In eukaryotes, transcription and RNA processing events are spatially separated from translation by the nuclear envelope. Although ribosomal subunits are synthesised and assembled in the nucleus, it is believed that they are kept inactive whilst in the nucleus. Yet, there were observations from this and other laboratories that suggest translation can occur in the nucleus. To further investigate whether translating ribosomes exist in the nucleus, I employed a bimolecular fluorescence complementation (BiFC) 80S reporter based technique previously developed in our laboratory that detects 80S assembly in \(Drosophila\) cells. The initial characterization indicated that the assay reports ribosomal subunit interaction, but other explanations could not be excluded. My first aim was to assess whether this technique is genuinely reporting translation-dependent subunits association. My results were consistent with the assay reporting 80S ribosomes formed as a consequence of translation. Following up from this, I developed a similar technique with Venus, which resulted in a more sensitive 80S reporter. While the 80S signal was more apparent in the cytoplasm, in both cell culture and fly tissue cells, a fraction of the cells showed a signal in the nucleus, particularly concentrated in the nucleolus. This signal was enhanced by translation elongation inhibitors in both cytoplasm and nucleus indicating that the detected 80S are engaged in translation. Notably, the nucleolar signal was prevented by RNA Pol II inhibition, suggesting that the 80S might be associated with mRNA in the nucleolus.

The Leukocyte Immunoglobulin-Like Receptors (LILRs) are a family of immunomodulatory transmembrane receptors with both activatory and inhibitory forms, and are primarily expressed on myelomonocytic cells. Group I LILRs include the well-characterised members LILRB1 and LILRB2, which bind MHC Class I and transduce inhibitory signals. Group II LILRs, by comparison, are poorly understood. Particular interest has arisen in the Group II receptor, LILRB4, and its role in the induction of immune tolerance via its actions upon dendritic cells and T cells. I attempted ligand-identification and structural studies on LILRB4. Staining of human PBMCs with multimeric LILRB4 revealed broad expression of the ligand, and upregulation on activated T cells. Investigations into candidate ligands by surface plasmon resonance excluded binding to known costimulatory receptors of the B7/CD28 families. X-ray crystallographic studies revealed LILRB4 has structural similarities to LILRA5, has novel structural features at the interdomain interface, is electrostatically and chemically unsuited to the recognition of MHC Class I, and identified potential ligand interaction sites. Finally, investigations were conducted into the structure and ligand recognition of other Group II LILRs. These studies define the distribution of the LILRB4 ligand and represent the first structural analysis of this important immunoregulatory receptors.

The endo-membrane system of eukaryotic cells is a dynamic network of membrane-bound organelles that exists in a constant state of flux as they exchange membrane and protein cargoes. The yeast vacuole is a highly dynamic organelle that changes shape in response to both hypo and hyper-osmotic stresses and so has been used to study both membrane fusion and fission. Fusion is relatively well characterised but fission is not well understood. This work focuses on the molecular events that co-ordinate vacuole fission during hyper-osmotic stress. One polyphosphoinositide pathway was known to regulate vacuole fission before this work began: the Fab1p pathway. The Fab1p pathway is activated during hyper-osmotic stress and causes the vacuole to fragment into many tiny sub-compartments by a process of membrane fission. I characterised a number of the components of this pathway to learn how they work together, including Vac7p, Vac14p, Fig4p and Ymr1p. The specificity and localisation of several of these proteins were characterised and a greater understanding of this protein network emerges from this work. I then focused on understanding how fission of the vacuole is maintained and fusion inhibited in hyper-osmotic media. It was previously reported that Yck3p, a vacuolar protein casein kinase, was activated by hyper-osmotic stress to phosphorylate Vps41p, a component of the vacuole fusion machinery. The effect of this phosphorylation and the upstream signalling pathway controlling Yck3p were unknown. I started by looking if any known signalling systems might control Yck3p and ruled out both the Fab1p and Hog1p osmotic stress response pathways as upstream activators. I then found that another polyphosphoinositide signalling system controls Yck3p: namely the Plc1p phospholipase pathway that produces a series of water soluble inositol polyphosphate second messengers. I further found that deletion of any of the inositol polyphosphate kinases in the Plc1p pathway prevented Yck3p signalling, suggesting that the inositol pyrophosphates control Yck3p activity in some fashion. I also identified the Yck3p phosphorylation sites on Vps41p by FT-ICR mass spectrometry and showed that phosphorylation of these sites was required to block vacuole fusion during hyper-osmotic stress. vps41 mutants lacking these sites showed aberrant behavior: their vacuoles underwent normal fission in response to hyper-osmotic stress but then immediately re-fused in an apparent “futile cycle”. Thus Plc1p controls a novel signalling pathway mediated by Yck3p that prevents vacuole fusion by phosphorylating thus inhibiting a vital component of the fusion machinery: Vps41p.

