The Ubiquitin Conjugating System (UCS) describes a system in which the 96-amino acid residue Ubiquitin can be selectively covalently linked to intracellular proteins. This endows cells with an indispensable level of regulation to determine protein fate in a wide range of basic cellular events. The abundant, neuron specific Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is intimately involved with the UCS – both in a hydrolase and ligase capacity. Mutations in UCH-L1 have clearly been associated with various neurodegenerative disorders, including Alzheimer’s, Huntington’s and particularly Parkinson’s disease. The main and unique objective of this study was to identify any common Caucasian sequence variants in UCH-L1’s promoter, and to investigate whether they are associated with neurodegenerative symptoms, and any change in UCH-L1 transcriptional activity. Seven novel UCH-L1 Single Nucleotide Polymorphisms (SNPs), as well as the C54A documented coding region polymorphism (Ser18Tyr), were identified using both denaturing High Performance Liquid Chromatography (dHPLC) and DNA sequencing analysis. In relation to the translational start site, the novel SNPs elucidated were: A-307G, A-306G, G-234A, A-24G, C-16T, G12A and G21A. Restriction Fragment Length Polymorphism (RFLP) genotyping analysis was then employed within Caucasian DNA sample sets of 31 and 480 individuals, to firstly elucidate the common UCH-L1 promoter haplotypes that exist within the population, and secondly, in an attempt to uncover any association between the polymorphic alleles and general neurodegenerative symptoms - no association was uncovered. Using pGEM-T Easy as an initial ‘holding vector’, the three common UCH-L1 promoter haplotypes elucidated – AAGAC, GAGGT and AGAAC - were incorporated into a modified pGL3 vector to ascertain transcriptional activity rates. This was done by Luciferase expression analysis, and the results identified the GAGGT promoter haplotype as having a significantly increased transcriptional activity in all human cell lines tested. It is my contention, that the pronounced increase in transcriptional activity elucidated for the GAGGT UCH-L1 promoter haplotypes, potentially indicates a primary genetic risk factor for sporadic Parkinson’s disease in the Caucasian population – a novel pathogenic model of which is proposed in this thesis. The fact that RFLP genotyping analysis uncovered no association of the promoter polymorphic alleles with more general neurodegenerative symptoms, indicates the need for further studies to be focused more specifically towards Parkinson’s disease.

As the global population continues to increase, so will the demand for timber (and other raw materials) for building, construction, and also for the pulping industry. The high demand for wood and the increasing human population mean that natural forests are being lost and degraded. A potential solution to this problem is to improve the productivity of our plantation forests to relieve the pressure on natural forests in terms of sustainable wood production. This project is the first to use newly available microarray technology to study differential gene expression in cambial tissue of high versus low wood density field grown samples from two contrasting species, Sitka spruce (gymnosperms) and poplar (angiosperms). Genes up-regulated in high-density Sitka spruce and poplar samples had functions in cell formation and expansion, with down-regulated genes having functions in lignin biosynthesis, stress-response and defence. Plantation trees could be screened at a young age to assess their expression of candidate genes to speed up the breeding and selection process.

The work presented in this thesis involves the characterization of a small plasmid, pHD2, of approximately 2 kb in size from Bacillus thuringiansis subspecies kurstaki HDl-Dipel. The plasmid was cloned and sequenced and compared to other plasmids from Gram-positive bacteria for which sequence information was available and for which replication functions had been assigned. Homology between the predicted amino acid sequence of an open reading frame within pHD2 and the rep gene products of the pT181 group of staphylococcal plasmids suggested a common method of plasmid replication. The further identification of possible plus and minus origins indicated that pHD2 was a member of a family of plasmids replicating via a single-stranded DNA intermediate. Plasmid replication control in the staphylococcal plasmids pT181, pC221 and pS194 involves a negative control circuit using countertranscripts. Replication control in the case of pHD2 may utilize an alternative system involving the gene product of a second small open reading frame with homology to RepA of pLSl in which replication control is achieved by the binding of the repA gene product to the promoter region of the replication protein repB. pHD2 is the first plasmid from a Bacillus thuringiensis isolate to which replication functions have been assigned. The work presented here suggests pHD2 to be a member of the ssDNA family of plasmids and extends the range of such plasmids which have been characterized with the suggestion that this group contains, in addition to the highly related staphylococcal plasmids, a number of more distantly related members. Additionally, chimeric plasmids containing pHD2 and pBR322 have been demonstrated to show structural instability, although not segregational instability, in an alternative Bacillus thuringiensis host. Consequently, the use of such constructs in the cloning of heterologous genes in Bacillus thuringiensis in such a system may prove impractical at this stage with further work being required in order to overcome these problems and extend the exploitation of this industrially important family of entomopathogenic bacteria.

