VictorJuiz/thesis huggingface dataset:
Genetic engineering of the primary/secondary metabolic interface in tobacco BY-2 cells

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway.
The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful.
The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

Keywords: Nicotiana tabacum
Tobacco
Metabolomics
Plant Cell Culture
Metabolic Engineering
Tobacco BY-2 cells
Citrate Synthase
13C MFA
Suspension Cultures
TCA cycle
HMGR
Isoprenoid Biosynthesis
Metabolic Flux Analysis
13C-FLUX
Phytosterols
GC-MS
Secondary Metabolism
Advisor: Sweetlove, L
Ratcliffe, R
Radiation damage in protein crystallography: susceptibility study

Protein structure models obtained from X-ray crystallography are subject to radiation damage. The resulting specific alterations to protein structures can be mistaken for biological features, or may obscure actual protein mechanisms, leading to misidentification or obscuration of biological insight. The radiation chemistry behind this site-specific damage is not well understood.
Radiation damage processes progress in proportion to the dose absorbed by the crystal in the diffraction experiment. Doses can be estimated using existing software, but these assume idealised experimental conditions.
To simulate complex diffraction experiments, including treatment of imperfect X-ray beam profiles and inhomogeneous dose distributions, a new program, RADDOSE-3D, was developed. RADDOSE-3D can be integrated into beamline software to provide convenient, more accurate, comparative, and publishable dose figures, also facilitating informed data collection decisions.
There is currently no method to automatically detect specific radiation damage in protein structure models in the absence of an 'undamaged' reference model. Radiation damage research therefore generally relies on detailed observation of a few model proteins.
A new metric, BDamage, is designed and used to identify and quantify specific radiation damage in the first large-scale statistical survey of 2,704 published protein models, which are examined for the effects of local environments on site-specific radiation damage susceptibility. A significant positive correlation between susceptibility and solvent accessibility is identified.
Current understanding of radiation damage progression is mostly based on a few consecutive structure model 'snapshots' at coarse dose intervals. The low sampling rate considerably limits the ability to identify varying site susceptibility and its causes.
Real space electron density data are obtained for crystals of different mutants of a RhoGDI protein with very high sequence identity, to determine sensitising and stabilising factors for radiation induced structural changes. Utilising a newly developed data collection and analysis protocol, these changes could be tracked with unprecedented time resolution.

Keywords: radiation damage
specific damage
preferential damage
atomic displacement parameters
atomic B factors
Advisor: Garman, E
Deane, C
Cbf1 regulates chromatin remodelling of the Saccharomyces cerevisiae genome at multiple binding sites

The centromere binding factor 1, Cbf1, of Saccharomyces cerevisiae is a bHLH/ZIP protein which has been described as a determinant of specific chromatin structures and as a tethering factor for activators of transcription at the promoters of genes of the Methionine Biosynthesis Pathway. Deletion mutants show various phenotypes, among them methionine auxotrophy, an increased rate of chromosome loss, modifications in the growth rate and modification of the chromatin structure at MET genes. Meiosis competence also becomes greatly reduced in cbf1 cells. The sequence motif (RTCACRTG) to which Cbf1p binds is found at multiple loci through the yeast genome. This thesis shows that the chromatin structure is reorganised at multiple Cbf1p binding sites in vivo, when yeast cells are starved to enter meiosis. Extensive remodelling occurs at the MET16, MET17(25), DRS2 and GDH3 loci and at the YAL060W open reading frame, as detected by in vivo digestion of chromatin with micrococcal nuclease and indirect end-labelling. The same kind of analysis showed that the remodelling of chromatin at Cbf1p binding sites is not specific for meiosis, it occurs also in similarly starved haploid cells. The lack of methionine is a key trigger of these changes. This reorganisation of chromatin is dependent on Cbf1p, since starved cbf1 cells do not display any modification in nuclease accessibility patterns at or around Cbf1p binding sites. Mutational analysis revealed that a negative charge at a putative phosphorylation site (serine residue 226) and the DNA-bindmg activity of Cbf1p are both required for the chromatin reorganisation to occur in response to starvation. CBF1 mutants which do not reorganise chromatin were also shown to be unable to enter meiosis, suggesting that the remodelling of chromatin at multiple Cbf1p binding sites may be required to enter pre-meiotic DNA replication, since such cells arrest before the initiation of this process. In summary, the results presented in this thesis are compatible with a model in which Cbf1p plays an active role as part of a mechanism sensing the nutrient availability and regulates the reorganisation of chromatin, at multiple loci through the yeast genome, in response to starvation conditions.
Laser wakefield acceleration in tapered plasma channels — theory, simulation and experiment

