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Heart failure (HF) associated with diabetes is a distinct clinical entity known as diabetic cardiomyopathy (DCM) without the prevalence of other risk factors such as hypertension. The human heart is a pool of different cell types, including cardiomyocytes, fibroblasts, endothelial cells and immune cells, interacting via various secretory factors. The cardiomyocyte acting as an active mediator of the inflammatory response is an emerging concept with limited mechanistic understanding. Superimposed effects of hyperglycaemia and hyperlipidaemia dysregulate the intercellular links, contributing to diabetes-induced cardiac dysfunction. DCM is instigated by various pathological insults, including endoplasmic reticulum (ER) stress response and ectopic fibroblast growth factor receptor 1 (FGFR1) signalling, resulting in detrimental impacts on cardiac structure and function. Hence, elucidating the paracrine and autocrine mediators of cardiac intracellular signalling can provide new mechanistic and therapeutic strategies for HF in diabetes. First, we demonstrate the opposing roles of the cardiac ER stress response in our time-course model of high fat high-sugar diet (HFHSD)-induced diabetes. Using cardiac P21-activated kinase 2 (PAK2) deficient mice, we found that PAK2, an ER-localised signalling kinase, decelerates the transition from an adaptive to a maladaptive ER response in DCM. A concurrent examination of the myocardial inflammation, particularly macrophage response, revealed chronic cardiac ER stress to be the initiating cascade of the alarmin high-mobility group box 1 (HMGB1) in DCM. HMGB1 expression and subsequent secretion into the circulation was promoted via C/EBP homologous protein (CHOP), an ER stress responder. Our in vivo DCM models of ER dysfunction, the remediated ER response and pharmacological treatments established the functional and therapeutic relevance of targeting cardiac ER stress-mediated inflammation for alleviating DM-induced cardiac complications. Secondly, we determined that the FGFR1 cascade is the upstream facilitator of the PAK2 response. In diabetes, we observed a lack of cardiac FGFR1 and beta-Klotho (β-KL) signalling. We found that restoring cardiac FGFR1/β-KL expression results in the increased secretion of anti-inflammatory and proangiogenic factors from cardiomyocytes. Using proteomic analyses, we identified key cardiac-derived signals that regulate angiogenesis and macrophage polarisation upon FGFR1/β-KL activation in diabetes. This provides a proof-of-concept to explore the novel therapeutic avenue of myocardial cell interactions to prevent cardiac dysfunction under metabolic stress. Type 2 diabetes is a chronic disorder; therefore, our time-course analyses provide a fascinating insight into the early mechanistic events in DCM. We address the knowledge gap of cardiomyocyte-mediated macrophage and endothelial cell response and reveal mechanistic links behind cardiac inflammation and vascular damage in response to diet-induced metabolic stress. Hence, further dissection of the identified molecular web between cardiomyocytes and endothelial cells or macrophages in DCM may lead to the development of efficacious treatments for diabetes-induced HF.

Cardiovascular disease, including myocardial infarction, remains the number one cause of worldwide morbidity and mortality. The major cause of myocardial infarction is arterial thrombosis, driven by platelet aggregation. Adenosine diphosphate (ADP)-induced platelet aggregation is mediated by the Gi-protein-coupled receptor (GPCR), P2Y12. Therefore, P2Y12 antagonists are clinically used to prevent thrombotic events. However, current antiplatelet drugs have several drawbacks such as the increased risk of bleeding, difficulty in fine-tuning the antiplatelet effects of irreversible antagonists, and variability in patient response. Furthermore, the nucleoside-based, reversible drug ticagrelor has been reported to cause dyspnoea due to off-target effects. Additionally, the binding modes of the P2Y12 ligands are not fully known. Interestingly, the recently solved crystal structure of P2Y12 has revealed that the orthosteric site is composed of two sub-pockets. This thesis had two complementary aims: 1) to further understand the mechanism of action of cangrelor - the most recently approved, and only intravenously acting P2Y12 antagonist; and 2) to discover novel, competitively acting, non-nucleotide-based reversible inhibitor(s) of ADP-induced platelet aggregation. A plate-based aggregometry assay and platelet-rich plasma (PRP) isolated from the blood of human donors were used to show that cangrelor (in nM and µM concentrations) may act in a non-competitive manner to ADP (up to mM concentrations). This is in contrast with reports in the literature that cangrelor is a competitive antagonist of the P2Y12 receptor. Interestingly, it acted in a competitive manner when the P2Y12 receptor was stimulated with the synthetic and more potent agonist, 2-methylthio-ADP (2MeSADP). The cangrelor analogue, AR-C66096, acted in a competitive manner with both agonists. Subsequently, a multiplexed flow cytometric assay assessing phosphorylated platelet vasodilator-stimulating phosphoprotein (pVASP) levels in platelets was successfully optimised. For this assay, a technique called barcoding was used with a novel combination of dye and fluorophore-conjugated antibody, opening a new avenue for barcoding. This assay further showed that ADP (up to 1mM) + cangrelor (100nM) Emax did not reach that of ADP (1mM) + vehicle, whereas AR-C66096 did. Electrostatic field potential analysis of the two compounds revealed that AR-C66096 had a field of negative electrostatic potential that was missing in cangrelor. Additionally, these results suggested that there may be mechanistic differences in the activation of the receptor by ADP and 2MeSADP. To achieve the second aim, ligand-based in silico tools were used to virtually screen over 440,000 molecules to identify novel scaffolds possessing reasonable similarities in 3D shape and electrostatic properties in reference to the experimental P2Y12 antagonist, AZD1283. Docking of the best hits was performed against the recently solved crystal structure of P2Y12. Following the meticulous inspection of docked poses, as well as similarity indices with the query ligand, 33 compounds were purchased for in vitro validation. From these, two competitively acting, novel scaffolds (namely compound B6 and B11) were identified, which showed consistent inhibition of ADP-induced aggregation of platelets from human blood donors. These compounds were predicted to have comparable interactions with the receptor to the co-crystallised antagonist, AZD1283. Of these two best hits, compound B6, which is a 2-aryl benzoxazole derivative, was chosen for further investigation. To establish the structure-activity relationship (SAR) analysis around the B6 scaffold, nine analogues of this compound were purchased and experimentally tested using the assays described above. This led to the identification of another novel inhibitor of ADP-induced platelet aggregation, namely compound S8. However, despite good docking profiles of the compounds against the crystal structure of P2Y12, the latter could not be confirmed as their target upon analysis of pVASP levels. Further work is required to confirm the mechanism by which these compounds inhibit platelet aggregation. To summarise, this thesis has increased our understanding of cangrelor's mechanism of action, and several 2-aryl benzoxazole derivatives are described as competitive and reversibly acting inhibitors of ADP-induced platelet aggregation.

