VictorJuiz/thesis huggingface dataset:
The activities of various antimalarial drugs on Plasmodium falciparum isolates in Kilifi Kenya and studies on mechanisms of resistance

Drug resistance is a significant challenge in the fight against malaria. Importantly, reduced efficacy has been reported against artemether (ATM)/Lumefantrine (LM) (LM-ATM), amodiaquine (AQ)/artesunate (AS) (AQ-AS), two important combination treatment regimens in Africa, and against piperaquine (PQ), a drug which has been evaluated as a potential alternative in Africa, in combination with dihydroarteminisin (DHA). Chloroquine (CQ) resistance in P.falciparum is associated with two main transporters PfCRT and PfMDR1. I investigated the mechanisms of resistance to PQ, LM and AQ, with the overall goal of identifying molecular markers that can be used to track resistance. I used CQ as a reference.
The key antimalarial drugs were highly active against clinical isolates from Kilifi, Kenya with median inhibitory concentrations (IC50s) of <5nM for DHA and <55 nM for CQ, AQ, PQ, LM and DEAQ (desethylamodiaquine, the active metabolite of AQ). pfcrt-76 and pfmdr1-86 mutations were associated with AQ, DEAQ and LM but not DHA or PQ activity. Interestingly, > 20% of analysed isolates had decreased susceptibility to LM (IC50 >100nM); these isolates were the most susceptible to CQ and carried wild type genotypes at pfcrt-76 and pfmdr1-86. I observed that CQ resistance had been declining in Kilifi since 1993 (prior to CQ withdrawal) to 2006 (7 years after its withdrawal), similar to observations in Malawi. My results support the hypothesis that susceptibility to antimalarial drugs returns when drug pressure is removed, and suggest that the use of LM-ATM may hasten the return of CQ susceptibility.
Continued monitoring of drug susceptibility is crucial. pfcrt-76 and pfmdr1-86 may be useful molecular markers of LM-ATM efficacy in Kilifi and other African sites. Using a microarray approach, I identified additional genes (including various transporters) that may contribute to LM resistance. I recommend further studies to clarify the exact roles of the identified genes.

Keywords: genomics
molecular markers
public health
drug resistance
malaria
pharmacology
Advisor: Marsh, K
Nzila, A
Cardiosphere-derived stem cell culture, characterisation and labelling for in vivo testing in the infarcted heart

Cardiac stem cells (CSCs), isolated from heart tissue explants and expanded via the formation of cardiospheres (Csp), are a promising candidate for cell therapy to prevent heart failure following myocardial infarction. To allow early administration to patients, isolation and expansion of CSCs must be performed in the shortest time possible. Hence, this project aimed to optimize culture conditions and characterize the cardiac explant-derived cells (EDCs), Csp and Csp-derived cells (CDCs) produced. Rat neonatal EDCs contained 4-7% c-kit+ cells, measured using flow cytometry. Optimal Csp growth conditions were determined, such that plating 3 x 10^4 EDCs per well of a 24-well plate coated with 16.7 µg/ml poly-D-lysine, in CGM containing 7% serum, improved Csp production and generated 1.5 x 10^7 CDCs in 16 days, a sufficient number for cell therapy. The CDCs expressed the stemness markers; c-kit, Oct3/4, SOX2, and Klf-4, and the cardiac differentiation markers; GATA4 and Nkx2.5. The therapeutic effect of CDCs may be limited by the low, 3 ± 0.1%, c-kit+ cell numbers. To increase c-kit+ cells in CDCs, an alternate culture method for Csp and different extracellular matrices (ECM) for cell expansion were tested. The hanging drop culture method produced Csp with higher levels of c-kit+ cells (9 ± 2%) than poly-D-lysine-coated and low-bind culture dishes. Of five ECM tested, collagen IV was found to enhance EDC migration and CDC proliferation, and produced 11 ± 0.4% c-kit+ cells, with Csp cultured in hanging drops. Intramyocardial injection of CDCs improved left ventricular ejection fractions of infarcted rat hearts by 9% and prevented the peri-infarct wall from thinning, measured in vivo using MRI over 16 weeks. To improve cell tracking using MRI, two MR positive contrast agents, gadolinium-DTPA and gadonanotubes were tested. Gd-DTPA had low sensitivity after labelling (1.4 x 10^5 cells/mm2); whereas gadonanotubes did not provide positive contrast at 11.7 T. Thus, neither contrast agent could be used for cell tracking using high magnetic field. In conclusion, CDCs were an effective source of stem cells that could be used for heart repair, although cells could not be tracked using positive MR contrast.

