VictorJuiz/thesis huggingface dataset:
Cyclic amine functionalisation by engineered cytochrome P450BM3
Keywords: C-H activation at non-activated carbons
CYP102A1
cytochrome P450
amine functionalisation
protein engineering

Cytochrome P450 enzymes are known for their characteristic activity of C–H bond oxy-functionalisation whereby one oxygen atom of atmospheric dioxygen is inserted in an available carbon-hydrogen bond of their substrates, such as drugs and natural products, to form the alcohol derivative. Cytochrome P450BM3 from Bacillus megaterium has been extensively engineered to develop biocatalytic pathways to bioactive compounds due to its self-sufficiency, high expression level and promiscuity in substrate recognition. In this thesis, a high proportion of available carbon-hydrogen bonds in cyclic amines and lactams have been shown to be oxidised by engineered P450BM3 variants with high turnover and reasonable regioselectivity (Figure 1). A combination of substrate engineering via introducing substituents (methoxy and methyl), N-protecting groups (phenyl, benzyl, acyl, Boc, mesyl, tosyl, and isopropylsulfonyl) and auxiliary carbocycles (cyclopentane and cycloheptane), and protein engineering by site-directed and site-saturation mutagenesis at active site residues identified by screening with a 48 P450BM3 variant library of diverse substrate pocket topology and polarity, have enabled the oxidative diversification of these core motifs in numerous FDA-approved drugs. Unusual activities of P450BM3 – the Povarov reaction with N-phenyl cyclic amines and N1-C8' coupling (amination) of 6-methoxy-1,2,3,4-tetrahydroquinoline – were also discovered. Biotransformation in vivo and in vitro of these substrates reached 1 g/L/day with 20 mM substrate conversion at turnover numbers of up to 15,000. The research has provided the basis for developing alternative routes to hydroxy and multi-functionalised cyclic amines and lactams, introducing synthetic handles for functional group elaborations of such compounds which are frequently used in fragment-based drug discovery.
On adaptive behaviour and its neural basis: Insights from computational models and the mouse auditory cortex
Keywords: neuroscience
auditory neuroscience
learning
perceptual decision making

The processes of learning and decision making are at the core of intelligence. Although these phenomena have been studied for centuries, their nature and bi- ological implementation are still unclear. In this thesis, I approach these issues from multiple perspectives. First, I developed a novel infrastructure for performing large-scale behavioural and optogenetics experiments in rodents. Additionally, I performed validating experiments showing that auditory cortex is necessary for sound detection in mice.
To gain further insights into mouse behaviour, I developed a novel model of head-fixed mouse behaviour. I applied this model to data from mice performing a sound detection task and showed that it accurately describes behaviour. Addition- ally, I used this method to obtain a deeper understanding of behavioural strategies. Next, I extended this model to account for changing strategies over learning and demonstrated that it is able to infer changing strategies on a trial-by-trial basis. By applying it to behavioural learning data, I showed that mice exploit simplicity in task-structure to rapidly acquire an effective behavioural strategy.
Finally, I investigated the neural basis of sound-driven behavioural strategies by performing two-photon calcium imaging in layer 2/3 of the mouse auditory cortex during behaviour. Neuronal activity, which covaried with environmental events, importantly covaried with behavioural choice, potentially reflecting its role in driving behaviour. Modelling of population activity showed that choice-related activity is best accounted for by both changes in population gain and local func- tional connectivity. These data suggest that local computations in layer 2/3 of mouse auditory cortex contribute to sound-driven behaviour.
Together, this work has generated novel tools and represents an almost end- to-end study of the processes of learning and decision making, providing a deeper understanding of behaviour and its neural basis.
G.K. Chesterton, natural theology, and apologetics
Keywords: imagination
incarnation
wonder
apologetics
Newman
natural theology
Chesterton
paradox