Spatiotemporal responses to extracellular signals have been documented in a wide variety of cells, such as neuronal synapses, cytotoxic T lymphocytes, germ cells and during embryo development. Selective release of a key molecule allows a cell to respond at a given moment, and cells ensure that the response can be initiated instantly by pre-producing and packaging the molecule, often storing the molecule as a granule. Differentially Expressed in FDCP6 is a guanine nucleotide exchange factor, primarily expressed in T cells, which has been previously shown to form cytoplasmic aggregates when phosphorylated by ITK. DEF6 also translocates to the immunological synapse following phosphorylation by LCK in response to antigenic presentation. As a result, DEF6 is a likely candidate in mediating a spatiotemporal response to an extracellular signal in T cells. Data presented in this work suggest that endogenous DEF6 forms cytoplasmic granules in a variety of T cell states and the DEF6 mutant Y210EY222E, which mimics ITK phosphorylation, interacts with mRNA. Moreover, DEF6 is hypothesised to have two unconventional RNA binding domains; a feature which has also been described in the literature within proteins that catalyse glycolysis. DEF6 is also shown to be in close proximity to PABP and eIF4E, both of which are translation factors, as well as active translation in resting Jurkat T cells and the immunological synapse. Furthermore, endogenous DEF6 co-localises with 4E-T, a P-body marker which is involved with miRNA mediated decay, in resting and stressed Jurkat T cells. These data corroborate that of Hey et al. (2012) and suggests that DEF6 does indeed interact with P-bodies. Finally, translocation of DEF6 appears to occur in response to an extracellular signal alternative to the T cell receptor during T-T communication and that the translocation may occur in vesicle-like structures in close proximity to LFA-1. Consequently, these data identify a novel link between DEF6 and active translation as well as mRNA turnover and that the extracellular signal required for this spatial response is not antigen presenting cell specific but rather a response to LFA-1 stimulation.

Pluripotency is conserved in the major trunk of Vertebrate evolution, but how the gene regulatory network (GRN) that governs it evolved is poorly understood. A central component of this network is the Homeodomain containing transcription factor Nanog. How Nanog evolved is not understood, as Nanog sequences have not been identified in invertebrate genomes. This study provides evidence of Nanog activity encoded in the homeodomain of the invertebrate Vent gene family. The Vent2 gene from Saccoglossus kowalevskii, a model hemichordate, successfully reprogrammed mammalian pre-iPS cells to pluripotency, as demonstrated by the activation of dormant pluripotency genes, and the ability to generate all three primary germ layers. A second property of invertebrate Vents was also characterised in the Vent gene found in Nematostella vectensis, a sea anemone and model for cnidarian development, expression of which was insufficient to activate the endogenous pluripotency network of pre-iPS cells, though it could induce the cells to a XEN-like state that demonstrated up regulation of extra-embryonic markers, and subsequently gained dependence on ERK signalling. A direct comparison between the Saccoglossus and Nematostella Vent homeodomains was used to provide insight into the step-wise changes that appear to have given rise to Nanog activity. Swapping the homeodomains from one CDS to another, to create hybrid molecules, I demonstrated that the respective reprogramming activities of these genes is conserved in the homeodomain. I then identified specific amino acid (AAs) differences in the homeodomains that conferred a Nanog-like capacity for reprogramming to the Nematostella gene. Identification of a Nematostella EsrrB ortholog, which demonstrated reprogramming activity in mammalian pre-iPS cells, suggests wider conservation of pluripotency factors. I therefore propose that an ancient GRN for pluripotent mesoderm evolved in vertebrates to form part of the ground state network.

