VictorJuiz/thesis huggingface dataset:
Eutherian-specific homeobox genes in the preimplantation embryo; function, evolution, and applications.

This thesis explores the evolution, function, and possible applications of a group of lineage-specific genes known as the Eutherian Totipotent Cell Homeobox (ETCHbox) genes. The ETCHbox genes duplicated from a highly conserved vertebrate eye-specific homeobox gene called Cone-rod homeobox (CRX) in the lineage leading to eutherian mammals. The ETCHbox genes are unusual in many respects. Firstly, they are eutherian-specific, meaning that they are only present in the genomes of one group of mammals and not in marsupials or monotremes, which only have CRX. Secondly, they are highly variable in copy number and sequence, both compared to the “parental” gene from which they diverged, and between different species of eutherian mammal. Thirdly, their expression is limited to a few stages in the very early preimplantation embryo, after which they are not known to be expressed in any tissue. How can duplicates of a gene conserved in sequence and in function become so divergent so quickly, and what can their role be?
I explore how the extreme divergence has evolved in eutherians, what their new functions could be, and whether their specificity for the very early embryo can be applied to the field of regenerative biology. I initially focus on the mouse genome, using ectopic expression to reveal unexpected functional overlaps between human and mouse paralogues, not orthologues, and hypothesise inferred ETCHbox function. I use sequence analysis and ectopic expression to explore the evolution of ETCHbox genes from CRX using comparisons of eutherian and marsupial homeobox genes and embryology, finding evidence for a nuanced subfunctionalisation occurring on the eutherian lineage between CRX and ETCHbox. I then continue the evolutionary approach specifically within the order Rodentia, where I find that stepwise gene losses and sequence divergences occurred. Finally, I examine the effect of ETCHbox genes on pluripotency before discussing a model for how ETCHbox complexities could evolve. Overall, I have used a blend of computational and experimental techniques to increase our understanding of ETCHbox gene evolution and function, before exploring potential applications.

Advisor: Holland, P
PARP1 resolves DNA replication defects in PCNA-associated DNA repair disorder

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair. The toroidal structure of a PCNA trimer makes it a sliding clamp protein, which utilizes its interdomain connecting loop (IDCL) interacting with PIP-box containing proteins to recruit these clients to sites of DNA replication to enable their functions. In 2014, the Crosby and Green laboratories reported a novel DNA repair disorder with clinical features including short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, which results from a homozygous missense (p.Ser228Ile) alteration of PCNA so it is named PCNA-associated DNA repair disorder (PARD).
Previous structural studies have shown that the S228I alteration of PCNA results in a dramatic conformational change of the IDCL, and biochemical studies indicated a profound perturbation of PCNA's interaction with many of its interaction partners, including FEN1, LIG1, Cdt1, DNMT1 and XPG. Patient-derived cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. However, no evidence for DNA replication defects has been found in these patient-derived cells. The aim of this thesis was to determine cellular pathways that are affected by the S228I alteration and to uncover mechanisms by which PCNAS228I cells tolerate the perturbed PCNA interactions in specific pathways.
An isogenic PCNAS228I knock-in hTERT RPE-1 cell line was generated using CRISPR-Cas9 technique. Phenotypes observed in patient-derived cells were recapitulated in the knock-in cell line. By applying survival assays with treatment of different chemical agents, we showed most DNA repair pathways unaffected, except that PCNAS228I cells were more sensitive to treatment with the PARP inhibitor olaparib than WT controls.
By comet assay as well as immunostaining of γH2AX, I showed that olaparib induced more DNA damage in cells with the PCNAS228I alteration, and FACS analysis showed perturbed cell cycle progression after PARP inhibition. By using an inhibitor of PARG, I detected high levels of PAR chains being formed at replication forks in PCNAS228I cells. Thus I proposed that the PCNAS228I mutation results in abnormal interactions between PCNA and key enzymes needed for normal Okazaki fragment maturation at replication forks. The resultant persistent single-strand breaks are processed via a back-up, PARP-dependent mechanism to ensure completion of lagging strand synthesis.
Overall, these data have implications both for the etiology of PCNA-associated repair disorder as well as suggesting novel circumstances in which PARP-inhibition might be of use in the cancer clinic.

