A protocol for the analysis of preferential gene expression was devised and validated. Analysis revealed possible sequence upregulation of RNA polymerase beta subunits, pyruvate formate lyase activating enzyme and a deoxyglucose epimerase enzyme at pH 7.0. The colonisation patterns in all sites were shown to be complex with many different clonotypes of a given species present. Interestingly, over time the colonisation patterns were not stable with significant changes in the species representation. The Streptococcus anginosus group has been show to display a significant amount of genomic diversity during in-vitro culture.

This thesis provides a detailed study of the diet of various procellariiformes using new molecular approaches. Dietary studies remove fundamental blocks to our understanding of the structure of food webs, and provide insights into the demographic regulation of populations and the structuring of communities. The study species were the Band-rumped Storm-petrel (Hydrobates castro), Bulwer’s petrel (Bulweria bulwerii), Cory’s shearwater (Calonectris borealis) and White-faced Storm-petrel (Pelagodroma marina). The breeding colonies of the Madeiran-archipelago are Important Bird Areas (IBA) in the North-Atlantic, but little is known about the predator-prey relationships of its seabird populations. This probably relates to difficulties associated with obtaining robust prey estimates and the need to develop new methodologies to improving the resolution of species identification. Here, new molecular approaches were developed to recover prey from faeces and stomach contents using DNA-barcoding and high-throughput sequencing (HTS). The results obtained show clear improvements to the identification of the diets of procellariiformes, considerably outperforming morphological analysis, and retrieving prey identities from non-invasive faecal remains. Such approaches further showed that sympatric small seabirds of the sub-tropical NE-Atlantic significantly segregated their resources, while showing similar prey types with the species distributed in the Pacific, indicating that these petrels maintain foraging specialization across their distribution range. Foraging efficiency in seabirds has been widely hypothesized to change according to the moon cycle. Predators either optimise foraging during moonlit nights or reduce foraging effort because less accessible prey migrate downward the water column to avoid visual predators. I tested whether prey composition and diversity differ between moon-phases. However, I found no evidence for a significant influence of the moon on the diet of Bulwer’s petrel, contradicting previous ecological assumptions. The results highlight the potential of DNA methodologies to the understanding of marine food webs and predator-prey relationships and will certainly make important contributions to marine community ecology.

The WASP family of proteins are nucleation-promoting factors that dictate the temporal and spatial dynamics of Arp2/3 complex recruitment, and hence actin polymerisation. Consequently, members of the WASP family, such as SCAR/WAVE and WASP, drive processes such as pseudopod formation and clathrin-mediated endocytosis, respectively. However, the nature of functional specificity or overlap of WASP family members is controversial and also appears to be contextual. For example, some WASP family members appear capable of assuming each other’s roles in cells that are mutant for certain family members. How the activity of each WASP family member is normally limited to promoting the formation of a specific subset of actin-based structures and how they are able to escape these constraints in order to substitute for one another, remain unanswered questions. Furthermore, how the WASP family members collectively contribute to complex processes such as cell migration is yet to be addressed. To examine these concepts in an experimentally and genetically tractable system we have used the single celled amoeba Dictyostelium discoideum. The regulation of SCAR via its regulatory complex was investigated by dissecting the Abi subunit. Abi was found to be essential for complex stability but not for its recruitment to the cell cortex or its role in pseudopod formation. The roles of WASP A were examined by generating a wasA null strain. Our results contradicted previous findings suggesting that WASP A was essential for pseudopod formation and instead demonstrated that WASP A was required for clathrin-mediated endocytosis. Unexpectedly, WASP A – driven clathrin-mediated endocytosis was found to be necessary for efficient uropod retraction during cell migration and furrowing during cytokinesis. Finally, we created a double scrA/wasA mutant, and found that it was unable to generate pseudopodia. Therefore, we were able to confirm that SCAR is the predominant driver of pseudopod formation in wild-type Dictyostelium cells, and that only WASP A can assume its role in the scrA null. Surprisingly, the double mutant was also deficient in bleb formation, showing that these proteins are also necessary for this alternative, Arp2/3 complex-independent mode of motility. This implies that there exists interplay between the different types of actin-based protrusions and the molecular pathways that underlie their formation.

