Myocyte stress 1 (MS1) also known as Striated Muscle Activator of Rho Signalling (STARS) and Actin-binding Rho activating protein is a stress responsive, muscle specific protein expressed in cardiac, skeletal and smooth muscle. MS1 was first observed to increase in mRNA levels during early states of pressure overload induced left ventricular hypertrophy, making MS1 sensitive to extracellular stress. MS1 is highly involved in actin dynamics, where polymerization of actin leads to the regulation of the myocardin related transcription factor-A and the serum response factor (MRTF-SRF). The SRF pathway plays a critical role in the regulation of the skeletal muscle. While in the heart, MS1 is thought to be implicated in hypertrophic signalling and cardiac remodelling. Previous studies in the lab have shown that MS1 increased in mRNA levels during simulated ischaemia/reperfusion injury but levels were attenuated with the addition of JNK inhibitor SP600125 during simulated ischaemia/reperfusion injury. Although we know MS1 is involved in actin dynamics due to its ability to bind actin at the C-terminal as a result of actin binding domains (located between 234-375 a.a) and also bind actin binding proteins ABLIM-2 and 3, there is limited information on how MS1 becomes upregulated and its specific function. In this study we wanted to investigate the effect of various stimuli on MS1 promoter activation with the use of luciferase reporter assays. The MS1 promoter was responsive to sorbitol induced osmotic stress, oxidative stress by serum deprivation and hypertrophic agonist phenylephrine. All of these are well known activators of the stress activated protein kinases (SAPKs); JNK and p38. There may be a link between MAPK activation and MS1 regulation. Investigation of other interacting partners of MS1 was proposed to give some insight into the function of MS1. Binding assays using purified MS1 fragments were used to look at potential interactions in the heart. Interestingly, novel myofibrillar proteins were pulled out of heart extract and identified by mass spectrometry as Myosin-6, troponin I, troponin T, α-tropomyosin, myosin LC2 and actin. We observed potential phosphorylation sites, located within the N-terminus of MS1. In vitro kinase assays using activated JNK, p38 and ERK, allowed for phosphorylation of MS1. Three novel phosphorylation sites Thr24, Thr62 and Ser77 were identified by mass spectrometry. Immunofluorescence studies were used to determine whether phosphorylation alters MS1 subcelluar distribution or interaction with actin. Co-localisation was observed between MS1 and HA-JNK at the cell membrane where there was evidence of membrane ruffling and actin stress fibres present at the periphery. All of these findings in this study are novel and imply that MS1 may be involved in the MAPK pathway and also play critical roles in contractile function, muscle development and cell motility, where phosphorylation may be responsible for its ability to interact with myofibrillar proteins.

The Bcl-2 family of proteins are the primary regulators of the intrinsic apoptotic pathway, with anti-apoptotic members up regulated in cancer. Bcl-X is of particular interest, as it can be alternatively spliced to yield antagonistic proteins, the proapoptotic Bcl-XS and the anti-apoptotic Bcl-XL. Heterogeneous ribonuclearprotein (hnRNP) F/H, which binds to G-tract RNA, was shown to shift splicing in favour of the XS 5’ss (Garneau et al., 2005). G-tracts can form G-quadruplexes (G4s), which have been implicated to effect various stages of RNA processing, including splicing. Structural studies of hnRNP F/H revealed that it prefers to bind single stranded G-tract RNA (Dominguez et al., 2010) and competes with G4 formation (Samatanga et al., 2013). It was proposed that G4s may also form within the Bcl-X pre-mRNA that will alter 5’ss selection. In this study, two main questions were asked: (1) Is the Bcl-X pre-mRNA capable of forming any G4s (2) if so, do these G4s alter the 5’ss selection. Once a better understanding of 5’ss selection in Bcl-X was attained, a novel non-intrusive method was developed that identified putative G4s regions in the presence of competing secondary structures. Three main regions were highlighted for further investigation. By screening over 30 G4 ligands for their effect on 5’ss selection in Bcl-X, it was revealed that G4 ligands not only cause a shift to favour the pro-apoptotic site, but that such an effect is restricted to certain structural classes of G4 ligands. By investigating the mechanism of action of the best G4 ligand from the screen, GQC-05, two independent non-canonical G4s were found to be stabilized by GQC-05 owing to its dual effect on 5’ss selection. This not only expands the range of potential G4 forming sequences in RNA but also offers a new therapeutic strategy for alternative splicing regulation of apoptosis.

