Claspin is a large, acidic, DNA binding protein required for the ATR mediated activation of Chk1 in response to DNA replication stress. Upon checkpoint activation Claspin is phosphorylated which allows binding to Chk1. This binding in turn promotes the phosphorylation and activation of Chk1 by ATR. Although these findings have led to the designation of Claspin as a mediator protein in the activation of Chk1, the broader biological functions of Claspin have not been extensively explored at the cellular level, in part due to the lack of a genetically tractable system in which to study Claspin function. The purpose of this study was to use reverse genetics in order to generate a Claspin knock-out cell line with which study Claspin function at the cellular level. This was to be achieved by exploiting the recombinogenic vertebrate DT40 cell line, a pre B-cell line of chicken origin. The chicken homologue of Claspin was successfully isolated and characterised. The chicken Claspin orthologue shares 56% identity with human Claspin and the overall domain structure of Claspin appears to be conserved based on sequence comparison. This information facilitated the construction of gene targeting vectors to disrupt Claspin expression in the DT40 cell line. One allele of Claspin was successfully targeted. Despite multiple attempts at disrupting the remaining allele homozygous Claspin knock-out cells were not obtained. This strongly indicates that Claspin is essential for the viability of DT40 cells. In order to circumvent this lethality, efforts were made to conditionally express Claspin in the hemizygous background to facilitate the generation of a conditional knock-out cell line. These efforts proved unsuccessful. Other studies using a polyclonal antibody raised against Claspin investigated phosphorylation of the protein under various stress conditions. Data is presented which shows that phosphorylation of Claspin in response to replication stress is dependent on Chk1 kinase activity. However results from an in vitro kinase assay suggest that Chk1 itself does not directly phosphorylate Claspin. In addition it is also shown that Claspin is phosphorylated following inhibition of protein synthesis. Using specific kinase inhibitors against p38, JNK and mTOR, protein kinases known to be activated during protein synthesis inhibition, the involvement of these kinases in mediating this response was ruled out. The significance of this observation is yet to be determined. Overall the work presented here provides evidence that a conditional DT40 knock-out cell line will be a valuable tool in revealing novel functions of Claspin once a successful rescue of lethality is achieved.

Ero1α is a resident ER oxidase and is an important member of the oxidative protein folding machinery. It generates disulphide bonds de novo and donates them to protein disulphide isomerase (PDI), which in turn oxidises nascent substrate proteins within the ER. Ero1 activity must be tightly regulated for two key reasons: (i) it must maintain the balance of oxidised PDI to ensure oxidative protein folding can occur, but cannot be so active that the ER becomes hyperoxidising and dysfunctional, and (ii) Ero1 activity must be regulated to prevent the accumulation of hydrogen peroxide, a reactive oxygen species (ROS), within the ER. The regulation of Ero1α comes principally from 3 intramolecular disulphide bonds which are reduced by substrate upon activation, and re-oxidised upon inactivation by an unknown mechanism. Using an SDS-PAGE based assay we tested three hypotheses: that sulphenylation by Ero1α-produced hydrogen peroxide could induce re-oxidation; that an internal disulphide exchange mechanism could generate and distribute disulphide bonds within Ero1α; and that ER oxidoreductases could act to inactivate Ero1α. Having successfully expressed, purified and characterised a recombinant version of Ero1α, this was tested in a number of assays to address the above hypotheses. In vitro findings show that Ero1α is specifically and rapidly oxidised by ERp46 and PDI. Sulphenylation and internal disulphide exchange-mediated oxidation of Ero1α provided a comparatively slow and incomplete method of re-oxidation. In vivo results suggest that ERp46 and PDI may have implications in Ero1α activity regulation. Overexpression of several ER oxidoreductases had no effect on Ero1α re-oxidation after DTT challenge, whereas Ero1α oxidation was impaired slightly in PDI- ERp46 double knockdown cells. Depletion of PDI from cells results in the DTT-resistance of Ero1α, suggesting that Ero1α, PDI and glutathione are involved in an intricate mechanism of sensing and reacting to ER redox conditions. Two key ER oxidoreductases, PDI and ERp57, are oxidised in semi-permeabilised cells. Oxidation coincides with permeabilisation of the plasma membrane and the removal of cytosolic glutathione, directly implicating glutathione in the maintainence of the redox states of ER oxidoreductases. Oxidation during the permeabilisation of cells is an enzymatic process which is mediated in part by Ero1α. Semi-permeabilised cells harbour a more oxidising environment than do microsomes. This study contributes significantly to the research field by complementing several previously reported findings, as well as providing a novel investigation into the molecular regulation of Ero1α and its relationship with PDI and glutathione in the cell.