In this dissertation I argue that G.K. Chesterton’s work is built around a distinctive natural theology, which gives coherence and substance to his thinking across the various genres in which he wrote. In chapter one I introduce his natural theology and explain why this central aspect of his work has been very largely neglected in Chesterton scholarship up till now. In chapter two I outline the basic structure of his natural theology. Then, in chapter three, I explore the ways in which he developed it, in the course of his apologetics, demonstrating that it is in fact at the heart of all his work, across the numerous different genres he worked in.
In chapter four, I analyse this natural theology in relation to his apologetics, showing how it shaped that apologetics, and was expressed in terms of apologetics. My analysis investigates both his overall strategies and also the linguistic expression of his ideas. Then, in chapter five, I consider the main exception to this rule, Chesterton’s treatment of the Incarnation. In chapter six, I review the arguments of the thesis, conclude that this understanding of Chesterton unites the seemingly disparate and unconnected aspects of his work in a coherent whole, and draw certain further conclusions.
This dissertation is original because the natural theology in Chesterton’s work has been very little studied. It is significant because Chesterton’s natural theology enabled him to pursue a very original and creative form of apologetics, and it has potential today to open up new possibilities in discussions between different religious and ideological views. Chesterton’s natural theology also included creative use of narrative theology and of the imagination in theology, among other innovations.
Investigating drug and vaccine resistance as a challenge for hepatitis B virus elimination in Africa
Keywords: HBV
Sustainable Development Goals
Hepatitis B virus
SDGs
RAMs
TDF
VEMs
NAs
TAF
Lamivudine
Resistance associated mutations
Vaccine espace mutations
3TC
Tenofovir
Nucleos(t)ide analogues

Background
The United Nations Sustainable Development Goals set out to eliminate viral hepatitis by the year 2030. However, hepatitis B virus (HBV) drug resistance-associated mutations (RAMs) and/or vaccine escape mutations (VEMs) are potential barriers to the achievement of this target. I have set out to describe the prevalence, distribution and impact of RAMs and VEMs in African populations infected with HBV, with a focus on cohorts in South Africa.
Methods
Through systematic literature reviews, analysis of published sequence data downloaded from GenBank, as well as sequence data generated from individuals infected with chronic HBV infection (CHB) recruited from South Africa, I have assessed the frequency, co-occurrence, distribution, evolution and transmission of HBV RAMs and VEMs. In addition, I have assessed the impact of HBV RAMs on HBV prevention of mother-to-child transmission (PMTCT) interventions through a cost-effectiveness analysis.
Results
In Africa, the most common RAM was M204I/V, occurring either alone or in combination with compensatory mutations, present among HIV/HBV coinfected and HBV monoinfected patients, and identified in both reportedly treatment-naïve and treatment-experienced adults. I also report the first case from Africa with strong evidence for HBV resistance to tenofovir (TFV), on the basis of high HBV DNA viral load (VL) despite 60 months of therapy, with a suppressed HIV VL, and a combination of sequence polymorphisms that have been reported in association with TFV resistance. Although screening and treatment of all women with CHB with TFV during pregnancy may be a cost-effective strategy for HBV PMTCT in Africa, increased selection of TFV RAMs could increase health-care costs.
Discussion
There is emerging evidence for polymorphisms that may reduce susceptibility to TFV. In order to drive progress towards 2030 elimination targets, there is a need for enhanced surveillance, scale-up of HBV prevention and treatment, and new therapeutic strategies that can bring about cure.
Defects in replicative DNA polymerases linked to cancer predisposition and tumour development
Keywords: ultramutator
polymerase kappa
polymerase epsilon
polymerase delta
cancer predisposition
hypermutator
germline
exonuclease
mutational signature
mutagenesis
POLE
mismatch repair

