The CaBPs are a family of small EF-hand-containing calcium binding proteins with limited homology to calmodulin (CaM). The family comprises seven genes with alternative splicing giving rise to a greater number of protein variants. Two distinct groups exist within the CaBP family based on differences in their cation binding properties and domain organization: CaBPs 1-5 and CaBP7 & 8. CaBPs 1-5 interact with a number of known CaM target proteins but generally exert unique regulatory roles and have been implicated in the regulation of numerous Ca2+-channels. A number of interacting binding partners have also been identified which do not overlap with those of CaM and knockout studies have revealed non-redundant roles for CaBP4 and CaBP5. Much less is known about the functions of CaBP7 and CaBP8 with only a direct interaction with phosphatidylinositol 4-kinase III β (PI4KIIIβ) having been characterized in detail. The research presented in this thesis focused on the less well characterised members of this protein family, CaBP7 and CaBP8. Bioinformatic analyses revealed that CaBP7 and CaBP8 form a distinct group of calcium sensors from the rest of the CaBP family and are highly conserved throughout evolution. Examination of CaBP sequences present in species representing important evolutionary nodes indicated that the CaBP family first arose with the appearance of early vertebrates. The primordial CaBP family members, CaBP1S and CaBP8, were identified in lamprey, the closest living relative to the common vertebrate ancestor. The expansion in the number of CaBP proteins from lamprey to mammals largely coincides with a whole genome duplication event occurring before the divergence of cartilaginous fish. A transient expansion of the group encompassing CaBPs1-5 between the divergence of cartilaginous fish and the emergence of bony fish is also apparent. Examination of the targeting mechanisms of CaBP7 and CaBP8 demonstrated that these proteins form a unique class of calcium sensors which associate with cellular membranes via a C-terminal tail anchor. The membrane topology of CaBP7 and CaBP8 was found to be characteristic of the tail anchored class of integral membrane proteins and was consistent with the previously reported interaction with the cytosolic enzyme PI4KIIIβ. Further examination of their subcellular distributions revealed that CaBP7 and CaBP8 localise to the trans-Golgi network and to vesicles which were positive for markers of the late endosomal and lysosomal pathway. This is the first reported instance of a CaM-related calcium sensor localising to membranes of acidic compartments. CaBP7 also co-localised with markers of late endosomes and lysosomes in dividing cells. These CaBP7 positive vesicles redistributed to the vicinity of the interzonal microtubules and opposite poles of the spindle during cytokinesis. In accordance with previous reports, knockdown of CaBP7 in HeLa cells resulted in an increase in the number of binucleate cells, inferring that CaBP7 plays an important role in the regulation of cytokinesis. CaBP7 was shown to co-localise with VAMP7, a protein known to be important for Ca2+-regulated lysosomal exocytosis and also for the completion of cytokinesis. In addition CaBP7 localised to VSVG positive vesicles which have been previously shown to directly dock and fuse with the plasma membrane at the midzone. Lysosomes are emerging as important platforms for Ca2+-signalling, and proteins important for the release of lysosomal Ca2+ stores have also been implicated in mitosis. This may suggest a role for CaBP7 in the regulation of Ca2+ signalling processes at the lysosomal membrane during both interphase and in dividing cells. Finally the use of nuclear magnetic resonance (NMR) spectroscopy led to the partial backbone and side chain assignment of a 100 amino acid N-terminal fragment of CaBP7. Using this data the secondary structure of this fragment was calculated and closely resembled that of Ca2+-bound CaM and CaBP1S N-termini. Spectra for apo CaBP7 were distinct from that of Ca2+-saturated CaBP7 implying that the protein undergoes a significant conformational change upon Ca2+ binding. In addition, the use of native PAGE demonstrated a shift in mobility of CaBP7 1-100 upon addition of Ca2+ but not Mg2+. Continued NMR analysis of CaBP7 will be required for the determination of a high-resolution structure for this protein.

