VictorJuiz/thesis huggingface dataset:
Biochemical studies on muscarinic cholinergic receptors

A novel solubilising agent (0.l% sodium cholate—lM NaCl) has been developed which will solubilise 10—30% of muscarinic cholinergic receptors from bovine caudate nucleus. Using the muscarinic antagonist quinuclidinyl benzilate (QNB), a single saturable binding component was found with an equilibrium constant of ZOOpM, approximately 10—fold higher than the membrane receptor and 4—fold higher than the ratio k-l/kl determined kinetically in the soluble material. This latter difference may indicate that the binding of QNB to the solubilised receptor is not a simple second—order process. Inhibition constants for a variety of muscarinic agonists and antagonists were 10 to 20—fold higher than in the membrane state and non-muscarinic ligands were without effect. The decrease in affinity was shown to be due to the presence of high salt.
Evidence was presented that the apparent increase in Hill coefficient for muscarinic agonist binding to soluble material was not due to a differential solubilisation of muscarinic receptors or to a conformational change of high to low affinity agonist sites during the solubilisation. Instead the Hill coefficient of the soluble material decreased as the percentage of total binding sites solubilised increased. The stability of receptor binding at different temperatures was shown to be dependent on the protein: cholate (w/w) ratio. Results from gel filtration, affinity chromotography and immunization studies are also reported. The results of this thesis are discussed in the light of the possible importance of phospholipids for receptor activity.

Keywords: biochemical
cholinergic
receptors
muscarinic
Advisor: Cuello, A
Retinitis pigmentosa GTPase regulator gene replacement - a potential treatment for x-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) is the most severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRORF15). Because of the relatively large prevalence and severe clinical phenotype, the responsible gene locus, RPGRORF15, is ideally suited for a tailored gene therapeutic approach. The overall objective of this work therefore was to develop an optimised gene replacement therapy for human XLRP disease.
The human RPGRORF15 coding sequence (cds) was codon optimised and both wildtype (wt) and codon optimised (co) sequences were spliced into a specific vector backbone designed for production of adeno-associated virus (AAV). In vitro studies in HEK293T, SHSY-5Y and 661W cell lines showed that the co construct features greater sequence stability and expression levels when compared to the wt construct.
After packaging the superior co construct into AAV8 capsids, subretinal injections were performed in a pilot study on relevant animal models with targeted disruption (Rpgr-/y) or natural mutation (C57BL/6JRd9/Boc) in the murine homologue Rpgr. Transgene expression and correct localisation to the physiological subcellular compartment could be demonstrated. A masked, randomised and controlled study in C57BL6/J wild type mice was able to show lack of toxic effects while application in Rpgr-/y and C57BL/6JRd9/Boc mice led to significant rescue as determined by electroretinography.
Overall the work in this thesis provides the basis for a clinical trial development to treat patients with XLRP due to RPGR mutations. A patent application to protect the intellectual property of this work has been filed on behalf of the University of Oxford.

Advisor: MacLaren, R
Synthesis of nitrogen containing heterocycles

This thesis is concerned with the development of synthetic methodology for the synthesis of nitrogen containing heterocycles, specifically 2,5-disubstituted pyrrolidines, 2,6-disubstituted piperidines and 3,5-disubstituted indolizidines.
Chapter 1 introduces current methods for the formation of 3,5-disubstituted indolizidines.
Chapter 2 describes studies into the asymmetric synthesis of both cis- and trans-2,5-disubstituted pyrrolidines via a diastereodivergent intramolecular aza-Michael reaction.
Chapter 3 describes attempts to apply the diastereodivergent intramolecular aza-Michael methodology, developed in chapter two, to the synthesis of cis- and trans-2,6-disubstituted piperidines. Investigations into extending the scope of the Davies' lithium amide conjugate addition methodology to the synthesis of polysubstituted piperidines is also described.
Chapter 4 describes the conversion of the cis- and trans-2,5-disubstituted pyrrolidines, synthesised in chapter two, into an indolizidine core. One of the diasteroisomers is subsequently elaborated into the natural product monomorine.
Chapter 5 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4.

