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The unlimited replicative potential of cells is one of the hallmarks of cancer. Telomeres, DNA structures found at the ends of chromosomes have attracted a great deal of interest in recent years as potential anti-cancer drug targets since they play an important role in cancer cell immortality. The repetitive TTAGGG sequences of telomeres are complexed to a group of six indispensible proteins, one of which is the protection of telomeres 1 (POT1) protein. This specialised protein binds to a ten nucleotide single stranded DNA sequence at the ends of chromosomes and plays an important role in telomere capping and length regulation. It has recently been proposed that the key function of POT1 is to suppress a potent DNA damage response at telomeres thereby protecting chromosome tips from being recognised as sites of DNA damage. Deletion of POT1 from telomeres in a variety of organisms including humans results in cytogenetic aberrations, senescence and cell death. These results indicate that POT1 is an integral telomere end-protection protein which is necessary for continued cellular proliferation and therefore POT1 is becoming a promising new target in cancer. Using a structure-based approach, several small molecule inhibitors of POT1 have been designed to affect telomere integrity by disrupting the binding interaction of human POT1 with its target DNA sequence thereby driving cancer cells into senescence/apoptosis. Using a range of computational tools, a suitable drug binding pocket in POT1 has been identified and the de novo design of a specific class of POT1 inhibitor was completed. Using this novel scaffold, a small focussed library of hit-like compounds were synthesised and screened in a new POT1 fluorescence polarisation displacement assay developed by scientists at the University of Nottingham. In total, over 90 small molecule inhibitors based on two different scaffolds: pyrido[1,2-a]pyrimidines and sulfathiazoles have been synthesized with some inhibitors effectively decreasing POT1-DNA binding between 10-54% at 100μM ligand concentration. The biological results have established that electron-withdrawing substituents on the pendent phenyl ring of the pyrimidine core are essential for strong binding. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of cancer.

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Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has proven its ability to characterise (in)organic surfaces, and is increasingly used for the characterisation of biological samples such as single cells. By combining ion imaging and molecular depth profiling it is possible to render 3D chemical images, which provides a novel, label-free way to investigate biological systems. Major challenges lie, however, in the development of data analysis tools and protocols that preserve the cell morphology. Here, we develop and employ such tools and protocols for the investigation of neuronal networks. One of the reasons 3D ToF-SIMS imaging of cells is underused is the lack of powerful data analysis tools as 3D ToF-SIMS measurements generate very large data sets. To address this issue, we developed a method that allows the application of principal component analysis (PCA) to be expanded to large 3D images making 3D ToF-SIMS image processing of whole, intact cells and cellular networks with multivariate analysis now accessible on a routine basis. Using this method, we are able to separate cellular material from the substrate and can then correct z-offsets due to the cells' topography resulting in a more accurate surface heightmap. The method also facilitates differentiation between cellular components such as lipids and amino acids allowing the cell membrane, the cytoplasm and the extracellular matrix (ECM) to be easily distinguished from one another. These developments permit us to investigate the intracellular localisation of specific native and non-native compounds label-free, not just in single cells but also in larger cellular networks. The visualisation of the cellular uptake of non-native compounds, namely fluorescent dyes, in primary rat cortical neurons and the chemical differentiation between cell types, namely primary rat cortical neurons and retinal pigment epithelium (RPE) cells, are presented as applications. Even though the dyes have distinct fragment ions in the high mass range, it was not possible to detect the fluorophores by 3D ToF-SIMS imaging of freeze-dried cells. However, it was possible to detect distinct differences in the kind of ions detected for freeze-dried primary rat cortical neurons and RPE cells albeit in the low mass range. To obtain meaningful results, however, it is paramount that sample preparation does not induce significant physical or chemical changes. We present the first comprehensive comparison between large 3D ToF-SIMS images of freeze-dried and frozen-hydrated cells using PCA to facilitate the data analysis of these large data sets. A higher degree of colocalisation of the K+ signal with cell regions is observed for frozen-hydrated cells, which indicates a lower degree of membrane damage and migration of diffusible chemical species. Frozen-hydrated cell samples are therefore considered to best reflect the native cell state, but freeze-dried cell samples allow far easier sample handling. The mass spectrum of frozen-hydrated cellular material also has increased ion intensities for higher-mass fragments, which is an additional advantage, because the poor signal-to-noise ratio of molecular species with m/z > 200 is a major bottleneck in the advancement of ToF-SIMS imaging as a diagnostic tool.

