Genomic imprinting, the parent-of-origin specific expression of alleles is an important area of research in evolutionary biology and human health (cancers and developmental syndromes). Haig’s kinship theory suggests that the maternally and paternally derived alleles of offspring resource allocation genes have evolved under different selectional pressures. Thus within different kin related individuals they are expressed unequally, each allele favouring their own inclusive fitness. Social insects provide the best independent model system to study the evolution of imprinting. However, imprinting has not been discovered in any social insect. My PhD lays the groundwork for a social insect model of genomic imprinting. Methylation is a common epigenetic tag of genomic imprinting in mammals and flowering plants. I found that a functional methylation system which is involved in the reproductive caste formation, development and social behaviour is present in the bumblebee, Bombus terrestris. Under queenless conditions, reproducing and non-reproducing worker castes show different brain methylome patterns. Alteration of methylation can cause a sterile worker to turn into a reproductive worker with increased aggressive behaviour and ovary development. Next I found monoallelic methylation associated with monoallelic expression in genes predicted to be imprinted by Haig’s theory. Also, differential allele specific expression that are apparently due to parent-of-origin effects is present in reproduction loci of B. terrestris. Reciprocal crosses at these loci is recommended as further work, to check whether these expression patterns are due to genomic imprinting. I assess the effects of maternally and paternally contributed sociobiological factors on worker male production and found that the paternity or the queen mating frequency has a significant influence on worker male production in eusocial Hymenoptera. Finally, I also studied the polyphenism involved in phase dependent behavioural plasticity of locusts. I found that the transition of solitarious to fully gregarious behaviour in the desert locust, Schistocerca gregaria begins without significant changes in the DNA methylation landscape of the CNS but subjected to the pronounced differences at a later stage.

This thesis investigates the feeding ecology of the lesser flamingo (Phoeniconaias minor). Studies took place at six endorheic alkaline-saline lakes within the East African Rift Valley, which varied in terms of taxonomic composition and biomass, both spatially and temporally. The lakes could be categorised as Arthrospira fusiformis dominated (lakes Bogoria and Sonachi), diatom dominated (Lakes Natron and Elementaita) or ‘other’ cyanobacteria dominated (lakes Oloidien and Nakuru). From 2009 to 2013, the lake levels were consistently rising with the average biomass reducing concurrently; maximum surface A. fusiformis recorded was > 800 mg L-1 in 2009 at Lake Bogoria, falling to < 50 mg L-1 in 2013. A reduction in average biomass was recorded at all lakes throughout the study period, with the exception of Lake Natron, where higher diatom abundance was recorded in 2011 than in 2009. This study identified 10 distinct feeding behaviours utilised by lesser flamingos at different times of day. Different behaviours were also employed to access different food resources from a variety of niches throughout the aquatic habitat: from the lake’s edge, the open water and the lake-sediment interface. Deep water feeding was occurring in higher frequency than recorded previously, at Lake Bogoria where flamingos were exploiting a highly concentrated food resource of sedimented A. fusiformis, discovered during this study. This confirms that deep water feeding behaviours are more common than only being utilised to access diatoms as an emergency food source, as has been suggested. The time spent feeding was negatively correlated with food abundance across the six lakes with lower biomass requiring longer feeding duration, however, at Lake Bogoria a positive correlation was identified, suggesting re-stocking of energy reserves. A positive correlation was identified between the distribution of food resources within Lake Bogoria and lesser flamingo distribution across the lake. Finally, a significant difference was found between flock density when feeding on different food sources (more densely flocked when feeding on A. fusiformis than on diatoms) or through different feeding behaviours.

Crocus sativus L., saffron Crocus, is a sterile triploid (2n=3x=24) species of unknown relationship to other diploid and polyploid species in the genus Crocus (Iridaceae). The species have large genomes (typically 3000Mbp 1C), much repetitive DNA, and show high morphological diversity within and between species, with no clear phylogenetic patterns below the level of section Crocus, series Crocus. I aimed to examine molecular diversity in C. sativus and related species by analysis of sequences and IRAPs (Inter Retroelement Amplified Polymorphisms). Repetitive DNA sequences and genomic DNA from various species were used for in situ hybridization, with chromosome morphology, to infer relationships and ancestry of saffron. The IRAP analysis, involving 63 primer combinations and 4745 polymorphic bands, revealed no polymorphism within 17 saffron accessions obtained from across the world from Kashmir through Iran to Spain. In contrast, high levels of polymorphism were identified between accessions of six wild Crocus series Crocus species, with further variation between the species. Analysis of 123 sequences of the ATP-synthase gene and 107 TC25 gene-SSR sequences from seven saffron accessions and eight wild species showed that the saffron accessions often carried three alleles, a result also found with clustering of published EST sequences. The analysis showed many alleles were shared by Crocus species and did enable a well-resolved phylogeny. Chromosome analysis grouped saffron chromosomes into 8 groups of 3, but one chromosome differed from the other two. It was concluded 1) Saffron crocus has minimal genotypic variation and the triploid hybrid species is most likely to have arisen only once; 2) Saffron is a allotriploid species, with the most likely ancestors being C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics and show the need for conservation and collection of wild Crocus.

