The co-ordinated degradation of proteins is vital to all aspects of cellular activity. The main mechanism of intracellular protein degradation is the ubiquitin proteasome system (UPS) which labels target proteins with ubiquitin, thereby marking them for degradation by the 26S proteasome. Protein ubiquitination is mediated by three enzymes; E1, E2, E3. The largest family of E3’s is the cullin-RING E3 ubiquitin ligases (CRLs). CRLs require the cyclic addition and removal of a ubiquitin-like protein called NEDD8 to and from the cullin subunit. Removal of NEDD8 (deneddylation) is mediated by the eight subunit COP9 signalosome (CSN; CSN1-8). Cullin deneddylation by the CSN has been demonstrated to prevent the autocatalytic degradation of the substrate recognition subunit (SRS) of CRLs. The CSN has also been shown to associate with deubiquitinase and kinase activity and has thus been identified as a highly conserved key regulator of protein degradation. CSN subcomplexes have also been identified which function in protein degradation, and a direct role for the CSN complex in transcriptional regulation has been posited. Although the COP9 signalosome (CSN) has been studied in human cells, little is known of its role in haematopoietic cells or of any potential contribution to leukaemogenesis. In this study the deneddylase catalytic subunit CSN5 and the non-catalytic subunit CSN2 were knocked down in the human haematopoietic cell line and chronic myeloid leukemia model, K562. Both knockdowns had similar consequences for CRL activity whilst having divergent effects on the levels of SRS mRNA. Knockdown of either subunit also resulted in a common sequential proteasome-dependent loss of SRS proteins, an observation that had not been previously described. Although both knockdowns resulted in reduced cell proliferation followed by significant cell death, the cellular phenotypes and mechanisms of cell death were distinct. CSN5 knockdown was associated with mitotic defects, G2/M arrest, and culminated in apoptosis. In contrast, CSN2 knockdown resulted in autophagy inhibition and non-apoptotic cell death. This is the first time the CSN has been associated with autophagy. CSN2 and CSN5 knockdowns also had divergent effects on the intact CSN complex. CSN2 loss resulted in significant reduction of the intact CSN whilst, for the first time, the intact CSN complex was shown to be retained in CSN5 knockdown cells with loss of only monomeric CSN5. The common effect on CRL activity by either knockdown suggests a common loss of deneddylase activity, which was explained in CSN2 knockdowns with the loss of the intact CSN complex. However, in the case of CSN5 knockdown, in which the intact complex remained, the reason for loss of deneddylase activity is less easy to explain. The results of this study may indicate for the first time that sustained deneddylase activity is dependent on a novel mechanism requiring a pool of CSN5 monomer. Finally, the significance of monomeric CSN5 function loss to the differential phenotype of CSN5 knockdown cells to cells lacking CSN2 was tested by re-expression of both wild type and deneddylase dead CSN5 in a CSN5 knockdown background. Importantly, both approaches rescued the cellular phenotype to the same extent. Overall, the findings of this study provide novel insight into both the function and potential regulation of the CSN complex, whilst further suggesting that the CSN may be a target worthy of investigation in the treatment of chronic myeloid leukaemia.

Current regulatory risk assessment strategies have several limitations, such as linking subcellular changes to higher-level biological effects, and an improved knowledge-based approach is needed. Ecotoxicogenomic techniques have been proposed as having the potential to overcome the current limitations, providing greater mechanistic information for ecotoxicological testing. In this thesis, metabolomics is explored as a novel method for toxicity testing using Daphnia magna. Initially I evaluated the potential application of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) based metabolomics for use in regulatory toxicity testing. Subsequently, I aimed to use this approach to discriminate between toxicant modes of action (MOA) and to link toxicant induced metabolic effects to reduced reproductive output in D. magna. FT-ICR MS metabolomics was determined to be a feasible approach for toxicity testing of both whole-organism homogenates and haemolymph of D. magna. It is capable of discriminating between life-stages of D. magna as well as determining toxicant-induced metabolic effects. Highly predictive multivariate classification models were capable of significantly discriminating between four different toxicant MOAs; achievable in both haemolymph and whole-organism extracts, with the latter being the more information-rich sample type. Multivariate regression models were predictive of reduced reproductive output in D. magna following toxicant exposure, and determined that a metabolic biomarker signature was significantly able to predict the reproductive output of D. magna. Ultimately this research has concluded that an FT-ICR MS metabolomics approach for use in regulatory toxicity testing using Daphnia magna is both viable and can provide valuable information.

