Homologous recombination is an integral part of meiosis and is essential for generating crossovers that ensure balanced segregation of homologous chromosomes and establish genetic variation within offspring. It is therefore exceedingly important that meiotic cells employ stringent control mechanisms to safeguard crossover formation. Work in yeast has indicated that the meiotic axis, a proteinaceous structure that tethers meiotic chromosomes into looped arrays, plays a crucial role in many aspects of homologous recombination, from double strand break formation to crossover interference. It has also been suggested that increased crossover interference helps to establish meiotic stability by inhibiting multivalent formation during autopolyploid meiosis. Using immunocytochemistry coupled with super-resolution microscopy, we have further investigated the role played by the meiotic axis protein ASY1 in stabilising meiosis in the established autotetraploid Arabidopsis arenosa. We have also used Arabidopsis arenosa as a model for studying how meiotic interference might operate within an autopolyploid context. Alongside this, experiments using transgenic lines of the model plant Arabidopsis thaliana have helped to shed light on how crossover formation and synapsis are affected by reduced expression of ASY1 and ASY3 and to determine what effect limiting meiotic crossover numbers might have on neopolyploid meiotic stabilisation.

GCN4 is a yeast transcriptional factor, which binds sequence selectively to DNA as a homodimer. Two domains are required to achieve this, an N-terminal basic domain, which interacts with the target site directly, and the C-terminal leucine zipper responsible for dimerisation. The latter can be replaced by chemically linking together two basic domains, to retain similar DNA binding. This work investigates using a ferrocene complex as the dimerization unit due to its flexibility and its characteristic electrochemical redox properties. A strategy was proposed which took advantage of coupling to the thiol side chains of two cysteine residues, and detailed studies on dimerising two equivalents of Cys, or the tripeptide glutathione, were performed. Design of five peptides based on GCN4, which were synthesized and dimerized with a ferrocene linker molecule, to combine the attractive redox electrochemistry of the stable ferrocene and the DNA binding ability of GCN4 dimerized basic domain. Molecular dynamic models were produced for four of the ferrocene peptide dimer complexes bound to target DNA over 10 ns. Their DNA binding ability was investigated by circular dichroism, and electrochemistry was studied in the absence and presence of non-specific and target DNA respectively.

QTL mapping with segregating populations results in poor map resolution which limits the applicability of mapped QTL in further research such as gene cloning. The current research project aimed mainly at developing STepped Aligned Recombinant Inbred Strains (STAIRS) covering the top region of chromosome 3 and demonstrating the feasibility of using STAIRS in high resolution mapping of QTL in Arabidopsis. The top region of chromosome 3 of Arabidopsis had been reported to house QTL related to flowering time. This region was first saturated with 24 polymorphic microsatellite markers and 23 narrow STAIRS were produced within the region via a marker-assisted backcross breeding programme using whole chromosome substitution lines. The analysis of QTL with the narrow STAIRS revealed a major pleiotropic QTL within 2-3 cM affecting flowering time, leaf number at day 20 and rosette and cauline leaf numbers at flowering. A second QTL with less but opposite effect on the same traits were located within 15-20 cM. The search for candidate genes within 2-3 cM of chromosome 3, to locate possible candidate genes revealed COL-2, CONSTANS-Like gene which affects flowering time. Microarray gene expression profiling was performed using the two genotypically closest lines which differ for flowering time to compare the two lines at the same chronological and physiological ages in two experiments respectively. The lists of differentially expressed genes were obtained from the two experiments. Differential expression was observed for the possible candidate gene in the latter experiment. The results emphasized the power of STAIRS in fine mapping of QTL and the possibility of using them in transcriptional profiling to study the expression of genes.

