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Hunger and pain are powerful homeostatic drives, which compete for a behavioural response when experienced simultaneously. This thesis set out to explore neural mechanisms underpinning this competition, and how appetitive visual and olfactory stimuli may modulate the effect of homeostatic energy manipulations on pain. Using well-established techniques including EEG source analysis and resting state fMRI, we consistently employed a within-subjects fasting vs. satiation paradigm to investigate the effects of appetite on subjective pain perception and neural pain processing. Pain stimuli which selectively activated nociceptive Aδ fibres were presented concurrently with appetitive stimuli, and the neural nociceptive responses were mapped with high-density (128-channel) EEG recordings and fMRI functional connectivity. Based on the results of previous research, we hypothesised that fasting would suppress subjective and neural pain processing, and that visual and olfactory appetitive stimuli may augment this effect. We first found that a relatively short overnight fast was sufficient to induce significant changes in resting state functional connectivity in areas that underlie both hunger/satiety and pain: insula cortex, hypothalamus, and regions of prefrontal cortex. Source analysis of EEG data revealed a small group of brain regions whose pain-related activation was suppressed by hunger and/or appetitive stimuli: anterior cingulate cortex, operculo-insular cortex, parahippocampal cortex, and cerebellum. Functional connectivity analysis of fMRI data further uncovered a widely-distributed network of brain areas whose pain-induced connectivity was enhanced by fasting or satiety. Of particular interest was a small network of areas involved in stimulus saliency processing (anterior insula, anterior cingulate cortex, and prefrontal cortex), which was stronger during fasting; presumably advantageous when searching for food. Lastly, in an experiment using a bread odour, we found that the suppressive effect of appetitive stimuli on nociception is not just confined to the visual modality. Brief, strong pain can also be suppressed by an appetitive odour during fasting. We conclude that fasting reliably interferes with pain processing, and that ambient appetitive stimuli might be of use in situations where short-lasting pain is likely to occur.

Objectives: This project was undertaken to investigate the potential of the human PLB-985 cell line to terminally differentiate, along the myeloid lineage into mature neutrophil-like granulocytes, and to identify the molecular properties of the differentiated PLB-985 cells in comparison with blood neutrophils, in order to establish their usefulness as model of neutrophil differentiation and functions. Methods: Human neutrophils were isolated from whole blood of healthy, consented, adult donors. The human PLB-985 cells were grown in routine culture media of RPMI 1640 (+10mM L-glutamine). Exponentially growing PLB-985 cells were induced to differentiate into neutrophil-like cells in differentiation culture media of RPMI 1640 (+10mM L-glutamine), supplemented with three differentiation and maturation-inducing agents: ATRA, DMF and sodium pyruvate. Granulocytic differentiation was measured by cell morphology using light microscope, apoptosis was determined by morphology or flow cytometry, cell viability, cell cycle parameters, expression of cell surface receptors and phagocytosis were determined by flow cytometry, oxidative burst activity was measured using flow cytometry and luminol-enhanced chemiluminescence assay, and expression of apoptotic proteins was determined by western blotting. Results: A modified differentiation protocol and optimisation procedure has been established. Terminally-differentiated, neutrophil-like PLB-985 cells, have been consistently cultured, that resemble mature blood neutrophil morphology, evident by the acquisition of multi-lobed nucleus and granulated cytoplasm, and which had an appreciably extended lifespan. It was discovered that supplementing the differentiation medium with either G-CSF or GM-CSF both improved differentiation and delayed apoptosis of the differentiated cells. These cells underwent cell cycle arrest and progression into apoptosis and they resembled mature blood neutrophils in terms of cell cycle parameters and apoptotic morphology. The differentiation induced expression of anti-apoptotic proteins, Mcl-1 and Bcl-XL in these cells and their expression levels correlated with cell survival status, whilst Bcl-2 expression was lost. The differentiated PLB-985 cells also displayed phagocytosis and oxidative burst activity, expressed cell surface receptors for CD11b, CD14 and CD16 and expressed strong chemotactic transmigration towards chemoattractants. Finally, JAK inhibitors baricitinib and tofacitinib suppressed the growth, differentiation and viability of the differentiated cells, increased their progression into apoptosis, and down-regulate their responses to G-CSF and GM-CSF signalling. Hence, they may inhibit in vitro granulopoiesis, and may have anti-inflammatory activity. Conclusions: This study has indicated that under appropriate conditions, PLB-985 cells can differentiate terminally into mature neutrophil-like phenotypes that resemble blood neutrophils morphologically and functionally, with an enhanced survival of the mature cells. These differentiated PLB-985 cells may therefore, provide an excellent neutrophil-model system for in vitro study of neutrophil differentiation and functions, such as apoptosis regulation.

