This thesis examined genetic and phenotypic aspects of production of farmed Red deer in the UK. Heritabilities for weight traits tended to be moderate to high. Selection on weight at a given age will tend to lead to a correlated increase in weight and all ages and has implications for increased calving difficulty and higher maternal overheads. Animals of Wapiti and Eastern European parentage tended to have higher liveweights than those of British parentage pointing to their possible use as 'terminal' sires. Care is needed when selecting hinds to cross with these stags. Older dams were more likely to have a successful pregnancy and calve earlier. Calving traits tended to have low genetic variation. A central performance test was set up to improve across herd linkages. It is concluded that in future the test should start earlier and a lower limit on the weight of animals going on test should be set. The traits that were included in the economic breeding objective for Red deer included number of calves weaned, hind and offspring food consumption, stag calf and hind carcass weight and hind calf liveweight at 15 months. It was concluded that antlercharacteristics should be excluded from the breeding objective as they have no monetary value in the UK deer industry, but they may be included in selection criteria if they can be shown to improve the accuracy of breeding value prediction. Sources of variation in carcass traits and weight traits were investigated using linear body measurements and photographic techniques. Heights and girths were found to be the best predictors of weight traits. Weight was found to be the best predictor of carcass composition. Recommendations are made for future research. These include the setting up of cross breeding and selection experiments for more accurate parameter estimation and the heterotic effects of using Wapiti and animals of European parentage. Farmers are encouraged to use artificial insemination and the BDFA and MAFF are advisedto set up a performance recording scheme.

The localisation of the prokaryotic metallothionein (MT) divergon smt (which includes the MT gene smtA and a divergently transcribed gene smtB] was examined, and smt deficient mutants of Synechococcus PCC 7942 (strain R2-PIM8) have been generated by insertional inactivation/partial gene deletion mediated by homologous recombination. The structure and homozygosity (of the smt region) of these mutants, designated R2-PIM8(smt), was confirmed by Southern analyses and plasmid recovery in Escherichia coli (involving the generation of a ca. 7.8 kb plasmid from Soil digested R2-PIM8(smt) DNA). Furthermore, smtA transcripts were not detected in R2-PIM8(smt) RNA. Viability of R2-PIM8(smt) reveals that smt performs no essential role in Synechococcus under these culture conditions. R2-PIM8(smt) has reduced tolerance to Zn(^2+) and Cd(^2+), and short term reduced resistance to Ag(^+). Restoration of Zn(^2+) tolerance was used as a phenotypic selection to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment containing an uninterrupted smt divergon. These smt-restored cells also exhibited restored Cd(^2+) tolerance. Hypersensitivity to Cu(^2+) or Hg(^2+)was not detected in R2-PIM8(smt) indicating independence of Cu(^2+) and Hg(^2+) resistance to smt-mediated metal tolerance. Sequences upstream of smtA (Including smtB and/or the smt operator-promoter) fused to a promoterless locZ, conferred metal-dependent β-galactosidase expression in R2-PIM8. At maximum permissive concentrations for growth, β-galactosidase assays revealed Zn(^2+) to be a more potent elicitor of metal-dependent expression from the smtA operator-promoter than Cd(^2+). Equivalent experiments, in R2-PIM8(smQ and R2-PIM8(smtA+/B-) (containing functional chromosomal smtA and non-functional chromosomal smtB), revealed that smtB encodes a repressor of smtA transcription. In addition, it is demonstrated that SmtB can act in trans. It is proposed that Zn(^2+) is the most potent (metal ion) inducer of SmtB mediated derepression of smtA transcription. Furthermore, β-galactosidase assays indicated that, in addition to SmtB, other regulatory elements (including a transcriptional activator) are involved in the regulation of expression from the smt operator-promoter. Restoration of Zn(^2+) tolerance was also used as a phenotypic selection to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment, containing functional smtA and non-functional smtB. The resulting transformants, R2-PIM8(smtA+/B-), exhibited increased (early) tolerance to Zn(^2+) and Cd(^2+) as compared to R2-PIM8(smt-. reintroduced ) (equivalent to R2-PIM8).The work presented in this thesis proposes a role for SmtA in Zn(^2+) homoeostasis/metabolism and Cd(^2+) detoxification. SmtB is confirmed to be a trans-acting inducer- (metal ion) responsive negative regulator of smtA. The phenotype of R2-PIM8(sm(A+/B-) (with respect to metal tolerance) has significance regarding previous work (Gupta et al., 1993. Molecular Microbiology 7, 189-195), in which analysis of the smt region of Synechococcus PCC 6301 cells selected for Cd(^2+) resistance, by stepwise adaptation, revealed the functional deletion of smtB. It was proposed that loss of smtB may be beneficial for continuously metal challenged cells. Loss of smtB, now shown to encode a repressor of smtA transcription, is shown to confer constitutive derepressed expression from the smtA operator- promoter and determine an (early) increase in metal (Zn(2+)/Cd(^2+)) tolerance.

