Ca2+ as a universal messenger participates in a great variety of physiological functions and biological events such as cell maturation, chemotaxis or gene expression. These diverse functions are controlled through complex spatio-temporal calcium patterns. To date it is known that these patterns depend on stimuli type and concentration. However, the majority of these observations were from constant or step change stimulation protocols. Under these conditions two leading hypotheses for the stimulus encoding into cytosolic calcium responses were proposed, namely amplitude and frequency modulation. Under physiological conditions, however, cells often experience time dependent stimuli such as transient changes in neurotransmitter or oscillations in hormone concentrations. How cells transduce such dynamic stimuli into an appropriate response is an open question. We exposed HEK293 cells and astrocytes to dynamically varying time courses of carbachol and ATP, respectively, and investigated the corresponding cellular calcium activity. While single cells generally fail to follow the applied stimulation due to their intrinsic stochasticity and heterogeneity, faithful signal reconstruction is observed at the population level. We suggest eight possible population representation measures and using mutual information measure show that the area under the curve and total number of spikes are the most informative ones. Next we provide simple transfer functions that explain how dynamic stimulation is encoded into area under the curve and ensemble calcium spike rates. Cells in a physiological environment often experience diverse stimulation time courses which can be reproduced experimentally. Furthermore, cell populations may differ in the number of cells or exhibit various spatial distributions. In order to understand how these conditions affect population responses, we compute the single cell response to a given dynamic stimulus. Single cell variability and the small number of calcium spikes per cell pose a significant modelling challenge, but we demonstrate that Gaussian processes can successfully describe calcium spike rates in these circumstances and outperform standard tools such as peri-stimulus time histograms and kernel smoothing. Having the single cell response model will allow us to compare responses of various sets of cells to the observed population response and consequently obtain insight into tissue-wide calcium oscillations for heterogeneous cell populations. Finally,in vivo astrocytes respond to a range of hormones and neurotransmitters. Furthermore these agonists can have different characteristics, for example glutamate is a fast excitatory transmitter, while ATP can be an inhibitory transmitter. Despite of this, how (or if at all) astrocytes differentiate between different agonists is still not clear. We hypothesize that astrocytes discriminates between different stimuli by exploiting the spatial-temporal complexity of calcium responses. We show how 2D A Trous wavelet decomposition combined with Bhattacharyya distance measure can be applied to test this hypothesis.

In eukaryotes, the removal of the poly (A) tail of cytoplasmic mRNA (deadenylation) is a crucial step in post-transcriptional gene regulation. A major enzyme involved in regulated mRNA deadenylation is the Ccr4-Not deadenylase, which contains two catalytic subunits: the Caf1 and Ccr4 ribonucleases. For both enzymes, two Mg2+ ions are required in the active site for activity. These enzymes in addition to six other non-catalytic subunits of the Ccr4-Not complex are possible drug targets in diseases such as metastatic cancer, osteoporosis and obesity. To facilitate the discovery, development and characterisation of small drug-like inhibitors of these enzymes, a biochemical approach was used. First, we investigated the biochemical characteristics of the highly similar CNOT6 and CNOT6L enzymes. Next, we evaluated two biochemical assays for characterisation of Ccr4-Not catalytic subunits and evaluation of N-hydroxyimide inhibitors of CNOT7. A chemiluminescence-based detection assay of AMP was used as the basis of a method and thermal shift assays were also used to characterize binding of compounds to deadenylase enzymes. In addition, cell based assays were used to study interactions between BTG2 variants and CNOT7 and CNOT8 in lymphoma. Our findings demonstrate that the CNOT6 and CNOT6L though highly similar, display different biochemical characteristics in vitro. The deadenylase activity of CNOT6 was higher compared to CNOT6L. The results also indicate that AMP detection is a highly sensitive assay that can be used as a secondary assay to the previously developed substrate-based assay for compound screening and IC50 determination. We conclude that thermal shift assays can be used to determine the binding mode of inhibitory compounds, specifically regarding the presence of the Mg2+ ions in the active site. Finally, we identified BTG2 variants that potentially regulate mRNA abundance in lymphoma via interaction with the Ccr4-Not deadenylase.

