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Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy characterized by renal failure and determined by genetic and acquired defects of alternative pathway (AP) of the complement system. Autoantibodies against factor H (anti-FHs), a regulator of the AP, were reported in 10% of patients, and are associated with the deficiency of factor H related 1 (FHR1), a FH homologous protein. The aim of this thesis was to evaluate the contribution of genetics to the development of anti-FHs in aHUS. Thirty patients affected by aHUS resulted positive for anti-FHs (9.8%) and FHR1 deficiency was present in 83.3% of them. A healthy control also showed anti-FHs in concomitance with FHR1 deficiency documenting that the lack of FHR1 strongly predisposed to anti-FH development also in healthy subjects although this condition was not sufficient for the disease manifestation. The presence of infectious prodromal signs and an age at the disease onset around 8 years indicated that common infections may trigger the development of autoantibodies in subjects with at risk genetic background. Likely pathogenetic variants in complement genes were observed in 37% of our patients with anti-FHs. At variance, common variants in complement genes did not seem to contribute to the disease, as documented by comparing patients with super controls, unaffected subjects carrying FHR1 deficiency. Finally, I report that the HLA-DRB1*11:04 allele could be a predisposing genetic variant for anti-FH associated aHUS. Further works will be necessary to confirm this finding and to explore the presence of other genetic susceptibility factors that, in combination with the HLA-DRB1*11:04 allele and the FHR1 deficiency, could increase the risk for anti-FHs.

The transcription factor MITF regulates many essential functions in both melanocytes and melanoma. It controls differentiation, proliferation and survival in a dose dependent manner; therefore it is under tight regulation. Many signalling pathways and transcription factors have been identified to control the expression and activity of MITF, however, there is little known about how the surrounding microenvironment could impact on MITF function. It is now well appreciated that stromal components also have an important role in determining disease progression, and in fact the extracellular matrix (ECM) is a key regulator of cellular differentiation, growth and survival, similarly to MITF. Therefore, we aimed to identify a potential crosstalk between ECM mediated adhesion signalling and MITF in order to better understand melanoma biology. By conducting transcriptomal analysis of cells in different microenvironments, we discovered that MITF levels and function is affected by collagen signalling in a matrix stiffness dependent manner. We demonstrated that increased matrix stiffness or collagen abundance leads to upregulation of MITF and consequently cells become more proliferative and differentiated and this is mediated by YAP1/PAX3. In addition, we found that in melanoma patients increased collagen abundance correlates with nuclear YAP1 localization. However, using large patient datasets we discovered that tumour-microenvironment mediated YAP1 signalling is more complex. In the absence of fibroblasts, YAP1/PAX3 drives MITF expression, but in the presence of fibroblasts, TGFβ induces a switch from YAP1/PAX3 driven transcription to YAP1/TEAD/SMAD driven transcription and consequently a de-differentiated phenotype. Furthermore, we show that high collagen expression is linked to poor survival, however the worst prognosis is seen in patients with high collagen expression, who also express MITF target genes such as the differentiation markers TRPM1, TYR and TYRP1 as well as CDK4. In summary, while a lot of effort has gone into identifying factors that induce the de-differentiated/invasive phenotype, it is not well understood how the switch to the differentiated/proliferative phenotype is controlled. Here we provide a novel mechanism whereby matrix signaling can induce MITF expression through YAP1 and show that this high matrix/differentiation/proliferation phenotype is predictive for poor survival.

BRCA-1 Associated Protein 1 (BAP1) is a nuclear deubiquitylase (DUB) frequently inactivated by mutation in specific cancers including malignant pleural mesothelioma (MPM). Mutations cause loss of nuclear BAP1 expression or catalytic inactivation and, as BAP1 regulates both ubiquitylation and transcription, its loss in cancer may dysregulate expression of specific proteins, and potentially non-coding RNAs such as miRNAs. To begin to address these questions, the proteome response to BAP1 mutation was previously profiled in isogenic MeT5A mesothelial cells, and an unbiased DUB siRNA screen revealed that BAP1 regulates cellular abundance of the histone deacetylase HDAC2. In this thesis, I set out to (1) validate BAP1-dependent protein expression in isogenic MeT5A BAP1-mutant cells and patient-derived MPM cell lines, (2) address the mechanism by which BAP1 regulates HDAC2, and (3) to identify BAP1-miRNA regulatory networks in MPM. Profiling the effect of BAP1-deficiency on the MeT5A proteome had revealed alterations in cytoskeletal and metabolic pathways. I verified reduced expression of glycolytic enzymes but increased expression of TCA cycle and associated anaplerotic pathway proteins in MeT5A BAP1-mutant cells. Altered expression of certain cytoskeletal proteins correlated with BAP1-status in a panel of patient-derived MPM cell lines, confirming responses to BAP1 loss in isogenic cell models may be evident on more complex genetic backgrounds. Altered expression of HDAC2 is reported in cancer and our siRNA screen suggested this may be linked to BAP1 status. I found that BAP1 depletion regulates HDAC2 transcript abundance rather than protein stability, and that BAP1 status influences the response of MPM cell lines to HDAC inhibitors. As BAP1 can influence transcription, I next explored the idea that BAP1 may also regulate expression of miRNAs. I used a NanoString platform to screen for BAP1-dependent miRNAs in isogenic MeT5A mesothelial cells. In total, ~400 unique miRNAs were identified and, compared with parental cells, 113 miRNAs were significantly modulated (P≤0.05, >1.5-fold) in BAP1-deficient cells. Unsupervised hierarchical clustering generated five distinct clusters, including a large group from the chromosome 14q32.31 miRNA tumour-suppressor locus that was downregulated in BAP1-deficient cells. Gene ontology analysis suggested these miRNAs may affect the cytoskeleton and transcriptional regulation. I then used the Nanostring platform to profile miRNAs in a panel of 20 MPM cell lines. I observed differential miRNA expression in MPM cell lines grouped by BAP1 status, including downregulation of the ch14q32.31 locus. As loss of BAP1 protein may be more frequent than the reported mutation rate in MPM, post-transcriptional regulation of BAP1 may also be of importance in cancer. To explore this idea, I used computational algorithms to predict miRNA target sites in the BAP1 3’UTR. Several candidate miRNAs were identified, including members of the miR-125 and miR-200 families. We found significantly increased expression of miR-125b-5p in BAP1-deficient isogenic cells (P≤0.01) and an inverse correlation between miR-125b-5p and BAP1 protein expression (R2=0.85) in MPM cell lines. In addition, a miR-125b-5p mimic could reduce BAP1 expression, suggesting that miR-125b-5p may act as a post-transcriptional modulator of BAP1, operating in a double-negative feedback loop to control BAP1 expression. Together, the data in this thesis show that BAP1 deficiency leads to a significant deregulation of proteins involved in cytoskeletal and metabolic pathways, a reduction in HDAC2 transcript abundance, and profoundly altered miRNA expression profiles. My data suggest for the first time that BAP1-dependent miRNAs may in part account for the BAP1-deficient MPM phenotype and could provide new biomarkers for BAP1 loss.

To promote survival and proliferation cancer cells re-programme their metabolism, altering both uptake and utilisation of extracellular nutrients. To examine correspondences between nutrient supply and viability, in order to identify targetable requirements, I individually depleted amino acid nutrients from diploid Human Mammary Epithelial (HME) isogenic cells expressing commonly activated oncogenes. Cystine deprivation was found to induce massive-oxidative stress associated-cell death of HME cells expressing an activated epidermal growth factor receptor (EGFR). Cell death occurred via an iron-dependent mode, known as ferroptosis associated with increased generation of reactive oxygen species and lipid peroxidation. Pharmacological inhibition of EGFR or mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) signalling was found to block ferroptosis and ROS production and was associated with increased expression of glutathione peroxidase 4 (GPX4). Suppression of GPX4 expression in wild-type or gefitinib-treated HME-EGFR cells was sufficient to sensitise cells to ferroptosis. Importantly, MAPK signals were also important in suppressing cell-cell contact and communication that was found to provide an essential line of defence against ferroptosis induction and spread. In this way, microscopic observation of ferroptosis in wild-type HME cells identified a cell death spread phenotype that is consistent with necrosis phenotypes observed in ischemic models. Inhibition of ROS generation and lipid peroxidation effectively blocked the progression of necrosis indicating that counteracting lipid peroxidation might be beneficial for degenerative conditions where lipid peroxidation is evident. Additional therapeutic application of my findings was modelled using non-small lung cancer cell (NSCLC) lines with overactive ERK signalling. These cells were found to be sensitive to ferroptosis following deprivation of cystine in vitro as well as in vivo where in systemic deprivation of cystine was achieved in xenografted mice following administration of a cystine-degrading enzyme. Taken together, my results show that the presence of common oncogenic mutations can render cells sensitive to the depletion of a specific nutrient, and further suggest potentially novel anti-cancer therapies based on the inability of some MAPK-driven cancer cells to overcome oxidative stress following nutrient depletion, as well as therapies to limit the spreading phenotype of ferroptosis in cells associated with other diseases such as Alzheimer’s Disease, or that occur in normal cells in response to ischemic reperfusion and acute kidney injuries.

