Feedback node genes (FNGs) are essential for negative feedback control of inflammatory responses. By definition, their expression is controlled by both pro- and anti-inflammatory stimuli, often in a cooperative manner. This thesis investigates three FNGs, namely Dual specificity phosphatase 1 (DUSP1), Tumour necrosis factor alpha inducible protein 3 (TNFAIP3) and Tristetraprolin (TTP, encoded by the \(Zfp36\) gene). DUSP1 is a negative feedback regulator of mitogen-activated protein kinases, TNFAIP3 negatively regulates the nuclear factor κB (NF-κB) signalling pathway and TTP is a destabiliser of pro-inflammatory mRNAs. All three FNGs were induced by pro-inflammatory stimuli, with very different dependence on NF-κB signalling. Further to this, the anti-inflammatory agonists dexamethasone, prostaglandin E\(_2\) (PGE\(_2\)) and transforming growth factor β (TGFβ) were able to impair NF-κB activity and yet cooperated with pro-inflammatory agonists to increase expression of all three FNGs. Experiments in primary mouse knock-out macrophages suggested that DUSP1 may be necessary for some anti-inflammatory effects of PGE\(_2\), and for the cooperative regulation of other FNGs by pro- and anti-inflammatory agonists. Three putative regulatory elements located upstream of the \(Zfp36\) locus were shown to mediate cooperative transcriptional regulation by various combinations of pro- and anti-inflammatory agonists. Chromatin immunoprecipitation experiments also demonstrated dynamic remodelling of the locus in response to a pro-inflammatory stimulus.

Introduction Most familial MTC is caused by a germline mutation of the RET proto-oncogene. Rare families exist with predisposition to MTC in whom no RET mutation has been identified. Identification of novel candidate genes within such families may inform the molecular behaviour of more common, sporadic disease. Whole exome sequencing (WES) enables all protein coding regions of the genome to be sequenced in parallel; a novel paradigm for MTC gene discovery. Methods Patients with MTC were recruited through internationally developed collaborations. WES was completed in three generations of the index family. Germline and tumour DNA were analysed for conformational mutations. In vitro functional analysis of candidate gens was completed to unpick biological pathways. Results Over 20,000 mutations were screened. A frameshift mutation in the oestrogen receptor 2 gene (ESR2) has been identified with familial segregation. Further alterations in ESR2 have been identified in germline DNA from a patient with young onset sporadic disease and tumour DNA from sporadic MTC. The functional protein of the ESR2 gene binds to a response element in the upstream pathway of the RET gene, controlling transcription. In-vitro studies show that ESR2 mutants lead to null proteins and up-regulation or RET at mRNA and protein levels. Further, loss of ESR2 protein is observed in patients with germline ESR2 mutations. Conclusions This study establishes a novel method of gene predisposition identification in the context of MTC. As well as the potential for a genetic test, and as a prognostic biomarker, the on-going functional work may elucidate targets for novel therapies that may include pre-existing anti-oestrogen.

CD4\(^+\) and CD8\(^+\) T cell populations can be divided into subpopulations based on expression of surface markers CCR7 and CD45RA. The resulting populations are referred to as naive, central memory, effector memory and effector memory RA\(^+\) (EMRA). The aim of this study was to identify potential lineage relationships between these subpopulations for both CD4\(^+\) and CD8\(^+\) T cells through microarray analysis. The genes found to distinguish between these subpopulations include many molecules with known functions in T cell differentiation, including CCR7, CD45RA, granzymes, L-selectin and TNF receptors. Several genes from the tetraspanin family of proteins were found to be differentially expressed at mRNA and protein level; suggesting a possible role for these genes in CD4\(^+\) and CD8\(^+\) T cell activation, migration and lysosomal function. Other genes identified, such as LRRN3 and CXCR5 which were expressed highest on naive and CM T cells respectively, provide interesting gene targets to follow up on their function in these T cell populations. Microarray data was validated through Real Time PCR and suggests that both CD4\(^+\) and CD8\(^+\) T cells differentiate along a linear pathway of naive to central memory to effector memory. The transcriptional programmes responsible for these differentiation steps were distinct between CD4\(^+\) and CD8\(^+\) T cells, although additional elements were common to both subsets.