Platelets release a variety of extracellular vesicles, including platelet-derived microparticles that expose phosphatidylserine on their surface. This thesis aims to resolve the mechanisms by which pro-thrombotic microparticles are released from platelets, which have not been well-characterised. Experimental methods used include flow cytometry, Western blotting, fluorescence-based microplate assays, confocal microscopy and electron microscopy. Intact cholesterol-rich lipid rafts were found to be required for the calpain-dependent release of microparticles from activated platelets. Microparticle release was prevented when cholesterol was depleted or sequestered from the platelet membrane. Membrane blebbing and scission in this process were independent of influx of hydrophilic ions or the endosomal sorting complex required for transport. It was found that microparticles were also released from platelets undergoing apoptosis, which would progress to secondary necrosis in vitro as they were not cleared. Microparticle release in apoptotic platelets was dependent on Ca2+ entry, intracellular Ca2+ mobilisation and caspase activity, but it was largely independent of calpain. During apoptosis, caspases downregulated microparticle release from platelets in response to pro-coagulant stimuli. This may limit the pro-thrombotic consequences by providing a period of reduced capacity for platelet activation. 2-Aminoethoxydiphenyl borate (2-APB), a non-specific modulator of ion channels, was found to inhibit microparticle release from both activated and apoptotic platelets. In activated platelets, the effect of 2-APB was not related to inhibition of plasma membrane ion channels or calpain activity. In apoptotic platelets, the effect of 2-APB might be associated with inhibition of Ca2+ entry, although the specific target remained unclear. Future studies in identifying the target of 2-APB might provide new insights into how microparticles are released from platelets. Moreover, 2-APB may provide a scaffold for developing a pharmacological inhibitor of microparticle release.

Cell membranes coordinate a large variety of biological processes by selectively recognising and responding to different external stimuli, and membrane-spanning proteins play a vital role in these signalling pathways. A large number of examples of synthetic membrane channels and transporters have been reported in the past four decades. However, signal transduction without mass transfer is considerably more challenging. In this dissertation, an indirect artificial transmembrane signalling mechanism which operates by controlled translocation of a synthetic transducer across the lipid bilayer was investigated. The fundamental concept of the translocation mechanism is the switching of membrane-permeability of the two head groups of the transducers. The recognition head group of the transducer becomes membrane-permeable in response to an external chemical stimulus, which leads to membrane translocation, exposing a catalytic head group to the interior of the vesicle. Catalytic hydrolysis of an internal substrate generates an amplified output signal. Previously reported examples showed that the transducer could respond to different external stimuli, such as pH (hydroxide ions) or metal ions. Herein, we present efforts towards generalising the concept and extending the scope of this mechanism into more biological systems. Firstly, a maleimide transducer was designed as a "stem transducer" to which a wide range of recognition groups could be attached in situ by using maleimide-thiol conjugation chemistry. Secondly, a carboxylic acid transducer was studied in an attempt to achieve controlled cargo-release in response to an acid input. Thirdly, a vesicle-to-vesicle communication system was assembled with a desthiobiotin transducer by leveraging the desthiobiotin-avidin-biotin system. This example represents a key step towards designing artificial cell-like compartments that communicate in a similar way to cells. Furthermore, we explored an analogue of the β-galactose transducer and the viability of using enzymatic cleavage as an input on the membrane surface. Finally, 19F NMR studies were conducted to provide insight into the positioning of the catalytic head groups of transducers in the membrane, and a fluorescence quenching assay was used to determine the ion transport ability of transducers. This work provides further understanding of the proposed membrane translocation mechanism and extends the scope of artificial transmembrane signal transduction from purely synthetic assemblies into more sophisticated biological systems. This opens up the potential for the future development of intelligent responsive vesicles in bionanotechnology.

Signal transduction is one of the most fundamental biochemical processes that occurs in living organisms and involves the transmission of molecular signals from a cell's exterior to its interior. Foldamers of 2-aminoisobutyric acid (Aib) have shown potential in the creation of a transducer of structural information across nm distances, through a variety of covalent and non-covalent interactions both in solution and aqueous environment. This thesis details the advancement of these helical systems towards a functioning synthetic GPCR mimic capable of signal transduction. Foldamers containing ZnII-recognition sites at the N-terminus were used to recognise chiral carboxylates that switch on signal transduction and conformation control. A C-terminal glycinamide spectroscopic reporter was used to detect this conformation control in solution. A [ZnII(BQPA)]-binding site was pivotal for the creation of a foldamer as a dual NMR and CD probe which cannot only sense helical screw-sense preferences but also sense enantioselectivity of the binding carboxylate. An attempt to activate this functioning foldamer by the binding of a messenger, produced from a desymmetrisation reaction, was also described. An alternative C-terminal reporter was also assessed, not only in solution but in artificial membranes. A highly sensitive bis(pyrene) reporter was exploited for the fluorometric detection of induced screw-sense preferences in covalently controlled Aib foldamers. The effect of phospholipid chirality on foldamer conformation was also explored through the synthesis and incorporation of enantiomeric foldamers bearing the bis(pyrene) reporter. Finally, the synthesis of a ruthenium transfer hydrogenation catalyst and its attachment to Aib foldamers was explored aiming to create a foldamer that can initiate remote catalysis once activated.

Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained "stemness" in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer.

