The mammalian central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. However, retinal stem cells have been isolated from the ciliary epithelium of the mammalian retina. ChxW is a paired-like homeobox transcription factor gene expressed in the presumptive neural retina of the invaginating optic vesicle. The Chx10 gene is expressed in the proliferating retinal progenitor cell population throughout retinal development hence is one of the earliest characterised RPC-specific markers. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, it was showed that in the ocular retardation mouse model lacking Chx10 (Chx1(frJ/orJ), dividing cells persist in the adult CNR, suggesting the existence of a dormant stem/progenitor population. The neurosphere-forming assay is a tool which has allowed scientists to study the behaviour of neural stem/progenitor cells in vitro. Here, I show that cells deriving from the CNR of the adult microphthalmic retina are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Muller glial cell markers. My results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR possible deriving from the abnormal appearance of GFAPpos Muller glia in late embryonic stages of the Chx1(frJ/orJ retina. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors. One of the cardinal features of stem cells is their differentiation potential and multipotency. My experiments illustrate that Chx10 rJ/orJ CNR-derived neurospheres are able to differentiate in a similar fashion to wildtype CE-derived neurospheres. Furthermore, when neurospheres lacking Chx10 are placed in conditions that promote differentiation, they significantly up-regulate the expression of photoreceptor genes in comparison to wildtype. Hitherto, the developmental origin of CE-derived neurosphere-forming retinal stem cell is unclear. The ciliary body, where the CE is located in adult mammals, includes cells of mesodermal, neural crest and neural ectodermal origin. Here, data collected from lineage tracing analysis and in vivo Brdll-tagging experiments suggest that neurospheres are formed from BrdUpos cells observed in vivo, and that these cells originate from the embryonic anterior forebrain. The comparative analysis of the microphthalmic CNR retinal progenitors and CE-derived progenitors provides valuable information on cell properties relevant for potential cell-based replacement therapies, as well for retinal regeneration potential in mammals.

Conservation studies have generally focused on the environmental and biological issues affecting the decline of a species. However, to ensure the sustainability of a species three aspects must be considered: biological, economic and sociological. This thesis focuses on a case study of salmon in the Exe catchment comprising three individual studies, and explores the temporal stability of the species across the South West region to assess the status of salmon in other catchments within the region. A panel of microsatellite markers modified from the West Virginia panel was utilised in exploring the population genetics of Atlantic salmon. Weak population structuring was found within the Exe supported by a number of population genetic statistics. This information was provided to hatchery managers who modified their protocols utilising two strains of fish rather than stocking the catchment with fish from only one tributary. The catchment level estimate of effective population size was consistently reported to be lower than the management target (MT). A questionnaire distributed to more than 200 anglers suggested that despite a wide variation in opinion the consensus was that as a group they are happy with current management practices but that there are still some changes that could be made, for example, many respondents felt that the some groups involved in decision making regarding salmon management should have a different level of input than they currently have. The majority of anglers were positive about the use of hatcheries as a management tool. The efficacy of practices at two South West hatcheries (Exe and Tamar) was assessed. Both hatcheries suffered from a decreased level of genetic diversity as a result of the use of a small number of adults and a bias in parental contribution. Finally, a spatio-temporal analysis of the region suggested that spatial distribution is a dominant force to temporal variation in shaping the population structure of Atlantic salmon in the region. A distinct divide was observed between populations of salmon in the easterly chalkstream rivers (e.g. the Test, Itchen and Frome) and those in the west of the region (e.g. the Exe, Taw and Fowey). The findings of these studies are discussed in a wider context and support the following recommendations: 1. If a hatchery is to be used on the Exe catchment multiple stocks should be reared to reflect the populations found. 2. Stakeholders, including representatives of angling groups should be involved in conservation and kept up to date with research undertaken and results found. 3. If the Exe and Tamar hatcheries are to be continued, more broodstock should be utilised to improve levels of genetic diversity in the offspring. 4. Effective population size should continue to be monitored at a number of key sites across the region. These sites should be selected following more in depth studies of population structure at a catchment level.

