VictorJuiz/thesis huggingface dataset:
Structural studies on determinants of receptor/ligand binding in the tumour necrosis factor and T cell receptor protein families
Keywords: T-Cell Receptor
TNF
X-ray crystallography

Protein-protein recognition plays a central role in the surveillance of self and non-self in the mammalian immune system and ultimately in cellular survival within the organism. Two systems of fundamental importance to the immune system are the Tumour Necrosis Factor (TNF) and the T cell receptor (TCR) families.
High-throughput methods developed within the Oxford Protein Production Facility have been successfully applied to the production of members of the TNF receptor and ligand superfamilies for structural characterisation. The TNF receptor DR6 was successfully refolded from E.coli inclusion bodies using a rapid-dilution technique and yielded diffraction quality crystals. Data collected from these crystals will be used to obtain an x-ray crystallographic model of DR6. Vascular Endothelial Growth Inhibitor (VEGI) was produced as a soluble recombinant protein in E.coli, and formed a number of poorly diffracting crystals, it is hoped that further trials and optimization of conditions will lead to improved data quality. Lymphotoxin β receptor was produced in a Eukaryotic system. This has shed light on the complications posed by signal peptide cleavage and glycosylation on the production of protein for crystallization trials.
TNF superfamily proteins are ideal targets for the design of novel therapeutic agents due to their involvement in a number of disease pathologies. Various methods of molecular docking and small molecule design were applied to the search for potential inhibitors of receptor binding for the TNF ligand proteins TRAIL and BAFF. A number of potential drug leads were identified from the National Cancer Institute drug database.
The Natural Killer (NK) T cell restricted TCRs recognise CD1d-presented glycolipid. Determination of the crystal structures of the invariant NK TCR and the NK restricted TCRs 5E and 5B shows that these proteins adopt the canonical structures of class I MHC restricted TCRs. This suggests that the binding of CD1d-glycolipid by these receptors will conform to the same model of binding seen for the class I MHC restricted TCRs.
Cell signalling in response to heat shock in Arabidopsis thaliana
Keywords: cell signalling
abiotic stress
arabidopsis
heat shock

Increases in temperature damage plant cells, and plants react to heat stress by inducing of a number of protective mechanisms. In this study, it has been shown that heat damages cells both directly while the plants are being heated, and indirectly through heat induced oxidative stress during recovery from heating. Different stress response pathways are induced in each case. Evidence implicates salicylic acid, abscisic acid, ACC (a precursor of ethylene), calcium ions and active oxygen species in pathways resulting in thermotolerance (i.e. increased survival at high temperature). Addition of these potential second messengers results in increased survival and decreased oxidative damage after heating (as measured using the TBARS assay), while silencing the pathways through use of mutants, inhibitors or transgenes results in decreased thermotolerance. In vivo calcium measurements show cytosolic calcium transients only at initiation of cooling after heat stress. Calcium chloride also induces thermotolerance when added after heating, as does ACC. These substances can induce early increases in ascorbate peroxidase activity after heating, and induce expression of antioxidant genes. Thus they may play a role in heat induced oxidative stress response pathways in recovery. Two different pathways induced during heating appear to result in the expression of genes for heat shock proteins. The predominant pathway induced at 30oC involves ABA, while that at 35-40oC involves an oxidative burst generated through the NADPH oxidase, atrbohB. Addition of SA can induce HSP (heat shock protein) expression, but there is little evidence that this occurs endogenously in plant cells, although nahG plants unable to signal via SA are thermosensitive. Results in this study indicate that there are several signalling pathways associated with heat shock. The pathway induced during recovery from heating involves calcium ions and ethylene, and results in increased antioxidant capacity. During heating two pathways induce expression of HSPs: one involving abscisic acid and possibly salicylic acid and one involving an oxidative burst. At least one further pathway is believed to exist, which involves protein kinases and phosphatases as heat shock causes up-regulation of expression of certain genes for these signalling components.
From the un-mixing to the re-mixing of peoples: Understanding the quest to “reverse ethnic cleansing” in Bosnia
Keywords: Bosnia
Refugees
Post-conflict Peace Building
Ethnic Conflict