In many eukaryotes, kinetochores may form over a variety of unrelated centromeric sequences. This fact has led to the idea that an epigenetic process determines kinetochore location. To investigate the nature of this epigenetic process, I have established a system that allows the manipulation of the chromosomal centromeric DNA in a genetically tractable model organism, Schizosaccharomyces pombe. The system enables the definition of the relationship between centromeric DNA size, sequence, chromosome position and function. I have used this system to measure how binding of the conserved kinetochore protein Cnp1 (CENP-A homolog) varies as a function of the amount, sequence and position of the centromeric DNA. In humans, cytogenetic data suggests that the number of centromere-specific CENP-A nucleosomes at each centromere is approximately uniform regardless of the sequence and length of the centromeric DNA. These observations suggest that the number of these centromere-specific nucleosomes is tightly regulated. I set out to test whether such mechanism is evolutionarily conserved in the fission yeast S. pombe. I did this by manipulating the amount of the centromeric DNA at one centromere and then measuring the amount of Cnp1 bound to it. The results showed that in S. pombe, the amount of bound Cnp1 is proportional to the amount of centromeric DNA and thus the relationship between centromeric DNA size and CENP-A binding differs from humans. During these measurements, I observed that the size and sequence of the centromeric DNA do have a role in determining Cnp1 binding to centromeric DNA but, as described by others, it is not sufficient and sometimes not necessary for functional centromere formation. The requirement of the kinetochore protein Swi6 for neo-centromere formation but not for the maintenance of a pre-established centromere was also confirmed.

Background: The chronic joint disease osteoarthritis (OA) represents a significant global problem, not only at the present time, but also for the future. Characterised by articular cartilage degeneration, inflammation of the synovium, and subchondral bone changes, it is the chronic pain associated with OA which presents the most serious consequences. There is a clear need to understand the mechanisms under lying chronic joint pain, and to identify novel therapeutic targets. Two targets which may have therapeutic potential are BDNF and cordycepin. Objectives: The objectives of this thesis are two-fold; firstly to establish a slow progressing murine model of OA that is representative of post-traumatic OA, and secondly to investigate peripheral targets which may modulate chronic OA pain. Methods: Surgical destabilisation of the medial meniscus (DMM) was carried out in adult C57BL/6 mice. Weight bearing asymmetry and hindpaw withdrawal threshold were measured up to 16 weeks post-surgery. Joint pathology was then assessed post-mortem at 16 weeks post-surgery. Another cohort of adult C57BL/6 mice underwent a modified surgical destabilization of the medial meniscus. Pain behaviour and joint pathology outcomes were measured 16 weeks and 20 weeks post-surgery. Osteoarthritis was induced in adult male Sprague Dawley rats via intra-articular injection of monosodium iodoacetate (MIA) or vehicle (50ul 0.9 % saline. A second cohort of male Sprague Dawley rats underwent either meniscal transection (MNX) or sham surgery. Rats then received intra-articular injections of either trkB-fc or human IgG. Pain behaviour was tested up to 3 hours post injection. Adult C57BL/6 mice underwent DMM or sham surgery. Pain behaviour was measured up to 16 weeks post-surgery. From 14 weeks post-surgery mice were orally dosed with either cordycepin or vehicle every two days for two weeks. Joint pathology was then assessed post-mortem at 16 weeks post-surgery. Results:There was a significant increase in weight bearing asymmetry from 13 weeks post DMM surgery in C57BL/6 mice, but no changes in hindpaw withdrawal thresholds. There were also significant increases in chondropathy and synovitis at 16 weeks post-surgery. When the surgical induction of the DMM model was modified there were still significant changes in joint pathology, but no significant changes in pain behaviour. Intra-articular injection of TrkB/fc chimera in rats with established MIA induced joint pain was found to acutely reduce weight bearing asymmetry and increase ipsilateral hindpaw withdrawal thresholds. There was also a significant reduction in pain behaviour in rats with MNX established joint pain when TrkB/fc chimera was injected into the knee joint. Following systemic administration of cordycepin in mice with DMM induced joint pain there was a significant reduction in weightbearing asymmetry when compared to vehicle treated mice. There was also a significant reduction in DMM induced chondropathy, subchondral bone thickening, and osteophytosis in mice treated with cordycepin compared to vehicle treated mice. Conclusions: The changes in pain behaviour outcomes between the traditional and modified DMM, despite similar joint pathology outcomes, suggests a role for meniscal damage as a peripheral driver of OA pain. Localised injection of TrkB/fc chimera into the knee joint of rats with both MIA and MNX induced joint pain was found to acutely reverse joint pain. This implies that peripheral BDNF may be involved in mediating OA joint pain. Oral administration of cordycepin was found to reduce both pain behaviour and joint pathology changes in the DMM model in mice. These results suggest a role for local protein translation underlying both OA chronic pain and joint damage.