Parasitic helminths are able to survive within their hosts due to their ability to dampen immune responses by secreting molecules with anti-inflammatory and tissue repair properties. Reflecting this, there is increasing evidence of an inverse correlation between parasitic worm infection and the incidence of allergic and autoimmune disorders on a global scale. Such epidemiological evidence has led to the “hygiene hypothesis” which postulates that the recent rapid eradication of parasitic worms in developed countries has resulted in unbalanced hyper-reactive immune systems and consequently, inflammatory disease. As “worm therapy” per se is not ideal, this in turn triggered the idea that exploiting the ability of helminth-derived “immunomodulators” to dampen pathological host inflammation would potentially allow identification of the key pathogenic events in models of human inflammatory disease and hence provide a starting point for development of new and safer therapeutics. Consistent with this, as a serendipitous side-effect of its anti-inflammatory actions, ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae exhibits therapeutic potential in mouse models of inflammatory disorders such as asthma, lupus and rheumatoid arthritis (RA). RA is a chronic autoimmune inflammatory disorder that affects 1 % of the population in industrialized countries, with no known cure. This disorder causes joint destruction and leads to reduced mobility and disability. Deregulation of T cell activation has long been considered to be a major force driving inflammation and thus to date, therapies have focused on systemic anti-inflammatory treatments, which generally leave individuals immunosuppressed and open to infection. Thus, interest has begun to focus on the role(s) that synovial fibroblasts (SF) in the joint play in the early onset of the disease, the maintenance of established inflammation and even in the spread of disease to unaffected joints. This reflects that despite not being part of the immune system, SF produce pro-inflammatory cytokines during the pathogenesis of RA and also directly mediate joint destruction by secreting matrix metalloproteinases (MMPs) that damage cartilage and bone. Indeed, there is increasing evidence that the local pro-inflammatory environment pertaining in the joints drives SF to become 3 imprinted pathogenic aggressors that initiate, drive and spread joint inflammation and bone resorption during development of collagen-induced arthritis (CIA), a mouse model of RA. Intriguingly, therefore, whilst it is established that protection afforded by ES-62 against joint inflammation and bone destruction in CIA is associated with reduced production of the pathogenic cytokine, IL-17 by  and CD4+ T cells, recent evidence suggested that ES-62 could also act directly to suppress the aggressive hyper-inflammatory phenotype of SF in the joint. The molecular mechanisms involved were not defined but interestingly, given that SF express the ES-62 target TLR4 and are the only cells in the joint to express the IL-22 receptor, the parasite product appeared to harness the inflammation-resolving and/or tissue repair actions of IL-22 to suppress SF responses during the established phase of disease. Thus the core goal of this thesis was to advance our fundamental understanding of how SF become imprinted pathogenic aggressors that initiate, drive and spread joint inflammation and bone resorption in the CIA mouse model, as a surrogate for the pathogenic events in the joints in RA. In particular, the primary major aim was to investigate the impact of the local pro-inflammatory environment pertaining during disease, specifically focusing on the signalling and epigenetic mechanisms by which IL-17 and IL-22 potentially (counter)regulate the pathogenic phenotype of SF. Complementing this, another major aim was to establish whether ES-62 acted directly to modulate the phenotype of SF and thus, to identify