Antibiotic resistance, particularly amongst Gram-negative bacteria, has emerged as a major global concern. Working synergistically with the permeability barrier created by the cell envelope, multidrug efflux pumps contribute significantly to the intrinsic resistance of Gramnegative bacteria. Site-directed mutagenesis, informed by computational analyses, has been used on the prototypical tripartite efflux system, AcrAB-ToiC from Escherichia coli. Likely AcrA-ToiC interactions and functional roles of their sub-domains have been assessed using mutants in a combination of in vivo and in vitro approaches. Results indicate that both the tip region and the equatorial domain, halfway up the ToiC channel, are involved in determining the compatibility of AcrA with ToiC. On the other side of the interaction, both the tip region and the helices of the AcrA hairpin are essential for normal function. The hairpin tip is required to maintain the permeability barrier, while the helices are necessary for a stable AcrA-ToiC interaction. Combining these results with available literature, the first dynamic model of tripartite complex assembly is presented here. This model postulates initial bundling of AcrA and ToiC coiled-coil domains to open the ToiC channel followed by transition to a tip-to-tip interaction to drive channel closing and complex disassembly in an energy-dependent manner.

Fibroblast growth factor receptors (FGFRs) regulate fundamental cellular processes, including proliferation, differentiation and angiogenesis and have emerged as growth factor receptors central to oncogenesis. This study developed a live-cell assay system for studying FGFR endocytosis and trafficking by employing both confocal and total internal reflection fluorescence (TIRF) microscopy in cells expressing a previously characterised GFP-tagged FGFR2 construct. Data from this work have demonstrated that endocytosis of activated FGFR occurs through clathrin-mediated endocytosis. Interestingly, FGF treatment also significantly increased the number of CCPs as well as the number of clathrin-mediated endocytic events. However, treatment of cells with the Src family inhibitor Dasatinib or depletion of Src kinase target Eps8, prevents the FGF induced increase in plasma membrane clathrin and reduces the internalization of FGFR. This study also shows that both Src and Eps8 are required for receptor to exit from EEA I positive peripheral compartment into the Rab 1 1 positive PNRC. Eps8 depletion also inhibits the early phases of ERK activation in response to FGFR activation, placing this signalling event early in the trafficking pathway of the receptor. Thus, these results have identified the endocytic pathway for endocytosis of FGFR2 and described Eps8 and Src as key mediators of the early phases of activated FGFR trafficking and signalling.

Natural products, particularly polyketides are among the most important sources of antimicrobial compounds. 20% of the top selling drugs are polyketide based. In recent years genetic engineering has played a critical role in modifying biosynthetic pathways of different polyketide compounds as a way to create novel structures with improved clinical properties. Further investigation and understanding of these giant multi-enzyme complexes is necessary to achieve efficient synthetic engineering. In many PKS systems including the mupirocin biosynthesis pathway, the thioesterase (TE) is normally considered as the end of the assembly line. However, expressing the CoA-ligase tmlU from the thiomarinol pathway in the mupirocin producer strain (Pseudomonas fluorescens NCIMB10586) revealed that TmlU could only release truncated pseudomonic acid when a TE domain was present. This finding led to the hypothesis that perhaps the TE domain could act as a tether for TmlU, in order for the latter to be able to capture the growing chain and perhaps load it onto the post TE pathway. This study also presents the first evidence of MmpB being involved in producing the 9-hydroxynonanoic acid in the mupirocin biosynthesis pathway.

The skin is continuously being shed and therefore it is vital that it is renewed. Epidermal self-renewal is dependent on a population of keratinocyte stem cells (KSC) that reside in the basal layer. During epidermal regeneration, KSC divide asymmetrically giving rise to more stem cells as well as committed progenitors. Committed progenitors exit the cell cycle before going on to differentiate to form the highly resilient cells that make up the outermost layer. It is thought that committed progenitors and KSC are differentially regulated therefore allowing for such different behaviors. Rnd3 is an atypical GTPase that is constitutively active and has been previously shown to regulate keratinocyte differentiation. However, the identification of the molecular mechanism is currently unknown. The work presented here shows that Rnd3 depletion enriches for keratinocytes with a number of ‘stem-like’ phenotypes including reduced differentiation, reduced cell size, increased adhesion to ECM proteins and a deregulation of putative stem cell markers. Furthermore, using a quantitative proteomic approach, it can be seen that Rnd3 regulated the abundance of proteins involved in regulating stem cell function. This work proposes a function for Rnd3 in the regulation of key proteins involved keratinocyte differentiation and self-renewal.

