This thesis consists of two parts, which deal with different topics in dynamical systems. Part I (DYNAMICS FROM GAMES) is the main scope of the work. There we study a family of flows which are often applied in studies of some game dynamics in animal competition and evolutionary biochemistry. These flows are the solutions, on simplexes, of cubic differential equations determined by "pay-off" matrices. The main result in this part is a proof for a classification of stable flows in this family, in dimension 2, first conjectured by Zeeman in 1979 (stability under small perturbations in the pay-off matrix). We add necessary and sufficient conditions for stability, which decide the exact class for each stable flow in the family. We also give as preliminary properties some simple expressions to calculate eigenvalues at fixed points and prove that hyperbolicity of these is necessary for stability, in all dimensions. In order to complete Zeeman's classification we had to adapt, in dimension 2, some techniques of structural stability for flows not satisfying the usually required transversality condition. We discuss some aspects and difficulties present when one attempts to study cases in dimension i3. One important three-dimensional example, involving a Hopf bifurcation, is discussed in detail. In the final chapter, we present some three-dimensional cases to which a discussion of stability is feasible. Part II (LIAPUNOVF UNCTIONSF OR DIFFEOMORPHISMSh) as as its purpose the construction of Liapunov functions for diffeomorphisms. A local construction is presented in neighbourhood of compact isolated invariant sets. A globalization is obtained for Axiom A diffeos with no cycles.

Ponds are diverse habitats that make major contributions to regional biodiversity, yet have received relatively little attention compared to other freshwater environments. This study investigated the physico-chemical characteristics and ecology of a nationally important concentration of temporary upland ponds in Radnorshire, Wales in both spatial and temporal dimensions (up to 80 ponds over 18 months). Plant and macroinvertebrate communities were typical of oligotrophic, acidic ponds and appeared to vary along gradients of pH and hydroperiod, whilst including several nationally rare/threatened taxa. A national classification based on the biological community of temporary ponds grouped Radnorshire ponds with a few others in western Britain. Ponds were generally small, shallow and acidic with low concentrations of nutrients and dissolved minerals. Water chemistry was associated with concentration by evaporation and dilution by rainwater, whilst the overall hydrology of ponds was successfully modelled using local meteorological data. Over the course of the study, large increases in macroinvertebrate abundance were observed, but assemblage composition and richness were broadly constant. Seasonality in the abundance of individual macroinvertebrate taxa was evident and suggested that autumn was the optimum time for invertebrate sampling in terms of abundance and diversity. Nearly 40% of the variation among ponds in macroinvertebrate and plant communities was explained by a combination of a species-area effect and differences in pH: no effect of pond isolation was detected within the context of the study region. A cumulative species-area analysis found that a group of small ponds support higher biodiversity than a single large pond of the same area, highlighting the importance of considering beta diversity. Taken together, the findings of this PhD project justify the designation of Radnorshire as an Important Area for Ponds: the ponds form a distinct ‘pondscape’ of >80 waterbodies, with a highly dynamic environment and biota, and which provide a major habitat resource for rare species in the UK.

Adult articular cartilage has a limited repair capacity. This leads to an increasing demand for optimised repair techniques. Furthermore, current procedures to regenerate articular cartilage fail to achieve sufficient results. Previous work within our group suggested that combination of functional tissue engineering and gene transfer represents a promising alternative approach. In this thesis, different viral gene transfer methods were investigated and optimised. A clinically relevant three dimensional transduction model was developed. These results were directly implemented in further work aiming to investigate the combined effect of multiaxial mechanical stimulation and adenoviral-mediated over-expression of bone morphogenetic protein 2 on human chondroprogenitor cell chondrogenesis and progression towards hypertrophy. Two cell sources were investigated, namely human mesenchymal stem cells and human articular cartilage progenitor cells. The combined approached enhanced human mesenchymal stem cell chondrogenesis. Yet, it was not possible to completely prevent progression towards hypertrophy. For human articular cartilage progenitor cells, over-expression of bone morphogenetic protein 2 did enhance their chondrogenic differentiation potential. However, mechanical stimulation alone, in the absence of exogenous growth factors, led to stable chondrogenic induction without signs of hypertrophic differentiation. This suggests these cells should be further investigated. Additionally, the potential of Dorsomorphin, as possible agent to block hypertrophic differentiation by inhibition of bone morphogenetic protein signalling, was investigated in a fibrin polyurethane composite system, using human mesenchymal stem cells. As opposed to the pellet culture model, application of Dorsomorphin led to a cytotoxic effect which decreased the general differentiation potential. Finally, the chondrogenic potential of the two cell types was directly compared, using the pellet culture model. Under serum-free conditions, human articular cartilage progenitor cells were not able to undergo chondrogenesis. The reasons for this remain to be elucidated. The combined results of the thesis can help to develop a novel one-step procedure to treat articular cartilage defects.