Macrophages are the largest cells in the immune system and they regulate inflammatory signalling and inform cell fate decisions. Many signals, including those mediated by Tumor Necrosis Factor alpha (TNFα) converge on a few key intracellular signalling pathways, including the Nuclear Factor kappa B (NFκB) network. The NFκB signalling pathway plays a vital role in the regulation of many different cellular responses, including the production of TNFα itself, which is required to sustain and propagate immune responses to, for example, infection or tissue damage. In this thesis we report on studies-both experimental and theoretical-of the NFκB signalling pathway in macrophages. Our collaborators stimulated these cells with various doses of Lipopolysaccharide (LPS), a molecule that forms the major component of the outer membrane of Gram-negative bacteria: in these experiments it serves as a proxy for bacterial infection. The macrophages, studied in vitro, respond as they are believed to do in tissues, by secreting certain signalling molecules called cytokines: the level of secretion proved to depend on the strength of the LPS stimulus. Further, heterogeneity of macrophage signalling was observed in response to a range of LPS doses. Within individual macrophages LPS stimulation results in oscillatory behaviour of NFκB localisation-NFκB shuttles in and out of the nucleus-with an amplitude (peak nuclear concentration) that also depends on the LPS dose. Heterogeneity was also observed in cells that were stimulated with the same dose intensity. This raises an important question about how immune cells coordinate inflammatory activity in the presence of this variability. In this thesis we aim to achieve an understanding of the system through the qualitative analysis of mathematical models of it. This work explores both the parametric sensitivity and bifurcation analyses for two mathematical models of NFκB in macrophages. Parametric sensitivity analysis is used to investigate the role of parameters on the model's output, especially on certain features of the signal-peak amplitudes, inter-peak intervals and areas beneath curves-that are commonly measured in single-cell experiments. Local bifurcation analysis is conducted in order to show all the possible behaviours produced when varying parameters.

Plasmodium spp. and Trypanosoma brucei spp. are protozoan parasites with complex lifecycles, each having to adapt to the diverse environments of their insect vector and mammalian host. Each has multiple developmental forms that differ in their morphology and metabolism. Differentiation between these forms is a tightly regulated and often synchronised process in response to changes in the parasite's environment. The molecular mechanisms by which they perceive and respond to such environmental changes are largely unknown. The Plasmodium and T. brucei genomes encode a homologue of the phylogenetically widespread GPR89 family of putative receptors or channels. The mammalian GPR89 homologue has been implicated in the regulation of Golgo acidification, whilst in plants it has been shown to be involved in G protein signally pathways. This study set out to characterise the Plasmodium and T. brucei GPR89 proteins in order to assess their potential role as environmental sensors. Bioinformatic analyses demonstrated that the GPR89 proteins are a highly divergent family of multi-transmembrane domain proteins that may perform a channel or transporter function. Several expression strategies were employed to evaluate the role of the GPR89 proteins. Functional insight was gained from the ectopic expression of both the T. brucei and Arabidopsis thaliana GPR89 proteins in T. Brucei cells. Over-expression of TbGPR98 causes premature stumpy formation in pleomorphic T. brucei cells. This phenotype was replicated by over-expression of a A. thaliana homologue in T. brucei despite signigicatnt sequence divergence. Furthermore, both were demonstrated to act on the same pathway as the putatuve RNA binding protein, RBP7 that was receontly identified in a genome-wide screen for components of the stumpy differentiation pathway. Hence, TbGPR89 is likely a compnent of the slender to stumpy differentiation pathway in bloodstream form trypanosomes and there appears to be functional complementarity between T. brucei and A. thaliana GPR89 proteins.

Two component systems (TCSs) are regulatory pathways in bacteria and lower eukaryotes that integrate multiple stimuli and bring about appropriate responses to promote adaptation of the bacteria to their niches. They are commonly insulated from cross-talk and form discrete regulatory systems where the sensor histidine kinase (SK) and the response regulator (RR) share high fidelity for one another. The GacS network controls the switch between acute and chronic virulence of P. aeruginosa. The network is unusual in having a 'core' SK, GacS, which is modulated directly by one other SK, RetS. Here the complex relationship between GacS and RetS is dissected to reveal three distinct mechanisms by which they interact. Two of these mechanisms involve the dephosphorylation of GacS-P by RetS and it is these mechanisms that are important in vivo for the regulation of biofilm formation, rsmY and rsmZ expression, swarming, and virulence in both Galleria mellonella and an acute model of infection in mice. This study reveals an unprecedented level of complexity in the ability of RetS to interact with GacS and suggests that RetS has a number of mechanisms by which it can downregulate the GacS network output. Furthermore, the interactions of additional SKs that have previously been linked to the GacS network were investigated. Here I demonstrate that many of these kinases can interact with one another but that RetS remained the only kinase tested that could directly interact with GacS. The interactions observed between kinases could be either stimulatory, having a synergistic impact on phosphorylation levels, or inhibitory. I also show that kinase-kinase interactions allow for the regulation of phosphorylation of downstream proteins. Finally, we searched for additional SKs that may be able to interact with the GacS network. Here I identify three new kinases, which show differing interactions with the kinases of the GacS network. The discovery of additional SKs in the GacS network indicates that the network is likely to respond to a far greater number of different signals than previously realised as it decides between acute and chronic virulence.

Prostate cancer (PCa) cells predominantly metastasize to bone and the complex crosstalk between PCa cells and osteoblasts (bone-forming cells) and osteoclasts (bone-destroying cells) leads to increased tumour growth and worsening of bone disease. Understanding the mechanisms of PCa bone metastasis can identify the aggressive fraction of PCa resulting in earlier intervention. The ability of PCa cells to express bone cell-specific features, termed osteomimicry, could potentially explain the osteotropic nature of PCa cells. The aim of this study was to determine the role of osteomimicry in the regulation of epithelial-mesenchymal transition (EMT) in bone metastatic prostate cancer cells. It was demonstrated that the osteoblast-specific marker alkaline phosphatase (bone/liver/kidney) (ALPL) was overexpressed in bone metastatic (ARCaPM), compared to non-metastatic (ARCaPE), human PCa cells. Knockdown of ALPL resulted in decreased cell viability, increased cell death and a change from mesenchymal to epithelial morphology in ARCaPM and PC3 cells, and increased CDH1 expression along with decreased migration in ARCaPM cells. Treatment with extracellular ATP also resulted in decreased viability, increased expression of epithelial markers (CDH1, KRT14) and decreased expression of mesenchymal markers (VIM, ZEB1), and reduced expression of ALPL in ARCaPM cells. Small RNA-sequencing identified microRNAs differentially expressed between ARCaPE and ARCaPM PCa cell lines: miR-373 expression was lower in ARCaPM compared to ARCaPE cells and its overexpression in ARCaPM cells resulted in a change to epithelial morphology, increased expression of the epithelial marker CDH1 and decreased expression of the mesenchymal markers VIM and ZEB1. Finally, the development of a high-throughput screening method to identify novel microRNA regulators of osteomimicry was described, which identified two microRNAs miR-199a-5p and miR-212 as positive regulators of ALP activity. Taken together, this thesis describes the identification of ALPL and ATP as novel regulators of epithelial-mesenchymal transition in PCa cells and high-throughput ALP-activity screening as a powerful tool to identify novel microRNA regulators of ALP expression.