Keywords: extracellular matrix
myocardial infarction
cardiospheres
Cardiac stem cells
Advisor: Clarke, K
Carr, C
Paramagnetic defects in CVD diamonds

Paramagnetic defects in free standing polycrystalline diamond films made by chemical vapour deposition (CVD) have been studied using electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and infrared absorption.
EPR experiments at a range of frequencies (1-35 GHz) confirm the 1H hyperfine parameters for the recently identified H1 defect (Zhou et al., Phys. Rev. B, 54:7881 (1996)). In the samples studied here, H1 is always accompanied by another defect at g=2.0028(1). Saturation recovery measurements are consistent with two defects centred on g=2.0028. The spin-lattice relaxation rate of H1 is a factor of 10-100 times more rapid than the single substitutional nitrogen centre (N0S), which is known to be incorporated into the bulk diamond.
1H matrix ENDOR measurements indicate that the H1 centre is in an environment with hydrogen atoms 2-10 A distant from the centre. The near neighbour hydrogen identified by the EPR was not detected in the ENDOR experiments. The concentration of H1 correlates with the total integrated C-H stretch absorption in the samples studied here. All the evidence is consistent with H1 being located at hydrogen decorated grain boundaries (or in intergranular material) rather than in the bulk diamond.
The affect of annealing the films in vacuo up to 1900 K has been studied. On annealing at 1700 K it was found that some of the hydrogen on internal grain boundaries became mobile but was not lost from the sample, and the intensity of the EPR absorption at g=2.0028 decreased. Annealing at 1900 K severely degraded the optical properties of the samples, and a new defect with g=2.0035(2) was created. Infrared measurements show that hydrogen is lost from most CVD diamond samples when annealed to 1900 K for four hours.
An EPR imaging (EPRI) probe was designed and built. This comprised a 3-loop, 2-gap loop-gap resonator and a pair of anti-Helmholtz coils providing a magnetic field gradient ∂Bz/∂z. Using this probe the distribution of N0S was measured in the growth direction of four CVD diamonds to a resolution of 20 μm. The distribution of N0S is shown to be different to the distribution of defects with g=2.0028.
Two-dimensional images of the spin density of N0S in single crystal type Ib diamonds made by the high temperature and pressure (HTP) method have been generated, demonstrating a resolution of 100 μm. A two-dimensional image of the spin density of g=2.0028 defects in a CVD sample is compared to a photograph of the same sample, showing the correlation between the distribution of the defects with the distribution of non-diamond material in the sample.
The distribution of the [N-N]+ defect in a natural diamond has been examined using ∂Bz/∂Bϰ field gradient coils.
Addressing the issue of equity in health care provision during the transition period in Bulgaria

The collapse of the communist regimes in Central and Eastern Europe in 1989-1990 heralded the beginning of an economic transition from central planning to market economies. The subsequent period was marked by malfunctioning of these countries’ social sectors, including their health care systems, raising serious issues of equity. This thesis examines the impact of the transition period and the introduction of social insurance on equity in health care provision in Bulgaria. Equity in health care is investigated with respect to function - i.e. financing (according to ability to pay) and delivery (according to need) - and outcomes - i.e. health status, income inequality and poverty. Differences in health, health care financing and delivery are explored by income, education, ethnic, employment, marital status, age and sex groups. Furthermore, the thesis outlines the impact of health care provision, in particular social insurance, on poverty and health inequalities. The thesis employs empirical analysis based on household data. Its methodology includes concentration and decomposition analysis, and provides new ways of modelling health care financing and delivery, as well as the link between health and health care delivery. The thesis concludes that social insurance does not provide a uniform means of improving equity and that the root cause of the problem lies in the large proportion of out-of-pocket payments and the rather limited size of the health insurance sector. Inequity in health care provision leads to poverty and untreated illness. The data suggests that there are differences between socio-economic groups as regards their likelihood to seek treatment for their ill health, which result in differences in their health status. The social factors that have impacted the most on health are low education and low income.