Introduction: miR-16 and miR-26a have been identified as key effectors of the p53 pathway in response to genotoxic stress. This work is focused on preliminary elucidation of regulatory mechanisms by which p53 controls expression of miR-16 and miR-26a and characterisation of their gene targets involved in the p53 network. Methods: Microarray expression analysis of miR-16 and miR-26a was followed by Q-PCR to confirm these miRNAs dependence on p53. We analysed the transcriptional regulation of these miRNAs by p53 via luciferase assay and ChIP assay. We investigated these miRNAs contribution to p53-dependent response to genotoxic stress. To validate miR-16 and miR-26a targets (Cyclin E, CHK1, and WEE1) we employed Q-PCR, western blotting and luciferase assay. We also analysed the transcriptional regulation of Cyclin E by SET9 via luciferase assay. Results: High miR-16 and miR-26a expression are associated with increased cancer survival. p53-dependent and -independent regulatory mechanisms exist for miR-16 and miR-26a, and p53 controls expression of miRNAs on several levels. p53 recruits Drosha complex to miR-16 and miR-26a to facilitate the processing of these miRNAs. miR-26a cooperates with p53 to induce apoptosis and miR-16 enhances p53-mediated cell cycle arrest. miR-16 and miR-26a regulates CHK1 and WEE1, in the presence or absence of p53. miR-16 also reduces Cyclin E levels, in the presence and absence of p53. SET9 controls expression of Cyclin E on a transcriptional level. Conclusions: Our results showed that in response to DNA damage, miR-16 and miR-26a expression levels are controlled by p53-dependent and p53-independent mechanisms, potentially involving other stress-response transcription factors such as E2F1. Our data also confirms that miR-16 and miR-26a directly target Cyclin E, CHK1, and WEE1 for down-regulation. Additonally, SET9 directly controls Cyclin E expression. Reduced CHK1 and WEE1 levels leads to decreased G2/M arrest, and reduced Cyclin E levels results in increased G1/S arrest. As a consequence, apoptosis occurs.

There are high economic, human health and environmental reasons for using Second Generation Anticoagulant Rodenticides (SGARs) but there is also widespread non-target exposure to a large number of bird of prey species, such as red kites and barn owls. My overall aim was to assess the potential biochemical and physiological impacts of sub-lethal exposure on birds. I determined environmentally realistic doses of two SGARs, brodifacoum (high acute toxicity) and difenacoum (most widely used and detected in wildlife), in a model species, Japanese quail (Coturnix coturnix japonica), and used these doses in subsequent studies. Anticoagulation profiles and liver residues associated with the doses were characterised. Unexpectedly, one dose (0.4 mg kg-1 body weight brodifacoum) induced persistent (> three weeks) residual anticoagulation. Half-life and repeat-exposure studies highlighted the risk to birds from multiple exposures of brodifacoum in particular. Liver half lives in quail were longer than in rats for brodifacoum but shorter for difenacoum. The magnitude and duration of anticoagulation was greatly increased by multiple exposures involving brodifacoum and was the result of a complex interplay (including inhibition of binding and replacement) between residues in the liver. Studies on quail chicks demonstrated that, while chicks were not especially sensitive to SGARs, exposure reduced growth by 5-10%. Overall, my results suggest that the most likely exposure scenario for UK wildlife, that of repeated exposures to difenacoum, is likely to pose relatively little increased risk compared to single exposures. This is not true for brodifacoum, but it is less widely used in the UK. Thus, exposure scenarios likely to be associated with greater risk, such as repeated exposures to brodifacoum or mixed exposure patterns, are probably less common in the UK. This may partially explain why the number of detected rodenticide-induced wildlife mortalities is low.