Reactive Oxygen Species (ROS) are known as important intracellular signaling molecules. These are also well known for their role in oxidative stress and cellular damage, leading to their involvement in several pathologies. Despite the widespread postulation of ROS mechanisms, little is actually known about the immediate response in living cells to the generation of these highly reactive compounds. The development of nanoplatforms incorporating photosensitizers would permit the generation of ROS at specific sub-cellular locations and determine the in situ cellular response. The work presented in this thesis describes the development of porphyrinic nanoplatforms for the controlled generation of ROS and investigates their impact on the surface marker expression of human Mesenchymal Stem Cells (hMSCs). Surface tailoring of polyacrylamide nanoparticles with alkyne and amine functionalities were exploited to achieve stable reactive chemical groups for further conjugation. Nanoplatforms surface was also modulated with trimethylammonium functionalities for the development of nanosystems for sub-cellular targeting and facilitated uptake. Physicochemical characterization of alkyne and alkyne/trimethylammonium functionalised constructs showed sizes in the range of 40 nm with a positive surface charge. Alkyne/trimethylammonium nanosystemswere found to be stable over long periods of time, whilst amino functionalized nanosystems were found to be prone to aggregation. Mechanisms of conjugation were exploited to create covalent linkage of porphyrinic photosensitizers to mono and dually functionalised constructs. Conjugation through "click chemistry" allowed stable coupling with alkyne and alkyne/trimethylammonium nanosystems. To overcome aggregation associated with amino functionalised nanoplatforms, porphyrin conjugated monomers were synthesised which resulted in stable polyacrylamide nanoparticles. The developed conjugated nanosystems showed final sizes in the range 40-100 nm, while conjugates with surface charges greater than + 20 mV have led to sizes higher than 100 nm. The effect of surface charge on cellular delivery was investigated and nanosystems with a surface charge in the range + 13 mV to + 18 mV proved optimal in terms of cell delivery and viability. It was found that highly charged nanosystems (above + 20 mV) remained attached to the cellular membrane and had a negative effect on cell viability. In addition, intracellular co-localisation studies showed preferential mitochondrial targeting of the delivered nanosystems. Production of ROS in nanoparticle treated hMSCs was achieved by exposure to light at wavelength of 575 nm. For porphyrin conjugated nanosystems a single light dosage resulted in a "blast zone" in the irradiated area where significant production of hydrogen peroxide was also observed. Titration of the amount of porphyrin conjugated at the surface of nanoparticles resulted in systems with different levels of ROS production. Control of ROS generation allowed development of a nanoplatform that was used to expose cells to repeated exposure of ROS over a time period of 100 minutes. The surface marker expression of hMSCs treated with porphyrin conjugated nanosystems was investigated. In the absence of light the surface marker expression of hMSCs was maintained, positive for CD29 and CD105 and negative for CD34 and CD45. Increased generation ROS in hMSCs did not produce alterations in the surface marker expression of cells, and over two generations of treated cells (light and nanoparticles) no changes were detected in surface marker expression. The developed nanoplatforms have the potential to be applied as a tool to investigate the cellular mechanisms and metabolism associated with different levels of oxidative stress. In addition, these nanosystems could also represent an innovative platform for theranostic applications (drug delivery/diagnostic).

The complicated task of interpreting the many ubiquitin signals is mediated by specific ubiquitin binding domains. The investigations discussed in this thesis focus on two very different ubiquitin binding domains. Chapters 3 to 5 detail the structural characterisation of the ubiquitin binding protein ZNF216. The structure of the ubiquitin binding Znf_A20 domain has been determined using multidimensional NMR techniques. A thermodynamic and structural characterisation of the interaction between the Znf_A20 and ubiquitin has been performed utilising chemical shift mapping, PRE based approaches, ESI-MS and ITC. The Znf_A20 domain forms a high affinity complex with Ub utilising a non-canonical binding site on ubiquitin centred at Asp58. The investigation was extended to the function of the Znf_A20 domain in the context of the full length protein. ZNF216, like many other ubiquitin receptors, has a ‘hook and line’ domain architecture with two independent domain separated by a long disordered linker. Chapters 6, 7 and 8 focus on the UBA domain of p62. The p62-UBA domain has been identified as a ‘hot spot’ for mutations linked to Paget’s disease of bone, a bone disorder which affects >3% of the over 55s. The dimerisation of the p62-UBA domain has been shown here to be a novel regulatory mechanism for the ubiquitin binding properties of p62. Modulation of both dimerisation and ubiquitin recognition are potential mechanisms by which mutations may disrupt p62 function and this prospect has been investigated here. The final chapter of this thesis examines the possibility of cooperation between different ubiquitin binding domains by simultaneous interaction with Ub to form ternary complexes. Using the Znf_A20 and the p62-UBA as an example the formation of a ternary complex has been demonstrated. By examining the available ubiquitin complexes it has been suggested that the formation of Ub mediated ternary complexes is limited to only a few UBD pairings. The cormation of Ub mediated ternary complexes may have interesting implications for the formation of larger multi-protein complexes utilising ubiquitin as an interaction hub on the recognition of poly-Ub with chain linkage specificity; and for Ub mediated signalling in general.

