VictorJuiz/thesis huggingface dataset:
Properties of recombinant HIV and SIV antigens

When expressed in Spodoptera frugiperda cells using recombinant baculoviruses, the HIV - 1 gag gene product, p55, self assembles to form immature HIV core like particles that are secreted into the culture medium. Using this system, the gag open reading frame was progressively truncated from the carboxy terminus, and each deleted Gag protein examined for its ability to produce core like particles. Deletions that removed the distal region of the Gag nucleocapsid domain, including both Cys - His motifs thought to function as RNA capture signals, did not disrupt particle formation. Analysis of the truncation mutants by north - western blot with a radiolabelled HIV - 1 RNA encapsidation signal, confirmed that only precursors with the Cys - His motif present were able to bind the probe. It was concluded that HIV - 1 Gag particle formation per se does not require RNA encapsidation. This finding clarifies uncertainties proposed previously regarding the role of RNA encapsidation in particle assembly. Further deletion mutants led to the delineation of the carboxy terminal boundary of a Gag assembly domain.
Properties of the SIV env encoded TM glycoprotein were also explored. Previous reports have found that the cytoplasmic tail of this membrane spanning protein was preferentially deleted in vivo when SIV was grown in a variety of cultured human cells. However, when propagated in cells of simian origin, the TM glycoprotein cytoplasmic tail was retained. Analysis of the function of this domain was addressed by expression of both the truncated and the full length proteins in insect cells using recombinant baculoviruses, coupled with a study of their biochemical characteristics. A role for the cytoplasmic tail was identified in the post - translational modification and localization of the glycoprotein.
The use of the Gag particles in designing vaccines was investigated. Acting as non - infectious carriers of immunogenic antigens, the particles represent a safe and efficient means for presentation of epitopes for eliciting a protective immune response. Two possible approaches for loading the particles with foreign antigen were examined. In the first, the deleted portion of Gag was replaced with a sequence encoding the foreign antigen. The second method involved co - expression of Gag with another recombinant virus that expressed a foreign antigen on the surface of the cell. Both methods were found to be feasible, although limitations were identified when addition of the fusion protein promoted excess degradation of the Gag precursor.

Advisor: Jones, I
Bishop, D
Structure of the essential malaria invasion protein RH5 in complex with its erythrocyte receptor and inhibitory antibodies

Invasion of host erythrocytes is an essential stage in the life cycle of Plasmodium parasites and in development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by the coordinated release of specialised apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and the erythrocyte-binding like (EBL) proteins, that mediate host-parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin. Indeed, antibodies targeting either PfRH5 or basigin can block parasite invasion with high efficiency in vitro, making PfRH5 an excellent candidate for a vaccine to protect against the most deadly form of malaria. Here I present crystal structures of PfRH5 in complex with basigin and with two distinct inhibitory antibodies. This is the first structure of any RH protein, revealing a novel fold in which two three-helical bundles come together to form a kite-like architecture. The two immunoglobulin domains of basigin and the inhibitory antibodies bind to one tip of the kite. These findings provide the first structural insights into erythrocyte binding by the Plasmodium RH protein family and identify novel inhibitory epitopes to guide the design of a new generation of vaccines against the blood-stage parasite.

Keywords: Plasmodium falciparum
RH5
malaria
X-ray crystallography
Advisor: Higgins, M
Rates of convergence of variance-gamma approximations via Stein's method

Stein's method is a powerful technique that can be used to obtain bounds for approximation errors in a weak convergence setting. The method has been used to obtain approximation results for a number of distributions, such as the normal, Poisson and Gamma distributions. A major strength of the method is that it is often relatively straightforward to apply it to problems involving dependent random variables.
In this thesis, we consider the adaptation of Stein's method to the class of Variance-Gamma distributions. We obtain a Stein equation for the Variance-Gamma distributions. Uniform bounds for the solution of the Symmetric Variance-Gamma Stein equation and its first four derivatives are given in terms of the supremum norms of derivatives of the test function. New formulas and inequalities for modified Bessel functions are obtained, which allow us to obtain these bounds. We then use local approach couplings to obtain bounds on the error in approximating two asymptotically Variance-Gamma distributed statistics by their limiting distribution. In both cases, we obtain a convergence rate of order n-1 for suitably smooth test functions.
The product of two normal random variables has a Variance-Gamma distribution and this leads us to consider the development of Stein's method to the product of r independent mean-zero normal random variables. An elegant Stein equation is obtained, which motivates a generalisation of the zero bias transformation. This new transformation has a number of interesting properties, which we exploit to prove some limit theorems for statistics that are asymptotically distributed as the product of two central normal distributions.
The Variance-Gamma and Product Normal distributions arise as functions of the multivariate normal distribution. We end this thesis by demonstrating how the multivariate normal Stein equation can be used to prove limit theorems for statistics that are asymptotically distributed as a function of the multivariate normal distribution. We establish some sufficient conditions for convergence rates to be of order n-1 for smooth test functions, and thus faster than the O(n-1/2) rate that would arise from the Berry-Esseen Theorem. We apply the multivariate normal Stein equation approach to prove Variance-Gamma and Product Normal limit theorems, and we also consider an application to Friedman's X2 statistic.