Cells depend on the actin cytoskeleton and the structures it forms for a wide range of processes. IRSp53 acts downstream of cytoskeleton master regulators Rac and Cdc42 in the formation of lamellipodia and filopodia, respectively. IRSp53 interacts with many other effectors in the formation of these structures, via various protein interaction domains. IRSp53 also contains an IMD (IRSp53/MIM homology domain), which is able to induce formation of filopodia-like structures when overexpressed in cells. Early reports suggested that the IMD exerts its effect by bundling actin filaments. However, the structure of the IMD revealed that it is related to BAR domains, which can induce membrane curvature by binding to membranes. It is still unsure whether IMD activity is dependent on actin or membrane interactions, or both. Data are presented here showing that actin destabilizing drug cytochalasin D prevents extension of IMD protrusions, suggesting a role for actin in this process. Fluorescence recovery after photobleaching experiments suggest that a large proportion of IMD is stably associated with the protrusions. Finally, in vitro lipid binding experiments suggest that IMD binds to membranes by mainly electrostatic interactions. These findings suggest that IMD activity may depend on interactions with both actin filaments and membranes.

During meiosis, axis formation and synapsis of homologous chromosomes are tightly co-ordinated with meiotic recombination. This study investigates the influence of chromosome axis and synaptonemal complex proteins on meiotic crossover formation. The study involved the characterization of a novel protein, AtASY3, required for normal meiosis in Arabidopsis. Analysis of Atasy3 mutants revealed that loss AtASY3 compromises chromosome axis formation, synapsis and normal levels of crossover formation. Further analysis revealed that loss of AtASY3 disrupts the axial organization of AtASY1. In separate studies, colleagues found that AtASY3 and AtASY1 are able to interact. Together, these results suggest that AtASY3 is a functional homologue of the budding yeast axis protein, Red1. Since studies in budding yeast indicate that Red1 and Hop1 (homologue of AtASY1) play a key role in establishing a bias to favour inter-homologue recombination, this study suggests that AtASY3 and AtASY1 may have a similar role in Arabidopsis. The study also involved the analysis of the putative phosphorylation site, residue T295, on AtASY1. This revealed that T295 is essential for AtASY1-mediated crossover formation during meiosis. Additionally, a potential meiotic role for the RECQ DNA helicase, AtRECQ4B was investigated, however, the protein does not appear to be essential for Arabidopsis meiosis.

CCRL2 is a member of the atypical chemattractant family. It has been proposed to bind the chemokines CCL19 and CCL5, as well as the adipokine chemerin. Unlike typical chemokine receptors, atypical chemoattractant receptors do not undergo conventional G protein signalling upon binding, but instead degrade, transcytose or present their ligands on the cell surface. This study aims to characterise the role of CCRL2 in B cells upon their activation and differentiation into either extrafollicular plasmablasts or germinal centre B cells. CCRL2 mRNA is upregulated upon plasmablast differentiation. Upon immunisation with NP-Ficoll, CCRL2 deficient mice produce more NP-specific antibody and larger numbers of NP-specific plasmablasts. Further assessment show this phenotype is due to B cell intrinsic effects. CCRL2 deficient plasmablasts tend to proliferate more and undergo less apoptosis than CCRL2 expressing plasmablasts. The role of CCRL2 in the germinal centre was also assessed. Germinal centres formed normally, including polarisation into light and dark zones in CCRL2 deficient mice. However, FDCs within the germinal centre appeared to extend further into the follicular mantle in CCRL2 deficient mice. This may be the cause of an increased proportion of germinal centres over the whole spleen in CCRL2 deficient mice. These differences did not result in significant changes in affinity maturation. Together, this shows a novel role for CCRL2 in the regulation of the extrafollicular plasmablast response to NP-Ficoll. However, minor differences in CCRL2 deficient germinal centres do not affect high affinity plasma cell output, suggesting a minimal role of CCRL2 in GC function.

Mass spectrometry-based metabolomics aims to study endogenous, low molecular weight metabolites and can be used to examine a variety of biological systems. To substantially increase the accuracy of metabolite identification and increase coverage of the metabolome detected by high-resolution (HR) mass spectrometry I developed, optimised and/or employed several analytical and bioinformatics methods. Biological samples contain thousands of metabolites that are related through specific substrate-product transformations. This prior biological knowledge together with a mass error surface, which represents the mass accuracy of peak differences within mass spectra, were employed to significantly reduce the false positive rate of metabolite identification. To maximise the sensitivity of the Thermo LTQ FT Ultra mass spectrometer, the existing direct-infusion SIM-stitching acquisition parameters (Southam et al., 2007) were reoptimised, yielding a ca. 3-fold increase in sensitivity. Finally, relative isotopic abundance measurements (RIA) using HR direct-infusion MS were characterised on the two most popular Fourier transform MS instruments (FT-ICR and Oribitrap) using the reoptimised SIM-stitching acquisition parameters. Several novel observations regarding RIA measurements were reported. Utilising these RIA characterisations within a putative metabolite identification pipeline increased the number of single true empirical formula assignments compared to using accurate mass alone. To conclude, analytical and bioinformatics methods developed in this thesis have successfully facilitated the putative identification of hundreds of metabolites in several metabolomics studies.