We urgently require novel antimicrobial therapies for TB infections as a third of the world’s population is infected with latent TB and cases of multidrug-resistant TB are increasing. Naturally occurring host defence peptides (HDPs), components of the innate immune system, provide protection against infections in vivo through their direct antimicrobial and immunomodulatory functions. The effect of one HDP, bovine bactenecin-5 (Bac5), on mycobacterial infections was investigated in this study using the M. marinum-zebrafish TB infection model. Bacterial two-component systems (TCSs) regulate gene expression in response to external environmental changes, controlling physiological processes including virulence. Generation of TCSs deletion mutants in Mycobacterium marinum was attempted, unsuccessfully, by mycobacteria-adapted recombineering and sucrose counter-selection, with a view to studying the effects of targeting these systems during mycobacterial infections using zebrafish as a model organism. The M. marinum-zebrafish embryo model permits the monitoring of pathogenesis in real time through live-imaging. Bacterial Distance Distribution and Fluorescent Pixel Count protocols were developed or optimised in Icy to quantify bacterial dissemination and bacterial burden from images of zebrafish embryo infections using fluorescently labelled bacterial strains. These techniques, coupled with zebrafish leukocyte recruitment quantification using transgenic zebrafish lines and our image processing protocol, permit simultaneous analysis of multiple infection progression parameters during longitudinal studies. Injection with exogenous Bac5 alone and co-injection with M. marinum led to a significant initial increase in macrophage recruitment to the zebrafish injection site. We also observed trends following injections of zebrafish embryos with Bac5 for increased initial neutrophil recruitment, increased expression of the chemokine cxcl-c1c and pro-inflammatory cytokines IL-1β and MMP-9, and reduced M. marinum burdens. These results provide a basis for further investigations into the efficacy of Bac5 treatment of mycobacterial infections, in particular the optimal timings and dosage, with a view to using this exogenous HDP to treat human TB infections.

Autophagy is a highly conserved intracellular degradation process crucial for cell-autonomous immunity against bacterial infection. However, some bacteria may subvert the host cytoskeleton to escape recognition from autophagy. In the cytosol, Shigella flexneri and Listeria monocytogenes use actin-based motility to avoid cell-autonomous immunity and spread from cell-to-cell. The host cytoskeleton plays a key role in autophagy and its ability to restrict or promote bacterial replication. Septins, a cytoskeletal component that interacts with cellular membranes and actin filaments, are GTP-binding proteins that polymerize into non-polar filaments and rings. Our lab has discovered that septin cage-like structures entrap actin-polymerising Shigella targeted to autophagy. The septin cage is recognized as a mechanism of host defence mechanism, however the precise fate of septin cage entrapped Shigella remains unknown. Evidence has suggested that mitochondria provide membrane for the biogenesis of autophagosomes during starvation. The role of the mitochondria during the autophagy of Shigella is unknown. Mitochondria are highly dynamic organelles characterised by events of membrane fission and fusion. Work has previously established a role for the host cell division machinery, i.e. actin and myosin, in the regulation of mitochondrial dynamics (a process called 'mitokinesis'). Whether septins are also involved in mitochondrial dynamics has not yet been tested. For my thesis, the entrapment of Shigella by septin cages was used as a paradigm to better understand host defence against bacterial infection and to discover new septin biology. Results have shown that mitochondria mediate the assembly of septins into cage that target Shigella to degradation by autophagy. We have also discovered that septins regulate mitochondrial fission. Finally, we have revealed a new link between septins, autophagy, and host cell metabolism. Together these results suggest that a more complete understanding of septin biology during bacterial infection can enable its manipulation for therapeutic purposes.

This thesis aimed to identify the trypanocidal mode of action of non-natural chamuvarinin analogs, and to assess the previously uncharacterized secondary alternative oxidase (AOX2) as a possible drug target of the trypanosomatids. The drugs used to treat infections with Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are highly toxic and are increasingly becoming less effective as the parasites develop resistance, therefore new drugs against the diseases are desperately needed. Non-natural analogs of chamuvarinin were tested for trypanocidal activity to determine the structure activity relationships of the compounds against insect-form T. cruzi and Leishmania spp. This identified several potent and selective analogs, which retained good activity against the medically relevant intracellular forms of the parasites. Photoaffinity labeling was utilized to identify the mode of action and protein target(s) of the chamuvarinin analogs. The analogs were shown to deplete ATP levels and to induce mitochondrial dysmorphia and mitochondrial oxidative stress. Photoaffinity labeling confirmed the mitochondrial localization of the protein target(s) of these compounds, however the exact protein target(s) were unable to be identified by protein pull-down and mass spectrometry. The previously uncharacterized secondary alternative oxidases (AOX2) are conserved throughout the human-infective trypanosomatids and are absent from mammalian cells, thus making an attractive drug target if the protein is essential. The AOX2 of T. brucei, T. cruzi and L. major were expressed in Escherichia coli to characterize the enzymatic activity of the proteins. T. brucei AOX2 was successfully purified and shown to be an ubiquinol oxidase, which contains bound iron (III). The role of AOX2 within the trypanosomatids was determined by biochemical phenotyping and genetic manipulation of T. brucei, T. cruzi and L. major, which indicated that AOX2 is an essential mitochondrial oxidase in the three trypanosomatids, with a putative role in energy production, and therefore is an attractive multi-trypanosomatid drug target.

The effect of human activity on the natural world is increasingly shaping the evolution of species. The capacity of evolution to occur in individuals of a species, via natural selection acting on the genotypes of local populations through successive generations, is known as local adaptation. In southwest England, historical mining activity has resulted in a patchwork of highly metal-contaminated rivers across the region. Where the ecological diversity in many of these rivers has been decimated, metal-tolerant brown trout (Salmo trutta L.) populations seem to thrive. What are the mechanisms underlying this apparent metal-tolerance? And can it be attributed to processes of local adaptation? This thesis takes a multi-faceted approach in assessing this, by exploring the patterns and processes involved in metal-tolerance in brown trout populations in southwest England. A series of investigations were undertaken, including the use of neutral genetic markers (microsatellites), reduced representation genome sequencing (RAD-seq), common-garden exposure experiments, and genome-wide analysis of hepatic gene expression (RNA-seq). The microsatellite analysis illustrated that metal-tolerant trout have a different genetic architecture compared to fish in clean rivers and, using Bayesian analysis, these demographic differences were correlated with key periods of mining history. We then developed an approach to facilitate robust screening of genome-wide polymorphic loci through a method of parameter optimisation for RAD-seq. This approach formed the basis for identifying loci for investigating the genomic processes of local adaptation in metal-tolerant trout. We present genome-wide (RAD-seq) data highly indicative that neighbouring trout populations, differently impacted by unique ‘cocktails’ of metal pollutants have evolved both parallel and convergent mechanisms of metal tolerance. Through a common garden experiment, exposing metal-tolerant and metal-naïve fish to a mixture of metals, we were able to hone in on the physiological mechanisms underlying metal-tolerance. Finally, through RNA-seq, we observed that metal-tolerant fish showed little to no changes in hepatic gene expression when exposed to metals, pointing to innate mechanisms of metal handling. Together, the marriage of these various investigations showcases the remarkable ability of local adaptation in conferring metal-tolerance to brown trout populations in southwest England, and, importantly, the resilience of species’ in the face of human-altered environments.

Previous multi-strain mathematical models have elucidated that the degree of cross-protective responses between similar strains, acting as a form of immune selection, generates different behavioural states of the pathogen population. This thesis explores these multi-strain dynamic states, to examine their robustness and stability in the face of pathogenic intrinsic phenotypic variation, and the extrinsic force of immune selection. This is achieved in two main ways: Chapter 2 introduces phenotypic variation in pathogen transmissibility, testing the robustness of a stable pathogen population to the emergence of an introduced strain of higher transmission potential; and Chapter 3 introduces a new model with a possibility of immunity to both strain-specific and cross-strain (conserved) determinants, to investigate how heterogeneity in the specificity of a host immune response alters the pathogen population structure. A final investigation in Chapter 4 develops a method of reverse-pattern oriented modelling using a machine learning algorithm to determine which intrinsic properties of the pathogen, and their combinations, lead to particular disease-like population patterns. This research offers novel techniques to complement previous and ongoing work on multi-strain modelling, with direct applications to a range of infectious agents such as Plasmodium falciparum, influenza A, and rotavirus, but also with a wider potential for other multi-strain systems.