Keywords: Variance-Gamma distribution
Stein's method
functions of multivariate normal random variables
rate of convergence
chi-square distribution
coupling
distributional transformation
product normal distribution
Friedman's statistic
Advisor: Reinert, G
Signal transduction in response to active oxygen species in Arabidopsis thaliana

Many environmental stresses result in increased generation of active oxygen species (AOS) in plant cells, leading to the induction of protective mechanisms. In this study, signalling components linking AOS perception to downstream responses were examined, with particular emphasis on H2O2 signalling.
All AOS investigated had an early [Ca2+]cyt peak in common, but differed in other aspects of their Ca2+ signatures, indicating that the plant is able to discriminate between different types of AOS. An early event in AOS signal transduction may involve changes in the cellular redox balance as reduction of glutathione levels prior to stress application increased the height of the first [Ca2+]cyt peak. Inhibiting or enhancing the height of the H2O2-triggered Ca2+ signature lead to inhibition or enhancement of GST1 and APX1 induction, respectively, demonstrating that the Ca2+ signature is required for induction of genes encoding antioxidant enzymes.
OX1, encoding a putative ser/thr kinase, was shown to be involved in signal transduction in response to H2O2-generating stresses. Transcript levels of OX1 were increased upon treatment with H2O2 and a range of abiotic and biotic stresses as well as ABA, all of which have been shown to result in H2O2 accumulation. Inhibition of stress-induced [Ca2+]cyt elevations inhibited OX1 induction, placing the OX1 kinase downstream of Ca2+ in the signalling chain. OX1 is required for full activation of AtMPKS and AtMPK6 in response to ozone fumigation, indicating that OX1 functions upstream of these MAP kinases. An ox1 null-mutant displayed enhanced susceptibility to infection with a virulent Peronospora parasitica isolate as well as reduced induction of several defence genes. In addition, the ox1 mutant exhibited shorter root hairs and an early flowering phenotype.
AOS treatment induced several genes encoding AtERF transcription factors, but did not have an effect on other members of this family. Induction occurred in an ethylene-independent but Ca2+-dependent manner.

Advisor: Knight, M
The design and development of an HIV-1 vaccine to elicit a broadly neutralising antibody response

Despite 30 years of research, a prophylactic vaccine against HIV-1 is still lacking and is urgently needed in order to control the global AIDS pandemic. The discovery of broadly neutralising antibodies (BNAbs) was an important step for HIV-1 research but no vaccine candidate tested so far has been able to reproduce responses containing such antibodies, and it remains unclear how this could be achieved via immunisation. In this thesis, I attempted to explore this gap of knowledge in two ways. First, certain features (‘signatures’) of the Env protein that were associated with a broadly neutralising response were identified through machine learning. Further characterisation of these signatures revealed several ways by which these naturally-occurring mutations might alter the immunogenicity of the Env protein that could result in the elicitation of a broadly neutralising response. The incorporation of such signatures in future vaccine design could be useful as the Env protein might adopt a conformation that encourages the elicitation of a broadly neutralising response. Second, 3 novel vaccination approaches were proposed aiming to induce a BNAb antibody response. The development of 2 approaches proved to be difficult and was not continued. For the third approach, non-neutralising immunogen-derived antibodies were used to mask immunodominant epitopes on the Env protein (i.e. ‘antibody-shielding’), thus allowing the antibody response to be focused to the highly conserved CD4 binding site (CD4bs). Subsequent immunisation of the antibody-shielded gp120 proteins in mice and rabbits demonstrated that antibody-shielding was able to significantly dampen the V3-specific antibody response while retaining the CD4bs-specific response. However, the antibody response to the V1/V2 loop was enhanced upon V3-dampening which indicates that further optimisation of the antibody-shield is needed in order to eliminate any antibody response towards the immunodominant regions. In conclusion, these results are the first description of a number of novel vaccination ideas and provide valuable insights into how these approaches could be optimised to become effective HIV-1 vaccines that can lead to the elicitation of a broadly neutralising antibody response.

