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Ageing is the major risk factor for the development of cardiovascular diseases. Endothelial cells play a major role in the normal physiology of vessel wall. Endothelial dysfunction contributes to the development of cardiovascular diseases. In particular, the presence of senescent endothelial cells in the atherosclerotic plaque. Association of senescent endothelial cells in the development of endothelial dysfunction is not clear. This thesis was aimed to investigate the phenotype of senescent (SIPS and REPS) HUVECs and perhaps develop their markers In vitro models of senescent endothelial cell were developed i.e. SIPS and REPS. Genes identified from previous transcriptomics data analysis, for the first time, notably included CST1, LRRC17 and CRTAC1 as differentially expressed in senescent HUVEC. Their expression was validated by quantitative PCR but the gene product of CST1, cystatin SN, was not identified in Western blotting. Subsequent bioinformatics re-analysis of differentially expressed genes described molecular pathways, molecular functions and diseases that were affected in senescent HUVECs. Senescent cells are presented with senescent associated secretory phenotype (SASP). Perhaps the pro-inflammatory factors in SASP contribute to the endothelial dysfunction. Characterisation of the secretome from senescent endothelial cells using mass spectrometry did not identify pro-inflammatory factors. Moreover, SASP from senescent HUVECs did not potentiate the binding of monocytic cells to endothelial monolayers. However, functional assays confirmed increased adhesion of monocytes to senescent HUVECs in both static and flow-based cell models in vitro. Further investigation of candidate genes involved in the adhesion of monocytes to endothelial cells in these scenarios, identified E-selectin and CD44 as potential mediators. Taken together, these new data suggest that senescent endothelial cells might represent a pro-inflammatory phenotype via expressing adhesion molecules that could recruit inflammatory cells to sites of vascular damage or endothelial dysfunction.

Cellular senescence is a state of permanent cell cycle arrest in which cells remain metabolically active, adopting characteristic phenotypic changes. Although senescence is a potent tumour suppressor mechanism, the accumulation of senescent cells in tissues over time has been shown to contribute to several pathophysiological conditions, including fibrosis, diabetes, cancer, Alzheimer's disease and ageing, making the targeting of these cells a potentially relevant therapeutic strategy for fighting these pathologies. Clearing senescent cells has been reported to protect against cancer and the onset of age-related pathologies. Furthermore, preventing the accumulation of senescent cells in tissues also prolonged healthspan and lifespan in mouse models. Currently available markers of senescence, however, lack specificity and selectivity, limiting their therapeutic use. This underscores the need for senescence-specific markers that could be targeted and translated into clinical use. Here, we describe the application of molecularly imprinted polymer nanoparticles (nanoMIPs) designed to target an extracellular epitope of B2MG, a membrane protein recently characterized as a novel marker of senescence, and propose this as a novel tool for the specific detection of senescent cells both in vitro and in vivo. We further show that B2MG nanoMIPs laden with a toxic payload selectively kill senescent cells in culture. Consistent with this, we show that antibody-drug conjugates (ADCs) against B2MG selectively kill senescent cells in culture. We also demonstrate that Ibrutinib, a clinically approved chemical inhibitor of BTK, reduced the accumulation of senescent cells in vivo, prolonging lifespan and healthspan in a mouse model of progeria. Zmpste24-/- mice treated with Ibrutinib showed an increase in maximum lifespan and a reduction in age-related fitness decline, evidenced by their increased physical strength, reduced anxiety and better long-term memory, with no evidence of spontaneous tumour formation. The results from this research altogether highlight new strategies for reducing the accumulation of senescent cells in vivo, which could find translational application in the clinical management of senescence-related and age-related ailments.

Stra6 is a protein that is upregulated in response to retinoids. Interestingly, we have found that Stra6 can induces p53 independently of DNA damage through ROS generation. Stra6 have shown a great induction of p53 protein levels with high stability as well. p53 stabilisation through Stra6 leads to stimulate downstream proapoptotic events such as increase the activation of caspase-3, caspase-9 and PARP cleavage. Stra6 have shown the capability of driving tumour cell to apoptosis after cell sensitized with ATRA and stimulated with DNA damaging agents. However, both Stra6 and p53 were important to achieve maximum percentage of cell death, as this was observed by detecting a positive feedback loop between Stra6 and p53. Pull-down assay of Stra6 protein have shown truncated forms of the Stra6 protein with molecular weights of 25 and 15 kDa. Basically, we have found that the small form of Stra6 with 25 kDa was translocated from cell membrane to cytosol in the absence of DNA damage and it was found to be located within the nucleus in response to DNA damage. Furthermore, this shorter form of Stra6 was capable of generating ROS and enhancing p53 dependent apoptotic cell death via the upregulation mechanism of the downstream cascade of p53 signalling pathway. Additionally, mass spectrometry data have identified some of cytosolic and nuclear proteins as Stra6’s protein binding partners. Remarkably, several of these identified proteins were related to apoptosis, as well were associated in the regulation of important cellular mechanisms. We propose that Stra6 can sensitize tumor cells to DNA damaging agents and function as an apoptotic protein in a p53-dependent manner.