the key mechanisms by which ES-62 could prevent SF from promoting inflammation and bone destruction and in this way render them insensitive to pro-inflammatory signals. From a therapeutic point of view, being a large immunogenic molecule, ES-62 is not suitable for use in the clinic and thus candidate small molecule analogues (SMAs) of ES-62, based around its active PC moiety have been designed, some which mimic its therapeutic potential in a variety of inflammatory disorders. Thus, it was also important to address whether ES-62 and its SMAs were similarly able to affect SF and prevent their pathogenicity. These studies revealed that the microenvironment of the joint during induction and progression of CIA did indeed result in remodelling of the epigenetic landscape of SF and that such cell reprogramming was associated with the acquisition of a hyperinflammatory, tissue destructive phenotype. Such 4 reprogramming could be recapitulated in vitro, at least in part, by chronic exposure of normal SF to pro-inflammatory cytokines such as IL-17 and IL-1 pathogenic mediators that are found at high levels in the arthritic joint. Such reprogramming was dependent on ERK and STAT3 signalling converging on miR-155-mediated regulation of inflammatory networks via global DNA hypomethylation. ES-62 was able to counteract this by suppressing the levels of ERK, STAT3 and miR-155 signalling but rather surprisingly, this did not result in abrogation of this hypomethylated epigenetic landscape. Rather, whereas the SMA 12b appeared to act simply by preventing/reversing global DNA demethylation to suppress the induction of genes that drive pathogenesis in CIA, ES-62 induced further global DNA hypomethylation and modulation of the epigenetic landscape by inducing HDAC1: collectively these findings suggested that ES-62 might additionally induce (homeostatic) inflammation-resolving and tissue repair genes that would have translational impact in established disease. In any case, these studies suggest that the proposal to use the global DNA methylation status of RA patients as a biomarker of disease should be treated with caution.

Studies were undertaken to examine immunoglobulin repertoire diversification in cattle. Diversification was examined in a number of organs from late first trimester bovine fetuses and from the ileal Peyer’s patch (IPP) follicles of young calves. To investigate the diversification in IPP follicles, individual IPP follicles were isolated by microdissection and diversification of the lambda variable region was examined by RT-PCR and subsequent cloning and sequencing. When intrafollicular sequences from a 4 week old calf were determined and compared, two major groups could be delineated. An examination of these groups revealed clear genealogical relationships that implicated both gene conversion and untemplated somatic hypermutation as the mechanisms responsible for diversification of VX within the IPP follicles. Diversification of Vλ was also examined in early (95–110 gestational day) fetal organs. The organs examined included fetal spleen, blood, liver, thymus, ileum and bone marrow. Sequences obtained from the various organs revealed that while Vλ sequences were highly diversified in spleen, very little VλX diversification was seen in the blood, liver, ileum or bone marrow. The sequences obtained from spleen indicated that both gene conversion and untemplated somatic hypermutation could be taking place in fetal spleen. Evidence for diversification in fetal spleen was also obtained by examining expression of recombination activating genes (RAG). An examination of fetal tissues for the expression of RAG-1 found that RAG-1 transcripts were present only in fetal thymus, bone marrow and spleen. The presence of both RAG-1 transcripts and a highly diversified population of Vλ sequences implicates the fetal spleen as an organ where both Vλ rearrangement and diversification might take place in cattle.