Deoxyribonucleic acid (DNA) is a long polymer consisting of two chains of bases, in which the genetic information is stored. A base from one chain has a corresponding base on the other chain which together form a so-called base-pair. Molecular-dynamics simulations of a normal DNA duplex show that breathing events – the temporary opening of one or more base-pairs – typically occur on the microsecond time-scale. Using the molecular dynamics package AMBER, we analyse, for different twist angles in the range 30-40 degrees of twist, a 12 basepair DNA duplex solvated in a water box, which contains the ’rogue’ base difluorotoluene (F) in place of a thymine base (T). This replacement makes breathing occur on the nanosecond time-scale. The time spent simulating such large systems, as well as the variation of breathing length and frequency with helical twist, determined us to create a simplified model, which is capable to predict with accuracy the DNA behaviour. Starting from a nonlinear Klein-Gordon lattice model and adding noise and damping to our system, we obtain a new mesoscopic model of the DNA duplex, close to that observed in experiments and all-atom MD simulations. Defects are considered in the inter-chain interactions as well as in the along-chain interactions. The system parameters are fitted to AMBER data using the maximum likelihood method. This model enables us to discuss the role of the fluctuation-dissipation relations in the derivation of reduced (mesoscopic) models, the differences between the potential of mean force and the potential energies used in Klein-Gordon lattices and how breathing can be viewed as competition between the along-chain elastic energy, the inter-chain binding energy and the entropy term of the system’s free energy. Using traditional analysis methods, such as principal component analysis, data autocorrelation, normal modes and Fourier transform, we compare the AMBER and SDE simulations to emphasize the strength of the proposed model. In addition, the Fourier transform of the trajectory of the A-F base-pair suggests that DNA is a self-organised system and our SDE model is also capable of preserving this behaviour. However, we reach the conclusion that the critical DNA behaviour needs further investigations, since it might offer some information about bubble nucleation and growth and even about DNA transcription and replication.

This PhD thesis describes research into the structure and function of lysostaphin (EC 3.4.24.75), a glycylglycine endopeptidase secreted by Staphylococcus simulans biovar staphylolyticus ATCC 1362. Lysostaphin is a member of the M23/M37 zinc metalloprotease family and is a pre-pro-enzyme. The mature form (after removal of the pro-region) contains two distinct domains, the C-terminal cell wall targeting domain of lysostaphin (termed LssTdom in the thesis) facilitates binding to Staphylococcus aureus cells. The endopeptidase domain (termed LssEdom in the thesis) cleaves the pentaglycine crosslinks in the peptidoglycan resulting in cell death through cell rupture of S. aureus. Lysostaphin is a potential therapeutic antibiotic for Methicillin-resistant Staphylococcus aureus (MRSA) for which new antibacterials are required owing to the widespread occurrence of multi-drug resistant strains. To date, the structural requirements for enzymatic activity and the target in the cell wall for lysostaphin have not been fully elucidated. Thus a structure might enable rationally guided design of lysostaphin variants for the generation of new enzymes which would also cleave the non-canonical crosslinks and would thus overcome bacterial resistance. Thus, one approach was to obtain the lysostaphin structure using homology modelling. Lysostaphin shares significant homology with the ALE-1 (83 % identity) and LytM (48 % identity) bacteriocins, and modelling the structure of lysostaphin was achieved using the recently released structures of LytM and ALE-1 derived from X-ray crystallography as templates. In addition, we report the successful production of active recombinant lysostaphin (as well as the endopeptidase and the targeting domains) and initial characterisation of their secondary structure by Fourier-transform infrared spectroscopy. Initially, advanced spectroscopic techniques including X-ray and NMR methods were investigated for molecular interaction studies between Lss and its putative ligands. Significant problems were encountered with these methods and mass spectrometry studies proved more amenable. Lysostaphin targeting domain-ligand complexes have been identified, along with their stoichiometry. The strength of the protein-ligand interactions has also been quantified. Lysostaphin was shown to bind in vitro Gly5 (mimicking the pentaglycine cross-bridge) and Lys-D-Ala-D-Ala (mimicking the stem peptide) with low affinity, but not NAM-L-Ala-D-iGln-Lys. It was also shown that lysostaphin targeting domain affinity for Gly5 was significantly reduced by addition of Gly-Gly-Ser-Gly-Ser (found in the host bacteria resistant to lysostaphin action – S. simulans) in solution. From these studies it could be concluded that resistance due to the incorporation of serine residues in the crossbridge were a result of the endopeptidase domain being unable to cleave this sequence and not due to the targeting domain being unable to bind it.

