Parasite life history theory predicts that lifetime reproductive success evolves through differential allocation of energy to life history traits constrained by trade-offs. These life history traits govern the characteristics of parasites such as their virulence, transmission and infection phenotypes, so understanding their evolution is a key concern for infectious disease prediction and management. This thesis uses the powerful tool of experimental evolution to gain a fuller understanding of the factors and constraints involved in parasite life history evolution, using bacteriophage Φ2 as a model. I found that the evolution of life history in this phage is sensitive to spatial structure, UV-C exposure and coparasitism with plasmids, and evolution can be mediated by co-evolution with the host. The high levels of variance I observed here suggest that evolution of parasite life history is more complex than a single trajectory towards a predicted optimum, and likely involves some degree of epistasis or pleiotropy with genes elsewhere on the genome. There was some degree of independent evolution of individual life-history traits, indicating that simple direct trade-offs were not in operation. I demonstrated that co-evolution with the host provided additional mutational input, resulting in a greater degree of evolution in co-evolved populations than those evolved to a static host. Furthermore, I note that co-parasitism with phage and plasmid may provide the necessary conditions for plasmid persistence under fluctuating selection for plasmid-encoded traits, and that the efficacy and suitability of phage as therapeutic agents against plasmid-encoded antibiotic resistance is complicated. No direct link between mutation and phenotype could be elucidated in this study, suggesting that evolution in life history is either governed by genes not examined in this thesis, or involves epistasis and pleiotropy with genes elsewhere on the genome. I concluded that it is important to consider the specific ecology of the focal parasite, its host and any co-occuring symbionts in order to make informed predictions of life history evolution, and general predictions may not be achievable.

Neutrophils are the major cellular constituent of blood leukocytes and play a central role in the inflammatory response, expressing an array of destructive molecules and antimicrobial processes that characterise the cells as front-line defenders of the innate immune system, thus neutrophils are crucial to host defence. It is now appreciated that neutrophils produce and respond to a variety of inflammatory signals and are able to regulate both the innate and adaptive immune response. The molecular changes that underlie this regulation are poorly defined, yet represent an attractive area of research to fully elucidate the role and regulatory capacity of neutrophils within the immune response. RNA-Seq provides an accurate and robust mechanism for global characterisation of cellular transcripts. Neutrophils were isolated from healthy donors and incubated with or without inflammatory cytokines for 1 h. RNA was extracted and analysed by RNA-Seq using the SOLiD or Illumina platforms. Raw data was quantified using a number of software packages which formed a bioinformatic pipeline for data analysis which was developed during the course of the research. Results were validated by a selection of traditional laboratory functional assays. Priming of neutrophils by GM-CSF and TNFα was found to induce differential gene expression and activation of transcription factors, which led to differential regulation of apoptotic pathways. Stimulation of neutrophils with inflammatory cytokines/chemokines (IL-1β, IL-8, G-CSF, IFNγ) resulted in expression of discrete gene sets and differential activation of signalling pathways. Stimulation of neutrophils with IL-6 did not induce any significant expression of genes but result in activation of STAT signalling. Comparison of gene expression of neutrophils isolated by density gradient and magnetic bead preparation revealed significant differences in gene expression and function, in part attributable to levels of contamination associated with each isolation method. Bead isolation was found to enrich a more heterogeneous neutrophil population including a subpopulation of neutrophils expressing transcripts previously associated with low density granulocytes. Thus, RNA-Seq and bioinformatic analysis has provided a full characterisation of neutrophil gene expression under inflammatory conditions and identified several new areas of research that could lead to targeted drug design for the treatment of inflammatory disease.

This thesis applies integrative and systemic approaches to gene expression experiments measuring responses to environmental stress. Methods were developed to identify systematic differences in response strength, functional pathway activation, and gene regulatory network structure. Results in three wild killifish populations revealed high population variability at the level of individual genes, consistent with the killifish’s genetic diversity and ability to adapt rapidly to anthropogenic pollution. Despite gene level diversity, modular network structures, patterns of pathway activation, and patterns of gene expression canalization were conserved in the three populations, demonstrating that gene regulatory networks are preserved by selective processes and may constrain killifish adaptation. The presence of arsenic during killifish acclimation to osmotic shock systematically reduced the magnitude of gene expression responses, and reduced coordination between genes that respond to osmotic shock. Results in the water flea suggested that cadmium tolerance is associated with systematically larger gene expression responses to cadmium stress, and greater network coordination among genes that respond to cadmium. In summary, environmentally responsive gene regulatory networks 1) shape the efficacy of biotic and abiotic stress responses, 2) are targeted by toxic effects, and 3) are shaped by selective forces.