Eusocial species show incredible variation in all aspects of social living, which has facilitated their ecological success. Investigating the mechanisms which regulate variation in social traits is an important goal for evolutionary biology, since understanding fundamental mechanisms underpinning variation can inform social evolutionary theory. In this thesis, I investigate aggressive behaviour and genome architecture as essential mechanisms in regulating variation in the polymorphic social phenotype of the multiple queened ant species L. acervorum. I investigated the role of enforcement behaviour in maintaining reproductive skew in functionally monogynous colonies. I show that in the absence of worker aggression (enforcement), functionally monogynous queens continue to engage in highly aggressive interactions and, crucially, high colony skew was not affected. Furthermore, I show that low skew is not affected by aggressive worker enforcement in polygynous colonies. Therefore, enforcement behaviour is likely to be important in regulating skew in functionally monogynous colonies but not in polygynous colonies, where potentially the loss of sensitivity to enforcement may be an evolved response to fitness benefits associated with the polygynous social phenotype. Furthermore, I investigated the role of genome architecture in regulating variation between the two social phenotypes. I scanned the genomes of four populations (two polygynous and two functionally monogynous) for extreme population differentiation (FST) at SNP loci, which were associated with a difference in the social phenotype. I found a large (6.2Mb) contiguous region associated with different social phenotypes (the social region), which mapped to LG2 on the S. invicta linkage map. The social region displayed some similarities with the social chromosomes in S. invicta and F. selysi. Furthermore, the region contained potential gene candidates, such as odorant binding proteins, which have been associated with divergent social phenotypes in S. invicta. The work presented in this thesis highlights the importance of different mechanisms, both behavioural and genomic, in regulating variation in fundamental social traits. Furthermore, it demonstrates the importance of understanding how mechanisms can bridge the gap between genotype and phenotype.

The Fanconi Anemia (FA) pathway is critical for repair of DNA damage interstrand crosslinks (ICL). Mutations in the pathway lead to a rare genetic disorder known as FA, where patient’s symptoms include a high predisposition to cancers, anemia and developmental defects. The pathway is complex, consisting currently of 15 proteins (in vertebrates). The key event of the pathway is the monoubiquitination of downstream targets FANCD2 and FANCI, which signals the recruitment of the DNA repair machinery. The E3 ligase activity that carries out the monoubiquitination event resides in the Fanconi Anemia Core Complex (FA CC), which consists of 7 proteins. Patient mutations in any of the FA CC proteins prevent the monoubiquitination of targets, FANCD2 and FANCI. Interestingly, E3 ligase activity is associated with only one of these 7 FA CC proteins, which is FANCL. The studies presented in this thesis unveil the structure of Human FANCL and the molecular details of FANCLs interactions, required for the key monoubiquitination event of the FA pathway. These observations provide an insight into the biochemistry underlying the FA pathway and the role of E3 ligases in selective monoubiquitination.

Entry into mitosis is accompanied by dramatic changes in the morphology and polarity of cells, both of which entail remodelling of the cortical actomyosin cytoskeleton. Here I identified Diaphanous as the main actin nucleator required for the formation of the mitotic actin cortex in the context of a developing epithelium. I also identify the pathway involved, which we show is dependent on Pebble/RhoA and on the apical polarity regulators aPKC/Cdc42/Par6. Strikingly, following the activation of Pbl upon entry into mitosis, Cdc42 RhoGTPase and the polarity proteins aPKC and Par6 extend their domain more basally driving the cortical accumulation of Dia, while Rho directs the accumulation of cortical p-Myosin II. Taken together, these data reveal a new molecular mechanism for the concerted activation and localization of Dia via RhoGTPases and the intimate link between cortical repolarisation and the regulation of actin filament organisation.

Skin integrity is essential for sustaining life and it is important to understand the processes involved in its maintenance and repair. There are several key stages involved in wound healing that rely on the complex communication through gap junctions and their connexins to ensure the resolution of the wound. Gap junctions are expressed in all cells linked with tissue repair and provide a regulated pathway linking the cytoplasm of neighbouring cells and allowing signals to pass freely between the two. In the skin there are three key connexins (Connexins 26, 30 and 43) that undergo dynamic changes and regulate the stages of wound closure. To date, extensive research has shown that inhibiting Cx43 expression can achieve significant improvements in wound repair. Synthetic connexin mimetic peptide Gap27 which possess a conserved homology to the second extracellular loop of Cx43 is now being considered as a candidate to improve the rate of wound repair. At low concentrations Gap27 has been shown to block hemichannels but can target gap junctional intercellular communication at higher concentrations and for longer incubation periods. By using Gap27 as a tool, this thesis explores the importance of connexins, hemichannels and gap junctions in tissue injury and repair. I have dissected out the relative contributions of connexins and their involvement with hemichannels and gap junctions in wound repair while investigating if and how Gap27 reduces other connexins. Further work using in vitro wound healing models has shown how Gap27 can enhance the rate of wound healing in early stages. In the second half of this thesis I continue to use Gap27 to investigate the connexin based communication involved in the spread of cell death and damage during ischemia reperfusion injury in vitro and in vivo. The potential therapeutic implications of the wound healing properties of Gap27 are exciting, novel and promising.

