A method was developed based upon soil dispersion using an ion exchange resin and differential centrifugation that allowed the selective isolation of Streptomyces spores rather than mycelia; this allowed the characterisation of a germination/sporulation cycle in situ. The method was able to detect relatively low numbers of streptomycetes in soil, through its concentrating action. The ecology of a temperate actinophage derived from 4'C31, containing the thiostrepton-resistance gene, was studied in conjunction with strains of Streptomyces lividans. In sterile amended soil, phage numbers showed initial increases due to a corresponding germination of the host spore inoculum; subsequently phage numbers declined when the host streptomycete was no longer in the mycelial state and hence receptive to phage infection. Lysogens were readily obtained in sterile amended soil; use of the spore extraction method and another method that isolated both spores and mycelia enabled lysogenic mycelia to be first detected after 2 days and lysogenic spores after 5 days. Phagemediated gene transfer of the thiostrepton-resistance gene from a lysogenic donor to a non-lysogenic recipient was also demonstrated in sterile amended soil. In nonsterile soil, host growth was considerably retarded with respect to sterile amended soil, although phage numbers showed a similar pattern with an initial burst of activity followed by subsequent decline. Lysogens were only rarely found in nonsterile soil and were also found to be less fit in situ than the parent strain. No difference was found in sterile amended soil.

The expression of eukaryotic genes is controlled by non-coding regulatory elements such as promoters and enhancers, which bind sequence-specific DNA-binding proteins (transcription factors). In multicellular organisms, the characterisation of these elements is required in order to understand how a single genome is utilised to generate a multitude of cell types, and how aberrant regulation of transcription contributes to disease processes. This involves the identification of transcription factor binding sites within regulatory elements that are occupied in a defined regulatory context. Digestion with DNase I and the subsequent analysis of regions protected from digestion followed by high-throughput sequencing (DNase-seq footprinting), allows for the quantification of genome-wide transcription factor binding. However, the handful of methods for analysing DNase-seq data has not been extensively validated or benchmarked. This thesis describes a novel footprinting algorithm, Wellington, which is presented in the context of a comprehensive comparison of several other DNase-seq footprinting algorithms on a multitude of datasets. Wellington outperforms other methods in almost all situations. An open-source software package, pyDNase, that facilitates interacting with DNase-seq data and provides many tools for DNase-seq analysis is also presented. Wellington is used to perform footprinting on clinical samples to validate cell lines as a model system, and to identify the binding partners of the RUNX1/ETO fusion protein in t(8;21) AML. By expanding the Wellington method, differential footprinting is shown to be able to link differences in transcription factor binding at promoters to changes in gene expression. Applying this methodology to a range of haematopoietic cell types illustrates the ability for differential footprinting to identify key regulators in the haematopoietic lineage. These results represent advances in the methods available to analyse DNase-seq data (all of which have been released as free, opensource software) and demonstrate the power of integrating DNase-seq footprinting with other functional genomic assays to study transcriptional regulation.

Intrinsically disordered proteins (IDPs) are functional proteins which lack a unique and stable tertiary structure. IDPs such as n16N are involved in biomineralisation, the process by which organisms produce mineral materials, such as shells. Here, the role that accelerated simulation can play in the study of IDPs is examined and furthered. The coarse-grained models PLUM and PRIME20 are implemented and refined based on existing single-chain n16N simulations. In conjunction with the replica exchange molecular dynamics technique, the models are used to simulate systems of 1, 2, 3 and 6 chains of n16N, and a mutant form n16NN. The modified PLUM model is in striking agreement with existing hypotheses regarding the structure of n16N, when simulations are run in multiplicity. The PRIME20 model has difficulty producing plausible backbone structure in every system size, though it does fulfil some expectations regarding residue interaction specificity. New hypotheses are offered on bulk n16N and n16NN aggregation based on the presented data. Future directions for development of accelerated simulation techniques for IDPs are suggested.