Somatic and germline mutations in the exonuclease domain of the replicative DNA polymerase epsilon (POLE-EDM) can contribute to cancer development. The ultramutator phenotype observed in the POLE-EDM tumours is thought to be due to alteration of the polymerase proofreading ability. Notably, while some POLE-EDM variants show a mutation rate similar to a 3’-5’ exonuclease-dead version, others have a mutation rate several orders of magnitude higher than exonuclease defective polymerases. Thus, it is likely that the mechanism of mutagenesis involves some processes other, or in addition to, loss of proofreading. As many POLE-EDM variants are unclassified, and to better assess their pathogenicity, we constructed in fission yeast carrying germline POLE-EDM variants identified in familial cases of colorectal cancer. Our combined in vitro and in vivo experimental approach confirmed that mutagenesis differs amongst POLE-EDM variants, and that this capability is not due to a simple loss of exonuclease activity. Although previous studies identified an unbalanced dNTP levels as the origin of hypermutator phenotypes, our data demonstrate that this does not apply in our study. Our exploration via deep-sequencing performed on hypermutated strains confirmed that concurrent POLE-EDM and mismatch repair (MMR) deficiency leads to a previously characterised specific mutational signature. Intriguingly, a second mutational signature related with coexisting polymerase delta alteration and MMR deficiency was also identified. Finally, further investigation via genetic interactions lead us to suspect the translesion synthesis polymerase polymerase kappa as a potential cause for the hypermutated phenotype and the mutational signatures observed. In summary, this work clarifies the tumour driver ability of germline POLE-EDMs, and has the potential to assist genetic counselling and POLE-EDM targeted treatments.
Biophysical characterisation of self-assembling nanoparticles
Keywords: lipid
viral like particles
membrane
peptide
biophysics
SMA
DIBMA
Lipodisq
peptide nanoparticles
styrene-maleic acid copolymer
PMA
nanoparticle
lipid-polymer nanoparticles

Biophysical characterisation of membrane proteins has been revolutionised through the development of membrane protein extraction with the use of a random co- polymer of styrene:maleic acid (SMA). Since the development of SMA, several novel polymers have been developed, in addition to modifications to the original SMA structure. However, a rigorous comparative characterisation of these polymers is missing.
This thesis presents the development of novel, high-throughput, solubilisation assays that can be used for the direct comparison of membrane solubilisation. These assays allow for the determination of optimal solubilisation conditions using the four polymers tested (SMA 3:1, SMA 2:1, DIBMA, and PMA). In addition, the effects of pH, ionic strength and divalent cation presence on the solubilisation of membranes by these polymers to form lipid-polymer nanoparticles was probed.
Characterisation of DMPC-polymer nanoparticles formed in ‘ideal’ conditions (pH 7.4, 100 mM NaCl) was performed to ascertain the lipid environment within these extracted bilayers. This showed that all lipid-polymer nanoparticles possess a higher lipid order than that observed in large unilamellar vesicles (LUVs) and further the thermal phase transition of the lipids within these nanoparticles is altered.
A previously discovered novel property of lipid-polymer nanoparticles, colli- sional lipid transfer, was also explored. Through the novel use of asymmetric membrane models it was found that lipid mixing occurs during membrane solubilisation. The rate of this mixing is variable according to the temperature and polymer used, and is a native property of lipid-polymer nanoparticles. This thesis goes on to apply this novel property for the specific titration of lipids into protein containing SMA-nanoparticles (Lipodisqs), using the well characterised cytochrome oxidase c dependence on cardiolipin as a test case. This is the first recorded instance of specific lipid:protein titration without the use of detergent or organic solvent addition.
Finally, the thesis explores the generation of a self-assembling peptide, ST1- QAA, which has been designed to spontaneously form viral-like particles for the purpose of gene transfection. It present a robust, optimised, purification protocol for synthesised ST1-QAA and biophysical characterisation of peptide self-assembly to form viral-like particles.
An analysis of axisymmetric receding contacts
Keywords: Orthogonality
Contact
Torsion
Shrink fit
Axisymmetric
Plane/anti-plane slip
Frictional slip