Keywords: income inequality
health inequalities
poverty
socio-economic inequalities
minority health
Advisor: Walker, R
Surender, R
An epidemiological study of pneumonia in migrant infants and children on the Thai-Myanmar border

Background: Pneumonia is the leading cause of death in children and the estimated incidence of childhood pneumonia in Myanmar is the third highest in the world. Effective interventions, including case-management and immunisations, have been established.However these focus on bacterial pneumonia and recent advances in molecular diagnostics have highlighted the substantial role played by viruses. An understanding of the local epidemiology and aetiology of childhood pneumonia is vital to ensure that current and future interventions are appropriate to the local population.
Objective: To study the severity and aetiology of childhood pneumonia, in migrant infants and children under five years old, presenting to a clinic on the Thai-Myanmar border.
Methods: A one-year prospective observational study was conducted in a rural clinic on the Thai-Myanmar border. All children attending the clinic, who were diagnosed with clinical pneumonia according to the World Health Organization definitions, were treated and investigated according to local paediatric guidelines.
Results: During the study period 175 episodes of clinical pneumonia were diagnosed. Of these 59% were male and half were infants (48%). The median age at presentation was 7.5 months (IQR 3 to 13). Clinically half (50%) had severe or very severe pneumonia. Using the WHO standardised method for interpretation, 38% of radiographs had primary endpoint pneumonia.
At least one virus was identified in 49% of patients. The most commonly identified virus was adenovirus, which was identified in 24% of cases, followed by RSV (22%), then hMPV (6%) and Influenza A (3%). The cases of pneumonia peaked at the end of the rainy season and this closely followed the RSV activity.
Conclusion: The majority of children presented with severe or very severe pneumonia, suggesting that in this population patients may present later. The substantial burden in infants suggests that future interventions should focus on this vulnerable age group. Viruses were frequently identified. Adenovirus was commonly identified, however its presence in nasopharyngeal secretions is of uncertain significance, especially in the presence of other viruses. The persistence of adenovirus and its role in acute pneumonia needs further investigation. RSV is an important and commonly found pathogen and prevention of RSV through a safe and effective vaccine could have substantial effect on the reduction of childhood pneumonia on the Thai-Myanmar border.

Keywords: Infants
Paediatric
Pneumonia
Epidemiology
Children
Aetiology
Advisor: Pollard, A
The importance of DNA replication termination and the MHF complex to genome stability

The final stages of replication fork termination requires the timely and orderly orchestration of catalytic and enzymatic activities. Given the complexity of this process, it is conceivable that the final stages of fork termination is susceptible to problems that could trigger recombination, which could lead to deleterious genomic rearrangements if ectopic homologous sequences are recombined. Using the site-specific RTS1 barrier in fission yeast, I demonstrated that fork termination is generally not a recombinogenic process, and that hyper-recombination-induced by fork blockage at RTS1 is largely a result of replication fork restart.
To investigate the actual mechanisms and proteins, which drive and influence recombination at a replication barrier, I studied the MHF proteins, which assist Fml1 in limiting crossovers during double-strand break (DSB) repair and promoting Rad51-mediated recombination at impeded replication forks, and are also components of the constitutive centromere-associated network (CCAN). Intriguingly, structural studies revealed that the MHF can exist as an octamer in vitro. I examined the biological significance of octameric MHF by employing three mutations that disrupt the octamer configuration in vitro. In fission yeast, these mutations cause hypersensitivity to methyl methanesulfonate (MMS), suggesting that the MHF octamer may have a role in DNA repair. One of the “octamerisation” mutants, exhibits greater hypersensitivity to MMS than the other two, and biochemical experiments indicated that this is because it confers an additional defect in MHF’s interaction with Fml1. Further genetic experiments on this mutant suggest that the ability of Fml1 to unwind D-loops depends more critically on its interaction with MHF than fork reversal. Additionally, I showed a synergistic interaction between Dcr1 and MHF, and demonstrated that in the absence of Dcr1, there is a greater need for recombination to tolerate/repair DNA damage. Lastly, I uncovered a novel function for the MHF in controlling the initiation of septation.

