Mutation in the human CHX10 homeobox gene causes microphthalmia, and the ocular retardation (Chx10^{orJ/orJ}) mouse models this phenotype. Lack of Chx10 causes reduced proliferation of retinal progenitor cells, resulting in an abnormally small neural retina, as well as loss of the optic nerve and failure of bipolar cells to differentiate. This study aimed to characterise the molecular mechanisms through which microphthalmia arises, to assess the importance of FGF signalling in Chx10-mediated control of early retinal development, and to assess whether Chx10 acts cell autonomously or regulates intercellular signalling. Real time RT-PCR and RNA in situ hybridisation was used to compare the expression levels and patterns of a set of genes potentially regulated by Chx10 in wild type and Chx10^{orJ/orJ} embryonic neural retinas. Increased expression of a number of RPE-specific genes was observed in the Chx10^{orJ/orJ} retina, indicating a role for Chx10 in maintenance of the neural retina/RPE boundary, whereas expression of several genes involved in cell cycle progression, cell differentiation and FGF signalling was decreased. Blocking FGF signalling in wild type embryonic retinal explants, cultured ex vivo for 24 hours, resulted in Chx10^{orJ/orJ}-like gene expression, implying FGF signalling is required for mediation of Chx10 activity. Addition of exogenous FGF to Chx10^{orJ/orJ} explants did not rescue the gene expression defect, indicating Chx10 is required to potentiate the FGF signalling mechanism. Finally, chimaeric embryos were generated combining wild type and Chx10^{orJ/orJ} cells, to assess the cell-intrinsic requirement for Chx10 expression. Defects in the timing of the onset of neuronal differentiation were not rescued in Chx10^{orJ/orJ} cells. However the presence of wild type cells was sufficient to reduce abnormal expansion of the ciliary body and to increase the number of Chx10^{orJ/orJ} cells in the neural retina, indicating a role for Chx10 in regulating extrinsic factors critical for normal eye development. This study shows that Chx10 plays an early role in maintaining the boundary between the neural retina and the RPE in the embryonic optic cup. It suggests a Chx10/FGF regulatory loop is important for maintaining retinal progenitor cell identity and regulating the onset of retinal histogenesis.

Campylobacter infections are the most common bacterial cause of human diarrhoeal illness. When stressed, C. jejuni can enter a viable but not culturable (VBNC) state, which may aid survival in harsh environments. Although resuscitation has been reported in live animal models and eggs, other investigators have been unable to replicate these experiments. The importance of the VBNC state is therefore still under debate. Development of a reliable protocol for inducing resuscitation would greatly aid further investigations. The Rpfs produced by Actinobacteria spp are molecular signals capable of promoting resuscitation. The hypothesis under investigation in this thesis was that the resuscitation of VBNC C. jejuni is controlled by a Rpf orthologue. Bioinformatics searches demonstrated that the predicted C. jejuni NCTC 11168 protein, Cj0645 shares: domain content and arrangement, sequence similarity, and genomic synteny, with M. luteus Rpf. This strongly suggests a shared ancestry. A two-pronged approach was taken to investigate whether Cj0645 was capable of resuscitating VBNC C. jejuni. Firstly, attempts were made to produce a recombinant protein for functional analysis. However, none of these attempts were effective in producing soluble protein. The second approach involved the construction and phenotypic testing of an isogenic mutant and a genetically complemented strain. Phenotypic testing demonstrated that mutation of cj0645 affects growth rate, and the process of cell division, causing the cj0645 mutant to form filaments. However, experiments testing the ability of conditioned media to promote growth from the VBNe state and low cell density found no evidence of a Rpf-like activity in exponential phase C. jejuni culture supernatants. Since the cj0645 is known to be expressed during this growth phase, it appears that, unlike M. luteus Rpf, Cj0645 does not promote growth and as such is not a orthologue.