Arsenic is an acute poison and its contamination in soil and water is widespread. Crambe abyssinica accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae. Being a non-food, high biomass crop that is naturally tolerant to heavy metals, crambe has significant potential for phytoremediation of arsenic. In order to identify the pathways involved in arsenic metabolism and detoxification in C. abyssinica, differentially expressed genes in response to arsenic exposure were isolated employing a PCR-Select Suppression Subtraction Hybridization approach. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The differential expression of transcripts corresponding to the subtracted cDNAs was confirmed by the semi-quantitative RT-PCR. ^ Arabidopsis homologs of two uncharacterized proteins from this subtracted cDNA library were further characterized for their role in As detoxification in plants. One of these two genes, AtChaC2-1 functions as a gamma-glutamyl cyclotransferase as evident from in vivo studies in yeast as well as in Arabidopsis. It plays a significant role in glutathione homeostasis and participates in gamma-glutamyl cycle to recycle Glu. T-DNA insertion AtChaC2-1 mutant plants were tolerant to arsenic toxicity due to the elevated glutathione contents. AtChaC2-1 over-expression lines were also tolerant to As presumably due to more active gamma-glutamyl cycle and an efficient Glu recycling. Furthermore, AtChaC2-1 overexpression increased the N utilization efficiency as it decreased the de novo synthesis of Glu and thereby N assimilation. ^ A second gene, AtMATE21, is an efflux protein of MATE family of secondary transporters. Heterologous expression in yeast RM1 mutant strain decreased the As accumulation in yeast presumably by efficient effluxing of As from yeast cells. Arabidopsis plants with T-DNA insertional mutation in the AtMATE21 locus were sensitive to arsenate. The AtMATE21 over-expression lines were more tolerant to arsenate and accumulated a significantly higher amount of arsenic in the aboveground parts. Both AtChaC2-1 and AtMATE21 genes have significant potential to be utilized for developing plant-based strategies for arsenic mitigation in the environment.^

Theileria annulata is an intracellular, tick-transmitted apicomplexan parasite, which causes tropical theileriosis in cattle. It undergoes a complex life cycle with several distinct stages occurring within the bovine host and tick vector. ApiAP2 proteins are key candidate transcription factors for regulation of stage specific gene expression across apicomplexans. They are differentially expressed in specific developmental stages and certain ApiAP2s bind specifically to unique DNA sequence motifs. Identification of stage-specific expression of putative transcriptional regulators, the motifs they bind to and potential target genes provided the rationale for this study to understand the molecular mechanisms that control stage differentiation to the merozoite in T. annulata. The results demonstrated that T. annulata ApiAP2s show marked differences in expression levels during the parasite life cycle. ApiAP2 target DNA motifs orthologous to those in Plasmodium and Cryptosporidium were also discovered in Theileria intergenic regions, indicating that the genes downstream are potential targets of Theileria ApiAP2s. These motifs were also found in upstream regions of up-regulated TaApiAP2 genes, suggesting possible auto-regulation and an interaction network of ApiAP2 transcription factors. Importantly ApiAP2 fusion proteins up-regulated during differentiation to the merozoite stage bound to their predicted specific DNA motifs validating that ApiAP2 DNA-binding domain structure is conserved across Apicomplexa genera. Evidence was also produced that AP2 proteins play important roles in steps that commit a cell to differentiate: TA13515D is the orthologue of the AP2G factor in Plasmodium that is a major regulator of gametocytogenesis: TA16485 may be involved in down-regulation of genes during merogony and expression of TA11145 at a higher level in a cell line competent for merogony relative to a line severelly attenuated indicated involvement in regulation of this differentiation step. Discovery of multiple nuclear factors binding to a 2x(A)CACAC(A) motif implicated in autoregulation of TA11145, together with phylogenetic evidence for a clade of related domains that bind this motif suggest that multiple competing ApiAP2s may operate to regulate stochastic commitment to merozoiteproduction. Based on this data an updated stage differentiation model has been generated, with up regulation of the TA11145 gene a key event. A C-box motif association with genes implicated in establishment of the transformed host cell (TashAT, SVSP) suggests it could be important for deregulation of this event as the parasite undergoes stage differentiation. In contrast the inverse G-box was found associated with genes up-regulated from merozoite to piroplasm. EMSA analysis of parasite nuclear extract with a G/Cbox motif probe showed that the motif is an active binding site for a stage regulated nuclear factor. Specific binding of candidate TA12015 protein to the G/C-box motif was unable to be confirmed. Taken together, these results provided evidence that ApiAP2 proteins are regulators of stage-specific gene expression in T.annulata. They also provide insight into probable ApiAP2 interaction networks and support the postulation of a differentiation mechanism conserved across the Apicomplexa. Finally, the data suggests that this mechanism is stochastic and is likely to occur via a positive feedback loop generating a threshold that commits the cell to differentiate to the next stage of the life cycle.

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.