High throughput sequencing can generate hundreds of millions of reads in a single day and is revolutionizing modern genetics. This project aimed to utilize next generation genetic approaches to analyze non-model but important agronomical plant species. A key feature of these species is their complexity. Mapping and SNP calling of these sequencing datasets is fundamental to many downstream analyses that have been implemented here including; mutant identification, comparative analyses between related organisms and epigenetic studies. The first objective in this project involved developing accelerated mutant identification techniques using mapping-by-sequencing analyses that combine whole genome sequencing with genetic mapping. Such methods have largely required a complete reference sequence and are typically implemented on a mapping population with a common mutant phenotype of interest. Here mutant identification was demonstrated on the model diploid plant Arabidopsis thaliana as a proof of principle of the methodology. It was also demonstrated on a simulated hexaploid mutant that was developed using the Arabidopsis reference genome. In species such as wheat, no finished genome reference sequence is available and, due to its large genome size (17 Gb), re-sequencing at sufficient depth of coverage is not practical. Therefore a genomic target enrichment approach was validated and used here to capture the gene rich regions of hexaploid bread wheat, reducing the sequencing cost while still allowing analysis of the majority of wheat’s genic sequence. A pseudo-chromosome based reference sequence was developed from this genic sequence with a long-range order of genes based on synteny of wheat with Brachypodium distachyon. Using the capture probe set for target enrichment followed by next generation sequencing; an early flowering locus was mapped in the diploid wheat Triticum monococcum and in hexaploid bread wheat Triticum aestivum, the stripe rust resistance gene was located. A bespoke pipeline and algorithm was developed for mutant loci identification and the pseudo-chromosome reference was implemented. This novel method will allow widespread application of sliding window mapping-by-sequencing analyses to datasets that are; enriched, lacking a finished reference genome or polyploid. The second main objective of this project involved a study of methylation patterns in wheat utilizing sodium bisulfite treatment, combined with target enrichment. An enrichment system was specifically designed, developed, validated and implemented here to perform one of the first studies of methylation patterns in hexaploid bread wheat across the 3 genomes that used a genome-wide subset of genes and can thus be used to infer genome-wide methylation patterns and observations. This investigation confirmed that differential methylation exists between the A, B and D genomes of wheat and that temperature is capable of altering methylation states.

Diseases caused by parasites are responsible for immense human and animal suffering, declines in biodiversity, and substantial economic losses across the globe. It is therefore important to understand how parasites spread through and persist within natural populations, so that control interventions that aim to reduce individual infection risk can be designed and implemented appropriately. Crucially, most parasites exist within multi-host communities, and often appear to infect multiple sympatric species, all of which potentially play a role in parasite persistence. However, certain species may contribute disproportionately to transmission and be nearly completely responsible for the persistence of a parasite within a community. Identifying such “key hosts” therefore offers a means to appropriately target control interventions to maximise success. However, assessing host species contributions to parasite transmission within multi-host communities is a challenging task, and much insight can be gained from studies of model host-parasite systems. In this thesis, the transmission dynamics responsible for the persistence of several endemic Bartonella parasites (bacterial flea-borne haemoparasites) are investigated within wild sympatric populations of wood mice (Apodemus sylvaticus) and bank voles (Myodes glareolus) in northwest England. Bartonella infections were first identified to species-level according to an existing method based on length polymorphism of a fragment of the 16S-23S internal transcribed spacer region (ITS). Broad patterns of prevalence suggested that some species were host generalists while others were host-exclusive, indicating that different transmission dynamics underlie the persistence of each Bartonella species within the rodent community. Attempts to identify key transmission hosts for each Bartonella species based on the effect of past host population densities on infection risk proved inconclusive. However, finer-scale characterisation of Bartonella infections, using DNA sequencing, found that Bartonella species that appear to be generalists actually comprise a complex of genetic variants, the majority of which are host-specific, suggesting that transmission between host species is uncommon and limited to a relatively few host-shared variants. Furthermore, detailed characterisation of the flea community infecting wood mice and bank voles found that these Bartonella vectors were host-generalists, and that at least two flea species were able to transfer between individuals of different host species. This suggests that a lack of between-species transmission is likely to arise through different compatibility between host species and Bartonella variants, rather than as a result of current ecological encounter barriers (e.g. through differential Bartonella-flea or flea-rodent specificity). The results of an experimental manipulation of between-species transmission within these wild communities support the notion that between-species transmission of Bartonella parasites is uncommon. Across three woodland sites, bank voles were treated with a veterinary insecticide to remove their fleas and therefore reduce the rate of transmission of Bartonella from treated bank voles to the rest of the rodent community. Following treatment, risk of bank vole infection with bank vole-exclusive Bartonella variants was reduced, but there was no affect on the risk of bank vole infection with host-shared variants, nor risk of infection in wood mice with either wood mouse-exclusive or host-shared variants. Importantly, these treatment effects were best identified by grouping the parasites on a ‘functional’ (i.e. host-exclusive versus host-shared variants) rather than a taxonomic (i.e. Bartonella species) basis. Together, these findings highlight the importance of characterising parasite infections to as fine a scale as possible, and the value of using a combination of observation, genetic and experimental approaches to understand parasite transmission within complex natural multi-host systems.

