The intracellular transport of organelles and vesicles is thought to utilise both microtubules and actin filaments, which mediate long and short-range transport, respectively. Melanosomes, synthesised in melanocytes, are a convenient model organelle to study intracellular transport, since they are visible using brightfield microscopy. They are believed to be transported from the perinuclear area to the actin cortex along microtubules, and then captured by the myosin-Va/melanophilin/Rab27a complex which traffics them along actin filaments to the plasma membrane. In contrast, data presented here demonstrate that anterograde melanosome transport relies only upon the actin cytoskeleton. Myosin-Va null melanocytes were used to test the importance of microtubules and actin on long-range organelle transport. In these cells, melanosomes cluster around the perinuclear area, but disperse into peripheral dendrites upon reintroduction of the myosin-Va gene. When this assay was repeated in the absence of microtubules, melanosomes still dispersed indicating that microtubule-based motors are not necessary for long-range anterograde trafficking. However, depolymerising F-actin, or freezing actin dynamics with latrunculin A or jasplakinolide inhibited the dispersion of pigment granules in myosin-Va null cells melanocytes and induced a clustered phenotype in WT melanocytes. This effect was abolished if microtubules were absent, suggesting that microtubules are only required for retrograde transport whilst dynamic actin is essential for anterograde melanosome transport. Moreover, when Kif5B was forcibly recruited to the melanosome membrane via an inducible dimerisation system, the melanosomes dispersed abnormally. An siRNA knockdown screen of over 120 actin binding proteins identified several proteins including formin-1, Arpc1b, cofilin-1, gamma-actin and spire1/2, which appear to be necessary for maintaining peripherally dispersed melanosomes. This evidence further underlines the importance of the actin cytoskeleton, rather than the microtubule network as previously thought, for the anterograde trafficking of melanosomes.

In gene regulation, mRNA polyadenylation, deadenylation and decay are closely linked processes whose full biological importance is only starting to emerge. Using thiouridine labelling as a method of capturing newly synthesised mRNAs, we have been able to measure poly(A) tail sizes of newly synthesised individual and total mRNAs in mouse NIH3T3 cells and global changes in mRNA turnover during the loss of pluripotency in human embryonic stem cells. These two systems, although very different, allow us to study changes in individual mRNAs during inducible gene expression. In order to track changes of poly(A) tail length during rapid gene induction we have used the serum response as a model in NIH 3T3 cells. Using poly(A) fractionation technique (Meijer et al, 2007), we have been able to study global changes of the poly(A) tail length during the serum response and have observed striking increases in rapidly induced mRNAs compared to control levels. Functional analysis reveals that these mRNAs are involved in transcriptional regulation, indicating that they are involved in reprogramming gene expression. Further analysis of the poly(A) fractionation microarray data, also indicates that certain mRNAs, such as Akt2, change in poly(A) tail length but are either down regulated of have no change in abundance. This group of genes areis enriched in regulators of signal transduction. When treated with Actinomycin D, some of these mRNAs still retain the increased poly(A) tail length during the serum response. This suggests a role for cytoplasmic polyadenylation, as inhibiting transcription has no effect on the elongation of the poly(A) tail. We also established by Western blotting and immunohistochemistry, that despite reduced mRNA levels, protein levels of Akt2 increase during the serum response. We conducted microarray analysis to determine whether miRNA regulation contributes to the changes we observed in poly(A) tails during the serum response. However, no major changes in abundance are found after 30 minute treatment, indicating that all changes in microRNA abundance are slow and unlikely to play a role in this system. As a second system, we studied the role of RNA binding proteins and mRNA stability in the early neuronal differentiation of human pluripotent stem cells, when pluripotency is lost. The role of mRNA processing and stability on the maintenance and establishment of pluripotency is poorly studied, despite the fact that these processes are important in germline development and early embryogenesis. We therefore studied the differential expression of RNA binding proteins involved in the regulation of polyadenylation and mRNA stability during early differentiation of human embryonic stem cells into neuronal precursors. Over an 8 day time period we have found large changes in mRNAs belonging to the CELF, CPEB, PUM and MSI families, which start early after the indication of differentiation, when pluripotency is lost. Using thiouridine labelling and microarray analysis on differentiating and pluripotent cells we were able to identify a group of mRNAs which appear to be destabilised during differentiation. This group includes known pluripotency factors such as OCT4 and LIN28B. Functional analysis showed that many ribosomal protein mRNAs also are within this group and also genes involved in the regulation of translation elongation, further exposing an important role for post-transcriptional regulation in the maintenance and loss of pluripotency. Our data indicate that the initial poly(A) tail size and mRNA turnover rate can vary greatly depending on both the individual mRNA and the state of the cell. Overall, our investigations give intriguing glimpses in the role of poly(A) tail metabolism and mRNA stability in two different models of inducible gene expression.