Keywords: AIDS
vaccine
HIV
Advisor: McMichael, S
Stewart-Jones, G
Child agency and economic circumstances: how does family economic status affect child agency in Kyrgyzstan's post-Soviet culture of transition?

This thesis explores how children’s experiences of childhood in Kyrgyzstan transformed after the collapse of the Soviet Union (1991) and the consequent transition to market economy. In particular it studies the interrelations of culture and economic circumstances and their effects on child agency in times of economic, social, cultural and political change which were not given enough attention in the relevant literature. I sampled 40 children (aged 12 and 16) from a state school in an underprivileged area and a prestigious private school (used as economic dividers) to study the complexity of child agency and structure in their daily lives. An ‘agency’ concept was applied as a theoretical framework conceptualised through the four components as action, freedom, purposiveness and outcome and was formulated as the setting-based ability of children to act in response to cultural and economic structures and relationships at home, school and the neighbourhood. The effects of low and high income on child agency are not straightforward due to the changing traditional culture in the Kyrgyz society, which makes agency not only a social and cultural construct, but one affected by economic conditions. The study demonstrated the nuances of child agency as freedom in high income, its conflicting purposes in low income and differentiated outcomes of short and long term wellbeing between the two groups. Economic circumstances do not only influence the dynamics of agency across settings, age and gender but challenge the very notion of the classic Western concept of agency as an independent ability to act. The findings elaborate on the concepts of the new sociology of childhood (Prout and James, 1997) and the cultural politics of childhood (James and James, 2004), as these theoretical frameworks do not account sufficiently for economic dis/advantages as a structural factor of agency, which emerges as a socially shared process of acting whose nature depends on material circumstances.

Keywords: child agency
poverty
Advisor: Walker, R
Bennett, F
Regulation of DNA replication during meiosis in fission yeast

The interval between meiotic nuclear divisions can be regarded as a modified mitotic cell cycle where DNA replication is blocked. Mechanisms regulating this critical aspect of meiosis that allows haploid cells to be generated from a diploid progenitor were investigated in this project. Licensing is restricted after meiosis I due to down-regulation of Cdc18 and Cdt1. Late meiotic expression of Cdc18 and Cdt1, which load the MCM helicase onto replication origins, can lead to partial DNA replication after meiosis I. This implies that block to initiation via licensing forms an important component of this regulation. As detecting any minor DNA re-replication after meiosis I requires a technique more sensitive than flow cytometry for detection of total cell DNA contents, I also investigated a procedure to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast.
Additional inactivation of Spd1 or stabilization of Dfp1 after MI when Cdc18 and Cdt1 are also expressed does not enhance re-replication, but cyclin-dependent kinase Cdc2 plays a role in preventing re-replication during the MI-MII interval. Unexpectedly, when the licensing block is subverted, replication forks only move a short distance in the interval between meiosis I and II, implying that the elongation step of DNA replication is also inefficient. In addition, I show that the regulation of entry into meiosis II is not delayed by a partial round of DNA replication or DNA damage, indicating that replication and DNA damage checkpoints do not operate in late meiosis.

Keywords: fission yeast
meiosis
DNA replication
Advisor: Kearsey, S
Transformations of representation in constraint satisfaction

In this thesis I study constraint satisfaction problems or CSPs. These require determining whether values can be assigned to variables so that all constraints are satisfied. An important challenge is to identify tractable CSPs which can be solved efficiently.
CSP instances have usually been grouped together by restricting either the allowed combinations of values, or the way the variables are allowed to interact. Such restrictions sometimes yield tractable CSPs. A weakness of this method is that it cannot explain why all-different constraints form a tractable CSP. In this common type of constraint, all variables must be assigned values that are different from each other. New techniques are therefore needed to explain why such CSPs can be solved efficiently.
My main contribution is an investigation of such hybrid CSPs which cannot be defined with either one of these kinds of restrictions. The main technique I use is a transformation of a CSP instance to the microstructure representation. This represents an instance as a collection of sets, and a solution of the instance corresponds to an independent set in the clause structure.
For the common case where all constraints involve only two variables, I show how the microstructure can be used to define CSPs that are tractable because their clause structures fall within classes of graphs for which an independent set of specified size can be found efficiently. Such tractable hereditary classes are defined by using the technique of excluded induced subgraphs, such as classes of graphs that contain neither odd cycles with five or more vertices, nor their complements. I also develop finer grained techniques, by allowing vertices of the microstructure representation to be assigned colours, and the variables to be ordered. I show that these techniques define a new tractable CSP that forbids an ordered vertex-coloured subgraph in the microstructure representation.