Advisor: Davies, S
Optical Bloch equations for simulating trapped-ion qubits

This thesis describes work on numerical modelling of the 43Ca+ ion in a Paul trap using the optical Bloch equations. This is a challenging system to study, with many states involved in the internal dynamics. A major outcome is the development of a cooling scheme for the 146.09 gauss atomic clock transitions which makes use of a dark resonance. It is much more effective than methods that avoid coherent effects. The scheme is realised in experiment. Complicated fluorescence data is modelled very well, and predictions for the ion's motional temperature show good agreement with measured values. Data and fits for an ion that has been Doppler cooled below the Doppler limit are presented.
I describe GLOBES, a set of routines that simulates an arbitrary ion in the presence of an arbitrary system of laser beams using the optical Bloch equations. Techniques used to efficiently calculate the steady state, analyse fluorescence scans and solve time-dependent problems for small and large times are discussed. A new routine SILVER IMPER that leapfrogs over the initial dynamics to model the approach to the steady state is introduced.
Doppler cooling in 40Ca+ is analysed and two extensions made to the basic theory. The ‘excursion method’ of calculation takes account of the non-linear variation with velocity of the scattering rate. The ‘dynamic method’ allows for the fact that the ion may not be in equilibrium with the incident radiation during its oscillations, a necessity as the timescale of the external motion is of order the characteristic timescale of the internal motion for standard secular frequencies. This 'dynamic effect' is a general property of trapped ion systems and is also observed in a two-state system.
A two-variable fluorescence scan taken from a four-laser, five-level system in 40Ca+ is analysed. Techniques to fit large data sets and automatically resolve resonant features are demonstrated. A general treatment of resonant behaviour in three, four and five level pump/probe systems is used to describe the data. This is verified by a second, tailor-made set of scans.

Keywords: Efficient atomic simulation
Fitting fluorescence scans
Doppler cooling
Atomic clock transitions
Multi-level resonance dynamics
Optical Bloch equations
Advisor: Steane, A
Beyond public reason liberalism: moderate perfectionism

Should the state undertake the task of promoting the good life? Perfectionism is the idea that the state should promote the good life. Many philosophers have answered in the negative to the above question, so they reject perfectionism. This thesis aims to develop a moderate version of perfectionism, and seeks to defend it against several influential anti-perfectionist arguments, in particular the argument from public reason liberalism.
I begin by examining public reason liberalism. John Rawls, Gerald Gaus, Martha Nussbaum, and many other political philosophers endorse public reason liberalism. They believe that state coercion should be publicly justified, and that perfectionism cannot meet the requirements of public reason. I argue that public reason is the object of reasonable rejection, so it cannot be realised in actual politics through state intervention in a publicly justified way. In addition, I argue that respect for persons is not a reliable basis for public reason. Thus we have good reason to reject public reason liberalism.
Then I develop a moderate version of perfectionism. I contend that the state should promote the good life through supporting a wide variety of perfectionist goods, and that it should do so by using moderate measures and by appealing to perfectionist judgements of a moderate kind. Some anti-perfectionists consider that perfectionism would be unnecessary when a fair distribution of resources is realised. Yet a fair distribution of resources is not foreseeable. I propose several kinds of moderate perfectionist policies that are of great importance for any neo-liberal society where the distribution of resources is far from fair, and these policies are ultimately important for the good life, not only for remedying unfairness. Contrary to the positions of many liberal philosophers including Ronald Dworkin and Jeremy Waldron, I argue that moderate perfectionism should not be rejected on grounds of paternalism and unfairness to different conceptions of the good.