Objectives: The aim of this thesis was to evaluate the potential of new technologies, including two stem cell technologies, mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), to understand the molecular basis of human diseases. These technologies were evaluated for their ability to restore diabetes-induced defects in wound tissue repair (MSCs) and to generate mature neutrophils after in vitro differentiation of iPSCs. The latter used iPSCs from a patient with abnormal function due to impaired WASp (Wiskott Aldrich syndrome protein) signalling. Other techniques evaluated were differentiation of the myeloid cell line, PLB-985 (expressing exogenous genes) into mature neutrophils and new advances in metabolomics, to identify altered neutrophil function in human diseases. Methods: The potential of MSCs and oral vitamin C to generate factors that could promote healing of diabetic wounds, was measured by RT-PCR for eight genes associated with either angiogenesis or extracellular matrix production, after incubation under normoglycaemic and hyperglycaemic conditions with and without vitamin C. The angiogenic effects of the MSC secretome on wound healing was measured using a tubular formation assay (in vitro) and a nude mice diabetic wound model (in vivo). The bilateral full-skin thickness wounds were created in an in vivo wound model using diabetic nude mice. Oral vitamin C (1.5 g/L) was administered in combination with topical MSC treatment (MSCs 1x 106 cells per wound). Diabetic wound models were divided into five groups; control (CON; n=6), diabetes (DM; n=12), diabetes treated with MSCs (DM+MSCs; n=12), diabetes treated with VitC (DM+VitC; n=6), and diabetes treated with MSCs and VitC (DM+MSCs+VitC; n=12). The capillary density was measured under in vivo fluorescent microscopy, and the tissue VEGF levels were measured. WAS dermal fibroblasts were reprogrammed using retrovirus transfection, and the corrected-WAS-iPSCs were differentiated into the neutrophil-like cells via the formation of iPS-sacs (the sac-like structure containing haematopoietic progenitor cells derived from iPSCs). Neutrophil (from WAS patients and healthy controls) chemotaxis was measured using transwell migration towards N-formylmethionine-leucyl-phenylalanine (fMLP). PLB-985 and KCL-22 cells were differentiated into neutrophil-like cells using RPMI-1640 media containing N,N-dimethyl formamide, sodium pyruvate, all-trans retinoic acid, human AB serum and dimethyl sulfoxide (with penicillin/streptomycin). Morphology was assessed by cytospin. PLB-985 cells were transduced with enhanced green fluorescent protein (EGFP)-tagged Myeloid Cell Leukaemia-1 (Mcl-1), sub-cloned into a pLVX-TetOne-Puro system. 1H NMR metabolomics was carried out using protocols optimised for neutrophils as part of this thesis. An intracellular metabolite extraction method was developed to minimise the loss of neutrophil metabolites and to avoid contaminants arising during the extraction procedure. The NMR analyses were also optimised to identify neutrophil metabolites and allow the comparison from resting and activated states and in health and disease (rheumatoid arthritis patients). Results: Upregulation of angiogenic genes, vascular endothelial growth factor-α (mVEGF-α) and platelet-derived growth factor-BB (mPDGF-BB), in response to TGF-β1 in MSCs was lower following incubation under hyperglycemia (compared to normoglycaemic controls), but vitamin C treatment re-sensitised the MSC response to TGF-β1. A diabetic mouse model showed that administration of oral vitamin C, as an adjunct to MSC therapy, resulted in accelerated wound healing that was associated with increased capillary density. Preliminary experiments with WAS neutrophils showed significantly lower rates of chemotaxis towards fMLP compared to healthy controls. iPSCs from WAS fibroblasts were cultured and differentiated into neutrophil-like cells. The efficiency of both PLB-985 and KCL-22 cells to differentiation into neutrophil-like cells was evaluated and PLB-985 cells differentiated more efficiently into neutrophil-like cells than the KCL-22 cells. PLB-985 cells, transfected with Mcl-1:EGFP in pLVX-TetOne-Puro system were generated. Nuclear magnetic resonance (NMR) metabolomics identified metabolites and pathways altered during in vitro activation with PMA (including metabolites of NADPH synthesis and inhibitors of reactive oxygen species (ROS)) and in vivo activation in rheumatoid arthritis identified metabolites of the ketosis pathway, citrullination pathway and tryptophan metabolism. Conclusions: A number of technologies have been evaluated to study the molecular basis of human disease, including metabolic (diabetes mellitus) and genetic (WAS) diseases. Vitamin C modulated the secretome of MSCs, increasing angiogenesis and accelerating wound healing, providing a potential new approach for designing adjuncts to existing therapies. Neutrophils from WAS patients demonstrated chemotactic defects, and the potential of WAS-iPSCs to differentiate into neutrophil-like cells was demonstrated. The approach could be applied in further studies to study genetic defects of leukocyte function. PLB-985 cells transduced with EGFP-tagged Mcl-1 in an inducible expression vector, was developed as a cell-line model of neutrophil differentiation, to facilitate further studies into the role of the Mcl-1 gene in regulating neutrophil survival. Protocols for human neutrophil metabolomics, using 1H NMR spectroscopy were developed and applied to the study of in vitro and in vivo activated neutrophils. The results demonstrated the potential of metabolomics for future studies of human diseases.