Staphylococcus aureus is a Gram-positive human pathogen that has developed resistance to all traditionally used antibiotics. Sortase A (SrtA) is a 'house-keeping' enzyme present in a number of Gram-positive organisms including S. aureus that is responsible for the covalent anchoring of proteins to the cell wall through the recognition of a highly conserved LPXTG motif. Many of the proteins it anchors are involved in virulence and immune evasion suggesting that it is an attractive target for potential anti-infective therapies. Moreover, as SrtA is not vital for bacterial growth or survival, inhibition may not select for the development of drug-resistance, unlike conventional antibiotics. This study evaluated different assays to assay SrtA activity and includes an in vitro Fluorescence Resonance Energy Transfer (FRET) assay using purified recombinant SrtA protein and an in vivo whole cell-based assay that measured SrtA-mediated anchoring of Gaussia luciferase (GLuc) in S. aureus. A further in vivo assay was evaluated, which measured SrtA activity using fluorescence as a reporter and analysis by flow cytometry and structured illumination microscopy. In this study three novel small molecules were identified as potential inhibitors of SrtA using in silico computational docking and SAR analysis; NCC-00014270, NCC-00076932 and NCC-00032784. These compounds were shown to inhibit SrtA in vitro in a dose-dependent manner with IC50s of 140 ± 24.6 µM, 172 ± 28.1 µM and 628 ± 122 µM respectively and were shown to act as competitive inhibitors of the SrtA. With the use of an in vivo reporter, it was shown that all three compounds negatively affected SrtA-mediated anchoring in S. aureus whole cells. Moreover, the cytotoxicity of these lead compounds against eukaryotic cells was assessed. Overall, these data suggest that two of the three lead compounds were potential 'hit' molecules for further structural modification for increased inhibitory activity against SrtA.

Replication of genetic information is a vital process across all domains of life. Bacillus subtilis is considered the gram-positive model bacterium for studying DNA replication (Escherichia coli has been studied extensively as the gram-negative model) and is most representative of the ancestral phylum of prokaryotes. DNA replication has three distinct stages; initiation, elongation and termination. Replication initiation is the focus of this research and this process occurs at a single origin conserved throughout bacteria, termed oriC. B. subtilis primosomal machinery is formed of replication initiator proteins DnaA, DnaD and DnaB, the helicase loader DnaI, replicative helicase DnaC and primase DnaG. The role of the initiator proteins is to melt the DNA double helix and enable loading of the hexameric ring helicase onto each strand of DNA for bidirectional replication. Initiation is the first stage in DNA replication and despite its importance the molecular mechanisms of replication initiation remain largely unclear. The work presented in this thesis has focussed on the essential interaction between replication initiator proteins DnaA and DnaD, with an aim to characterise their binding interface and reveal molecular details of their mechanisms of interaction during DNA replication initiation. The direct interaction between isolated DnaA domain I and DnaD DDBH2 domain was detected by NMR spectroscopy which was subsequently used to identify the specific residues involved and characterise the nature of the binding interface. The kinetics of the interaction were investigated by SPR and computational techniques were used to model the DnaA-DnaD complex. This structural characterisation of the DnaA-DnaD interaction provides greater understanding of the molecular mechanisms of DnaA and DnaD during DNA replication initiation.