Keywords: Okazaki fragments
PARP1
DNA repair
PCNA
DNA replication
Advisor: Green, C
Towards practical applications of quantum optics

This DPhil thesis presents two key works towards practical applications of quantum optics. Both works are novel and achieve competitive state-of-the-art results.
Today's most widely used method of encoding quantum information in optical qubits is the dual-rail basis, often carried out through the polarisation of a single photon. On the other hand, many stationary carriers of quantum information | such as atoms | couple to light via the single-rail encoding in which the qubit is encoded in the number of photons. As such, interconversion between the two encodings is paramount in order to achieve cohesive quantum networks. In the first part of this thesis, we demonstrate this by generating a hybrid entangled resource between the two encodings and using it to teleport a dual-rail qubit onto its singlerail counterpart. Our key results yield an average fidelity of F = (92:8±2:2)% for the teleportation and F = (89:7 ± 2:1)% for entanglement swapping, thus confirming the applicability of this scheme towards a real-world implementation. This work completes the set of tools necessary for the interconversion between the three primary encodings of a qubit in the optical field: single-rail, dual-rail and continuous-variable.
A remarkable aspect of quantum theory is that certain measurement outcomes are entirely unpredictable to all possible observers. Such quantum events can be harnessed to generate numbers whose randomness is asserted based upon the underlying physical processes. In the second part of this thesis, we formally introduce and experimentally demonstrate an ultrafast optical quantum randomness generator that uses a totally untrusted photonic source and whose idea we have patented. While considering completely general quantum attacks, we certify randomness at a rate of 1:1 Gbps with a rigorous security parameter of 10-20. Our security proof is entirely composable, thereby allowing the generated randomness to be utilised for arbitrary applications in cryptography and beyond.

Keywords: Quantum Random Number Generator
Quantum Teleportation
Advisor: Lvovsky, A
Neural network models of hierarchical map-based planning in the brain

Many researchers have tried to model how model-based behaviour is produced in the brain, and specifically how environmental knowledge is learned and used in the form of cognitive maps. Previous work has proposed various cellular substrates that might encode a cognitive map, as well as several possible mechanisms for self-organising these maps. Current models also agree to some extent on the planning mechanism that produces goal-directed actions using a cognitive map, although they propose subtly different implementations of this planning mechanism, and a variety of mechanisms for extending it. We felt that this previous work was limited in several important ways: there was little consensus on how these cognitive maps were represented and how they were formed, the planning mechanism that was used seemed to be inherently limited to performing relatively simple tasks, and there had been little consideration of how these mechanisms would scale up beyond laboratory tasks.
This thesis makes several important advances. Firstly, the planning mechanism that is used by the majority of current neural models on this subject propagates a decaying signal through the environment to create a gradient that points towards the goal. However, this decaying signal will eventually decay to nothing, limiting the scale and complexity of tasks that can be solved in this manner. Based on the idea, originally proposed by Ponulak et al. 2013, that a wavefront propagating from a goal state can carry information about the direction of that goal state, we propose a novel planning mechanism that does not require decaying activity.
Unlike the model proposed by Ponulak et al., the mechanism that we propose is: able to work in a state-action map, able to output explicit actions to move towards the goal, able to perform planning without altering synaptic weights, able to plan in nondeterministic environments, and able to interface with a hierarchical mechanism. We also propose several ways in which a network which implements this planning mechanism can self-organise during unsupervised exploration. We demonstrate that the neural substrate for the cognitive map, the mechanisms required to plan using this map and the cognitive map itself can all be learned using local Hebbian learning, in an unsupervised manner.We compare and contrast these mechanisms in terms of both their effectiveness and their biological plausibility.
Finally, we extend the model with a hierarchical planning mechanism: a layer of cells that can identify frequently-used sequences of actions and reuse them to significantly increase the efficiency of planning. This increase in efficiency is largest for tasks with longer, more complex solutions. We speculate that this may explain the apparent ability of humans and animals to perform model-based planning on both small scales (laboratory experiments) and on large scales (outdoor travel) without a noticeable loss of efficiency.

Keywords: MDP
Cognitive Maps
Navigation
Hierarchical Planning
Advisor: Stringer, S
Mechanism of action studies of the utrophin modulator ezutromid for the treatment of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle wasting disease arising from mutations in the dystrophin gene, affecting about 1 in 5000 boys. Whilst there is currently no available cure for DMD, a number of therapeutic strategies are under development. One such therapy aims to promote expression of utrophin, an autosomal paralogue of dystrophin. Utrophin upregulation rescues the dystrophic phenotype in disease model mice. A novel small molecule utrophin modulator, ezutromid (Summit Therapeutics; formerly SMT C1100), which was identified using a phenotypic screen, progressed to Phase II clinical trials in DMD patients. Interim 24-week data demonstrated reduced muscle fibre damage and increased levels of utrophin, providing the first evidence of ezutromid target engagement and proof of mechanism. However, these effects were not seen after the full 48 weeks of the trial. This work aims to define the mechanism of ezutromid in order to help understand the trial results, and to aid development of new generations of utrophin modulators.
Target identification through affinity based protein profiling (AfBPP) has been carried out in this work alongside phenotypic profiling studies. AfBPP requires bioactive analogues suitable for affinity purification. Biotinylated and photoaffinity-labelled analogues of ezutromid designed to be positive and negative control probes have been synthesised and bioactivity confirmed. These probes were used in AfBPP experiments with disease model mouse myoblasts followed by LC-MS/MS. The results from this experiment and phenotype profiling converged on the aryl hydrocarbon receptor (AhR) as a target of ezutromid. Target validation studies demonstrated that ezutromid binds AhR potently, behaving as an AhR antagonist. Moreover, reported AhR antagonists also upregulate utrophin, showing that this pathway could be exploited in future DMD therapies.