Bolted joints are one of the most used types of fixture in industry and when the contact area is small compared with the joint’s thickness, they represent an example of receding contacts, which are characterised by the reduction of the contact area when a loading is applied. Despite their ubiquity, axisymmetric receding contacts are not fully understand, and the current literature on the problem is limited either to plane problems or to frictionless cases.
This thesis presents an analysis of the frictional behaviour of fundamental axisymmetric receding contacts. The geometry of the problem is represented in an idealised format, through the contact of a semi-infinite thin layer of material and an elastic half-space. This allows for a precise solution to be found.
The methodology consists of assuming that the bodies are fully adhered and applying circular dislocation densities as nuclei of strain to correct the stresses when conditions of partial slip and separation are violated. Singular integral equations with Cauchy kernels are posed and solved in order to recover Signorini and orthogonality conditions.
In Part I, the methodology is implemented and verified through the solution of ring cracks in a half-space and the frictional behaviour of a shaft/hub system under partial slip subjected to an extraction force and to a torque.
Part II presents the solution for the thin layer resting against an elastic half-space under three different normal loadings: uniform pressure outside a disk (stamping loading); concentrated force; and uniform pressure over a disk.
It was shown that the receding contacts modelled present the basic properties that the contact snaps upon the application of any load, the interfacial tractions are proportional to the applied load, the extent of the contact size and slip zone are independent of the load, and the tractions are independent of the material parameters (namely the Poisson’s ratio).
Charge-carrier dynamics in hybrid metal halide perovskites for next-generation solar cells
Keywords: SnF2
ultrafast spectroscopy
tin-lead perovskites
upconversion spectroscopy
solar cells
lead-tin perovskites
time-resolved photoluminescence
charge-carrier cooling
charge-carrier dynamics
photovoltaics

Hybrid metal halide perovskites have emerged over the last decade as highly promising materials for use in solar cells. Their advantages include low production costs, and rapidly increasing efficiencies to compete with those of commercially dominant silicon cells. Recently, increasing attention has been devoted to the use of perovskites in next-generation photovoltaic devices in order to overcome the efficiency limit for conventional single-junction cells. Work is still needed to identify the most promising perovskite materials for these applications, and to fully understand their relevant properties. This thesis contributes to both goals.
The challenge of identifying the true timescale of charge-carrier cooling in metal halide perovskites, and whether certain compositions in fact show exceptionally slow cooling, is addressed. This is central to assessing the viability of hot-carrier extraction in perovskite solar cells. Key sources of error in previous approaches to determine charge-carrier temperature, which have produced an orders-of-magnitude range of reported cooling times, are identified. An improved model, which accounts for the full photoluminescence lineshape and enables a consistent approach that can be applied to studies across different perovskite compositions, is then described.
Applying this improved model, charge-carrier cooling in FASnI3 is studied, revealing that observations of a secondary regime of slow charge-carrier cooling are in fact attributable to electronic relaxation, state-filling and recombination in this energetically disordered material. This result highlights that careful analysis of charge-carrier cooling dynamics is vital to assessing the realistic prospects for hot-carrier solar cells. Photoluminescence polarisation memory in FASnI3 is also examined, finding no persistent polarisation anisotropy within 270 fs. This points to a need for further study to identify the factors that lead such memory to arise from structural changes in certain perovskite compositions.
Finally, the effects of the SnF2 additive commonly used to improve material quality of tin-containing perovskites are explored for FA0.83Cs0.17SnxPb1-xI3 across the lead-tin range. Significant beneficial effects from the suppression of tin oxidation and associated background hole doping, including reduced energetic disorder and increased charge-carrier mobilities and lifetimes, are found with as little as 1 mol% SnF2 added. The larger amounts of additive that have commonly been used are found to contribute to detrimental non-radiative recombination. These results, unravelling the impacts of SnF2 on both structural and optoelectronic properties, provide a foundation for control over the properties of mixed tin-lead perovskites for use as low-bandgap absorbers in all-perovskite tandem cells.
Sharing infectious disease related data and biological samples from low resource settings: perspectives on ethics and governance
Keywords: Global health ethics
Sample sharing
Infectious disease
Data sharing

