Our immune system must be able to rapidly fight against pathogens, but at the same time be tightly regulated to prevent harmful autoimmune and inflammatory responses. This intricate balance is controlled in part by T lymphocytes. Therapies targeting T cells have the potential to revolutionise the ways in which inflammation and autoimmune diseases are treated. However, before this can be achieved, a better quantitative understanding of the molecular processes controlling the functions of these cells is required. T cell signalling is tightly regulated by a series of complex molecular networks, which converge on key transcription factors, including Nuclear Factor-κB (NF-κB), Nuclear Factor of Activated T cells (NFAT), and Activator Protein 1 (AP-1). Using a combination of single-cell time-lapse imaging, and genome-wide assays probing for chromatin accessibility and gene expression, this study provides a better understanding of the mechanisms underpinning T cell activation and signalling. One central tenet of T cell activation is that activation-associated gene expression is triggered by the binding of the cognate antigen to the T cell receptor (TCR), and enhanced by co-stimulatory receptors, including CD28, which act to augment TCR signalling. This study shows that activation- associated gene expression programmes (induced by calcium ionophore ionomycin and phorbol 12-myristate 13-acetate (PMA) in Jurkat T cells) are closely associated with specific chromatin landscapes. Further to this, data shown here indicate that the integration between TCR and co- stimulatory receptor signalling occurs at the chromatin level, and plays a pivotal role in regulating T cell activation. Using live-cell imaging, this study also shows that information about the diverse external signals received by T cells could be encoded within the dynamic nuclear translocations of key transcription factors. In particular, TCR signals appear to be processed by the duration of NFAT nuclear occupancy. TCR stimulation in the presence of a co-stimulatory signal resulted in the rapid nuclear import and export of NFAT proteins. In contrast, when TCR stimulation was applied without a co-stimulatory signal, prolonged nuclear occupancy of NFAT was observed. Further investigation suggested that the sustained activity of NFAT could confer a ‘signal memory’ within the TCR signalling network, thus providing a potential mechanism for preventing premature T cell turn-off during transient T cell-Antigen presenting cell interactions. This new detailed picture of T cell biology moves the field towards better therapeutic strategies for numerous diseases.

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The PI3K/Akt/mTOR and MAPK pathways are frequently altered in ovarian cancer cells, making them potential candidates for targeted therapy. A more complete understanding of the complex interactions between the different proteins within the two pathways would assist in developing more effective treatment strategies to help overcome therapy resistance. The purpose of this project was to understand the phosphoproteomic changes in response to PI3K/mTOR inhibition in ovarian cancer cells and to identify potential mechanisms that may lead to targeted therapy resistance. To investigate the effect of inhibiting PI3K/mTOR at the cellular level in ovarian cancer, PI3K (LY294002), mTOR (rapamycin) and dual PI3K/mTOR (BEZ235) inhibitors were used to treat a panel of ovarian cancer cell lines. All tested cells, irrespective of PI3K/Akt/mTOR and MAPK pathways mutational status, responded to the three inhibitors. BEZ235 treatment produced greater cell inhibition than the monotargeted agents, while PTENmutated cell lines were more responsive to mTOR blockade than inhibition of PI3K alone. The phosphoproteomic changes in the cell lines were evaluated over a time course after treatment with the inhibitors, stimulated by heregulin, and studied using reverse phase protein array analysis. The results revealed that the decreased expression of pAkt (Thr308) appears to be a biomarker of sensitivity for LY294002 and BEZ235 in both PEO4 and A2780 cells, while upregulation of pAkt (Ser473) is an indicator for effective rapamycin treatment within the same cell lines. Increased pAkt (Ser473) expression after rapamycin treatment in PEO4 cells is believed to be due to the S6K1-mTORC2-Akt feedback loop. It was observed that pERK was upregulated upon BEZ235 treatment, which suggested the presence of cross talk between the PI3K/Akt/mTOR and MAPK pathways. A combination of BEZ235 and PD-0325901 (MEK inhibitor) treatments inhibited both pAkt (Ser473) and pERK, which also produced significant inhibition in cell proliferation compared to monotherapy treatment. The data also revealed a novel finding in ovarian cancer that prolonged (24h) treatment with rapamycin sensitises mTORC2 in PEO4 cells under heregulin stimulation. Moreover, network correlation and clustering analysis using the phosphoproteomic data identified significant correlations between the expression of pmTOR (Ser2481), and both p-cRaf (Ser259 and Ser338). Sin1 knockdown was performed in PEO4 cells and showed significant downregulation in the expression of pAkt (Ser473) and upregulation in pERK expression, indicating the role of Sin1 to regulate both the PI3K/Akt/mTOR and MAPK pathways potentially via mTORC2 and Ras. Phosphoproteomic profiling was performed on 469 ovarian cancer tissue samples using TMA and IHC analysis. Several significant associations were discovered between the phosphoproteomic data and the different clinicopathological parameters. High expression of pmTOR (Ser2448) was correlated with poorer overall survival in patients with ovarian endometrioid carcinoma compared to patients with low expression (p < 0.024). This implies that pmTOR (Ser2448) expression may potentially be a prognostic marker for patients with ovarian endometrioid carcinoma. In conclusion, I present dynamic phosphoproteomic profiling of the PI3K/Akt/mTOR and MAPK pathways in ovarian cancer, suggesting novel feedback loops and cross talk that could play a role in resistance mechanisms to these therapies. Combination treatment showed an additive effect on cell growth offering an approach to overcome drug resistance.