Advisor: Mcdermott, D
Alpha-synuclein in Parkinson's disease: molecular pathogenesis and development of genome engineering-based silencing approaches

The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Parkinson’s disease (PD) is one such example, in which the dopaminergic neurons that manifest pathology are embedded in an inaccessible region of the midbrain, the substantia nigra pars compacta, making it unfeasible to obtain samples of the affected region from living patients. Overabundance of alpha-synuclein, encoded by the SNCA gene, has long been implicated in the pathogenesis of PD. However, the precise mechanism by which overexpression of alpha-synuclein leads to the demise of dopaminergic neurons remains elusive. Neurons differentiated from PD patient-specific iPSCs carrying multiplications of the SNCA gene may provide a means to recapitulate molecular phenotypes of the disease in vitro. The application of CRISPR/Cas9 to mammalian systems is likewise revolutionizing the utilization of genome editing in the study of molecular contributors to the pathogenesis of numerous diseases, including PD. In this body of work, I have utilized the double-nicking CRISPR/Cas9 system to mediate site-specific mutagenesis of SNCA in PD patient-specific iPSCs harboring a triplication of the SNCA gene locus, resulting in isogenic cells which can be used to phenocopy normal alpha-synuclein expression from two alleles. I have further demonstrated the utility of nuclease null or "dead" Cas9, for transcriptional silencing of alpha-synuclein expression. Finally, I have used these systems to interrogate phenotypic outcomes of SNCA triplication, demonstrating that alpha-synuclein induces endoplasmic reticulum stress and aberrant activation of a highly conserved arm of the unfolded protein response. The tools generated in these studies can be applied in future efforts to dissect the contributions of alpha-synuclein overexpression to PD pathogenesis and as a platform for the discovery of disease-modifying therapeutic approaches for PD.

Keywords: Parkinson's disease
Induced pluripotent stem cells
Alpha-synuclein
CRISPR/Cas9
Unfolded protein response
Genome Engineering
Endoplasmic reticulum stress
Advisor: Wood, M
Reversible myocardial depression in critical illness

Introduction: Reversible myocardial depression (RMD) is a transient impairment of left ventricular systolic function. This has been described in sub populations of patients experiencing critical illness, but it is unclear if the epidemiological features are generalisable to the broader populations of critically ill adults. Previous work has identified that RMD is not benign and has been associated with a range of adverse sequelae such as arrhythmia, left ventricular outflow tract obstruction, and intra-ventricular thrombus. Consequently, studies were designed to determine the incidence, time course, and associated risk factors for the development of myocardial depression in the general population of adults experiencing critical illness.
Methods: Myocardial depression was defined as a decrease in left ventricular ejection fraction ≥5% from AICU baseline and was assessed using serial transthoracic echocardiography. Three studies were conducted - two prospective observational cohort studies and a retrospective analysis. The incidence, absolute decrease in ejection fraction, and time course of myocardial depression was described. Routinely available demographic and clinical variables were collected. These were then rationalised and trialled as candidate explanatory variables in a logistic regression model to predict the development of myocardial depression.
Results: The incidence of myocardial depression was between 16.3 – 34%, which occurred around day four of AICU admission. The median decline in LVEF at the onset of myocardial depression was between 6.5 – 14.7%, which progressed to between 10 – 17.5% at the nadir. Myocardial depression was not entirely reversible, with between 43.7 – 71.4% of participants demonstrating some degree of recovery, with LVEF improving between 13.3 – 20.2% across the studies. The probability of development of myocardial depression can be determined using five routinely collected variables, expressed as a factor; heart rate, systolic blood pressure, the presence of severe sepsis, cardiovascular organ dysfunction and sinus rhythm. Increasing systolic blood pressure, the presence of severe sepsis, and cardiovascular organ dysfunction were associated with increased risk. Increasing heart rate and the presence of sinus rhythm were associated with decreased risk. Model diagnostics indicated that the model was a good fit of the data. The model had a modest discriminating ability, with the area under the receiver operating characteristic curve = 0.69.
Conclusion: The incidence of myocardial depression was between 16.3 – 34% of participants, and usually developed around day four of AICU admission. The decreases in left ventricular ejection fraction were considerable and were not always reversible. Myocardial depression can be predicted using routinely collected haemodynamic and clinical variables that are available within the first 24 hours of AICU admission.

