Understanding the origin of biodiversity is a central question in evolutionary biology. Ecological specialisation, including the repeated rapid and parallel evolution of ecological specialists (‘ecotypes’), is a major source of biodiversity. The parallel evolution of ecotypes in salmonid fishes, such as Arctic charr, brown trout and European whitefish, has resulted in extensive diversity in northern postglacial freshwater ecosystems. Despite their ecological diversity and importance for northern ecosystems, the knowledge on the genetic basis of ecological specialisation, the evolutionary history of adaptive divergence, and the factors shaping the underlying genetic architecture are still not well understood in salmonids. Over the last decade many studies have investigated the genetic basis of ecologically relevant phenotypic traits in a wide range of salmonid species using genetic mapping approaches. However, knowledge on the conservation of the genetic basis for particular traits, or suits of traits, across species is limited, mainly due to a lack of genomic resources. Similarly, little is known about the genomic architecture of phenotypic diversity within species, such as the organisation of species-specific quantitative trait loci across the genome and the frequency of potential pleiotropy or genetic linkage. To understand how conserved the genomic basis for particular traits is across species and how quantitative trait loci (QTL) are organised within the genome, we analysed the genetic basis for a wide range of phenotypic traits (N=18) in six salmonid species using a dataset comprising of 943 QTL markers. We developed a novel analytical approach to analyse the colocalisation and synteny of QTL within and across species using a hetero-specific reference genome, in this case the Atlantic salmon (Salmo salar) genome. We found that QTL were not randomly distributed across the genome and that gene-density determined the distribution of QTL across chromosomes. By comparing QTL across species, we further identified genomic regions that were enriched for QTL for morphological and physiological traits (synteny blocks) in a range of species. Within three of the species, we also detected the significant colocalisation of QTL for different traits. Overall, the detection of synteny blocks and colocalised traits suggests a small but detectable role of pleiotropy and genetic linkage in trait evolution in salmonids and a conserved genetic basis for some traits across species. However, the observed patterns of conserved genetic basis and colocalisation were relatively weak, as QTL were mostly not conserved across species or colocalised within species. In general, the repeated evolution of similar ecotypes across populations and species implies a certain predictability of evolution. However, it is not well understood how phenotypic evolution overcomes the contingencies of heterogeneous genomic backgrounds of natural populations. To investigate the repeatability and predictability of parallel evolution, we used eco-morphological, genome-wide SNP and transcriptome data within and across lakes and evolutionary lineages of Arctic charr (Salvelinus alpinus). We found significant parallelism across replicated ecological specialists in foraging-associated traits. This phenotypic parallelism evolved despite population-specific variation in demographic histories, varying genomic response to selection and the non-parallel genetic basis of ecotype divergence. However, the regulatory molecular basis of ecological specialisation, inferred from gene expression and biological pathways, was highly parallel across ecotypes, bridging non-parallel genomic patterns and parallel eco-morphology. These findings suggest that parallel phenotypic evolution is possible despite non-parallel evolutionary routes when the functional molecular basis of ecological specialisation compensates for non-parallel genomic basis and histories. Evolutionary and genomic contingencies, such as demographic histories and genomic features can strongly influence the genomic architecture of adaptive divergence and reproductive isolation. To investigate how genomic features and demographic history influence the genetic architecture of adaptation and reproductive isolation, we reconstructed the demographic history and analysed the genetic architecture of divergence in brown trout (Salmo trutta) from the Maree Catchment in Scotland. Brown trout display reproductively isolated and divergent life histories and ecological specialisation, including a large piscivorous life-history form (ferox trout) and a smaller benthivorous life-history form. We found that ferox trout and benthivorous brown trout most likely diverged under a secondary contact of at least two distinct postglacial lineages and identified 33 genomic islands across the genome differentiating life-history forms. We demonstrated that some of these genomic islands formed under selection, and contained genes and biological pathways related to growth, development and immune response. Overall, we found strong genomic signals of divergence that were partially driven by selection on divergent phenotypes, and not only caused by genetic drift or through underlying genomic features, such as reduced recombination. The identification of the underlying evolutionary history and genetic architecture highlights the strength of genomic studies using species pairs for understanding the driving factors of adaptive divergence and reproductive isolation. Despite extensive knowledge on the genomic mechanisms underlying adaptive divergence over longer time scales and under the influence of phases of geographic isolation, less is known about the mechanisms underlying rapid ecological and phenotypic divergence. Rapid evolution plays an important role in the adaptation of species