In eukaryotes, the removal of the poly (A) tail of cytoplasmic mRNA (deadenylation) is a crucial step in post-transcriptional gene regulation. A major enzyme involved in regulated mRNA deadenylation is the Ccr4-Not deadenylase, which contains two catalytic subunits: the Caf1 and Ccr4 ribonucleases. For both enzymes, two Mg2+ ions are required in the active site for activity. These enzymes in addition to six other non-catalytic subunits of the Ccr4-Not complex are possible drug targets in diseases such as metastatic cancer, osteoporosis and obesity. To facilitate the discovery, development and characterisation of small drug-like inhibitors of these enzymes, a biochemical approach was used. First, we investigated the biochemical characteristics of the highly similar CNOT6 and CNOT6L enzymes. Next, we evaluated two biochemical assays for characterisation of Ccr4-Not catalytic subunits and evaluation of N-hydroxyimide inhibitors of CNOT7. A chemiluminescence-based detection assay of AMP was used as the basis of a method and thermal shift assays were also used to characterize binding of compounds to deadenylase enzymes. In addition, cell based assays were used to study interactions between BTG2 variants and CNOT7 and CNOT8 in lymphoma. Our findings demonstrate that the CNOT6 and CNOT6L though highly similar, display different biochemical characteristics in vitro. The deadenylase activity of CNOT6 was higher compared to CNOT6L. The results also indicate that AMP detection is a highly sensitive assay that can be used as a secondary assay to the previously developed substrate-based assay for compound screening and IC50 determination. We conclude that thermal shift assays can be used to determine the binding mode of inhibitory compounds, specifically regarding the presence of the Mg2+ ions in the active site. Finally, we identified BTG2 variants that potentially regulate mRNA abundance in lymphoma via interaction with the Ccr4-Not deadenylase.

Understanding root development and patterning is important for both nutrient and water uptake. The Arabidopsis thaliana primary root has a di-arch vascular pattern consisting of a central xylem axis, perpendicular phloem poles and intervening procambial cells. Governance of this pattern involves a dynamic, antagonistic interaction between domains of auxin and cytokinin signalling bias. Here, one element of this auxin-cytokinin relationship; cytokinin’s indirect transcriptional regulation of the auxin PIN-FORMED 7 (PIN7) efflux transporter, has been investigated. Two complementary strategies were employed; transcriptomic profiling of an Type-B ARABIDOPSIS RESPONSE REGULATOR (ARR) response (the last known components in the core cytokinin signalling machinery) via an inducible glucocorticoid system, and an EMS mutagenesis based forward genetic screen of reduced PIN7::PIN7:GFP expression and subsequent genomic resequencing to identify potential causative agents. Both workflows produced novel candidate PIN7 regulators and the ensuing candidate validation revealed ETHYLENE RESPONSE FACTOR 104 (ERF104), CYTOKININ OXIDASE/DEHYDROGENASE 5 (CKX5) and the ECA1-like AT5G36520 from its vascular over-expressor DOUBLE PROTOXYLEM (DPX) phenotype, in particular as strong contenders for components involved in the regulation of PIN7 and patterning of the vascular cylinder.

The control of cellular morphogenesis and differentiation at the molecular level in Rhodomicrobium vannlelii is investigated by biochemical and genetic analysis. Chemical characterization indicates that the R.vannlelll genome is 2.19 x 109 daltons, and is made up of 95 per cent unique DNA sequence. Five per cent of the DNA consists of short inverted repeat sequences, 400 bp in length. No extrachromoscmal DNA is detectable. The DNA from each of the cellular expressions in R.vannielil does not show any major differences in sequence composition. Kinetic analysis of nucleic acid synthesis during the obligate differentiation of the swarm cell shows a 'lag' or maturation period prior to the onset of DNA replication, whereas no lag occurs in RNA synthesis. The initiation of DNA replication during swarm cell differentiation occurs towards the completion of stalk synthesis. Studies with the protein synthesis inhibitor chloramphenicol during swarm cell differentiation demonstrate a requirement for protein synthesis in the initiation, but not the elongation step of DNA replication. Nalidixic acid inhibits DNA synthesis in the swarm cell, and although a new daughter cell is produced, cell division does not occur. This implies that chromosome replication and cell division cure directly linked in a 'dependent pathway' of events. Daughter cell synthesis is under the control of the mother cell genome, the daughter cell genome becoming metabolically active just prior to cell division. Further 'cells' produced in nalidixic acid-treated cultures show gross cellular distortion and no stalk formation. The development of a genetic system for R.vannielll is discussed. The promiscuous plasmid R.68.45 is transferred by conjugation from E.coll to R.vannlelll, where it expresses only one of three plasmid-bome antibiotic resistances, but may be maintained intact in the bacterium. This plasmid may now be used in mapping the R.vannielll chromosome. Analysis of the R.vannlelli genome by restriction enzyme cleavage is described. Restriction fragments containing the coding sequences for 16s rRNA and 23s rRNA are Identified by 'Southern' hybridization, and their sizes estimated. Attempts to locate the origin of replication on a single restriction fragment are discussed. Preliminary data on the physiology of nitrogen fixation in R.vannlelli shows that the enzyme system is inducible. The potential of this organism as a model system for further study on the molecular biology of microbial morphogenesis and differentiation is discussed.