Keywords: DNA replication termination
DNA repair
MHF
genome stability
Advisor: Whitby, M
Ethical practice and objective reasons

This thesis develops a 'response-dependent' account of the objectivity of ethical judgment. It uses the Kantian device of the Categorical Imperative (CI) as the critical reflective test for convergence in judgment that is genuinely objectivity-sustaining. After a discussion of the challenges to ethical objectivism, it is argued that Wittgenstein's consideration of rule-following provides a compelling case for rejecting attempts to ground objectivity in a manner that is external to normative practices. This removes one potent source of hostility to ethical objectivity, but it does not establish the truth of this position. Within the constraints of a broadly Wittgensteinian conception of objectivity, plausible accounts of ethical objectivity will be 'response-dependent'. The key issue facing such theories is whether they can provide a model of practical rationality that delivers suitably demanding, objectivity-sustaining standards of rational criticism. I examine two approaches to this issue. The first (suggested by John McDowell) takes Wittgenstein's rulefollowing considerations as implying a virtue-centred approach to ethical deliberation. On this view, virtuous agents' uncodifiable responses determine correct judgment. But the case for this theoretically modest model of objectivity has not been successfully made, and the approach has insufficient resources to yield genuine objectivity. Instead, appeal must be made to some more articulated account of practical rationality. The second, Kantian, model delivers this because the CI test provides a mechanism for objectivity-sustaining reflective criticism of agents' reasons for action, without importing any illicit codification of rationality. The CI can also be used for critical reflection on agents' appreciation of the ethical features of their situation. In this way the model can avoid the problem of empty formalism and offer a weak form of ethical cognitivism. This model also suggests a potential route to plausible approaches to some other problematic issues in ethical theory, such as akrasia and the categorical nature of moral reasons.
Fibrewise CoHopf spaces

A fibrewise coHopf space X over a base B is a sectioned space for which the diagonal map X —> X x BX may be compressed into X VBX up to fibrewise pointed homotopy. Such spaces have been investigated by I. M. James in the case where X is a sphere bundle over a sphere. The purpose of this thesis is to demonstrate some of the properties of fibrewise coHopf spaces over more general bases. Particular attention is given to sphere bundles and fibrations with spherical fibre.
The fibrewise reduced suspension of a sectioned fibrewise space with closed sec- tion is fibrewise coHopf with associative comultiplication (up to fibrewise pointed homotopy) and a fibrewise inversion. Examples of fibrewise coHopf spaces not of this form are exhibited, and sufficient conditions are given to ensure that a fibrewise coHopf space has the primitive fibrewise pointed homotopy type of a fibrewise re- duced suspension, in terms of the dimension and connectivity of the space, its base and the fibres. It is shown that these conditions may be relaxed if the fibrewise coHopf structure on the space is assumed to be homotopy-associative. An example of a non-associative fibrewise coHopf sphere bundle is given.
It is shown that, if q > 1 is odd, a sectioned orientable q-sphere bundle over a finite connected complex is fibrewise coHopf if and only if its fibrewise localisation at the prime 2 is fibrewise coHopf. Moreover, the fibrewise rationalisation of an odd-dimensional sphere bundle over a finite polyhedron whose fibrewise unreduced suspension is fibrewise coHopf is shown to be a trivial fibration. As an application, it is shown that new fibrewise coHopf spherical fibrations may be constructed by mixing.
The Thorn space is used to determine the cohomology ring of the total space of a fibrewise coHopf sphere bundle in terms of that of its base, and a generalised Hopf invariant is constructed which vanishes on fibrewise coHopf sphere bundles.