In addition to their role in fighting infection, mast cells have long been implicated in the pathogenesis of allergic and autoimmune inflammatory diseases and cancers. Increasingly, however, there is recognition that these cells may also play a part in protecting against development of pathologies. Indeed, there is increasing evidence that mast cells comprise heterogeneous phenotypes that exhibit functional plasticity to allow them to play both pro- and anti-inflammatory roles during an immune response. This plasticity appears to reflect that immature mast cells are tailored by their particular microenvironment not only to trigger protective inflammatory responses but also to limit pathology by resolving inflammation and promoting wound healing and tissue repair. Mast cells can be activated by a range of stimuli including (pathogen-derived) antigen/allergen-mediated crosslinking of antibody-bound to Fc receptors, most notably FcεRI, pathogen-associated molecular patterns (PAMP) such as bacterial lipopolysaccharide (LPS) acting on TLR4, inflammatory cytokines such as IL-33 (via the IL-1R/TLR-like receptor ST2) and tissue-derived signals such as SCF (via cKIT). During infection these stimuli provoke a response optimised for pathogen clearance however in autoimmune or allergic disease such responses can initiate and exacerbate host pathology. Thus the challenge for therapeutic targeting of mast cells in inflammatory or malignant disease is to limit mast cells with pathogenic phenotypes whilst preserving those contributing to protective homeostatic and anti-pathogen responses. Thus, as a first step, it was a core aim of these studies to generate in vitro mast cell models representing the phenotypic and maturational heterogeneity of mast cells in vivo, as these are difficult to isolate and purify due to their limited numbers in tissue. Distinct murine mast cell phenotypes, namely mature serosal peritoneal-derived mast cells (PDMC), connective tissue-like mast cells (CTMC) and mucosal-like mast cells (BMMC), the latter two subtypes both derived from bone marrow progenitors, were found to differentially respond, in terms of cytokine production and degranulation, to important immunoregulatory receptors in health and disease, namely FcεRI and TLR4. Consistent with their distinct functional responses, these mast cell subtypes were also found to display differential calcium signalling profiles in response to FcεRI and TLR4 signalling, further highlighting the importance in investigating phenotypically relevant and microenvironment-specific (serosal versus mucosal) mast cells in drug discovery programmes. Recently there has been great interest in IL-33, a pro-inflammatory cytokine increasingly recognised as playing an important role in a variety of mast cell responses associated with allergic inflammatory disorders and tumour pathogenesis. Consistent with this, whilst IL-33 can stimulate mast cell cytokine production, but not degranulation, via the IL-1R/TLR-like receptor ST2, responses to this cytokine are amplified following IgE sensitization and/or exposure to SCF or serum. Such augmented responses reflect increased calcium mobilization, PLD, SphK, ERK and NF-κB signalling and mTOR activation and can be suppressed by existing therapeutics targeting the costimulatory signal, for example, Imatinib or Dasatinib for SCF/cKIT and potentially Omalizumab for IgE/FcεR1. Moreover, IL-33/ST2 signalling can modulate mast cell responses resulting from antigen-mediated crosslinking of FcεRI and LPS-TLR4 signalling. Indeed, ST2 signalling can differentially modulate LPS/TLR4 responses depending on the presence (enhances) or absence (inhibits) of IL-33, as in the latter case ST2 acts to limit LPS cytokine production, potentially by sequestering MyD88. This receptor crosstalk is likely to occur under pathological conditions, thus targeting of such cooperative signalling may allow the downregulation of hyper-inflammatory responses, whilst leaving protective and homeostatic mast cell responses intact. ES-62 is an immunomodulator produced by filarial nematodes to dampen immune responses in order to promote parasite survival and prevent tissue damage without immunocompromising the host to infection. As a serendipitous side effect of its anti-inflammatory actions, ES-62 exhibits therapeutic potential in both allergic and autoimmune inflammatory disease and thus to further explore the potential for safe, targeted downregulation of pathogenic mast cell responses, the parasite product was exploited in order to identify signals regulating mast cell activation. ES-62 was found to be able to induce hypo-responsiveness of all three mast cell phenotypes in terms of degranulation and cytokine production in response to stimulation of FcεR1-, TLR4- or IL-33/ST2, either alone or in combination. ES-62 mediated these effects, at least in part, by mechanisms involving downregulation of PKCα (and in BMMC, MyD88) expression and calcium mobilisation and, in PDMC, potentially by subverting the negative feedback interactions of ST2 on TLR4. The precise mechanism of modulation varies both with receptor usage and mast cell phenotype as ES-62 exhibits differential effects in PDMC and BMMC. Nevertheless, collectively these data support the role of calcium-, PKCα- and MyD88- as key regulatory intersection sites in the functional crosstalk amongst these important immunoregulatory receptors and importantly, suggest they are potential targets for therapeutic intervention in pathogenic mast cell responses.

