VictorJuiz/thesis huggingface dataset:
Investigating SAMHD1 restriction of HIV-1 in stem cell-derived macrophages

SAMHD1 is potent in restriction of lentiviruses in primary bone marrow-derived monocytes, macrophages, and dendritic cells and is variably neutralised in these cells by the SIV accessory protein Vpx. However, the dynamics of SAMHD1 restriction of lentiviruses in yolk sac derived tissue-resident macrophages has not been formally demonstrated. Here I use a human stem cell differentiation protocol, competent in the production of macrophages that are developmentally related to, and thus a good model for, tissue-resident macrophages such as microglia, Langerhans cells, and alveolar macrophages. This system produced stem cell origin Yolk-sac-Derived-like Macrophage Precursor cells (scYDMPs) and Macrophages (scYDMs) over extended periods, which were subsequently used as a tool to investigate SAMHD1 restriction of HIV-1 and antagonisation by Vpx. Using a loss-of-function approach, I showed that the phenotype of SAMHD1 expression, hydrolysis of dNTPs, restriction of HIV-1, and antagonisation by Vpx in scYDMs is very similar to that reported in blood MDMs. I also showed that Vpx-mediated rescue of HIV-1 infection was more potent than the shRNA knockdown of SAMHD1. I, therefore, hypothesised that either Vpx facilitates HIV-1 infection via a SAMHD1 independent mechanism or the SAMHD1 protein remaining after knockdown was of sufficient potency to facilitate HIV-1 restriction and dNTP hydrolysis. I used a CRISPR-Cas9 knockout strategy to show that Vpx did not rescue HIV-1 infection by a SAMHD1 independent mechanism in scYDMs. However, the application of Vpx to SAMHD1 knockout cells was concomitant with an increase in dNTPs. This showed that in scYDMs, Vpx functions to increase dNTPs by a mechanism independent of SAMHD1. Using a dNTPase defective SAMHD1 mutant scYDM cell line, I was able to show for the first time that the dNTPase function of SAMHD1 is dispensable for HIV-1 infection in scYDMs. I also showed for the first time that Vpr of SIVmac degrades SAMHD1 and rescues HIV-1 infection in scYDMs. Moreover, and in contrast to Vpx-mediated rescue of HIV-1 infection at the steady-state in scYDMs, Vpx (and Vpr) rescued HIV-1 infection from the antiviral state established by type I interferons and LPS in scYDMs. This result implies that in scYDMs, Vpx and Vpr may interact with additional components of the innate immune system that are independent of SAMHD1. Taken together, these results highlight the experimental flexibility and integrity of data generated using a stem cell to macrophage differentiation strategy combined with genetic modification, and show that these cells represent a good model for investigating SAMHD1 restriction of HIV-1.

Keywords: HIV-1
HIV-2
SIV
Macrophage
Stem Cell Differentiation
Vpr
SIVmac
Lipopolysaccharide
Interferon
SAMHD1
Vpx
Advisor: James, W
Moore, M
Τhe burial rite of Enchytrismos in Attica, Euboea and Boeotia during the Late Geometric and Archaic periods. Towards an understanding of its social significance

Inhumation inside ceramic vessels, conventionally termed “enchytrismos” in modern scholarship, is a long-lasting practice attested in the Aegean world and beyond. The present study examines in detail the evidence relating to enchytrismos burials from the regions of Attica, Euboea and Boeotia during the Late Geometric and Archaic periods (760-480 BC), which it uses as case studies. During this time frame, enchytrismos becomes the prevalent burial rite for young individuals in all three regions, significantly enriching the archaeological record and thus offering plentiful material for a comprehensive study of this funerary ritual.
The decision to provide any distinct mortuary treatment is an active decision of the living when confronted with the death of a particular group of individuals. Based on the above premise, the present study focuses on illuminating three distinct but mutually dependent aspects of the rite of enchytrismos: the acts entailed when practicing an enchytrismos, the individuals chosen to be interred inside these burials, and, finally, how these individuals were treated by the living in the event of their death.
The systematic examination which has been undertaken clearly demonstrates that the rite of enchytrismos was a time-consuming and carefully thought social act that placed particular emphasis on the protection and preservation of the dead body. Since the rite of enchytrismos concerned mainly the biologically youngest members of Attic, Euboean and Boeotian communities, the present study brings this—often largely neglected—age category to the foreground. The choice of affording such an attentive funerary treatment to the young, who have for long been considered as an “insignificant” social category, clearly suggests that their untimely demise did not provoke a minor social reaction. Therefore, in the context of the present study, “non-adults” emerge as a complex social category whose death could initiate a series of social reactions that emphasised the need for protection and connection to their family in perpetuity and which were largely imbued with sentimental value.