Cell signalling determines physiological responses to many cellular stimuli and environmental changes. The transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP) signalling pathways begin by binding of ligand to the heterodimeric receptor complex, followed by activation of Smads that translocate to the nucleus to regulate transcription of genes that further mediate cellular physiology. The TGFβ/BMP pathways are very important for proper tissue development and homeostasis, thus precise spatial and temporal regulation of the signalling pathway is required and achieved by many positive and negative signalling regulators. This thesis work identified the liver kinase B1 (LKB1) pathway as a negative regulator of TGFβ/BMP signalling pathways. In the first paper, we established LKB1 as a negative regulator of TGFβ signalling and TGFβ-induced epithelial to mesenchymal transition (EMT). LKB1 impairs Smad4 binding capacity to DNA leading to suppressed TGFβ-activated gene transcription. The second paper describes further the mechanism of LKB1 negative regulation on BMP signalling, by mediating BMP type I receptor degradation resulting in inhibition of BMP-induced cell differentiation. Downstream of LKB1, salt inducible kinase 1 (SIK1) is a TGFβ target gene and its expression is up-regulated by Smad2/3/4-mediated gene transcription. The third paper elucidates the mechanism of SIK1 transcriptional induction via an enhancer element located 3’ of the gene and SIK1-mediated type I TGFβ receptor degradation, which requires the activity of Smad7 and of the Smurf2 ubiquitin ligase. The fourth manuscript finds sucrose non-fermenting (SNF) 1-like kinase 2 (NUAK2) as another TGFβ target gene and its up-regulation results in modification of the mammalian target of rapamycin (mTOR) pathway that controls protein synthesis. NUAK2 cooperates with LKB1 leading to Raptor phosphorylation and inhibition of mTOR-mediated protein synthesis. Collectively, this thesis work has provided a functional link between two important signalling pathways, the metabolic LKB1 pathway and TGFβ/BMP pathway.

A significant advance in understanding skeletal muscle adaptation to physical training has been the observation that nutrients and exercise work in synergy to enhance muscle protein synthesis. Physical activity triggers an adaptive response to which nutrition provides the necessary building blocks for an optimal response. The aim of the present work was to contribute to the understanding of the molecular events induced by exercise or nutrients (creatine and amino acids) to create the adaptive environment and to induce the cellular adaptation and growth, respectively. From a methodological point of view, two experimental models were used: muscle biopsies taken from the vastus lateralis of human volunteers and myotubes cultured from C2C12 cells. The transcription of a series of genes involved in muscle remodelling (MAFbx, MHCIIA, PGC-1á, PCNA and IL-6) was increased immediately after the completion of a resistance exercise session performed in the fasted state. The phosphorylation state of p38 and ERK1/2 was also increased, whereas the Akt/PKB pathway was negatively regulated. This contrasted with the high phosphorylation state observed on p70s6k and 4E-BP1 when subjects received a large amount of amino acids during the recovery period. Our results suggest that the MAPK pathway can be triggered by contractile activity alone, whereas the Akt/PKB pathway requires nutrients to be activated. Certain amino acids regulate the phosphorylation state of mTOR and its downstream targets, as demonstrated by one of our in vitro studies. However, that modulation did not lead to a systematic modification in the rate of protein synthesis. Amino acids were also able to influence the expression of muscle-specific genes, highlighting their importance in the control of muscle protein synthesis. Protein anabolism was largely enhanced and cell differentiation was accelerated by creatine in our in vitro model. We have identified the p38 and Akt/PKB pathways as mediators of these effects. Nevertheless, we were unable to confirm the existence of similar events in human skeletal muscle in vivo.