The transcriptional co-repressor Sin3a is a ubiquitous eukaryotic protein complex that has a multitude of critical functions, including the regulation of embryonic development, cell division and maintenance of genomic integrity. It incorporates the highly related HDAC1 and HDAC2 enzymes as its catalytic subunits, which interact with the complex through the HID domain of the Sin3a co-repressor. Sin3a is responsible for deacetylating lysines of Histone tails, condensing chromatin and consequently repressing the transcription of genes. The enzymatic activities of HDAC1 and 2 within Sin3a depend on the association of the Sin3aspecific SDS3 protein, which also interacts with the complex via the HID domain. The mechanism by which Sin3a recruits HDAC1, HDAC2 and SDS3 remains unknown, and elucidating it would represent a big step forward in understanding the epigenetic regulation of genes through the deacetylation of chromatin. The aim of this thesis is to understand how Sin3a recruits its catalytic subunits in to the complex as well as to get a deeper insight into the role of SDS3 in the HID domain by using both structural (X-ray crystallography) and biochemical approaches. Our data suggests that HDAC1 may interact with Sin3a through an extended surface of the co-repressor and that SDS3 stabilizes this interaction by simultaneously binding to HDAC1 and Sin3a. Enzymatic and kinetic assays indicate that Sin3a may be the only Class I HDAC containing complex that is not regulated by IP4. IP4 is a co-factor that regulates the activity of the Class I HDACdependent complexes NuRD and SMRT-NCoR. Thus, our results suggest that Sin3a may have followed a separate evolutionary pattern and its activity may be regulated in a different way.

The human serine/threonine NIMA-related protein kinase family comprises eleven members, named Nek1 to Nek11. Of these, Nek1, Nek4, Nek8, Nek10 and Nek11 are implicated in the DNA damage response (DDR) with Nek11 playing a central role in the G2/M checkpoint. In response to DNA damage, Chk1 is activated by the ATM/ATR kinases. Chk1 then phosphorylates and activates Nek11 before both Chk1 and Nek11 phosphorylate Cdc25A. This promotes binding of SCFB-TrCP and subsequent degradation of Cdc25A resulting in cell cycle arrest at G2/M. Nek11 protein expression is also increased in colorectal cancers. This study focuses on the role of Nek11 in colorectal cancer cells to examine whether targeting Nek11 in combination with DNA damaging agents may have a clinical benefit for colorectal cancer patients. Using RNAi to deplete Nek11 in HCT116 colorectal cancer cells we show that Nek11 is required for the G2/M arrest in response to ionizing radiation and chemotherapeutically relevant drugs, oxaliplatin and irinotecan. Furthermore, depletion of Nek11 alone and in combination with IR results in apoptosis and loss of cell survival. Nek11 exists as several closely related but distinct isoforms in colorectal cancer cells: Nek11 Long (L), Nek11 Short (S), Nek11C and Nek11D. We reveal that depletion of Nek11S results in a more substantial abrogation of the G2/M checkpoint, as compared to depletion of the Nek11L or D isoforms. Furthermore, through the use of stable cell lines, we show Nek11 isoforms exhibit distinct localisation patterns and all isoforms are able to undergo nucleocytoplasmic shuttling, regulated by the C-terminal domain. Excitingly, we observe Nek11 localisation at sites of DNA damage foci in response to IR exposure. Overall, our findings reveal an essential role for Nek11 at the G2/M checkpoint in HCT116 cells contributing not only to their arrest but also their survival after DNA damage. Hence, Nek11 could be an exciting target for the development of novel anti-cancer drugs.

Sam68 is a member of the STAR family of proteins, linking post-transcriptional gene regulation with signal transduction pathways. Sam68 has been shown to specifically regulate the alternative splicing of many genes through interactions with the pre-mRNA and spliceosomal machinery. Selection of particular isoforms of several of these genes has been shown to contribute to neoplastic transformation and aberrant Sam68 expression and function has been implicated in the development of various genetic diseases and cancers. It is therefore important to understand Sam68 RNA recognition at the molecular level in order to design next generation drug therapies. This thesis describes the structural and biophysical techniques used to define the bipartite RNA consensus sequence specifically recognised by Sam68 and the mechanisms of interaction. This data provides a model of Sam68 contribution to alternative splicing regulation. Splice site selection is also influenced by posttranslational modifications of Sam68 including serine and threonine phosphorylation. In several cases, the phosphorylation state of Sam68 directly influences the outcome of splicing and leads to cancer development. Identification of phosphorylation sites in the STAR domain of Sam68 by NMR and radiolabelled kinase assays reveals how this post-translational modification may affect RNA binding at the molecular level.