Keywords: enchytrismos
Archaic period
Early Iron Age
burial rites
child burials
Advisor: Lemos, I
Consequence-based reasoning for the Description Logic SROIQ

Consequence-based (CB) reasoners combine ideas from resolution and (hyper)tableau calculi to solve the problem of ontology classification in Description Logics (DLs). Existing CB reasoners, however, are only capable of handling DLs without nominals (such as SRIQ), or DLs without disjunction (such as Horn-SROIQ). In this thesis, we present a novel CB calculus for concept subsumption in the DL ALCHOIQ+, a logic that extends ALC with role hierarchies, inverse roles, number restrictions, and nominals. To the best of our knowledge, ours is the first CB calculus for an NExpTime-complete DL. By using standard transformations, our calculus extends to SROIQ, which covers all of OWL 2 DL except for datatypes. A key feature of our calculus is its pay-as-you-go behaviour: the calculus is worst-case optimal for many well-known fragments of ALCHOIQ+, including ELH, Horn-ALCHOIQ+, and ALCHIQ+. Furthermore, the calculus is worst-case time exponential for the full logic ALCHOIQ+, except for cases where nominals, inverse roles, and number restrictions interact simultaneously, which are very rare in practice. Our calculus can also decide DL reasoning problems other than subsumption, such as ontology classification, instance retrieval, and realisation. We have implemented our calculus as an extension of the reasoner Sequoia, which previously supported ontology classification for SRIQ. Our implementation includes novel optimisation techniques that overcome some of the performance limitations affecting the previous version. We have carried out an empirical evaluation of our implementation which shows that the performance of Sequoia is competitive with other state-of-the-art systems. Our results also show that Sequoia nicely complements tableau-based reasoners, as it can more easily classify some ontologies. Thus, the calculus and implementation presented in this thesis provide an important addition to the repertoire of reasoning techniques and practical systems for expressive DLs.

Keywords: Description Logics
Consequence-Based Reasoning
Advisor: Horrocks, I
Cuenca Grau, B
Lewis acid activated electrophilic alcohols as alkylating agents for the stereoselective synthesis of heterocycles

Oxygen- and nitrogen-containing heterocycles are precursors of many valuable synthons in both synthetic and medicinal chemistry, which facilitate the synthesis of a broad array of natural products with biological activities such as antitumor, anthelmintic, antimalarial, antimicrobial, and antiprotozoal. I have found that the polar and protic bonding solvent hexafluoroisopropanol (HFIP) plays an important role in the stereocontolled synthesis of complex oxygen- and nitrogen-containing heterocycles from simple linear precursors.
My novel method for the stereoselective functionalisation of homoallyl alcohols/amines uses readily available allylic or benzyl alcohols as alkylating agents. Only one equivalent of the electrophilic partner is required for the cyclization process, and most of the products are obtained selectively as 2,3-trans diastereomers. Mechanistic studies reveal that Ti(OiPr)4 in HFIP plays a unique role in this reaction, as the in-situ formation of the complex Ti(Oi-Pr)2(OHFIP)2, was found to initiate the cyclization, leading to a formal 5-endo-trig cyclization that involves the stereoselective formation of both a C-C and a C-O/C-N bond in a single synthetic step. This method has been proven to be efficient and atom economic especially because only a catalytic amount of Ti(OiPr)4 is used and water is the only by product. Moreover, a wide range of the homoallyl alcohol or amine and electrophilic initiators show excellent compatibility with the system; finally, further functionalization of the products enables access to the synthetically useful heterocyclic molecules.