Malaria is a major public heath disease with over 3.4 billion people at risk globally. High coverage of pyrethroid-treated long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS) have played a key role in reducing transmission over the last decade. Unfortunately, resistance to pyrethroids is now widespread and increasingly being reported to the few other WHO-approved alternative insecticides. The problem might be critical in Côte d’Ivoire, especially in the southern rice-growing area of Tiassalé where mosquitoes have been found to be resistant to pyrethroids and DDT. In this thesis, I aimed to investigate the profile of resistance to WHO-approved insecticide classes in Anopheles gambiae from Côte d’Ivoire, with a particular emphasis on Tiassalé, where I conducted in-depth investigation resistance characterisation and investigation of the genetic basis of extreme and multiple insecticide resistance. I first demonstrated the presence of resistance to all four WHO-approved classes of insecticide in wild population of in An. coluzzii from Tiassalé. This was the first demonstration of such unprecedented multiple insecticide resistance, representing a real concern for implementation of control measures based on current insecticide classes. Target site mutations in the voltage-gated sodium channel were significantly associated with DDT, but not pyrethroids, yet a meta-analysis of published and unpublished data spanning twenty years of testing in Côte d’Ivoire suggested that significant increases in DDT and pyrethroid resistance have occurred, more strongly in the South, and are likely linked to increase in the kdr 1014F mutation in A. coluzzii. Nevertheless contemporary data suggest that overexpression of metabolic genes might be more important in pyrethroid resistance; a speculation supported by significant PBO-enhancement of Tiassalé A. coluzzii mortality to pyrethroids and other insecticides tested, suggested primary importance of P450s detoxification enzymes. In addition, using dose-response assays, females were found to exhibit an extreme level of bendiocarb resistance, with some surviving even at 8h exposure. Whole genome microarrays were used to investigate the genes potentially responsible for this extreme resistance in a stringent, multiply-replicated design, detecting overexpression of several CYP6 P450s and the ACE-1 target site genes as resistance linked. The latter association arises via duplication of ACE-1 119S resistant alleles, providing the first direct evidence in Anopheles for a link between target site duplication and insecticide resistance. Synthesis of the results from several experiments suggests that the ACE-1 G119S substitution is the primary determinant of variation in survival at 60 minutes (WHO standard) exposure to bendiocarb, whereas overexpression of ACE-1 is the primary determinant of survival at an exposure duration corresponding to the population LT50. However, at an LT80 level elevated expression of both ACE-1 (resistant alleles) and CYP6 P450s enable survival. Interestingly, this work also highlighted how specific mosquito genes such as CYP6M2 and CYP6P3 were able to contribute to resistance across insecticide classes with contrasting modes of action, providing a key novel insight into how metabolic mechanisms can lead to cross-resistance in mosquitoes. Unfortunately, results from wider testing and meta-analysis suggest that multiple resistance may be present across Southern Côte d’Ivoire. The results presented in this thesis have shed new light on the extent of multiple and cross-resistance in Anopheles and the underlying mechanisms and should help national malaria control programmes, health departments and decision-making stakeholders to better plan the resistance surveillance programmes in order to combat multiple insecticide resistant vectors in African countries.