Calcium is a universal messenger that regulates many biological and pathological processes by interacting with crucial calcium-binding proteins like the calcium-sensing receptor (CaSR). CaSR is a prominent protein that regulates various signaling pathways and diseases and detects changes in extracellular calcium concentration and metabolites to cause calcium changes in the intracellular environment. The study detailed in this thesis elucidates CaSR's presumed function in calcium homeostasis regulation and explores the binding and functional cooperativity of bacterially expressed CaSR using Trp-sensitized FRET assay. The second section of this thesis explores the optimization of TNCA a natural allosteric modulator of CaSR as our lead compound with the aid of structure-based drug design to develop novel CaSR therapeutics. These investigations offer valuable insights into the molecular basis for CaSR-related diseases to facilitate the discovery of new therapeutics.

Many molecules and the complex interactions between them underlie plasticity in the cerebellum. However, the exact relationship between cerebellar plasticity and the different signalling cascades remains unclear. Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but very little is known about its function during plasticity induction in the cerebellum. The aim of this thesis is to contribute to a better understanding of the molecular mechanisms that regulate the induction of synaptic plasticity in cerebellar Purkinje cells (PCs). The focus of the thesis is to investigate the role of CaMKII isoforms in the bidirectional modulation of plasticity induction at parallel fibre (PF)-PC synapses. For this investigation, computational models that represent the CaMKII activation and the signalling network that mediates plasticity induction at these synapses were constructed. The model of CaMKII activation by calcium-calmodulin developed by Dupont et al (2003) replicates the experiments by De Koninck and Schulman (1998). Both theoretical and experimental studies have argued that the phosphorylation and activation of CaMKII depends on the frequency of calcium oscillations. Using a simplified version of the Dupont model, it was demonstrated that the CaMKII phosphorylation is mostly determined by the average calcium-calmodulin concentration, and therefore depends only indirectly on the actual frequency of calcium oscillations. I have shown that a pulsed application of calcium-calmodulin is, in fact, not required at all. These findings strongly indicate that the activation of CaMKII depends on the average calcium-calmodulin concentration and not on the oscillation frequency per se as asserted in those studies. This thesis also presents the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. The results of computer simulations of a simple mathematical model suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors determines whether LTD or LTP occurs in cerebellar PCs. This model replicates the experimental observations by Van Woerden et al (2009) that indicate that CaMKII controls the direction of plasticity at PF-PC synapses. My computer simulations support Van Woerden et al’s original suggestion that filamentous actin binding can enable CaMKII to regulate bidirectional plasticity at these synapses. The computational models of intracellular signalling constructed in this thesis advance the understanding of the mechanisms of synaptic plasticity induction in the cerebellum. These simple models are significant tools for future research by the scientific community.

For years, it has been appreciated that embryos have remarkable abilities to heal wounds efficiently and perfectly, without scar formation. However, the molecular mechanisms underlying embryonic wound healing, especially how they coordinate and function in an efficient way, remains poorly understood. The primary aim of my PhD thesis was to use Xenopus as a model system to investigate the molecular and cellular mechanisms which are responsible for the regulation and coordination of embryonic wound healing. More specifically, my thesis includes the study of three signalling pathways during embryonic wound healing; namely the Erk MAPK pathway, PI3K pathway and inositol phosphate pathways. Erk and PI3K signalling are sequentially activated post injury, during separate phases of wound closure. The initial activation of Erk signalling governs the initial stage of wound closure, by mediating myosin-2 phosphorylation and actomyosin contraction through Rho activity. PI3K signalling increases in the late stage of wound closure, promotes leading edge migration and zippering via Rac and Cdc42 activity (Manuscript #1). From the findings of this study, I proposed a novel model, which suggests a cooperation of these two signalling pathways in orchestrating distinct cytoskeletal events during in tissue morphogenesis. In the second part of my thesis, I studied the role of inositol phosphate signalling during wound healing. In particular, I studied the role of the enzyme Itpkb and its product InsP4, in promoting rapid wound healing (Manuscript #2). Itpkb colocalizes with F-actin cable and promotes its formation at the wound edge in both single cell and multicellular wounds, enhancing the activity of three Rho GTPases Rac, Cdc42 and Rho at the same time. In addition, itpkb is required for calcium propagation from the wound edge to distant cells, suggesting a role in transmitting the wound signal across the tissue, resulting in the coordination of healing in multicellular wounds. Together, these PhD work provided more insights into the in vivo regulation of intracellular and intercellular signals in coordinating cell behavior in tissue movement during embryonic wound healing.

Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.