Keywords: heterocyclization
alkene functionalisation
HFIP
Advisor: Donohoe, T
The mechanisms of sleep restriction therapy for insomnia disorder

Insomnia is one of the most common health burdens world-wide. While pharmacological treatment is available, the current recommended first-line intervention is a multi-component psychological therapy called cognitive behavioural therapy for insomnia (CBT-I). Although randomised-controlled trials support the efficacy of CBT-I, its treatment mechanisms are not well understood.
This thesis sought to addresses this gap by focusing on the mechanisms of one of the chief components of CBT-I, Sleep restriction therapy (SRT). The first chapter introduces the subject matter and familiarises the reader with the complexity of insomnia. A systematic literature review then summarises theoretical and empirical evidence for putative mechanisms of SRT. This work concludes by introducing a new testable model of the SRT mechanisms-of-action followed by practice points and a research agenda (chapter two).
Chapter three describes the first randomised-controlled trial to systematically test the mechanisms of SRT. The restriction element of SRT was isolated by comparing a restricted time in bed with consistent bedtimes, showing treatment effects for insomnia severity and quality of life in favour of SRT. High-resolution measurements of sleep and daytime functioning further reveal improved sleep continuity, increased sleep pressure, diminished cognitive arousal, reduced sleep-related mentation and normalised subjective-objective sleep discrepancy, again in favour of SRT.
The fourth chapter extends this work by focusing on the impact of SRT on circadian phase. The presented study is the first to obtain dim light melatonin onset (DLMO) before and after two weeks of SRT and the first to conduct repeated measurements of subjective and objective vigilance. The study shows that SRT increases the phase angle between attempted sleep and DLMO. Furthermore, medium to large impairments are reported for subjective and objective vigilance measurements.
Finally, chapter five synthesises and critically reflects on results from the preceding chapters. Emphases are placed on methodological considerations, future research and implications for clinical practice.

Keywords: Sleep restriction therapy
Behavioural therapy
Sleep
Insomnia
Advisor: Kyle, S
Espie, C
The effect of light pollution on orientation in Manx shearwaters (Puffinus puffinus)

Humans introduce artificial light at night (ALAN) to the landscape for many reasons - to facilitate commuting to work, to improve their safety and to enhance the nocturnal aesthetics of places. The resulting change in light levels distorts a cue which living organisms use. A cue which, otherwise, has been predictable for billions of years. While some animals adjust to or even take advantage of ALAN, other species respond negatively, with populations suffering due to the presence of artificial light. In particular, burrow-nesting seabirds struggle with light pollution located near to their colony, with thousands of juvenile birds landing in lit-up areas during fledging season – termed ‘grounding’. The aim of this thesis is to investigate the behaviour of seabirds towards light pollution and, ultimately, to understand mechanistically the processes involved in grounding of seabirds. I explore, through observation and experimentation, the behavioural responses of an ALAN-susceptible seabird, the Manx shearwater (Puffinus puffinus) to artificial light. These experiments span the life-history stages of the Manx shearwater, from the orientation of juvenile shearwaters, before and during fledging, to the responses of breeding adults at the colony. I discover that the magnitude, but not direction, of behavioural reactions to artificial light is consistent across these life stages. At each life stage, reactions to ALAN appear to be related to the intensity and wavelength of light, the latter of which implies disruption of the visual system as a root cause of seabird grounding. Finally, in an applied study, I explore the effectiveness that updating old sodium pressure lamps with LEDs has had on shearwater grounding in a coastal town. Overall this thesis highlights the importance of understating the animals’ behaviour towards light pollution at different life stages and locations, contributing to the evidence base for mitigation measures that might be implemented in future.