Strong coordinated uterine contractions are needed in labour and the mechanisms that govern this are well understood. However when these contractions are weak or uncoordinated, labour cannot progress normally. This is termed dysfunctional labour and it accounts for ~20% of all caesarean sections (CS) in the UK. Currently the only treatment available is oxytocin, however this does not reduce the incidence of CS. Lactate is significantly increased in myometrial capillary blood during dystocia suggesting it may be impairing force production, but there are no functional data on the effect of lactate on myometrial contractility. The aims of this work were to a) investigate the effect of lactate and its mechanism of action on myometrial contractility using both human myometrium and an animal model. The effect on spontaneous and oxytocin-stimulated contractions was also examined and b) investigate the presence of lactate transporters MCT-1 and MCT-4 in rat myometrium throughout gestation and in human myometrium was looked at. Lactate, dose-dependently and significantly, decreased spontaneous contractility in rat and human myometrium. Weak acids butyrate, propionate, and pyruvate also significantly reduced contractions in a dose-dependent manner. The effects of lactate were reduced in the presence of oxytocin but not abolished. The effect on pregnant myometrium was greater than in non-pregnant, and was shown to increase towards term in the animal model. In labouring myometrium, there was little effect of lactate on contractility. Using the animal model to investigate lactate’s mechanism in the myometrium, tissue strips were loaded with either Indo-1 AM (a Ca2+ sensitive indicator) or Carboxy SNARF-AM (a pH sensitive indicator). Lactate inhibited Ca2+ transients and had little effect on force when a Ca2+ channel agonist was present. Lactate decreased pHi in a dose dependent manner and this drop in pHi was reduced when the tissue was stimulated by oxytocin or in labour. Both MCT-1 and MCT-4 were found to be present in rat and human myometrium, with MCT-1 at a higher amount in pregnant myometrium. From this work I have shown that lactate in the physiological range potently decreases spontaneous contractility in both rat and human myometrium. The effects of lactate were reduced in the presence of oxytocin or labour but still produced significant decreases and this may be due the difference in alteration of pHi. Other weak acids produce similar effects to lactate suggesting its mechanism of action is not via lactate’s role in metabolism. Lactate inhibited Ca2+ transients, which could be due to a fall of pHi, as it has previously been demonstrated that intracellular acidification decreases Ca2+ current through L-type Ca2+ channels. The difference in MCT-1 and MCT-4 expression between non-pregnant and pregnant tissue may indicate a role in labour. I suggest that differences in myometrial lactate in women can lead to accumulation of extracellular lactate, which as we have shown, will reduce myometrial contractions and could therefore contribute to dysfunctional labour.

Galectins are galactoside-binding proteins that are expressed by various types of human cells. Recent studies have shown that the levels of circulating galectins are significantly higher in the bloodstream of cancer patients and promote cancer cell adhesion and aggregation by interaction with the cancer-associated mucin protein MUC1. However, previous studies have also shown that circulating galectins have MUC1-independent actions in metastasis promotion. This thesis further explores these MUC-1 independent actions. The studies reported here show that galectin-2, 3, -4 and -8, whose serum concentrations are all increased in cancer patients, induce secretion of various cytokines from endothelial cells in vitro and in vivo: (G-CSF, GM-CSF, IL-6 and sICAM-1 by galectin-3; G-CSF, IL-6 and GROα/CXCL1 by galectin-2; G-CSF, IL-6, GROα/CXCL1 and MCP-1/CCL2 by galectin-4 and -8). The secretion of these cytokines autocrinely/paracrinely enhances the endothelial expression of cell surface adhesion molecules, causing adhesion of cancer cells to the blood vascular endothelium. The galectin-induced secretion of cytokines is also shown to promote endothelial cell migration and tubule formation in angiogenesis. Intravenous introduction of a pathological galectin concentration into nude mice resulted in significant increase of circulating cytokine concentrations within 24 or 48 hours. Higher serum levels of these galectins correlated with higher serum levels of these cytokines in colon and breast cancer patients. Thus the increased circulation of galectins in the bloodstream of cancer patients promotes secretion from the blood vascular endothelium of metastasis-promoting cytokines that enhance circulating cancer cell adhesion and angiogenesis. These MUC1-independent actions of circulating galectins likely make an important contribution to the metastasis-promoting action of circulating galectins. Targeting the actions of circulating galectins in cancer patients therefore represents a promising therapeutic strategy to reduce metastasis and improve survival.