Phagocytosis is a first line defense of the innate immune system. It proceeds in a sequential manner: (1) phagocyte migration towards sites of infection; (2) recognition of PAMP by PRR; (3) engulfment and internalisation of pathogens into an endocytic, membrane-bound compartment called a phagosome; (4) phagosome maturation to form the microbicidal phagolysosome; and (5) killing of pathogen or host cells by a combination of oxidative and nitrosative stresses. In previous studies, cell viability, morphology, cell wall glycosylation status, hyphal length, and spatial orientation were identified as factors that impact on the dynamics of Candida albicans phagocytosis. However, little is known about how the engagement of specific receptors influences these dynamics, and whether or not these dynamics differ in other organisms. To address these questions, a series of in vitro experiments were performed using murine bone-marrow derived macrophages from wild-type mice or mice deficient in specific C-type lectin receptors (CLR) that couple to the adaptor protein FcR?, including dectin-2 and mincle. This stems from a previous report where an effect for dectin-2 deficiency on defense against systemic C. glabrata infections was documented, wherein distinct components of phagocytosis were compromised. In this study, I showed that deficiency in FcR? itself, dectin-2, or mincle correlated with significant impairment of specific components of the phagocytic process. This varied in magnitude, but was largely replicable with fungal species from diverse taxons, including ascomycetous, basidiomycetous, and zygomycetous fungi. Specifically, I observed impairment of macrophage migration, displacement, engulfment, and phagosome maturation. I then specifically explored the impact of dectin-2 deficiency on phagocytosis of C. albicans glycosylation mutants, and showed partial effects on upstream stages of phagocytosis. Together, these data highlight previously unappreciated roles for the engagement of FcR?-coupled CLR in shaping the dynamics of fungal phagocytosis. These effects could identify potential therapeutic targets that may be exploited for enhanced control and clearance of fungal infections in clinical settings.

Long lifespan of evolutionary higher organisms including humans is associated with the challenge to maintain viability of post mitotic cells, such as neurons, for decades. Autophagy is increasingly recognized as an important prosurvival pathway in oxidative and proteotoxic stress conditions. Autophagy degrades cytosolic macromolecules in response to starvation and is involved in the selective degradation of damaged/toxic organelles, such as mitochondria. With age autophagy function declines, and is also compromised in several neurodegenerative diseases. We identified a novel role for autophagy in the maintenance of mitochondrial health, specifically respiratory complex I. Intriguingly, galactose-induced cell death of autophagy deficient cells was rescued by preventing ROS production at complex I or bypassing complex I-linked respiration. We propose that aberrant ROS production via complex I in response to autophagy deficiency could be pathogenic and result in neurodegeneration and preventing this could be an interesting therapeutic target. Furthermore, we found that vertebrates have evolved mechanisms to induce autophagy in response to oxidative stress. This involves the oxidation of the autophagy receptor p62, which promotes autophagy flux and the clearance of autophagy cargo, resulting in increased stress resistance in mammalian cells and survival under stress in flies. In addition, we obtained data revealing an important role for redox-regulated cysteines in NDP52 for the degradation of mitochondria via mitophagy and tools were created to study the role of other autophagy receptors in autophagy initiation and selective autophagy.

Macroautophagy (‘autophagy’ hereafter) mediates the capture of aberrant cytoplasmic material into vesicles called autophagosomes, which then shuttle to lysosomes for degradation. Autophagy is implicated in numerous diseases, largely in a pro-survival role. However, autophagy has also been suggested as a form of programmed cell death (PCD), from cases of dying cells showing autophagosome accumulations. Debate occurs between whether these vesicles drive the lethality, or are instead a failing rescue attempt. This study aimed to provide clarity on this issue. Via the use of chemical and genetic strategies of inducing autophagosome accumulations, we found combining stimulators of autophagosome biogenesis with lysosomal degradation inhibitors gave rise to toxicity. Notably, this effect was dependent on the autophagy machinery and independent of other PCD routes. Research into the underlying mechanisms revealed an energy deficit under these conditions. Since autophagosomes cannot be recycled at lysosomes here, their continued synthesis affords no survival benefits, and instead just serves to deplete cellular energy further. For this reason, we designate this event ‘Futile Autophagosome Synthesis’ (FAS) toxicity. Other contributors to this toxicity include the persistence of harmful agents like Reactive Oxygen Species (ROS). Having established our FAS model, we explored its relevance in both cancer and neurodegeneration. Importantly, we found FAS inducing strategies to be effective in tumour treatment. Also, inhibiting FAS reduced the toxicity seen in neurodegenerative disease. Therefore, not only does this study improve our knowledge of autophagy in PCD, but also indicates it may have important medical implications.

Thousands of chemical pollutants enter the environment continuously, each with the potential to cause adverse effects in both terrestrial and aquatic organisms. As a result, organisms are often exposed to a mixture of stressors within their habitat. Populations of fish inhabiting most aquatic environments are exposed to time-varying or repeated pulses of exposure, driven by run-off events or spills, or due to their mobility between polluted and clean waters. Therefore, the sustainability of fish populations is critically dependent on their ability to adapt to frequent changes in their local environment. Despite this, legislation to protect the environment from chemical contamination are generally based on toxicological measurements following exposures to single stressors, conducted under optimal laboratory conditions, and that do not take into account the variation in susceptibility of wild populations, or the potential consequences of exposure for the susceptibility of the population during future exposures, including across generations. Increasing evidence is suggesting that a number of chemicals may interact with the epigenome, and that differential responses to pollutants may be modulated, at least in part, via epigenetic mechanisms. However, our understanding of the role of epigenetic mechanisms in normal development in fish models or its susceptibility to exposure to environmental stressors is currently very limited. This thesis aimed to document the mechanisms of genetic and epigenetic responses to industrial pollutants in fish, and to explore the extent to which differential responses can be induced in the lab following exposure during the critical window of embryonic development or in adults. To address these objectives, I performed a series of experiments using both the zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus) as fish models. I first used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. I provide evidence of the sex-specific transcription of genes involved in reproduction and their regulation by epigenetic signalling in this commonly used vertebrate model and highlight important considerations regarding the use of whole tissues comprised of multiple cell types in epigenetic and transcriptomic studies. I then investigated the potential for exposure to Bisphenol A (BPA) to cause adverse effects on reproduction and to disrupt the expression profiles and promotor DNA methylation of target genes important for reproductive function and epigenetic signalling in the zebrafish. To do this, I exposed breeding zebrafish to a range of BPA concentrations over 15 days and found that BPA disrupted reproductive processes in zebrafish, likely via estrogenic mechanisms, but only at high concentrations. Importantly, exposure to environmentally relevant concentrations of BPA resulted in altered transcription of key enzymes involved in DNA methylation maintenance, and caused changes in promoter DNA methylation. I also conducted a series of repeated exposures to copper in the three-spined stickleback to investigate the extent to which differential susceptibility can be induced in the lab. This work provides evidence that pre-exposure to copper results in differential responses in future exposure scenarios both when the initial exposure occurred in adults and during embryogenesis. For adults, fish appeared to recover completely from the initial exposure following a period of depuration of 30 days, but displayed decreased susceptibility upon re-exposure. In contrast, for fish exposed during the critical windows of embryonic development when epigenetic reprogramming are hypothesised to occur, differential copper accumulation was maintained throughout life. Importantly, the initial exposure caused increased tolerance in the offspring, which was inherited up to the F2 generation. This work provides valuable information regarding potential critical windows of development which may be more susceptible to effects associated with pre-exposure, highlighting that early life exposure to a low concentration of copper can induce differential responses to copper across generations. These data highlight the extent of differential responses to chemical stressors likely to be present in wild populations, and point towards the possibility that effective population management will likely require an in-depth understanding of the exposure history of a given population in order to manage restocking initiatives, and to inform conclusions drawn from toxicity testing studies conducted using individuals originating from wild populations. In addition, these data suggest that it is likely that both epigenetic and genetic changes can contribute to the adaptation of individual populations to their local environment. Finally, other vertebrates including humans have been shown to be exposed to the chemicals tested in this thesis. Therefore, this highlights the potential for these chemicals to also cause toxic effects in humans, potentially via (epi) genetic mechanisms, and advocate the testing of the potential for inheritable phenotypes, such as those described in this thesis, to occur in mammalian models.

Plants provide a cost-effective and scalable technology for production of therapeutic antibodies, with the potential for precise engineering of antibody glycosylation. Altering glycan structures in the antibody Fc region influences binding properties to Fc receptors and thus opens up new opportunities for modulation of antibody effector functions. To test the impact of plant glycosylation and effects of glycan modification on binding to human Fc receptors, different glycovariants of VRC01, a broadly-neutralising HIV antibody, were generated in Nicotiana benthamiana using glycoengineering tools and characterised. These include glycovariants lacking plant characteristic [alpha]l,3-fucose and [beta]l,2-xylose residues and extended with terminal [beta]l,4-galactose. Several surface plasmon resonance-based assays were inspected for kinetic/affinity evaluation of antibody - FcγR interactions. Protein A capture of antibodies was found to provide appropriate assay conditions and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FcγRI, FcγRIla, FcγRIIb and FcγRIIIa, however the binding characteristics can be restored and even improved with glycoengineering. Functional consequences of distinct FcγR-binding activities were further investigated in cell-based assays using flow cytometry. Fluorescent latex beads were coated with HIV-1 gpl40 antigen and different VRC01 glycovariants were compared for their ability to mediate phagocytosis of the beads by THP-1 human monocytes. The results demonstrate that typical plant glycosylation had a negative effect on binding of antibody-coated antigen-beads to cell surface receptors, and consequently may limit their uptake by the cells. However, plant glycoengineered antibodies are equally effective in enhancing cell-mediated phagocytosis as a human cell-made variant. Furthermore, a novel SPR-based assay was developed to investigate the influence of plant glycosylation on antibody affinity to neonatal Fc receptor (FcRn), which is involved in determining IgG half-life in the blood. The results suggest that FcRn binding is independent of plant glycosylation, however all plant-made glycovariants had a slightly lower affinity to the receptor, which correlates with higher oxidation levels of methionine residues involved in binding. Plant-based production and purification procedures, that may promote antibody oxidation leading to lower affinity to FcRn, point towards a need for process optimisation to control oxidation levels and improve the quality of plant-produced antibodies.