Rapid phenotype identification and screening is a relatively unexplored field compared with genotype screening probably owing to a lack of appropriate technology. The Lensless microscope has a large field of view and allows the capture of the diffraction pattern from a large number of cells simultaneously, its potential to screen growth phenotypes will be evaluated in this thesis. A simple algorithm has been developed to measure intensity changes in the Airy Disc First Fringe (ADFF) from which length and width dimensions can be derived from scattering objects with an accuracy of 5%, except for those lengths below 6 microns which have diffraction-limited measurements. A low refractive index growth medium was developed to allow growth phenotypes under normal and silver-stressed conditions to be measured for the three model organisms, S. pombe, E. coli and S. aureus. Phenotype classification parameters were derived from the growth curve of these organisms from which a total of 18 growth phenotypes were identified. All three cell populations exhibit survival phenotypes for both transitions from planktonic to surface growth, typically 98%, and from natural to stressed growth conditions at sub-lethal concentrations of silver. In S. pombe growth phenotypes of interest involve the movement into a possible G0 growth phase of the cell cycle on exposure to silver and a skewed ratio of monopolar to bipolar growth rate increase not previously observed. S. aureus growth under silver stress displayed asymmetric growth of the colonies under silver stress. Analysis of the lag period parameter in the normal growth population of S. aureus identified 4% of the population which have the characteristics of a known growth phenotype, Small Colony Variants. The lag period parameter also identified two cell populations of E. coli under normal conditions, with 20% of the colonies demonstrating a significantly shorter lag period length than the remaining 80%. More importantly, a high sub-lethal dose of silver ions induces two growth phenotypes in E. coli, called here ‘sub-bug’ with parameters indicating an increased resistance to the silver stress growing slowly and a second sub-population with similar enhanced silver resistance that grew rapidly, a ‘super-bug’, which has a shorter lag period, a faster growth rate and reaches a much larger colony size. Genomic analysis demonstrated that these two growth types were genetically identical and are therefore a silver resistant growth phenotype.

The genome of an organism is not uniformly mutagenic. The overall aim of this project was to identify cis and trans factors that may contribute to such differential mutagenic activities within the genome using Saccharomyces cerevisiae. A well-characterised recombination reporter construct, hisG-URA3-hisG, was separately introduced into five different locations of chromosome III. Each locus had differing features with respect to their replication dynamics: three replication termination sites, two of which coincided with “Replication Slow Zones” (RSZ; Cha and Kleckner, 2002), one replication origin and a region of no discernable feature. Fluctuation analysis was used to assess the rate of URA3 inactivation at each locus. First, the effects of temperature, a replication inhibitor hydroxyurea, and ploidy were assessed. Significant differences in mutation rates existed in diploid strains heterozygous for the construct in these conditions, but not in respective haploids. The effects of inactivating various genes known to be involved in genome stability were also examined. Elimination of an essential signal transduction protein, Mec1p, or a DNA helicase required for efficient replication, Rrm3p, led to an increase in mutation rates only in diploid strains. No statistically significant effect was seen when a top2 temperature-sensitive allele was used in either haploids or diploids. In general, no cis effect was observed in any of these mutant backgrounds. The nature of genetic alterations associated with URA3 inactivation was also determined by Southern analysis for the five loci. The analysis revealed that the nature of genetic alteration is regulated in a cis manner, as URA3 inactivation was either exclusively via recombination or by small changes depending on the location of the reporter construct. These findings reveal some unexpected ways in which cis and trans factors may regulate mutagenic events in budding yeast. These will be discussed in context of eukaryotic genome instability in general.

The centrosomal pericentrin-related proteins play pivotal roles in various aspects of cell division, however their underlying mechanisms have remained largely elusive. Here we show that fission yeast Pcp1, a pericentrin-like protein, regulates multiple functions of the spindle pole body (SPB) by recruiting two crucial factors: γ-tubulin complex (γ-TuC) and polo kinase (Plo1). We isolated two temperature-sensitive pcp1 mutants, pcp1-15 and pcp1-18, that display similar abnormal spindles but with remarkably different molecular defects. pcp1-15 is defective in recruiting γ-TuC to the mitotic SPB, and crucially restoring γ-TuC localisation to the SPB suppresses the mutant. In contrast, pcp1-18 fails to recruit Plo1, which results in defects in mitosis-specific reorganisation of the nuclear envelope (NE) and consequently, impairment of SPB insertion into the NE. Strikingly, pcp1-18 is rescued by overproducing nuclear pore components or advancing mitotic onset. Consistent with these findings, Pcp1 forms a complex with both γ-TuC and Plo1 in the cell. Lastly, we also show that Pcp1 is phosphorylated by Plo1 during mitosis. Our results therefore verify Pcp1’s speculated role in γ-TuC-mediated spindle assembly and unveils its unanticipated function in Plo1-dependent mitotic entry and structural reorganisation of the NE. The central role of Pcp1 in orchestrating multiple SPB functions provides mechanistic insight into how centrosomes regulate multiple cellular pathways, and may be relevant to cancer development due to centrosomal aberrations.

