity in wild populations and are able to examine how genetic variation is maintained in the face of natural selection. The aim of this thesis is to investigate the selection on, and genetic variation underlying, immunity in a wild Soay sheep population by looking at antibody responses to the prevalent parasite Teladorsagia circumcincta. Anti- T. circumcincta antibody levels (IgA, IgE, IgG) were measured in neonatal plasma samples taken soon after birth, representing maternally-derived antibodies, and in samples from August yearly from four month old lambs and adults, representing endogenous antibodies. All three endogenously produced antibody measures in lambs and adults were repeatable and heritable. In addition, a genome wide association study run on the three antibody traits on August lamb and adult measures found associations between anti-T. circumcincta IgA levels and single nucleotide polymorphisms in a region on chromosome 24. There was evidence for age- and isotype- dependent negative associations between antibody isotypes and strongyle faecal egg counts (FEC). Further, there was evidence for age-dependent selection via positive associations between anti-T. circumcincta IgG and survival in females and annual fecundity in males. In comparison, there was no additive genetic variance underlying maternally-derived (neonatal) anti-T. circumcincta antibody levels in neonates, but maternal and maternal genetic effects explained a considerable proportion of the variance in these traits. There was evidence for associations between neonatal anti-T. circumcincta IgG and later offspring phenotype and fitness, independent of total antibody (IgG) transferred. We found that neonatal anti-T. circumcincta IgG levels positively predicted survival to four months old, as well as weight in August. In addition, neonatal anti-T. circumcincta IgG levels were associated with reduced strongyle FEC in August, and were associated with improved survival over the first winter. In early life, maternally-derived anti-helminth antibodies are important for early growth, survival, and parasite resistance, as well as first winter survival, while fitness benefits in adulthood were associated with higher endogenous anti-helminth antibody levels. This thesis illustrates that maternal effects and genetic variation can have strong effects on variation in immunity in the wild, and this variation in turn can have health and fitness consequences for individuals.

Many parasites have evolved to release products that inhibit host defence mechanisms such as enzymes in the mammalian host, in order to promote and sustain their survival within the host. The human filarial nematode Brugia malayi produces larval microfilariae, which circulate in the blood stream. Their most abundant secreted product is a serine protease inhibitor Bm-SPN-2. Serine protease inhibitors (Serpins) are reported to be involved in how the nematodes avoid host immune defences, and in the case of Bm-SPN-2, the protein was found to specifically inhibit the enzymatic activity of human neutrophil elastase and cathepsin G in a dose-dependent manner. More recently, these two enzymes have been linked to the activation of a major innate cytokine IL-33, which is stored as a full-length 270-aa protein in the cell nucleus, and released as an active C-terminal domain upon stimulation. As full-length (FL) human and murine IL-33 are not commercially available, soluble murine and human FL-IL-33 were produced in transfected HEK 293T cells, following mutation of the nuclear binding motif. In this form, IL-33 is no longer retained in the nucleus and can be purified as a soluble protein. It was confirmed that once cleaved, recombinant human IL-33 was able to induce significant IL-6 secretion by mast cells. Bm-SPN-2 was then shown to block human full-length IL-33 cleavage by inhibiting human neutrophil cathepsin G in a dose dependent manner, supporting the hypothesis that Bm-SPN-2 may act in vivo to prevent IL-33 activation and the promotion of the TH2 immune response. However, in the in vivo setting, it was unexpectedly found that IL-33R (ST2) gene deficiency did not enhance the survival of B. pahangi microfilariae. Furthermore, in the absence of IL-33R, murine immune responses to microfilariae were not significantly altered compared to wild-type BALB/c mice, other than in a significant increase in IL-33 expression. Hence while Bm-SPN-2 can act in vitro to forestall one of the key events in TH2 induction, this has not yet been shown to be crucial to the immune response to the parasite in vivo.