Chronic lymphocytic leukaemia (CLL) is a heterogeneous B-cell malignancy with multiple genetic abnormalities, which have been used to stratify patient’s treatments and prognosis. For instance, genetic alterations have been identified in CLL patients, such as SF3B1, Notch1, TP53, and Myd88, and they have been related to prediction of progression and survival. Moreover, mitogen activated protein kinase (MAPK) signalling is crucial in the pathogenesis of a number of cancers, and it has also showed increased expression in a large subset of CLL patients. In this project, we investigated the sensitivity of five leukaemic cell lines to 22 targeted inhibitors. We found that cells with V600EBRAF mutations showed greater sensitivity to MAPK inhibition, while cells with K601NBRAF mutations were more resistant. Wild type BRAF CLL cells showed varied sensitivity to 16 different inhibitors but not to MAPK inhibitors. Combination of a MAPK inhibitor (Sorafenib) and CUDC-907 (CDK/HDAC inhibitor) showed strong synergism in CLL cells at nanomolar concentration, suggesting a potential therapeutic application of blocking both pathways simultaneously. BRAF exon 15 mutations had low frequencies in our cohort (1.052%), but we also found Notch1 mutations (14.81%), Myd88 (1.35%) and SF3B1 (16.21%, 20.8% of which were first-time reports of non-coding regions). Notch1 mutants were more resistant to pladienolide B and more sensitive to MK-0752. On the other hand Myd88 mutants were more sensitive to IRAK1/4 inhibitors. In conclusion, our results showed that therapies for CLL can be stratified based on in vitro information of drug sensitivity in relation to mutations present in the cells and suggest that inhibiting the MAPK pathway could be an effective approach in combination therapies. These preclinical data could be applied in CLL management in the near future.

Cellular senescence is a reversible cell cycle arrest that has been shown to play a role in aging and cancer. Senescent cells accumulation can occur prematurely, as seen in response of stress and oncogenic insults, or normally, as observed in organismal aging. Thus, identification and studying senescent cells in vivo and in vitro have an important diagnostic and therapeutic potential. In addition, the molecular mechanisms involved in establishing and maintaining senescence are not fully understood. Consequently, the current approach that is used for senescent identification has limitations. For this reason, we characterized a list of potential markers of senescence from a proteomic screening of plasma membrane of senescent cells. From the list, we have validated 10 of them, namely, DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, VPS26A and PLD3 that are differently expressed in different model of cell senescence. These markers can be combined to detect senescent cells in vitro or in tissue sections. We also proposed a FACS based method using two markers (DEP1 and B2MG) with known extracellular epitopes. This could facilitate the senescence detection and studying. From the proteomic screening, we also found that BTK is elevated in senescent cells. BTK was induced in response of p53 activation due to different stimuli. Moreover, phosphorylation of ATM, p53 at ser15 and γH2AX was increased after BTK overexpression, which suggested the involvement of BTK in DNA damage pathways. BTK was able to phosphorylate p53 directly at N-terminus. BTK inhibition with chemicals or RNAi depletion was sufficient to bypass senescence. In addition, BTK inhibitors were also able to extend life span in flies. p53 KO flies did not show any difference in life span after using BTK inhibitors, which showed the importance of BTK in p53-mediated senescence. BTK inhibition also decreased the senescent cells accumulation in flies. All these data collectively suggest the possibility of using BTK inhibitors to ameliorate aging-associated disorders.

