VictorJuiz/thesis huggingface dataset:
The role of receptor interacting protein kinase 3 in inflammatory signalling

Inflammatory signalling and programmed cell death are fundamental processes employed by higher order organisms as defence mechanisms against foreign substances. Necroptosis, a proinflammatory type of programmed cell death mediated by the kinase RIPK3, has emerged as an important innate immune defence mechanism against viruses. The necroptosis process is accompanied by de novo synthesis of cytokines and chemokines within the dying cells. However, how inflammatory signalling is induced in the necroptotic cell remains poorly understood.
In this study, I describe that RIPK3, independently of its cell death-inducing activity, stimulates inflammatory signalling pathways mediated by the kinases TAK1 and IKKs. I provide evidence that RIPK3 can stimulate inflammatory signalling through two distinct mechanisms, the scaffolding-dependent pathway and the RIPK3 kinase activity-dependent pathway. In the scaffolding-dependent pathway, RIPK1 connects RIPK3 to the ubiquitin ligases, cIAPs and LUBAC, for the activation of TAK1 and IKKs through K63- and M1-linked polyubiquitin, independent of RIPK3’s kinase activity. In contrast, the RIPK3 kinase activity-dependent pathway does not require cIAPs or LUBAC, but is mediated by RIPK3’s kinase activity and its substrate, MLKL, and is suppressed by the RIPK1-recruited protease caspase-8.
Taken together, this study indicates that RIPK3, like its family members RIPK1 and RIPK2, is a scaffolding kinase able to promote inflammatory signalling. RIPK3 promotes inflammatory signalling through kinase-dependent and -independent mechanisms. Given the role of necroptosis in the host defence against viruses that often encode caspase inhibitors, RIPK3 kinase activity-dependent signalling may serve not only as an additional mechanism to ensure the activation of inflammatory signalling, but also as a regulatory checkpoint between RIPK3-mediated inflammatory signalling and cell death.

Advisor: Gyrd-Hansen, M
Investigation of K2P channel permeation and gating

The ability to conduct potassium ions (K+) selectively in the abundant presence of other ion species is crucial to the function of K+ channels. While the mechanism of ion selectivity has been an in- tensely studied and debated topic, the protein structure responsible for ion-selectivity has long been identified as the so called selectivity filter (SF). The SF also doubles as the principal regulatory element (gate) in many K+ channels.
In this project several phenomena related to the permeation, selectivity and gating at the SF of two-pore-domain K+ channels (K2P channels) were investigated. Firstly, the effect of mutations at the intracellular entrance of the TASK3 (TWIK-related acid sensitive K+ 3) channel SF were studied. Secondly, the mechanism of pharmacological activation of the TREK2 (TWIK related K+ channel 2) SF gate was probed on a single molecule level. Channel activation was found to be conferred by a change to the electrostatic milieu at the SF entrance.
Following from this electrostatic mechanism the dependence of gating on salt concentration (ionic strength) was probed. Here an unusual property emerged: TREK channels close in the presence of high ionic strength.
Finally, evidence was found that TREK2 may change its ion- selectivity in the context of voltage-dependent gating. This result suggests that TREK2 may have a stimulating effect on cellular exciteability, which is a novel and atypical property for a K+ channel

Advisor: Tucker, S
Carpenter, L
Refractory body-centred cubic high-entropy alloys for nuclear fusion