Macrophages belong to the mononuclear phagocyte system and they perform fundamental roles to maintain homeostasis in the organism. Macrophage development, survival, proliferation and functionality depend upon the colony stimulating factor 1 (CSF1) and interleukin-34 (IL-34), which signal through the CSF1 receptor (CSF1R). CSF1R is a type III tyrosine kinase receptor that is present in the plasma membrane of monocytes and macrophages. Mutations in Csf1r in mice produce the loss of many tissue macrophage populations and multiple developmental abnormalities. In humans, abnormal enhancement of CSF1R expression has been correlated to adverse prognosis in a subset of carcinomas; and mutations in the human CSF1R are associated with an autosomal-dominant neurodegenerative disease. CSF1R is encoded by the c-fms proto-oncogene and its expression is partially controlled by the fms-intronic regulatory element (FIRE). The FIRE sequence is highly conserved across species and contains binding motifs for multiple transcription factors, which are relevant for haematopoiesis. Previous results from murine Csf1r transgenes showed that FIRE is essential for driving Csf1r expression, and that interactions between FIRE and multiple myeloid transcription factors contribute to maximal regulatory activity. This project aimed to study the role of FIRE in its normal chromatin context, in vivo. A FIRE knockout (FIRE-/-) mouse model was generated using the CRISPR/Cas9 technology in mouse embryonic stem cells (ESCs) and in mice. In ESCs, the deletion severely compromised the differentiation of macrophages from embryoid bodies generated in vitro. In mice, the frequency of the FIRE- /- genotype in the progeny does not follow a Mendelian distribution and about 5% of the offspring developed hydrocephalus. Unlike Csf1r -/-mice, which die before weaning, most surviving FIRE-/- mice grew normally and were fertile. The impact of the mutation on macrophage populations is selective. FIRE-/- mice are not monocyte deficient (identified as F4/80+ Csf1r+ cells in peripheral blood), although these cells have reduced levels of Csf1r mRNA and do not bind porcine CSF1 Fc fusion protein. The development of peritoneal macrophages and Iba-1+ microglia was abolished, but Adgre1+ (F4/80+) macrophage populations in liver and spleen were unaffected. Csf1r was greatly reduced in bone marrow progenitors, but about 30% of these cells were able to differentiate into macrophages in vitro, upon exposure to recombinant human CSF1 (rhCSF1). This study shows that FIRE is essential for the development of a subset of tissue-resident macrophage populations. In FIRE-/- mice, potential compensation from additional regulatory elements within Csf1r might underlie the development of unaffected tissue-resident macrophages.

Macroautophagy, here referred to as autophagy, is an intracellular degradation pathway cells use to maintain their homeostasis. Autophagy is also required for cell survival during nutrient deprivation, as well as development and immunity in higher eukaryotes. Aberrations in autophagy can lead to pathologies including cancer, neurodegeneration and diabetes. Autophagy is characterised by the formation of a double membrane phagophore, which sequesters cytosolic cargo and forms a vesicle termed an autophagosome. The autophagosome eventually fuses with the lysosome, resulting in the degradation of the cytosolic cargo. Although autophagosome formation is orchestrated by the sequential action of the core autophagy proteins, a key question remains - what gives rise to a double membrane phagophore? The key event in phagophore biogenesis is the production of PI3P at the phagophore formation sites. WIPI2b, a PI3P effector protein, directly interacts with ATG16L1 and is recruited to the omegasomes, which is the basis for LC3 recruitment to the forming phagophore. To address how the function of WIPI2b at the phagophore is regulated, I focused on phosphorylation, as there have been reports about potential phosphorylation sites on WIPI2b. I confirmed an interactive relationship between WIPI2b and ULK1 that was reported previously and identified a number of phosphorylation sites on WIPI2b upon overexpression of ULK1 kinase. I found that phospho-mutants of WIPI2b S68 exhibit reduced interaction with ATG16L1 and WIPI4. I generated and characterised a WIPI2 CRISPR knockout cell line and found that WIPI2b S68 phospho-mutants are unable to rescue LC3 lipidation in WIPI2 CRISPR knockout cells. I also found that WIPI2b S284 phosphorylation is important for the regulation of WIPI2b association with membranes. I propose WIPI2b phosphorylation by ULK1 provides a feedback loop during autophagy to control the amount of functional WIPI2b at phagophores and therefore allows phagophore elongation.