The apicomplexans are causative agents of human and animal infections including malaria, toxoplasmosis and theileriosis and have a huge economic and social impact. A number of apicomplexan genomes have been sequenced. However, the annotation of gene functions remains challenging. A semi-automatic approach was used to systematically assign genes to their functions within pathways/networks through the integration of genomic information with biochemical evidence from the literature. This method has resulted in the evidence-based annotation of metabolic functions and the development of organism specific metabolic pathways. A web database named Library of Apicomplexan Metabolic Pathways (LAMP, www.llamp.net) was developed to host the metabolic mappings for Toxoplasma gondii, Neospora caninum, Cryptosporidium and Theileria species and Babesia bovis at present. A comparative analysis of the overall metabolic capabilities of apicomplexan species showed that the metabolic adaptations has evolved for different ecological niches and led to the identification of putative drug targets. The identification of missing enzymes that are essential to complete the metabolic pathways and the identification of a subsection of these missing enzymes from raw genomes demonstrated probable inaccuracies in gene model predictions. The utilisation of T. gondii and N. caninum proteomic datasets and their mapping to alternative gene models showed regions of genes that require further refinement. The evaluation of the quality of two different gene model releases with this peptide evidence showed the importance of integration of RNA-Seq datasets in improving gene models and further improvements that can be made with proteomic datasets. A global post-translational modification (PTM) analysis was carried out for T. gondii and N. caninum via the utilisation of non-enriched proteomic datasets available for these species. This analysis identified proteins of functional importance that have undergone PTMs, particularly methylation, acetylation, phosphorylation and oxidative modifications. All these analyses helped in the improvement of gene models and functional annotation of genes from Apicomplexa genomes.

The transcription factors STAT3 and NF-κB play key roles in inflammation, immunity and cell fate. In the liver, they are responsible for transcribing hundreds of genes in response to combinations of IL-6, TNFα and IL-1β, and so together co-ordinate the acute phase response to infection. Dysregulated STAT3 and NF-κB signalling leads to chronic inflammation and is implicated in the development of many cancers. A variety of highly context-dependent intercellular and intracellular mechanisms have been discovered which facilitate both positive and negative cross-talk between STAT3 and NF-κB. Whilst the long-term signalling dynamics of NF-κB have been characterised in single cells, and were found to be oscillatory, imaging studies on STAT3 have focused upon the short-term mechanisms of nuclear transport rather than the long-term dynamics. STAT3 has been shown to oscillate in a population of synchronised cells so it is possible that STAT3 will exhibit oscillatory spatio-temporal signalling dynamics in response to cytokine stimulation. The primary aim of this thesis was to characterise the long-term signalling dynamics of STAT3 in response to IL-6, using fluorescent fusion protein reporters for STAT3 and its inhibitor SOCS3, in conjunction with live single cell fluorescence microscopy. Towards these aims, STAT3 and SOCS3 fluorescent fusion proteins were constructed. The responses of a candidate cell line to IL-6 and TNFα were investigated, and then the fluorescent reporters were characterised in that cell line. The N-terminal tagged EGFP-STAT3 reporter was found to be the most accurate reporter of IL-6 signalling. The EGFP-STAT3 was then used to investigate the single cell spatio-temporal dynamics of STAT3 in response to differently timed lengths of IL-6 stimulation. STAT3 was found to oscillate with a period of approximately 90 min in response to continuous IL-6 stimulation, but only underwent a transient nuclear translocation in response to a 30 min IL-6 pulse. Furthermore, the patterns of gene expression were characterised for the timed IL-6 treatments. The quantified single cell dynamics were used to constrain an existing generic model of STAT:SOCS signalling; the model was able to capture the observed single cell dynamics using a minimal ordinary differential equation approach. The secondary aim of the thesis was to study cross-talk between STAT3 and NF-κB using live cell microscopy techniques. The effects of co-stimulation of NF-κB and STAT3 were investigated using combinations of TNFα and IL-6 stimuli. Combinations of single or dual transfections, and single or dual stimulation were performed as controls in order to tease apart the effects of co-expression and co-stimulation. The importance of the timing of cytokine stimulation was also investigated. Finally, the effects of IL-1β upon IL-6 induction of STAT3 were investigated, as this was shown elsewhere to inhibit STAT3 signalling and so was expected to produce interesting spatio-temporal signalling effects. This preliminary study revealed distinct subpopulations of cells with different p65 and STAT3 response patterns. The STAT3 response was knocked down or significantly delayed in many cells but a small subset exhibited atypical oscillatory dynamics. Interestingly, the p65 dynamics were also significantly perturbed by IL-6 and IL-1β co-stimulation, indicating that there are cross-talk events occurring in both directions. Consequently these studies represent a very important area for future investigation.