Polyadenylation is a universal step in the production of all metazoan mRNAs except histone mRNA. Despite being universal, previous experiments have implicated it in the regulation of inflammation. An inflammatory system using RAW 264.7 murine macrophage cells was established with bacterial lipopolysaccharide (LPS) used as a stimulus. After improving the poly(A) tail test (PAT) method of measuring poly(A) tail lengths, it was applied to inflammatory mRNAs during the inflammatory response. Poly(A) tail length was shown to vary over the course of the inflammatory response, and for Tnf, this was even true of initial poly(A) tail size, which is widely believed to be uniform for the majority of mRNAs. The adenosine analogue cordycepin (3’-deoxyadenosine) was shown to have anti-inflammatory effects on mRNA, in line with existing literature, and is likely to be the anti-inflammatory component of Cordyceps militaris ethanol extract. Inhibition of either import of cordycepin into cells or phosphorylation of cordycepin was sufficient to abolish its anti-inflammatory effects. Adenosine treatment led to repression of Il1b mRNA, but did not repress other mRNAs tested that were cordycepin-sensitive. This suggests that cordycepin does not simply act by mimicking the effect of adenosine, and that the two compounds have distinct modes of action. Inhibiting deamination of cordycepin potentiated its effects. We also observed that pre-mRNA levels of inflammatory genes were decreased by cordycepin treatment, indicative of effects on transcription. Other groups have reported that cordycepin interferes with NF-B signalling. As NF-B is an important transcription factor for the induction of inflammatory genes, this would provide a basis for explaining our observation that cordycepin represses at the transcriptional level. However, we did not observe any changes in NF-B signalling, with degradation of IB completely unimpeded by cordycepin treatment. Notably, cordycepin did shorten the Tnf poly(A) tail, and the observed inhibition of polyadenylation is consistent with observations that cordycepin led to decreased efficiencies of mRNA 3’ cleavage and transcription termination for Tnf. Such effects on polyadenylation and 3’ processing of mRNA were hypothesised to particularly affect unstable mRNAs that depend on longer poly(A) tails for avoiding decay and/or mRNAs with a high rate of transcription. However, comparison of microarray data to data from RNA-seq of RNA from 4-thiouridine labelling experiments showed that cordycepin-sensitivity did not correlate with mRNA stability or transcription rate. Long noncoding RNAs (lncRNAs) were found to be enriched in cordycepin-treated cells. If some of those lncRNAs have regulatory roles in inflammation, cordycepin’s effects may be mediated through them. Lastly, cordycepin significantly altered pain behaviour in a rat model of osteoarthritis (OA), supporting its continued use as a lead compound for exploration of new OA therapeutics.

The immune system comprises highly sophisticated networks of cells and signalling molecules which function in concert to protect the body against pathogens. Within this system a role for the extra-cellular microenvironment as a crucial mediator of immune responses is becoming increasingly apparent. Conventional in vitro cultures lack physiologically relevant extra-cellular cues, such as extracellular matrix (ECM) and shear flow. Tissue engineering can be used to simulate features of the natural microenvironment for the development of biologically relevant platforms. It is anticipated that this will enable the study of the influence of the extra-cellular environment on immune responses. This thesis describes the development and characterisation of tissue-engineered platforms for immune cell culture which incorporate the ECM and shear flow. This work goes on to apply these platforms for the study of the effect of the extra-cellular environment on dendritic cells and their interactions with T cells in the context of immunological stimulation. The ECM defines the three-dimensional architecture of the natural microenvironment. It provides structural support and also promotes cell motility in tissues. This is important for the function of the immune system as it directs the organisation and interactions of immune cells which ultimately contributes to the modulation of immune responses. Candidate synthetic and natural biomaterials were assessed for their suitability to provide an in vitro extracellular matrix (ECM) platform for human immune cell culture. The suitability of these materials to provide an artificial ECM platform was based on the viability, resting immune state and immune competence of the cells. The synthetic biomaterials tested were a thermo-responsive colloidal gel and electrospun PET and PLGA scaffolds coated with a thermo-responsive polymer. An important finding from the work done with the colloidal gel was that the human dendritic cells, which were incorporated into the gel at the beginning of the experiment, could not be separated from the material for flow cytometric analysis. Therefore, characterisation of the colloidal gel for immune cell culture could not be completed. Regarding the characterisation of the electrospun PET and PLGA scaffolds, although they did not significantly impair cell viability of dendritic cells they were found to induce cell maturation. As a result, none of the synthetic biomaterials were found to be a suitable ECM surrogate. A semi-natural biomaterial, gelatin methacryloyl (GelMA) hydrogel, was included in the investigation. The results from the characterisation of GelMA for human immune cell culture indicated that the hydrogel induced a pro-inflammatory immune response due to the profile of secreted cytokines. Based on this, GelMA was also discounted as an appropriate material for the development of the ECM platform. The final ECM candidate was a collagen hydrogel, which is a naturally-derived biomaterial. The collagen hydrogel was shown to support immune cell survival and human dendritic cells maintained an immature phenotype in culture. In addition, typical responses to immunological stimuli by human dendritic cells and T cells were observed in collagen hydrogel cultures. This work demonstrated that out of the biomaterials which were characterised, the collagen hydrogel was the most suitable biomaterial for the development of the ECM platform. The influence of the collagen hydrogel ECM platform on antigen-specific immune responses was investigated in the context of autologous human dendritic cell and T cell co-cultures stimulated with the model antigen Mycobacterium tuberculosis purified protein derivative, also referred to as PPD. The results from these experiments indicated that the presence of the collagen hydrogel increased the sensitivity and specificity of the immune response, compared to conventional tissue culture conditions. An attempt was made at utilising the ECM platform to investigate immune responses to chemical sensitisers to address the requirement for in vitro alternatives to replace current animal testing methods. In this work, innate and adaptive immune responses to sensitisers were detected using the ECM platform. However, the reproducibility of these experiments was low due to large donor variation. Therefore the effect of the ECM platform on immune responses to sensitisers could not be evaluated. This difficulty likely reflects the complexity of the molecular and cellular mechanisms which lead to the acquisition of chemical sensitisation. Shear flow is a type of physiological stress to which immune cells are exposed in vivo due to the movement of blood and lymph fluid. Recent studies have implicated flow as an immunologically relevant stimulus, capable of inducing changes in the expression of receptors and chemokines involved in regulating immune cell migration, and activating immune receptor signalling. A fluidic cell culture platform was developed to recapitulate the effect of shear flow. Two different prototypes were constructed, one of which was taken forward and characterised for immune cell culture applications. The fluidic platform taken forward had a paper-based cell culture scaffold which was coated with collagen hydrogel. The scaffold was found to induce maturation of human dendritic cells which was attributed to the possibility of incomplete coverage of the scaffold by the collagen hydrogel. The viability of dendritic cells was slightly impaired by flow, however not significantly. Interestingly, when exposed to shear flow, dendritic cells maintained a less mature phenotype compared to their static counterparts. Antigen-specific immune responses were studied on the fluidic platform by setting up co-cultures comprising PPD-stimulated autologous human dendritic cells and T cells. Typical T cell activation was observed on the platform and the sensitivity and specificity of immune responses was found to be greater under flow conditions, compared with static cultures. In conclusion, this thesis demonstrates the value of developing biomimetic platforms for studying the influence of the extra-cellular environment on immune responses. Finally, the ability to mimic extra-cellular cues to which cells are exposed in vivo has the potential to generate more realistic immune responses in the lab. This presents huge opportunities for advancing understanding in immunology. It also has implications for methods used in research, drug discovery and safety testing, where currently only animals provide a representative system for the study of immune reactions. It is anticipated that enhancing the physiological relevance of in vitro cell culture will ultimately contribute to the reduction of animals used in research and testing.