Ribosome-inactivating proteins (RIPs) are produced by many plants and inhibit ribosome function through an N-glycosidase activity that removes a single adenine residue from a universally-conserved stem-loop structure close to the 3' end of the large subunit rRNA. This site specific action is also retained on 'naked' rRNA, but usually with a much lower catalytic efficiency. Although all known RIPs are active on mammalian ribosomes, their activity on ribosomes from other sources varies considerably. In the work reported, the action of two RIPs with different substrate specificities has been studied on ribosomes and sub-ribosomal derivatives from Escherichia coli. The RIPs are pokeweed antiviral protein (PAP), a single chain RIP from the leaves of Phytolacca americana and the catalytically active A-chain (RTA) from the heterodimeric toxic lectin ricin from the endosperm of the castor oil bean. The former RIP is active on native E. coli ribosomes, whereas the latter is inactive but both are active on naked rRNA. Hence, it is postulated that the ribosomal proteins in the native ribosome either allow, in the case of PAP, or prevent, in the case of RTA, action on the target site in the rRNA. The aim of the work is to use ribosome dissociation and reconstitution techniques to study the relationship between ribosomal proteins and the activity of PAP and RTA. The initial part of the work concerned establishing conditions under which both PAP and RTA show high levels of discrimination in activity between native ribosomes and naked rRNA substrates. A buffer that contained Ca2+ in place of the more usual Mg2+ was shown to produce such discrimination. However, in the case of RTA action, the relatively mild treatments resulting in ribosome dissociation were sufficient to allow action. Various subparticles were prepared from purified 50S subunits through the successive removal of ribosomal proteins by increasing ionic strength. As more proteins were split off, the activity of PAP decreased, whereas the activity of RTA remained nearly constant. This is consistent with the hypothesis that ribosomal proteins modulate the activity of the two RIPs differently. In an attemot to use a small defined RNA substrate with which to studv the [influence?] of ribosomal proteins on RIPs' activity the region encoding domain VI of 23S rRNA (containing the RIP target site) was sub-cloned from a cloned rmB operon using PCR and used as a template for the synthesis of transcripts in vitro. These transcripts were susceptible to depurination by PAP, but for unknown reasons were refractory to RTA. Using gel retardation analysis, it was shown that total 50S ribosomal proteins (TP50) bound to domain VI transcripts in an RNA sequence specific manner, and that the reconstituted complex was relatively stable. However, the activity of PAP on this reconstituted RNP was equivalent to that on the transcript alone, and RTA was inactive on both. These results are discussed in relation to the influences of Mg2+ and Ca2+ ions and to the possible role of ribosomal proteins L3 and L6.

Rinderpest virus (RPV) is a morbillivirus which causes a highly contagious disease affecting members of the order Artiodactyla. The viral L protein is the catalytic subunit of the RNA-dependent RNA polymerase, but requires the P protein for activity. In previous studies it was found that, in addition to a direct L-P interaction, both the C and V non-structural proteins bind to L. The L proteins of morbilliviruses consist of three long highly conserved domains separated by short unconserved sequences. The interaction of P, C and V with these three domains was studied. Using co-immunoprecipitation, it was shown that P interacts with the first domain, whilst C and V were each shown to interact with the central domain. Further mutational analysis using the yeast two-hybrid system (Y2HS), showed that the P binding site lies in the amino-proximal domain of L, between amino acids 1 and 233, which fits with the co-immunoprecipitation data. However, the Y2HS suggested that the binding site for C and V includes a region between amino acids 1 and 363 of L, i.e. within the first domain. These data indicate (i) that the P binding site is distinct from that ofC and V, and (ii) that the C and V binding site(s) may be complex. To search for host cell proteins with which L interacts, a library screen was performed using the Y2HS and a porcine macrophage cDNA library. Three host cell proteins were recovered from the library screen as putative L interactors. The interaction with one of these, striatin, was confirmed by co-immunoprecipitation, and co-localisation of the two proteins was observed by confocal microscopy. The L sequence with which striatin interacts was investigated. Like the C and V proteins, striatin was shown to interact with the second conserved domain of L by co-immunoprecipitation and Y2HS data indicated that a possible second binding site for striatin includes a region of L sequence between amino acids 1 and 363.

The twin arginine tranlocation (Tat) pathway is responsible for the transport of folded protein across the membrane. In bacteria, this occurs at the cytoplasmic membrane. In Gram-negative bacteria, Tat forms a 3-component machinery named TatABC. The current hypothesised mechanism is compiled from the model organism Escherichia coli. In Gram-positive bacteria the Tat machinery lack the TatB component and so raises the question on the validity of the mechanism assumed from the TatABC system. To date there is limited research available on the TatAC system with in Gram-postive bacteria. Recent characterisation has been focused on the Bacillus subtilis system, which contain two TatAC systems; the TatAdCd and TatAyCy. This thesis focuses on the structural characterisation of the TatAC system of B. subtilis and their similarity to the TatABC system in E. coli. The TatAdCd complex was studies by electron microscopy (EM) to show structure similarity to the TatBC complex. Mutation within the N-terminus region of the TatAy protein showed functional involved in complex assembly. The TatAyCy was also analysed by EM show conserved round complexes similar to the TatAdCd and TatBC. The similarity in structure may suggest the Gram-negatives and the Gram-Positives share a similar mechanism of transport despite difference in the components.

The identification and treatment of bacterial infections remains a major healthcare challenge, especially to ensure appropriate application of a limited spectrum of antibiotics. Therefore the development of alternatives to antibiotics and new analytical tools to probe pathogenic infection processes and as point-of-care biosensors is crucial to combat the spread of infectious diseases. Glycopolymers offer many opportunities for interfacing synthetic materials with biological systems. However, the nature of the interactions between glycopolymers and their biological targets, lectins, and the structural features necessary to obtain high-affinity materials are not fully understood. The application of synthetic glycopolymers to anti-adhesive therapies has so far been limited by their lack of lectin specificity. Herein a number of tandem post-polymerisation modification methods are utilised to probe the multivalent inhibition of a bacterial toxin as a function of linker length, carbohydrate density, and glycopolymer chain length. Guided by structural-biology information, the binding-pocket depth of the toxin was probed and used as a means to specifically improve inhibition of the toxin by the glycopolymer. Glycosylated gold nanoparticles that change colour due to lectin-mediated aggregation may find use as biosensors to aid in the detection of infectious diseases and biological warfare agents such as ricin. Here, carbohydrate-functionalised, gold nanoparticles have been used to discriminate between lectins and bacterial phenotypes. Optimisation of the particle coating is required to ensure stability in complex media, but still allow for rapid detection readouts.