This thesis explores the interactions between adaptor proteins containing Src homology domain 3 (SH3) domains and their potential binding partners. Biochemical, biophysical and in vivo studies were used to investigate the structure, function and dynamics of two SH3-containing adaptors CIN85 and Grb2 in apo and ligand bound states. The project aimed to understand the role of CIN85 and Grb2 as key modulators in signalling cascades and the significance of their interactions with other proteins at the physiological level. The interaction between CIN85 and Cbl/Cbl peptides was investigated. The global architecture and dynamics of CIN85 in apo and Cbl bound states were reported for the first time. NMR studies reported that all three SH3 domains of CIN85 binds to Cbl derived peptides in CIN85ABC, however the interaction have a complex mechanism due to existence of complex dynamics between the SH3 domains and linker regions. However, the study identifies that the SH3 domains in CIN85 works in cooperation with each other with the linkers contributing towards the dynamics and global conformation of CIN85 in the presence and absence of Cbl, which is yet to be understood. The interaction between CIN85 and ubiquitin was investigated by in vitro and in vivo approaches. Ubiquitin binds to all three isolated SH3 domains of CIN85 with diverse affinities in vitro. However the complex between CIN85ABC and ubiquitin was weak and physiologically insignificant. No interactions were observed in cell studies between CIN85ABC and CIN85FL with ubiquitin. Finally, the complex between Grb2 SH3C domain with FGF receptor 2 peptide shows a novel mode of interaction investigated using NMR and docking studies. This work shows that SH3-adaptors have discrete preference for ligands and cannot be generalised as linkers in signalling cascades. Each SH3 domain in adaptors displays their function and subtle regulatory character that is interwoven with their structure and dynamics.

In this work, I studied the in vivo functions of Kidins220 (Kinase D-interacting substrate of 220kDa, also known as ARMS; ankyrin-repeat rich membrane spanning), by phenotypic analyses of full and conditional Kidins220 knockout mice. Kidins220 is a large transmembrane protein involved in neurotrophin and Eph/ephrin signalling, with roles in neurite outgrowth, neuroprotection, and synaptic modulation. I demonstrate pleiotropic functions of Kidins220 during development of the nervous and cardiovascular systems, and a novel function downstream of vascular endothelial growth factor (VEGF)/neuropilin-1 (Nrp1) pathway. Kidins220-/- embryos develop smaller than wildtype, and die around birth. Analysis of Kidins220-/- brains revealed increased apoptosis in specific brain regions. Increased apoptosis was also observed in the peripheral nervous system, suggesting that it mediates the survival of specific cell populations. Kidins220 also regulates proliferation in the cortex. Enlargement of the heart and outflow tract defects are observed in Kidins220-/- mice, suggesting its function in neural crest cells. In vitro experiments confirmed Kidins220 as mediating neurotrophic response. Vascular defects are also observed in Kidins220-/- brains. Glomeruloid vascular tufts formed in the Kidins220-/- brains are similar to that of Nrp1-/- mice. Nrp1 is a coreceptor to VEGF receptors and I observed that Kidins220 coimmunoprecipitates with VEGFR2 and VEGFR3. The presence of angiogenesis defects specifically in the brain point to the spatially and temporally restricted functions of Kidins220. In addition, I have also identified the presence of alternative splice isoforms of Kidins220 only found in the adult. The splicing will yield a shorter Kidins220 protein lacking many of its protein-protein interacting domains, suggesting that it plays a different function from that of the conventional Kidins220 isoform. This work therefore reports novel functions of Kidins220 during embryonic development, likely through its involvement in the neurotrophin, ephrin and VEGF receptor pathways, and novel splice isoforms of Kidins220.

Death associated protein kinase (DAPk) is a multidomain protein kinase that plays a key role in the promotion of programmed cell death. A number of different stimuli such as TΝF-α, TGF-β, IFN-γ, ceramide, c-Myc oncogenic transformation and matrix detachment are all known to activate DAPk, suggesting that DAPk lies at an important convergent point for the different cell death signalling pathways. DAPk-induced cell death is not entirely dependent on the presence of functional p53 or caspases and is highly conserved in C. elegans, rodents and human. The pro-apoptotic functions of DAPk are dependent on its kinase domain as well as the death domain (DAPk-DD). The structure of the kinase domain has been solved, but no experimental structural information is available for the DAPk-DD. The death domain belongs to the death fold superfamily that also includes the death effector domain, the caspase recruitment domain, and the Pyrin domain. These domains all share a common architecture of a six α-helix bundle arranged in an antiparallel manner. The death domain is a protein-protein interaction motif found in different signalling proteins involved in apoptosis, inflammation and NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) signalling. The role of the death domain is to promote the assembly of large multimember protein complexes, such as the DISC and the apoptosome, required for signal transduction purposes. This project aimed to tackle the specific structural parameters of the DAPk-DD; an important step in understanding the biological function of this enzyme. An efficient protocol was developed for the production of milligram quantities of human DAPk-DD in a stable and soluble form. Purification and buffer optimization provided the necessary sample homogeneity and high concentration solubility required for downstream applications, respectively. The multimeric state of DAPk-DD was assessed hydrodynamically, while biophysical techniques such as NMR, CD spectroscopy and fluorimetry were used to explore the 3D shape of DAPk-DD. Further analysis was performed on the in vitro interaction of the DAPk-DD with a newly identified partner, extracellular signal-regulated kinase (ERK2). GST pull-down assays highlighted the necessity of an intact DAPk-DD for interaction with ERK2, while ITC estimated the ratio and affinity of the complex. CD spectroscopy was also performed on the complex to monitor structural changes upon mutual binding. Overall, the DAPk-DD proved to have an unusual structural behaviour not previously seen in other members of the death domain superfamily that puts into question its classification as a classic DD.

