Modelling the 3D-Genome: The development of network theory approaches to characterise and predict active enhancers

Ph. D. Thesis.Gene regulation is an important mechanism that ensures the correct functioning of a cell and is generally orchestrated by gene regulatory elements such as transcriptional enhancers. Identification of these genomic regions are important in understanding a wide range of phenomena such as evolution, homeostasis and disease. During gene regulation, signals pertaining to transcriptional activation are transferred across the chromatin regulatory network from enhancers to genes in the form of transcription factors and cofactors that in turn, recruit transcriptional machinery such as RNA Polymerase II to increase the rate of gene transcription. Conceptually, we describe this as a flow of information from enhancers to genes, mediated by the chromatin conformation. We exploit this relationship in order to decode the regulatory landscape of genes and identify active enhancers. This thesis outlines the difficulties associated with identifying pathogenic mutations in the non-coding genome due to a lack of robust enhancer annotations. We use network theory to annotate these regions and develop a new method, 3D-SearchE, that serves to predict the location of novel putative active enhancers. 3D-SearchE achieves this by reverse engineering the flow of information between enhancers and genes to calculate an imputed activity score (IAS) at intergenic loci. We show that intergenic loci with a high IAS are also present for other enhancer associated features including the histone marks H3K27ac, H3K4me1 and H3K4me2, P300, CAGE-seq, Starr-seq, eQTLs and RNA Polymerase II. 3D-SearchE successfully leverages and summarises the relationship between the 3D organisation of chromatin and global gene expression and represents a novel enhancer associated feature that can be used to predict active enhancers

AMBRA1 as a Biomarker and its Functional Crosstalk with Autophagy and Epidermal Differentiation in Cutaneous Squamous Cell Carcinoma Tumourigenesis

Ph. D. ThesisCutaneous Squamous Cell Carcinoma (cSCC) is a skin cancer with an increasing worldwide incidence. While most patients have an excellent prognosis, a subset of patients develop disease recurrence/metastasis, emphasising the need for novel reliable prognostic biomarkers, as well as an improved understanding of the cellular signalling mechanisms underlying cSCC tumourigenesis and progression. Autophagy is essential for cellular homeostasis and keratinocyte differentiation, with the deregulation of both processes being associated with cSCC tumourigenesis. As a key protein to both autophagy and keratinocyte differentiation, the aim of the current study was to define crosstalk between AMBRA1 and the deregulation of these processes in cSCC development and progression and its potential, together with the associated autophagy cargo protein SQSTM1 (p62), as prognostic biomarkers. Biomarker assay development and analysis in a cohort of primary cSCC tumours revealed that loss of cytoplasmic AMBRA1 expression in the tumour growth front, in combination with loss of cytoplasmic p62 expression in the peritumoural epidermis, as putative prognostic biomarkers for cSCC reoccurrence and metastasis, independent of tumour differentiation status. Importantly, the combined loss of these proteins also identified moderately/poorly differentiated primary cSCC tumours at high risk of metastasis. Studies of the potential contribution of cullin E3 ligase-mediated degradation or TGF-β2-mediated downregulation of AMBRA1 in cSCC cell lines revealed only increased levels of TGF-β2 secretion correlated with loss of AMBRA1 expression. Furthermore, although chemical inhibition of TGF-β signalling inhibited cSCC cell proliferation in vitro, no effect on AMBRA1 expression levels was observed, suggesting an undefined TGF-β2 independent-mediated mechanism of AMBRA1 loss in cSCC. Studies investigating AMBRA1 involvement in keratinocyte differentiation and autophagy further demonstrated AMBRA1 expression in keratinocytes initially relies on autophagy activation but is later maintained by epidermal differentiation-related calcium signalling. Additional studies also revealed that this calcium-signalling mediated regulation of AMBRA1 expression is lost during cSCC tumourigenesis, likely resulting in the maintenance of a dedifferentiated cell phenotype, facilitating sustained tumour cell proliferation. This further highlights that loss of AMBRA1 expression as a key event in the uncoupling of autophagy and keratinocyte differentiation in cSCC development. Collectively these data highlight the tumour suppressive role of AMBRA1 in cSCC and its loss of expression in the tumour growth front, in combination with the loss of peritumoural epidermal p62 expression, as a novel prognostic biomarker for cSCC reoccurrence and metastasis.European Regional Development Fund, Northern Powerhouse and AMLo Biosciences Ltd