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Signals via the T cell receptor (TCR) are critical for the development of T cells in the thymus, maintenance of a self-tolerant peripheral T cell repertoire and the activation of T cells in secondary lymphoid organs. A dynamic balance between tyrosine phosphorylation and dephosphorylation is essential for the maintenance of homeostasis and proper regulation of the immune system. The cytoplasmic phosphatase, PTPN22 (protein tyrosine phosphatase non-receptor type 22) is involved in negative modulation of signal transduction through the TCR and plays a central role in regulating lymphocyte homeostasis. Genome wide association studies reveal that point mutations in PTPN22 confer an increased risk of developing multiple autoimmune diseases in humans. The precise function of PTPN22 and how mutations contribute to autoimmunity is controversial. Loss-of-function mutations in PTPN22 are associated with elevated T effector cell expansion and autoreactive B cells in both humans and mice. A thorough dissection of the molecular involvement of PTPN22 and its allelic variant R619W is important to delineate its role in autoimmunity, to this end we utilised the Ptpn22-/- mice generated in our laboratory. In order to address whether R619W allelic variant is a gain- or loss-of-function mutation, we expressed both PTPN22WT and PTPN22R619W constructs in primary activated Ptpn22-/- T lymphocytes using lentiviral transduction. Surprisingly expression of either wild type or variant phosphatase showed no affect on cytokine production. Preliminary results from bone marrow chimeras generated by retroviral expression of PTPN22WT and PTPN22R619W in Ptpn22 deficient mice showed reduced T cell activation compared to Ptpn22-/- T cells. PTPN22WT appeared to be more suppressive of T cell responses than variant PTPN22R619W. Consistent with studies conducted in comparable knock-in mouse models, our data point to the variant PTPN22R619W as being a partial loss of function allele. To elucidate the mechanism of PTPN22 action in context of an autoimmune disease, we investigated the effect of Ptpn22 deficiency on the phenotype of SKG mice. The SKG mouse harbours a point mutation (W163C) within the carboxyl terminal SH2- domain of ZAP-70, which results in decreased TCR signalling and impaired thymocyte development with defective positive and negative selection. These mice are prone to developing CD4+ T cell mediated autoimmune arthritis that closely resembles rheumatoid arthritis in humans. We found that thymus differentiation was partially restored in SKG Ptpn22-/- thymocytes and Ptpn22 deficiency enhanced TCR mediated signalling in SKG Ptpn22-/- thymocytes relative to SKG thymocytes. Consistent with increased signalling observed in the thymocytes, there was improved in vitro proliferation and IL-2 production of CD4+ T lymphocytes from SKG Ptpn22-/- mice compared to SKG mice. By contrast to SKG mice, SKG Ptpn22-/- mice developed less severe mannan-induced arthritis and showed decreased proportions of Th17 and higher numbers of regulatory T cells. These results show that removal of PTPN22 can compensate, at least partially, for the deficient ZAP-70 activity in the SKG mouse, thus linking PTPN22 and ZAP-70 to the same signalling pathway. This study advances our understanding of how manipulating TCR signals impacts on downstream T cell functions, suggesting PTPN22 may be a valuable target for the treatment of autoimmune diseases. Further studies to determine physiological role of the phosphatase and its disease-associated variants could provide insight into mechanism of immune activation, tolerance and autoimmunity.

The ability of cells to react to changes in their environment is critical to their survival. Effective decision making strategies leading to the activation of specific intracellular pathways hinge on cells sensing and processing extracellular variation. We will only be able to understand and manipulate how cells make decisions if we understand the “design” of the mechanisms that enable them to make such decisions, in terms of how they function, and in terms of their limitations and architecture. In this thesis, using mathematical modelling, I revisited classical signal sensing and transduction mechanisms in light of recent developments in methodological approaches and data collection. I studied the sensing characteristics of one of the simplest of sensors, the allosteric sensor, to understand the limits and effectiveness of its “design”. Using the classical Monod-Wyman-Changeux model of allostery, I defined and evaluated six engineering-inspired characteristics as a function of the parameters and number of sensors. I found that specifying one characteristic strongly constrains others and I determined the trade-offs that follow from these constraints. I also calculated the probability distribution of the number of input molecules that maximizes information transfer and, as a consequence, the number of environmental states a given population of sensors can discriminate between. Next, I proposed a new general model of phosphorylation cycles that can account for experimental reports of ultrasensitivity occurring in regimes that are far away from Goldbeter and Koshland’s zero-order saturation, the classical ultrasensitivity-generating mechanism. The new model exhibits robust ultrasensitivity in “anti-zero-order” regimes. The degree of ultrasensitivity, its limits, and its dependence on the parameters of the system are analytically tractable. The model can, additionally, explain in an intuitive way some puzzling experimental observations. Finally, I addressed the problem of integrating different types of signals from multiple sources, and performed some preliminary exploration of how cells can “learn” to associate and dissociate correlated signals in non-evolutionary time-scales. This work provides insights into the function and robustness of signal sensing and transduction mechanisms and as such is applicable to both the study of endogenous systems and the design of synthetic ones.