The NF-κB transcription factor p65/RelA controls hundreds of genes involved in inflammation, immunity, cell proliferation and survival. Cells treated with the inflammatory cytokine TNF-α show repeated nuclear-cytoplasmic translocations of p65 maintained by multiple negative feedback loops. Application of pulses of TNF-α at various set intervals can entrain the translocation frequency and cause different patterns of NF-κB-dependent gene transcription. Translocation frequency can also be altered by biological and environmental factors such as temperature and diclofenac treatment. These observations have led to the hypothesis that the dynamics of NF-κB play a role in the transcriptional responses dependent on particular cellular states. NF-κB can physically interact with members of other molecular networks including glucocorticoid signalling, the circadian clock and the cell cycle. In a previous study, two tumour cell lines showed differential p65 dynamics when treated with TNF-α at different cell cycle stages which concomitantly also altered cell cycle length. Evidence suggested that physical interactions between p65 and the cell cycle regulators E2F1 and E2F4 could, at least in part, be responsible for these effects. This thesis describes studies designed to investigate if similar responses exist in more normal cells. Although transcript levels of NF-κB target genes were suppressed in MEF cells treated with TNF-α during S phase relative to G1/S, NF-κB translocations appeared unaffected. Knowledge of the interactions with other molecular networks is central to understanding the context-dependent role of NF-κB, and the complexity of these dynamic interactions requires a Systems Biology approach. Within this study, a model of the interaction between NF-κB and the cell cycle was tested for its ability to recapitulate biological data from HeLa cells. Also, the effect of cell cycle timing of TNF-α treatment on NF-κB dynamics in HeLa cells was examined using ImageStream flow cytometry. It is suggested that a Systems Biology approach could be used to extend a study, initial results of which are presented here, of the interaction between NF-κB and glucocorticoid signalling. Understanding the interactions between NF-κB signalling and other networks should have clinical benefits, for example for cancer treatment dependent on circadian timing.

Immune cells must accurately interpret environmental signals to make robust cell fate decisions and control inflammatory signalling. Many signals (e.g. Tumor Necrosis Factor alpha (TNFα) or interferon gamma (IFNγ)) converge on just a few key signalling systems such as Nuclear Factor kappa B (NF-κB) or Signal Transducers and Activators of Transcription (STAT), which exhibit complex activation dynamics that control patterns of downstream gene expression. Often, seemingly identical cells show heterogeneous or random behaviour to a common stimulus. Therefore, a key question is how can immune cells coordinate inflammatory signalling in the presence of this noise. The NF-kB system dynamics were studied in response to rapidly changing inflammatory signals. It was shown that pulsed TNFa cytokine stimulations induced digital single-cell NF-kB responses, with only a fraction of cells able to respond to repeated pulses. These responses appeared to be reproducible in individual cells, but heterogeneous in the population. Mathematical models of the NF-kB signalling network suggested that single-cell responses were governed through a refractory state potentially encoded via 'extrinsic' noise in the levels of signalling molecules related to the TNFa signal transduction pathway. Such signal processing enabled robust and reproducible single cell responses and maintained acute tissue-level signalling, with fewer cells responding to shorter pulsing intervals. The NF-kB system is involved in effector cytokine propagation in response to pathogen infection. It was shown that in macrophages, the dose of TLR4 stimulation (mimicking the pathogen infection) was encoded in graded (yet heterogeneous) NF-kB dynamics in single cells. This resulted in analogue inflammatory gene expression patterns in the population. However, individual cells substantially differed in their ability to encode TLR4 signal and to regulate TNFa expression, which was explained by extrinsic noise in the NF-kB system. Quantitative mathematical modelling showed that tissue-level environment modulates heterogeneous single-cell TNFa outputs; by effectively removing it from circulation. This may determine the interaction distance between tissue-resident immune cells to enable propagation of cellular inflammation. Heterogeneity of single cell macrophage signalling was also observed in NF-kB and STAT1 system responses to a range of IFN stimulation doses. Although each system showed substantial variability between cells, their responses were surprisingly well correlated in individual cells. It was however apparent (based on gene expression studies) that individual cells may not be able to precisely discriminate different IFNg doses. Overall, this work suggests that heterogeneity in the NF-kB (and other) regulatory networks might be a part of an inherent design motif in the inflammatory response, which enables robust control of the tissue-level inflammatory response by preventing homogeneous and thus potentially harmful activation.