Portfolio of Evidence Paper 1: Thompson, K.G., Haljand, R., Cooper, S-M. and Martin, L. (2000) The relative importance of selected kinematic variables in relation to swimming performance in elite male and elite female 100m and 200m breaststroke swimmers. Journal of Human Movement Studies, 39, 015-032. Paper 2: Martin, L. and Thompson, K. (2000) Reproducibility of diurnal variation in sub-maximal swimming. International Journal of Sports Medicine, 21, 387-392. Paper 3: Martin, L. and Whyte, G.P. (2000) Comparison of critical swimming velocity and velocity at lactate threshold in elite triathletes. International Journal of Sports Medicine, 21, 366-368. p14 Paper 4: Martin, L., Doggart, A.L. and Whyte, G.P. (2001) Comparison of physiological responses to morning and evening submaximal running. Journal of Sports Sciences, 19, 969 – 976. Paper 5: Martin, L., Nevill, A. M. and Thompson, K. G. (2007) Diurnal Variation in Swim Performance Remains, Irrespective of Training Once or Twice Daily. International Journal of Sports Physiology and Performance, 2, 192-200. Paper 6: Martin, L., Lambeth-Mansell, A., Beretta-Azevedo, L., Holmes, L.A., Wright, R. and St Clair Gibson, A. (2012) Even between-lap pacing despite high within-lap variation during mountain biking. International Journal of Sports Physiology and Performance, 7 (4), 261-270. Follow links to access papers in the portfolio of evidence.

The covalent post-translational modification of selected substrates with the ubiquitin protein has emerged as a central regulatory mechanism, governing protein stability, activity and localisation, and accordingly an array of cellular processes. Ubiquitin signalling versatility arises owing to the diverse nature of (poly)ubiquitin modification, with distinct modifications subsequently transduced in a specific manner by ubiquitin-binding domains found in ubiquitin-binding proteins. In recent years the notion that ubiquitin exerts influence solely via the covalent modification of substrates has been challenged, with unanchored, or substrate-free polyubiquitin chains emerging as key regulators of cellular physiology. The investigations described in this thesis seek to exploit the inherent selectivity of natural and engineered ubiquitin-binding proteins, to afford the purification of endogenous unanchored polyubiquitin, probing the molecular composition and interactions of this biologically significant ubiquitin pool. In chapter 3 by utilising the deubiquitinating enzyme USP5, which contains multiple ubiquitin-binding domains, and is normally responsible for the selective disassembly of unanchored ubiquitin, we purify unanchored polyubiquitin from mammalian cell extracts. Subsequently, we apply both ubiquitin-selective antibodies and mass spectrometry-based analyses to examine the polyubiquitin profile of the mammalian unanchored ubiquitin pool. In chapter 4 we then assess the mechanisms of ubiquitin recognition by USP5, presenting a structural mass spectrometry-based framework to probe and quantify ubiquitin: ubiquitin-binding domain interactions. Finally in chapter 5, based on the conclusions we draw from USP5-ubiquitin recognition in chapter 4, that multiple domains in suitable arrangement yield specificity for polyubiquitin chains, we design and synthesize a synthetic protein to favour the capture of unanchored polyubiquitin chains of defined topology, namely Lys-48 linked diubiquitin (and longer polyubiquitin chains), from mammalian cell extracts. We conclude that strategies for the rational design and engineering of polyubiquitin chain-selective binding in non-biological polymers are possible, paving the way for the generation of reagents to probe the unanchored polyubiquitin chains of defined topology, and more widely the ‘ubiquitome’.

