VictorJuiz/thesis huggingface dataset:
MFS transporter superfamily: modelling and dynamics

The aims for this thesis were threefold. First, is it possible to develop a numbering protocol to aid homology modelling, despite the diversity of MFS sequences? Given these methods, apply these to improve the homology model of a putative MFS protein, SV2A. Finally, can Molecular Dynamics (MD) simulations be used to elucidate the transport mechanism in an MFS protein?
There are several solved X-ray crystal structures of secondary active transporter MFS proteins, all of which have a core 12 transmembrane (TM) helix fold. Despite this highly conserved fold, the sequence identity between the transporters is low (15 – 25 % identity between structures) and so this provides difficulties when exploring homology modelling of target proteins based on known structures of MFS transporters. To overcome this, exploration of both conservation of amino acid type in multiple sequence alignments (MSAs) and conservation of contacts between helices elucidate 'anchor points' in the structure. This analysis can be used to aid alignment between template and target in homology modelling, such as for SV2A, which is the binding site for an anti-epileptic drug, levetiracetam. The binding site was thought to be in the central cavity of the TM region of the MFS protein and so drug-protein interactions were explored using MD simulations.
Finally, MD simulations were conducted on FucP, the X-ray crystal structure of which is solved in an outward open conformation (open to the perplasmic cavity). The aim for this work was to investigate the movement from the outward open state to the occluded state and determine whether there is any effect on placing static hydrogen atoms on two key titrable residues in the central TM cavity, D46 and E135.

Advisor: Biggin, P
Processing and properties of polymer-based composites for metamaterials applications

Transformation optics offers a theoretical tool for the design of electromagnetic devices that can manipulate the flow of microwaves in an unprecedented way, but also places demanding requirements on the permittivity ε and permeability μ of materials used to form the devices, along with their spatial variations in three dimensions. Polymers that are light-weight and with high processability are considered potentially useful in realizing transformation optic designs, but only where the effective ε and μ can be increased controllably by loading the polymer with inorganic particles. This thesis focuses on the development of bespoke polymer-based composite materials and robust manufacturing approaches for potential applications in electromagnetic devices.
Three main manufacturing approaches have been explored. Firstly, casting was demonstrated to produce bulk materials with equal permittivity and permeability by embedding NiZn ferrite particles in an epoxy matrix. Hybrid composites with a combination of soft magnetic ferrite and metallic magnetic fillers, anisotropic composites with magnetic field aligned fillers, and high magnetic anisotropic materials with multi-layered structures were all produced successfully. Developed materials showed superior electromagnetic properties to many competitor approaches for miniaturization and low profile antenna, especially for the case of magnetically anisotropic cast tiles. Secondly, a spray deposition approach was developed for the fabrication of large-scale nanocomposite polymer-based films. By incorporating Fe3O4 nanoparticles or multi-wall carbon nanotubes, as a thin sandwich layer into a perfluoroalkoxy (PFA) matrices respectively, 30-50 μm thick flexible multilayer nanocomposite films with enhanced dielectric and magnetic properties were produced. A broadband permittivity and permeability measurement technique was also developed for these films. Finally, a 3D printing technique was employed to produce electromagnetic materials with complex 3D shape. Feedstock on a ferrite/acrylonitrile butadiene styrene (ABS) composite filament was demonstrated successfully and may facilitate the realization of complex design transformation optics devices.