Advisor: James, I
On the regularity of holonomically constrained minimisers in the calculus of variations

This thesis concerns the regularity of holonomic minimisers of variational integrals in the context of direct methods in the calculus of variations. Specifically, we consider Sobolev mappings from a bounded domain into a connected compact Riemannian manifold without boundary, to which such mappings are said to be holonomically constrained. For a general class of strictly quasiconvex integral functionals, we give a direct proof of local C1,α-Hölder continuity, for some 0 < α < 1, of holonomic minimisers off a relatively closed 'singular set' of Lebesgue measure zero. Crucially, the proof constructs comparison maps using the universal covering of the target manifold, the lifting of Sobolev mappings to the covering space and the connectedness of the covering space. A certain tangential A-harmonic approximation lemma obtained directly using a Lipschitz approximation argument is also given. In the context of holonomic minimisers of regular variational integrals, we also provide bounds on the Hausdorff dimension of the singular set by generalising a variational difference quotient method to the holonomically constrained case with critical growth. The results are analogous to energy-minimising harmonic maps into compact manifolds, however in this case the proof does not use a monotonicity formula. We discuss several applications to variational problems in condensed matter physics, in particular those concerning the superfluidity of liquid helium-3 and nematic liquid crystals. In these problems, the class of mappings are constrained to an orbit of 'broken symmetries' or 'manifold of internal states', which correspond to a sub-group of residual symmetries.

Keywords: quasiconvexity
holonomic
variational methods
harmonic maps
regularity
Advisor: Kristensen, J
Studies of enzyme kinetics and aspects of enzyme structure in vivo using NMR and molecular genetics

A quantitative understanding of metabolic control depends on a knowledge of the enzymes involved. The extrapolation of studies in vitro to the intact cell is controversial because the intracellular environment is relatively poorly characterised, particularly with respect to the interactions between weakly-associated enzymes.
There is a clear need to study enzymes directly in the cell, yet there are few suitable techniques. Metabolites have been very successfully studied in cells by the non-invasive technique of nuclear magnetic resonance (NMR). NMR studies of enzymes in the cell have, however, been prevented by difficulties in assigning the resonances from the many proteins within the cell.
A method for studying a specific enzyme in the cell has been developed, using Saccharomyces cerevisiae and phosphoglycerate kinase (PGK) as a model system. Using an inducible expression system, PGK was synthesised in the cell without significant synthesis of other proteins. With 5-fluorotryptophan in the growth medium, fluorine-labelled PGK was formed in situ. Fluorine is an excellent label for NMR since it is absent from most cells and has a high receptivity to NMR detection.
19 F NMR was used to study PGK in the intact cell. Comparisons with measurements in vitro showed that PGK was exposed to only a small fraction of the total intracellular [ADP], implying some form of compartmentalisation. The NMR relaxation properties observed in vivo and in vitro were compared with theoretical predictions. This showed that PGK was not part of a complex in the cell and that the viscosity of the cytoplasm, relative to water, was c. 4 at 30 °C. Fluorine-labelled pyruvate kinase and hexokinase have also been prepared; the spectra of these proteins in vitro are responsive to their ligands, and further work will study these proteins in vivo.
NMR techniques were also applied to study the kinetics of PGK in the cell. PGK and GAPDH catalyse an ATP↔Pi exchange which is near-equilibrium in wild-type cells. 31P magnetisation transfer experiments in genetically manipulated cells showed that the reaction becomes unidirectional if the PGK activity is reduced by 95 %. Net flux is reduced by less than 30 %. In low-PGK cells, the ATP↔Pi exchange from oxidative phosphorylation can be isolated from that of glycolysis, facilitating direct measurements of the P:O ratio. In the cells studied, the P:O ratio was 2 to 3.