Keywords: critical illness
Intensive Care
Critical care
Takotsubo
neurogenic myocardium
cardiomyopathy
sepsis cardiomyopathy
myocardial depression
critical therapy
sepsis
Advisor: Young, J
Griffiths, J
Orme, R
Issues related to the economic analysis of genomic diagnostic technologies in the UK National Health Service: an exploration of methods

This thesis explores the empirical and methodological issues that arise when conducting economic analyses of genomic technologies in the UK National Health Service. The main focus of the thesis is the identification of the most appropriate economic evaluation approach to use in this context. Health economists are currently uncertain as to whether existing extra-welfarist approaches are sufficient to evaluate genomic technologies. Clarification is required because alternative methods, such as the welfarist approach, may lead to different adoption decisions, which will impact on population health.
The advantages and disadvantages of alternative approaches are demonstrated using a clinical case study: the use of genomic testing to guide treatment decisions in chronic lymphocytic leukaemia (CLL). Two forms of extra-welfarist economic evaluation are conducted – a cost-effectiveness analysis (CEA) and a cost-utility analysis (CUA) – along with a welfarist economic evaluation which took the form of a cost-benefit analysis (CBA), informed by a discrete choice experiment (DCE). These analyses consider several test-treatment strategies, reflecting different combinations of testing and the use of both existing therapies and new treatments such as ibrutinib. Information is also generated on patient preferences in this context, via the DCE, and the results of a survey are presented in which stakeholders are asked to consider which form of economic evaluation best captures the true costs and benefits of genomic testing in CLL.
The results of the CEA, CUA and CBA all indicate that the optimal strategy is to not adopt genomic testing in this context. The different approaches are then compared to assess their relative merits. The key finding – which is supported by the stakeholder survey – is that the extra-welfarist approach to economic evaluation is most appropriate when assessing genomic interventions. However, an evolution in methods is required to ensure that the results of these analyses provide decision-makers with sufficient information to facilitate the efficient allocation of scarce healthcare resources in this context.

Keywords: Leukemia
Chronic lymphocytic leukemia
Genetics
Economics
Chronic lymphocytic leukaemia
Health economics
Leukaemia
National Health Service
Genomics
Advisor: Wordsworth, S
Schuh, A
Factors associated with ventilatory failure in obesity

INTRODUCTION: Only a third of obese patients develop chronic ventilatory failure. This cross-sectional study assessed multiple factors potentially associated with chronic ventilatory failure.
MATERIALS/PATIENTS AND METHODS:
Participants had a body mass index (BMI) &GT;30 kg/m(2), with or without chronic ventilatory failure (awake arterial partial pressure of carbon dioxide >6 kPa or base excess (BE) =2 mmols/L). Factors investigated were grouped into domains: (1) obesity measures, (2) pulmonary function, (3) respiratory and non-respiratory muscle strength, (4) sleep study derivatives, (5) hypoxic and hypercapnic responses, and (6) some hormonal, nutritional and inflammatory measures.
RESULTS:
71 obese participants (52% male) were studied over 27 months, 52 (SD 9) years and BMI 47 (range 32-74) kg/m(2). The best univariate correlates of BE from each domain were: (1) dual-energy X-ray absorptiometry measurement of visceral fat (r=+0.50, p=0.001); (2) supine forced expiratory volume in 1 s (r=-0.40, p=0.001); (3) sniff maximum pressure (r=-0.28, p=0.02); (4) mean overnight arterial oxygen saturation (r=-0.50, p&LT;0.001); (5) ventilatory response to 15% O2 breathing (r=-0.28, p=0.02); and (6) vitamin D (r=-0.30, p=0.01). In multivariate analysis, only visceral fat and ventilatory response to hypoxia remained significant.
CONCLUSIONS:
We have confirmed that in the obese, BMI is a poor correlate of chronic ventilatory failure, and the best independent correlates are visceral fat and hypoxic ventilatory response.