Pancreatic ductal adenocarcinoma (PDAC) is noted for its resistance to therapy and poor prognosis. PDAC is characterised by extensive local invasion and metastases at distant organs. Having previously shown that the cAMP cascade had regulatory effects on cell migration/invasion in PDAC cells, which are vital processes in metastasis formation, we decided to characterise the cAMP signalling machinery in PANC-1 cells. In this study we adopted a live-cell imaging approach taking advantage of genetically engineered probes that use Fӧrster resonance energy transfer (FRET) to reveal cAMP concentration and PKA activity at a single cell level. Using the Epac-based cAMP sensor H134 we found that PDE3 and PDE4 are the principal cAMP destroyers, whereas β-adrenoceptors are the main physiological cAMP-cascade activators, in PANC-1 cells. Downstream, using Boyden chamber assays we found that PDE3 has the biggest role in cell migration under ‘basal’ conditions, whereas PDE4 has a bigger role in the presence of isoproterenol. However, isoproterenol on its own did not influence PANC-1 cell migration despite having the ability to increase cAMP concentration inside the cell. This part of the study puts forward PDE3 and PDE4 as potential targets for reducing migration/invasion of PDAC. In the second part of the study, we found that these cells have an efficient Ca2+ signalling system; in which ORAI1 is the main mediator of store-operated Ca2+ entry (SOCE) in PANC-1 cells. To explore the possibility of a Ca2+-cAMP crosstalk in PANC-1 cells, we used the AKAR4 probe to measure PKA activity during Ca2+ responses. The application of neurotensin (NT), a well-known IP3-producing agonist in this cell type, induced a Ca2+ response which was accompanied by an increase in PKA activity. SOCE is likely to play an important role in this process since the activation of SOCE by thapsigargin-mediated store depletion consistently and robustly increased PKA activity. To further investigate the downstream roles of PKA signalling in PANC-1 cells we utilised immunofluorescence to visualise the distribution of PKA activity. Using antibodies specific for phosphorylated PKA substrates, we found that phosphorylated PKA substrates are preferentially concentrated in the nucleus; and notably at the leading edges of PANC-1 cells displaying a migratory phenotype. At the leading edges phosphorylated PKA substrates colocalised with actin-rich ruffles. Interestingly, utilising the rapamycin-inducible heterodimerisation system to reveal endogenous ER-PM junctions in migrating PANC-1 cells, we found that ER-PM junctions are also situated in close proximity to the leading edge. These results suggest that SOCE activates PKA responses in the region strategically important for PDAC cell migration.

Debilitating age-associated neurodegenerative disorders (NDs) are a major public health challenge in increasingly ageing societies. Currently approved therapeutics are successful in slowing the progression of NDs but not in reversing or preventing the symptoms. A major goal of neurodegeneration research is therefore to identify potential new therapies for these devastating and eventually fatal disorders. In this study, multiple well-defined C. elegans neurodegeneration models were integrated to uncover effective therapeutic interventions that target shared pathogenic pathways. Using locomotion behaviour and lifespan as phenotypic readouts, the well-established anti-epileptic drug ethosuximide was identified as a promising compound with the potential to combat more than one ND. It not only rescued the short lifespan and chemosensory defects of a C. elegans null mutant model of the rare autosomal dominant human ND known as adult-onset neuronal ceroid lipofuscinosis (ANCL), but also ameliorated the mobility defect and short lifespan of worm tauopathy and polyglutamine models based on transgenic expression of mutant human disease proteins. The ability of ethosuximide to rescue these phenotypes did not correlate well with levels of aggregated Tau and polyglutamine protein, suggesting that ethosuximide suppresses proteotoxicity without preventing protein aggregation. Although the main proposed therapeutic target of ethosuximide in epilepsy is the T-type calcium channel, deletion of its worm homologue, cca-1, did not affect the increase in locomotion and lifespan in the tauopathy model. This suggests that ethosuximide counteracts proteotoxicity via a novel mechanism. To further investigate how ethosuximide might exert its neuroprotective properties, we conducted global gene expression analyses using the Affymetrix C. elegans whole genome microarray platform and compared the transcriptome of ethosuximide-treated ANCL models and wild-type worms to that of unexposed controls. Various downstream bioinformatic investigations including gene ontology analyses, regulatory motif discovery, publication enrichment analysis, comparative analyses with curated data sources and literature were performed and revealed a wide range of DAF-16-dependent transcriptional alterations. C. elegans DAF-16 is the sole orthologue of the mammalian FOXO family of transcription factors (TFs) that are implicated in the regulation of a wide range of physiological processes. Genes commonly regulated in ethosuximide-treated animals have varied roles in lipid metabolism, redox homeostasis, longevity/ageing, chromatin remodelling and ubiquitination. Many ethosuximide-responsive genes also contained DAF-16 regulatory elements within their promoter regions and were known to be among the top most responsive DAF-16 TF targets. The importance of DAF-16 in ethosuximide-mediated protection was further substantiated by RNA interference and cell culture experiments. Ethosuximide was shown to consistently induce the transcriptional activity of a subset of mammalian FOXO target genes and conferred protection against expanded polyglutamine peptides-induced aggregation in mammalian neuronal cells. These findings should encourage further screening and characterisation of other neuroprotective compounds, and ultimately may assist in expediting translational drug research and clinical testing for new therapeutic targets to combat protein conformational disorders in general.