Skeletal muscle comprises about half of the body weight in mammals and its diverse roles in metabolism and innate immune functions makes cultured muscle as a useful tool in biomedical research. In this thesis, some of the major technical obstacles in porcine primary muscle cell cultures such as inadequate differentiation of myoblasts into myotubes and the premature detachment of the formed myotubes have been overcome with the achievement of high differentiation and myotube fusion rate of over 85% along with prolonged maintenance of myotubes in excess of 70 days. Myosin heavy chain (MyHC) expression profile of differentiated myotubes recapitulated adult muscle fibres and displayed phenotypic plasticity of gene expression in response to different media. Growth factor ractopamine (Ract) treatment (1h and 6h) of myotubes followed with subsequent bioinformatics analysis of a stable isotope labelling by amino acids in cell culture (SILAC) based proteomics study suggested differential expression of proteins associated with anti-viral innate immune response and increased protein accretion. Porcine muscle cells were infected with influenza A viruses to evaluate their immune function. Porcine muscle cells expressed influenza virus sialic acid α-2,3 and α-2,6 receptors and were fully permissive to influenza virus infection. Myoblasts produced more virus particles than myotubes. Muscle cells expressed the pro-inflammatory genes tumor necrosis factor alpha (TNF-α) and antiviral gene Mx-1 and the infected cells had elevated caspase 3/7 activity to indicate apoptosis. However, myotubes pre-treated with Ract appeared to confer no reduction in influenza virus output. Evidence presented herein suggests that the functional myotubes developed by this work can be used as a tool to study the molecular mechanisms of growth and innate immune pathways in muscle.

Pattern recognition receptors (PRRs) are localized at the cell surface to recognize conserved microbial patterns and activate plant immunity. The activation status regulates the localization of ligand-bound PRRs and routing to late endocytic trafficking for vacuolar degradation. This ligand-induced endocytosis is conserved across PRR families, and best characterized for the receptor kinases flagellin-sensing 2 (FLS2) and EF-Tu receptor (EFR), both mediating anti-bacterial immunity. However, the molecular determinants of FLS2 and EFR endocytic trafficking, as well as the biological relevance of these processes remain poorly understood. In this study, I have dissectedlolo l the molecular code underlying ligand-mediated endocytosis. I show that flagellin induced the interaction of FLS2 with vacuolar protein sorting (VPS) 37-1, a subunit of the endosomal complex required for transport-I (ESCRT-I) that recognizes internalized ubiquitinated proteins for vacuolar sorting. This led me to investigate the role of ubiquitination in FLS2 endocytosis. Using mass-spectrometry and mutational approaches, I identified ubiquitinated FLS2 lysine residues potentially involved in FLS2 endocytic trafficking. Additionally, I revealed an involvement of the E3 ligases keep on going (KEG) and the two redundant plant U-box (PUB) 12 and PUB13 in FLS2 trafficking. I showed that PUB12/PUB13-mediated monoubiquitination of FLS2 is a key regulatory process for internalisation of activated receptors, while EFR endocytic degradation is regulated by receptor phosphorylation on the tyrosine residues 875 and 877. For both FLS2 and EFR, altering ligand-induced endocytosis did not impact the initiation of downstream signalling, demonstrating that these processes are uncoupled. Instead, my results showed that ligand-mediated endocytosis of FLS2 and EFR plays a pivotal role in maintaining chronic immune signalling responses upon long term ligand stimulation. Overall, my results uncover an important molecular mechanism regulating the subcellular trafficking of the central immune components FLS2 and EFR, and extend our understanding on how plant responsiveness to its surrounding pathogens is maintained.

This is a case study of diagrams in a field of biological mechanism research (apoptosis), revealing that mechanism diagrams play a crucial role in the practice of developing mechanistic explanations for cell biology. This thesis supports and extends the existing literature in the following aspects: the relationship between scientific representation and practice (Daston and Galison, 2007), inter-field and inter-level integration in biological practice of mechanism research (Bechtel, 2006; Craver and Darden, 2013), and the assertive and engaging power of diagrams (Bender and Marrinan, 2010; Wood, 1992, 2010). The methodology is composed of two parts: quantitative and qualitative. The quantification draws the comprehensive patterns of diagram use via analysing the coverage of diagrams. The qualitative part analyses three layers of the diagrams: visual element, composition, and style. This part contextualises the diagrams in four senses: source of ideas, perspective, adjacent text, scope of research. The results and the interpretation of results are also composed of quantitative and qualitative parts. The quantitative part shows a noticeable prevalence of two themes of diagrams: object and mechanism. The former reflects an interest in manipulating entities. The latter reflects an interest in integration of, and interaction between, different perspectives. The relative changes in the coverage of these two themes suggest a shift in the focus of practice from manipulation of biological entities toward inter-field interaction between heterogeneous perspectives. The qualitative part contains a central argument and several interesting discoveries. The central argument is that mechanism diagrams synthesise heterogeneity and thus have the power to assert novel ideas and engage real-world practice. The heterogeneity of perspectives is embedded in the practice of developing the cell models. The term “synthesis” means that novel meanings emerge from the integration of existing perspectives. This novelty of meanings attributes to the assertive power of mechanism diagrams. The engaging power facilitates interaction amongst the component perspectives, which is an important feature of mechanism research. In sum, this argument can explain the increasing reliance upon diagrams found in the quantitative results. The other interesting qualitative discoveries include but are not limited to the following. Firstly, biological diagrams can go beyond visual resemblance to entities. Secondly, there are many creative ways of making diagrams, such as importing visual vocabulary from non-specialist areas and modular use of visual elements. These creative ways show that visual conventions in biological diagrams are not given but undergo evolution, probably responding to the growing complexity of ideas. Thirdly, the evolution of biological visualisation is not merely driven by development of technology but embodies the interaction between ideas and technological advancement. The conclusion of this study treats mechanism diagrams as both epistemological and communicative devices acting in the research dynamics. The communication is part of the processes of knowing and intervening, taking place both horizontally and longitudinally. The horizontal communication is amongst different research groups in the field. The longitudinal communication is between different stages of model developing by the same individuals. Diagrams serve in the constant defining and redefining of boundary of research arenas through bringing about new problems and activating future research.

Macroautophagy (hereafter: autophagy), cell signalling and cellular metabolism are tightly linked processes. Multiple examples of metabolites and metabolic enzymes have recently been found to regulate signalling pathways and autophagy. Metabolic reprogramming is one of the hallmarks of cancer and often mediated via aberrant signalling pathways, such as receptor tyrosine kinase (RTK) signalling. Growth factor receptor-bound protein 2 (Grb2) is a key RTK signalling adaptor and is involved in a number of downstream signalling cascades, such as mitogen-activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) pathways, which in turn regulate metabolism through alterations in gene expression or by directly modifying enzymatic activity. This study investigates the role of ribose-5-phosphate isomerase (RPIA), a key metabolic enzyme in the pentose phosphate pathway (PPP), in the regulation of autophagy and Grb2-mediated signalling. RPIA expression induces Grb2 translocation from an even cytoplasmic distribution to unknown sub-cellular structures, possibly through direct protein-protein interaction. Interestingly, this effect is independent of the catalytic activity of RPIA, suggesting a non-canonical role in signalling. Neither RPIA over-expression, transient knockdown or deletion by CRISPR/Cas9 genome editing resulted in differences in metabolic activity or the MAPK pathway as tested by extracellular signal-regulated kinases (ERK) 1/2 phosphorylation and MTT assay. Furthermore, knockdown of RPIA by shRNA or genomic deletion resulted in an increase of LC3 processing and LC3-positive autophagosomes, suggesting that endogenous RPIA is an inhibitor of basal autophagy. Data fromSaccharomyces cerevisiae, mass spectrometry of sugar phosphates and pharmacological treatment assays suggest that RPIA may inhibit autophagy through a non-canonical function. Although the molecular mechanisms by which RPIA acts on Grb2 signalling and how it contributes to the regulation of autophagy are currently not fully understood, this study presents some interesting observations that may have implications in the development of therapeutics that target cancer metabolism or aim to modulate autophagy.