Signal transduction pathways are mainly depending on phosphorylation events, which are controlled by the activity of phosphatases and kinases. Although kinases have been widely studied, however, much less is known about the contribution of phosphatases to the regulation of cell signalling pathways. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is involved the regulation of a number of receptor tyrosine kinases (RTKs) including platelet-derived growth factor receptor (PDGFβR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. The differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP) was analysed. A significant change in abundance of phosphorylation on 270 phosphorylation sites from 205 proteins was associated with the lack of LAR phosphatase activity. Gene ontology analysis revealed an enrichment of LAR-mediated phosphorylation events on proteins involved in many signalling transduction pathways including those regulating the actin cytoskeleton, cell adhesion, endocytosis and cell metabolism. Analysis of putative kinases upstream of LAR-dependent phosphorylation events revealed a role for LAR in regulating signalling through mTOR and JNK. In summary, this thesis identifies an important role for LAR phosphatase in the regulation of signal transduction, cell adhesion and cell metabolism.

The aim of this work is to provide geneticists with appropriate statistical methods and computer programmes for the analysis of human pedigree data in view of mapping genes on the human chromosomes, and discovering the origin of chromosomal abnormalities such as the autosomal trisomies, the 47,XXY Klinefelter's syndrome and the 46,XX men syndrome. J.H. Edwards' marker algebra is presented in detail as used in his computer programme (MARK III) that analyses linkage with Morton's lod method for normal diploids. The programme is also described with all its specifications. The cytological mechanisms leading to autosomal trisomy are described to show that the proportion of trisomics carrying three alleles from three of their grandparents is bound to be greater than zero for any locus anywhere on a trisomic chromosome. The use of A.W.F. Edwards' method of support is then demonstrated on various sets of data to definitely exclude the ABO, MN, P, Jk, Gc and Lp loci from chromosome no. 21, and the theory is extended to show that about 401 of 47,XXY men receive an extra X from their fathers and 60% from their mothers, and that in general 46,XX men are more likely to arise from 47,XXY zygotes that lose their Y chromosomes than by an interchange between the X and Y chromosomes of their fathers.

Biocatalysis processes use biologically-derived enzymes to perform fine-chemical synthesis. Whole-cell biocatalysis, using live microorganisms, offers protection against buffer conditions and denaturation, and allows turnover of effective enzymes. However, cells may still be damaged by reaction conditions. In nature, cell populations protect themselves by attaching to surfaces and producing a multi-component protective extracellular matrix. This multicellular mode of growth is termed a biofilm. Biofilms offer many advantages over individual free-floating cells which may be beneficial in whole-cell biocatalysis. The primary aim of this work was to develop a biofilm platform using non-pathogenic Escherichia coli strains as a generic host for various biocatalysis enzymes. To this end, a simple, inexpensive and reliable biofilm generation method was developed and optimised using quantitative assays and confocal laser scanning microscopy. Reporter gene technology was used to provide insight into the expression of the matrix component curli. Flow cytometry was employed to reveal curli expression heterogeneity in biofilm-forming populations. Biofilm-modulating plasmids were used to determine whether improvements could be made to the biofilm-forming strains and their relevant effects were observed. Lastly, three biocatalysis processes were tested in the biofilm biocatalyst with observation of effects on biofilm formation, curli expression and biocatalytic potential.

Alternative splicing occurs in more than 90% of human genes and is particularly abundant in the nervous system. It has been recognized that toxicity can be caused at the level of pre-mRNA processing and potentially lead to age-dependent neurodegeneration upon low-dose chronic exposure. ELAV (Embryonic Lethal Abnormal Visual system)/Hu family proteins are prototype RNA binding protein and gene specific regulators of alternative mRNA splicing in the nervous system. Analysis of mutants in ELAV family proteins shows overlapping and distinct functions during development and age-dependent neurodegeneration. Overexpression of ELAV family proteins further revealed that cytoplasmic localization of ELAV family proteins in associated with enhanced neurotoxicity. Intriguingly all Drosophila ELAV family proteins and mammalian Hu proteins can regulate neuron-specific alternative splicing of Drosophila neuroglian gene- a known ELAV target. The blood brain barrier (BBB) and efficient excretion are protective mechanisms making delivery of many drugs to the brain difficult in vivo. Therefore, I analyzed the roles of a number of key Organic Anion Transporter Protein (OATP) and Multi- Drug Resistance (MDR) proteins and established a sensitized genetic background for CNS drug delivery. To assess if xenobiotics can interfere with ELAV function leading to neurodevelopmental/neurodegenerative defects, I assessed ELAV regulation of its major target erect wing (ewg) using an ewg fluorescent reporter, which recapitulates endogenous ELAV-mediated splicing and allows rapid visualization of potential modulators. From a compound screen in a sensitized genetic background, I identified a number of xenobiotics that cause changes in ewg splicing, indicating interference with ELAV function. Importantly, these compounds also phenocopy specific characteristics of ELAV mutants. My approach demonstrates the potential for using Drosophila in drug screening and neurotoxicity assessments.