Chromatin is the complex of DNA, histones and non-histone proteins that make up chromosomes and the region of dispersed chromosomes during the interphases and S phase of the cell cycle. It is found inside cell nuclei in eukaryotic cells. There are many important proteins in the nuclei which are involved in DNA replication, gene expression, DNA repair, etc. Core histones, linker histones and HMGs are the most important proteins in this group. In this thesis, a new, quick and efficient method is developed, to extract and purify proteins from cell nuclei, which is named "forward technology". By using the "forward technology", ultra-pure native core histone octamers, dimers and tetramers were extracted from chick erythrocytes. By using the pure histone dimers and tetramers, the complexes of NAP1-dimer and NAP1-tetramer were obtained in collaboration with others. Crystals of pure histone octamers with a higher resolution than before were produced (Chapter 2). A low KCI/phosphate wash of chick erythrocyte nuclei removed up to 1 g of proteins without nuclei lysis (cePNE1 proteins). The high KCI/phosphate soluble fraction of the cePNE1 is rich in HMG proteins, peptidyl-prolyl isomerases (FKBP3) and heatshock protein 70 which were fractionated by cation-exchange chromatography and anion-exchange chromatography (Chapter 3). Valuable (in terms of function) high-molecular-weight proteins were enriched, and another group of nucleoproteins called cePNE5 is fractionated by the "forward technology" from chick erythrocyte nuclei. It was confirmed that HMGB proteins prefer to bind to core histone H2A-H2B dimers (Chapter 4). It has proved possible to fractionate HMG-rich PNE1 proteins separate from a linker- histone rich nucleoprotein extract, separate from pure core histones (not as oetamers) from human tissue culture cells. The method has been applied to human leukocytes obtained from the National Blood Service. This enrichment of particular groups of proteins will be useful for proteomic studies (Chapter 5).

Freshwater environments are threatened worldwide by external stressors and biodiversity decline, with major implications for ecosystem resilience. The genetic consequences so far have been neglected, especially for freshwater invertebrates, though their abundance, diversity, ease of sampling and functional importance renders them ideal candidates for genetic appraisal. For three freshwater invertebrates (Amphinemura sulcicollis, Isoperla grammatica and Baetis rhodani) novel microsatellite markers were developed so that genetic structure, and genetic diversity could be assessed throughout upland Wales. The aim was to investigate dispersal and the genetic response to environmental stressors. Genetic diversity in these species was compared to species diversity across whole macroinvertebrate assemblages to investigate what factors might cause a correlation between these fundamental levels of biodiversity. The demographic history of each species was also investigated with the aim of assessing whether reduced genetic diversity was due to bottlenecks and more broadly, what this indicates in terms of the populations’ resilience. Species differed in their genetic structure and genetic diversity. All three species showed effective dispersal and geneflow, with each species displaying panmixa across catchments in southern and mid-Wales. However, A. sulcicollis and I. grammatica revealed genetic isolation and reduced genetic diversity at specific northern sites. Genetic and species diversity were correlated positively only in A. sulcicollis, where isolation combined with a common driver were the likely cause. There was evidence of recent bottlenecks in all three species. All these results could be explained by an underlying genetic response to post-industrial acidification: reduced genetic diversity correlated significantly with acidity for A. sulcicollis, while reduced species diversity and genetic bottleneck signatures was consistent with chronic and episodic acidification across the Welsh region. Overall, these results show how a positive correlation between species and genetic diversity can never be assumed, and illustrate how assessments of genetic health expand insights available from traditional biodiversity assessment.