Advisor: Grant, P
Effects of beta-2 adrenergic receptor agonists in DOK7 congenital myasthenic syndrome

Congenital myasthenic syndromes (CMS) are a rare group of heterogeneous disorders, characterised by compromised neuromuscular transmission and symptoms of fatiguable muscle weakness. CMS is caused by mutations in genes that affect the structure and function of the neuromuscular junction (NMJ). In about 20% of CMS cases, patients have mutations in the gene DOK7; the protein product, DOK7, is crucial for maintaining the dense aggregation of acetylcholine receptor (AChR) clusters at the NMJ. DOK7-CMS patients do not respond to treatment with acetylcholinesterase inhibitors which are the first line treatment for most forms of CMS. Instead, a dramatic response to beta-2 adrenergic receptor (ADRB2) agonists, such as salbutamol, is observed. The aim of this project was to investigate the molecular mechanisms that underlie the beneficial effects of ADRB2 agonists.
Firstly, NMJ functioning was modelled in vitro by studying AChR clusters formed on cultured C2C12 mouse myotubes in the presence of WT DOK7. Overexpression of mutant DOK7 led to a significant reduction in the number of AChR clusters, explaining the pathogenic effect of the mutation. Importantly, incubation of myotubes with salbutamol increased the number of AChR clusters and their stability. The results provide the first evidence that ADRB2 agonists directly affect proteins located at the NMJ. However, this disease model suffers from limitations.
The rest of the thesis focussed on developing alternative cell culture models to explore the AChR clustering pathway. The first model combined optogenetics and fluorescence lifetime microscopy to study the effects of ADRB2 activation on AChR cluster stability in single live cells. The second used CRISPR/Cas9 genome editing tools to directly introduce Dok7 mutations to the genome of C2C12 cells, thereby overcoming some of the drawbacks associated with DOK7 overexpression. Further manipulations of these novel model systems will be used in the future to examine in more detail the molecular events underlying the pathogenic effects of DOK7 mutations and the mechanisms of ADRB2 agonists.

Keywords: DOK7
beta-2 adrenergic receptor agonist
salbutamol
neuromuscular junction
congenital myasthenic syndrome
acetylcholine receptor
Advisor: Beeson, D
Cossins, J
The role of religion in shaping women's family and employment patterns in Britain and France

The current study examines the influence of religious affiliation and practice on family patterns and labour market activity for women in Western Europe, focusing on Britain and France. While both countries have experienced a sharp decline in institutionalized forms of religion over the past decades, differences in family and fertility behaviour on the basis of religiosity seem to persist. Although previous studies documented a positive correlation between religion and both intended and actual family size, there is still uncertainty about the different routes through which religion affects fertility, how structural factors are involved in this relationship and whether and how this relationship has changed along with the process of religious decline. This study aims to fill this gap by exploring the interrelationships between religion, educational attainment, female labour force participation, union formation and fertility levels. The data come from the British Household Panel Survey (BHPS), which contains 18 waves from 1991 to 2008, and the French survey of the Generations and Gender Programme (GGP), which was initially conducted in 2005. By following trends in fertility differences by religious affiliation and practice across birth cohorts of women, it is found that religious differences in fertility are not only persistent across birth cohorts, there is also a growing divide between non-affiliated and religiously practicing women who maintain higher fertility levels. Religious differences in family formation patterns and completed fertility are also explored, taking into account the interaction between education and religiosity. It appears that the effect of education on fertility differs by level of religiosity, as higher education is less likely to lead to childlessness or to a smaller family size among more religious women. The findings on the relationships between family and work trajectories by level of religiosity also point to a reduced conflict between paid employment and childbearing among actively religious women, although these patterns vary by religious denomination and by country.

Keywords: fertility
western Europe
religion
Advisor: Sullivan, O
Progress towards a demonstration of multi-pulse laser wakefield acceleration and implementation of a single-shot wakefield diagnostic