Incoherent effects in single-electron quantum optics

Ph. D. Thesis.Dynamic quantum dots as sources of single electrons open up new avenues to explore fundamental issues of solid-state physics. These types of single electron source allow for injection of electrons at energies high above the Fermi level. These high energies increase the spatial separation between bulk and injected electrons, reducing electron-electron interactions which are the dominant source of decoherence for cold electrons. Unfortunately, although these high energies reduce one source of decoherence, they introduce the potential for others to become dominant. In this thesis, we investigate two of these sources of decoherence on electrons which reduce their ability to act quantum mechanically, and explore the conditions required to mitigate these effects. First we investigate the effect of phase averaging, which is caused by the uncertainty in the injection energy of an electron. We calculate the phase contributions from beamsplitters, path lengths and the AharonovBohm phase, as well as the energy dependence of the transmission and reflection coefficients of the beamsplitters. We find that optimum conditions such that visibility can be maximised are obtained not at zero path length difference as in optics, but with an offset in the length of the interferometer arms. At the higher energies in hot-electron quantum optics, longitudinal-optical (LO)-phonon emission becomes the dominant source of decoherence. In this thesis we derive a complete quantum master equation to describe the rate of emission of LO-phonons and the behaviour of electrons undergoing this emission. The findings in this thesis are vital to the successful implementation of quantum optics-like experiments with hot electrons. These results can be used as input into both experimental architectures and dynamical simulations, and combined with previous results provide a complete quantum picture of the incoherent effects in hot electron quantum-optic

Image-informed numerical modelling of particulate systems with irregular grains

PhD ThesisGranular materials are everywhere around us. Their omnipresence makes our interaction with them on a daily basis a certainty, and yet our understanding of their mechanical behaviour is far from complete. Regarding geotechnical applications, most natural granular materials, such as silts, sands, gravels and ballast, feature irregular particle shapes, a fact that makes their mechanical behaviour all the more complex across scales, from micro to meso and macro. A multitude of experimental and numerical studies have demonstrated the importance of particle morphology in the shear strength of particulate materials, although rarely demonstrating a direct link or mechanisms of causality between them. This is mainly due to the high complexity of the problem but also partially due to the lack of intelligible and accessible tools to quantify the morphology of three-dimensional irregular particles. This thesis aims to contribute to the current state-of-art studying the characterisation of granular materials by providing analytical and numerical tools for shape characterisation. Regarding analytical tools, this thesis attempts a critical review of existing indices to characterise and classify particle form, while introducing a new set of indices. Regarding numerical tools, this thesis provides novel software solutions for automatic particle shape characterisation and for the generation of image-informed numerical models. These open-source tools are meant to shed light on the inherent subjectivity of performing shape characterisation on a practical level. Regarding the generation of numerical models based on imaging data, algorithmic implementations are offered to create simplified polyhedra and multi-sphere particles at user-defined fidelity levels of resolution, the morphology of which can also be characterised and compared to that of the original fidelity level. Combining the produced analytical and numerical tools, this thesis demonstrates a seamless workflow between particle imaging data and numerical modelling, using the discrete element method and non-spherical particles. This workflow is utilised to develop a methodology for the generation of Representative Element Volumes (REVs) of non-spherical particles, which represent the polydispersity of both particle size and shape, aiming to link quantitative morphology characterisation at the particle scale and mechanical characterisation at the level of a representative assembly of particles. The methodology is then applied to systematically generate REVs of railway ballast using image-informed multi-sphere particles of various levels of simulation fidelity, allowing for a parametric study of the effect of several modelling parameters on the shear strength of the material