Plasma membrane calcium/calmodulin-dependent calcium ATPases (PMCAs) are high affinity calcium pumps regulating many calcium-dependent processes and advances in its characterisation have discovered that it may play a novel role in signal transduction pathways. It was the aim of this work to further characterise and confirm the role PMCA plays in regulating calcium/calmodulin-dependent signal transduction pathways. PMCA4 has already been shown to inhibit the NFAT family of transcription factors by its interaction with calcineurin A in mammalian cells when ectopically expressed. This prompted the investigation into other isoforms of PMCA that may interact with the calcium/calmodulin-dependent calcineurin, to determine if this interaction was isoform-specific in a variety of cell lines. Endogenous proteins were isolated by immunoprecipitation with calcineurin A antibody and the presence of PMCA isoforms was determined by western blot using isoform-specific antibodies. This work has demonstrated that the PMCA and calcineurin interaction occurs in vitro at endogenous levels in MCF-7 human breast adenocarcinoma cells and endothelial cells and is isoform specific, predominantly for PMCA2. The characterisation of the PMCA2b-calcineurin A interactive domain was performed and it was demonstrated that PMCA2b significantly inhibits the NFAT/calcineurin pathway. These results indicate that PMCA2 is important in regulating the calcineurin/NFAT pathway in tissues where it is highly expressed. This work also demonstrates that the Flag-tagged, characterised interaction domain of PMCA2 with calcineurin, F-PMCA(462-684) when overexpressed, can disrupt the inhibitory PMCA2/calcineurin interaction in endothelial cells and significantly increase calcineurin activity. The expression of PMCA in endothelial cells prompted the investigation of calcium/calmodulin-dependent proteins in endothelial cells as evidence for the important role of PMCA in regulating signal transduction pathways. Nitric oxide synthases have been shown to be regulated by PMCA4 in cardiac cells. To further characterise the regulation of NOS by PMCA, this work shows that there is a novel molecular interaction between endogenous eNOS and the plasma membrane calcium ATPase (PMCA) in HUVEC primary endothelial cells. PMCA2 has been identified as the major isoform interacting with eNOS in endothelial cells. The interaction between the two proteins has been mapped to the region 735-934 of eNOS and 462-684 of human PMCA2b. NO production was found to be inhibited by ectopic expression of PMCA2b in HUVEC cells. Moreover, disruption of the interaction between endogenous PMCA and eNOS by overexpression of theFlag-tagged, PMCA2b interaction domain, F-PMCA2(462-684), significantly increased NO levels in activated HUVEC endothelial cells. In summary, these results offer strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA2 as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways. Overall this is a novel discovery which clearly demonstrates that PMCA is an important regulator of calcium/calmodulin-dependent signal transduction pathways in various cell types. Parts of this work have been published; ‘Holton, M., Yang, D., Wang, W., Mohamed, T.M., Neyses, L. and Armesilla, A. (2007) The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells. FEBS letter. 581(21), 4115-4119.’ and presented at ‘The 14th congress of calcium binding proteins, La Palma, Canary Islands, Spain. 2007’ and ‘The 25th Conference of the European Society on Microcirculation (August 26-29, 2008, Budapest, Hungary).’

The common human GNB3 825C > T variant, which is present in 50% of the world’s chromosomes, has previously been shown to predispose individuals to hypertension, cardiac and neural disorders. This variant causes the production of a stable and gain of function protein Gβ3S- This thesis describes the discovery of a novel D153del mutation that produces an unstable, loss of function, protein Gβ3D in the recessively inherited, retinopathy globe enlarged (rge) chickens. This thesis also demonstrates that the normal Gβ3 downstream phosphorylation signalling pathways are significantly altered in a tissue specific manner in rge chicken organs and in a human GNB3 825TT lymphoblast cell line. In rge tissues expressing Gβ3D protein, the cAMP induced GRK2 phosphorylation activity is significantly altered. Moreover MAPK1 (ERK2) phosphorylation is significantly decreased compared to normal tissues. In contrast human 825TT cell lines expressing the Gβ3S protein, showed enhanced cAMP induced GRK2 and MAPK (ERK1 and ERK2) phosphorylation activity. These results confirm previous findings of 825C > T Gβ3 studies, that Gβ3S is indeed a hyper-activating structural variant, in contrast to the D153del Gp3D is a classical recessively inherited non-functional mutation.

Bacteria use conserved signal transduction pathways, called sensory systems, to sense environmental stimuli. Most of our understanding of sensory systems in bacteria, however, comes from the chemotaxis system of Escherichia coli, which senses chemical gradients to control the direction of flagellar-based motility (chemosensing). Importantly, bacteria can also sense mechanical stimuli to actively shape their physiology. An in-depth mechanistic understanding of mechanosensory systems, when compared to their chemosensory counterparts, is however lacking. This dissertation presents work towards understanding mechanosensing in the important opportunistic human pathogen Pseudomonas aeruginosa. This Gram-negative bacterium uses Type IV pili (TFP), retractile polarly localized appendages, to sense mechanical forces generated during surface contact at one cell pole. We and others have demonstrated that spatially resolved mechanical stimuli transmitted by TFP activates the Pil-Chp mechanosensory system. Upon surface contact, TFP transmits mechanical stimuli to the Pil-Chp receptor, PilJ, thereby altering the autophosphorylation state of ChpA and thus the phosphorylation of PilG and PilH, the antagonistic Pil-Chp response regulators. PilG and PilH inversely control two outputs of the Pil-Chp system in P. aeruginosa: cAMP production and twitching motility. Sensing of surface contact by the Pil-Chp system activates the membrane bound CyaB adenylate cyclase, which catalyzes the production of the second messenger, cyclic adenosine monophosphate (cAMP). cAMP binds to the Vfr transcription factor, leading to altered transcription of >200 genes involved in acute virulence as well as selected TFP regulatory proteins. Signal processing through PilG and PilH is critical for surface-dependent cAMP production. PilG promotes cAMP production and upregulation of the surface dependent transcriptional program while PilH has the opposite effect. The Pil-Chp mechanosensory system is required for twitching motility, partially independently of cAMP levels. In Chapter 2, we demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from TFP, in a process called mechanotaxis. The Pil-Chp system controls the balance of forward and reverse twitching motility of single cells in response to the mechanical inputs. We show that the Pil-Chp response regulators PilG and PilH control the polarization of the TFP extension motor PilB. PilG localizes to both poles, but shows greater accumulation at the leading pole, where it stimulates polarization favoring forward migration. In contrast, PilH, is primarily cytoplasmic, thereby globally antagonizing PilG. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local-excitation, global-inhibition in higher order organisms.In Chapter 3, we demonstrate that PilG and PilH enable dynamic cell polarization by coupling their antagonistic functions on TFP extension. By precisely quantifying the localization of fluorescent protein fusions, we show that phosphorylation of PilG by the histidine kinase ChpA controls PilG polarization. Although PilH is not inherently required for twitching reversals, upon phosphorylation, PilH becomes activated and breaks the local positive feedback established by PilG so that forward-twitching cells can reverse. To spatially resolve mechanical signals, the Pil-Chp system thus locally transduces signals with a main output response regulator, PilG. To respond to signal changes, Chp uses its second regulator, PilH, to break the local feedback. In Chapter 4, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system. Bacterial sensory adaptation has primarily been studied in flagellar-mediated chemotaxis, where reversible methylation of sensory receptors by a methyltransferase and a methylesterase “tune” their sensitivity of signaling. The Pil-Chp system encodes the PilK methyltransferase, predicted to methylate PilJ, and the ChpB methylesterase, predicted to demethylate PilJ; however, whether sensory adaptation occurs in response to surface contact remained underexplored. Using biochemistry, genetics, and cell biology, we discovered that PilK and ChpB are segregated to opposing cell poles as P. aeruginosa explore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local PilJ methylation in vivo. We propose a model in which spatially resolved mechanical inputs transmitted by TFP not only alter PilG and PilH signaling mechanisms but locally controls PilJ methylation to modulate twitching motility reversal rates and surface-dependent cAMP production. Despite decades of chemosensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to mechanical sensory stimuli.Acinetobacter species are opportunistic pathogens that are ubiquitous throughout the environment and are emerging as a public health threat around the world due to their widespread multidrug resistance. Intriguingly, many Acinetobacter strains encode homologs of the P. aeruginosa Pil-Chp mechanosensory system. In Chapter 5, we demonstrate that A. nosocomialis strain M2, a pathogenic member of the Acinetobacter calcoaceticus-baumannii complex, has a robust surface-dependent transcriptional response. We speculate that the homologous Pil-Chp mechanosensory system is responsible for the surface-dependent transcriptional response that we report in this dissertation. Overall, this dissertation demonstrates that mechanosensing through the Pil-Chp system takes advantage of the intricate internal organization of bacteria to sense spatially resolved mechanical information. As medically Acinetobacter species exhibit a surface transcriptional response, defining the mechanosensing mechanism of Acinetobacter species represents an exciting area of investigation. Understanding the mechanisms of bacterial mechanosensing may lead to the generation of desperately needed therapeutics to treat multi-drug resistant infections, such as the ones typically caused by P. aeruginosa and medically relevant Acinetobacter species.