to human-induced environmental changes. However, it has been shown that in some cases human-driven environmental changes can lead to rapid loss of species and functional diversity, e.g. through species collapse and hybridization. Even though theoretical models predict that species can rapidly re-diverge under the right conditions following a species collapse and hybridization, the underlying mechanisms of rapid re-divergence remain to be elucidated. Empirical evidence for re-divergence following a species collapse is also lacking. We found evidence for the rapid evolution of ecologically-relevant phenotypic diversity in a European whitefish subspecies from Lake Constance, the gangfisch (Coregonus lavaretus macrophthalmus) after the recovery of pristine ecosystem conditions, following human-driven eutrophication, and speciation reversal. We found that a key functional trait, gill raker number, rapidly diversified within less than 10 generations following ecosystem recovery, allowing the use of vacant trophic niches. Variation in gill raker number is controlled by a sparse genetic architecture, as predicted by theory, and we further found evidence suggesting that introgression potentially provided the underlying adaptive variants. Several biological pathways that are known to be involved to ecological specialisation in fishes, such as metabolism, immune response and neural development, were identified based on coexpressed gene modules and genes under selection associated with gill raker number. Overall, our results demonstrate that functional diversity can rapidly re-emerge, given the right combination of genetic architecture, genetic diversity, and selection. In summary, this thesis demonstrates the evolutionary and genomic routes underlying phenotypic evolution and ecological specialisation in salmonid fishes. Comparing across different study systems, we find that secondary contact and historical gene flow played an important role in the evolution of salmonid species. Despite strong variation in the genomic basis of phenotypic traits across species and the genomic patterns of divergence across populations within species, we find some molecular parallelism across populations and species. Parallel ecotypes most likely evolved through parallel regulatory evolution and involvement of similar functional biological pathways. Furthermore, we find biological pathways that are repeatedly involved in adaptive divergence in different species. / Overall, our results indicate that despite the flexibility of rapid and parallel phenotypic evolution on the genomic level, it is relatively conserved on the level of regulatory mechanisms and functional biological pathways.

Working equids, vital to many of the world’s most economically vulnerable people, face many challenges to their health, welfare and productivity. In The Gambia, West Africa, appropriate nutrition, husbandry and veterinary assistance are limited, while infectious disease is a constant threat, particularly the parasitic disease trypanosomosis. The prevalence of generalised trypanosomosis in working equids attending the Gambia Horse and Donkey Trust show in 2013 using PCR was 55.4%. Trypanosoma congolense was most prevalent (47.0%), followed by T. vivax (15.7%) and T. brucei s.l. (2.4%). Mixed infections were common (9.4%) and T. congolense/ T. vivax coinfection appeared to have the greatest clinical effect. Spread of T. brucei parasites to the central nervous system (CNS), confirmed using immunohistochemistry and PCR, causes severe CNS dysfunction. Horses showed spastic paraparesis that rapidly progressed to recumbency, while donkeys more often displayed somnolence and cranial nerve dysfunction with a slower deterioration. The disease was fatal in all cases. Histopathology revealed diffuse lymphocytic-plasmacytic meningoencephalo-myelitis with marked perivascular cuffing, particularly in the white matter. T cells were prominent in this first study of lymphocyte distribution in equine CNS trypanosomosis. Extensive reactive astrocytosis was also demonstrated. Currently, a reliable diagnosis of equine CNS requires post mortem samples. The loop-mediated isothermal amplification (LAMP) assay was assessed for the diagnosis of equine T. brucei infection for the first time in both blood and cerebrospinal fluid (CSF). An entomological survey showed that Glossina morsitans submorsitans was common in dry woodland areas while G. palpalis gambiensis was found in riverine habitats. The prevalence of T. brucei in the midguts of Glossina specimens was 1.7% and equine DNA was found in tsetse bloodmeals, providing evidence for ongoing interaction between host, parasite and vector. Atylotus agrestis, vector of T. vivax and T. congolense, was present in large numbers in village areas. Equine DNA was detected in one A. agrestis specimen, however, no evidence of T. brucei in association with these flies was found. Finally, microsatellite genotyping was used for the first time to investigate T. brucei populations in equine trypanosomosis in The Gambia. The results revealed a heterogenous population, providing further evidence for a tsetse-transmitted mode of transmission. No evidence of population clustering by disease type or host species was detected, suggesting that host factors determine pathogenesis. Initial evidence for the involvement of the tsetse vector supports evaluation of vector control methods although further analysis of T. brucei populations in insect vectors and their relationships with those infecting equids is recommended. The clinicopathological descriptions will be of use in further study of equine CNS trypanosomosis and the development of new therapeutics and LAMP has the potential to facilitate research, especially in the study of CNS infection which has, up to now, relied on post mortem confirmation.

Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease caused by the absence of the sarcolemmal protein dystrophin. The skeletal muscles of DMD have disrupted dystrophin-glycoprotein complex (DGC) and impaired sarcolemma integrity. In this study, we show that clonally derived dystrophin-deficient myoblasts PD50A are differentiation impaired. Coculture with osteoblasts improves the differentiation efficiency of PD50A myoblasts. We also establish that supplementation of combinations of IGF-1/IGF-2, IGF-1/LIF and IGF- 2/LIF in cultured PD50A myoblasts ameliorates the differentiation impairment. We establish that there are elevated levels of Cav-3 and Cav-1 proteins in dystrophin-deficient myoblasts and mdx mouse embryos and that Cav-3 and Cav-1 form heterooligomers in adult skeletal muscles. We show that overexpression of Pax7 suppresses Cav-3 in dystrophin-deficient myoblasts. Using a genetic mouse model (mdx/cav3\(^{+/-}\)) embryo we further establish that immunohistochemistry staining of Cav-1 and Cav-3 coincides with the mouse heart development. The DGC of skeletal muscles plays a role in signal transduction and mechanical response. Here we show that AKT/mTOR and IGF-2/p57\(^{kip2}\) (but not ERK) signalling pathways are upregulated in dystrophin-deficient myoblasts and mouse embryos. Using atomic force microscope we show that Cav-1 helps maintain the stiffness of dystrophindeficient myotubes while Cav-3 help maintain that of dystrophin-deficient myoblasts. This study suggests that Cav-1 and Cav-3 have both compensatory and compromising roles in mdx.

The microflora of two acidic environments was investigated using analysis of 16S rDNA amplified by the polymerase chain reaction (PCR) from environmental DNA. These environments had different chemical characteristics from most of the acidic environments studied by others. The first sample site, a coal spoil (Birch Coppice, Warwickshire), might have been expected to produce niches enriched in humic matter. The second, comprising geothermal vents on the Island of Vulcano, was unusual for natural acidic environments since it was saline. Three vent regions of different temperatures (30°C, 45°C and 80°C) were examined. Prior to the 16S rDNA analysis of the sites, a brief investigation into selection of a suitable method of DNA extraction was carried out. A bead-beating method and a chemical lysis/freeze-thaw method were compared. With regard to clone types found via each method, there was little qualitative difference. DNA was extracted from the two sites and 16S rRNA genes were amplified by PCR. PCR products were ligated and competent E. coli cells were transformed to produce clone libraries. Restriction fragment length polymorphisms (RFLPs) were examined and representatives of each RFLP type were sequenced and analysed with reference to RNA gene sequence data bases. The coal spoil clone library was dominated by sequences related to those from uncultured actinobacteria, particularly those found previously in peat bogs and various soils. Representatives of some well-known acidophiles were also found (e.g. Leptospirillum species). The clone bank from the saline, geothermal site DNA comprised sequences from acidophiles capable of growth at the respective temperatures of different samples. The lowest temperature samples produced sequences from a novel Acidithiobacillus species and also indicated a novel species probably related to Thiobacillus prosperus (which was isolated previously from Vulcano). A high temperature sample gave sequences from archaeal acidophiles, Acidianus brierleyi and, previously isolated from Vulcano, Acidianus infernus and Thermoplasma volcanium. Where the clone banks revealed the presence of novel organisms, attempts were made to isolate and characterise them. The novel actinobacteria did not appear to grow in laboratory enrichment cultures. The novel Acidithiobacillus species and two novel Thiobacillus prosperus-like species were characterised.

The rotaviruses are a recently defined ubiquitous group of viruses responsible for causing acute-gastroenteritis in human infants and young animals. Biochemical studies have shown that the rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA). This thesis concerns an investigation of the nature and extent of genomic variation in rotaviruses. A rapid and sensitive method for analyzing the genome profiles of rotavirus field isolates was developed. This is based on the direct extraction of dsRNA from faecal samples followed by radiolabelling with [³²P] pCp using T4 RNA ligase. This procedure has been further developed to produce a method for generating diagnostic fingerprints from individual species of dsRNA. A detailed structural study making use of this fingerprinting method has been undertaken on bovine, porcine and human rotavirus isolates. These analyses show that genome segment mobility variations are always associated with detectable changes in nucleotide sequence. They also show that corresponding genome segments with no mobility variation can have sequence-changes at least as great as those found in segments showing electrophoretic mobility variation. These results also revealed evidence for genome segment specific regions of terminal sequence conservation. Evidence for the occurrence of genome segment reassortment between viruses in the field was obtained. Finally evidence for the existence of a 'new' porcine rotavirus which is antigenically unrelated to previously described rotaviruses and has an unusual pattern for its 11 genome segments is presented.