p53 is a tumour suppressor protein that has a crucial role in cellular responses to stress signals. p53 regulates a wide range of cellular functions that cover cell cycle arrest, apoptosis, gene repair, metabolism, fertility, cellular reprogramming and autophagy, among others. p53 regulation is a complex process that occurs via different mechanisms, including post-translational modifications (PTMs). The PTMs of p53 are achieved through various modulators. Here, we demonstrate the regulation of p53 through PTM by Burton’s Tyrosine Kinase (BTK), and Euchromatic histone-lysine N-methyltransferase 2 (G9a). We found that BTK expression is induced in response to DNA damage. Consecutively, BTK phosphorylates both p53 and MDM2, which results in p53 stabilization and increase in activity. Additionally, our experiments showed that BTK-mediated phosphorylation of p53 regulate other PTMs of p53. Specifically, BTK inhibits MDM2-dependent p53 ubiquitination, whereas augments both SET9-mediated K372 methylation, and CBP-mediated K373 acetylation. As a consequence, BTK enhances up-regulation of p53 stability, nuclear localization, and activity. On the other hand, we examined the effect of different forms of G9a on p53 activity, and we showed that they differentially regulate p53. The activity of p53 is enhanced by short isoform of human G9a (hG9a(S)), while we demonstrated the opposite effect d for mouse G9a (mG9a). In addition, the function of hG9a(S) towards p53 does not depend on K373-methylation. We propose that hG9a(S) acts as a coactivator of the p53-CBP interaction. Thus, our data suggest that both BTK and G9a play an important role in p53 regulation using both post-translational modification.

This thesis contains two distinct parts: Current models of Chronic Lymphocytic Leukaemia (CLL) pathogenesis invoke specialised anatomical microenvironments that harbour proliferating cells. Such proliferating CLL cells are more resistant to current immuno-chemotherapeutic regimens than cells in the peripheral blood and are thought to be the cause of disease relapse. Using a system to recapitulate CLL proliferation centres in vitro, I have observed that CLL cells undergo proliferation. Unexpectedly, under these conditions an induction of wild-type TP53 protein was also observed in all cases of CLL analysed. The results reported here were undertaken to understand how CLL cells upregulate TP53 protein and proliferate. For reasons that remain unclear, TP53 is unable to transactivate its classic target genes to induce cell-cycle arrest or apoptosis. However, it remains able to trigger a full apoptotic response after further DNA damage and a higher threshold of protein levels is reached. We propose a model whereby oxidative stress induced by proliferation in CLL triggers TP53 protein expression. Hairy Cell Leukaemia (HCL) represents approximately 2% of all leukaemias, follows an indolent course and remains an incurable disease. Recently, virtually all HCL patients shown to carry the BRAFV600E mutation, thought to be a disease-defining event. The BRAF V600E mutation results in constitutive activation of the MEK-ERK pathway resulting in aberrant proliferation, and targeted inhibitors have shown efficacy in BRAFV600E positive tumours. We wanted to test whether this efficacy can be extrapolated to HCL. Here we report in vitro studies using PLX4720 and in vivo trial of Vemurafenib in a patient with refractory HCL. While BRAF inhibition showed no effect on HCL survival in vitro, it resulted in rapid loss of viability of hairy cells in vivo. The results obtained show that efficacy of BRAF inhibition achieved did not occur via the expected inhibition of MEK-ERK activation.

Cells have evolved sophisticated mechanisms to maintain genomic stability after cellular stress. Activation of DNA damage response pathways, and most importantly p53, leads to adaptive responses that can be influenced by different extracellular factors. The aim of this project was to study how extracellular factors modulate p53 cell-fate decisions after DNA damage, with particular interest in oxygen tensions and vitamin A metabolites. First, we focused on how physiological oxygen tensions (5% 02) may influence cellular responses to genotoxic stress. We showed that normal and cancer cells cultured at 5% 02 had a reduction in p53-mediated apoptosis after exposure to different genotoxic stresses. This was not due to a decrease p53 protein levels or its transactivation activity, and the oxidative damage caused by DNA damaging agents was not affected by oxygen tensions. We also found a p53-independent activation of MAPK at 5% 02, which when inhibited restored levels of p53-induced apoptosis. HIF-1α, a transcription factor induced at lower O2 concentrations, was present and active at 5% 02. However, this did not affect MAPK activation and HIF-1α was not involved in the resistance to apoptosis under these conditions, although MAPK was necessary for HIF-1α expression and activation. We next explored the effect of the vitamin A (retinol) pathway on the cellular responses to DNA damage. We showed that Stra6, a retinol-inducible gene, is upregulated by p53 after DNA damage. While overexpression of Stra6 sensitized cells to p53-induced apoptosis independently of the retinoic acid signalling, its inhibition resulted in decreased apoptosis after DNA damage and less induction of oxidative stress. This shows that both oxygen tensions and vitamin A metabolites, through Stra6, are potent modulators of the p53 responses to DNA damage.