Reversible phosphorylation of proteins is a vital means of regulation in signal transduction processes. The PKC family of lipid dependent serine/threonine kinases is known to play key roles in a whole range of cellular functions. To comprehend the diverse roles of the different family members, it is important to define the regulation of each PKC isoform. While activation of PKC isoforms is critical for their function in targeting downstream substrates/effectors, of equal importance is the desensitisation of the active kinases in determining their consequential effects. The overall aim of this thesis was to explore the mechanism(s) causing the downregulation and degradation of PKCδ. The degradation of this novel isoform of PKC has been examined in NIH 3T3 cells, in response to chronic activation by the phorbol ester TPA. A significant part of this study focused on the dephosphorylation of PKCδ as a primary event predisposing the enzyme to degradation. This was addressed in vivo using cell permeable inhibitors of protein phosphatases. In addition, in vitro studies using purified forms of PP1, PP2A and PP2C were carried out to investigate the potential effects of the different protein phosphatases towards PKCδ as a substrate. Consistent with other data, PP2A was identified as the putative protein phosphatase involved in the dephosphorylation of PKCδ. Interestingly, studies revealed that either nucleotide binding or substrate binding to PKCδ protected the enzyme from the action of phosphatases. To further investigate the requirement of phosphorylation/dephosphorylation during the downregulation process in cells, phosphorylation site mutants of PKCδ were generated and studied in transient transfection experiments. While these provided useful information, it was concluded that ectopic expression of PKCδ did not entirely represent the behaviour of the endogenous protein. Nevertheless, information from mutant analyses together with data from in vivo studies of endogenous PKCδ, in contrast to the previous speculation, provides strong evidence for the role of phosphorylation in the targeting of PKCδ to degradation via the ubiquitin/proteasome pathway.

The application of the proteomic approach has facilitated efforts directed toward the mapping of the entire Arabidopsis cell wall proteome. Proteins were sequentially extracted from purified cell walls using 0.2 M CaC1(_2) followed by a urea buffer. The extracts were resolved via large format two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were visualised via Coomassie brilliant blue staining. The aim was to identify and characterise as many cell wall proteins as possible, with the hope of identifying novel cell wall proteins. Out of 325 spots visualised on the 2-D polyacrylamide gel, 144 gave a positive protein identification representing 104 different proteins. The identified proteins were divided into 3 categories. The first category included proteins that have been previously identified as plant cell wall proteins. The second category was designated to include novel cell wall proteins (hypothetical proteins) and the third category was made up of proteins, which had recognised functions, but had never hitherto been known to be secreted to the extracellular matrix. Among the identified novel cell wall proteins there were several that shared high homology with protein kinases. These proteins possessed all the characteristics of secreted polypeptides, such as the cleavable N-terminal signal peptide, and were found to lack both the transmembrane domain and the endoplasmic reticulum retention tetrapeptides (HDEL and KDEL). These observations suggested that, as in animal cells, plant cells had extracellular protein kinase activity (phosphorylation). This was supported by the recent discovery that plant cells secrete ATP to the extracellular matrix (Thomas et al., 2000). Verification of the occurrence of extracellular protein kinase activity was further strengthened by the identification of phosphorylated bona fide cell wall proteins and stress responses caused by the depletion extracellular ATP.

Mono-ADP-ribosylation is a post translational modification of cellular proteins, which has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking and secretion. However, the physiological role of the ADP-ribosylation reaction in intact cells remain to be clarified. We demonstrated that the β subunit of the heterotrimeric G proteins is endogenously mono-ADP-ribosylated in intact cells. This modification occurs when the βɣ dimer is in its active dimeric conformation (the addition of increasing concentrations of GDP-αi3 in the ADP-ribosylation assay mixture results in the inhibition of the β subunit labelling), while the enzyme catalyzing this reaction is a membrane-associated ADP-ribosyltransferase that modifies arginine 129. Interestingly, this amino acid is located in the N-terminal region, which is involved in the interaction with the α subunit and with βɣ effector enzymes. The experiments described in this thesis demonstrate that the βɣ dimer, once modified, loses its ability to inhibit the calmodulin-stimulated type 1 adenylyl cyclase (Lupi et al., J. Biol. Chem., 2000) and to stimulate phospholipase Cβ2 in in vitro assays. ADP-ribosylated β subunit is deribosylated by a cytosolic hydrolase completing an ADP-ribosylation/deribosylation cycle that might thus modulate the interaction of βɣ with specific effectors. Heterotrimeric G proteins play a key role in cell regulation, with a growing body of evidence demonstrating that the βɣ complex participates in a wide range of G protein function. Our finding of an ADP-ribosylation/deribosylation cycle acting on the βɣ subunits delineates a novel molecular mechanism that could provide crucial control of G protein-mediated signalling pathways.