TRIB2 belongs to the TRIBBLES family of serine/threonine pseudokinases (TRIB1, TRIB2 and TRIB3). TRIBBLES regulate proliferation, survival, differentiation, metabolism and stress responses, and their expression is frequently deregulated in cancer, including Acute myeloid leukaemia (AML). AML is a heterogeneous group of blood malignancies characterised by differentiation block and uncontrolled proliferation of myeloid precursor cells. Aberrant TRIB2 expression drives myeloid transformation through downregulation of the tumour suppressor CCAAT/enhancer-binding protein (C/EBPα). However, the mechanism of TRIB2 gene deregulation in cancer, and the role of TRIB2 in the oncogenic pathways of other AML oncogenes have not been fully investigated. Moreover, there is still limited information regarding the expression and function of TRIB2 and its family members, in the haemopoietic system, and how this is altered in different AML subtypes. This work aims to address these biological questions, by employing in silico, in vitro and in vivo experimental strategies.

The world population is continually growing with an increasing food demand to match whilst pests represent a major loss to agricultural production. The aim of this thesis was to assess the overwintering survival and activity potential of three candidate biological control agents, \(Euseius\) \((Amblyseius)\) \(ovalis\) Evans; \(Euseius\) \(stipulatus\) Athias-Henriot and \(Euseius\) \(gallicus\) Kreiter and Tixier (Acari: Phytoseiidae). Cold tolerance assessments indicated that both \(Euseius\) \(ovalis\) and \(Euseius\) \(stipulatus\), as non-native species, were physiologically ill-adapted to UK winter temperatures. \(Euseius\) \(gallicus\), as a species native to temperate European countries, was found to have an acclimation and diapause trait, being well-adapted to survive a UK winter. Thermal activity thresholds supported all three Euseius spp. as efficient agents against \(Tetranychus\) \(urticae\) under low temperature conditions, with \(Euseius\) \(gallicus\)’ acclimated lower activity threshold supporting its use in open-field releases of native countries. The data from the cold tolerance of \(Euseius\) spp. and \(Tetranychus\) \(urticae\) have further enhanced the reliability of the winter survival regression (LTime\(_50\) at 5°C) as an essential component of arthropod cold survival assessments. Finally, this thesis presents a study on the role of the media in public understanding and awareness of pest control strategies; discussed with regards to food security.

Apoptosis-induced Proliferation (AiP) refers to an evolutionary conserved process that stress-induced apoptotic cells stimulate neighbouring cells to undergo extra proliferation to compensate for the loss of apoptotic cells. It is therefore involved in tissue regeneration in multi-cellular organisms. I found that dAtg1 (Drosophila autophagy-related gene 1), a gene best known in activating autophagy, is required for AiP. dAtg1 is transcriptionally induced by the JNK pathway, a stress response signalling pathway, during AiP and it regulates AiP by acting upstream of growth signals e.g. Wg/Wnt. Surprisingly, other Atg genes involved in autophagy are not required for AiP. Therefore, dAtg1 regulates AiP independent of its canonical roles in mediating autophagy. In parallel, I investigated the non-apoptotic cell death induced by Eiger (Egr), the Drosophila tumour necrosis factors (TNFs). In mammals, TNF induces apoptosis and, when apoptosis is blocked, necrosis. In Drosophila, the type of cell death induced by Egr remains elusive. I found that expression of egr in the developing Drosophila eye primarily induces apoptosis through the canonical apoptosis pathway. Intriguingly, when apoptosis is blocked by inactivation of effector caspases DrICE and Dcp-1, Egr induces necrosis instead. Therefore, mechanisms underlying TNF-induced cell death are more conserved in Drosophila than previously thought.

Cell number plasticity drives organismal growth, and is coupled in the CNS to the emergence of neural circuits, ensuring appropriate function. In mammals, neurotrophins promote cell survival via Trk and p75\(^{NTR}\) receptors or induce cell death via p75\(^{NTR}\) and Sortilin. In \(Drosophila\), DNTs bind Toll receptors promoting cell survival, but whether they regulate cell death within the CNS remains unknown. I show Toll receptors have distinct and overlapping spatial and temporal expression and functions. Driving RNAi knockdown and overexpression of each Toll, I show that different Toll receptors are required in glia for adult locomotion; in neurons for the regulation of VNC size; and to induce cell survival or death in distinct contexts. I focused on the signalling mechanisms downstream of Toll-6. My data show DNT-Toll-6 signalling switches between promoting cell survival or death via NFkB, ERK, or JNK signalling. These outcomes depend on the cleavage state of the DNT, time and available downstream adaptors. Toll-6 induces cell survival via MyD88 and cell death via dSarm, and these alternative outcomes depend on Weckle. Altogether, my data contribute to showing that the Toll receptors, DNTs and downstream signalling adaptors constitute a novel mechanism of cell number plasticity within the CNS.