Keywords: artificial light
Random Forest Classification
Fledging
seabirds
generalised additive models
ALAN
repulsion to light
magnetoreception
Oxford, Bodleian Library, Trinity College MS 93: a study and partial edition

This thesis examines the Middle English Declaracion on the Bible contained uniquely in Oxford, Bodleian Library, Trinity College MS 93. The text represents a fascinating and methodologically varied response to the Later Version of the Wycliffite Bible (LV), written very soon after its completion.
The thesis is in two parts. The opening chapter of Part I provides a brief overview of the manuscript and its contents, and considers evidence concerning how, where and when the manuscript might have been produced, proposing that the text was likely the product of a scholar or group of scholars working in Oxford in the 1390s. Chapter 2 develops the themes of the opening chapter by examining the evidence for authorship and intended audience, suggesting that the text was almost certainly produced by and for the scholarly community, but perhaps also to facilitate small group learning outside the university. Chapter 3 considers the context in which the Declaracion was compiled, suggesting that, while it would have been condemned by some, the text might have appealed to a relatively wide audience, possibly including some moderate opponents of scriptural translation. The chapter then considers the Declaracion from an ideological standpoint, suggesting that, though it is not polemical, its themes and concerns are consistent with Wycliffite ideas. Chapter 4 examines the Declaracion as a work which balances exegesis and paraphrase. Comparing the text to analogous Wycliffite works, it argues that the Declaracion is the most varied and experimental Wycliffite response to scripture. Chapter 5 provides an in-depth examination of the Declaracion’s use of sources. It argues that the main biblical source is LV, but that the level of engagement with other sources, notably the Vulgate, Lyra’s Postilla, the Magna, and the Glossa, has long been underestimated. A brief conclusion suggests that the Declaracion is an exceptional and intricate example of Wycliffite biblical scholarship deserving of further study. Part II includes a selection of text edited from the manuscript, along with Explanatory and Textual Notes, brief introductory material for each biblical book edited, and an Editorial Note. The edited text consists of all the books of Ezra (including 3 and 4 Esdras), the Penitential Psalms, Isaiah, the Gospels and Acts.

Advisor: Sutherland, A
Magnetic field effects on Drosophila melanogaster and avian cryptochromes

One of the major challenges in the field of sensory biology is to understand the process by which migratory songbirds sense the direction of the Earth’s magnetic field. This phenomenon, which has fascinated biologists for decades, has seen a growth of interest in the recent years. This poorly understood “sixth sense” has focused the attention of scientists from a variety of disciplines, from biology to theoretical physics. This multidisciplinarity is reflected in the work presented in this thesis, which spans animal behaviour, biochemistry, and spectroscopy. The common thread that links these experiments is the need to gain a better understanding of the radical pair mechanism of magnetoreception. According to this hypothesis, animals perceive magnetic fields by means of magnetically sensitive radicals formed by light-excitation of photosensitive flavoproteins called cryptochromes.
The first project, presented in Chapter 2, was an investigation of magnetic field effects on Drosophila melanogaster negative geotaxis. Two sets of equipment were built, experiments were conducted in a meticulously controlled environment, and strong statistical tests were applied to a large-scale dataset, but it proved impossible to reproduce the magnetic field effects reported in the literature.
The second project, presented in Chapter 3, was to test whether avian cryptochromes 1a and 1b could be the magnetoreceptor molecules. The proteins were expressed and purified and their binding affinity for flavin adenine dinucleotide, the chromophore responsible for light activation of the protein, was determined. It was concluded that cryptochromes 1a and 1b cannot be the magnetoreceptors, even though they have been considered ideal candidates for many years.
Finally, the third project, described in Chapter 4, was a comparative study of the spectroscopic properties and magnetic sensitivity of avian cryptochrome 4s from different species using Broadband Cavity-Enhanced Absorption Spectroscopy. This technique has the advantage of detecting the change in the optical absorbance of photo-induced radicals when the proteins are exposed to an external magnetic field. Striking differences were found between cryptochrome 4s from migratory and non-migratory birds.