Brassica crops are grown worldwide for food, oil, medicinal and crop rotation properties. They suffer from insect pests which cause large yield and economic losses. Application of insecticides is the preferred way of dealing with insect problems, but it is not only hazardous to the environment, it also affects humans as the chemicals easily get incorporated into the food chain. As a result, new more resistant varieties are urgently needed to meet the demand of growing populations. A set of 200 accessions were classified as resistant (non-preferred) or susceptible (preferred) in response to cabbage aphid feeding in the field. Fifteen accessions were further assessed to characterize and identify the level and location of resistance factors by investigating feeding behaviour of cabbage aphid using the Electrical Penetration Graph (EPG) technique. The feeding behaviour assessment revealed the presence of interspecific & intraspecific variation and presence of resistance factors at multiple levels. The transcriptional response of these accessions under presence and absence of aphid feeding for 24h showed that gene expression is highly regulated in response to aphid feeding. Gene ontology (GO) enrichment study helped identify strong candidate genes for aphid resistance. In addition to this, the gene expression differences between CWR and landraces indicated adaptations of landraces during the process of domestication. Lastly, Gene expression data was used to develop models to predict insect resistance status. In conclusion, the combination of EPG and transcriptomics provides an opportunity to assess brassica germplasm for further research into defence mechanisms of cabbage aphids.

The work described in this thesis concerns the design, synthesis, DNA binding and biological activity of palladium(II) supramolecular cylinders that might be capable of recognizing a DNA four-way junction. An introduction to DNA structure will be presented, as well as the different binding modes of natural and synthetic agents that can recognise and bind to DNA. Since this work is focused on the design of large metallo-structures, the general principles of supramolecular chemistry will be summarised with particular emphasis on metallo-supramolecular structures. Palladium(II) supramolecular cylinders have already been reported and these show promising cytotoxicity against different cancer cell lines. However, these complexes present poor solubility in aqueous solutions. It is therefore the aim of this thesis to improve the water solubility of palladium(II) supramolecular cylinders without significantly changing their structure or quenching their cytotoxic activity. The DNA binding properties of the newly synthesised palladium(II) complexes will be presented. Several spectroscopic techniques, such as circular and linear dichroism and ethidium bromide displacement assays, as well as electrophoresis experiments were carried out and these will be discussed. Initial DNA four-way junction experiments and cytotoxicity studies will also be discussed.

The process of angiogenesis in which new blood vessels form from pre-existing vessels, can be intensively studied through the use of in vitro and in vivo models. The in vitro co-culture tube formation assay is used to assess the ability of endothelial cells to develop into three dimensional tubular structures which mimics the growth of capillaries. Different fluorescent labelling techniques were developed and utilised alongside confocal microscopy to visualise endothelial tubulogenesis and investigate the mechanisms of lumenogenesis. Imaging the actin cytoskeletal organisation by expressing the lifeact peptide conjugated to fluorescent proteins revealed that Factin fibres outline lumens within endothelial tubules and enabled clear visualisation of filopodia formation. Further studies presented in this thesis aimed to develop, test and evaluate computational tools for analysing endothelial sprouting from fluorescently labelled spheroids generated using the in vitro hanging drop spheroid assay and quantify blood vessel formation in the in vivo zebrafish model. The results confirmed that both analysis tools were able to rapidly quantify a wide range of angiogenic images and generated comparable results to frequently used manual methods. The developed computational analysis tools are user friendly and can be used to assess the effects of inhibitor compounds and silencing vascular related genes.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The mycobacterial cell wall consists of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are adorned with a complex polysaccharide, arabinogalactan (AG), via a unique α-L-rhamnopyranose–(1→3)-α-D-GlcNAc-(1→P) linker unit. Whilst the cytoplasmic steps of mycobacterial cell wall biosynthesis have been largely delineated, the molecular processes that govern the flux of PG intermediates and the mechanism by which PG and AG pathways converge has remained elusive. We identified key conserved serine/threonine residues of MurC, as potential candidates for phospho-regulation by the cognate protein kinase, PknA. Pseudo-phosphorylated MurC mutants exhibited differential enzyme activity, suggesting that M. tuberculosis is capable of tight control of PG biosynthesis through phosphorylation of MurC. In addition, we have identified Lcp1, a mycobacterial orthologue of the LytR-CpsA-Psr (LCP) family of proteins found in Gram-positive bacteria, responsible for ligating cell wall teichoic acids to PG. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is a phosphotransferase capable of ligating AG to PG in a cell free radiolabelled assay.