An ongoing experiment is described to demonstrate the principle of multi-pulse laser wakefield acceleration, in which a plasma wakefield is resonantly excited by a train of laser pulses, spaced by the plasma wavelength. Particle-in-cell simulations of the initial single-pulse experimental setup are presented, in order to calculate the expected signal. Preliminary results are presented and future plans, based on work done so far, are discussed.
Part of this work involves the implementation of a single-shot wakefield diagnostic — frequency-domain holography, which records the phase shift caused by passage of a probe pulse through the plasma. This implementation is described in detail, along with the associated analysis procedure. Practical difficulties encountered while implementing the diagnostic are discussed, along with possible ways of mitigating them in the future. A method is presented by which the noise level in the resulting phase measurements can be predicted, much more accurately than any previously published method for this technique.
Methods of generating pulse trains for use in future multi-pulse laser wakefield acceleration experiments are presented. These include techniques proposed for use in this demonstration experiment, as well as one intended for use in a dedicated high-efficiency, high repetition-rate, multi-pulse driver laser. This last method, based on programmable pulse shaping using a spatial light modulator, requires a suitable mask to be computed based on the parameters of the required pulse train; an algorithm is described to perform this computation.

Keywords: frequency-domain holography
Wakefields
pulse trains
LWFA
FDH
Advisor: Corner, L
Walczak, R
Precipitation pattern as a proxy for protein crystallization

X-ray crystallography has been the workhorse behind most 3D protein structures, which are crucial in the understanding of their biological function and interaction with other molecules. However, a major rate-limiting step in X-ray crystallography remains obtaining suitable protein crystals. The best approach to crystallise a protein can be summarized as a random-screen-and-wait procedure, with little to no readout from experiments that do not produce crystals. This project aims to make the most out of present screening practice, by establishing objective analyses of sparse-matrix screening experiments that extract informative readouts from this standard front-line experiment, independent of whether it yields visible crystals or not.
We have developed methods to objectively characterize crystallization outcome based on image analysis, enabling several things. Firstly, the ranking of droplets based on their likelihood of crystallinity to increase the efficiency and accuracy of human visual identification of crystals. Secondly, fingerprints of the collective precipitation behaviour of a protein across standard sparse-matrix can be generalised, and compared objectively to fingerprints of historical experiments, to assess crystallizability and infer optimization strategies based on past successes. Thirdly, clear drops can be automatically identified, and mapped to chemical components in a sparse-matrix screen to suggest alternative buffers for protein formulation. Fourthly, TeXRank, a user interface could be developed to present and make all algorithm output accessible for daily use. Fifthly, the associated data mining led us to evaluate the strategies for setting up screening experiments with limited protein samples, based on over ten years of crystallization data at the Structural Genomics Consortium, Oxford. Our methods capitalizes on present day standard screening procedure and hardware to extract useful information, bypassing laborious and subjective evaluation of each droplet.

Keywords: machine learning
pattern recognition
protein crystallization
precipitation pattern
Advisor: von Delft, F
Osborne, M
Dekker, C
Kroemer, M
The role of HPV E6 protein in the induction of autophagy

Non-Melanoma skin cancer is the most common cancer in Caucasians. Accumulating evidence suggest a strong role for β-HPV (mainly HPV5 and HPV8) in the development of NMSC alongside UV, the main etiologic agent. The HPV genome encodes two potent oncoproteins, E6 and E7. HPV E6 protein has been shown to inhibit mitochondrial apoptosis following UVB exposure by specifically targeting BAK, a key apoptotic protein for proteolysis. A cross talk exists between the apoptotic pathway and the cellular survival pathway, autophagy. Where both autophagy and apoptosis can be triggered by common upstream signals. Additionally, cell lacking BAK and BAX respond with autophagy following the receipt of an apoptotic stimulus.
The aim of this work was to investigate if HPV E6 may induce autophagy in response to an apoptotic stimulus.
Cells expressing HPV5 E6 protein were exposed to UVB and autophagy levels, as well as autophagic flux, were investigated using endogenous LC3 staining, LC3 immunoblots and electron microscopy. Results showed an increase in autophagic flux and autophagy levels in HPV5 E6 expressing cells upon UV irradiation. Furthermore, the modulation of autophagy was demonstrated to be a conserved function amongst high-risk HPV (8, 20, 38 and mucosal HPV 16 and 18). Moreover, inhibition of autophagy resulted in a marked decrease in the viability of cells expressing E6 from HPV types 5 and 8. Furthermore, examining the respiration status of HPV5 E6 expressing cells showed a marked increase in both the glycolytic and the oxidative phosphorylation pathways.
These results indicate that HPV E6 may promote autophagy following an apoptotic stimulus allowing the cells to survive in the background of DNA damage and that may contribute towards tumorigenesis.