The epithelial monolayer of cells surrounding the animal, the hypodermis, Synthesises five cuticles during the nematode life cycle. The first cuticle is formed within the egg, prior to hatching, and the remainder towards the end of each larval stage. Because of the structural role of the cuticle, mutations in genes involved in assembly of this ECM can cause a spectrum of effects from lethality late in embryogenesis to alterations in the nematode shape. The severity of phenotype correlates with the severity of cuticle synthesis defects. Accordingly, two distinct mutant alleles that cause death after embryonic elongation, possibly due to failure in synthesising an intact cuticle, were characterised . One mutant, ij15, was isolated from a forward genetic screen previously performed (I. Johnstone, Glasgow University, Glasgow, UK). ij15 defines mutationally the gene stc-1, which encodes a HSP70-like protein possibly localised in the secretory pathway. The other mutant, h402, defines mutationally the gene let-607. A second let-607 allele, h189, which results in larval lethaity at the L2 stage was also analysed in this study. let-607 corresponds to the predicted gene F57B10.1, which encodes a putative bZIP transcription factor. Both stc-1 and let-607 are expressed in the hypodermis at all developmental stages. Furthermore, disruption of the function of either stc-1 or let-607 by mutation or RNAi affects cuticle synthesis in different ways. Thus, stc-1 and let-607 encode for a HSP70-like protein and a putative bZIP transcription factor required for synthesis of the cuticular ECM in C. elegans. In addition, this study defines C. elegans mutant phenotypes that can be used as indicators for gene products with controlling roles in the synthesis of this ECM.

The inflammatory autoimmune diseases rheumatoid arthritis, psoriatic arthritis and psoriasis have seen a break through in therapy by targeting cytokines in the last decade. Interleukin-17A, a potential new target, is considered as a crucial player in rheumatoid arthritis, and has been suggested to be produced by CD4+ T cells (Th17 cells). I explored the cellular sources of IL-17A in human established RA synovium. Surprisingly, only a small proportion of IL-17 positive cells were T cells without expression of a Th17 marker CCR6. Unexpectedly, the majority of IL-17A expression colocalized within mast cells. These data do not contradict a crucial role for IL-17A in RA pathogenesis, however, suggest that in addition to Th17 cells, cells of the innate immune system, particularly mast cells, may be an important component of the effector IL-17A response. Psoriasis is a common chronic autoimmune disease of the skin characterized by hyperplasia of epidermal keratinocytes with associated inflammation. IL-33 is a new member of the IL-1 superfamily that signals through the ST2 receptor and was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently broader immune potential has been discovered for IL-33 particularly via mast cell activation. With its expression at body barrier surfaces it is assumed to act as an alarmin. In this thesis I demonstrate that IL-33 expression is up-regulated in the epidermis of psoriatic lesions, compared to healthy skin, thus indicating that IL-33 may be a mediator regulating crosstalk between keratinocytes and infiltrating immune cells in psoriatic plaques. In a phorbol ester-induced model of skin inflammation ST2-/- mice exhibited reduced cutaneous inflammatory responses compared to WT mice. Furthermore, consecutive injections of IL-33 into the ears of mice induced a psoriasis-like inflammatory lesion. This was partially mast cell dependent and cellular analysis demonstrated recruitment of neutrophils to the ear. This concludes that IL-33, via activation of mast cells and recruitment of neutrophils, may play a role in psoriasis plaque inflammation. In the last part of the thesis I tested if nanoparticles can be utilized to image cytokine driven inflammation. Bio-linkages with protein-nanoparticles have been established and in vivo detection of nanoparticles performed. This final interdisciplinary outlook demonstrates a still to be established/finalized method with great potential.