The paraventricular nucleus (PVN) is a region of the hypothalamus considered the “master controller” of the autonomic nervous system. A subregion of the PVN, the parvocellular subnucleus, is believed to be involved in autonomic control, but its physiological importance is not fully understood. This thesis aimed to investigate the role of the parvocellular PVN in autonomic control and the underlying mechanisms responsible. In slice cell-attached patch action current recordings showed that putative parvocellular neurones are sensitive to osmolality and that a member of the mechanosensitive transient receptor potential ion channel (TRP) family TRPV4 plays a role in this osmosensing. TRPV4 agonists gave a similar reduction in action current frequency (ACf) to hypotonic challenge, which was reversed by selective TRPV4 inhibitors. Single-channel recordings identified a TRP-like channel on parvocellular neurones, and the activity of this channel was increased by the TRPV4 agonist 4αPDD. Intracellular calcium recordings showed increases in Ca2+ in response to either hypotonic challenge or 4αPDD. Furthermore, a role for TRPV4 was verified in central osmosensing at the whole animal level; central injections of hypotonic solution decreased blood pressure; an effect ablated by a TRPV4 inhibitor. Functional coupling between TRPV4 channels and Ca2+-activated K+ (KCa) channels was also explored. The effect of hypotonic challenge was reversed by inhibition of the small-conductance KCa (SK) channel. Since the effects of TRPV4 could also be blocked by an SK inhibitor, it is proposed that TRPV4 is coupled to SK to modulate neuronal activity. During calcium recordings Aaplication of a TRPV4 agonist in the presence of an SK inhibitor showed a reduced, but sustained Ca2+ rise compared to TRPV4 agonist application alone, suggesting feedback mechanisms are also in play. These mechanisms were also verified, quantitatively, with a mathematical model written in the NEURON simulation environment and incorporating experimentally derived parameters. A role for this area of the PVN in temperature sensing was also discovered, with ACf decreasing with an increase in temperature from 25oC to 37oC. Pharmacological investigation identified another TRP channel, TRPM2, to be central for the PVN response to temperature. ECG recordings from rats implanted with telemeters confirmed roles for neurokinin 1 receptor (NK1) expressing neurones in the PVN in the cardiovascular response to psychological stress and in the setting of circadian heart rate. Heart rate variability analysis showed that increases in the sympathetic activity indicator, “LF/HF”, in response to handling stress were ablated by specific lesioning of the NK1 neurones in the PVN. In addition these animals had a significant shift in the daily variation of their average day/night heart rate. In conclusion this thesis identifies the mechanisms underlying several different functional roles for parvocellular PVN neurones and indicates the PVN may be a multifunctional homeostatic “detector”.