Advisor: Storey, A
Simon, K
Novel approaches for targeting BRCA2-deficient tumour cells

Defects in homologous recombination (HR) repair are associated with significant mortality and improved therapies for HR-deficient tumours are therefore urgently needed. This work aimed to address this unmet need and utilised pharmacological approaches to investigate several methods for the targeting of BRCA2-deficient tumour cells. Parallel lines of investigation demonstrated the selective and effective targeting of BRCA2-deficient cells via both chemical G-quadruplex stabilisation and ERK inhibition. These studies provide impetus for the further development of specific and clinically relevant inhibitors. Further, pharmacological screens generated a body of data relevant to the selective targeting of BRCA2-deficiency. In particular, screening studies demonstrated that targeting the kinase GSK3 selectively kills BRCA2-deficient cells. Moreover, pharmacological screens revealed for the first time that disulfiram, an aldehyde dehydrogenase inhibitor currently in clinical use as an alcohol-aversive agent, induces replicative stress and DNA damage, selectively reducing the viability of BRCA2-deficient human tumour cells. Together, these data reveal a greater dependency of BRCA2-deficient human tumour cells on certain pro-proliferative pathways than their wild type counterparts and demonstrate the vulnerability of BRCA2-deficient cells to replicative stress. Importantly, this work delivers clear rationale for the further study and clinical development of several novel approaches relevant to the treatment of HR-deficient tumours and thus has the potential to help reduce the burden of these devastating diseases in the future.

Keywords: BRCA2-deficient tumours
Targeted cancer therapies
HR-deficient tumours
BRCA2
Advisor: Tarsounas, M
Ryan, A
Youth and crisis: discourse networks and political mobilisation

This thesis explores the meaning of "youth" and the political mobilisation of young people in key moments of crisis in Europe. Between 2005 and 2011, youth became critical for the consolidation of the authoritarian regime structures in Russia. I show that this process included the restructuring of the discourse about youth, the physical mobilisation of young people, and the isolation of oppositional youth. How valid are these findings for regime crises more generally?
I answer this question through an analysis of the breakdown of the authoritarian Soviet Union during perestroika, the breakdown of unconsolidated democracy during the last years of the Weimar Republic, and the crisis of the democratic regime in France around 1968. The cross-regional and cross-temporal comparison of these episodes demonstrates that regimes lacking popular democratic support compensate for their insufficient legitimacy by trying to mobilise youth symbolically and politically.
By developing a new method of textual analysis which combines qualitative content analysis and network analysis, the thesis offers a novel social science perspective on the meaning of youth in the four cases. My study shows how discursive structures about youth condition the possibility of political mobilisation of young people.
The thesis makes three contributions to comparative politics. First, on an empirical level, my study offers new insights into social movements at moments of regime crisis in different political settings. Second, on a conceptual level, I refine our understanding of the symbolic significance of the terms "youth" and "generation" in moments when society is reorienting itself. I also examine the significance of "crisis" and argue that the term expresses openness and the possibility to remake the past and future. Third, on a methodological level, my thesis builds on the growing interest in textual analysis by developing a novel multi-level approach in three linguistic contexts, which offers insights into the structure of public discourse and the actors involved.