In this work refractory High-Entropy Alloys (HEAs) have been investigated for application as Plasma Facing Components in nuclear fusion reactors. Sample fabrication was guided by optimising composition in order to maximise the Ω parameter, which has been previously identified as correlating with the stability of solidsolution microstructures. Arc melting was used to fabricate billets of TiVZrHfTa, TiVZrTa, and TiVTa, each of equiatomic and optimised compositions. These samples were analysed by XRD and SEM in the as-cast condition and after being annealed at 1400 ◦C for twenty four hours. The matrix of each sample was found to be BCC, and each contained secondary phases with either BCC, HCP, or rocksalt crystal structures. The latter of these phases was found to be a carbonitride, formed from an air leak that occurred during melting. Finite-Element Analysis of thermal transport in arc-melted billets has shown that cooling rates generated are rapid, and even more so in smaller billets. Smaller 10 g billets of equiatomic alloys were found to have much lower volume fractions of secondary phases than as-cast 50 g counterparts, and this has been attributed to the differences in solidification and cooling conditions. The modelling also demonstrated large variations in cooling rate throughout larger 50 g billets, and so the effect of this on as-cast microstructures was also investigated. It was found that the differences in cooling rate was sufficient to create variations in microstructures, with an increase of 10% volume fraction of the carbonitride phase between the bottom and top of the billet, along with a large increase in porosity. The main conclusion of this portion of the thesis is that arc melting plays a larger role in determining as-cast microstructures than previously thought. Two ion-implantation experiments were run into four different alloys of TiVZrHfTa up to damages of 0.6 and 3.5dpa. Nanoindentation has then been used to show there is no measurable change in hardness, despite a strong reaction being found in a vanadium control sample. Separately, these alloys were used to demonstrate the efficacy and limitation of using a new nanoindentation technique called Express indentation, which rapidly makes thousands of indents in order to map the hardness and modulus of materials.

Keywords: High-entropy alloys
Nuclear Fusion
Advisor: Wilkinson, AJ
Armstrong, DEJ
Insights into the mechanisms of asymmetric phase-transfer catalysis

This thesis describes work conducted towards the study of the mechanisms of asymmetric cation-directed phase-transfer catalysis (PTC).
Chapter 1 introduces the field of PTC, including its general mechanisms, utility in an asymmetric context, and previous kinetic and mechanistic studies. Despite being a well-established technique in organic chemistry, the precise mechanisms of enantioselective phase-transfer reactions, particularly those effected by cinchona alkaloid-derived catalysts are not fully understood.
In Chapter 2, we found that studying enantioselective phase-transfer reactions effected by cinchona alkaloid-derived catalysts proved to be both practically and intellectually demanding. The manner in which the reactions were practically conducted had significant bearings on the kinetic behaviour that was exhibited. Primarily, we developed robust methods that enabled the collection of reliable, reproducible kinetic data from polyphasic heterogeneous reaction systems.
Chapter 3 describes the kinetic and mechanistic study of a phase-transfer catalysed intramolecular C-acylation reaction. Deconvolution of the mechanism involved a series of practical kinetic investigations, evaluation of theoretical models, and further experimental interrogation. The final mechanistic hypothesis highlighted a series of processes concerning catalyst activation and regeneration, which were rate-determining in this system.
Chapter 4 presents a concise summary and conclusion to the thesis, and Chapters 5 to 10 describe experimental details and provide supporting information.

Keywords: spirobiindanone
kinetics
phase-transfer catalysis
organic chemistry
enantioselective
Advisor: Smith, M
Hypersonic boundary layer instability measurements at low and high angles of attack

This research experimentally investigated the effects of angle of attack, wall-to-total temperature ratio, and freestream disturbance levels on hypersonic boundary layer instabilities at Mach 7. The motivation was to improve the understanding of hypersonic boundary layer instabilities and inform numerical transition models so that transition can be predicted in flight. Although many studies exist on this topic, there is little high resolution data available on instabilities for high angles of attack. The study used a 7° half angle cone with small nose bluntness, and included a heater cartridge to test multiple wall-temperature distributions. Measurements are presented using two experimental facilities with varying wall-to-total temperature ratio: the low total enthalpy Oxford High Density Tunnel (HDT) and the high total enthalpy T4 Stalker Tunnel at The University of Queensland. Models were heavily instrumented with high-frequency PCB132 pressure sensors, to investigate the development of instabilities on the cone surface.