Adverse growing conditions resulting from abiotic stresses e.g. pathogen attack results in large losses in crop yields. Understanding and improving plants tolerance to an unfavourable environment remains one of the objectives in the study of plant biotechnology. One growing theory for reducing plant susceptibility to a broad range of stresses is modulation of cellular energy homeostasis. NAD+ (along with ATP) is the most important of cellular energy transducers in the form of a hydrogen ion donor and acceptor. However, a bigger picture is emerging as its role broadens to include a coenzyme, precursor for secondary messengers and a substrate for protein modifications. Three groups of proteins use NAD+ as a substrate, the NAD+ protein deacetylases, poly ADP polymerases and ADP ribose cyclases. Work on these groups of proteins in model organisms have identified their involvement in many biological roles including DNA repair, increasing longevity, initiating apoptosis and regulation of transcription. However, their role in plants is largely unknown. The aim of this study has been to identify the genes involved which use NAD+ and to further characterise their role in plant stress responses. A summary of the main results follows, a) AtPARG2 and sirtuin At5g55760 null lines were more sensitive to DNA damaging agents. b) AtPARG2 lines showed a disruption in circadian rhythm resulting in early flowering. c) Sirtuin At5g09230 null lines were more sensitive to UV-B exposure d) AtPARP3 was highly upregulated with exogenous Abscisic acid application.

Biological agents, including peptides and gene delivery systems (recombinant adenoviral vectors), suffer from a relative lack of native targeting capacity. Improvement of their targeting capacity could significantly increase the efficiency of these agents to provide therapeutic effects. Selective targeting of an anti-oxidant peptide to the vasculature in the SHRSP and administration of a variety of adenovirus (Ad) 35-based vectors into transgenic mice has lead to a significant enhancement in both the effectiveness of the anti-oxidant treatment and increased understanding of the requirements needed for the generation of a successfully targeted Ad35 vector. This helps understanding of the requirements for a successful treatment for cardiovascular disease and cancer.

The connections between cell cycle exit and terminal differentiation remain poorly understood. Cyclin dependent Kinase Inhibitors (CKIs) provide a possible link between entry into the quiescent state and differentiation. The initial aim of this project was to further investigate if the CKI p27Xic1 could promote differentiation in addition to, and independently of, its well characterised cell cycle exit function. p27Xic1 has been shown to be involved in cell fate determination during gliogenesis, neurogenesis, myogenesis and cardiogenesis and many mammalian Cip/ Kip CKI homologues of p27Xic1 have been described as important regulators of cellular processes beyond control of cell division. We aimed to investigate these roles during development of the embryonic kidney, the pronephros. We discovered that p27Xic1 does not affect differentiation during pronephrogenesis, but instead controls pronephric organ size through its cell cycle exit function. In addition we identified a previously unrecognised role for the cell cycle exit function of p27Xic1 in allocation of the somites during paraxial mesoderm segmentation. Preliminary results had suggested p27Xic1 expression in the pronephros was under the control of the Notch signalling pathway. Over-expressing a constitutively active form of Notch, Notch-ICD, and a dominant negative form of the Delta ligand, DeltaSTU, showed that both mis-activation and suppression of Notch signalling inhibited p27Xic1 expression. However, when investigating the effects these overexpressions had on pronephros development, we identified novel results indicating the Notch signalling pathway, which has previously been implicated in pronephros development, is essential for the separation of the proximal lateral and medial pronephric mesoderms. This process we propose is mediated by the Notch signalling pathway through the establishment of a boundary between these two distinct populations of cells, permitting both compartments to develop in isolation. The results in this thesis suggest novel mechanisms by which cell division controls X. laevis segmentation and organ size and how the Notch signalling pathway is able to pattern the pronephros anlagen such that the different compartments of the mature pronephros are able to develop, and thus function.