Keywords: microstructure
hybrid tractability
constraint satisfaction
Advisor: Jeavons, P
Long path fourier transform absorption spectroscopy for investigating pollution in the urban boundary layer

The control of atmospheric pollution in the urban environment has, in recent years, taken on important local, national and international significance. Legislation has been introduced in many countries to improve air quality in urban areas. Monitoring air pollution is an important part of understanding and improving air quality. Current standard methods of measuring air pollution only monitor at a single point in space which can lead to ambiguities when assessing air quality over an extended region, such as a city centre or a road intersection. Optical remote sensing techniques, such as long path Fourier transform absorption spectroscopy, overcome the limitations of point measurements by integrating over long paths and thus measuring the average ambient pollutant concentrations.
A commercially available Fourier transform infrared (FTIR) spectrometer, together with custom built external optics has been used to make measurements of air pollution in Oxford city centre. It has been shown that it is possible to measure the concentrations of several pollutants simultaneously and in short measurement times. Issues relating to the performance of the spectrometer have been studied, their effects quantified and solutions proposed.
Optimal estimation techniques have been applied to the analysis of the single beam spectra recorded by the FTIR spectrometer. This technique has previously only been applied to radiance and transmission spectra and so extensions were necessary.
High resolution laboratory measurements of the absorption cross-sections of benzene and 1,3-butadiene have been made and the potential for detecting them in ambient urban air determined.

Keywords: FTIR
air pollution
Advisor: Barnett, J
Morris, P
Antibodies directed against AMPA and GABAB receptors in neurological diseases and identification of novel antigen targets

Antibodies directed against AMPAR and GABABR subunits have been implicated in forms of limbic encephalitis (LE), a disease characterised by memory loss and seizures. Patients with LE show clinical improvement with immunomodulatory treatment, suggesting that the associated antibodies are pathogenic. To explore further the AMPAR and GABABR antibodies, an in house cell based assay (CBA) was established for screening and potential pathogenicity was explored using a series of in vitro experiments.
Human embryonic kidney (HEK) cells transfected with AMPAR and GABABR subunits and primary neuronal cultures were used to detect antibodies in patient sera and CSF. In total, 15/1361 (1.1%) AMPAR antibody positive samples and 24/1438 (1.7%) GABABR antibody positive samples were identified.
The predominant antibody subclass for AMPAR and GABABR antibodies was shown to be IgG1. Interestingly, on transfected cells, only AMPAR antibodies showed complement deposition, and therefore had the potential to activate the classical pathway of the complement cascade. Application of IgG purified from AMPAR antibody positive patients, but not GABABR antibody positive patients caused a down regulation of the receptor from the cell surface of transfected HEK cells and primary hippocampal cultures. Electrophysiological analysis showed changes in Up state duration and spike rate in the entorhinal cortex following application of purified GABABR antibody IgG on brain slices. These findings suggest that GABABR antibodies are having a direct short term effect on GABABRs, and by extension cortical networks.
Finally, we attempted to study whether or not viral infection could be a trigger for antibody production to known and novel antigen targets in a cohort of Japanese encephalitis viral (JEV) samples. A JEV cohort of 44 children was screened for antibodies to neuronal surface proteins by CBA. Twenty-seven percent of patients had antibodies to known neuronal surface antigens, with NMDAR and CASPR2 as the most common antigen targets. Interestingly, screening the cohort on primary neuronal cultures revealed that 65.9% of children with JEV have neuronal surface antibodies. The identification of the novel antigen target was attempted using immunoprecipitation and mass spectrometry techniques. In total, 4 neuronal surface proteins were identified that could be potential targets in JEV patients.
In summary, antibodies directed against previously described antigenic targets were explored further for pathogenic potential, and a cohort of patients with a viral infection with potential novel antigen targets was investigated.

Keywords: autoimmune
Neurological diseases
neuronal surface proteins
Advisor: Lang, B