Advisor: McGilvray, M
The effects of physical activity on hip development

In recent decades the sports opportunities available to young athletes has risen significantly. The effect of such high levels of physical exertion on the developing skeleton is largely unknown. Retired football players have significantly greater rates of hip osteoarthritis than control groups, and a higher prevalence of morphological abnormalities known to predispose to future osteoarthritis, such as cam morphology, acetabular dysplasia and retroversion, and varus leg alignment. Cam morphology is likely acquired during adolescence, and dysplasia and leg alignment develop from birth throughout childhood and adolescence. High activity is a recognised risk factor for the development of these morphologies. However, their aetiology is insufficiently understood to recommend intervention. The aim of this thesis is to explore the impact of high levels of activity during adolescence on the development of the skeletally immature hip and knee. This is achieved via a longitudinal prospective observational cohort study in elite level footballers and age matched controls age 9-18 years.
The first three studies in this thesis investigate the effect of activity on hip morphology and leg alignment. The results provide strong evidence that elite level football and high activity during adolescence significantly influences skeletal development in these areas. High activity individuals are more likely to develop cam morphology, dysplastic and retroverted acetabula, and varus leg alignment. The most significant effects appear to occur between 11 and 14 years of age. At present, it is not possible to recommend intervention such as activity modification due to our limited understanding of aetiology, and the cardiovascular benefits of exercise are likely to outweigh potentially detrimental effects on morphology. However, high activity individuals between the ages of 11 and 14 years likely represent a high-risk cohort warranting surveillance for early joint failure.
The effect of exercise on cartilage in weight bearing joints in young healthy individuals is poorly understood. The final study in this thesis uses compositional MRI to determine the effect of high activity on cartilage composition during youth. The results suggest that elite level footballers and individuals with high activity levels may have differing cartilage composition and architecture to low activity individuals during adolescence.

Advisor: Glyn-Jones, S
Palmer, A
Evaluation of CRISPR-Cas9 approaches to investigate microRNA targeting in human chondrocytes

Micro RNAs (miRNAs) are small, non-coding, negative regulators of genes that play a major role in a wide variety of biological and pathological processes. The chondrocyte-selective miR-140 is required for normal cartilage development, as demonstrated by the developmental defects in miR-140 knockout (KO) mice. However, only a few studies have been conducted to explore the mechanism of action of miR-140 at the cellular level.
In the present study, we describe a CRISPR-Cas9 system to identify functional miR-140 targets in isolated primary human osteoarthritic articular chondrocytes. CRISPR-Cas9 editing offers a highly target-specific and stable method for exploring functional miRNA targets, without perturbing other miRNA levels. Developing a systematic approach of double transfection using CRISPR-Cas9, we produced rapid, efficient, and reproduceable genetically engineered populations of primary human articular chondrocytes, without prior need for clonal selection.
Stable knockdown of miR-140 (> 90 %), with no detectable off-target activity, identified several miR-140 regulated targets, of which Septin 2 (SEPT2), bone morphogenetic protein 2 (BMP2), and fibroblast growth factor 2 (FGF2) had previously been reported. The major aggrecanase ADAMTS-5 was not a miR-140 target in our study, even though this had been reported previously. A number of novel targets were identified including Agrin (AGRN), a gene encoding for a heparan sulfate proteoglycan with chondrogenic potential, two regulators of the retinoic acid pathway, retinoic acid receptor gamma (RARG) and cytochrome P450 26B1 (CYP26B1), and two molecules critical for primary cilium function, intraflagellar transport 88 (IFT88) and Tau Tubulin Kinase 2 (TTBK2).
In order to attempt to differentiate between direct and indirect effects of the miRNA, I adopted a novel CRISPR-Ca9 based technology, in which the miRNA response element (MRE) of the target gene, rather than the miRNA itself, was targeted. I report the challenges associated with this approach in primary human chondrocytes.
Taken together, I describe a highly efficient and reproduceable CRISPR-Cas9-mediated protocol, in which I identified novel miR-140 targets of disease relevance using primary OA chondrocytes.

