The okapi (Okapia& johnstoni) is an endangered, evolutionarily distinct giraffid, endemic to the Democratic Republic of Congo (DRC). The okapi is a flagship species for the DRC,a country that contains some of the greatest biodiversity in the world. The okapi is currently under major threat from habitat fragmentation, human encroachment and poaching, yet to date, very little is known about the species in the wild, and no genetic study in the wild or captivity has ever been carried out. This thesis aims to use genetics to aid conservation efforts of okapi,a species that due to its elusive nature, is highly challenging to study using alternative methods.

Plants as sessile organisms have adapted highly sophisticated cellular processes to cope with environmental stress conditions, which include the initiation of complex transcriptional regulatory circuits. The heat shock transcription factors (HSFs) have been shown to be central regulators of plant responses to abiotic and biotic stress conditions. However, the extremely high multiplicity in plant HSF families compared to those of other kingdoms and their unique expression patterns and structures suggest that some of them might have evolved to become major regulators of other non-stress related processes. Arabidopsis thaliana HSFA1b (AtHSFA1b) has been shown to be a major regulator of various forms of plant responses to abiotic and biotic stresses. However, it has also been suggested that overexpression of AtHSFA1b results in a subtle developmental effect in Arabidopsis thaliana and Brassica napus in the form of increased seed yield and harvest index. Through genome-wide mapping of the AtHSFA1b binding profile in the Arabidopsis thaliana genome, monitoring changes in the AtHSFA1b-regulated-transcriptome, and functional analysis of AtHSFA1b in Saccharomyces cerevisiae under non-stress and heat stress conditions, this study provides evidence of the association of AtHSFA1b with plant general developmental processes. Furthermore, the outcome of this research shows that AtHSFA1b controls a transcriptional regulatory network operating in a hierarchical manner. However, in an agreement with a previously suggested model, the results from this study demonstrate that the involvement of AtHSFA1b in the regulation of heat stress response in Arabidopsis thaliana is possibly limited to the immediate and very early phases of heat stress response which also results in a collapse in its transcriptional network which seems to be accompanied by a general shutdown in plant growth and development.