To gain insights into the endogenous role of flavin-containing monooxygenase 5 (FMO5), the phenotype of male Fmo5 knockout mice was investigated. Up to 22 weeks of age Fmo5 -/- male mice have a similar body weight to wild-type animals. However, from 30-weeks of age the knockout mice have a reduced weight gain. Gonadal fat deposits are smaller in young (4-week old), but are greatly reduced in older knockout animals. Despite the lower body weight and reduced fat deposits, the older Fmo5 -/- mice show increased food intake. They do not exhibit increased activity but have an increased metabolic rate. Proteomic results of 30-week old mice revealed downregulation of cytosolic malic enzyme, which is important in the production of NADPH, a major cofactor required in the biosynthesis of fatty acids. Older mice have reduced plasma glucose and the distribution of glycogen in the liver of the Fmo5 -/-mice is changed. Plasma total cholesterol, HDL and LDL are reduced in the knockout mice from 15 weeks of age. The rate limiting enzymes of cholesterol metabolism were analysed in 30-week old mice by real time PCR. The mRNAs for HMG CoA reductase and Squalene Synthase were increased, suggesting that the feedback mechanism of cholesterol regulation is responsive. However, the level of cholesterol cannot be increased to that of wild type mice. HMG CoA synthase protein, but not mRNA, is down-regulated in the Fmo5-/- mice. This suggests a restriction of a substrate for cholesterol biosynthesis. The results of the studies on the knockout mice reveal that FMO5 plays a role in regulating endogenous fat and cholesterol metabolism. The glucose phenotype might be a consequence of the inability to maintain long-term energy storage deposits. The exact mechanism of how FMO5 is involved in the fat and cholesterol metabolism is not known.

Methicillin resistant Staphylococcus aureus (MRSA) is the resistant subpopulation of the bacteria S. aureus that causes a large number of hospital-associated infections worldwide. These infections could be prevented if the mechanisms of transmission and isolate or clonal success were understood. The work presented in this thesis aims to link experimental measures of fitness (biological success) with mathematical models of clinical populations to generate insights into what makes a successful clone and thereby suggest novel infection control measures. Using a new experimental fitness assay it was shown that carriage of large antibiotic resistance plasmids had little effect on fitness, but that lineage and clonal background did. This implies antibiotic resistances are likely to be maintained long term and that CC22 SCCmecIV may be the dominant MRSA clone in the UK as it is the fittest. In the light of these differences, and in an epidemiological study using data from a UK hospital (St George’s Healthcare NHS Trust), three novel reasons for clonal success are suggested: high relative fitness, flexibility of resistance carriage and resistance to fluoroquinolones. The importance of these to clonal dynamics was confirmed in a deterministic model of clonal populations. The reasons for clonal success also generated a hypothesis that use of ciprofloxacin (a fluoroquinolone) was responsible for maintaining MRSA populations. This was tested, and the magnitude of the effect, compared to other infection control methods, estimated in a new stochastic model of MRSA transmission. In conclusion, due to the small fitness costs associated with antibiotic resistance carriage, a general decline in antibiotic use may not restore susceptibility. However, using a better understanding of near universal ciprofloxacin resistance, a new infection control method is proposed: decreased use of this antibiotic will decrease the main selective pressure on MRSA populations and lead to a decline in infection incidence.