Keywords: Proteomics
Target identification
Affinity-based Protein Profiling
Mechanism of action
Photoaffinity labelling
Duchenne muscular dystrophy
Advisor: Russell, A
In silico tools to aid medicinal chemistry: optimising bromodomain inhibitors

Development of ligands for bromodomains has expanded our understanding of gene regulation and epigenetic links to disease. These acetylated lysine ‘reader’ domains are found within larger macromolecules that are involved in gene transcription and degradation. This thesis looks to employ a range of computational tools to compliment the development of inhibitors to a selection bromodomain targets. The first target was TRIM33b, an E3 ubiquitin ligase that also contains a methylated lysine reader, known as a plant homeodomain. After identifying putative binding locations for ligands identified in a previously performed high-throughput screen through docking and molecular dynamics studies, one ligand was taken forward for further optimisation. Subsequent free energy calculations on both the ligand and water molecules were used to justify a proposed binding model, in the absence of a co-crystal structure. This model identified a stable water molecule in the ZA channel and was used to design the highest affinity ligands known for the TRIM33b bromodomain. The model was also scrutinised to understand why vector elaborations above the binding site did not yield a higher affinity probe molecule.
The second set of targets were bromodomains within parasites responsible for a range of tropical diseases. In the absence of co-crystal structures, homology models and molecular dynamics studies were used to design binding models for a set of existing bromodomain ligands. These models were used to aid development of tool compounds, based on the BRD4(1)/PLK1 inhibitor BI-2536, and propose future modifications to increase affinity for parasite bromodomains. Two of the homology models were later confirmed by a subsequent co-crystal structure. Finally, several computational methods are employed to understand the trends in binding affinity for a range of ligands against the CREBBP bromodomain and BRD4(1). These studies highlighted the importance of an intramolecular hydrogen bond in analogues of OXFBD02, the role of a ZA channel water molecule on imposing selectivity for CREBBP ligands, and supported the role of a proposed cation-p interaction.

Advisor: Biggin, P
Conway, S
Morris, G
Investigating the links between N2 fixation and metabolism in rhizobia

In this thesis the metabolic flow of carbon and electrons enabling N2 fixation in the agriculturally relevant model organisms Rhizobium leguminosarum and Azorhizobium caulinodans was investigated. Building upon functional genomic data-sets including transcriptomics, metabolomics and mutagenic screens, the core metabolic reactions needed for dicarboxylate ORS571 and RL3841. Isocitrate dehydrogenase was found to be essential for N2-fixation and operation of the TCA-cycle in RL3841, contradicting previously published INSeq data. This was due to TA insertions in gene termini misleading the Hidden Markov Model used to asses INSeq data. As a result of this, new bioinformatic pipelines have been developed to avoid such errors. The redox sink poly-3-hydroxy butyrate (PHB) was shown to be non-essential for N2 fixation in ORS571. Previously published results showing PHB synthesis to be essential are actually due to missregulation of phaR translation in phaC mutants used to block PHB synthesis, rather than the loss of PHB as a redox store. This bring ORS571 in line with other species of rhizobia, where PHB defective mutants retained their ability to fix N2. Two alternative metabolic pathways, the malonate shunt in ORS571 and the isocitrate shunt in RL3841 have both been investigated and shown to be inoperative at biologically relevant levels in their respective organisms. In ORS571, the entirety of the carbon flux between succinate and acetyl-CoA has been demonstrated to proceed via NAD+ dependent malic enzyme (DME) and phosphoenolpyruvate carboxykinase, allowing existing models of carbon flux to be revised to remove the erroneous malonate shunt. In addition, DME has been demonstrated to be essential for N2 fixation, but dispensable for growth on succinate, indicating a disconnect between the requirements of N2 fixation and dicarboxylate metabolism.