In vertebrates, haematopoietic stem cells (HSCs) maintain the blood system throughout life. HSCs are believed to arise during embryogenesis from haemogenic endothelial cells (HECs) that undergo an endothelial-to-haematopoietic transition (EHT). Here, in order to trace the progeny of the embryonic ECs in zebrafish, an inducible CreERT2-LoxP system was used. Following short-term induction of the Cre recombinase during early embryonic stages, Cre reporter gene expression was observed in early larval haematopoietic cells (HCs). At adult stages, PCR revealed the presence of the recombined Cre reporter gene in HCs, demonstrating that adult HCs had originated from embryonic ECs. In zebrafish, HECs of the ventral wall of the dorsal aorta (vDA) are thought to form HCs by basal epithelial-to-mesenchymal transition (bEMT), a process that depends on the transcription factor Runx1. Here, making use of the recently identified gfi1aaqmc551Gt line, confocal microscopy showed that qmc551: GFP+ cells were HECs. While most GFP+ cells underwent bEMT, some displayed a novel type of apical departure. In runx1morphants, bEMT was abrogated and most GFP+ HECs remained in the vDA. Apical departure, however, was still observed in the absence of Runx1, suggesting a fundamental difference in the underlying mechanism. While gfi1aa expression was lost in vDA HECs of qmc551 homozygous embryos, EHT of GFP+ HECs was completely unaffected. Up regulation of its paralogue Gfi1ab suggested functional redundancy. To study this redundancy, the CRISPR/Cas9 system was used to mutate the gfi1ab gene. Here, the mutant gfi1ab alleles qmc552 and qmc553 were identified. Both alleles, as well as a third allele sh320 that was generated by a collaborator, encode truncated, most likely non-functional proteins. Initial data on gfi1aaand gfi1ab double mutant embryos showed a defect in definitive haematopoiesis. Whether HECs were affected and blocked in their ability to undergo EHT remains to be determined.

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.

Human schistosomiasis is one of the helminthic neglected tropical diseases. It leads to serious health problems and imposes a huge burden on communities. All interactions between the parasite and the human body occur at a molecular and cellular level. Therefore, the study of the molecular aspects of infection and the immune response is a very active area of research. It has been known for decades that there is a direct relationship between protection against infection, or reinfection after treatment, and parasite-specific Immunoglobulin E (IgE) antibody in infected individuals. This study aims to investigate the allergenicity of S. mansoni antigens using RS-ATL8 reporter cell line assay. Towards this goal, I first identified the best reporter cell line for allergenicity assessment. This was done by the characterisation of the transgenic human FcԑRIα chain’s gene copy number and by comparing the levels of human FcԑRI receptor surface expression. The second goal was the optimisation of RS-ATL-8 reporter cell line. This was achieved by the optimisation of fundamental conditions such as the cell density, sensitising agent’s optimum dilution and the stimulant optimum concentration. Once a robust standard operating procedure (SOP) had been established, I investigated the allergenicity of the four expressed S. mansoni antigens using the optimised reporter cell line RS-ATL8.

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.