This dissertation focuses on international actors' response to the ethnic cleansing perpetrated during the 1992 – 1995 Bosnian War. The work illuminates the multilateral attempt to reverse one of the outcomes of ethnic cleansing following the war, through the return of displaced people. The policy emphasis on "re-mixing" people, interpreted through a strategy of minority returns, and supported and coordinated on an international scale, was unprecedented.
This dissertation asks: why did powerful states and international organizations pursue a re-mixing policy as a response to ethnic cleansing in Bosnia? At first glance, the choice seems counterintuitive. The policy was expensive. Post-1989, the West no longer needed "to keep Yugoslavia afloat." Furthermore, reversal required a degree and duration of international involvement that, at the time, was thought to be politically, militarily, and financially impossible.
There are two existing explanations for this surprising phenomenon: international moralism and norm evolutionism. International moralists posit that international actors were moved to re-mix Bosnians out of a sense of guilt. Norm evolutionists argue that international norms governing appropriate responses to ethnic cleansing have shifted during the twentieth century towards support for re-mixing.
In contrast to these two dominant views, this dissertation argues that the re-mixing policy initially emerged as a practical fix to a series of pressing, context-specific political challenges. State policymakers justified the re-mixing policy, however, on normative grounds. Though not the original incentive for action, international organizations on the ground then adopted the policy, empowered by states' normative justifications and thereby transformed the political rhetoric into concrete action.
This dissertation corrects a common assumption that the origins and motivations behind the re-mixing policy were normative in nature, it contributes to a better understanding of how normative discourses emerge, mature, and transform into policy and it offers policy recommendations based on lessons learnt from this important and seemingly contradictory case.
Single-molecule DNA sensors and cages for transcription factors in vitro and in vivo
Keywords: single-molecule
drug-delivery
ALEX
FRET
biosensors

Gene regulation is vital to the success of all living organisms. Understanding this complex process is crucial to our knowledge of how cells function and how in some cases they can lead to debilitating or even fatal disease. In this thesis I focus on a set of DNA-binding proteins known as transcription factors (TFs), proteins fundamental to the process of gene regulation at the level of transcription. I develop assays and techniques for the detection and quantitation of TFs in vitro and in vivo as well as a method for TF encapsulation and release.
The advantages of the TF detection assays in this thesis are made possible through the use of single-molecule (sm) fluorescence. This methodology enables detection of individually labeled molecules allowing discrimination of sample heterogeneities inaccessible with ensemble techniques. Here I present two different TF assays based on two sm observables: relative probe stoichiometry and Förster resonance energy transfer (FRET).
The first assay design, based on stoichiometry, detects TFs using TF-dependent coincidence of two distinctly labelled DNA ‘half-sites’. I demonstrate sensitive detection (~ pM) in solution and on surfaces, multiplexed detection of multiple TFs, and detection in cell lysates. A kinetic model of the system is also developed, verified experimentally and used to quantify TF concentrations without the need for a calibration curve.
The second assay design, based on FRET, is a novel approach to TF detection using TFmediated DNA bending. TFs are detected by bending the sensor and monitored with FRET at the single-molecule or ensemble level. I demonstrate TF detection in purifed form and expressed in cell lysates. As this sensor was designed for use in vivo, methods to hinder nuclease degradation are explored.
For TF detection in vivo, I describe a successful strategy to internalise fluorescently labeled molecules into live E.coli. Viability and internalisation efficiency are characterised and ensemble measurements with FRET standards are demonstrated. Importantly, sm FRET measurements in vivo are achieved opening many exciting possibilities. The FRET based TF sensor is then internalised as a step towards real-time in vivo monitoring of TF concentrations.
Finally a system based on DNA nanotechnology is presented for the non-covalent encapsulation and release of TFs. Such a system could be delivered into a cell to alter levels of gene expression using external stimuli as inputs.
We believe these tools will generate valuable information in the study of prokaryotic gene expression as well as providing a potential commercial avenue towards diagnostics.
The estimation of recombination rates from population genetic data
Keywords: genetic recombination