Peroxiredoxins are small ubiquitous cysteine-containing proteins that exhibit high reactivity to hydrogen peroxide. Apart from their role as antioxidants, detoxifying hydrogen peroxide to water, peroxiredoxins have been implicated in other cellular processes, such as protein folding and signalling. Using S. cerevisiae as a model organism, we utilised a variety of techniques to examine previously unexplored links between peroxiredoxins and mitochondrial function. Firstly, we characterised the role of Gpx3 in yeast mitochondria. Proteomic work revealed the presence of Gpx3 in the mitochondrial intermembrane space (IMS) and we characterised when, how and why Gpx3 can be found within the mitochondria. We showed that cells lacking Gpx3 have aberrant mitochondrial morphology and defective protein import capacity and inner membrane potential upon H2O2 stress. Gpx3 translocates to the IMS via a targeting sequence encoded from a non-AUG codon. This provides a novel and unique molecular mechanism that protects mitochondria from the exceptional oxidative stress which their activity imposes. Secondly, we focused on the role of Tsa1 upon protein aggregation-induced stress. Previous studies using the proline analogue AZC to cause protein misfolding revealed that protein aggregates are localised adjacent to mitochondria and mitochondrial ROS are generated in response. We questioned what effect this might have on mitochondrial function and we showed that upon AZC treatment there is a drop in respiratory rate, dependent on Tsa1. We questioned whether Tsa1, like other peroxiredoxins, is involved in regulating signalling cascades and we showed that cells that are lacking Tsa1 have alterations in the activity of the cAMP/PKA pathway. In parallel, we looked for differences both in the proteome and the transcriptome to understand what is the cause of the lethality of a tsa1 strain upon protein aggregation stress. We propose a mechanism where Tsa1 mediates a transcriptional response to protein misfolding stress via the activity of the heat shock transcription factor, Hsf1. Finally, we focused on the role of the mitochondrial peroxiredoxin Prx1. Under conditions where the mitochondrial matrix is oxidised, either genetically or by chemical addition, we showed than an apoptotic pathway is activated, dependent on the redox state of thioredoxin, Trx3. We showed that Trx3 can interact with Prx1 and loss of Prx1 also stops the induction of cell death. Analysis of the interactome of Trx3 unraveled the involvement of Bxl1/Ybh3, the yeast BH3 domain-containing protein and Aim9, a previously uncharacterised protein with kinase-like motifs, in the progression of cell death. The data presented in this thesis widens our understanding of the function of peroxiredoxins and their involvement in the regulation of cellular cascades that ensure correct mitochondrial function and responses to stress.

Autophagy is a core cytoplasmic degradation process. It is established that KRas-mutant lung cancer cells require basal autophagy for survival. However, the mechanisms that govern this are poorly understood. It has recently been suggested that selective autophagic degradation of signalling complexes may regulate downstream cell signalling pathways. Primarily, this thesis aims to uncover molecular mechanisms through which selective autophagy can regulate signalling pathways that may impact upon cancer cell proliferation. Previous work in the lab identified a putative interaction between the signalling protein TRAF3 and the autophagy protein Ndp52 via mass spectrometric screening. In this thesis I have identified TRAF3 as a target of selective autophagy in both KRas-mutant lung cancer cells and in in vitro transformed MEFs. TRAF3 is a negative regulator of a gene expression regulation pathway called alternative NF-κB. As such, autophagy of TRAF3 promotes basal activation of the alternative NF-κB signalling pathway. This basal activity supports the proliferation of cancer cells. Investigation of TRAF2, a protein closely related to TRAF3, revealed that it too associates with the autophagy pathway, but is not degraded. This is promoted by the activity of TBK1, which itself can phosphorylate TRAF2. Both TBK1 and TRAF2 promote alternative NF-κB signalling, and I investigate possible mechanisms underlying this, including changes in TRAF3 mRNA and protein levels and binding to other alternative NF-κB regulators. This thesis therefore identifies mechanisms through which basal alternative NF-κB signalling is regulated in KRas-mutant lung cancer cells, with implications for cell proliferation. Ultimately, this work provides valuable mechanistic insight to inform the use of autophagy and/or TBK1 inhibition in future cancer therapies.