A novel version of the CSA (Chemical Shift Anisotropy) amplification experiment which results in large amplification factors is introduced. Large xa (up to 48) are achieved by sequences which are efficient in terms of the number of π pulses and total duration compared to a modification due to Orr et al. (2005), and greater flexibility in terms of the choice of amplification factor is possible than in our most recent version. Furthermore, the incorporation of XiX decoupling ensures the overall sensitivity of the experiment is optimal. This advantage has been proved by extracting the CSA tensors for a novel vinylphosphonate-linked nucleotide. The application of CSA amplification experiment to six nucleosides is also discussed. The measured principal tensor values are compared with those calculated using the recently developed first-principles methods. Throughout this work, the NMR parameters of all nucleosides are presented. Finally, high-resolution multi-nuclear solid-state NMR experiments are used to study some novel vinyl phosphonate-linked oligo-nucleotides.

The corpus luteum (CL) is a tissue formed from the remnants of an ovulated follicle in the ovary, and it produces the progesterone needed for a healthy pregnancy. CL growth is highly dependent on a growing nutrient supply, and can be compared with the most aggressive vascular tumours. Angiogenesis, the growth of new blood vessels from existing ones, plays a key role in the growth and function of the CL. Inadequate angiogenesis has been linked to infertility in cows. The CL is composed of several vascular(e.g. endothelial cells (ECs), pericytes (PCs)), and avascular (e.g. luteal cells (LCs), immune cells) cell types, and several pro-angiogenic factors (e.g. Fibroblast Growth Factor 2, FGF2) found to be important in the angiogenic process. The objective of this thesis is to shed light on the cellular and extracellular level determinants of angiogenesis in the bovine CL. We begin with the relevant biological and mathematical literature in Chapter 1. In Chapter 2, an ordinary differential equation model of CL growth is introduced. We assume that the CL volume is a continuum of three cell types, ECs, LCs, and stromal cells (such as PCs). The fourth variable in the model, FGF2, enhances the EC proliferation rate. The model is able, by varying parameters such as the maximal proliferation rate of the ECs, to distinguish cases where the CL shifts from a ‘normal’ to a ‘pathological’ growth. In Chapter 3, we present in vitro CL published and novel studies from Robinson’s Lab. Preliminary results demonstrate interesting endothelial and pericyte behaviours regarding cell aggregation and sprout formation, which are the motivation for the next two Chapters. In these experimental studies, all the CL cell types were incorporated in the same in vitro culture, hence providing a closer approximation to the in vivo environment compared to other in vitro cultures which use only a single cell type (mainly ECs). However, this complicates matters in terms of distinguishing cell behaviours and factors which contribute on the overall cell dynamics. Therefore, in the Chapters 4 and 5 we use data from literature. In Chapter 4, by using the Cellular Potts Model (CPM) framework, we focus on EC-PC interactions, and particularly on the mechanism which is responsible for the EC growth inhibition. Our model incorporates two possible mechanisms for inhibition. That is, the mechanical cell-cell contact inhibition, and the inhibition mediated from diffusive TGF-b secreted once the two cell types come in contact. Interestingly, our model results suggest that the effective range of TGF-b is a crucial determinant of the degree of EC growth inhibition. Chapter 5, by using a CPM, is devoted to sprouting angiogenesis (the formation of new blood vessel). The dynamic interchange between stalk and tip EC phenotype is incorporated through the Notch signalling pathway, with the leading tip cell moving up macrophage-mediated VEGFA gradients in a non-uniform matrix environment. The model reproduces phenomena in sprouting angiogenesis, including sprout morphology, tip competition, and explains knockout experiments on the Notch signalling pathway. Finally, we close with Chapter 6 where we summarise the ain results from each chapter and propose model extensions for future directions.