Members of the Pneumoviridae virus family are responsible for severe respiratory tract disease in their hosts. Human respiratory syncytial virus (hRSV) is responsible for over 200,000 deaths worldwide each year and bovine respiratory syncytial virus (bRSV) causes major economic loss to the cattle industry worldwide. The current model for all nonsegmented negative-sense single stranded RNA virus gene expression, is that mRNA is generated in a polar gradient, with decreasing levels of mRNA transcribed from genes further along the genome from the 3 ́ end. With the exception of translation of ORF-2 located on the bicistronic M2 mRNA, translation of Pneumoviridae mRNAs is thought to be regulated through the levels of mRNA abundance. Translation of M2 ORF-2 has been characterised as being regulated by the non-canonical mechanism of coupled translation termination/initiation in pneumonia virus of mice (PVM), hRSV and avian metapneumovirus (APV). This mechanism is reliant on a proportion of the elongating ribosome translating the upstream M2 ORF-1, terminating and reinitiating translation of M2 ORF-2. Although the initiation site for M2 ORF-2 is similar in bRSV to other members of this family that use the mechanism of coupled translation, the mechanism has not been characterised. Using the technique of ribosomal profiling to analyse steady state viral mRNA abundance and viral translation in both hRSV and bRSV-infected cells, it was observed that for certain viral mRNAs, levels of mRNA abundance did not follow the standard polar transcription model. This was characterised by an increase in the levels of mRNA abundance between the mRNA’s respective gene and its upstream neighbour. The increase was observed in the same group of mRNAs in both viruses suggesting that factors other than the transcription polar gradient influence levels of viral mRNA abundance. It was also observed that levels of proportional translation did not match the respective proportional levels of mRNA abundance for certain viral mRNAs in both viruses. This would suggest that translation of viral genomes is not primarily controlled by mRNA abundance and instead other translational regulatory factors influence levels of translation. The mechanism of bRSV M2 ORF-2 translation was also characterised using reporter plasmids assays. It was identified that the mechanism of initiation of translation of M2 ORF2 used, was not that of coupled translation termination/initiation used by other members of this family. Instead it was observed that translation of M2 ORF-2 used an internal initiation mechanism located inside M2 ORF-1 to initiate translation. The mechanism of coupled translation termination/initiation used for translation of PVM M2 ORF-2 was also further characterised. It was observed that translation of M2 ORF-2 was reliant on upstream sequence in the M2 ORF-1 sequence. A predicted mRNA secondary structure was identified in this region and when disrupted, inhibited translation of M2 ORF2. This was similar to the mechanism of coupled translation used in hRSV, suggesting that the mechanism used by this family is reliant on a mRNA secondary structure located upstream of the initiation site.

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.