Keywords: cryptochrome
magnetoreception
drosophila
Advisor: Hore, P
Mouritsen, H
Mapping networks of alpha-Synuclein proteostasis in cellular models of Parkinson's disease pathology

Parkinson’s disease (PD) is the second most common neurodegenerative disorder for which there is no disease-modifying therapy. Pathologically, the neuronal loss correlates with abnormal accumulation, aggregation and toxic gain of function of the small pre-synaptic protein alpha-Synuclein (aSyn). Whether neurons degrade aggregated aSyn or, only its soluble monomers, remains elusive. This thesis combines hypothesis-driven and unbiased genetic screens to identify pathways that specifically regulate the pathological fraction of aSyn. Initial data in cellular PD models show that intracellular aSyn inclusions that form over a period of days are conjugated to K48-linked ubiquitin (Ub) chains and that the pharmacological activation of the proteasome, but not of macroautophagy, reduces the relative levels of aggregated aSyn. Subsequent studies involving large-scale CRISPR/Cas9 knockout (KO) screens targeting the human ubiquitination machinery (1,247 genes: almost all E2s, E3s and DUBs) aim to map novel Ub effectors whose KO modify either pathological levels of aSyn or seeded aggregation. A major hit from these screens is ARIH1, a RBR type E3 ligase. ARIH1 KO increases the levels and aggregation of aSyn. ARIH1 interacts with aSyn in a neddylation-dependent manner suggesting that an ARIH1-cullin-RING ligase (ARIH1-CRL) regulates the ubiquitination of aggregated aSyn. Such complexes typically form K48-linked Ub chains. Accordingly, upon inhibition of NEDD8 transfer, the co-localisation of K48-linked Ub chains and the recruitment of p97 to aSyn inclusions is reduced. Furthermore, the knockdown of either p97 or ARIH1 prevents the clearance of aggregated aSyn by the proteasome. Thus, in this cell-based model, a Ub-dependent and p97-mediated proteasomal pathway appears to be an early attempt to clear misfolded proteins, that upon further validation may constitute a target for the development of novel therapeutics against aSyn aggregation and toxicity.

Keywords: seeded aSyn aggregation
Parkinson's disease
p97
ARIH1
CRISPR/Cas9 knockout screens
Advisor: Tofaris, G
D'Angiolella, V
The influence of lipid binding on the oligomeric state of membrane protein complexes

A large fraction of membrane proteins assemble into long-lived, non-covalent complexes, in a process called oligomerisation. Oligomerisation confers many benefits on membrane proteins, from increased thermal stability and access to a greater diversity of potential functions, to greater opportunities for cellular regulation. Despite this ubiquity and importance, membrane protein oligomerisation, and the role of the lipid environment in determining oligomeric state are poorly understood.

Non-denaturing mass spectrometry (MS) is a versatile method for probing both oligomeric state and protein-lipid interactions and hence is an ideal technique for investigating the relationship between these two phenomena. In this thesis, we report the application of a novel MS-based methodology to examine the oligomerisation of two dimeric membrane proteins: the bioamine transporter LeuT and the sugar transporter SemiSWEET. For SemiSWEET, we used both high-resolution and high-energy MS methods to characterise a small cohort of lipids which co-purify with the protein. Then, we performed subunit exchange experiments with dimeric SemiSWEET, establishing that it exists in a monomer-dimer equilibrium, the quaternary dynamics of which can be monitored in real time. Finally, by adding exogenous cardiolipin to SemiSWEET, we found that lipid binding shifts the monomer-dimer equilibrium towards the dimeric state.

For LeuT, we observed by MS that the dimeric form, but not the monomeric form, of the protein co-purifies with a larger cohort of lipids, including diacylphospholipids and cardiolipin. Using biochemical experiments and molecular dynamics simulations we ascertained that these lipids bind specifically at the LeuT dimer interface, via a bridging interaction between cardiolipin phosphate groups and cationic residues on the protein subunits, and that these protein-lipid interactions are essential for LeuT dimerisation.

Overall, the results show that the oligomeric states of membrane proteins can be strongly influenced by the local lipid environment, suggesting a potential mechanism for spatial and temporal regulation of protein function.

Advisor: Robinson, C