In cyanobacteria, state transitions serve to regulate the distribution of excitation energy delivered to the two photosystem reaction centres from the accessory light harvesting system, the phycobilisome. The trigger for state transitions is the redox state of the cytochrome bf complex/plastoquinone pool. The signal transduction events that connect this redox signal to changes in light harvesting are unknown. In order to identify signal transduction factors required for the state transition, random cartridge mutagenesis was employed in the cyanobacterium Synechocystis PCC6803 to generate a library of random, genetically tagged mutants. The state transition in cyanobacteria is accompanied by a change in fluorescence emission from PS2. By using a fluorescence video imaging system to observe this fluorescence change in mutant colonies it was possible to isolate mutants unable to perform state transitions. Five such mutants were isolated and in all cases disruption of the gene slll926 found to account for the phenotype. slll926 has only one significant homologue in current databases, a gene in Anabaena PCC7120. A hydropathy analysis would suggest that both proteins are membrane associated. An insertional inactivation mutant of slll926 (δslll926) is specifically unable to perform state transitions: no impairments in electron transport, in light harvesting, or in reaction centre/phycobilisome assembly or function are evident. The product of slll926 may therefore be a signal transduction factor. slll926 inactivation in mutants lacking photosystem 1 or photosystem 2 leads to changes in phycobilisome coupling with the remaining photosystem. This strongly supports a mobile phycobilisome model for state transitions and suggests that state transitions are brought about by changes in the affinity of the phycobilisomes for both photosystems. δslll926 has a doubling time similar to the wild-type at all but very low light intensities. This implies that a major physiological role of state transitions is to maximise the efficiency of light utilisation.

The aim of the work has been to investigate the applicability of certain computational models to the modelling and simulation of bacterial chemotaxis. It is hoped that the model will provide insights into the information processing capabilities of the system and replicate the bacteria's ability to monitor their surroundings and adapt to changes. The chemical stimuli are detected by membrane-spanning receptors, transmitted through the signal transduction pathway, and then the swimming behaviour of the cell is adapted in response. In effect the system is taking in a great deal of information and processing it, in order to determine whether it continues moving in one direction, or rotates in order to search for a more favourable one. In the individual-based method that has been followed, components in the signal transduction system are represented (in correct proportions) as individual program objects that can interact with each other through the simulation of chemical reactions. Existing systems analysis and design techniques have been adapted and applied to the modelling of biological intracellular signalling pathways after the existing Structured Biological Modelling method proved to be insufficient. Then the Swarm simulation package was used to create an individual-based model of an intracellular signalling pathway for the first time. Existing methods for stochastic simulation were applied and found to be inappropriate, leading to the development of a new method suited to individual-based modelling, which closely matched the output of the deterministic model. Finally a spatial element was introduced, thus making this the first individual-based, spatial simulation of the complete bacterial chemotaxis intracellular signalling pathway. The overall system is essentially a robust control system, able to choose one of only two courses of action in response to a great number of inputs. From the point of view of adaptable computer-based systems, the computational abilities of the biological system are very powerful and when represented appropriately in software could be usefully incorporated into many real-world applications.

In order to identify light responsive cis-acting DNA sequence elements of the Arabidopsis thaliana chalcone synthase gene (AtCHS) concerned with induction by UV-B and UV-A/blue light, we developed a UV/blue light inducible transient expression system. This system involved transfection of chimaeric AtCHS promoter constructs into Arabidopsis cell culture protoplasts. The reporter enzyme beta-glucuronidase (GUS) was used to monitor light responsive promoter activity. Development of this system involved defining and optimising procedures for preparing protoplasts from the Arabidopsis cell culture, transfection of chimaeric AtCHS promoter constructs into the protoplasts and incubation under defined illumination conditions. An efficient homologous protoplasts transient expression system was developed which subsequently enabled us to undertake functional analysis of the AtCHS promoter. Application of the transient expression system allowed us to define the light responsive cis-acting elements concerned with the transcriptional activation of AtCHS. This analysis showed that a 1972 bp promoter conferred UV-B and UV-A/blue light induction of GUS activity. Deletion of the promoter to 164 bp resulted in reduced promoter strength but retention of responsiveness to UV-B and UV-A/blue light. Further deletion abolished transcriptional activity. The 164 bp promoter contains sequences closely resembling the light-responsive unit (LRUPcCHS) of the Petroselinum crispum CHS promoter. This Arabidopsis CHS promoter region, designated LRUPcCHS, was sufficient to confer UV-B and UV-A/blue light responsiveness to a heterologous core promoter. Mutation of sequences in corresponding to the ACGT element and the MYB recognition element of LRUPcCHS resulted in inactivation of 164 bp and 335 bp promoter deletions. However, the mutant 668 bp promoter retained residual UV-B and UV-A/blue light-induced expression, indicating the presence of additional functional sequences upstream of -335. Mutation of a single G-box-like sequence around -442 had no effect on light responsiveness, indicating that it does not function in the light regulation of this promoter. Furthermore, in this analysis no difference in responsiveness to UV-B and UV-A/blue light was observed with any promoter variant tested. This suggests that the UV-B and the UV-A/blue phototransduction pathways regulate transcription factors which interact with common promoter elements. These results are discussed. We employed several different approaches to investigate the role of cryptochromes in the regulation of AtCHS promoter activity. Gain of function transient expression analysis suggests that CRY1 is the primary photoreceptor mediating UV- A/blue light induction of promoter activity. In addition, northern analysis using various cryptochrome mutant lines, including a cry1/cry2 double mutant, supports this hypothesis and, furthermore, suggests that (an)other photoreceptor(s) able to respond to UV-A/blue light, exist in Arabidopsis. Possible reasons for these observations are discussed. We used the transient expression system to investigate the role of sugar regulation in the control of AtCHS promoter activity in Arabidopsis protoplasts. This revealed that sucrose, glucose or fructose was required to stimulate AtCHS promoter activity. Furthermore, studies using glucose analogues were performed which suggest that the initial signal for sugar stimulation of AtCHS promoter activity is consistent with signalling by hexokinase phosphorylation of hexose sugars. Evidence for this hypothesis is discussed.