In gene regulation, mRNA polyadenylation, deadenylation and decay are closely linked processes whose full biological importance is only starting to emerge. Using thiouridine labelling as a method of capturing newly synthesised mRNAs, we have been able to measure poly(A) tail sizes of newly synthesised individual and total mRNAs in mouse NIH3T3 cells and global changes in mRNA turnover during the loss of pluripotency in human embryonic stem cells. These two systems, although very different, allow us to study changes in individual mRNAs during inducible gene expression. In order to track changes of poly(A) tail length during rapid gene induction we have used the serum response as a model in NIH 3T3 cells. Using poly(A) fractionation technique (Meijer et al, 2007), we have been able to study global changes of the poly(A) tail length during the serum response and have observed striking increases in rapidly induced mRNAs compared to control levels. Functional analysis reveals that these mRNAs are involved in transcriptional regulation, indicating that they are involved in reprogramming gene expression. Further analysis of the poly(A) fractionation microarray data, also indicates that certain mRNAs, such as Akt2, change in poly(A) tail length but are either down regulated of have no change in abundance. This group of genes areis enriched in regulators of signal transduction. When treated with Actinomycin D, some of these mRNAs still retain the increased poly(A) tail length during the serum response. This suggests a role for cytoplasmic polyadenylation, as inhibiting transcription has no effect on the elongation of the poly(A) tail. We also established by Western blotting and immunohistochemistry, that despite reduced mRNA levels, protein levels of Akt2 increase during the serum response. We conducted microarray analysis to determine whether miRNA regulation contributes to the changes we observed in poly(A) tails during the serum response. However, no major changes in abundance are found after 30 minute treatment, indicating that all changes in microRNA abundance are slow and unlikely to play a role in this system. As a second system, we studied the role of RNA binding proteins and mRNA stability in the early neuronal differentiation of human pluripotent stem cells, when pluripotency is lost. The role of mRNA processing and stability on the maintenance and establishment of pluripotency is poorly studied, despite the fact that these processes are important in germline development and early embryogenesis. We therefore studied the differential expression of RNA binding proteins involved in the regulation of polyadenylation and mRNA stability during early differentiation of human embryonic stem cells into neuronal precursors. Over an 8 day time period we have found large changes in mRNAs belonging to the CELF, CPEB, PUM and MSI families, which start early after the indication of differentiation, when pluripotency is lost. Using thiouridine labelling and microarray analysis on differentiating and pluripotent cells we were able to identify a group of mRNAs which appear to be destabilised during differentiation. This group includes known pluripotency factors such as OCT4 and LIN28B. Functional analysis showed that many ribosomal protein mRNAs also are within this group and also genes involved in the regulation of translation elongation, further exposing an important role for post-transcriptional regulation in the maintenance and loss of pluripotency. Our data indicate that the initial poly(A) tail size and mRNA turnover rate can vary greatly depending on both the individual mRNA and the state of the cell. Overall, our investigations give intriguing glimpses in the role of poly(A) tail metabolism and mRNA stability in two different models of inducible gene expression.

Mesenchymal stem cells (MSCs) are a multipotent cell population which have been described to exert renoprotective and regenerative effects in experimental models of kidney injury. In addition, it was recently shown that human MSCs are able to contribute to the development of both renal tubules and glomeruli. These results suggest that MSCs might be potential candidates for stem cell-based de novo renal tissue generation. The current study was aimed at re-evaluating the renogenic capacity of mouse and human bone marrow-derived MSCs. In order to elucidate the renogenic potential of MSCs, a novel method of embryonic kidney culture was used that is based on disaggregation of mouse kidney rudiments and their subsequent re-aggregation in the presence of cells from different origins to form kidney chimeras. Initially, MSCs did show expression of some genes involved in renal development; however, neither mouse nor human cells expressed important renal development genes, such as Wt1 and Pax2. Accordingly, MSCs were demonstrated to have low renogenic potential in the chimeric kidney model as they did not engraft into ureteric buds, the precursors of collecting duct system, and were only occasionally found in the condensing metanephric mesenchyme, which gives rise to nephrons. In addition, the incorporation of MSCs into embryonic kidneys had some detrimental effect on metanephric development. This effect was mediated through a paracrine action of the cells, as conditioned medium derived from mouse MSCs was demonstrate to reduce ureteric bud branching in in vitro kidney rudiment culture. On the contrary, mouse neonatal kidney cells did engraft into the condensing mesenchyme of chimeric kidneys and were subsequently found in some developing nephron-like structures. Regarding the potential of mouse embryonic stem cells to contribute to renal development in the re-aggregated kidney chimeras, the cells were found to some extent in both the condensing mesenchyme and the laminin-positive tubular compartment of chimeric kidneys, possibly the ureteric buds. No negative effect on kidney development was observed using the neonatal kidney cells as well as the embryonic stem cells. Ultimately it has been shown that the pre-conditioning of mouse MSCs with medium derived from mouse neonatal kidney cells facilitated the engraftment of MSCs into condensing mesenchyme of chimeric kidneys. It also prevented the negative action of MSCs on kidney development confirmed in the in vitro kidney rudiment culture. MSCs were demonstrated to up-regulate GDNF expression upon the pre-conditioning which is important factor for outgrowth and branching of ureteric buds. In conclusion, although pre-conditioning of the MSCs with medium derived from kidney cells was able to improve considerably the renogenic potential of the cells in the chimeric kidney, MSCs demonstrate a relatively low renogenic potential and for this reason are not good candidates for regenerative approaches aimed at recapitulation of nephrogenesis.