Targeted degradation of cytoplasmic mRNA is of fundamental importance for regulated gene expression in eukaryotic cells. The shortening and removal of the poly(A) tail (deadenylation) is the initial and often rate-limiting step in this process. Two key components in deadenylation are the PAN2-PAN3 and the Ccr4-Not complexes. PAN2-PAN3 is a trimer composed by two regulatory subunits (PAN3) and a catalytic enzyme (PAN2). The Ccr4-not complex is composed of at least eight subunits, constituting four modules, that bind the scaffold protein CNOT1: the N-terminal module (CNOT10, CNOT11); the catalytic nuclease module (Caf1, Ccr4); the CNOT9 module (CNOT9); and the NOT-module (CNOT2, CNOT3). A cellular and biochemical characterisation of the PAN2-PAN3 complex was planned, to further understand its role in eukaryotic cells and determine the deadenylation rates. Similarly, we have started to reconstitute the human Ccr4-Not to understand the contributions of the individual modules to the activity of the complex. Initially, we purified a recombinant nuclease module composed of Caf1, Ccr4, and the anti-proliferative protein BTG2. Next, we reconstituted a core complex including the nuclease module, CNOT9 and a segment of CNOT1 encompassing the MIF4G and DUF3819 domains (CNOT1M). Then, we compared the activity of the core complex and the nuclease module using substrates containing short (A9) and longer (A20 or A50) poly(A) tails, as well as poly(A) tails coated with the poly(A)-binding protein PABPC1. We observed that the CNOT9 module contributes to enhanced deadenylation and increased selectivity towards terminal adenosine residues. Finally, the influence of the melanoma-associated P131L amino acid substitution of CNOT9 was investigated with structural and biochemical approaches.

Understanding the biology and mechanisms as to how the heart contracts has long been a point of interest for biologists and mathematicians alike. Since inconsistent beating of the heart has been linked to multiple pathological conditions, research into this area has been extensive but we still only have some of the answers. One of the key findings over the last century has been the role of calcium in activating the machinery within the heart that drives contraction. Further studies have shown that when calcium is mishandled by the heart's myocytes, it can lead to some of these pathological conditions. Since such discoveries a major point of research into the heart has focused on the possible avenues that calcium mishandling can occur. This thesis explores some of these avenues using a mathematical model of the ventricular myocyte developed by Thul and Coombes in their 2010 paper 'Understanding cardiac alternans: A piecewise linear modeling framework'. The chosen model contains key components involved in the movement of calcium within the myocyte. Moreover, the model used is piecewise linear and the stability of some important behaviours can be studied exactly without the need for approximations and reductions. This is often an issue in many other models used to study the calcium dynamics within a ventricular myocyte. The avenue towards calcium mishandling that this thesis predominantly focuses on is that of intracellular calcium diffusion between the building blocks of ventricular myocytes known as sarcomeres. Our research extends previous research into how strong diffusion between sarcomeres can cause unwanted calcium dynamics. Further to this, we explore how the balance in the strength of different forms of calcium diffusion between sarcomeres can drive a variety of spatial patterns in terms of how the calcium is distributed throughout the cell. Throughout these studies we also investigate the role of other parts of the myocyte, particularly the sarcoplasmic reticulum Calcium-ATPase pumps and sarcoplasmic reticulum release in relation to diffusion driven instabilities. As well as intracellular diffusion of calcium, this thesis considers the role of intercellular diffusion of calcium through gap junctions. This form of diffusion has historically been considered to a lesser extent than intracellular diffusion. As such this thesis introduces new ideas concerning gap junctions. These include a role in driving the mishandling of calcium as well as altering behaviours driven by intracellular diffusion. An important message is that calcium diffusion within the myocyte is far more important in terms of how unwanted behaviours can appear than previous studies suggest.