VanS is a sensor histidine kinase which forms part of a Two-Component System, with the response regulator, VanR. This system regulates inducible transcription of genes responsible for glycopeptide resistance in enterococci and actinomycetes. In the presence of an antibiotic inducer, VanS autophosphorylates at a conserved histidine residue, and transfers this phosphate to VanR. This phosphorylated form of VanR activates transcription of vanHAX genes, which confer resistance to glycopeptide antibiotics. This research investigates the structure and function of VanS proteins derived from E. faecium and S. coelicolor, which exhibit different antibiotic resistance phenotypes. The focus is on improving understanding of how glycopeptide antibiotics can either, directly or indirectly, induce VanS activity in each species, and if direct, to identify any ligand binding sites. To date, only one study has shown a direct binding between VanS (derived from S. coelicolor) and a vancomycin glycopeptide (Koteva et al., 2010), so this protein (termed VanSSC) was chosen as a control in ligand binding assays, alongside VanS proteins derived from E. faecium (termed VanSA). Full-length VanS proteins were purified in a functionally-active state and analysed for their structure and ligand binding properties by NMR spectroscopy. High resolution 2D NMR spectra of the isolated VanS sensor domains in each protein have been collected for the first time, and provided a platform for conducting NMR-based ligand titrations. Chemical Shift Perturbation Analysis of the resulting NMR titration data indicates several residues in VanS involved in binding to vancomycin. In conjunction with fluorescence assays, this data newly suggests that both VanSA and VanSSC can interact directly with vancomycin. This challenges consensus belief in the literature that VanSA should only be activated indirectly. NMR-based assays outlined here now pave the way for further in-depth studies of the ligand binding mechanism and putative identification of other ligands in this Two-Component System.

Cell migration is of crucial importance for many physiological processes, including embryonic development, wound healing and immune response. Defects in cell migration are the cause of chronic in ammatory diseases, mental retardation and cancer metastasis. Cell movement is driven by actin-mediated cell protrusion, substrate adhesion and contraction of the cell body. The emergent behaviour of the intracellular processes described above is a change in the morphology of the cell. This inspires the main hypothesis of this work which is that there is a measurable relationship between cell morphology dynamics and migratory behaviour, and that quantitative models of this relationship can create useful tools for investigating the mechanisms by which a cell regulates its own motility. Here we analyse cell shapes of migrating human retinal pigment epithelial cells with the aim to map cell shape changes to cellular behaviour. We develop a non-linear model for learning the intrinsic low-dimensional structure of cell shape space and use the resultant shape representation to analyse quantitative relationships between shape and migration behaviour. The biggest algorithmic challenge overcome in this thesis was developing a method for efficiently and appropriately measuring the shape difference between pairs of cells that may have come from independent image scenes. This difference measure must be capable of coping with the widely varying morphologies exhibited by migrating epithelial cells. We present a new, rapid, landmark-free, shape difference measure called the Best Alignment Metric (BAM). We show that BAM performs highly within our framework, generating a shape space representation of a very large dataset without any prior information on the importance of any given shape feature. We demonstrate quantitative evidence for a model of cell turning based on repolarisation and discuss the impact our proposed framework could have on the continued study of migratory mechanisms.

In this project I have undertaken an analysis of the enzymes and pathways of energy metabolism in Eimeria, with a particular view to determining how it is affected by, and adapted towards, the different environmental conditions encountered during its life cycle. The study has been dominated by two main approaches: characterisation of glycolytic enzymes, and analysis of end-products. In some areas it has been possible to carry out comparative studies using the related coccidial species Cryptosporidium parvum and Toxoplasma gondii. The activities of selected enzymes of energy metabolism were measured in an attempt to determine which pathways of energy metabolism were present in the parasites, and to asses their relative significance in different species and stages. Eimeria, Toxoplasma and Cryptosporidium all contained high levels of the glycolytic enzymes phosphofructokinase and pyruvate kinase, as well as lactate dehydrogenase. The TCA-specific enzyme succinate dehydrogenase could not be detected in Eimeria or Cryptosporidium but was present in tachyzoites of T. gondii. T. gondii and E. tenella contained an NADP+-specific isocitrate dehydrogenase but no NAD+-dependent enzyme; C. parvum contained neither isocitrate dehydrogenase variety. The activities of pyruvate kinase and lactate dehydrogenase were significantly higher in bradyzoites than in tachyzoites of T. gondii, suggesting that the former may be more reliant on fermentative modes of energy generation. In E. tenella, the enzyme activities investigated were generally much lower in sporulated than in unsporulated oocysts, probably reflecting the dormant nature of the latter. Sporozoites contained high levels of most enzymes indicating that significant activation or synthesis of enzymes occurs during, or shortly after, excystation.