The effect of analogues of abscisic acid (ABA) on stomatal aperture and GUS activity driven by an ABA-responsive gene promoter (CDeT6-19) in guard cells was examined. The ABA analogues used were (-)-ABA, (+/-)-trans, trans-ABA, and (+)-and (-)-dihydroacetylenic abscisyl alcohol (PB1-63 and PBI-51, respectively). (+)-ABA and (+/-)-ABA were included in the investigations for comparison. (-)-ABA, hitherto believed to have little or no effect on stomatal aperture, significantly inhibited stomatal opening in Commelina communis, Vicia faba, Nicotiana tabacum and Arabidopsis thaliana. In contrast, PBI-51 [a competitive inhibitor of ABA-induced gene expression] had no effect on ABA-induced inhibition of stomatal opening (determined in both C. communis and N. tabacum) or ABA-induced promotion of stomatal closure (determined in C. communis only). Differences in the effectiveness of PBI-63, PBI-51 and (-)-ABA at inhibiting stomatal opening were discovered between plant species. These data demonstrate the importance of comparing the effect of ABA analogues on stomatal opening and CDeT6-19/GUS activity in guard cells in a single plant species. (+/-)-ABA enhanced CDeT6-J9/GUS activity in guard cells of A. thaliana but not N. tabacum. The ABA analogues that inhibited stomatal opening in A. thaliana also enhanced CDeT6-J9/GUS activity in guard cells of this species. The relative biological activity of the ABA analogues in detached epidermis of A. thaliana was (+)-ABA > (+/-)-ABA > (-)-ABA > PBI-63. PBI-51 and (+/-)-trans, trans-ABA had no effect on stomatal opening or CDeT6-19/GUS activity in guard cells of A. thaliana. Interestingly, a differential effect of 10 -5 M (-)-ABA on the two ABA-induced responses in A. thaliana was observed; 10 -5 M (-)-ABA enhanced CDeT6-J9/GUS activity in guard cells but had no effect on stomatal opening. Preliminary measurements of guard cell cytosolic free calcium ([Ca2+]i) showed that (-)-ABA induced oscillations in [Ca2+]i in guard cells of C. communis. In addition, these studies demonstrate the possibility of microinjecting guard cells of A. thaliana with fura-2 and provide a measure of resting [Ca2+]i in this species. The data presented in this thesis from A. thaliana suggest that the "receptor" utilized in the signalling pathway by which ABA inhibits stomatal opening is similar but not identical to that by which ABA enhances CDeT6-19/GUS activity in guard cells.

HL60 cells cultured in 10% fetal calf serum (FCS)-supplemented medium were induced to differentiate to the granulocyte phenotype with 1μM retinoic acid. The molecular species compositions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from LH60 granulocytes were analysed by HPLC. Control HL60 granulocyte PC and PE had characteristic molecular species compositions with PC containing mostly saturated and monounsaturated species and PE containing more unsaturated species. Comparison of PC and PE species compositions of unstimulated HL60 granulocytes with the composition of P[³H]But synthesised in response to 100nM PMA showed that the P[³H]But formed resembled PC more than PE. However, the P[³H]But composition was not identical to PC with negligible formation of P[³H]But16:0/16:0. Supplementation of the cell culture medium with 30μM 18:2n-6 or 20:4n-6 fatty acid-albumin mixtures dramatically altered PC and PE molecular species compositions with significant increases in PC molecular species containing the supplemented fatty acid. Stimulation of 18:2n-6 or 20:4n-6 supplemented cells with PMA resulted in the production of mostly unsaturated P[³H]But species which were major components of the PC. The results of this study outline the modifications to the PLD activation products caused by culture of the cells in the presence of exogenous fatty acids and demonstrate that unsaturated species can be produced by PLD when they become available in the PC composition. The results suggest that PLD acted either on a discrete pool of substrate PC separate from the bulk of cellular PC or it exhibited a degree of selectivity for certain molecular species of the substrate PC. It is possible that modulation of the activity and selectivity of PLD can lead to generation of PA/DAG with altered compositions and consequent altered characteristics of PKC activation. This may be one mechanism underlying the dietary-induced changes which can occur in granulocyte responses.

Recent work on Drosophila limb development has indicated that short-range interactions between distinctly specified populations of cells (compartments) establish organizing centers at compartment boundaries. These organizing centers direct pattern formation and growth in the developing limbs. In the Drosophila wing imaginal disc, there are at least two such organizing centers, located at the anterior/posterior (A/P) and dorsal/ventral (D/V) compartment boundaries. The genetic hierarchies which establish these organizers are starting to be understood, and it also appears that the key mediators of some of the organizers have been identified. Thus Decapentaplegic (Dpp, a secreted signalling molecule of the TGF-B family) is the mediator of the A/P organizer, while Wingless (DWnt-l, a secreted molecule of the Wnt family) is a key mediator of the D/V organizer. In this thesis, several aspects of Wingless function in the wing imaginal disc are examined. Two regulatory mutations, spadeflag (spdfg) and Sternopleural (Sp), that affect Wingless expression in the wing imaginal disc are characterized. The analysis of the mutation spdfg, together with other data, identifies a role of Wingless as a localized mitogen in the developing wing hinge, and also indicates that cells in different regions of the wing disc respond very differently to the Wingless signal. The mutations spdfg and Sp are also among the tools used to examine the position of Wingless in the genetic hierarchy that establishes and mediates the activity of the D/V organizer. These experiments extend the evidence suggesting that Wingless mediates both short-range and long-range effects of the D/V organizer. Wg does so by controlling the expression domains of different target genes, including the acheate-scute genes, Distal-less and vestigial. Finally, the mechanism by which Wingless mediates the activity of the D/V organizer is examined. The results obtained suggest that Wingless functions as a long-range morphogen.