Calcium is a signalling messenger that is crucial to cellular function, controlling a diverse range of processes such as apoptosis, cell proliferation and muscle contraction. Store operated calcium entry (SOCE) is a specific pathway coupling depletion of the calcium stores within the endoplasmic reticulum (ER) to calcium influx through Orai channels on the plasma membrane. SOCE occurs in small sub-cellular regions called 'ER-PM junctions' which are typically less than $300$nm in diameter. The small size of these domains prevent direct measurement of the calcium signals as current calcium imaging techniques cannot resolve the local signals within ER-PM junctions. The calcium signals associated with SOCE control many downstream cellular processes, such as gene expression and immune responses. There is substantial evidence demonstrating that the placement of the calcium signalling machinery, including Orai channels and SERCA pumps, is vital to the generation of spatially distinct calcium signals which then enhance the selectivity of the calcium signal. However, experimental techniques cannot investigate the local calcium dynamics occurring on a spatial scale of micrometres so mathematical modelling techniques can be used to close this gap in understanding how the local calcium dynamics affect the experimentally observed global calcium dynamics. In this thesis, we construct a three dimensional spatio-temporal model of calcium dynamics and investigate the relationship between the placement of core components of the calcium signalling machinery, e.g. Orai channels and SERCA pumps, and the spatial calcium profiles generated as well as the rates of ER refilling observed. The model includes a spatially extended ER-PM junction to examine the spatial signature of the calcium profiles generated and a spatially extended sub-PM ER to examine the impact of Orai channel and SERCA pump placement on ER refilling dynamics. The model is the first to include spatially extended versions of both the ER-PM junction and sub-PM ER. In this thesis, we first focus on the construction of the spatio-temporal model and the solution techniques used to solve the model. We implement a semi-analytical solution using Green's functions to calculate the analytical solution of the spatial component of the diffusion equation and use numerical time stepping methods in MATLAB to evolve the spatial calcium profile over time. We compare the predictions of the model to expected biological outcomes and then use the model to investigate how the placement of Orai channels, and in particular how clustering of Orai channels, creates spatially distinct calcium profiles. We then examine whether the spatial calcium profile affects ER refilling and what factors control ER refilling. We find that Orai channel clustering creates spatially distinct calcium profiles within the ER-PM junction but does not enhance ER refilling. ER refilling is more strongly controlled by the proximity of SERCA pumps to Orai channels. In fact, the placement of SERCA2b pumps weakly affects ER refilling but the major regulator of ER refilling is the placement of SERCA2a pumps within the ER-PM junction. However, ER refilling continues, albeit at reduced rates, regardless of Orai channel and SERCA pump placement which suggests that other factors, such as the geometry of the ER-PM junction, could be important regulators of ER refilling. This work is relevant to experimental biologists and mathematicians within the calcium signalling community as the calcium signals generated within the ER-PM junction are crucial for advancing the understanding of how calcium signals regulate cellular function. The local calcium dynamics are important regulators of whole cell calcium dynamics and so mathematical methods allowing rigorous investigation of the mechanisms controlling local calcium signalling will be invaluable to furthering our understanding of how SOCE regulates cell function.

There has been considerable recent interest in models for epidemics on networks describing social contacts. This thesis considers a stochastic SIR (Susceptible - Infective - Removed) model for the spread of an epidemic among a population of individuals, with a random network of social contacts, that is partitioned into households and in which individuals also make casual contacts, i.e. with people chosen uniformly at random from the population. The behaviour of the model as the population tends to infinity is investigated. A threshold parameter that governs whether or not the epidemic with an initial infective can become established is obtained, as is the probability that such an outbreak occurs and, if so, how large it will become. The behaviour of this model is then compared to that of a finite population using Monte Carlo simulations. The effect of the different transmission routes on the final outcome of an epidemic and the effect of introducing social contacts and clustering to the network on the performance of various vaccination strategies are also investigated.

No description available.

Pluripotency is conserved in the major trunk of Vertebrate evolution, but how the gene regulatory network (GRN) that governs it evolved is poorly understood. A central component of this network is the Homeodomain containing transcription factor Nanog. How Nanog evolved is not understood, as Nanog sequences have not been identified in invertebrate genomes. This study provides evidence of Nanog activity encoded in the homeodomain of the invertebrate Vent gene family. The Vent2 gene from Saccoglossus kowalevskii, a model hemichordate, successfully reprogrammed mammalian pre-iPS cells to pluripotency, as demonstrated by the activation of dormant pluripotency genes, and the ability to generate all three primary germ layers. A second property of invertebrate Vents was also characterised in the Vent gene found in Nematostella vectensis, a sea anemone and model for cnidarian development, expression of which was insufficient to activate the endogenous pluripotency network of pre-iPS cells, though it could induce the cells to a XEN-like state that demonstrated up regulation of extra-embryonic markers, and subsequently gained dependence on ERK signalling. A direct comparison between the Saccoglossus and Nematostella Vent homeodomains was used to provide insight into the step-wise changes that appear to have given rise to Nanog activity. Swapping the homeodomains from one CDS to another, to create hybrid molecules, I demonstrated that the respective reprogramming activities of these genes is conserved in the homeodomain. I then identified specific amino acid (AAs) differences in the homeodomains that conferred a Nanog-like capacity for reprogramming to the Nematostella gene. Identification of a Nematostella EsrrB ortholog, which demonstrated reprogramming activity in mammalian pre-iPS cells, suggests wider conservation of pluripotency factors. I therefore propose that an ancient GRN for pluripotent mesoderm evolved in vertebrates to form part of the ground state network.