Studies on the molecular organisation and properties of the first three enzymes of pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPTase), aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase), in various organisms have been reviewed. The molecular organisation of these three enzymes has been investigated in wheat using gel filtration chromatography. CPSase activity could not be detected in gel filtered extracts and in crude extracts from wheat seedlings was shown to be highly labile. ATCase and DHOase activity was detected and the molecular weights of these enzymes were estimated to be 1.03 x 105 ( 1.4 x 10^4) and 8.6 x 10^4 (6 x 103), respectively. At no time during these investigations were high molecular weight species (consistent with the presence of a multifunctional complex containing these enzymes) detected. During the course of these investigations, a protease was detected which was shown to co-migrate with ATCase and DHOase activities. This protease was shown to be insensitive to the serine protease inhibitor PMSF, but was partially inactivated by iodoacetamide, consistent with the protease being a member of the cysteinyl protease family. Inclusion of iodoacetamide during chromatography also failed to reveal high molecular weight species of these enzymes. Antisera were raised against purified wheat ATCase and were characterised by their ability to inactivate the enzyme. These antisera were then used to probe western blots of crude extracts from wheat seedlings and screen a wheat cDNA expression library to ATCase sequences. Western blotting failed to show any immunoreactive species in extracts prepared under conditions which suppressed protease activity (SDS, -mercaptoethanol), although a low molecular weight (approximately 3.7 x 10^4) ATCase could be detected in samples obtained after gel filtration chromatography. Antisera also showed very little cross-reactivity with the ATCase from E.coli a result consistent with studies on the enzyme from B. subtilis.

Endosomal trafficking and autophagy are two fundamental processes of eukaryotic cell biology, from unicellular organisms such as yeast to multicellular metazoans such as C.elegans and Humans. Both processes are involved in a diverse number of physiological processes and implicated in a number of pathologies. A recent study has exhibited a mutation on the SM protein Vps45 as a cause of severe congenital neutropenia in humans. The same mutation in yeast causes defects in endosome to vacuole trafficking in S.cerevisiae as well as a temperature sensitive lethality at the non-permissive temperature. A null allele of vps-45 in C.elegans results in developmental arrest during the highly secretory phase of moulting in a similar temperature conditional manner to yeast and defects in yolk protein trafficking. The work presented in this thesis aims to provide basic understanding in an animal model of the impact of loss of Vps45 function that might be informative of the reason for the death of the highly secretory neutrophil cells under the absence of a functional Vps45 protein. The vps-45 and unc-51 mutants as well as a novel unc-51 vps-45 double mutant where possible, were characterised for lifespan, duration of post- embryonic development as well as moulting duration. Reduced embryonic viability, reduced lifespan as well as delays in the moulting process were identified. Data suggested that both autophagy and protein trafficking play a role in C.elegans development through unc-51 and vps-45 respectively. In addition to this, the seam cells of both the vps-45 and unc-51 defective C.elegans were observed during the moult using an autophagy marker. An increase in autophagic activity during the moult was observed, which was more pronounced in the case of the vps-45 mutant. As such the obtained data suggest autophagy and endosomal trafficking play an important role in the moulting process. Following up to previous work conducted in our lab in yeast defective for Vps45 trafficking which exhibited increased sensitivity to oxidative stress, the redox state of the vps-45 and unc-51 animals as well as their sensitivity to oxidative stress was assessed using a set of ER and cytosolic GFP markers and killing assays. Both the vps-45 and unc-51 mutants showed a higher sensitivity to oxidative stress, with the unc-51 exhibiting the more pronounced phenotype overall. These results came in agreement with the shorter lifespan phenotypes exhibited by both mutants in the previous experiments, possibly as a result of accumulation of ROS, as well as the severe defects of the double mutant. Finally, a suppressor identified for the moulting death of the vps-45 mutant was characterized for a set of phenotypes, in order to exclude suppression of any of the other phenotypes identified for the vps-45 mutant. Furthermore, the suppressor was identified as being autosomal and recessive and as thus an SNP full genome sequencing technique was employed, which gave rise to two suppression loci in two different chromosomes, along with two different subpopulations corresponding to these loci which exhibited different growing patterns.