Microarray technology allows the simultaneous measurement of the abundance of thousands of transcripts in living cells. The high-throughput nature of microarray technology means that automatic analytical procedures are required to handle the sheer amount of data, typically generated in a single microarray experiment. Along these lines, this work presents a contribution to the automatic analysis of microarray data by attempting to construct protocols for the validation of publicly available methods for microarray. At the experimental level, an evaluation of amplification of RNA targets prior to hybridisation with the physical array was undertaken. This had the important consequence of revealing the extent to which the significance of intensity ratios between varying biological conditions may be compromised following amplification as well as identifying the underlying cause of this effect. On the basis of these findings, recommendations regarding the usability of RNA amplification protocols with microarray screening were drawn in the context of varying microarray experimental conditions. On the data analysis side, this work has had the important outcome of developing an automatic framework for the validation of functional analysis methods for microarray. This is based on using a GO semantic similarity scoring metric to assess the similarity between functional terms found enriched by functional analysis of a model dataset and those anticipated from prior knowledge of the biological phenomenon under study. Using such validation system, this work has shown, for the first time, that ‘Catmap’, an early functional analysis method performs better than the more recent and most popular methods of its kind. Crucially, the effectiveness of this validation system implies that such system may be reliably adopted for validation of newly developed functional analysis methods for microarray.

The enteric nervous system (ENS) in vertebrates is derived from neural crest cells which emerge during embryogenesis from the hindbrain and, following stereotypical migratory pathways, colonize the entire gastrointestinal tract. Assembly of enteric ganglia and formation of functional neuronal circuits throughout the gut depends on the highly regulated differentiation of enteric neural crest stem cells (eNCSCs) into a plethora of neuronal subtypes and glia. The identification of eNCSCs and the lineages they generate is fundamental to understand ENS organogenesis. However, the study of the properties of eNCSCs has been hindered by the lack of specific markers and genetic tools to efficiently identify and follow these cells in vivo. Although previous in vitro studies have suggested that Sox10-expressing cells of the mammalian gut generate both enteric neurons and glia, the differentiation potential of these Sox10+ cells in vivo is currently unclear. Here, we have developed a genetic marking system which allows us to identify Sox10+ cells and follow their fate in vivo. Using this system we demonstrate that Sox10+ cells of the gut generate both enteric neurons and glia in vivo, thus representing multilineage ENS progenitors. To examine whether the neurogenic potential of Sox10+ eNCSCs is temporally regulated over the course of gut organogenesis, we generated additional transgenic mouse lines expressing a tamoxifen-inducible Cre recombinase (iCreERT2) under the control of the Sox10 locus (Sox10iCreERT2). Activation of iCreERT2 in Sox10iCreERT2 transgenic mice at specific developmental stages and analysis of enteric ganglia from adult animals showed that the pool of Sox10+ cells progressively lose their neurogenic potential. These findings raise the question of the origin of multilineage ENS progenitors isolated from cultures of post-neurogenic gut. By combining genetic fate mapping in mice, cultures of enteric ganglia and an ENS injury model, we demonstrate that glial cells in the adult ENS retain neurogenic potential which can be activated both in vitro and in vivo, in response to injury. The signals that lead Sox10+ progenitor cells to become either neurons or glial cells remain unclear. We hypothesized that the receptor tyrosine kinase RET may be part of the molecular fate switch between the two lineages being able to divert differentiation of eNCSCs away from the glial lineage and towards the neuronal fate. Here, we describe a genetic strategy to attain persistent expression of RET in vivo, in a temporally and spatially controlled manner. Such a strategy will allow us to assess the role of RET in ENS differentiation during development. Taken together, our data provide a framework for exploring the molecular mechanisms that control enteric neurogenesis in vivo and identify glial cells as a potential target for cell replacement therapies in cases associated with congenital absence or acquired loss of enteric neurons.