Antigenic variation processes play a central role in parasite invasion and chronic infectious disease, and are likely to respond to host immune mechanisms and epidemiological characteristics. Whether changes in antigenic variation strategies lead to net positive or negative effects for parasite fitness is unclear. To improve our understanding of pathogen evolution, it is important to investigate the mechanisms by which pathogens regulate antigenic variant expression. This involves consideration of the complex interactions that occur between parasites and their hosts, and top-down and bottom-up factors that might drive changes in the genetic architecture of their antigenic archives. Increasing availability of pathogen genomic data offers new opportunities to understand the fundamental mechanisms of immune evasion and pathogen population dynamics during chronic infection. Motivated by the growing knowledge on the antigenic variation system of the sleeping sickness parasite, the African trypanosome, in this thesis, we present different models that analyze antigenic variation of this parasite at different biological scales, ranging from the within-host level, to between-host transmission, and finally the parasite genetics level. First, we describe mechanistically how the structure of the antigenic archive impacts the parasite population dynamics within a single host, and how it interplays with other within-host processes, such as parasite density-dependent differentiation into transmission life-stages and specific host immune responses. Our analysis focuses first on a single parasitaemia peak and then on the dynamics of multiple peaks that rely on stochastic switching between groups of parasite variants. We show that the interplay between the two types of parasite control within the host: specific and general, depends on the modular structure of the parasite antigenic archive. Our modelling reveals that the degree of synchronization in stochastic variant emergence (antigenic block size) determines the relative dominance of general over specific control within a single peak, and can divide infection scenarios into stationary and oscillatory regimes. A requirement for multiple-peak dynamics is a critical switch rate between blocks of antigenic variants, which depends on host characteristics, such as the immune delay, and implies constraints on variant surface glycoprotein (VSG) archive genetic diversification. Secondly, we study the interactions between the structure and function of the antigenic archive at the transmission level. By using nested modelling, we show that the genetic architecture of the archive has important consequences for pathogen fitness within and between hosts. We find host-dependent optimality criteria for the antigenic archive that arise as a result of typical trade-offs between parasite transmission and virulence. Our analysis suggests that different traits of the host population can select for different aspects of the antigenic archive, reinforcing the importance of host heterogeneity in the evolutionary dynamics of parasites. Variant-specific host immune competence is likely to select for larger antigenic block sizes. Parasite tolerance and host life-span are likely to select for whole archive expansion as more archive blocks provide the parasite with a fitness advantage. Within-host carrying capacity, resulting from density-dependent parasite regulation, is likely to impact the evolution of between-block switch rates in the antigenic archive. Our study illustrates the importance of quantifying the links between parasite genetics and within-host dynamics, and suggests that host body size might play a significant role in the evolution of trypanosomes. In Chapters 4 and 5 we consider the genetics behind trypanosome antigenic variation. Antigen switch rates are thought to depend on a range of genetic features, among which, the genetic identity between the switch-off and switch-on gene. The subfamily structure of the VSG archive is important in providing the conditions for this type of switching to occur. We develop a hidden Markov model to describe and estimate evolutionary processes generating clustered patterns of genetic identity between closely related gene sequences. Analysis of alignment data from high-identity VSG genes in the silent antigen gene archive of the African trypanosome identifies two scales of subfamily diversification: local clustering of sequence mismatches, a putative indicator of gene conversion events with other lower-identity donor genes in the archive, and the sparse scale of isolated mismatches, likely to arise from independent point mutations. In addition to quantifying the respective rates of these two processes, our method yields estimates for the gene conversion tract length distribution and the average diversity contributed locally by conversion events. Model fitting is conducted for a range of models using a Bayesian framework. We find that gene conversion events with lower-identity partners are at least 5 times less common than point mutations for VSG pairs, and the average imported conversion tract is short. However, due to the high frequency of mismatches in converted segments, the two processes have almost equal impact on the rate of sequence diversification between VSG sub-family members. We are able to disentangle the most likely locations of point mutations vs. conversions on each aligned gene pair. Finally we model VSG archive diversification at the global scale, as a result of opposing evolutionary forces: point mutation, which induces diversification, and gene conversion, which promotes global homogenization. By adopting stochastic simulation and theoretical approaches such as population genetics and the diffusion approximation, we find how the stationary identity configuration of the archive depends on mutation and conversion parameters. By fitting the theoretical form of the distribution to the current VSG archive configuration, we estimate the global rates of gene conversion and point mutation. The relative dominance of mutation as an evolutionary force quantifies the high divergence propensity of VSG genes in response to host immune pressures. The success of our models in describing realistic infection patterns and making predictions about the fitness consequences of the parasite antigenic archive illustrates the advantage of using integrative approaches that bridge between different biological scales. Even though quantifying the genetic signatures of antigenic variation remains a challenging task, cross-disciplinary analyses and mechanistic modelling of parasite genomic data can help in this direction, to better understand parasite evolution.

The traditional paradigm relying on drug discovery to treat and heal the body is changing. Medicine for the 21st century is moving towards using the body’s internal language of DNA and RNA to cure disease and repair injuries to the body. We now appreciate the complexity of signalling through the genome and its transcribed RNA. The role of micro RNAs and short interfering RNAs are gaining much interest as potential therapeutics. This interest has been sparked by the discovery that the dysregulation of micro RNAs is the origin for a spectrum of diseases from cancer through to osteoporosis. Small regulatory RNAs have been shown to influence stem cell maintenance, proliferation and differentiation, offering the potential to produce new tissue by manipulating RNA levels. However delivery of these molecules is fraught with difficulties. Without protection these molecules are quickly degraded in vivo and in vitro before reaching their intended target. With this in mind, this thesis aims to investigate the potential role for gold nanoparticles to deliver small regulatory RNAs and in turn produce a non-toxic and physiologically significant effect upon the cells. Initial investigations revealed the importance of PEG density and AuNP concentration; with lower PEG densities, allowing attached therapeutic siRNA against C-Myc to reduce C-Myc protein levels and cell proliferation. Subsequently we determined that modulating the expression of osteo-suppressive miRNA, with a nucleic antagonist sequence was able to influence osteogenesis in two cell models (MG63s and hMSCs). This thesis has shown that AuNPs can be used to effectively deliver therapeutically active small molecules to cells in vitro.

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.