Genetic recombination is an important process that generates new combinations of genes on which natural selection can operate. As such, an understanding of recombination in the human genome will provide insight into the evolutionary processes that have shaped our genetic history. The aim of this thesis is to use samples of population genetic data to explore the patterns of variation in the rate of recombination in the human genome. To do this I introduce a novel means of estimating recombination rates from population genetic data. The new, computationally efficient method incorporates a model of recombination hotspots that was absent in existing methods.
I use samples from the International HapMap Project to obtain recombination rate estimates for the autosomal portion of the genome. Using these estimates, I demonstrate that recombination has a number of interesting relationships with other genome features such as genes, DNA repeats, and sequence motifs. Furthermore, I show that genes of differing function have significantly different rates of recombination. I explore the relationship between recombination and specific sequence motifs and argue that while sequence motifs are an important factor in determining the location of recombination hotspots, the factor that controls motif activity is unknown.
The observation of many relationships between recombination and other genome features motivates an attempt to quantify the contributions to the recombination rate from specific features. I employ a wavelet analysis to investigate scale-specific patterns of recombination. In doing so, I reveal a number of highly significant correlations between recombination and other features of the genome at both the fine and broad scales, but find that relatively little of the variation in recombination rates can be explained. I conclude with a discussion of the results contained in the body of the thesis, and suggest a number of areas for future research.
Reassessing civil conflicts in Genoa, 1160-1220
Keywords: Genoa
political violence
economic development

This thesis examines the phenomenon of civil violence in Genoa (1160-1220). Genoese civil conflicts in the period are victim to a historiographical paradox. While their importance for the survival of communal institutions has been frequently underlined, they have often been misrepresented in the historiography. Our current understanding of civil conflicts in Genoa is in need of a reassessment if we want to deepen our comprehension of the history of a city that is considered a key centre and fundamental building bloc in the rise of the European continent to economic prominence.
This thesis studies civil violence from a perspective that takes into account the shifting form of Genoese conflicts and their protagonists. The civil conflicts in Genoa saw constant development in their intricacy, nature and participants (Chapter one). I distance myself from the issue of motives and causation, a pursuit which has misled scholars. Instead I focus my attention on the underlying patterns that made conflicts in Genoa possible -- the web of relationships among the families of the Genoese elite – in order to study how the individuals and families that were involved in civil violence made their decisions (Chapter two). The understanding of these links and of the development of conflict in Genoa is an important thread to follow in order to reassess several aspects of the political history of the city between the twelfth and the thirteenth century (Chapter three). In light of my findings, the institutional transition of the city from a commune led by consuls to one led by a foreign podestà (Chapter four) and the Genoese involvement in the Mediterranean scenario (Conclusion), appear shaped by the maturing phenomenon of civil violence. This thesis aims to fill the current gap in academic studies on civil conflict in Genoa and to turn the phenomenon from a footnote to the current historiography into a rich vein of historical understanding of the fundamental dynamics of the city and its development.
Characterization of mouse two-pore channels (TPCs) in NAADP-mediated Ca(2+)  signalling
Keywords: Ca(2+)
NAADP
Two-Pore Channels
calcium signalling
TPC