In computational biology, one often faces the problem of deriving the causal relationship among different elements such as genes, proteins, metabolites, neurons and so on, based upon multi-dimensional temporal data. In literature, there are several well-established reverse-engineering approaches to explore causal relationships in a dynamic network, such as ordinary differential equations (ODE), Bayesian networks, information theory and Granger Causality. To apply the four different approaches to the same problem, a key issue is to choose which approach is used to tackle the data, in particular when they give rise to contradictory results. In this thesis, I provided an answer by focusing on a systematic and computationally intensive comparison between the two common approaches which are dynamic Bayesian network inference and Granger causality. The comparison was carried out on both synthesized and experimental data. It is concluded that the dynamic Bayesian network inference performs better than the Granger causality approach, when the data size is short; otherwise the Granger causality approach is better. Since the Granger causality approach is able to detect weak interactions when the time series are long enough, I then focused on applying Granger causality approach on real experimental data both in the time and frequency domain and in local and global networks. For a small gene network, Granger causality outperformed all the other three approaches mentioned above. A global protein network of 812 proteins was reconstructed, using a novel approach. The obtained results fitted well with known experimental findings and predicted many experimentally testable results. In addition to interactions in the time domain, interactions in the frequency domain were also recovered. In addition to gene and protein data, Granger causality approach was also applied on Local Field Potential (LFP) data. Here we have combined multiarray electrophysiological recordings of local field potentials in both right inferior temporal (rIT) and left IT (lIT) and right anterior cingulate (rAC) cortices in sheep with Granger causality to investigate how anaesthesia alters processing during resting state and exposure to pictures of faces. Results from both the time and frequency domain analyses show that loss of consciousness during anaesthesia is associated with a reduction/disruption of feed forward open-loop cortico-cortical connections and a corresponding increase in shorter-distance closed loop ones.

Live Cell Imaging and High Throughput Screening are rapidly evolving techniques and have found many applications in recent years. Modern microscopy enables the visualisation of internal changes in the cell through the use of fluorescently tagged proteins which can be targeted to specific cellular components. A system is presented here which is designed to track cells at low temporal resolution within large populations, and to extract fluorescence data which allows relative expression rates of tagged proteins to be monitored. Cell detection and tracking are performed as separate steps, and several methods are evaluated for suitability using timeseries images of Hoechst-stained C2C12 mouse mesenchymal stem cells. The use of Hoechst staining ensures cell nuclei are visible throughout a time-series. Dynamic features, including a characteristic change in Hoechst fluorescence intensity during chromosome condensation, are used to identify cell divisions and resulting daughter cells. The ability to detect cell division is integrated into the tracking, aiding lineage construction. To establish the efficiency of the method, synthetic cell images have been produced and used to evaluate cell detection accuracy. A validation framework is created which allows the accuracy of the automatic segmentation and tracking systems to be measured and compared against existing state of the art software, such as CellProfiler. Basic tracking methods, including nearest-neighbour and cell-overlap, are provided as a baseline to evaluate the performance of more sophisticated methods. The software is demonstrated on a number of biological systems, starting with a study of different control elements of the Msx1 gene, which regulates differentiation of mesenchymal stem cells. Expression is followed through multiple lineages to identify asymmetric divisions which may be due to cell differentiation. The lineage construction methods are applied to Schizosaccharomyces pombe time-series image data, allowing the extraction of generation lengths for individual cells. Finally a study is presented which examines correlations between the circadian and cell cycles. This makes use of the recently developed FUCCI cell cycle markers which, when used in conjunction with a circadian indicator such as Rev-erbα-Venus, allow simultaneous measurements of both cycles.