The aim of this project was to explore the role of the tyrosine kinases JAK1 and JAK3 in cytokine signalling, focusing on interleukin-2 signalling in CD8⁺ effector T lymphocytes. Initial experiments compared the effects of the pan JAK1/JAK3 inhibitor tofacitinib, the selective JAK1 inhibitor GSK186, and the selective JAK3 inhibitor GSK192 on IL-2 control of effector CD8+ cytotoxic T cells (CTL). On the basis of these preliminary data, a detailed analysis of the effect of tofacitinib on effector CD8⁺ T lymphocytes was performed. Phosphorylation events regulated by tofacitinib were identified using mass spectrometry analysis of SILAC (stable isotope labelling with amino acids in cell culture) labelled CTL. Tofacitinib regulated a selective number of phosphorylation sites, with less than 1.2% of the CTL phosphoproteome significantly regulated by tofacitinib treatment following 4hrs tofacitinib treatment. Proteins with downregulated phosphorylation sites were enriched in functions related to the Jak-STAT signalling, regulation of gene expression, and MAPK signalling cascades. Proteins with upregulated phosphorylations were also enriched in functions related to regulation of gene transcription. The proteome of tofacitinib treated CTL was defined by label free mass spectrometry. Approximately 4.5% of the CTL proteome was significantly regulated following 24 hours tofacitinib treatment, suggesting tofacitinib regulates the expression of a selective subset of proteins. Tofacitinib treatment resulted in the downregulation of proteins involved in ribosome biosynthesis, steroid biosynthesis, regulation of transcription and the cell cycle; and the upregulation of proteins with hydrolase activity, and with roles in the lysosome and extracellular exosomes. The phosphoproteomic and proteomic data demonstrates that JAK kinase dependent IL-2 signalling regulates essential processes in CTL by controlling a selective number of phosphorylation events and proteins. Validation of proteins identified as regulated following tofacitinib treatment identified new targets of IL-2 signalling in CTL, including the transcription factor NFIL3. NFIL3 was shown to be upregulated in CD8⁺ T lymphocytes following stimulation with IL-2 and regulated perforin and CD62L expression, suggesting a role in the regulation of CTL effector function.

Macrophages are sentinels present in most tissues of the body, where they recognise and respond to biological dangers. Recognition and uptake of particles is mediated through phagocytic receptors which upon activation induce appropriate responses. These responses need to be tightly regulated in order to destroy pathogens but prevent uncontrolled inflammation. Phagocytosis is an evolutionarily conserved process required for host defence and homeostasis. During phagocytosis, particles are recognised by cell surface receptors that trigger rearrangement of the actin cytoskeleton and internalization of the bound particle into a de novo, membranous organelle known as the phagosome. Regulation of phagocytosis and phagosome maturation can be achieved through changes in transcription/translation and differential recruitment of proteins but also through their non-translational modifications. Here I explored the role of ubiquitylation in the phagosome biogenesis of Interferon-gamma (IFN-ɣ) activated macrophages. Ubiquitylation is a diverse, reversible post-translational modification which is not only involved in protein degradation but also in vesicle trafficking and immune signalling. My data shows that phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ. I applied a targeted AQUA peptide approach by which we quantified ubiquitin chain linkage peptides from phagosome samples by PRM. This data shows that all chain linkages apart from M1/linear chains are present on phagosomes. Furthermore, IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, I characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. I demonstrated that most ubiquitin chains are on the cytoplasmic site of the phagosome enabling an interaction of ubiquitin chains with cytosolic proteins such as Rab7. Rab7 a major regulator of vesicle trafficking could be shown to be ubiquitylated on phagosomes. I further showed that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation and RNF115 is responsible for most of the increase of K63 polyubiquitylation of phagosomal proteins. Knock-down of RNF115 promotes phagosome maturation and induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome and disruption of this pathway induces pro-inflammatory responses in macrophages. In conclusion, this is the first study showing unbiasedly that ubiquitylation plays an important role in vesicle trafficking to the lysosome.