The effects of pupal cold shock on the life cycle and wing morphology of the Speckled Wood butterfly are examined and their genetic assimilation is investigated. Metamorphosis is modelled in terms of changes in stability, and the mediation of cold shock effects by hormones is considered. Current theories of pattern formation are evaluated for the species, and pattern is analysed using manual, photographic and digital methods. The development of wing morphology is modelled, and cold shock effects understood by comparison with normal development. Developmental canalisation is estimated as variability and fluctuating asymmetry. An index is developed that predicts the extent of assimilation. Likely modes of inheritance are suggested, and the possibility of natural cold shock and assimilation in the species is considered. Recent trends in biology indicate that neo-Darwinian concepts cannot adequately account for certain developmental and hereditary phenomena and that a new paradigm is emerging. The two schools are compared with particular reference to Weismann and Waddington, and the phenomenology is re-examined in the light of the new findings.

Parasitic helminths are able to survive within their hosts due to their ability to dampen immune responses by secreting molecules with anti-inflammatory and tissue repair properties. Reflecting this, there is increasing evidence of an inverse correlation between parasitic worm infection and the incidence of allergic and autoimmune disorders on a global scale. Such epidemiological evidence has led to the “hygiene hypothesis” which postulates that the recent rapid eradication of parasitic worms in developed countries has resulted in unbalanced hyper-reactive immune systems and consequently, inflammatory disease. As “worm therapy” per se is not ideal, this in turn triggered the idea that exploiting the ability of helminth-derived “immunomodulators” to dampen pathological host inflammation would potentially allow identification of the key pathogenic events in models of human inflammatory disease and hence provide a starting point for development of new and safer therapeutics. Consistent with this, as a serendipitous side-effect of its anti-inflammatory actions, ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae exhibits therapeutic potential in mouse models of inflammatory disorders such as asthma, lupus and rheumatoid arthritis (RA). RA is a chronic autoimmune inflammatory disorder that affects 1 % of the population in industrialized countries, with no known cure. This disorder causes joint destruction and leads to reduced mobility and disability. Deregulation of T cell activation has long been considered to be a major force driving inflammation and thus to date, therapies have focused on systemic anti-inflammatory treatments, which generally leave individuals immunosuppressed and open to infection. Thus, interest has begun to focus on the role(s) that synovial fibroblasts (SF) in the joint play in the early onset of the disease, the maintenance of established inflammation and even in the spread of disease to unaffected joints. This reflects that despite not being part of the immune system, SF produce pro-inflammatory cytokines during the pathogenesis of RA and also directly mediate joint destruction by secreting matrix metalloproteinases (MMPs) that damage cartilage and bone. Indeed, there is increasing evidence that the local pro-inflammatory environment pertaining in the joints drives SF to become 3 imprinted pathogenic aggressors that initiate, drive and spread joint inflammation and bone resorption during development of collagen-induced arthritis (CIA), a mouse model of RA. Intriguingly, therefore, whilst it is established that protection afforded by ES-62 against joint inflammation and bone destruction in CIA is associated with reduced production of the pathogenic cytokine, IL-17 by  and CD4+ T cells, recent evidence suggested that ES-62 could also act directly to suppress the aggressive hyper-inflammatory phenotype of SF in the joint. The molecular mechanisms involved were not defined but interestingly, given that SF express the ES-62 target TLR4 and are the only cells in the joint to express the IL-22 receptor, the parasite product appeared to harness the inflammation-resolving and/or tissue repair actions of IL-22 to suppress SF responses during the established phase of disease. Thus the core goal of this thesis was to advance our fundamental understanding of how SF become imprinted pathogenic aggressors that initiate, drive and spread joint inflammation and bone resorption in the CIA mouse model, as a surrogate for the pathogenic events in the joints in RA. In particular, the primary major aim was to investigate the impact of the local pro-inflammatory environment pertaining during disease, specifically focusing on the signalling and epigenetic mechanisms by which IL-17 and IL-22 potentially (counter)regulate the pathogenic phenotype of SF. Complementing this, another major aim was to establish whether ES-62 acted directly to modulate the phenotype of SF and thus, to identify the key mechanisms by which ES-62 could prevent SF from promoting inflammation and bone destruction and in this way render them insensitive to pro-inflammatory signals. From a therapeutic point of view, being a large immunogenic molecule, ES-62 is not suitable for use in