Advisor: Vincent, T
Systematics of Andean Lupinus L. and the origin of L. mutabilis Sweet

This thesis contributes to the central goals of systematics - the inventory of species, building the tree of life and understanding evolutionary processes - through a descriptive taxonomic study, molecular phylogenetic analysis and morphological survey of the genus Lupinus L.
Lupinus comprises ca. 290 annual and perennial species placed in the Genistoid clade of the Papilionoid legumes. In this study three new DNA sequence datasets are used to reconstruct species relationships. The new phylogeny is used to investigate character evolution, species diversification, domestication and as the basis for a new infrageneric classification.
Approaches to plant species-level phylogeny reconstruction and criteria for selecting DNA sequence loci are evaluated from a theoretical standpoint, and experimental work to develop hypervariable nuclear DNA sequence loci is presented.
The comparative morphology of Lupinus species is investigated and presented. This includesstudies of seed coats, cotyledons, leaves, and chromosomes. Optimisation of these data onto the 3-locus phylogeny reveals congruence between chromosome number and geographyand shows that non-digitate leaves have evolved twice independently within Lupinus.
Evolutionary rates analysis is used to estimate divergence times for clades and to investigate the tempo of species diversification. Notably, a strongly supported clade representing ca. 81 Andean species is estimated to be 1.18-1.76 Myr, implying a diversification rate of 2.49-3.72 species per Myr. This exceeds previous estimates for plants providing the most spectacular example of explosive plant species diversification documented to date.
Lupin species have been independently domesticated as grain legume crops in the Old and New Worlds. The origins of the Andean domesticate, L. mutabilis, are investigated. Lack of resolution amongst the Andean species, attributable to exceptionally rapid species diversification, meant that the progenitor(s) of L. mutabilis could not be firmly established. However, four Andean species are tentatively identified as closely related to L. mutabilis based on morphology. A taxonomic account of these species is presented.
Finally, a new infrageneric classification based on morphological diagnosability monophyly is proposed.

Keywords: Radiation
Lupinus
Phylogeny
Species diversification
Molecular phylogeny
Character evolution
Plant diversification
Leaf morphology
Andes
Lupin
Classification
Seed size
Domestication
Taxonomic accounts
Evolution
Advisor: Hughes, C
Structural investigation of the interaction between glypican-3 and uncoordinated-5

Uncoordinated-5 (Unc5) receptors are known for their function to induce repulsion in axonal and cell migration. Another proposed function is as dependency receptors where withdrawal of their ligands results in apoptosis. Unc5’s interactions with its ligands Netrin and fibronectin leucine-rich transmembrane (FLRT) have been extensively investigated; however, the molecular mechanisms of Unc5 as a switch for repulsive signalling and as a dependency receptor remain elusive. Recent work identified a novel interaction between Unc5 and Glypican-3 (GPC3), for which there are no known biological functions. GPC3 is a GPI-anchored heperan sulfate proteoglycan that has been studied in its role of modulating several morphogen signalling pathways. Using X-ray crystallography, this thesis reveals the structure of the core protein of human and mouse GPC3 at 2.6 and 2.9 Å resolution respectively. From this, the crystal structure of the GPC3/Unc5 complex was solved in two different crystal forms, at 4.0 to 4.6 Å. The crystal structure revealed an octameric assembly, showing that GPC3 clusters several Unc5 receptors. Biophysical studies attempted to validate the identified stiochiometry to further understand the molecular mechanisms of this interaction. The work presented provides the foundations for further functional studies for the GPC3/Unc5 interaction.

Advisor: Seiradake, E
Aksu, M
Rational verification in multi-agent systems

Rational verification problem is concerned with checking which temporal logic properties will hold in a system composed of multiple agents which are assumed to behave rationally and strategically in pursuit of individual objectives. Unfortunately, the problem is generally hard from computational point of view, and for the purpose of practical implementations, usually requires specialised techniques.
This thesis aims to develop algorithms and study computational complexity results for rational verification in multi-agent systems. Firstly, a practically amenable technique which relies on a reduction to the solution of a collection of parity games is proposed. The technique in this thesis uses a model of strategies that is bisimulation invariant—that is, in which individual strategies for system components are valid across all bisimilar systems, and which satisfy the same temporal logic properties in equilibrium. This approach has been implemented in the Equilibrium Verification Environment (EVE) system. Secondly, some cases in which the problem of rational verification is computationally tractable are investigated. In particular, it is shown that the complexity of rational verification can be reduced from 2EXPTIME-complete to fixed-parameter tractable. Furthermore, improved complexity results when considering quantitative goals, namely mean-payoff utility functions, are also studied. In doing so, a concept called equilibrium design is proposed. This concept is concerned with the design of incentives so that a desirable equilibrium is obtained.