The neuronal nucleus is highly specialized with a unique and characteristic organization of the genome, transcription and epigenetic regulation. Little is known about its chromatin organization as most studies have focused on rapidly growing and dividing cells. This study has determined the ultrastructural and molecular organization of the mouse striatal neuronal nucleus and to correlate this with the distribution of a range of epigenetic modifications. The relationship of the structural organization to a functional assay of in vivo transcription detecting nascent RNA together with the localization of RNA pol I, II and III have been explored. These provide strikingly different results to the literature, foremost is the highest level of transcription mediated by RNA pol III within the perinucleolar heterochromatin. A novel mechanistic theory of the neuronal nucleolar complex is postulated which may underlie the establishment and maintenance of the post-mitotic, non-regenerating and growth restricted status of neurons. Surprisingly, this unique neuronal nucleolar complex appears to be a focus for disorganization upon HDAC inhibition and in various neurodegenerative disease models. The cellular process observed for the extrusion of TDP-43 aggregates out of the neurons in the novel TDP-43M337V transgenic mouse models of ALS/FTLD further implicates a novel mechanism for the extracellular spread of transmissible proteinopathies. In agreement with recent literature, it is speculated that prion-like propagation involving RNA-binding proteins with putative prion domains might underlie the cell-to-cell emanation of neurodegenerative pathology throughout the nervous system.

The elaborate cytoarchitecture of the mammalian neocortex requires the timely production of its constituent pyramidal neurons and interneurons, and their disposition in appropriate layers. Numerous chemotropic factors present in the forebrain throughout cortical development play important roles in the orchestration of these developmental events. The Roundabout (Robo) family of receptors, together with their ligands, the Slit proteins, are expressed in the developing forebrain, which play important roles in the formation of major axonal tracts and commissures. More recently, Robo1 has been implicated in cortical interneuron proliferation and migration, but few studies have investigated its role in the development of pyramidal neurons. Here, I investigated the functions of Robo and Slit genes in the generation and disposition of pyramidal neurons during cortical formation. I first observed expression of Robo1 and Slit genes (Slit1, Slit2) in cells lining the telencephalic ventricles. I, then, found significant increases in basal progenitor cells at E12.5, and both apical and basal progenitors at E14.5 in the developing cortex of Robo1-/-, Slit1-/- and Slit1-/-/Slit2-/-, but not in mice lacking the other Robo or Slit genes. Further, analysis of Robo1-/-/Robo2-/- double mutants indicated that Robo2 is unlikely to be involved in these proliferative events. Using layer-specific markers, I found that both early and late-born pyramidal neuron populations were significantly increased in the cortices of Robo1-/- mice at the end of corticogenesis (E18.5). The observed increase in pyramidal neurons was likely due to prolonged proliferative activity of progenitors and not to changes in cell cycle events. This finding, confirmed by delivering, through in utero electroporation, Robo1 shRNA or control constructs into apical progenitor cells along the VZ, as well as in dissociated cortical cell cultures, points to a novel role for Robo1 in regulating the proliferation and generation of pyramidal neurons. The excess number of pyramidal neurons generated prenatally appear to die in early postnatal life, but the lamination of the cortex is altered, especially in the upper layers.

The seasonal timing of plant development is regulated by environmental cues. Flowering time is influenced by the temperature and photoperiod experienced during vegetative growth, while germination timing is affected by temperatures during seed maturation and after dispersal. The timing of each developmental transition also determines seasonal conditions experienced during subsequent life stages, however the significance and stability of these interactions are not well understood. This work aimed to further an understanding of the environmental regulation of plant phenology by creating a multi-stage life history model based on Arabidopsis thaliana. Laboratory and field studies were used to inform predictive models of seed development and seed dormancy. The time required to complete seed development was mainly affected by temperature, and was therefore sensitive to seasonal flowering time. Mean daily temperatures at the end of seed maturation had the greatest influence on rates of primary dormancy loss, and post-dispersal temperatures determined rates of secondary dormancy induction. Germination probabilities were predicted by modelling frequencies of primary and secondary dormancy within the seed population. This revealed an abrupt switch from low to high germination when mean daily temperatures exceeded 14°C. Thermoinhibition was also predicted at high temperatures due to rapid secondary dormancy induction. Combining models with a previously described model of flowering time provided a framework for investigating the effects of perturbations on entire life history phenology. Seed set timing in spring and winter annuals was consistently predicted to coincide with mean daily temperatures of 14°C in locations across Northern Europe, resulting in the production of both dormant and non-dormant offspring. Phenotypic plasticity at each growth phase also served to buffer against modest perturbations in germination date, flowering date, and climate in order to maintain these specific dispersal conditions. This result was interpreted as evidence for a robust bet-hedging germination strategy.