I conducted a series of studies which looked at influences on avian clutch size. Firstly I examined the traditional view that the demands of rearing chicks create a bottleneck at which clutch size is shaped by natural selection. I considered whether instead other stages such as incubation might also be important. I proposed that reproductive demands at each stage of the breeding season may be interdependent, and by developing a mathematical model, I formalised the argument and showed that data on the relationship between the number of offspring and the expenditure of resources at many stages of the season could reveal the importance of natural selection on clutch size at each stage. I then reviewed the literature on the importance of incubation for clutch size determination. Results indicated that metabolic demands of incubation were appreciable and that the incubation of enlarged clutches imposed penalties on the adults. In a field study of kittiwakes I found that breeding success was depressed during incubation and chick rearing by enlargement of clutches and broods respectively. I measured metabolic rates of kittiwakes during incubation and found them to be comparable with those during chick-rearing. Secondly, I examined whether individual adults within populations differed in their reproductive capacities (i.e. whether there was a range of 'adult quality') and whether this could then affect clutch size. In a study of kittiwakes I found clusters of birds with similar breeding performance, but found that these clusters did not persist between years. In a study of swifts, I found that some individuals were consistently good breeders but that this had negligible effects on the distribution of lifetime reproductive success between individuals. I then examined whether the low clutch sizes and high survival of swifts might reflect a bet-hedging strategy in a fluctuating environment, but found little evidence of this. I looked at whether differences in the amount of space available at the nest site could account for differences in clutch sizes of kittiwakes, but could find no such evidence. Lastly I developed a theoretical model to look at how clutch size might be affected by changes in reproductive effort with age. I examined whether the predictions of optimality models were borne out by the more appropriate population genetics approach and found that in birds the optimality models are robust.

The technique of continuous culture has been used to study the segregatlonal stability of plasmids in Escherichia coli in the absence of selective pressure. Conditions were established that allowed the detection of plasmid-free cells no matter how low their Initial frequency. Using these conditions the segregational stability of two related multicopy plasmids (pDSII09 and pBR322) was examined. pDSII09 was found to be stably Inherited throughout 120 generations of nutrient limited growth despite the observation that the plasmid copy number fell 4 to 5 fold during the culture period. By contrast, pBR322 was lost from chemostat culture after a lag of between 30 and 40 generations, a period during which (by analogy with pDSII09) its copy number had fallen about 2 fold. The functional basis of the differential segregation exhibited by these plasmids was ascribed to the presence (on pDSII09) or absence (on pBR322) of a functional par (partition) signal that ensured the efficient segregation of plasmid molecules into daughter cells at division. Based on this hypothesis, experiments were done to examine the possibility for correction of the defective partitioning of pBR322 by complementation in cis and in trans. In only two cases (both in cis) was complementation achieved. The first using a previously characterised par function from plasmid pSCIOI and the second using a fragment of plasmid pDSII09 that was considered (by argument) to be the region involved in its observed segregational stability. Supportive evidence for the existence of partition elements amongst multicopy plasmids is cited, as is confirmatory work since published by other workers. A possible mechanism of par action is discussed. Chemostat culture has also been used to examine the possibility of plasmid transfer by transformation within the chemostat. This study examined the effects of both growth rate and nutrient limitation on the transformability of Escherichia coli grown in continuous culture. The results obtained are discussed in relation to previously published transformation work using batch grown cells and a possible mechanism of plasmid transformation is suggested.