An important problem in developmental biology is to understand how precise patterns of cell types are maintained during development. Eph receptor tyrosine kinases and ephrins have key roles in stabilising these patterns of cell organisation and segregation during development and can restrict the movement of cells by promoting cell repulsion. Previous work by Alexei Poliakov in the Wilkinson lab has shown that Eph-ephrin signalling leads to directional persistence of migration, and modelling suggests that this can contribute to cell segregation. In order to test experimentally the contribution of directional persistence in cell segregation, I have used and developed in vitro assays to dissect the roles of EphB2-ephrinB1 signalling in cell segregation, boundary sharpening and directional persistence. In these assays, stable HEK293 cell lines expressing EphB2 or ephrinB1 are mixed in cell culture and this leads to segregation of the two cell populations. Plating these cells either side of a removable barrier and allowing migration of cells towards each other leads to the formation of a sharp boundary on interaction. Analysis of cell behaviour shows EphB2 cells to move more persistently after interaction with ephrinB1 cells. To analyse how EphB2-ephrinB1 interactions lead to directional persistence of migration, my studies have focussed on the role of components potentially involved in directional persistence that act downstream of EphB2-ephrinB1 signalling, including the planar cell polarity (PCP) pathway (Dishevelled and Daam1) and core polarity components such as the PAR proteins (PAR-3 and PAR-6B). The PCP and PAR components were all found to have roles in cell segregation, as siRNA-mediated knockdown of each of these components disrupted EphB2-ephrinB1 mediated cell segregation and boundary sharpening. However, cell behaviour studies showed that only Dishevelled and PAR-6B have roles in EphB2-ephrinB1 mediated directional persistence, whilst Daam1 knockdown has no effect on the migratory response of cells. PAR-3 knockdown affects the basal ability of cells to migrate, potentially due to its role in establishing front-rear polarity. Taken together, these findings can be explained by a model in which Dishevelled and PAR-6B have a role in EphB2-ephrinB1 mediated directional persistence required for cell segregation and boundary sharpening. I propose that Daam1 may function in the contact inhibition of locomotion between cells also required for segregation.

Compartmentalisation is a fundamental feature of biological systems. The organism as a whole can be seen as a single compartment of the wider ecosystem. At lower scales, we observe biological processes compartmentalised into organs, cells, cell subtypes and organelles. In the highly complex discipline of immunology, compartmentalisation is key in order to respond e ciently to foreign antigens and to maintain the balance between immunity and tolerance. Recent studies have raised questions about the role of compartmentalisation in lipid membranes, from the relatively well described immunological synapse, to the smaller, more transient lipid raft or microdomain. This thesis asks whether, and how, microdomains could in uence the formation of small receptor complexes. Speci cally, we approach what appears to be a simple surface reaction-di usion problem from multiple viewpoints: explicitly simulating particle di usion using a probabilistic pixel-based model, and deriving a deterministic relation between spatial parameters and the timecourse of chemical concentrations throughout the model space. We also show the equivalence between the predictions of these two models, further supporting the validity of our approach. We also embed the results of our model output in an existing model of the immunological response in order to determine the downstream consequences of enhanced receptor organisation. The study gives a broader understanding of the mechanisms involved in microdomain-mediated protein sorting, highlights the degree of interdependence on multiple spatial and chemical parameters and suggests numerous avenues for future research.

Cellular metabolism is one of the core processes for cell growth and proliferation. This process is altered in cancer cells as most solid tumours exhibit increased glucose uptake and lactate secretion, a feature known as the Warburg effect. These metabolic changes are the consequence of oncogene activation, loss of tumour suppressor function and/or mutations in metabolic enzymes. However, cancer cell metabolism is not limited to the Warburg effect and the exact role the metabolic machinery plays in facilitating proliferation and cell survival in different cancer types is still poorly understood and requires further study. Breast cancer is a complex and heterogeneous disease at the molecular level. In addition, the PI3K/AKT signalling pathway is frequently activated in breast cancers due to loss of the PTEN tumour suppressor, oncogenic activation of PIK3CA or overexpression of certain growth factor receptors. This study aimed to investigate whether the metabolic requirements of breast cancer cell lines are determined by their molecular alterations. By using RNA interference (siRNA), the expression of 231 metabolic enzymes, transporters and metabolic regulators of the cellular glucose and lipid metabolism were ablated in a panel of 14 breast cancer cell lines and 3 non-malignant breast cell lines with distinct molecular characteristics. Solid breast tumours are known to have regions of high/low delivery of nutrients and oxygen that facilitate changes in the metabolic dependencies of cancer cells that reside within these areas. Moreover, these solid tumours that contain regions of poor oxygen delivery are associated with cancers refractive to treatment and that have poorer overall survival. Thus, to examine the metabolic dependencies of cells that reside in these regions, an environment of low oxygen was recapitulated and the effect of silencing of metabolic genes on cell survival was assessed. Crucially, this approach has led to the identification of previously known and novel metabolic genes that are essential for survival of breast cancer cells for each of the defined breast cancer subgroups. In addition, the characterisation of the metabolic requirements and processes revealed that each subgroup displays a distinct metabolic phenotype that might provide potential novel molecular targets that could be exploited therapeutically.