Mammalian cell cultures are nowadays adopted as industrial platforms for monoclonal antibody (mAb) manufacture. Although improving product quality and expression stability becomes predominant these days, enhancement in mAb production still remains one of the greatest challenges facing the industrial biotechnology community. One of reasons behind this is the synthesis of mAb is restricted by the decline phase occurring in the mammalian cell cultures as a result of cell death. Prolongation of culture viability via suppression of cell death appears as a potential strategy to improve mAb production and productivity. Understanding of cell death in the mammalian cell cultures is, therefore, crucial to the improvement of the strategy toward mAb production and productivity enhancement. Apoptosis, or programmed cell death, is found to be the major cause of culture viability loss in the mammalian cell cultures. It is a highly-organised physiological process of cell death that can be triggered through activation of the genetic programme underlying a signal cascade that governs particular morphological and physiological changes in response to extracellular and intracellular stimuli. In order to devise strategies to optimise degrees of apoptosis in the mammalian cell cultures, understanding of apoptosis is inevitable. However, to establish the understanding of apoptosis is an extremely challenging task given the complexity arising from the dynamic nature of apoptosis and its association with internal and external milieu, including other cellular processes and environmental conditions. This thesis presents a combined experimental and modelling approach for a study of apoptosis in GS-NS0 cell cultures, aiming to identify relevant metabolic stresses, evaluate their impact on apoptosis induction, and investigate kinetics of apoptosis in response to the identified stresses. The novelty of this study is a systemic view of apoptosis where apoptosis is studied in accordance with dynamic changes in culturing conditions, as well as cell proliferation and metabolic activities. Batch culture experiments of GS-NS0 cell line demonstrate interplay between apoptosis and other cellular processes, including cell cycle progression and metabolism. In addition, the batch cultures allow evaluating impact of metabolic stresses and culture conditions on induction of apoptosis. Fed-batch culture experiments help validate links between apoptosis and metabolic stresses as suggested based on the batch cultures, and also shed some light on feeding strategies for a delay in apoptosis induction. The first-principles mathematical model for apoptosis in the GS-NS0 cell cultures demonstrates a good reproduction of the experimental data. Model analysis reveals model characteristics through global parameter sensitivities and helps guide further model modification toward development of a predictive apoptosis mathematical model.

A key aspect underpinning life-history theory is the existence of trade-offs. Trade-offs occur because resources are limited, meaning that individuals cannot invest in all traits simultaneously, leading to costs for traits such as growth and reproduction. Such costs may be the reason for the sub-maximal growth rates that are often observed in nature, though the fitness consequences of these costs would depend on the effects on lifetime reproductive success. Recently, much attention has been given to the physiological mechanism that might underlie these life-history trade-offs, with oxidative stress (OS) playing a key role. OS is characterised by a build-up of oxidative damage to tissues (e.g. protein, lipids and DNA) from attack by reactive species (RS). RS, the majority of which are by-products of metabolism, are usually neutralised by antioxidants, however OS occurs when there is an imbalance between the two. There are two main theories linking OS with growth and reproduction. The first is that traits like growth and reproduction, being metabolically demanding, lead to an increase in RS production. The second involves the diversion of resources away from self-maintenance processes (e.g. the redox system) when individuals are faced with enhanced growth or reproductive expenditure. Previous research investigating trade-offs involving growth or reproduction and self-maintenance has been equivocal. One reason for this could be that associations among redox biomarkers can vary greatly so that the biomarker selected for analysis can influence the conclusion reached about an individual’s oxidative status. Therefore the first aim of my thesis was to explore the strength and pattern of integration of five biomarkers of OS (three antioxidants, one damage and one general oxidation measure) in wild blue tit (Cyanistes caeruleus) adults and nestlings (Chapter 2). In doing so, I established that all five biomarkers should be included in future analyses, thus using this collection of biomarkers I explored my next aims; whether enhanced growth (Chapters 3 and 4) or reproductive effort (Chapter 5) can lead to increased OS levels, if these traits are traded off against self-maintenance. I accomplished these aims using both a meta-analytic and experimental approach, the latter involving manipulation of brood size in wild blue tits in order to experimentally alter growth rate of nestlings and provisioning rate (a proxy for reproductive expenditure) of adults. I also investigated the potential for redox integration to be used as an index of body condition (Chapter 2), allowing predictions about future fitness consequences of changes to oxidative state to be made. A growth – self-maintenance trade off was supported by my meta-analytic results (Chapter 4) which found OS to be a constraint on growth. However, when faced with experimentally enhanced growth, animals were typically not able to adjust this trade-off so that oxidative damage resulted. This might support the idea that energetically expensive growth causes resources to be diverted away from the redox system; however, antioxidants did not show an overall reduction in response to growth in the meta-analysis suggesting that oxidative costs of growth may result from increased RS production due to the greater metabolism needed for enhanced growth. My experimental data (Chapter 3) showed a similar pattern, with raised protein damage levels (protein carbonyls; PCs) in the fastest growing blue tit chicks in a brood, compared with their slower growing sibs. These within-brood differences in OS levels likely resulted from within-brood hierarchies and might have masked any between-brood differences, which were not observed here. Despite evidence for a growth – self-maintenance trade off, my experimental results on blue tits found no support for the hypothesis that self-maintenance is also traded off against reproduction, another energetically demanding trait. There was no link between experimentally altered reproductive expenditure and OS, nor was there a direct correlation between reproductive effort and OS (Chapter 5). However, there are various factors that likely influence whether oxidative costs are observed, including environmental conditions and whether such costs are transient. This emphasises the need for longitudinal studies following the same individuals over multiple years and across a wide range of habitats that differ in quality. This would allow investigation into how key life events interact; it might be that raised OS levels from rapid early growth have the potential to constrain reproduction or that high parental OS levels constrain offspring growth. Any oxidative costs resulting from these life-history trade-offs have the potential to impact on future fitness. Redox integration of certain biomarkers might prove to be a useful tool in making predictions about fitness, as I found in Chapter 2, as well as establishing how the redox system responds, as a whole, to changes to growth and reproduction. Finally, if the tissues measured can tolerate a given level of OS, then the level of oxidative damage might be irrelevant and not impact on future fitness at all.

Hypoxia is a global and increasingly important stressor in aquatic ecosystems, with major impacts on biodiversity worldwide. Hypoxic waters are often contaminated with a wide range of chemicals but knowledge is limited about the interactions between hypoxia and chemical stressors. Therefore, during this PhD I set out to investigate how the concentration of oxygen in the water influences chemical toxicity in fish. My work focused on two groups of chemicals: toxic metals, of which copper was used as an example, and anti-androgenic chemicals. Copper is an essential metal, widespread in the aquatic environment, but it can become toxic to aquatic organisms when environmental levels become too high. Copper and hypoxia are likely to co-occur but despite this our understanding of the interactions between these two stressors is limited. To address this, I performed a series of experiments using both the zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus) as fish models. I first investigated the effects of hypoxia on copper toxicity to zebrafish embryos during development. Copper toxicity was reduced by over 2-fold under hypoxia compared to normoxia during the first day of development, and I demonstrated that this protective effect was associated with the activation of the hypoxia inducible factor pathway. In contrast, hypoxia increased copper toxicity in hatched larvae, which was deduced to be associated with differential copper uptake. To test if the interactions between copper and low oxygen observed for the zebrafish also occurred for other fish species, exposures were conducted in a species with a lower tolerance to hypoxia, the three-spined stickleback. The results obtained showed that hypoxia suppressed copper toxicity prior to hatching, but after hatching this effect was reversed, similarly to that observed for the zebrafish. This suggests a potential conserved effect of hypoxia on copper toxicity during embryogenesis across fish species. To investigate if life stage influences the interactions between low oxygen and copper toxicity, the effects of combined exposures were assessed in the adult male three-spined stickleback. The critical oxygen level (Pcrit) was determined to allow appropriate experimental design. The combined exposures to copper and low oxygen resulted in a decreased ability to acclimate to low oxygen. Fish were able to lower their Pcrit in response to low oxygen conditions when exposed to hypoxia alone but not when exposed to hypoxia in combination with copper. Together, these datasets support the hypothesis that the life stage influences the effects of the combined exposure, as hypoxia protects from copper toxicity during early embryogenesis but increases copper toxicity in hatched embryos and in adults. I then investigated whether hypoxia can affect the toxicity of another widespread group of pollutants, anti-androgenic chemicals. Male three-spined sticklebacks were exposed to three anti-androgenic chemicals, flutamide, linuron and fenitrothion, under different air saturations. Each chemical had a unique transcriptional response alone and in combination with reduced oxygen saturation. Under both air saturations, spiggin transcription was strongly inhibited by exposure to flutamide. In contrast, exposure to fenitrothion did not result in a significant effect on spiggin transcription. Interestingly, linuron strongly inhibited spiggin under 100% air saturation, but this effect was absent under low air saturation, potentially as a result of interactions between the hypoxia inducible factor pathway and the aryl hydrocarbon receptor pathway. This work illustrates the potential mechanisms responsible for interactions between reduced oxygen and chemical toxicity, especially for aryl hydrocarbon receptor agonists, and highlights how hypoxia can modify the effects of a variety of chemicals with diverse modes of action. My research highlights the importance of considering the interactions between multiple stressors, and the need to take into account the type of chemical, life stage, and the species tolerance. Understanding these interactions is essential to facilitate the accurate prediction of the consequences of exposure to complex stressors in a rapidly changing environment.