Liquid extraction surface analysis (LESA) is an ambient ionisation technique which allows direct analysis of surfaces coupled with mass spectrometry. LESA mass spectrometry has been used successfully to analyse small molecules, but there are a limited number of examples where the approach has been applied to protein analysis. The work presented here aims to develop novel applications of LESA mass spectrometry of proteins. LESA mass spectrometry was used to analyse intact proteins from polymeric membranes. The rationale for these experiments was the potential application to analyse proteins electroblotted following polyacrylamide gel electrophoresis, i.e. top-down proteomics, and in air monitoring. The subsequent focus was dried blood spot (DBS) analysis. An automated LESA based trypsin digestion protocol was developed and coupled with liquid chromatography tandem mass spectrometry to enable DBS proteomics. i.e., untargeted global protein identification via a bottom-up approach. Approaches for DBS proteomics (in the absence of LESA) were explored further using conventional digestion procedures coupled with protein depletion. LESA was also applied for targeted analysis of proteins from DBS, to determine variants of alpha-1-antitrypsin. Finally, native LESA mass spectrometry was developed to analyse non-covalent complexes from dried surfaces. Native LESA mass spectrometry successfully identified the haemoglobin tetramer directly from DBS.

The chromatin provides a structural organization in which the DNA can be compacted up to 10,000-20,000 fold. Nevertheless, this compaction achieved by the chromatin structure has to be highly dynamic and controlled in order to allow the different vital processes of the DNA to occur such as transcription, replication, DNA repair, chromosome segregation and recombination (mitosis and meiosis). Nucleosomes are the basic unit of chromatin compaction that are positioned throughout the vast genomic DNA in higher eukaryotes. A nucleosome consists of a pair of each histone protein H2A, H2B, H3 and H4 and the associated 147 base pairs (bp) of DNA. They are important contributors to overall chromatin organization. Structure Specific Recognition Protein 1 (SSRP1) is an HMG protein that has been investigated in Human. We have also carried out a Small interfering RNA (siRNA) strategy to reduce the expression of hSSRP1 in endothelial cells. The knocked down cells showed a clear reduction of beta-tubulin microtubules in the mitotic spindle and errors in their organization that led to a poor alignment of the chromosomes and missegregation. Furthermore, DNA repair and cytokinesis were also affected in the siRNA knockdowns. Immunolocalization of hSSRP1 and hSPT16 have shown that both could be involved in DNA repair when localising to the chromatin forming the FACT complex but also they could be deeply involved in spindle formation and organization in higher eukaryotes. Especially, since hSSRP1 localises in the centrioles.

This thesis reports the Structure Activity Relationship study that was performed upon the 5-substituted-indole core as a means to identify Negative Allosteric Modulators of the human 5-HT3A receptor for the development of potential drugs for the treatment of IBS-d. Herein is reported the successful identification of a PAM to NAM switch and three novel NAMs which provide the basis for further study into the treatment of IBS-d and insight into the identity of the allosteric site of the human 5-HT3A receptor. The design, synthesis and testing of a novel fluorescent analogue of the orthosteric antagonist Quipazine is also described for the application of an improved competitive binding experiment without the need for radio-labelled ligands. Furthermore, the design and synthesis of novel diazirinyl-indoles for photo-affinity binding studies towards the identification of the allosteric site of the 5-HT3A receptor. Finally, the design and synthesis of novel BODIPY-BAPTA based fluorescent PET sensors for the detection of larger than usual ranges in concentration of cellular Ca2+ levels are described.