Recent studies have identified Two-Pore Channels (TPCs) as the channels activated by NAADP. To date, most studies that characterized these channels have employed heterologous expression or overexpression systems. The research reported here has focused principally on endogenous TPC activity by using single and dual gene knockout (KO) in a mouse system and has yielded insights into TPC expression levels, subcellular localisation, NAADP binding, and channel function.
Mouse models that had been generated by both the “gene-trapping” and the “genetargeting” techniques were obtained and validated. These included a knock-down strain (“hypomorph”). Surprisingly, all TPC mutant mice showed no gross phenotypes. In addition to the two known isoforms in mouse, TPC1 and TPC2, the expression of a shorter variant of TPC1 was discovered; this has an alternative (truncated) N-terminus, and has been termed (DELTA)N-TPC1. All TPC variants/isoforms were widely expressed in all mouse tissue types tested. Overexpression of mouse TPCs in mouse embryonic fibroblasts showed that (DELTA)N-TPC1 and TPC2 were expressed primarily in late endosomes/lysosomes while TPC1 was expressed in both endosomes and lysosomes. Dileucine sorting motifs target TPCs to late endosomes/lysosomes; it was shown that truncation or mutation of dileucine motifs significantly reduced localization in late endosomes/lysosomes. Furthermore, TPCs were shown not to be the direct binding target of NAADP, as the high affinity NAADP binding was retained in hepatic membranes from TPC double KO (DKO) mice. It is concluded that NAADP binds to an (as yet, unidentified) accessory protein.
The functional role of TPCs was studied in depth using mouse pancreatic acinar cells. NAADP is known to release Ca2+ from the acidic stores in response to the stimulation by the hormone cholecystokinin (CCK). In all TPC mutant mice, CCK was still able to evoke Ca2+ oscillations, but with slower and attenuated oscillations in the TPC1 hypomorph, and with slower oscillations in TPC DKO. In all TPC KOs, oscillations were disrupted by known inhibitors of the NAADP-signalling pathway (Ned-19, GPN and bafilomycin A1), indistinguishable from the responses with wild-type cells. This suggests that TPCs are not involved in CCK signalling, although it is possible that functional compensation masked the phenotype arising from the impaired signalling.
Enrichment at the claimant's expense: attribution rules in unjust enrichment
Keywords: corrective justice
attribution
causation
remoteness
at the expense of
law
enrichment
tracing
unjust enrichment
valuation
restitution

This thesis presents an account of attribution in unjust enrichment. Attribution refers to how and when two parties – a claimant and a defendant – are relevantly connected to each other for unjust enrichment purposes. It is reflected in the familiar expression that a defendant be 'enriched at the claimant's expense'. This thesis presents a structured account of attribution, consisting of two requirements: first, the identification of an enrichment to the defendant and a loss to the claimant; and, secondly, the identification of a connection between that enrichment and that loss. These two requirements must be kept separate from other considerations often subsumed within the expression 'enrichment at the claimant's expense' which in truth have nothing to do with attribution, and which instead qualify unjust enrichment liability for reasons that should be analysed in their own terms. The structure of attribution so presented fits a normative account of unjust enrichment based upon each party's exchange capacities. A defendant is enriched when he receives something that he has not paid for under prevailing market conditions, while a claimant suffers a loss when he loses the opportunity to charge for something under the same conditions. A counterfactual test – asking whether enrichment and loss arise 'but for' each other – provides the best generalisation for testing whether enrichment and loss are connected, thereby satisfying the requirements of attribution in unjust enrichment.
The law is stated as at 15 March 2014.
Developing P(MMA-co-NVP) hydrogels for use in self-inflating, anisotropic tissue expanders
Keywords: Hydrogels
Tissue Expansion