Chemical and physical transformation of aerosol droplets in Raman tweezers

Ph. D. Thesis.Calcium carbonate (CaCO3) particles are one of the most abundant mineral aerosol totalling to approximately 8% of the total mass of mineral aerosols in the atmosphere, amounting to a loading of ~1.3 Tg. It is possible to produce CaCO3 via a “simple” ion exchange reaction between calcium chloride (CaCl2) and sodium carbonate (Na2CO3). This reaction seems simple, however as a mineral CaCO3 can also have multiple polymorphs, such as calcite, vaterite and a hydrated crystal ikaite (calcium carbonate hexahydrate, CaCO3∙6H2O). This reaction and its mechanism is important in both inorganic and biological chemistry, as CaCO3 is major component of bones and shells of living organisms, and the exact mechanism of formation and transformation of the different polymorphs is not known. In the atmosphere it can react with various reactive gases, such as NOx and SOx gases. This thesis can be split in to two major parts: a comparison of the hygroscopic responses of CaCl2, sodium chloride and a hydrocarbon (2-Hydroxypropyl)-βcyclodextrin using aerosol optical tweezing and dynamic vapour sorption, and the other focusing on the investigation of the formation and transformation of CaCO3 polymorphs in an optically tweezed aerosol droplet using Raman spectroscopy. In this thesis the formation and transformations CaCO3 is looked at from different perspectives: a theoretical model of the changes in the equilibrium size of the particle at a set relative humidity as a function of depletion of CaCl2 available for the reaction, and a spectroscopic point of view focusing on the evolution of the Raman spectrum of the droplet as the CaCO3 forms and transitions between polymorphs. The thesis also looks at potential ways of overcoming the issues encountered in the previously mentioned parts of the thesis, such as the use of Brownian motion of the droplet in a video recording for sizing

Characterisation of anucleate cells

PhD ThesisBacteria are beneficial in industry as they are easily genetically manipulated to express proteins, enzymes or full pathways while remaining cheap to grow in large batches with simple feed stocks. However, there are limitations associated with this as overproduction can lead to interference from complex cellular processes or metabolic burden. Metabolic burden is caused when essential cellular resources, such as energy and carbon, are diverted to the engineered pathway used for bio-production. To overcome limitations, this project aims to compartmentalise biosynthetic pathways into anucleate compartments of Bacillus subtilis, Escherichia coli and Synechococcus elongatus while regulating expression in nucleated cells. Division mutants were constructed in each chassis for the production of minicells or maxicells. Analysis of these anucleate producing strains was done to determine the properties of plasmid replicons with respect to gene expression and segregation into anucleate cells. Here the Plac/LacI regulation system was used so expression in nucleated rod cells was repressed while expression occurred within anucleate compartments. Good repression was present in the minicell and maxicell producing strains of B. subtilis, however, only 50% of anucleate cells were expressing GFP. There were efforts in B. subtilis to alter copy number to understand the pLS20 replicon and increase the amount of anucleate cells gaining a plasmid, however, there was no single mutant which worked best in minicells. There was a dramatic increase in maxicell expression with copy number mutants but there was still a cost to the regulation system in nucleated cells. In E. coli, the systems regulation does not repress as efficiently in nucleated mother cells compared to B. subtilis. Studies from this work revealed that the RSF1010 driven plasmids were better for minicell expression where ~30-35% minicells were GFP positive. Although a minicell producing mutant was constructed for S. elongatus, integration of a plasmid and repression system remained problematic. However, the minicell producing phenotype was characterised in greater detail using widefield fluorescence and transmission electron microscopy. This work contributes to the wider Portabolomics project aiming to bridge the gap between academia and industry