In the last 40 years, the population of European eel (Anguilla anguilla L.) has declined dramatically and is now considered to be outside safe biological limits. In 2007 the European Union implemented an “Eel Recovery Plan” regulation, in order to assist in the recovery of the species back to the previous sustainable levels. The major factors regulating eel populations are still unknown and until there is an understanding of the factors causing the low recruitment of eels, the success of any management plans and conservation measures may be limited. One factor considered important in regulating the eel population is pollution. The major aim of these studies was to investigate the impact of environmental contaminants on eel throughout their life cycle. Laboratory and field studies were carried out to assess the impact of environmental levels of pesticides (atrazine, fenitrothion, pendimethalin, chlortoluron, flusilazole, copper oxychloride, metaldehyde and chlorpyrifos), metals (copper, lead, zinc and chromium) and flame retardants (tributyl phosphate and hexabromocyclododecane) on the transition of juvenile and adult eels between freshwater and the marine environment and growth and feeding during freshwater residency. Exposure to tributyl phosphate (TBP) in freshwater had some effect on physiological (plasma levels of glucose, sodium and chloride and kidney Na+/K+ ATPase) parameters associated with the silvering process in the eel but not on the morphology or the migratory behaviour during the transition from freshwater to the marine environment. Exposure to a mixture of pesticides (pendimethalin, chlortoluron, flusilazole, copper oxychloride, metaldehyde and chlorpyrifos) did modify the migratory behaviour of eels during the early stages of the freshwater migration but did not have any effects on the physiology of saltwater adaptation. Exposure to atrazine did not impair the olfactory system of eels and they were able to detect compounds released by prey items. Exposure of juvenile (glass eels) to 0.5 μg l-1 of tributyl phosphate did not have an impact on their freshwater adaptation as they were able to survive the movement between salt and freshwater. Glass eels exposed to a range of low concentration of metals (copper, lead, zinc and chromium) all demonstrated significant damage to their DNA. The long term impact of DNA damage is not known or whether this would reduce survival in the eels. The results of the study indicate that exposure to contaminants as the eels migrate between the freshwater and marine environments has probably only a minor role in regulating the eel population.

A number of pharmaceuticals have been shown to have adverse effects on key biological processes of aquatic organisms at low concentrations (ng/l range). Key questions for chemical risk assessment are whether such pharmaceuticals can produce adverse effects on organisms when present in the environment in combination and at low concentrations, whether these can be classified as additive, and under what circumstances can they be predicted. The main purpose of this study was to assess the potential for combination effects of a multicomponent steroid pharmaceutical mixture of dissimilarly acting compounds on an ecologically relevant end point – reproduction, using the existing predictive toxicity models Concentration Addition (CA) and Independent Action (IA). Concentrations of steroids close to those reported in the environment were shown to produce adverse effects on reproduction when present in combination with other steroids. Clear combination effects significantly larger than the effects of the individual compounds were observed when each compound was present at a concentration below the detection limit of the assay, demonstrating a ‘something from nothing’ mixture effect. Furthermore, IA predicted more pronounced effects on egg production that CA, an observation previously unreported from the literature. Actual observed effects were closer to the IA prediction. Additional biomarker and molecular endpoints were examined in subsequent studies to establish the mechanisms of disrupted reproduction in pair-breeding FHMs exposed to the steroid mixture. Results from this study indicate that reproductive impairment of fish exposed to the steroid mixture is likely due to the masculinisation of female fish due to the androgenic activity of the mixture. These results have implications for chemical risk assessment, and in particular, highlight the need for caution when using CA as a worst-case approximation of mixture effects.

Microsporidia are a group of obligate intracellular parasites of economic and medical importance. Many aspects of microsporidian genomes and cell biology are a result of an extensive reductive evolution during adaptation to their intracellular parasitic lifestyle. Microsporidian mitochondrial homologues called mitosomes have only a single known conserved metabolic function in biosynthesis of the essential iron-sulfur clusters. Based on genomic data an additional function of the mitosome in the alternative respiratory pathway (ARP) was proposed for some microsporidians including human pathogenic Trachipleistophora hominis. This thesis provides the first direct experimental evidence for a mitosomal localization of the two components of the ARP in T. hominis. Quantitative analyses of the immunofluorescence data together with western blotting experiments provided results consistent with the life cycle-stage specific function of the organelle. In the proliferative stages of the T. hominis life cycle, capable of stealing ATP from the host, mitosomes seem to function mostly in the biosynthesis of the essential iron sulfur clusters. The ARP proteins are enriched in the T. hominis spores, which is consistent with the hypothetical functions of the mitosome in energy metabolism of the spore that is unable to rely on its host for ATP production. This thesis also provides the first bioinformatics characterization of the molecular machineries involved in the processes required for inheritance of the microsporidian mitosomes: organelle fission and segregation during the cell division. Specific antibodies were generated and used to detect the microsporidian spindle pole body (SPB), an organelle hypothesized to play a role in inheritance of the microsporidian mitosomes. Double labelling experiments using the specific antibodies against the SPB and mitosomal markers provide evidence for a stable connection between the two organelles throughout the life cycle of the parasite.

Microsporidia are an enormously successful group of obligate intracellular fungal parasites that infect most eukaryotes including humans. In my thesis I have analysed and compared microsporidian genomes to identify which genes have been conserved, and which lost, during the transition of the group to parasitism, sequenced the transcriptome of a mixed infection of Trachipleistophora hominis in a rabbit kidney cell line, and provided a description of the T. hominis intracellular lifecycle. My results demonstrate that microsporidian genome evolution is extremely dynamic; with huge loss of genes in the microsporidian common ancestor balanced by group and lineage-specific gene family expansion and innovation. Genes that are conserved among microsporidians are generally expressed at higher than average levels in the transcriptome of T. hominis. Lineage-specific genes show greater variation in expression, but some are very highly expressed suggesting that they play important roles in T. hominis biology. The transcriptomics data for T. hominis confirmed that it contains one of the largest microsporidian genomes in terms of gene content and also identified previously unannotated genes, some of which may have important roles in the parasite. Detailed analysis of my gene expression data demonstrated that differential expression of duplicate genes is a general feature of T. hominis gene families, including nucleotide transport proteins that are already known to have important roles in the microsporidian lifestyle. A method for partially synchronising the T. hominis infection was established and used to investigate the development of the infectious cycle using light microscopy and antibodies to T. hominis proteins. My observations suggest that the infection proceeds in a reproducible and predictable pattern under the experimental conditions used; providing a tool for future detailed study of how T. hominis infects and exploits eukaryotic host cells.

Post-translational protein modifications (PTM) vastly increase the complexity and functional diversity of the proteome, to precisely regulate crucial cellular processes. The plant immune system is composed of complex signalling networks that are influenced by various PTMs. Activation of plant immunity is associated with a rapid burst of nitric oxide (NO), which can covalently modify cysteine thiols within target proteins by a process termed S-nitrosylation to form S-nitrosothiols (SNOs), constituting a redox-based PTM. Another key PTM involved in plant immunity is SUMOylation, an essential mechanism involving the conjugation of the small ubiquitin-like modifier (SUMO) peptide to lysine residues within target proteins. Although the targets and mechanisms of S-nitrosylation and SUMOylation are becoming evident, how these key PTMs are themselves regulated remains obscure. Work presented in this thesis reveals that during plant immune signalling, the sole Arabidopsis thaliana SUMO conjugating enzyme, SUMO CONJUGATING ENZYME 1 (SCE1), is S-nitrosylated at a highly conserved, but previously uncharacterized cysteine. S-nitrosylation of SCE1 was shown to inhibit its SUMO conjugating activity in vitro and mutational analysis revealed that the site of this modification, Cys139, is not required for enzyme activity but rather constitutes a redox-sensitive inhibitory switch. Generation and characterization of transgenic Arabidopsis plants overexpressing both wild-type and mutant forms of SCE1 revealed that Cys139 is required for efficient immunity against bacterial pathogens. Furthermore, after immune activation, S-nitrosylation of this residue inhibits global SUMOylation of proteins. These results provide evidence of a novel means of crosstalk between S-nitrosylation and SUMOylation in the context of plant immunity. The abundant cellular antioxidant, glutathione (GSH), is S-nitrosylated to form S-nitrosoglutathione (GSNO), which is thought to constitute a stable reservoir of NO bioactivity. In Arabidopsis, GSNO levels are controlled by the enzyme S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which indirectly influences the levels of protein SNOs. In this study, transgenic plants overexpressing FLAG-epitope tagged GSNOR1 were generated in various mutant backgrounds, including nitric oxide overproducer 1 (nox1), to further investigate the roles of GSNOR1 and NO in plant immunity. It was shown that ectopic GSNOR1 expression completely recovers developmental and disease susceptibility phenotypes of gsnor1, but not nox1 mutant plants, highlighting in vivo differences between accumulation of GSNO and free NO. Surprisingly, elevated NO levels in nox1 plants promote S-nitrosylation of GSNOR1, inhibiting its enzymatic activity. This suggests a previously unreported means by which NO might regulate its own bioavailability. Further work in this study revealed that recombinant GSNOR1 can be SUMOylated in vitro, which appeared to increase its enzymatic activity. Several potential SUMO modification sites were identified within GSNOR1 and mutational analysis revealed that at least one of these, Lys191, is SUMOylated. Co-immunoprecipitation experiments revealed that transgenic GSNOR1 might be SUMOylated in vivo, although the site(s) and biological function of SUMOylation were not identified. Nonetheless, these results reveal another possible layer of interplay between S-nitrosylation and SUMOylation.