Neutrophils have traditionally been studied in the context of acute infection, forming the first line of defence against invading pathogens, but with a restricted set of proinflammatory functions. More recently, it has become appreciated that neutrophils are, in fact, more complex cells that exhibit heterogeneity and functional plasticity, and capable of a wide array of specialised functions that contribute to adaptive immune responses and pathology of chronic inflammatory conditions. These later roles of neutrophils arise largely as a result of changes in their transcriptional activity in response to changes in the microenvironment in vivo and in vitro. However, relatively little is known of the mechanisms regulating this gene expression. Recent work has shown that, in response to long-term exposure to the TLR8 agonist R848, human neutrophils undergo chromatin remodelling and de novo synthesis of TNFa to express the cytokine, IL-6, expression of which by human neutrophils was previously controversial. In this thesis, the effect of R848 on global gene expression and on changes in the chromatin architecture that may control gene expression of neutrophils, alone or in combination with the proinflammatory factors TNFa or GM-CSF was investigated. Neutrophils were freshly-isolated from healthy individuals and treated with R848, either alone or in combination with TNFa or GM-CSF for different time periods. Histone post- translational modifications (e.g. pan and residue-specific acetylation and citrullination) were investigated by western blotting. RNA-seq was also performed on ultrapure neutrophils (>99%) stimulated with the above agents for 6 h, followed by computational and bioinformatics analysis to predict activation mechanisms. All major findings were validated by qRT-PCR at the mRNA level, and, in some cases, at the protein level. Further functional assays, such as apoptosis, ELISA and measurement of ROS, were performed as appropriate. R848, alone or in combination with the cytokines, induced acetylation of H3 after 6 h of treatment while it decreased H3 citrullination. These effects were more pronounced after 18 h of stimulation. Acetylation and transamination correlated with activation of gene expression in response to R848, but the effects of R848 used together with the cytokines were not simply additive. Unlike the cytokines, R848 induced the expression of interferon- response genes (IRGs) in an interferon-independent, but endogenous TNFa-dependent, manner. Exogenous TNFa failed to have any further effect on R848-induced expression of IRGs, while GM-CSF inhibited their induction. In contrast, R848 plus TNFa synergistically increased the expression of members of the IL-12 family cytokines. IL-23 was found, for the first time, to be expressed and secreted by neutrophils under such co-stimulation. On the other hand, R848 plus GM-CSF synergistically induced the expression of chemokines of the CC family and this effect was entirely dependent on endogenous TNFa. Finally, R848 and the cytokines had a pro-survival effect on neutrophils, but this effect was the outcome of different and complementary patterns of regulation of the anti-apoptotic proteins Mcl-1 and Bfl-1. The absence of an additive effect in apoptosis delay in response to the combined treatments excluded the possibility that TNFa or GM-CSF further increased gene expression in R848-treated neutrophils, simply because they further enhance neutrophil survival. In conclusion, these data show, for the first time, that chromatin remodelling agents, such as R848, can induce substantial changes in the patterns of gene expression of human neutrophils, when used alone or in combination with pro-inflammatory cytokines. The data predict that such chromatin re-modelling may occur in vivo in certain inflammatory conditions, substantially changing how neutrophils can respond to local cytokines. The data also show the importance of endogenous TNF expression and how this may result in autocrine signalling to regulate neutrophil function in human disease.