Entrepreneurial Education: Northumberland 1869 -1889

Ph. D. Thesis.This thesis analyses the impact of the ground-breaking 1870 Education Act on entrepreneurial education in Northumberland. Some regulation and funding of mass education was already in place by then but the 1870 Act introduced direct state provision in the form of elected School Boards and school buildings. The Act promised to support the voluntary sector and use state provision to fill the gaps between existing schools and the total number of school-age children. In 1876 however, an Amendment Act changed and enhanced the delivery of state education. The remit of school boards was considerably extended from their responsibility to educate disadvantaged children to become the primary vehicle for delivering universal education. The 1870 Act required local governments to assess educational need in their areas. Where provision was inadequate, they were required to form a democratically elected school board. Any ratepayer could serve on their committee or vote, including women and elections were to be held every three years to ensure accountability. Boards were intended to work alongside the voluntary sector, encouraging the growth of all school accommodation thus relieving pressure on state provision. By 1876, however, it was clear that in some areas, such as Newcastle, the democratic process of the boards had broken down. Furthermore, the 1876 Reform Act which introduced additional powers to enforce compulsory attendance was used to force closures of ‘undesirable’ schools. This included Private Adventure Schools charging 9d or less per week. The history of education in nineteenth-century Britain has attracted considerable scholarly scrutiny but relatively little attention has been paid to the function and contribution of Private Adventure Schools. Particularly those charging 9d or less per week which could be considered affordable to the working class. A surge of research in 1970, inspired by the centenary of the Education Act, did little to evaluate the diversity and quality of entrepreneurial education. All too often, flawed reports, observations and skewed statistics of government inspectors were accepted without due diligence. Aside from the early work of authors such as E.G. West (Education and the State, 1965) and Philp Gardner (The Lost Elementary Schools of Victorian England, 1984), and the G.R. Grigg’s 2005 case study of Welsh Private Adventure Schools, this lacuna in the literature has still not been addressed. This study offers an entirely new approach to identifying and analysing the impact of entrepreneurial education. By using the whole of Northumberland as a defined geographical area and concentrating on the two decades between 1869 and 1889 this study compares urban and rural educational development in the wake of the 1870 Education Act. A mixed-methods approach combines big data, GIS and a quantitative survey to map out school structures with demographic context. In addition, a broad selection of qualitative historical material has been used to excavate individual school histories and changes in attitudes to education from a variety of perspectives. The key findings conclude that entrepreneurial education was much more resilient in Northumberland than previously thought. It was also far more diverse, quality-driven and impactful than current literature suggests. This thesis argues that the decline of entrepreneurial education in the late nineteenth-century was not inevitable. These schools warrant more attention both as a neglected aspect of educational history and for its significance to contemporary global debates on the role of low-cost private schools in developed and developing economies.Economic and Social Research Counci

Proof of Concept of a Tailored Rehabilitation Programme for Interstitial Lung Disease, including Idiopathic Pulmonary Fibrosis

Ph. D. Thesis.Although the National Institute for Health and Care Excellence (NICE) recommended pulmonary rehabilitation program (PRP) for subjects with interstitial lung disease (ILD), tailored services and research in this key treatment area are limited. In this project UK Biobank data was retrieved and analysed for 122 subjects with ILD. A regional service of PR for ILD was established and 18 subjects with ILD were recruited. This was followed by a proof of concept RCT for 20 subjects with ILD. Similar methodology for the clinical regional service and the RCT was used and in the RCT subjects were allocated/ and or randomised to intervention inspiratory muscle training (IMT) with PRP or PRP alone. The subjects attended an 8 week program in a hospice care setting with one supervised and two unsupervised sessions. The supervised session was conducted in a novel hospice care partnership setting and included education, exercise, and relaxation sessions. After PRP, feedback was collected from all subjects. The UK Biobank data provided limited data for ILD, however, sedentary time showed correlation with weight (r=0.39, n=51, p=0.004), and with moderate and vigorous activities (r=- 0.32, n=51, p=0.021). The regional PRP service and RCT were shown to be feasible and appreciated by patients and carers, and attendance for RCT was 81%. In the RCT in general, when IMT was used, improvement was seen in maximum inspiratory pressure (MIP and sixminute walk test (6MWT). Circulating (Matrix Metallopeptidase 7 (MMP7) levels and 6MWT data showed improvement regardless the use of IMT. Variability was seen in other outcomes, where they either maintained, dropped, or improved. The feedback showed an appreciation for ‘exercise’, ‘information’ and ‘group’, stressing the importance of the education talks in the group therapy. There were also suggestions/requests for longer PRP, showing subjects appreciation for the program. In conclusion, UK Biobank contained limited data specifically for ILD research. A novel tailored PRP was established and was feasible in collaboration with a hospice healthcare partner. This was valued by subjects, carers and healthcare colleagues. Response to outcomes varied considerably in the RCT and regional service but the data suggested potential end points for further research trials. These could be investigated in future studies with larger sample sizes

Using theoretical chemistry to understand the properties of Polyoxometalates and their potential as energy storage materials