The RET receptor tyrosine kinase (RTK) is crucial for embryonic and adult development of multiple organs, tissues and neurons. Gain-of-function mutations in the RET gene are found in human cancer, while loss-of-function mutations are associated with congenital anomalies of the kidney and urinary tract (CAGUT) and Hirschsprung's Disease (HSCR). Previous work has identified that some HSCR RET mutations result in a bottleneck in RET folding and a subsequent loss of RET export. This thesis presents work examining the characteristics of wild type (WT) and HSCR RET maturation, export and signalling in stably transfected mammalian cell lines. High throughput siRNA screening was used to identify components involved in WT and HSCR RET maturation and export; preliminary validation has implicated Endoplasmic Reticulum associated degradation (ERAD), autophagy and the N-glycosylation pathway. RET is also a validated cancer target, as a driver of cancers including multiple endocrine neoplasia (MEN) 2A and B. While there are several FDA-approved RET inhibitors available, their lack of specificity and potency has resulted in high levels of off-target toxicity and low life expectancy extensions. As such, a new generation of more optimal inhibitors is required. This thesis presents the investigation of the molecular basis of RET kinase inhibition, through the elucidation of the RET kinase domain (KD) structure bound to several ATP-competitive chemical inhibitors that are known to inhibit RET in vitro. Preliminary development of an updated RET pharmacophore is described, defining key residue interactions and combining observations with biochemical and thermal stability data.

Integrin α7β1 plays an important role in maintaining adult skeletal muscle integrity and like dystrophin, provides anchorage and bidirectional signaling as a laminin receptor. The expression of α7β1 integrin was upregulated upon dystrophin deficiency arguing for the molecular compensation and thus considered as potential candidate for treatment of Duchenne Muscular Dystrophy (DMD). The existence of developmentally regulated alternative splice variants makes the α7β1 integrin a complex integrin to study its function in skeletal muscle. In this study we show that increased levels of the adult extracellular variant X2 interfere with muscle integrity, while the presence of embryonic integrin α7 extracellular variant X1 results in normal skeletal muscle architecture. Furthermore, detailed analysis of mdxα7tg mice suggests that overexpression of integrin α7 made no difference on the dystrophic phenotype, in fact mdx α7X2 mice show a more severe phenotype compared to mdx mice. Our study also shed light on the importance of integrin α5 during the development of the skeletal muscle by means of generating conditional knockout (cKO) mice using HSA-Cre and Pax3-Cre promoter systems. Our findings show no obvious difference in the Itga5 cKO when the HSA promoter drives Cre recombinase, however conditional loss under the control of the Pax3 promoter leads perinatal lethality. In addition we investigate the dosage effect of integrin α5 in integrin α7 knockout (KO) mice to understand the cross talk between these two integrins and to correlate with previous data suggesting a gain of function phenotype by that existence of integrin α5 at the myotendinious junction (MTJ) in α7KO muscle (Nawrotzki et al.,2003) From our data we know that gene therapy with integrin α7 is a challenge and is not a suitable alternative to cure dystrophy, at least not in mdx mice, we therefore switch our focus on looking into cell based therapies for DMD by investigating the potential role of perivascular cells (PVCs) using transplantation experiments in mice by artificially inducing muscle damage.

Cartilage, which lines joint surfaces to allow near-frictionless movement, lacks the ability to adequately repair itself and there are currently no effective, disease-modifying drugs to halt or repair the damage. Development of powerful in vitro models to investigate gene expression changes during osteoarthritis and chondrogenesis is key in understanding how the disease develops and how cartilage might attempt to repair itself. In this thesis, an enhanced model of chondrogenesis of the murine ATDC5 chondroprogenitor cell line was developed with cells cultured in micromass. Results revealed not only an increase in chondrogenesis markers, but markers of growth plate differentiation, including type X collagen, were either restricted or repressed, whilst expression of genes rich in articular cartilage were upregulated. This suggests that the enhanced ATDC5 model is more reminiscent of articular cartilage, making this model suitable for investigations into osteoarthritis - a disease of articular cartilage. The role of WNT5A signalling was then investigated (which is up-regulated in osteoarthritic cartilage) in a disease-like context by stimulating cultures with cytokines. Microarray analysis unearthed interesting and novel results, including a decrease in WNT5A signalling and in expression of members of the CCN family. Pathway analysis allowed further exploration of the interrelationship between cytokine and WNT signalling. Some changes in gene expression were reminiscent of those observed previously in in vivo models of early osteoarthritis. Finally, migration studies revealed that non-induced, undifferentiated ATDC5 cells have a migratory phenotype reminiscent of chondroprogenitor cells, which have the capacity to migrate to sites of cartilage injury in vivo. A novel model of cartilage invasion was also developed, with results suggesting WNT5A may be a potential inducer of chondroprogenitor invasion. Together, this thesis shows that the ATDC5 model is a good model for investigating articular cartilage both in a physiological and pathological setting.