The increasing application of nanoparticles (NPs) has led to increased occurrence of engineered NPs in the aquatic environment. Understanding the toxicity of NPs in the aquatic environment is dependent on linking NP physicochemistry with toxicological responses and while research has been moving towards this direction, the link has not been fully understood yet. The present study critically reviewed adsorption and desorption processes of organic environmental contaminants on TiO2 NPs and evaluated interactions of NPs with compounds of different physicochemistry already existing in the aquatic environment as contaminants. Aquatic toxicity of the new generation lead-halide perovskite NPs was evaluated relative to lead ion dissolution. Finally, the sedimentation of NP agglomerates during a traditional fish early-life stage toxicity test, a major limitation of assessing NP toxicity in the aqueous phase, was addressed by development of an exposure chamber designed to keep NP agglomerates in homogeneous dispersion. The model organisms used in the present study to evaluate NP toxicity were larvae and adult zebrafish Danio rerio and the unicellular green fresh water alga Chlorella vulgaris. The main findings were: 1) sorption of environmental contaminants on NPs can change the bioavailability of the contaminant in the aqueous phase. Specifically, sorption of copper and benzo(a)pyrene (under fluorescent light) on NPs reduced the adsorbent bioavailability. On the contrary, benzo(a)pyrene and anthracene, when adsorbed on TiO2 or Si NPs, were photo-catalysed under UVA and in the case of benzo(a)pyrene, highly toxic photoby-products showed increased bioavailability in larval zebrafish; 2) lead-halide perovskite acute toxicity was attributed to lead ion dissolution based on induction of metallothionein 2 gene expression through aqueous and dietary exposure, and 3) the perovskite-spiked diets did not disrupt zebrafish gut microbiome after a 14-d exposure while disruption of gut microbiota by equivalent Pb(NO3)2 diets was observed; finally, 4) higher toxicity was found when NPs were tested using an exposure chamber that allowed continuous NP dispersion, indicating toxicity is depended on the dispersion state of NPs. This study has expanded our knowledge on NP surface physicochemistry and interactions with surrounding compounds in the aqueous phase; has confirmed metal ion dissolution out of metallic NPs and linked perovskite NP toxicity to lead ion dissolution as well as linked NP toxicity to NP dispersion in the aqueous phase contributing to a better understanding of NP properties and reactivity relation to toxicity in the aquatic environment.

Macrophages are a dynamic cell type and represent a key component of the immune response, with a broad range of activities throughout the body. They respond to external cues, including microbes, alarmins, and growth factors, to eliminate invading pathogens through initiation of inflammation. Subsequently, they carry out several regulatory roles, including clearance of cellular debris, the resolution of inflammation, and wound healing to restore tissue homeostasis after an inflammatory response. The ability of macrophages to change their phenotype in this manner must be tightly regulated, as dysregulated macrophage activity is central to the pathogenesis of both inflammatory and fibrotic autoimmune disorders, such as Rheumatoid Arthritis (RA) and Idiopathic Pulmonary Fibrosis (IPF), respectively. One of the mechanisms by which regulation of macrophages occurs is the microRNA network. MicroRNAs (miRNAs) are a class of short, non-protein coding RNAs that post-transcriptionally regulate gene expression through translational repression, destabilisation or degradation of target mRNA. miR-155 is a multi-functional miRNA that has roles in regulating the development and function of many immune cells, including macrophages. Abnormal expression of miR-155 is associated with a number of autoimmune disorders and cancers. We reported previously that miR-155 is elevated in RA and contributes to the chronic, pro- inflammatory response of macrophages by repressing key anti-inflammatory proteins. However, the role of miR-155 in the regulation of remodelling responses by macrophages is less well characterised. Modulation of miRNA activity in cells through the use of mimics and inhibitors has emerged as a potential therapeutic strategy for the treatment of diseases. Technologies involving the use of lipid vesicles as delivery agents for introducing therapeutics into target cells have shown potential in increasing the drug efficacy.