Hepatic protoporphyria is a disorder of liver haem metabolism characterised by inhibition of the enzyme ferrochelatase (FK) and consequent accumulation of protoporphyrin. Two compounds that induce such a disorder are ATMP [1-[4-(acetyl-2,4,6-trimethylphenyl)-2,6-cyclohexanedionyl]-O-ethyl propionaldehyde oxime] and griseofulvin. These drugs are thought to be metabolised by cytochrome P450 as suicide substrates so that a fragment of the drug (a methyl group) becomes attached to the haem moiety of the enzyme, producing a powerful inhibitor of FK. The present work was undertaken to elucidate the mechanism of action of ATMP and further characterise the N-alkylated porphyrins (green pigments) produced by feeding mice griseofulvin. ATMP causes hepatic protoporphyria in mice but not in similarly treated rats, guinea-pigs, hamsters and chick embryos. A green pigment was isolated from the liver of mice treated with ATMP and identified by its electronic absorption spectrum and HPLC chromatographic properties as N-methylprotoporphyrin (N-MePP). The ATMP pigment markedly inhibited the enzyme FK in vitro, thus supporting its identification as N-MePP. The possible involvement of cytochrome P450 in the production of N-MePP after treatment with ATMP was also investigated. Loss in cytochrome P450 was not demonstrable in mice treated with ATMP. However, a role for cytochrome P450 was suggested by the findings that two inhibitors of cytochrome P450, SKF525-A and piperonyl butoxide, both afforded protection against ATMP-induced porphyria and production of N-MePP. In the mouse griseofulvin causes suicidal destruction of cytochrome P450 giving rise to green pigments in the liver. Two such pigments have been characterised here, using fast atom bombardment (FAB) mass spectrometry. One of them has been conclusively identified as N-MePP; the second is also an N-alkylated protoporphyrin and has been tentatively identified as the adduct of griseofulvin to protoporphyrin (N-griseofulvin protoporphyrin).

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.

Heme peroxidases are an ubiquitous family of catalytic iron-containing proteins. These enzymes are responsible for the catalysis of hydrogen peroxide removal from the cell through the formation of high valent transient states of the heme cofactor. The purpose of the present research project is to investigate the reaction pathway of cytochrome c peroxidase. The study of hydrogen related chemical features in the active site of this enzyme in the resting state and in the transient species is of paramount importance for the clarification of the reaction mechanism. In these circumstances neutron crystallography is the technique of choice. Neutron crystallography allows the direct localization of deuterium substituted hydrogen atoms in macromolecules in absence of radiation-induced damage. The structures of the resting state and of cryo-trapped compound I of cytochrome c peroxidase were determined by neutron crystallography. The nature of the catalytic center was investigated and in particular the protonation state of the heme iron axial ligand and of the key catalytic residues was established. These findings contributed to the understanding of the reaction pathway from the resting state to the intermediate species compound I. Complementary spectroscopic techniques were employed to assess compound I formation in single crystals and its stability in the conditions of the neutron crystallography experiment. The temperature dependence of the key catalytic features of cytochrome c peroxidase in the resting state was investigated by neutron crystallography. Significant alterations of protonation states were found in the resting state at cryogenic temperature. In addition the nature of the heme iron distal ligand was found to be affected by temperature in the resting state and it was possible to relate this phenomenon to the temperature induced spin state change observed by spectrophotometry in the visible region. As part of the present project, contribution was given to the development of the cryogenic temperature sample environment at the neutron macromolecular diffractometer LADI-III of the Institut Laue-Langevin broadening the experimental capabilities of the instrument. A wide range of experiments are made possible by the new set-up including the cryo-trapping of catalytic intermediates, the study of the temperature dependence of structural features and the investigation of species and complexes that are not stable at ambient temperature.