D-type cyclins in plants are represented by seven conserved subgroups and play a major role in controlling cell division. Relatively little is understood of their role during seed development, although their expression pattern has been characterized and ectopic expression of CYCD3;1 has previously been shown to disrupt normal embryo development. Here the consequences of ectopic expression of CYCD7;1 using the early endosperm-specific promoter FWA in developing Arabidopsis seeds were investigated. Ectopic CYCD7;1 expression in the maternal central cell prior to fertilization, and in the endosperm from fertilization until cellularization resulted in seeds up to 45% larger. Seed enlargement was accompanied seed lethality, shown to be due to a defect of development during early and mid stages of seed development. As expected from the maternal specific expression of the imprinted FWA promoter, seed size and lethality was dependent on maternal origin of the transgene. Larger seed size was correlated to mature embryo and seed coat outgrowth, and was due to cell proliferation rather than cell elongation. In particular, embryo development was accelerated during the early stages, suggesting these may be dependent on cell division rate, whereas later stages progressed at the same rate as WT seeds. Seed-targeted CYCD7;1 expression phenocopies (1) the nucleus proliferation in the endosperm prior to fertilization observed in rbr and fis-class mutants and (2) the seed enlargement observed in paternal genome excess interploidy crosses. These suggest that CYCD7;1 may act through the RBR pathway to promote cell proliferation and modify imprinting in the endosperm, thereby influencing the parental genome balance. Mechanistically, CYCD7;1 did not interact directly with CDKA;1 but the interaction was promoted in presence of the inhibitor of CDK, ICK1/KRP1 or ICK2/KRP2 in a yeast-three-hybrid assay. However, loss of either KRP1 or KRP2 in respective mutant backgrounds did not prevent the seed enlargement phenotype.

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.

Most of our understanding of immunity has been gained through studies of humans or laboratory rodents. However, such studies do not allow the immune system to be studied in the ecological context in which it has evolved and, as such, they provide a poor model for studying the variation in infectious disease resistance and immune function observed in natural settings. Studies of natural populations have provided fresh insights into the evolution and phenotypic consequences of immunogenetic variation, but have thus far concentrated almost exclusively on genes of the major histocompatibility complex (MHC). As these genes form only a fraction of the vertebrate immune repertoire, there is a need to broaden research in natural populations to include non-MHC genes, in order to gain a more comprehensive understanding of natural selection and immunity. In this thesis, the genetic diversity of a range of non-MHC immune genes was examined in a natural population of field voles (Microtus agrestis L.) in Kielder Forest, UK, which are subject to infection by a range of pathogens. Cytokine genes were the primary focus of this study as they play a central role in regulating the immune response but have rarely been studied in wild species. I examined the hypothesis that, as cytokines are crucial to immunity, variation within these genes may be under selection within populations and between species, and may mediate phenotypic differences between individuals in parasite resistance and immune function. Coding regions from nine cytokine and three other non-MHC vole genes were sequenced, yielding 6.6 Kb of sequence data and 26 SNPs (1 per 255 bp). Three cytokine genes (Il1b, Il2, and Tnf) exhibited patterns of polymorphism consistent with balancing selection maintaining genetic diversity, including an excess of intermediate frequency mutations and more even allele frequencies than one would expect under neutrality. Polymorphism within Il1b and Il2 was also consistently associated with variation in parasite resistance, providing evidence that pathogens are the selective force driving the maintenance of genetic diversity at these loci. In addition, Il1b and Il2 exhibited repeated associations with variation in host immune phenotype, while the Il12b gene was associated both with variation in pathogen resistance and with altered expression levels of Il1b and Il2. Variation in immune function, mediated through the cytokine network, is therefore likely to contribute to parasite resistance in the field vole. This work is the first to show that variation in cytokine genes of a natural population can be maintained by selection, and that this variation can lead to phenotypic variation in parasite resistance and immune function. More broadly, this thesis demonstrates that wild rodents are an excellent model to help us bridge the gap in our understanding between the mechanistic insights gained through studies on laboratory rodents and the variation in infectious disease susceptibility and immune function observed in nature.

The circadian clock is an internal mechanism found in most organisms generating a 24h rhythm, evolved to anticipate predictable environmental changes thus making best use of resources. Intensely studied in the model plant Arabidopsis thaliana (L.) Heynh, the circadian clock was found to control a large number of physiological traits and the expression of more than a third of the plant genes. Clocks therefore play a central role in the life of organisms, in fact plants lacking clock functionality lose their ability to anticipate the dawn showing a reduced fitness. In a perspective of raising global temperatures, impact on crops production of plants with a non optimised clock could be of major importance. The present study therefore investigated the importance for Arabidopsis plants of having a functional clock at high temperatures. Arrhythmic plants over-expressing the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene were compared to wild-type Col-0 plants: while at 17°C wild-type plants showed more leaf area, biomass and chlorophyll content than CCA1ox, at 27°C the difference was even greater. This increased difference at high temperature was also confirmed in successive transcript and metabolite profiling of the two lines. Not only a functional but also an accurate clock was previously found to be important for a good plant performance. For this reason investigation on the impact of high temperature was extended to plants with an internal clock period not matched to the external light/dark cycle. Period mutant lines ztl (30h) and toc1 (20h) along with their wild-types (24h) were grown in 12LD, 15LD or 10LD cycles. ztl line showed no difference from Col-0 at 17°C but a marked difference at 27°C, where the lines with a period matching the external light/dark cycle performed better. Similarly the toc1-1 line performed better at high temperature when its clock period matched the environment. Another key feature of circadian clocks is temperature compensation, a mechanism able to maintain an accurate and robust rhythm with a period close to 24h over a broad range of temperatures. To quantify the importance of temperature compensation at high temperatures, the mutant line gi-11, defective for the temperature compensation mechanism, was used. Little differences were found at both 17°C and 27°C between the gi-11line and its wild-type WS. Finally, a screen was performed to identify new components of the clock specifically required for function at high temperatures. At the end of the screen pipeline, four putative circadian mutants were identified, which will need to be further characterised to confirm their altered rhythmicity and eventual position in the current clock model.

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.