This thesis is concerned with speciation and population genetics in the plant genus Silene (section Elisanthe). The introductory chapter is a literature review covering characteristics of the species studied, and the current literature on their evolutionary dynamics and population genetics. The second and third chapters cover techniques used in all experiments, such as DNA extraction, sequencing and genotyping protocols, and explain the rationale behind the initial experimental design. The fourth chapter focuses on the multi-locus analysis of autosomal gene sequences from S. latifolia and S. dioica. The relationship between the two species was investigated using various analyses such as isolation modeling and admixture analysis providing estimates of evolutionary distance and extent of historical gene flow. The maintenance of the species despite frequent hybridization at present-day hybrid zones is discussed. The fifth chapter discusses S. diclinis, a rare endemic found only in Valencia, Spain. The nature of population structuring and the evolutionary history of this species were investigated using a multilocus approach incorporating individuals from S. diclinis populations. The causes of the restricted distribution and low population size of this species is discussed The concluding chapter discusses how the species evolved from a common ancestor amidst changing climatic and environmental conditions.

Since the human genome project was completed in 2003, extraordinary progress has been made in the field of genomics with the development of new sequencing technologies and the widespread introduction of next generation sequencing (NGS). The application of NGS initiated a new era in genomics by massively increasing the number and diversity of the sequenced genomes at lower cost. Human Molecular Genetics has greatly benefited from the use of NGS-based strategies to identify human disease genes. In this thesis, I investigated the application of genetic techniques to investigate the molecular basis of autosomal recessively inherited disorders of unknown etiology. A range of disease phenotypes, including oligodontia and fetal akinesia/multiple pterygium syndrome (FA/MPS), were investigated in patient cohorts that included many cases with parental consanguinity. Using an autozygosity linkage analysis-based approach and Sanger sequencing of candidate genes resulted in the identification germline RYR1 mutations in FA/MPS. Subsequently, using exome sequencing techniques, the molecular basis of FA/MPS was further elucidated by the identification of germline mutations in RYR1, NEB, CHRNG, CHRNA1 and TPM2. The application of NGS in genetically heterogeneous disorders such as fetal akinesia/multiple pterygium syndrome can enable better and less expensive molecular diagnostic services aimed at specific mutation spectra, though more extensive sequencing can lead to the identification of larger numbers of variants of uncertain significance.

Work described in this thesis shows the design, synthesis, DNA binding activity and gold nanoparticle interaction of functionalised triple stranded supramolecular helicates. DNA structures and the way molecules recognise and bind to them are reviewed, with specific emphasis on supramolecular compounds. Supramolecular helicates are discussed in detail with consideration of how they may be used as anticancer agents. Nanoparticles, specifically gold nanoparticles are studied, with specific reference to how they are able to enhance anticancer properties of drug molecules and how they may be used to develop potent anticancer therapies. With the formation of three novel supramolecular iron helicates and the incorporation of supramolecular chemistry and nanotechnology, through binding such complexes to the surface of gold nanoparticles, foundations of work in this area are discussed, showing promising results for future research.

Single nucleotide polymorphisms (SNPs) are single base variations in DNA which give genetic variation. However, SNPs can also be linked to the development of certain diseases. Modified oligonucleotides used to probe biological changes and processes have become an important focus of scientific research. Fluorescent tagging of DNA can be used to sense SNPs in DNA targets through differences in emission intensity on the formation of a duplex. An anthracene-tagged DNA probe developed by Tucker \(et\) \(al\). is able to discriminate between a fully complementary DNA target sequence and one with a single base difference. This thesis describes how SNP sensing with anthracene-tagged DNA has been extended to SNPs in RNA targets and sequences associated with Alzheimer's disease. Finally, a new dual fluorophore DNA probe was designed for SNP sensing via FRET.