the clinic and thus candidate small molecule analogues (SMAs) of ES-62, based around its active PC moiety have been designed, some which mimic its therapeutic potential in a variety of inflammatory disorders. Thus, it was also important to address whether ES-62 and its SMAs were similarly able to affect SF and prevent their pathogenicity. These studies revealed that the microenvironment of the joint during induction and progression of CIA did indeed result in remodelling of the epigenetic landscape of SF and that such cell reprogramming was associated with the acquisition of a hyperinflammatory, tissue destructive phenotype. Such 4 reprogramming could be recapitulated in vitro, at least in part, by chronic exposure of normal SF to pro-inflammatory cytokines such as IL-17 and IL-1 pathogenic mediators that are found at high levels in the arthritic joint. Such reprogramming was dependent on ERK and STAT3 signalling converging on miR-155-mediated regulation of inflammatory networks via global DNA hypomethylation. ES-62 was able to counteract this by suppressing the levels of ERK, STAT3 and miR-155 signalling but rather surprisingly, this did not result in abrogation of this hypomethylated epigenetic landscape. Rather, whereas the SMA 12b appeared to act simply by preventing/reversing global DNA demethylation to suppress the induction of genes that drive pathogenesis in CIA, ES-62 induced further global DNA hypomethylation and modulation of the epigenetic landscape by inducing HDAC1: collectively these findings suggested that ES-62 might additionally induce (homeostatic) inflammation-resolving and tissue repair genes that would have translational impact in established disease. In any case, these studies suggest that the proposal to use the global DNA methylation status of RA patients as a biomarker of disease should be treated with caution.

Theileria annulata is an intracellular, tick-transmitted apicomplexan parasite, which causes tropical theileriosis in cattle. It undergoes a complex life cycle with several distinct stages occurring within the bovine host and tick vector. ApiAP2 proteins are key candidate transcription factors for regulation of stage specific gene expression across apicomplexans. They are differentially expressed in specific developmental stages and certain ApiAP2s bind specifically to unique DNA sequence motifs. Identification of stage-specific expression of putative transcriptional regulators, the motifs they bind to and potential target genes provided the rationale for this study to understand the molecular mechanisms that control stage differentiation to the merozoite in T. annulata. The results demonstrated that T. annulata ApiAP2s show marked differences in expression levels during the parasite life cycle. ApiAP2 target DNA motifs orthologous to those in Plasmodium and Cryptosporidium were also discovered in Theileria intergenic regions, indicating that the genes downstream are potential targets of Theileria ApiAP2s. These motifs were also found in upstream regions of up-regulated TaApiAP2 genes, suggesting possible auto-regulation and an interaction network of ApiAP2 transcription factors. Importantly ApiAP2 fusion proteins up-regulated during differentiation to the merozoite stage bound to their predicted specific DNA motifs validating that ApiAP2 DNA-binding domain structure is conserved across Apicomplexa genera. Evidence was also produced that AP2 proteins play important roles in steps that commit a cell to differentiate: TA13515D is the orthologue of the AP2G factor in Plasmodium that is a major regulator of gametocytogenesis: TA16485 may be involved in down-regulation of genes during merogony and expression of TA11145 at a higher level in a cell line competent for merogony relative to a line severelly attenuated indicated involvement in regulation of this differentiation step. Discovery of multiple nuclear factors binding to a 2x(A)CACAC(A) motif implicated in autoregulation of TA11145, together with phylogenetic evidence for a clade of related domains that bind this motif suggest that multiple competing ApiAP2s may operate to regulate stochastic commitment to merozoiteproduction. Based on this data an updated stage differentiation model has been generated, with up regulation of the TA11145 gene a key event. A C-box motif association with genes implicated in establishment of the transformed host cell (TashAT, SVSP) suggests it could be important for deregulation of this event as the parasite undergoes stage differentiation. In contrast the inverse G-box was found associated with genes up-regulated from merozoite to piroplasm. EMSA analysis of parasite nuclear extract with a G/Cbox motif probe showed that the motif is an active binding site for a stage regulated nuclear factor. Specific binding of candidate TA12015 protein to the G/C-box motif was unable to be confirmed. Taken together, these results provided evidence that ApiAP2 proteins are regulators of stage-specific gene expression in T.annulata. They also provide insight into probable ApiAP2 interaction networks and support the postulation of a differentiation mechanism conserved across the Apicomplexa. Finally, the data suggests that this mechanism is stochastic and is likely to occur via a positive feedback loop generating a threshold that commits the cell to differentiate to the next stage of the life cycle.