Microbial communities play important roles in organismal and ecosystem health. High throughput sequencing has revolutionized our understanding of host-associated microbial communities, but the viral component of these communities remains poorly characterized relative to microbes such as bacteria, particularly in non-human hosts. This knowledge gap has implications for global health, as viruses originating in wildlife are responsible for recent disease outbreaks in humans and domestic animals. Although studies have identified factors differentiating viral communities between species, we have little understanding of the variability of viral communities within species. Comparative studies of viral communities are therefore necessary to characterize novel taxa and to evaluate the ecological factors influencing intraspecific viral diversity and distribution. Bats are recognized as “special” reservoirs for viruses because they are associated with diverse viral communities and display deep evolutionary relationships with individual viral taxa. Common vampire bats (Desmodus rotundus) represent a particularly interesting system in which to investigate viral communities, as they are obligate blood feeders that interact ecologically with many different host species, providing opportunities for the acquisition of diverse viruses. The overall objective of this thesis was to advance our understanding of intraspecific wildlife-associated viral communities using an established field network of common vampire bat colonies across Peru. Specifically, I developed a novel method for comparative viral community studies, characterized the viral communities of vampire bats, and examined the ecological correlates of vampire bat viral diversity across Peru. Metagenomic sequencing is a promising technique for comparative studies of viral communities in wildlife, but there is a need to first develop standardized methods that can be applied to samples collected in the field. In Chapter 2 I developed a shotgun metagenomic sequencing approach to characterizing viral communities from non-invasive samples. Specifically, I optimized extraction and sequencing protocols using fecal and oropharyngeal swabs collected from common vampire bats in Peru. Two preliminary sequencing runs were performed, the results of which motivated four pilot studies in which I tested how different storage media, nucleic acid extraction procedures, and enrichment steps affect the viral community detected. Metagenomic sequencing revealed viral contamination of fetal bovine serum, a component of viral transport medium, suggesting that swabs should be stored in RNALater or another non-biological medium. Extraction and qPCR tests were performed on swabs inoculated with known concentrations of virus, which revealed that nucleic acid should be directly extracted from swabs rather than from supernatant or pelleted material. Metagenomic sequencing of paired samples was used to test enrichment by ribosomal RNA depletion and light DNAse treatment, which both reduced host and bacterial nucleic acid in samples and improved virus detection. A bioinformatic pipeline was developed specifically for processing vampire bat shotgun viral metagenomic data. Finally, the optimized protocol was applied to twelve pooled samples from seven localities in Peru, and read subsampling demonstrated that the viral communities detected were consistent at commonly attained depths of sequencing. The protocol developed in this chapter enables minimally biased comparative viral community studies in non-invasive samples collected from wildlife. Having a detailed understanding of viral diversity in key wildlife hosts is an important first step in evaluating the risk of zoonotic disease emergence, but we still lack a holistic view of viral communities in many species including vampire bats. In Chapter 3, I used the metagenomic sequencing protocol developed in Chapter 2 to thoroughly characterize viral communities in the saliva and feces of vampire bats captured across Peru. Viruses were detected from a range of natural host groups including vertebrate-associated taxa that were potentially infecting vampire bats, bacteriophages associated with gut bacteria, and plant- or insect-infecting viruses potentially acquired from the environment. There were broad differences between fecal and saliva viral communities, showing evidence of body habitat compartmentalization. Overall, results established that vampire bat viral communities differ between body habitats and suggested that, for the vertebrate-infecting families analyzed, novel viruses mostly fall within bat-specific clades, without evidence of livestock or humans acting as a major source of viral diversity in vampire bats. Interspecific differences in ecological and life history traits are known to impact viral richness in bats, but the factors structuring viral communities within bat species are less well understood. In Chapter 4, I examined the spatial, demographic and environmental correlates of intraspecific viral diversity in vampire bats. Three measures of viral diversity were calculated at the colony level: richness, a novel measure of taxonomic diversity, and community composition. Generalized linear models were then used to test the effects of broad scale and local ecological variables on saliva and fecal viral diversity. The results showed for the first time that ecological variables can influence intraspecific viral diversity. In summary, the work presented in this thesis advances our understanding of wildlife-associated viral communities in an ecologically important bat host. Future directions in comparative wildlife viral metagenomics, as discussed in Chapter 5, will include exploring the determinants of viral communities across host species, environments and time.