The notochord is the defining structure of all chordates. A semi-­‐flexible elongated tube of cells, it forms along the central axis of the embryo and provides axial support during development. It also acts as a signalling centre during early embryogenesis, controlling the patterning of a number of tissues and establishing the early body axis of the embryo. In vertebrates, the function of the notochord expands beyond early development. It creates morphogenic gradients for the patterned formation of the vertebral bodies and, in adults, the remnants of the notochord form the nucleus pulposus, a gel-­‐like structure with an integral role in the distribution of vertebral pressure in the intervertebral disc. Little is known about how the notochord copes with damage during embryogenesis, but degeneration of the nucleus pulposus can lead to debilitating spinal disorders. In this thesis, I use a zebrafish model system to present new data that describes the cellular behaviours associated with how the notochord copes with external damage and how this damage can influence the future development of the vertebrae. I have uncovered a novel damage response in the notochord of zebrafish larvae and characterised the morphogenetic changes involved in the process using transgenic fluorescent lines. I have explored the damage in the context of the Wilms’ Tumour 1 (Wt1) gene, a vertebrate-­‐conserved transcription factor, which has recently been associated with several regenerative responses, and discovered that one of its zebrafish orthologues, wt1b, becomes upregulated in the notochord damage response. I have used fluorescent confocal imaging and immunohistochemistry to present new evidence that shows that upon injury, the outer notochord sheath cells upregulate the expression of wt1b. Additionally, I have used time-­‐lapse microscopy to show that damage to the notochord induces novel morphological changes in the injured organ, which include the loss of cellularity of the inner vacuolated cells and the movement of the wt1b-­‐positive outer sheath cells into the injured lumen. Long-­‐term imaging experiments have also demonstrated the capacity of the notochord to heal the damage over time, which ultimately leads to the formation of an extra, smaller vertebra in the wounded area. Skeletal staining of these fish has revealed a previously unknown putative cartilage switch at the site of damage, which leads to the formation of the new vertebral body. This finding has been supported by the microarray analysis of the injured area, which shows the unexpected de-­‐novo expression of cartilage markers at the site of damage The work in this thesis identifies for the first time an endogenous repair mechanism in the notochord of zebrafish larvae and describes the cellular, genetic and molecular processes cotrolling this novel wt1b-­‐associated damage response.

This thesis considers the field of ecological immunology, specifically considering life-history trade-offs between reproductive investment and self-maintenance. Classical immunology considers the physiological mechanisms behind the function of the immune system; both in a state of disease and at times of health. Ecological immunology makes the transition from the study of biochemical pathways and the molecular mechanisms involved, to an integrated study of these components in an ecological context, having been shaped by evolution. Variation in immune investment is observed across taxa and within populations. Despite the clear fitness benefits to efficient immune function, immune investment is costly. Therefore, investment in immunity must be balanced with investing in other life-history traits. This research considers how immune function and reproductive investment are balanced in different scenarios. Using burying beetles (Nicrophorus vespilloides) as a model system, this study investigated immune and reproductive strategies in a species that exhibits costly biparental care and also exploits a microbe rich environment. This thesis includes experiments designed to contribute to areas that are currently understudied in the literature: social immunity, how specifically parental care is balanced with immune function, changes in immune function with age, and the fitness consequences of changes in the balance of trade-offs.

Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), initiating a broad-spectrum defense response against pathogens, known as PRR-triggered immunity (PTI). However, immunity comes at a cost; and immune responses need to be tightly regulated. How PTI signalling is negatively regulated in plants is not fully understood. PRRs are present at the plasma membrane in dynamic kinase complexes that heavily rely on trans-phosphorylation to initiate signaling. The Arabidopsis cytoplasmic kinase BIK1 associates with different PRRs and plays a central role in the activation of downstream immune signaling. In this study, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the regulatory receptor kinase (RK) BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Notably, upon PAMP perception, PP2C38 is phosphorylated on serine 77, most likely by BIK1, and dissociates from the PRR-BIK1 complex. We suggest that this mechanism relieves the negative regulation imposed by PP2C38 to enable efficient BIK1 activation. This study uncovers an important regulatory mechanism of this central immune component, and extends our knowledge on how plant immunity is appropriately controlled.

Light-Oxygen-Voltage (LOV) domains are flavoproteins that are part of photoreceptors found in plants, prokaryotes and algae. LOV domains act as biological switches in response sensors to oxygen, redox potential or light, making them ideal for use as an optogenetics tool. The protein-switch is activated by formation of a covalent adduct between the flavin cofactor and an internal cysteine residue, causing changes in the hydrogen bonding network in the core of the protein. This results in a conformational change in the LOV domain leading to undocking of an amphiphilic helix that is generally coupled to a catalytic or transcriptional activation domain. In this investigation the LOV2 domain of phototropin 1 from Avena sativa has been modified to control protein-protein interactions in apoptosis by incorporating the Bcl homology region 3 (BH3) of a key pro-apoptotic protein (Bid) to the mobile helix of AsLOV2 (J). The design, cloning, production and structure of these hybrid proteins (AsLOV-Bid1-4) are discussed and their photo-switching characteristics are examined using UV/Vis and CD spectroscopy. Half-lives of the proteins varied between 13 min and 7.5 min, with small deviation between UV/Vis and CD half-life measurements for each protein. A further investigation on the binding of AsLOV2-Bid proteins to Bcl-xL, the natural binding partner of Bid, has been conducted in the dark and light states using fluorescence anisotropy measurements. The results verified that AsLOV-Bid1-4 bound to Bcl-xL effectively in the light state with KD values at less than 300 nM. However, AsLOV-Bid2 also bound in the dark state with KD at 1 mM. A second LOV domain, YtvA from Bacillus subtilis is characterised using UV/Vis and CD spectroscopy. YtvA is a homodimeric photo-switch, with a longer relaxation half-life than Avena sativa LOV2, making it more suitable for use as an optogenetics tool. Here, investigations on amino acid residues key for the dimerisation of this protein were performed, before any hybrid proteins could be engineered. Residues V27 and I113, were replaced with aspartate residues by site-directed mutagenesis to explore the effects on the protein quaternary structure using size-exclusion chromatography. Comparing against standards mutant I113D is considered as monomeric, however further experiments need to be conducted to verify this.

Since 2007 CEH and the Environment Agency are building a Fish Tissue Archive by annually collecting roach (and in 2007/08 also some eels and bleak) from a number of English river sites and storing them long-term at -80°C. This provides a resource for retrospective monitoring of bioaccumulative pollutants in the fish tissue - allowing future scientists to answer questions that cannot yet be answered or are not yet asked. By the end of 2014, 1684 fish had been collected of which 179 have so far been analysed for one or more groups of chemicals: metals, organochlorine pesticides, PCBs and PBDEs. The results from the individual fish were compared to each other as well as to regulatory standards and previously published UK and European data. Some of the results are: With the exception of lead in 3% of analysed individuals, no food standards were exceeded, but the environmental quality standard (EQS) for mercury was exceeded in the majority of samples (111/144) and the very low EQS for PBDEs was greatly exceeded in all samples. Some patterns found were:  Mercury and selenium increased with size of the fish and to some extent with the distance of the sampling site from the river source.  PBDE concentration correlated well with the modelled concentration of treated sewage at the sampling site  A hotspot was found for DDTs (banned in 1981) and to a lesser extent lindane, chlordane and copper. Further investigations revealed that a pesticide factory had been located close to the sampling site for much of the 20th century. This shows how unexpected results can point to previously unknown issues, which warrant further investigation.  Compared to previous European data, eels were generally less contaminated with organic pollutants and roach were low in mercury and cadmium, but relatively high in lead.