The tumour microenvironment consists of a diverse range of cell types, which work together to create favorable conditions to support tumour growth and metastasis. It has been repeatedly reported that bone marrow-derived mesenchymal stem cells (MSCs), also referred to as multipotent stromal cells, infiltrate into the site of developing tumours. These adult multipotent cells show significant therapeutic potential in wound healing, tissue regeneration, immunosuppression and the treatment of cancer. However, there have been numerous contradictory reports on the role and fate of MSCs following engraftment into the tumour microenvironment. Evidence suggests that MSCs may constitute >20% of the stromal component of some solid tumours. In addition, there is evidence that paracrine signalling from cancer cells may cause MSCs to differentiate into cancer-associated myofibroblasts (CAMs), Soluble factors produced by CAMs have been shown to enhance further MSC recruitment and promote tumour progression and metastasis. In light of this information, the aims of this project were: (1) to investigate the changes that occur in MSCs following exposure to cancer cells or CAMs, and conversely, (2) to study the effects that MSCs may have on cancer cells and stromal cells using in vitro co-culture techniques. Exposure to the secretome of the AGS gastric adenocarcinoma cell-line induced a distinct gene expression profile in MSCs that was strongly associated with immune and inflammatory responses. Exposure to primary CAMs did not induce similar changes and exhibited little correlation across MSC and CAM cell-lines. Furthermore, exposure to AGS- or CAM-CM did not promote the acquisition of CAM-like characteristics in MSCs. However, extended exposure to AGS-CM or TNFα prompts the formation of a tumour-promoting MSC (tMSC) phenotype that is supportive of AGS growth via direct cell contact. Nrf2 is a transcription factor that is classically associated with cellular responses to stress that also induces the transcription of various anti-inflammatory genes and can be chemically induced by the synthetic triterpenoid CDDO-Me. Although CDDO-Me could inhibit various pro-inflammatory genes in MSCs that are induced in response to AGS-CM, it also propagates the formation of a tMSC phenotype. Finally, a key trait associated with MSCs is their ability to modulate the immune response. It is widely reported that MSCs have the ability to strongly repress the immune response. We found that MSCs can differentially modulate the maturation of dendritic cells depending on the ratio of cells used, and whether MSCs have been pre-exposed to AGS-CM. These findings suggest that the role and fate of MSCs is heavily dependent on the tissue niche in which they reside. MSCs are a highly responsive set of cells that can be directed towards various functional phenotypes that may inhibit or promote local cellular growth and activity. This highlights the need for high-throughput studies to define MSCs responses to external stimuli that may assist in increasing the efficacy of MSC-based therapies.

Globally, gastric cancer claims around 800,000 lives per year. As many patients present at an advanced stage of disease, prognosis remains poor for most patients, with five-year survival rates of less than 30%. As many patients show only limited short-term benefits from current therapeutic regimes, there is a clear need for improved understanding of the molecular mechanisms that drive the development and spread of gastric cancer. In this context, the role of the tumour microenvironment in cancer development and the potential for new forms of therapeutic intervention has become a field of increasing interest in many areas of cancer research. It is now well established that the development and progression of gastric tumours is facilitated by reciprocal communication between cancer cells, and cells within the surrounding tissue. In this study we focus our investigation on the mechanisms and consequences of paracrine communication between gastric cancer cells and different populations of stromal myofibroblasts, which are prevalent within the cancer microenvironment and form a significant proportion of many solid tumours. Previous studies show that myofibroblasts derived from gastric tumours (CAMs) have inherently different profiles of gene expression, compared to patient matched adjacent tissue myofibroblasts (ATMs), or normal tissue myofibroblasts (NTMs). Given these differences, we were interested to know if specific myofibroblast populations respond differently to signals from cancer cells; or conversely, if they exhibit differential ability to facilitate pro- tumourogenic changes in gastric cancer cells. Using a combination of bioinformatics and experimental techniques we demonstrate that CAM-conditioned media induces distinct changes in the gene expression profiles and metabolic activity of AGS gastric cancer cells. Significantly, these changes were not observed following exposure to conditioned media derived from either ATM or NTM cells. Conversely, CAM cells were found to have higher levels of GLUT1 and MCT4 expression with a corresponding reduction in mitochondrial activity compared to NTM cell lines. Finally, initial analysis of CAM imposed changes in AGS gene-expression suggests changes may reflect patient prognosis or stage of tumour development, implying future potential for patient stratification. In conclusion, data from this study shows that activated CAMs are robustly programmed by cancer cells to facilitate optimal conditions for tumour growth. Therefore, further analysis of this system may provide much needed options for improved therapeutic intervention and precision medicine.