Keywords: Metabolism
Rhizobia
Advisor: Poole, P
Essays in applied microeconomics

This thesis consists of three self-contained chapters on topics in applied microeconomics. In Chapter 1 "The Impact of Economic Shocks on Wage Dynamics in Indonesia" I study the impact of negative productivity shocks on agricultural wages in Indonesia. I study whether and when downward nominal wage rigidity prevents labor market adjustments to negative productivity shocks. I provide evidence that the cost to the economy and to agricultural households caused by downward wage rigidities are limited in magnitude. I use transitory labor demand shocks in agricultural labor markets induced by plausibly exogenous rainfall uctuations to show that downward adjustment of nominal wages is a function of the cost of maintaining the nominal wage at the prevailing level. I also develop a theoretical framework with reference-dependent utility over past wages, in line with fairness norms, that is consistent with the observed wage rigidities.
In Chapter 2 "Income Shocks and Suicides: Causal Evidence From Indonesia" (coauthored with Cornelius Christian and Chris Roth) we explore the role of income shocks on the prevalence of suicides in Indonesia. We find that a cash transfer program reduces the yearly suicide rate by 0.36 per 100,000 people, corresponding to an 18 percent decrease. To come to this conclusion, we use a difference-in-differences approach, exploiting a cash transfer's nationwide roll-out, and corroborate the findings using a randomized experiment. Agricultural productivity shocks similar to those in Chapter 1 also affect the suicide rate. Encouragingly, the cash transfer program reduces the causal impact of negative agricultural productivity shocks, suggesting an important role for policy interventions. We only observe these effects for households engaged in agricultural production. Finally, we provide evidence for depression as a psychological mechanism through which income shocks affect the suicide rate.
In Chapter 3 "Political Activists as Free-Riders: Evidence from a Natural Field Experiment" (coauthored with Anselm Hager, Johannes Hermle, and Chris Roth) we explore how a citizen's decision to participate in political activism depends on the participation of others. We examine this core question of collective action in a natural field experiment in collaboration with a major European party during a recent national election. In a seemingly unrelated party survey, we randomly assign canvassers to true information about the canvassing intentions of their peers. We find that treated canvassers significantly reduce both their canvassing intentions and behavior when learning that their peers participate more in canvassing than previously believed.

Advisor: Quintana-Domeque, C
Quinn, S
Improving the measurement and treatment of infant pain

Infants undergo numerous medically necessary painful procedures while in hospital, and this early life pain can lead to adverse effects. A range of pain relief interventions are available for use in neonatal units, including pharmacological analgesics, and non- pharmacological comfort measures. However, the literature regarding the efficacy of many of these pain relief interventions is inconclusive and inconsistent, and infant pain is consequently undertreated.
To assess infant pain, behavioural and physiological surrogate measures are utilised in the absence of verbal report. However, as these measures are not direct measures of pain, and can be influenced by other factors such as procedure-related distress, their use can lead to inconsistent conclusions regarding the efficacy of pain relief interventions. The perception of pain manifests in the brain, therefore it is plausible that measures of brain activity can provide specific and objective indications of the infant pain experience, and that these measures can be used to assess the efficacy of pain relief interventions. In this thesis, noxious-evoked brain activity is measured and used to investigate the effect of a pharmacological analgesic, paracetamol, and a non- pharmacological pain relief intervention, gentle touch targeted towards C-tactile (CT) fibres.
Noxious-evoked brain activity has been well characterised following medically required heel lances and experimental noxious stimuli, but brain activity evoked by alternative clinically relevant stimuli must also be characterised to assess the efficacy of a wider range of pain relief interventions for these procedures. In this thesis, brain activity evoked by immunisation is characterised, enabling the analgesic efficacy of paracetamol to be tested during routine infant immunisations.
Between-subject comparisons are often necessary when investigating the efficacy of pain relief interventions for medical procedures. However, there is great individual variability in nociceptive sensitivity, and controlling for this variability could enable better between-group comparisons. In the last section of this thesis, infant nociceptive sensitivity is measured and accounted for, in order to explore how this approach can be used to assess intervention efficacy, using the example of gentle touch targeted towards CT fibres.
Overall, the aims of the research described in this thesis are to advance knowledge regarding how infants respond to noxious clinical procedures, how pain relief interventions can modulate these responses, and how individual variability can be accounted for to enable better assessment of intervention efficacy. Research in the area of infant pain management is critical to inform clinical practise and improve the treatment of infant pain.

Keywords: brain imaging
paracetamol
electroencephalography
touch
neonate
CT fibre
pain relief
pain
newborn infant
Advisor: Slater, R
Hartley, C