Polyoxometalates (POMs) are very appealing compounds as these transition metal oxide nanoclusters exhibit the ability to store multiple electrons in a reversible manner. Recently, POM-based energy storage devices, like redox flow batteries (RFBs) and alkali-ion batteries, have been extensively employed, since they meet equally the need of higher energy demand and low impact on the environment. However, POM-based technologies are still at early stage of development, mostly because of the difficulties to understand POM electronic behaviour. In this PhD thesis, it has been presented a theoretical study of POMs in different environments in order to understand the basic mechanisms behind their behaviour. In the first part, a general overview of electronic structures of POMs is given. Furthermore, the advantages and limits of POM-based batteries are discussed in detail. In Chapter 2 the state-of-the-art theoretical approaches used to study POMs are discussed, in particular, strong emphasis is given to the density functional theory (DFT), and both classical and quantum molecular dynamics (MD). In the third Chapter, the redox properties of POMs are investigated by means of simulations in an implicit and explicit environment. The implicit solvation is semiquantitative, and uncertainties arise due to the limit of the model. Quantum MD simulations reveal that an explicit environment can improve the calculated redox potentials of POMs, providing useful insights into their molecular nature. A spectroscopic study of x-ray absorption near-edge spectra (XANES) and extended x-ray absorption fine structure (EXAFS) is presented in Chapter 4. These techniques alongside with first-principles calculations have shown to be powerful tools to unveil the structure-property relationships of super reduced POMs. Chapter 5 is devoted to the study of self-assembly process of POMs. Classical MD simulations show that a rich network of hydrogen bonds mediates the POM-POM interaction, and their agglomeration strongly depends on total charge. Furthermore, first-principles calculations illustrate the effect of POM agglomeration on their redox potential, and catalytic efficiency towards the hydrogen evolution reaction. The results from this study show that it is now possible to adopt a range of computational approaches to understand the properties of POMs in different physical contexts. Specifically, the advantages and limits of DFT have been highlighted when computing the redox potentials of POMs, showing that further accuracy and insights into their electronic structure can be achieved by explicitly including the solvent molecules. For instance, it has been shown that the POM ability to undergo multiple redox reductions is due to possibility of delocalizing further electron density over all metallic atoms, regardless of the POM type. This behaviour is linked to their molecular structure, which undergoes an elongation of metal-oxygens bond lengths and formation of metal-metal bonds when further electrons are added to POMs. Furthermore, the inclusion of an explicit environment was shown to be an important factor to understand other properties of POMs, like the profile of their x-ray spectra or the self-assembly process. In the first case, QM/MM calculations shows that the polariazation of POM electron density returns more realistic molecular structures with respect to static DFT calculations, thus influencing the sensitivity of their simulated x-ray spectra. On the other hand, MD simulations revealed that the dynamical behaviour of POMs and the formation of long-lived agglomerates depends on several factors, like the total charge of POM, its counter ions, and solvent

Multiphase wireless dynamic charging systems for electric vehicles

PhD ThesisElectric vehicles (EVs) have been intensively developed as an attempt to reduce carbon-dioxide emissions caused by fossil-fuel vehicles. EVs require expensive batteries and power electronics for charging and discharging the battery. Unfortunately, battery technology, such as lithium-ion batteries requires substantial improvements to effectively compete with fossil-fuel cars in price. Also, batteries are usually heavy, take up large space and still have range limitation. Wireless Dynamic Charging (WDC), while the vehicles are in motion, is seen as an alternative to overcome the drawbacks associated with batteries. Due to the continues charging when driving, batteries can become smaller as most of the traction energy comes from the grid directly. WDC is fundamentally developed based on inductive power transfer (IPT) technology, where a time-varying magnetic field is generated by transmitter coils, which are installed underneath the road surface, to wirelessly power receiver coils, that charge the EV’s battery continuously. Presently, there are several technical challenges associated with WDC, which hinders commercialization. The output power fluctuation along the driving direction is one of the most serious problems. These fluctuations cause reduction in constant energy transfer thus requiring larger batteries. Also, batteries lifetime is significantly reduced as a result of increasing internal heating. Several studies attempted to realise constant output power for WDC. However, proposed methods so far, have disadvantages such as high cost, complexity or unable to sustain constant output power throughout the charging process. The work in this thesis proposes a multiphase WDC system to simultaneously achieve constant and high output power for EV applications. The proposed WDC system utilizes multiple primary windings that guarantee a homogeneous mutual magnetic flux for the receiver along the driving direction. This results in a constant induced voltage across the receiver and hence constant output power to charge the EV battery. High output power capability is attained by using multiple transmitter windings arranged in a novel winding method. The effectiveness of the proposed system is analytically described, simulated and demonstrated experimentally using a 3-kW laboratory prototype with the three-phase transmitter. The proposed system requires only simple control, eliminates communications between the primary and secondary sides and delivers 125% higher power transfer capability compared to conventional single-phase WDC systems