Artificial tissue expansion is required to generate new skin prior to reconstructive surgery, in order to compensate for a deficit of healthy tissue. Hydrogel tissue expanders, which expand anisotropically, show great promise in overcoming clinical limitations in the field, thus allowing the technique to be used in a wider range of surgeries. These devices consist of pellets of dry poly(methyl methacrylate-co-vinylpyrrolidone), compressed into discs through a hot compression moulding process. However, a number of significant problems still exist in these devices, and this thesis aims to address these issues.
To date, there has been a lack of investigation of the factors governing the behaviour of anisotropic swelling. For this reason, a range of different compression ratios have been investigated, with particular focus on the relationship between the material flow during compression and the swelling behaviour of the resulting device. It was found that samples of the same initial size expand to the same reference swelling dimensions, regardless of compression ratio. During hot pressing, the material flow was found to be governed by slip stick behaviour at the interface between the hot press and the device, affecting the properties and swelling behaviour of the devices.
Based on these findings, devices were developed which could expand from a disc into a non prismatic shape (dome or wedge). Such devices could reduce complication rates and allow the growth of new tissue with anisotropic resting tension. The devices were tested in a small in vivo trial, where it was shown that there were no adverse effects on the tissue produced, and that the shape of the expander (dome) was retained.
As devices are being produced for medical use, understanding the effect of sterilization by γ irradiation is essential, but to date this has been overlooked in the literature. It was found that γ-irradiation caused an increase in cross-linking in the P(MMA-co-NVP). Whilst this produced little change in swelling behaviour for isotropic devices, in the case of anisotropic devices it caused a change in the shape of expansion, reducing the area of new skin which could be generated by the device. It was found that by reducing the concentration of impurities (residual molecules from the polymer synthesis) the impact of γ-irradiation could be greatly reduced.
Finally, controlling the rate of expansion is essential in order to avoid clinical complications. In order to control the rate of expansion, particularly during the initial period of swelling, semi permeable PDMS coatings were applied to the compressed devices. Coatings of thickness greater than 0.375mm were found to effectively control the rate of swelling, for both cylindrical and non-prismatic shapes. As the coating thickness increased, the maximum swelling size decreased. However, it has been shown that change in height (the parameter which governs the area of skin produced) is affected less than the change in mass or diameter.
Linguistic studies in Euripides' Electra
Keywords: Euripides
textual criticism
Greek linguistics
Greek Tragedy
Electra

Euripides’ Electra has long been one of the playwright’s most controversial works. This book offers a reading of the play concentrating on its language, which is analysed by applying a variety of modern linguistic approaches: conversation analysis, pragmatic theories of speech acts and inference, politeness theory, the study of the interplay of gender and language, paroemiology, and the study of discourse cohesion.
The first three chapters argue for the Peasant, Electra and Orestes, respectively, that their linguistic behaviour constitutes a vital part of their characterisation. The Peasant’s (ch. 1) sturdy morality is established by the way his language becomes more forceful when he touches on ethical questions; it is then tested in his conversations with Electra, where his language is suggestive of a conflict between his morals and his desire to please his royal wife.
Electra herself (ch. 2) is characterised initially by the inability to communicate successfully with those around her — a disconnect which is suggestive of the fundamental incongruity of her circumstances. This adds a dimension to her motivations, which, as a force driving Electra’s linguistic behaviour, remain highly stable throughout the play up until the matricide. Another consistent feature of Electra’s language is the way it is patterned by her gender.
Orestes’ characterisation in the early part of the play is ingeniously kept to a minimum through his sustained disguise. Various aspects of his language, but particularly his use of gnomai, contribute to that disguise, which involves a suppression of emotion, an avoidance of self-reference, and the exertion of control over the flow and topic of his conversation with Electra.
We can only interpret a dramatic text if we know what it says, and if we know who says what. In chapter 4, I argue that the linguistic approaches I adopt can also help us in making a determination about textual-critical problems, particularly concerning the issue of speaker-line attribution (two notorious cases are discussed: 671-84 and 959-87).
The final two chapters deal with longer speeches. In the messenger scene (ch. 5), Euripides uses linguistic devices to create an ebb and flow of suspense, and to manipulate audience expectation. In the agon (ch. 6), differences in the way Clytemnestra and Electra structure their speeches, particularly their narrationes, reveal much about their different (and fundamentally irreconcilable) viewpoints and approaches.