Les plasmodesmes sont des nanopores membranaires qui traversent la paroi des cellules végétales. Ces nanostructures jouent un rôle central en communication intercellulaire et agissent comme des centres de signalisation capables de générer et de relayer le signal de cellule à cellule via l’activité de récepteurs. En tant qu’éléments clés de la communication intercellulaire, les plasmodesmes coordonnent les processus liés à la croissance et au développement des plantes ainsi que les réponses aux stress environnementaux. Dans cette étude, nous avons identifié trois récepteurs de la famille des récepteurs riches en leucine (LRRRLK), capables de relocaliser de manière dynamique au niveau des plasmodesmes après un stress abiotique. L'association plasmodesmale est rapide et survient en moins de 2 minutes. Cette association dynamique n’est pas un comportement général des protéines associées à la membrane plasmique ou aux microdomaines membranaires. En focalisant notre étude sur SAK1 (Sucrose Activated Kinase) nous avons démontré que l'association dynamique aux plasmodesmes est indépendante de la composition en stérols ou en sphingolipides et est partiellement dépendante du statut de phosphorylation de cette protéine. Nous avons identifié un strech d'acide aminé polybasique dans le domaine Juxtamembrane (JMD) de SAK1, décrit chez l’homme comme interagissant avec les lipides anioniques, qui est indispensable à l'association conditionnelle aux plasmodesmes. Au total, nos données indiquent que les changements dans la membrane signature moléculaire des domaines spécialisés plasmodesmes accompagne les réponses aux stimuli externes. / Plasmodesmata pores sustain intercellular communication in plants. They act as specialized signalling hubs clustering receptor activities, and are capable of generating and relaying signalling from cell-to-cell. As key elements in intercellular communication, plasmodesmata coordinate processes related to plant growth, development and environmental stresses responses. In this study, we identified three PM-located Leucine Rich Repeat Receptor Like Kinases (LRR-RLKs) that are able to dynamically and conditionally relocate to plasmodesmata upon abiotic stress. Plasmodesmal association occurs within 2 minutes and is not a general behaviour of PM or microdomain-associated proteins. Focusing on SAK1 (SUCROSE ACTIVATED KINASE) we demonstrated that the plasmodesmal dynamic association is neither dependent of sterol or sphingolipid composition and nor driven by the protein phosphorylation status. Importantly, we identified a polybasic amino acid motif in the Juxtamembrane Domain (JMD) of SAK1 predicted to bind anionic lipids that is critical for conditional plasmodesmal association. Altogether our data indicate that changes in the membrane molecular signature of plasmodesmata specialized domains accompanies responses to external stimuli.

Hedgehog (Hh) signalling from the notochord induces the slow muscle cell fate in the adaxial cells of the developing zebrafish embryo. Slow muscle formation is disrupted in zebrafish ??you-type?? mutants resulting in U-shaped somites. In many you-type mutants, genes encoding components of the Hh signalling pathway are mutated. scube2, a gene not previously known to be involved in Hh signalling, is disrupted in the you-type mutant ??you??. you mutants are deficient in several Hh dependent cell types and show decreased expression of Hh target genes. The Scube (signal peptide-CUB domain-EGF-related) family of proteins act as secreted glycoproteins or cell-surface proteins and are thought to be involved in protein-protein interactions and ligand binding. At the protein level, the Scube family resembles the endocytic receptor Cubilin. Cubilin is known to interact with another endocytic receptor Megalin, which can function as an endocytic receptor for Sonic Hedgehog (SHH) in vitro. Megalin endocytosis of Shh may be an important part of the Hh signal transduction pathway. An anti-Scube2 antibody was developed during this work to investigate the intracellular localization pattern of Scube2 and facilitate the identification of potential Scube2 binding partner(s). In addition, this work identified and characterized two homologs of scube2 in zebrafish, scube 1 and scube 3. The high level of similarity amongst the Scube family of proteins and the weak phenotype of the you mutant suggested scube1 and scube3 might also be involved in slow muscle development. Loss of function experiments performed by antisense morpholino knockdown of scube1 and scube3 in the you mutant decreases the expression of Hh target genes to levels seen in embryos lacking Hh signalling and dramatically enhances the loss of slow muscle fibres compared to you mutants alone. Thus, injecting both scube1 and scube3 morpholinos into you blocks Hh signalling and these embryos fail to develop slow muscle. Inhibition of the three partially redundant scube genes inhibits Hh signalling in zebrafish embryos, thereby demonstrating the essential requirement for scube gene function in the Hh signalling pathway.