Trypanosoma brucei, the eukaryotic parasite that causes human African trypanosomiasis in humans, evades the immune system through antigenic variation. T. brucei antigenic variation involves the periodic switching of the variant surface glycoprotein (VSG) coat to an antigenically distinct variant. A single VSG is expressed on the cell surface at any one time, but the T. brucei genome contains a vast number of silent VSGs. To be expressed, a VSG must be located in a specialised VSG blood stream form expression site (VSG BES). Silent VSGs are copied into VSG BES by homologous recombination. Several proteins have been demonstrated to be involved in this process but how VSG switching is initiated remains unclear. Four putative DNA repair factors were identified in T. brucei, whose eukaryotic homologues play a range of roles in DNA repair and other aspects of genome maintenance. These were two RecQ-like helicases, a Mus81 endonuclease and a Pif1 family helicase (PIF6). To examine whether these factors play a role in DNA repair and VSG switching, mutants were generated in blood stream form T. brucei cells. Analysis of RecQ1 by RNAi knockdown revealed it to be an essential gene in bloodstream form T. brucei, possibly involved in nuclear DNA replication. Phenotypic analysis of recq2 mutants suggests that RECQ2 is involved in the repair of a range of DNA damaging agents. Furthermore, analysis of survival following DSB induction suggests RECQ2 is involved in the repair of DNA DSBs, including those in the active VSG BES. VSG switching analysis showed that recq2-/- mutants have an elevated VSG switching rate and increase in recombination events upstream of the active VSG. These analyses suggest that RECQ2 suppresses VSG switching in T. brucei by suppressing recombination events near the active VSG. Analysis of mus81 mutants showed mus81-/- mutants to be sensitive to agents inducing replication stalling and DNA breaks, and that MUS81 is important in the repair of DSBs. PIF6 appears to be a complicated DNA repair factor, different from MUS81 and RECQ2. pif6+/- and pif6-/- mutants appear to be more resistant to MMS than wild type cells, though more sensitive to the replication stalling agent hydroxyurea. pif6 mutants do not appear to be more sensitive to DSBs than wild type cells and may even be more resistant. It is unclear whether PIF6 is involved in VSG switching and more work is required on this factor to attempt to understand its DNA repair and VSG switching function in T. brucei. These analyses shed light on the DNA repair functions of four previously uncharacterised T. brucei proteins. In particular, observations that RECQ2 is deficient in repairing DSBs upstream of the active VSG and mutants exhibit an elevated VSG switching rate cannot be reconciled with current thinking that direct formation of DSBs in this location initiates VSG switching. This suggests that the initiation of VSG switching is more complex than currently thought and requires careful further study and consideration of the relevance of using direct DSBs in this location to model VSG switching.

In commercial barley cultivars meiotic crossover (CO) distribution is skewed to the distal regions of the paired chromosomes. This restricts recombination to these regions thereby reducing the potential genetic variation that can be exploited in plant breeding programs. The aim of this project was to develop experimental strategies that will enable the frequency and distribution of meiotic crossovers to be modified in order to generate progeny with novel gene combinations. Treatment with the histone deacetylase inhibitor trichostatin A, led to significant modifications in crossover frequency in a concentration-dependent manner with lower concentrations not greatly impacting fertility, allowing for the extraction of fertile seeds. The genetic screening of a treated marker population at The James Hutton Institute (JHI), demonstrated subtle but significant shifts in the distribution of meiotic recombination, indicating that modifying recombination through chemicals applied via the transpiration stream is indeed feasible in barley and hence, possibly in other cereals. The cytological study of a barley desynpatic mutant \(des8\) in collaboration with JHI revealed that synapsis is normal despite reduced chiasma frequency. Genetic mapping studies are in progress to identify the mutant gene responsible for this phenotype which will help us to improve our current knowledge of meiosis in barley.

Two major reasons for the failure of central nervous system axon regeneration are (i) lack of neurotrophic factors available to CNS neurones and (ii) the presence of molecules that inhibit the growth of axons. In this study a gene therapy approach using adeno-associated virus 8 (AAV8) was used to manipulate these two factors. The following major aims were addressed: (i) confirm the bioactivity of transgenes that would be packaged into the AAV8 vector; (ii) assess the cellular tropism of AAV8 in the dorsal root ganglion (DRG); (iii) evaluate the inflammatory responses of the nervous system to AAV8 after intra-DRG and intrathecal injection; (iv) determine the axon regenerative effect of AAV8-mediated delivery of nt-3 (a neurotrophic factor) and shRNA\(_{RhoA}\) (a disinhibitory therapy) to dorsal root ganglion neurones after spinal cord injury in the rat. Delivery of the nt-3 transgene in vitro resulted in production of high levels of NT-3 protein. Transfection of shRNA\(_{RhoA}\)-containing plasmids into cell lines resulted in a marked decrease in the amount of RhoA detectable in cell lysates. AAV8 was found to preferentially transduce large diameter, proprioceptive DRG neurones (DRGN) but in the context of a significant inflammatory response after intra-DRG injection 28d following intra-DRG injection. Axon regenerative effects of AAV8-mediated transgene delivery before lesioning were ambiguous and further work need to be undertaken to clarify this matter.