Advisor: Sasse, G
Natural history and pathogenesis of IgG4-related disease

IgG4-related disease (IgG4-RD) is a systemic fibro-inflammatory condition characterised by elevated serum IgG4 and an abundance of IgG4 plasma cells in involved organs. The natural history of disease and pathogenic mechanisms are poorly understood, and are explored in this thesis.
The diagnosis of IgG4-RD is a challenge. Evidence to support a serum IgG4 level of 2.8g/l in differentiating IgG4-RD from non-IgG4-RD conditions, a serum IgG1:IgG4 ratio of 0.24 in differentiating IgG4-sclerosing cholangitis from primary sclerosing cholangitis with elevated serum IgG4, and the role of serum IgE in those with a history of allergy and atopy is provided. Furthermore, observational data highlighting new environmental risk factors and disease associations are revealed. Despite being considered a benign corticosteroid-responsive condition, evidence for disease relapse, organ dysfunction and failure, malignancy and mortality in a prospective cohort is shown. Patterns of disease presentation and levels of serum IgG4 and IgE at diagnosis are used to identify those who relapse and develop multi-organ disease.
A single antigen initiating disease has yet to be found. Polyclonal IgG4 responses to multiple environmental antigens in IgG4-RD are reported, and evidence against Helicobacter pylori plasminogen binding peptide as a microbial antigen is shown. Novel HLA class II associations, linked to disease susceptibility in a UK cohort provide support for immune-mediated pathogenesis. Gene expression analysis implicates cytokines in driving IgG4 switch and proliferation, chemokines in trafficking and homing of lymphocytes to end organs, complement proteins in the classical and lectin pathways, and members of the TGF-beta pathway as putative immune drivers of disease. Differences in the phenotype of IgG1 and IgG4 B cells in health and IgG4-RD are reported, including responses to complement activation and immune complexes. Finally, elevated IgE levels, the presence of IgE-positive mast cells in involved tissues, and up-regulation of the Fc-Epsilon receptor on the surface of IgG4 cells, support the role of an IgE-mediated response.

Keywords: IgG4
IgG4-related disease
Plasma cells
B cells
Advisor: Barnes, E
Klenerman, P
The genetic control of agricultural pests (Plutella xylostella, L. and Tribolium castaneum, Herbst)

The interaction between an increasing global human population and a number of important long-term trends and issues are putting strain on food production. Insects represent a significant food security concern causing up to 15% of global crop losses. Conventional chemical methods are ineffective; inducing resistance and degrading the environment. Sustainable alternatives are sought.
The sterile insect technique provides a sustainable solution. Genetic engineering can augment this historic technique by replacing radiation-induced sterilization with sperm-specific nuclease expression to introduce double-stranded DNA breaks in the gametes of mass-reared and released males. This paternal-effect system is dependent on elucidation of appropriate sperm-specific promoters and suitable chaperone-nuclease combinations. This thesis develops this technology in the SIT neglected insect orders, the Lepidoptera and Coleoptera. Specifically the Plutella xylostella (Plutella xylostella, L.) and the red flour beetle (Tribolium castaneum, Herbst).
I provide the foundations for a paternal-effect genetic-control-system in both species by developing a conditional sperm-specific expression system in Plutella xylostella and a female-specific expression system in Tribolium.
Mass-rearing insects for the genetic control of a species can be augmented by recent developments in RNAi. I show that the sex ratio of Tribolium can be adjusted by treatment with dsRNA transformer, producing pseudomales as an additional bonus.
In addition, an exploratory data analysis of producing transgenic lines in insects using piggyBac was undertaken. As well as providing a comprehensive compendium and assessment of the transgenic literature, something not yet published elsewhere, a predictive model was produced that could be very useful to a wide diversity of researchers in insect molecular biology, developmental biology, disease biology and genetics.
It is hoped that this work will contribute towards the effective control of the Plutella xylostella in the near term, and form a model for the sustainable control of other lepidopteran and coleopteran species through genetic pest management.

Keywords: Paternal effect
RNAi
Female lethal
Sperm lethal
Spermiogenesis
Tribolium castaneum
Markov Chain Monte Carlo Method
Food production
transformation
dsRNA
Plutella xylostella
Diamondback moth
Red flour beetle
Sterile insect technique
Spermatogenesis
piggyBac
Advisor: Shimeld, S
Morrison, N
Alphey, L