The mixed-lineage leukaemia 1 protein is a histone methyl-transferase that deposits the gene activating H3K4 trimethyl mark, and is often mutated in leukaemia. MLL1 is normally associated with a cohort of cofactors, but the mechanisms regulating the histone methyl-transferase activity remain unclear. Here I examine the role of Msk1, a downstream kinase of the MAP-kinase pathway, in regulating MLL1 activity. Msk1 is known to deposit the H3S10 phosphorylation mark, which was found to stimulate MLL1’s methylation activity in vitro. Here I demonstrate that MLL1 and Msk1 can be immunoprecipitated and their patterns of genomic binding show an overlap at ~30 of sites, suggesting a direct functional interaction. In transient MLL1 and Msk1 knock-down cells, known MLL1 target genes were down-regulated and at a global level, 30% of all responding genes were regulated in the same manner. Furthermore, key histone modifications at MLL1 target genes change in Msk1 knock-down cells, suggesting that histone cross-talk within the MLL1 complex acts as a means of gene regulation. Finally, cell cycle studies suggest MLL1-Msk1 cross-talk may stimulate MLL1-driven gene expression after mitosis. These findings suggest that MLL1 is regulated by Msk1 and therefore by extracellular signals via the MAP-kinase pathway.

The study was aimed at applying molecular marker techniques to locate QTL and determine the efficiency of the marker-assisted selection. The research was done using Brassica oleracea and Arabidopsis thaliana. The Brassica DH lines represented a population of homozygous individuals while the F\(_2\) and F\(_3\) generations of Arabidopsis represented a segregating population. Marker-assisted selection was applied after the detection of QTL which allowed the identification of markers linked to the QTL and hence the selection for such markers. In Brassica, 40 QTL were detected using the marker regression method. Between 1 and 6 QTL were located per trait, which individually explained 2-49% of the additive genetic variance. In Arabidopsis the marker regression method detected 23 QTL in the F\(_2\), whereas 40 QTL were detected by the interval mapping method in the F\(_3\) generation. 1 7 QTL mapping to similar positions and showing similar modes of action were detected by both methods. Alleles for various QTL were dispersed between parents in both crosses. The efficiency of MAS was determined using various approaches, based on the number of top ranks, number of lines in a group, phenotypic value and as the ratio between response based on MAS and response obtained in the F\(_3\) by applying phenotypic selection to the F\(_2\) generation. The MAS gave generally better response compared to phenotypic selection, particularly when heritability was low. MAS for single QTL was always more effective while multiple QTL and QTL showing linkage posed some practical problems in MAS applications. Overall, MAS has to be applied in conjunction with phenotypic selection to get best results as QTL of minor effect cannot be tackled through marker/QTL associations.

Crop wild relatives have been identified as ecologically and economically important plant genetic resources but are often a neglected resource. The recognition of the need for their specific conservation and their value for future use has been strengthened by the Convention on Biological Diversity and the International Treaty on Plant Genetic Resources for Food and Agriculture, both of which have been ratified by the UK. This thesis provides a detailed view of the ecological, geographic and genetic background to three crop wild relative species, Trifolium dubium, T. pratense and T. repens, of which the latter two are amongst some of the most economically important legume species in the UK. Assessments of ecogeography, amplified fragment polymorphism and single nucleotide polymorphism markers were employed to investigate the distribution of variation in these species across the UK, including outlying island sites. Based on this information it was possible to look for isolation by distance in populations in UK; identify areas containing unique variation; assess the conservation importance of island sites surrounding the UK and speculate on the causes of the observed patterns of diversity. Conservation recommendations were based on the cumulative data from this research to identify how the recommendations change with an increased focus on genetic diversity. These results provide insights into the use of different types of background information when setting conservation plans in widespread species, contributing to the development of conservation strategies for widespread species in general.