In the face of climate change and increasing anthelmintic resistance, sustainable control strategies for nematodes in livestock are needed. A prerequisite to the application of these strategies is an understanding of the ecology of the major gastrointestinal nematode (GIN) species, which forms the theme of this study. This thesis investigates the effect of microclimatic factors on the ecology and epidemiology of GIN in small ruminants. It focuses on the two most important climatic variables that determine the behaviour of the free-living stages of GIN : temperature and moisture. The rationale for the work and the methods used are summarised in Chapters 1 and 2. The effects of constant and fluctuating temperatures and moisture availability on the development and survival of infective larvae (L3) of Haemonchus contortus and Teladorsagia circumcincta were first investigated by conducting controlled experiments (Chapter 3). A significant part of the thesis then investigates the effect of moisture on the migration of L3 from faeces, which has barely been studied previously. This was first examined using controlled experiments on the effect of artificial rainfall on larval migration in laboratory environments (Chapter 4). These experiments revealed that a threshold faecal moisture content (FMC} was needed to release L3, and that adding more rainfall over this threshold had no effect on larval migration. The effect of current and future climate warming scenarios on opportunities for migration of L3 was examined using glasshouse experiments (Chapter5). Results indicated no significant difference in the rate of migration between baseline and warmed (+3°C} conditions, with migration being possible for 3-6 hours after a single rainfall event. Results of the controlled experiments were used to devise a simple model to predict the larval availability of H. contortus, which was tested in the field by comparing faecal egg counts (FEC} on sheep farms with meteorological data (Chapter 6). The field studies provided some qualitative validation of the predictions, but were limited by ability to differentiate H. contortus from other nematode species in faecal samples. Two rapid and simple methods for the diagnosis of H. contortu5 were consequently developed, based on egg morphology and differential development success in culture, to support future and more precise field studies (Chapter 7). There were three sequential steps to achieve the overall aims of the thesis, starting with laboratory experiments, then experiments on turf in controlled environments (experimental greenhouses) and finally validation with field data. The final goal of these efforts was to develop a mathematical model to predict the time course of larval availability under manipulated and natural climates, thus providing evidence to support the design of climate-driven GIN control strategies. While achieving robust and useful models for this purpose is an ongoing endeavour, the results presented in t his thesis provide new understanding of the effects of climate on larval availability, especially the role of rainfall in supporting migration of infective nematode larvae from faeces. In particular, it is clear that in drier regions of the world, water availability through rainfall will impose stronger effects on larval translation onto pasture than will higher temperatures. This thesis presents a renewed focus on micro-climatic conditions for parasite transmission, understanding of which will be essential to predicting effects of climate change on livestock disease from GIN.

Stomata are microscopic pores located in the epidermis of most terrestrial plants. As well as serving as gateways for gas exchange and transpiration, stomata have recently become known to have increasingly complex relationships with pathogens, with many pathogens utilising stomata as entry portals. However, the research so far has largely focussed on bacterial and fungal pathogens, leaving a gap in the knowledge about the interactions between plant viruses and stomata. This study aims to investigate the interactions and relations between stomata and viruses by investigating potential for stomata to act as entry portals for viruses and the developmental changes which occur under virus infection. In order to test the hypothesis that stomata can act as entry portals for plant viruses, a series of experiments was performed which altered stomatal apertures before applying purified virus suspended in solution in an aerosol. It was found that Nicotiana tabacum plants were more likely to become infected when TMV virus solution was applied when stomata were open. Natural and chemical factors were used to manipulate stomatal apertures prior to virus application. Stomatal development was investigated following a virus infection. A range of host-virus systems was used and it was found that susceptible host types had a general reduction in stomatal index and density when infected with a virus. Transcripts of genes involved in stomatal development were also tested for changes in healthy and infected plants and were found to vary upon infection. Knock-out mutants of various stomatal developmental or functional genes showed varying developmental responses to a virus infection, with notable changes in rin4 which resulted in an increase in stomatal development post infection. The results presented in this project provide an insight into a relatively new field of research which has so far been neglected in the field of plant pathology.

Cancer and neurodegeneration are detrimental conditions associated with an inappropriate regulation of cell survival and cell death, causing compromised cells to evade death or excessive death of healthy neurons. The mammalian target of rapamycin complex 2 (mTORC2) has been implicated in the regulation of cell survival by phosphorylating the protein kinase Akt. This is dependent upon the scaffold protein Sin1, a core component of mTORC2. The requirement of Sin1 in cell survival, and in particular in neuronal survival, has not been established due to the early embryonic lethality of mice with a targeted deletion of the Sin1 gene. To circumvent this issue, a novel conditional mouse knockout model was established. The role of Sin1 in regulating cell survival was evaluated in fibroblasts and cortical neurons. The loss of Sin1 significantly affected the phosphorylation and activity of Akt in fibroblasts and caused a reduction in cell survival by potentially inducing premature senescence. In contrast, the loss of Sin1 caused an increase in caspase-independent cell death in cortical neurons. Gene-expression analysis of Sin1 knockout cortical neurons demonstrated an important down-regulation of transcription factors, cytoskeletal proteins and components of signalling pathways involved in neuronal survival, aiding to uncover the mechanism by which Sin1 promotes neuronal survival. Taken together, the results presented in this study show a key role of the scaffold protein Sin1 in regulating neuronal survival.

The model angiosperm Arabidopsis thaliana produces viable seed through coordinated growth of three constituents; seed coat, embryo and endosperm. During development the embryo grows to fill the space defined by the seed coat. The growing embryo is surrounded by endosperm, an ephemeral, nutritive structure. The process of programmed cell death (PCD) is essential for endosperm consumption by the embryo however very little is known about developmental PCD in the endosperm. ZOU is a transcription factor expressed specifically in endosperm adjacent to the growing embryo in the Embryo Surrounding Region (ESR) (Yang et al., 2008). zou seed likely have reduced PCD resulting in abnormally persistent endosperm and a small embryo at seed maturity which results in seed shriveling. A second zou phenotype is an impairment of cuticle development in the embryonic leaves (cotyledons), suggesting that ZOU may mediate a signal from endosperm to embryo. The ESR expressed gene ALE1 is down-regulated in zou. When ALE1 is artificially expressed in zou ESR by the construct pSUC5::ALE1 the zou epidermal phenotype is rescued but not the seed shriveling phenotype of zou (Xing et al., 2013). Fixed and resin-embedded sections of zou and pSUC5::ALE1 lines herein confirm that zou-like endosperm is exhibited in pSUC5::ALE1 lines. This confirms that the two phenotypes of zou are genetically separable. The involvement of ZOU in epidermal processes is further confirmed through genetic studies showing that ZOU acts in the same pathway to impart embryonic cuticle as the embryo-expressed Receptor Like Kinases GSO1 and GSO2. In order to quantify PCD in the endosperm of wild-type and zou seed, PCD expression marker and TUNEL analysis were conducted. One PCD marker, pCEP:H2A-YFP is shown to be expressed in wild-type ESR, it is not clear if expression is lost in zou. To identify candidate genes under the control of ZOU active in endosperm PCD the results from several transcriptional profiling experiments were analysed and validated; this detailed gene expression in wild-type, ale1 and zou siliques which allowed for the identification of targets of ZOU but not of ALE1, targets predicted to be PCD effectors. In silico expression and ontology analysis confirmed likely roles for some candidates in endosperm PCD processes (particularly cell wall modification). Selected targets were cloned under pSUC5 and expressed in the ESR of zou seed as part of a molecular screen for the rescue of the zou endosperm phenotype. The ZOU target FRINGE-Like, a Glycosyl Transferase which shows strong endosperm expression is shown to partially rescue the zou phenotype but does not rescue the epidermal phenotype, suggesting that it may mediate PCD processes under ZOU control. The initial discovery that a Glycosyl Transferase may be active in a developmental PCD process in plants is exciting and novel and benefits understanding of developmental PCD and endosperm breakdown, two poorly characterized processes in plants.

The cell survival pathway autophagy, sequesters and degrades long lived and damaged proteins, recycling for reuse as biosynthetic building blocks. Autophagy is often dysregulated in disease pathologies, evidences shows both loss or gain of autophagy function can provide survival advantage. Constitutive activation of the KRas pathway occurs in cancer, with mutant Ras inducing autophagy for survival. Microarray datasets reveal that KRas mutant cancers increased MAP4K3 mRNA, which encodes a serine/threonine specific protein kinase that regulates mTORC1 signalling, in response to nutrients. Since nutrient signalling and autophagy are closely linked, the potential role for MAP4K3 in autophagy regulation, through mTORC1 in KRas mutant colorectal cancer cells, was investigated. MAP4K3 protein expression was elevated in KRas mutant (HCT116) cells compared to isogenic KRas wild type (HKH2) cells. Although silencing of MAP4K3 using shRNA decreased cell proliferation and metabolic activity in both cell lines, MAP4K3 silencing specifically diminished basal autophagic flux in KRas mutant cells, revealing a potential role for MAP4K3 in the regulation of autophagy. Loss of MAP4K3 expression after shRNA silencing led to decreased hVps34 activity and LC3 processing, both of which are essential for autophagy activation and progression. Furthermore, inhibition/silencing of MAP4K3, revealed a feedback mechanism that led to increased ERK1/2 phosphorylation in both cell lines. Combined Silencing of MAP4K3 with the MEK inhibitor PD184352 resulted in preferential sensitisation of KRas mutant cells to apoptotic cell death. This thesis identifies the novel function of MAP4K3 in autophagy control, through control of the hVps34 complex, furthermore the autophagy pathway is up-regu lated in KRas mutant cells and its inhibition sensitises KRas mutant cells towards MEK inhibitors. Thus, therapeutic targeting of MAP4K3 could be combined with Ras pathway inhibition to selectively target KRas mutant cancers.