[Truncated abstract] Androgens inhibit the growth of human breast tumours and have been successfully used to treat breast cancer in women. Expression of the androgen receptor (AR), which mediates androgen action, is upregulated in breast cancer cells and the AR is the most frequently expressed steroid hormone receptor in breast tumours. AR levels and activity are modulated by the activity of other signalling pathways, however interactions between the AR and signalling pathways and the consequent alterations to the androgen responsiveness of breast cancer cells are largely uncharacterised. The extracellular signal regulated kinase (ERK1/2) pathway is hyperactivated in ~30% of breast tumours and these tumours are often associated with low oestrogen receptor-a (ERa) levels, reduced responsiveness to antioestrogen therapies and an overall poorer prognosis. In this thesis, the MCF-7 human breast cancer cell line which expresses ERa, progesterone receptor (PR) and the AR, was used to investigate ERK1/2-mediated regulation of the AR and the androgen responsiveness of cells. Inhibition of ERK1/2 signalling was achieved by treatment of cells with U0126, an inhibitor of MEK1/2, the upstream activator of ERK1/2. Hyperactivation of ERK1/2 signalling was achieved by stably transfecting cells with a plasmid encoding a constitutively active form of the MEK1 protein (¿MEK1), resulting in the isolation of two clonal cell populations stably expressing ¿MEK1, ¿C3 and ¿6B, and a monoclonal cell line stably expressing the empty vector, MT3-1. Steady state AR mRNA levels, quantitated using real-time RT-PCR, were increased following U0126 treatment of MCF-7, MT3-1 and ¿6B cells. Conversely, treatment of cells with 10-8M 5a-dihydrotestosterone (DHT) for up to 72 hours decreased AR mRNA levels, indicating that ERK1/2 hyperactivation did not alter the androgenresponsiveness of AR mRNA. '...' Overall levels of AR phosphorylation were enhanced in ¿6B cells in the absence and presence of ligand, indicating that ERK1/2 hyperactivation either directly or indirectly induced receptor phosphorylation. The AR is localised in the cytoplasm in the absence of ligand and was more rapidly translocated to the nucleus in the presence of DHT in ¿C3 cells, an effect that was abrogated in the presence of U0126, thereby indicating an ERK1/2-specific mechanism. AR transcriptional activity, measured using androgen responsive reporter plasmids was not significantly altered in ¿6B cells in either the absence or presence of DHT, although the trend towards enhanced AR activity may be confirmed in future studies using optimised reporter assays. Consistent with the cell cycle regulatory functions of ERK1/2 signalling, proliferation of ¿C3 cells and ¿6B cells was increased in comparison to that of MT3-1 and MCF-7 cells. Treatment of ¿C3 cells and MCF-7 cells with 10-10 – 10-8M DHT produced similar inhibition of proliferation (~40%) during 8 days of culture, with no evidence of cytotoxicity. The results obtained in this thesis demonstrate that while ERK1/2 signalling regulates AR phosphorylation, processing and intracellular localisation, ERK1/2 hyperactivation in breast cancer cells does not inhibit the anti-proliferative effects of androgens. These findings support the development of tissue-specific androgenic treatments for breast tumours including poor prognosis tumours exhibiting ERK1/2 hyperactivation.

Emerging evidence suggests that an important mechanism for the negative feedback control of insulin signalling involves the inhibition of tyrosine phosphorylation of IRS-1 by its prior serine/threonine (ser/thr) phosphorylation. IRS-1 ser/thr phosphorylation has been linked to the dissociation of IRS-1 from the insulin receptor and PI3K, and its degradation via a proteasome-dependent pathway. Studies in animal models have shown that increases in plasma free fatty acids (FFAs) are associated with reduced IRS-1-signalling, and so it has been postulated that elevated FFA cause insulin resistance by activating pathways that negatively regulate insulin action, including hyper-phosphorylation of ser/thr residues in IRS-1. We have shown that in the case of linoleate-induced insulin resistance in L6 rat skeletal muscle cells, the inhibition of IRS-1-dependent signalling arises via effects on both the phosphorylation status and degradation of IRS-1, which are mediated, in part, by IKKb. In addition, the reduction of IRS-1 mRNA levels allude to transcriptional effects of linoleate treatment that also contribute to the observed reduction in the total levels of this protein. PtdOH, particularly dilinoleoyl PtdOH, was found to be significantly increased in linoleate treated L6 cells, and sufficient to induce at least some of the effects on insulin-signalling that are observed upon linoleate treatment. It is unlikely, however, that IKKb and PtdOH are components of the same inhibitory pathway, since inhibiting IKKb activity did not alleviate the effects of PtdOH on IRS-1 tyrosine (tyr) phosphorylation. Moreover, although an integral component of the mechanism by which linoleate induces insulin-resistance in L6 cells, it appears that restoring IRS-1 function in linoleate treated cells is not sufficient to reverse insulin resistance. Hence, we hypothesise that linoleate induces multiple inhibitory pathways in L6 cells, with at last two of these involving IKKb- and PtdOH-dependent inhibition of IRS-1 signalling, which act in parallel to reduce glucose disposal and cause insulin resistance in this model.