Leishmania spp. are protozoan parasites that infect humans and other vertebrates to cause a spectrum of disease, ranging from cutaneous ulceration to visceral dissemination dependent on the species. Leishmaniasis is prevalent across the developing world and is a major global health issue, yet difficulties in the efficacy and administration route of current anti-leishmanial treatments means the existing drug repertoire is inadequate. To address this, further research and development measures are necessary to identify Leishmania proteins representing useful targets for drug inhibition. Essential genes encode proteins that are necessary for parasite survival and therefore represent suitable drug targets, but the study of such genes is limited by the absence of a conditional deletion system. A family of proteins which has previously been shown to regulate crucial aspects of Leishmania biology are the protein kinases. Protein kinases have been validated in mammalian systems as drug targets in cancer therapy, therefore they represent a promising avenue for research into anti-leishmanial drugs. The cdc-related kinases CRK3 has been studied in particular depth in Leishmania, and current reverse genetic techniques have implicated expression of CRK3 as essential to promastigote survival. CRK3 regulates the cell cycle as demonstrated by treatment of cdc2 inhibitors, but a lack of a system to regulate expression prevents more specific phenotypic dissection of the role of CRK3. In addition the validation of CRK3 as a drug target has been limited by an absence of a conditional genetic system to ablate the gene in mammalian infective amastigotes. To regulate CRK3 expression in a conditional manner to assess its function in the cell cycle of promastigotes and validate it as essential for amastigotes, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) for use in L. mexicana. Cre recombinase mediates the excision of DNA sequences flanked by 34bp loxP sites (‘floxed’). diCre is encoded as two separate subunits each linked to rapamycin binding domains (FRB and FKBP12); therefore recombinase activity is induced by rapamycin treatment which causes dimerisation of the subunits. Our method involves replacing both CRK3 alleles with a ‘floxed’ CRK3 open reading frame and the diCre coding sequence through promastigote transfection and homologous recombination. Induction of diCre through rapamycin treatment of promastigotes results in highly efficient deletion of CRK3 and a distinct growth arrest phenotype corresponding to a block in G2/M. Induced loss of CRK3 can be complemented by expression of a CRK3 transgene but not by expression of an inactive site (T178E) CRK3 mutant, showing that protein kinase activity is crucial for CRK3 function. Significantly, inducible deletion of CRK3 in stationary phase promastigotes prevents the establishment of murine infection, thereby demonstrating an essential role in the amastigote cell cycle to further validate CRK3 as a drug target. Promisingly, inducible deletion is functional in lesion-derived amastigotes and will enable direct phenotypic assessment following essential gene loss in this life cycle stage. To establish a basis for future in vivo application of diCre in Leishmania, a murine infection model was developed with which to track bioluminescent parasite burden by in vivo imaging and assess innate immune cell recruitment to the site of infection by flow cytometry analysis. The combination of functional gene regulation in amastigotes and measures of parasite burden and immune response will yield a powerful tool for the further study of Leishmania genes encoding suitable drug targets. The application of the diCre technique to Leishmania would be greatly benefitted by targeting genes where there is evidence of a regulatory role of orthologous genes in model organisms. The utilisation of genome or protein family-wide RNAi screens in Trypanosoma brucei has identified a number of protein kinases which regulate the differentiation of the parasite between life cycle stages. The repressor of differentiation (RDK1) protein regulates bloodstream form to procyclic form differentiation in T. brucei, and the identification of a protein in L. mexicana with high sequence identity suggested a potentially analogous role in preventing Leishmania from undergoing amastigote to promastigote differentiation in vivo. To assess this, a cell line was generated deficient in RDK1 but no effect on differentiation was identified, as parasites were able to maintain murine infection and differentiate between life cycle stages. This study represents an important addition to the reverse genetic toolkit to study aspects of cell cycle regulation in vitro, and further assess essential genes as drug targets by deletion in amastigotes. The application of the diCre conditional deletion method will enhance the discovery and evaluation of suitable drug targets in Leishmania by phenotypic analysis.

Dosage compensation serves to equalise the levels of X-linked gene products between males and females. In mammals this occurs through the transcriptional silencing of the majority of the genes on one of the two female X chromosomes. The inactive X chromosome (Xi) differs from its active homologue in a number of ways, including the hypoacetylation of core histones, a common property of genetically inactive chromatin. This study has used Xi to explore the functional significance of hypoacetylation and patterns of histone methylation in silent chromatin. Xi was shown to be depleted for di- and tri-methylated lysine 4 of H3, but retained di-methylated lysine 9 of H3. I have examined the temporal order of these modifications as they become established using an in vitro model system for X inactivation; differentiating female embryonic stem cells. The results showed that the loss of tri-methylated lysine 4 of H3 preceded the loss of its di-methylated equivalent, which occurs during a time period of concurrent core histone deacetylation supporting a functional role to the level of lysine methylation. I have used cases of X;autosome translocation to examine how these modifications relate to late replication and transcriptional silencing. Results show that whilst the spread of X inactivation can occur in the absence of both of these properties, histone modifications are a more reliable indicator of the extent of spread of X inactivation than late replication. To explore mechanisms that drive changes in histone modification I have analysed the distribution of histone deacetylases across a region of defined histone deacetylation. The results showed a ubiquitous distribution that did not correlate with acetylated H3 or H4 suggesting that the global association of the Hdacs might serve to provide a rapid return the basal level of histone acetylation following specific targeting events.