Nowadays, there are many statistical methods available for genetic association analyses with data various designs. However, it is usually ignored in these analyses that an analytical method must be appropriate for an experimental design from which data is collected. In addition, association study is a population-based analysis and, thus its inference is highly vulnerable to many population-oriented confounding factors. This thesis starts with a comprehensive survey and comparison of those methods commonly used in the literature of genetic association study in order to obtain insights into the statistical aspects and problem of the methods. On the basis of these reviews, we managed to calculate the optimal trend set for the Armitage’s trend test for different penetrance models with a high level of genetic heterogeneity. We introduced two new strategies to adjust for the population stratification in association analyses. We proposed a maximum likelihood estimation method to adjust for biases in statistical inference of linkage disequilibrium (LD) between pairs of polymorphic loci by using non-random samples. In the process of the analysis, we derived a more sophisticated but robust likelihood-based statistical framework, accounting properly for the non-random nature of case and control samples. Finally, we developed a multi-point likelihood-based statistical approach for a genome-wide search for the genetic variants that contribute to phenotypic variation of complex quantitative traits. We tested these methods through intensive simulation studies and demonstrated their application in analyses with large case and control SNP datasets of the Parkinson’s disease. Despite that we have mainly focused on SNP data scored from microarray techniques, the theory and methodology presented here paved a useful stepping stone approach to the modeling and analysis of data depicting genome structure and function from the new generation sequencing techniques.

Cytoglobin (CYGB), a novel member of the globin family, has been shown to be upregulated in response to hypoxia, oxidative stress and fibrogenesis. Presented here is evidence of CYGB expression within cells of fibrotic lesions taken from patients with Idiopathic Pulmonary Fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD). CYGB staining was observed in fibroblasts, endothelial cells, type II pneumocytes, type I pneumocytes, haematopoietic stem cells and inflammatory cells, which were identified using cell specific markers. Cell types which express other members of the globin family, including smooth muscle and red blood cells were negative for CYGB. Fibroblasts were consistently positive for CYGB. CYGB expression was consistently positive within the lesion, and more variable at the edge. This study also provides evidence of an increase in CYGB expression in response to hypoxic and oxidative stress in vitro; however there was no evidence of cytoprotection with over expression of CYGB in response to these insults. There is evidence presented here that the increase in CYGB expression with fibrosis previously reported in the literature, is likely to relate to the hypoxic environment of the lesion and the influx of fibroblasts which are consistently CYGB positive. CYGB is likely to have a role in oxygen and redox homeostasis.

Drought is arguably one the greatest future challenges for agriculture. The response of phloem feeders, such as aphids, to increased drought expected under climate change is still relatively undefined. The effect on aphid feeding of drought stress in plants can be viewed as potentially positive and negative. It is currently accepted that under drought conditions, host plant sieve elements will become more concentrated; hence increases in available amino acid concentrations will potentially benefit aphids. However, the increased need for aphid osmoregulation under drought, to deal with the osmotically challenging diet, may be detrimental to aphid performance. Using the electrical penetration graph technique (EPG), the probing behaviour of two clones of Myzus persicae (O Clone and B Clone), Brevicoryne brassicae, Rhopalosiphum padi and Sitobion avenae on the host plants Brassica nigra and Lolium perenne (respectively), were analysed under a defined drought regime. Drought treatment had a significant effect on the behaviours of all aphid species and clones, except S. avenae. In B. brassicae and B clones of M. persicae, xylem feeding was significantly increased on droughted host plants. Furthermore, both clones of M. persicae and R. padi spent significantly less time ingesting sieve element sap, and more time not probing or in plant pathway activities whilst on droughted host plants. These results suggest that drought stress may cause a reduced palatability of host plants. In addition, fecundity measurements showed that drought resulted in a reduction in aphid reproductive performance in M. persciae (O and B clone), B. brassicae, R. padi and S. avenae. However, fecundity was only significantly reduced in the M. persicae (O clone only), B. brassicae and R. padi. To understand the aphid response to droughted B. nigra at the genetic level, the gene expression of M. persicae exposed to different drought regimes was analysed using microarrays. Gene expression analysis showed up regulation of the osmoregulation associated enzyme, sucrase, as well as the up regulation of other enzymes such as amylases, cytochrome P450s, Heat Shock Proteins, and an aquaporin when exposed to droughted hosts. Furthermore, it was found that the level of drought had a noticeable effect on gene expression in M. persicae, showing aphids have a very adaptable response to drought stress. The combined physiological and transcriptomic approach of this study gives a complementary whole organism assessment of aphid responses to drought. This study has helped to confirm xylem feeding and sugar polymerisation as important mechanisms of aphid osmoregulation, as well as providing support for the hypothesis of water cycling within the body of the aphid. This study has highlighted that aphids respond heterogeneously to water stress, and although it has been possible to identify some general trends, this study has emphasised that the adaptability of aphids to stress is species and even ecotype specific.