Drug toxicities represent a major problem in drug discovery and development; therefore there is a push to develop new technologies to detect these early on. In this thesis I investigated the utility of zebrafish embryos and larvae in evaluating the biological activity of novel compounds and developed new methods for assaying the potential toxic effects of drugs in vivo. An electrocardiogram (ECG) recording set-up for zebrafish embryos and larvae was developed to assay drug-induced cardiotoxicity. The set-up was validated by testing drugs known to induce cardiotoxicity in humans in zebrafish larvae. The results obtained were in agreement with those documented in humans demonstrating the utility of the zebrafish larva in detecting drug-induced cardiotoxicity. The zebrafish embryo was also found to be a useful model for probing the biological activity of novel and marketed compounds providing an insight into the relationship between chemical properties and biological effects. Additionally, the assessment of the anti-inflammatory activity of a set of reference drugs revealed that the zebrafish larva also presents a promising model for therapeutic drug screens. Overall, the results described in this thesis show that the zebrafish presents an effective, reliable and rapid model for assessing the biological activity of drugs in vivo.

This thesis investigated reconstitution of CMV-specific T-cells in two cohorts of HSCT patients and studied the potential role of Tumour Necrosis Factor Receptor 2 (TNFR2) in regulation of CMV-specific T-cell expansion post HSCT. The first cohort included patients of a randomized phase II trial of adoptive cellular therapy for CMV-specific CD8\(^+\) T-cells. Cellular therapy resulted in earlier and greater expansion of CMV-specific CD8\(^+\) T cells and also reconstitution of CMV-specific CD4\(^+\) and non-infused CMV-specific CD8\(^+\) T-cells. The number of infused therapeutic T-cells and circulating levels of Alemtuzumab were found to influence immunotherapy. Additionally, reconstitution of CMV-specific CD4\(^+\) T-cells was studied using HLA-class II tetramers. CMV-specific CD4\(^+\) T-cell count of >0.7x10\(^3\)/ml was found to protect from recurrent CMV reactivation. One third of specific CD4\(^+\) T-cells were perforin and granzyme-B positive indicating cytotoxic potential, whilst the majority expressed T-bet. Expression of CD57 molecule on CD4\(^+\) T-cells was demonstrated as a potential biomarker of immune response to CMV. Also, distinct cytokine receptor expression patterns in naïve versus memory T-cells were observed. The results showed rapid decrease in IL-6R and increase in expression of TNFR2 after T-cell differentiation from naïve to effector cells and engagement of TNFR2 led to the apoptosis of CMV-specific T-cells.

Self-incompatibility (SI) is the major mechanism used by flowering plants to prevent self-pollination and thus avoid inbreeding. It is a genetically controlled mechanism that encodes a highly specific recognition system that inhibits the growth of incompatible pollen, whereas compatible pollen from an unrelated plant of the same species is able to grow and effect fertilization. In Papaver rhoeas, inhibition of incompatible pollen is mediated by a signal transduction pathway that activates a complex network of intracellular events including Ca\(^{2+}\)-dependent phosphorylation of p26, an inorganic pyrophosphatase required for pollen tube growth, depolymerisation of the pollen actin cytoskeleton and ultimately programmed cell-death (PCD). To further understand the mechanisms involved in Papaver rhoeas SI it is important to identify and characterize components of the signalling network that mediate the SI response. Recent studies have revealed that p56, a mitogen-activated protein kinase (MAPK), is involved in activation of PCD during SI. MAPKs have been shown to be important signalling components in a range of cellular responses in eukaryotes. In Arabidopsis thaliana there are 20 MAPKs with roles including cell division, abiotic stress response, wounding response and hormone signalling. Preliminary studies suggested an MPK3-like gene might encode p56, as an anti-AtMPK3 antibody cross-reacts with a protein corresponding to p56. Experiments carried out here suggest this is not the case. Using a combination of bioinformatics and proteomics data it has been possible to identify and clone a candidate p56 gene from Papaver rhoeas pollen. The putative p56 gene has homology to AtMPK9 and is a member of the T-D-Y class of MAPKs. It has been designated PrMPK9-1. An antibody raised against recombinant PrMAPK9-1 protein is being used to confirm that this gene/protein does indeed correspond to p56.