Placental growth factor (PlGF) is a pro-angiogenic and inflammatory mediator that promotes many pathological conditions including, diabetes, atherosclerosis and cancer. In mouse models, the loss of PlGF or inhibition of vascular endothelial growth factor receptor-1 (VEGFR-1) activity suppresses these disorders. Hyperglycaemia plays a fundamental role in the pathogenesis of type-2 diabetes and associated conditions, resulting in a loss of PI3 kinase (PI3K) signalling and dysfunction in endothelial cells. Using pharmacological inhibitors, siRNA, and adenoviral constructs to modulate the PI3K/Akt signalling activity, I found that the induction of PlGF expression in human umbilical vein endothelial cells (HUVEC) by hyperglycaemia is PI3K/Akt-dependent. Using similar approaches, the FOXO1 transcription factor was identified as the downstream target of Akt involved in the regulation of both PlGF and VEGFR-1 expression. FOXO1 was found to interact directly with the VEGFR-1 gene promoter in vitro, and over-expression of constitutively-active FOXO1 promotes PlGF expression in vivo. Although VEGF activates PI3K/Akt, it stimulates robust PlGF release in endothelial cells. Here I show that this effect is both VEGFR-2 and PKC-dependent, but independent of PI3K/Akt. The PI3K/Akt/FOXO1 axis is an important regulator of vascular homeostasis and stress responses and the identification of its involvement in PlGF expression may provide new therapeutic targets for disorders characterised by endothelial dysfunction.

Since the completion of the human genome project in 2001, the field of genomics has advanced exponentially, largely due in part to the introduction of next generation sequencing (NGS); a technique that has revolutionised the ways in which genetic disease is investigated. NGS enables the simultaneous sequencing of multiple reads in parallel, which provides researchers with the opportunity to interrogate vast numbers of candidate genes in order to establish the genetic eitiology and key components of disease. Exome sequencing in particular offers an efficient method to investigate disease, as the exomic regions make up 1% of the whole genome, but can contain up to 85% of functional variants responsible for disease. Next generation sequencing has been employed to investigate and identify the genetic cause of Acrocallosal syndrome (a rare autosomal recessive disorder). Exome sequencing was then also applied to investigate the genetic associations with both familial and sporadic pheochromocytomas and paragangliomas (neuroendocrine tumours). This study describes the various applications, challenges and potential benefits that can be achieved by using exome sequencing as a tool to investigate rare autosomal recessive disorders in addition to more complex disorders including familial and sporadic cancer. This study aims to employ cutting edge technology to investigate human disease, in order to enhance current understandings of disease biology and pathogenesis. Through this, it is hoped that these findings may help to contribute to on-going efforts to develop novel therapeutic strategies and improve the clinical management of these disorders.

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.

Nodules and lateral roots are both key organs for the uptake of nutrients by plants. During nodulation, leguminous plants form root nodules, housing symbiotic Rhizobial bacteria able to fix atmospheric nitrogen, allowing the plant to utilise it. Lateral roots are formed by all plants and allow the root system to be extended laterally, increasing the region of soil from which nutrients may be taken up. Formation of lateral roots and nodules share developmental features such as single cell-type origins of the primordia, and hormonal and nutrient regulatory mechanisms, so it is hypothesised that the evolution of nodulation co-opted elements of pre-existing genetic mechanisms of lateral root formation. To test this hypothesis, Arabidopsis thaliana (non-legume) genes similar to known Medicago truncatula (legume) nodulation genes were screened for phenotypic effects. Mutants of Arabidopsis NODGS and a GRAS-domain SCR-like transcription factor were found to confer lateral root phenotypes, suggesting evidence for the co-option hypothesis. The mutants were examined further using cell-type specific transcriptomics through Fluorescence-Activated Cell Sorting (FACS) to identify genomic components underlying the possible co-option. For the purposes of future research, the translation of FACS transcriptomics to Medicago was evaluated, validating microarray probe design for the most recent genome annotation but also highlighting challenges faced in analysing more complex plant roots. The GRAS-domain SCR-like transcription factor mutant was found to modulate lateral root development through pathways involving the phytohormone gibberellic acid (GA). Treatment with GA rescued some components of the GRASdomain SCR-like transcription factor phenotype, indicating a potential role for the gene in activating GA biosynthesis. A second mutant, of NODGS, was also found to affect lateral root development with some dependence on nitrate level. Existing knowledge suggested a role in root morphogenesis and flagellin-triggered signalling, and this work implies a level of cell-type specificity in gene function.