Advisor: Stradling, J
Cyclic lateral loading of monopile foundations in cohesionless soils

The monopile is the dominant foundation type for offshore wind turbines, with current design guidance based on knowledge transferred from the oil and gas industry. Whilst there are some similarities between wind turbine and oil and gas pile design, there are also a number of key differences. Notably, offshore wind turbine monopiles are subjected to many cycles of large horizontal loads during their lifetime, whereas such loading conditions are not as prevalent in oil and gas design. As a result, the pile response due to this cyclic loading is poorly accounted for in current practice.
This thesis presents experimental and theoretical research, aimed at improving the understanding of the behaviour of rigid monopiles in cohesionless soils, when subjected to lateral cyclic loading. The experimental work involves laboratory floor model tests, scaled to represent a full-scale wind-turbine monopile. The test programme is designed to identify the key mechanisms driving pile response. It is divided into four main parts, investigating loading rate effect, hysteretic behaviour during unloading and reloading, as well as pile response to long-term single and multi-amplitude cyclic loads. In particular, the results show that the pile response conforms to the extended Masing rules, with permanent deformation accumulated during non-symmetric continuous cyclic loads. This ratcheting behaviour is characterised by two features: first, the ratcheting rate decreases with cycle number and depends on the cyclic load magnitude and secondly, the shape of the hysteresis loop tightens progressively, involving increased secant stiffness and decreased loop area. Tests investigating multi-amplitude loading scenarios prove that the interaction between these mechanisms describes the pile response. Finally, the continuous cyclic test results are interpreted using the p-y method combined with the Degradation Stiffness Model, and this shows a good fit to the observed pile deformation.
The key experimental findings are used for the development of a constitutive model that captures ratcheting while conforming to the observed Masing behaviour. The model, called HARM, is rigorous yet simple, and is framed within the hyperplasticity approach presented by Houlsby and Puzrin (2006). The model is tuned to capture the macro response of the pile under monotonic and cyclic loading, and is calibrated using the experimental data. The results demonstrate that HARM can successfully reproduce the main elements of the pile response with high accuracy. The method could easily be within common design approaches, such as the p-y method.

Keywords: civil engineering
offshore foundations
1-g model test
monopile
plasticity
laboratory testing
constitutive modelling
Advisor: Byrne, B
Networked creativity: ethnographic perspectives on chipmusic

This thesis examines creativity as manifested in an online and transnational network of musicians who compose chipmusic, a kind of electronic music characteristic of 1980s early home computers and videogame sounds. The primary argument is that creativity in chipmusic worlds is networked, meaning that it is dispersed across various activities that are labelled as creative: chipmusic-making, technology-hacking practices that underpin the music, digital cultural practices such as use of social media, online releases, crowdsourcing, staged and screened performances, and any other activity related to chipmusic. The thesis examines the ways in which networked creativity is mediated in the chipscene from an interdisciplinary methodological viewpoint informed by ethnomusicology, anthropology, and sociology.
Although the chipscene is geographically dispersed across more than thirty countries worldwide, the chipscene network is well-connected. Communication and music circulation practices of chipmusicians are enabled by the internet. This thesis primarily discusses chipmusic culture that suggests a rich context where creativity discourse is as intensely diverse as the chipscene itself, in which it is embedded.
In looking at the creative process and performance practices, I employ a mixed methods approach based on ethnographic research methods and social network analysis, to examine how intrinsic and extrinsic aspects of chipmusic-making, such as ideology, cultural values, network infrastructure, chiptune poetics and aesthetics, distribution of creative roles, authenticity, differentiation, genre dynamics, and intellectual property issues, shape creativity.