The sharing of data and samples are increasingly promoted as crucial to the timely response to infectious disease outbreaks by bodies such as the World Health Organization. Data and sample sharing are often discussed as an ethical imperative that should occur in order to better understand diseases, and facilitate the development of timely innovations to respond to infectious diseases. However, data and sample sharing present a number of macro- and micro-level ethical considerations which need to be reconciled in practice. These include issues of consent, privacy, and the distribution of benefits. Furthermore, the urgent need to share and respond to infectious disease emergencies may also strain certain areas of best practice. Crucially, the perspectives of stakeholders involved in sharing data and samples in infectious disease contexts have not been well documented.
This study aims to address the lack of emphasis on stakeholders’ views by foregrounding their perspectives and practical experiences of ethical issues and views on the role of ethics governance. To achieve these aims, I employed a qualitative methodology, including participant observation and semi-structured interviews to capture the experiences of 28 key stakeholders who were purposively sampled to represent a range of different roles and positions. The primary focus of this research was on gaining insights about sharing data and samples from low- and middle-income countries, given the disproportionate burden of infectious diseases in these settings.
The findings, and subsequent analysis, illustrate that data and sample sharing in the infectious disease context take place within a complex moral world. The importance of sharing data, and the optimal use of samples, was underscored by interviewees in this study. However, the nature of the data, samples, disease and the modes of sharing were all reported to have particular ethical implications. A further finding is that sharing often takes place between a diverse set of individuals and organisations, who can be unfamiliar with one another and who at times have differing priorities, and make divergent arguments about how sharing ought to take place for it to be considered ethical. Outbreaks were noted to present particular challenges. Timeliness of data and sample sharing was considered ethically important in outbreak situations, but there were otherwise conflicting views on whether and how the urgent nature of the response ought to change practice. The pressures of an outbreak response also present practical limitations to meeting the usual requirements of good practice, such as obtaining informed consent.
The findings show that aspects of practice that have ethical implications for data and sample sharing in the infectious disease context are insufficiently captured in current guidance documents.
Medicines for London : the trade, regulation and lifecycle of London apothecaries, c. 1610-c. 1670
Keywords: medicine
regulation
pharmacists
trade
London
England
lifecycle
medicines
apothecaries

Apothecaries formed a crucial part of early modern London's resources against the disorders and diseases that assailed its population, supplying medicines and a range of other goods and services - some medical, some not - to medical practitioners and patients alike. Despite their importance, this essential element in London's service economy has largely been neglected by historians.
This thesis is a study of the regulation, lifecycle and work of apothecaries from the beginning of the seventeenth century to around 1670. It demonstrates the importance of the personal, political and commercial histories of seventeen-century apothecaries to an understanding of medicine and work in early modern London. The thesis begins with a consideration of the politics and policing of medicine in seventeenth century London. The apothecaries' guild, the Society of Apothecaries, was established in 1617. The process by which apothecaries came to be separated from the Grocers' Company is analysed in Chapter Two. The Society's fortunes over the next five decades, in resisting the efforts of the College of Physicians to increase its authority over apothecaries are considered in Chapter Three. Two aspects of the internal operation of the company are discussed next. Chapter Four examines the extensive - but moderate - control of the craft by the Society, influenced by legal norms and the difficulties of assessing the quality of medicines. Chapter Five focuses on apothecaries' individual and collective identities through their lifecycle, backgrounds, corporate careers, and, particularly, apprenticeships and apprentice-taking. Chapters Six to Eight explore several aspects of the trade of apothecaries and the economy of medicine in early modern London. The complex ways medicine were sourced, manufactured and supplied; the interactions of medical practitioners, as associates, partners or competitors; the customers of apothecaries; and the material culture of apothecaries' shops, and its relation to the problem of reassuring consumers about the medicines they purchased.