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.

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.

In this thesis, we develop some novel methods for univariate and multivariate analyses of additive genetic factors including heritability and genetic correlation. For the univariate heritability analysis, we present 3 newly proposed estimation methods—Frequentist ReML, LR-SD and LR-SD ReML. The comparison of these novel and those currently available approaches demonstrates the non-iterative LRSD method is extremely fast and free of any convergence issues. The properties of this LR-SD method motivate the use of the non-parametric permutation and bootstrapping inference approaches. The permutation framework also allows the utilization of spatial statistics, which we find increases the statistical sensitivity of the test. For the bivariate genetic analysis, we generalize the univariate LR-SD method to the bivariate case, where the integration of univariate and bivariate LR-SD provides a new estimation method for genetic correlation. Although simulation studies show that our measure of genetic correlation is not ideal, we propose a closely related test statistic based on the ERV, which we show to be a valid hypothesis test for zero genetic correlation. The rapid implementation of this ERV estimator makes it feasible to use with permutation as well. Finally, we consider a method for high-dimensional multivariate genetic analysis based on pair-wise correlations of different subject pairs. While traditional genetic analysis models the correlation over subjects to produce an estimate of heritability, this approach estimates correlation over a (high-dimensional) phenotype for pairs of subjects, and then estimates heritability based on the difference in MZ-pair and DZ-pair correlations. A significant two-sample t-test comparing MZ and DZ correlations implies the existence of heritable elements. The resulting summary measure of aggregate heritability, defined as twice the difference of MZ and DZ mean correlations, can be treated as a quick screening estimate of whole-phenotype heritability that is closely related to the average of traditional heritability.

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.

Xenopus laevis erythrocyte nuclei have been used as a source of inactive ribosomal RNA genes to study the regulation of gene activity during early embryogenesis. Addition of an oocyte extract to the erythrocyte nuclear transcription assay brings about transcription by RNA polymerase I. This is not seen If an egg extract is used even though the amounts of RNA polymerase I are the same in both preparations. The oocyte-treated nuclei synthesize ribosomal RNA as defined by RNA-DNA hybridizations and sucrose gradient separation techniques. The active component of the oocyte extract has been semipurified to a single peak by Sephadex G100 column chromatography and DEAE cellulose salt elution. Its presence and absence during oogenesis and early embryogenesis parallels the transcription of the ribosomal genes in vivo at these stages. Within the oocyte itself, the active component is located exclusively in the germinal vesicle. The structure of the rlbo6omal genes in treated nuclei has been studied by DNase I digestion. The ribosomal genes in erythrocyte nuclei are Insensitive to DNase I, but treatment by an oocyte extract or egg extract plus the semipurified active component causes the entire gene repeat to become DNase I sensitive. An egg extract alone has no effect. The semipurified active component by itself causes only the 5' end of the transcribed sequence to become DNase 1 sensitive. The component therefore appears to act on the non-transcribed ribosomal genes of the erythrocyte nucleus to alter their chromatin conformation to a more DNase I sensitive one which permits RNA polymerase 1 transcription. The DNase I sensitivity of histone H4, globln, tRNA and 5Sooc RNA genes was also studied. The extracts had no effect on the DNase I sensitivity of any of these genes, but those genes transcribed by RNA polymerase 111 are DNase I sensitive whatever their transcriptional state.