Keywords: Model checking
Automata
Temporal logic
Games
Synthesis
Computational complexity
Nash equilibrium
Advisor: Gutierrez, J
Wooldridge, M
Offender agency at sentencing: exploring the grounds and scope for engaging offenders in the determination of punishment

Punishment is a grave intrusion into individual liberty, yet in most liberal criminal justice systems, including England and Wales, those punished are rarely directly engaged in determining their sentence. The thesis challenges this status quo and asks on what grounds and to what extent offenders should exercise agency at sentencing, and to what extent offenders can exercise agency in current English sentencing practice. First, it examines possible theoretical arguments for offender agency on a state-centred account of sentencing. It does so in respect of several theories of punishment: communicative variations of desert theory, a forgiveness-based theory of punishment, and therapeutic jurisprudence. Secondly, the thesis develops a normative framework for offender agency. It argues that there may be good justifications for allowing offenders to exercise agency at sentencing, to be more respectful of the offender as a responsible moral agent and to foster rehabilitation. It explores conceptually what an ‘agentic’ sentencing system might entail and argues it should be based on the paradigm of a dialogue between the judge and offender. The offender’s agency should be non-dispositive and in principle should have no guaranteed determinative influence on the sentence. However, the offender’s input may alter the disposition, insofar as it is aligned with the public interest. It explores other constraints on offender agency to preserve the state’s fundamental role in sentencing. The proposed model embodies a different conception of sentencing as a decision-making process to be done with offenders, rather than to offenders. The thesis examines objections that could be levelled against an agentic sentencing model. Finally, the thesis undertakes a doctrinal enquiry into instances of offender agency in current English sentencing practice. It concludes that, while current sentencing practice does not respect offender agency in a principled manner, there is both value in and scope for allowing offenders to exercise agency in a state-centred sentencing system.

Advisor: Zedner, L
Genetic variation contributing to autoimmune disease

Genetic variants influencing the IL-2 signaling pathway, including IL2RA , which encodes CD25, the alpha chain of the IL-2 receptor, are enriched in autoimmune diseases. Regulatory T cells (Tregs) are vital to the maintenance of a balanced immune system. They require IL-2 for their survival and by expressing high levels of CD25 are exquisitely sensitive to this cytokine. My thesis has focused on (1) accurate quantification and understanding of the heterogeneous nature of Tregs and (2) analysing phenotypes determined by IL2RA variants associated with autoimmune disease.
FOXP3 is the lineage-defining transcription factor of Tregs and stable expression is dependent on the demethylation of the Treg Specific Demethylated Region (TSDR) within intron 1 of the FOXP3 gene. I developed and validated a high-throughput and sensitive method to measure the TSDR methylation and used the assay to define the heterogeneous nature of Tregs.
The genetic association of IL2RA with autoimmune disease is complex and is explained by five independent signals, which have been termed the A, C, D, E and F haplotypes. Genotype-to-phenotype correlations have previously been identified for the A and D haplotypes but here I dissected IL2-RA gene polymorphisms and its mRNA expression in detail. I showed that the C haplotype results in decreased IL2RA mRNA expression, seen only in activated CD4 + T cells and the F haplotype correlates with decreased expression in ex vivo and activated B cells, and activated monocytes. The sequence relationship between disease-protective haplotypes was assessed across divergent populations. Finally, I have identified IL2RA mRNA expression phenotypes not associated with disease serving as a reminder that not all changes in gene expression correlate with a known disease haplotype.

Advisor: Wicker, L
Todd, J