Laser-plasma accelerators are of great interest because of their ability to sustain extremely large acceleration gradients, enabling compact accelerating structures. Laser-plasma acceleration is realized by using a high-intensity short pulse laser to drive a large plasma wave or wakefield in an underdense plasma.
This thesis considers the effect of axial plasma density upramps on laser wakefield acceleration. Theoretical groundwork shows that tapered plasma channels can be used to mitigate one of the main limitations of laser plasma acceleration, that is, dephasing of an electron beam with respect to the plasma wave. It is shown that it is possible to maintain an electron bunch at constant phase in the longitudinal electric fields of the laser wake field. This leads to an increased energy gain of an electron trapped in the wakefield. The required shape of the density slope is difficult to implement in experiments. Therefore, a linear density ramp is also considered which is predicted to also increase the energy gain beyond that possible in a uniform density plasma.
Towards an experimental implementation it was studied how a suitable gas density profile can be established in a capillary. This was done employing simulations using the computational fluid dynamics tool kit OpenFoam and comparing these to measurements of the axial density profile based on Raman scattering. It was demonstrated that a linear density ramp could be established by applying different pressures on the capillary gas inlets. The dependence of the density profile on the capillary parameters, such as, capillary diameter and length and inlet diameter were also studied. The results of the simulations and the measurement showed excellent agreement and demonstrate that approximately linear density ramps can be generated by flowing gas along a capillary of constant cross-section
Laser wakefield acceleration in plasmas with longitudinally varying density was investigated in an experiment at the Astra Laser at Rutherford Laboratories. The experiment utilised ionisation injection in order to operate in the mildly non-linear regime of laser-wakefield acceleration. The measured electron energies agree well with the theoretical predictions. It was demonstrated that an increase in the energy gain can be obtained by driving the accelerator in a ramped plasma, the electron spectrum is more narrow and the injected charge increases significantly.
Measurements of the X-ray spectrum emitted by the betatron motion of the accelerated electron bunch allowed the transverse radius of the bunch to be deduced. These measurements showed that retrieved electron bunch radius is inversely proportional to the longitudinal density gradient, that is a plasma density upramp (downramp) has a decreased (increased) electron bunch radius.

Keywords: Physics
Dephasing
Laser Wakefield Acceleration
Plasma density ramps
Tapering plasma channels
Particle Acceleration
Advisor: Hooker, S
A history of the Molemas, African notables in South Africa, 1880s to 1920s

This thesis is a family history of Silas Molema and his three children from the late 1880s to the late 1920s. The Molemas were a family of devout Methodists and educated chiefs in Mafikeng north of British Bechuanaland (part of the Cape colony in 1895) but they held extensive landholdings across the border in the Bechuanaland Protectorate. The thesis explores education, landholding and political office as strategies through which the Molemas attempted to maintain their position of class, status and power. Chiefs perceived formal annexation by Britain in 1885 also as opportunity to pursue greater self-determination, preserve the institutions of chiefly rule, and sustain respectable livelihoods. These aspirations had come to be experienced and understood as sechuana, which was a fluid reconstruction of tradition that helped Molemas and other Bechuana notables straddle incongruous cultural spheres along a racially and ethnically diverse colonial frontier. The thesis argues that nationhood was a key identification through which Molemas and other educated Bechuana saw themselves, and considers why they imagined their nation within the British Empire. The thesis also points to the various historical transformations and private entanglements that enmeshed various conceptions of nationhood into the everyday experience of the family as an emotive and socialising institution. These sentiments of nationhood profoundly shaped this family’s self-understanding, and mediated the choices children made about work, marriage and other significant relationships. The challenge to transfer inherited privilege across generations shaped identities, intersected with the reconfiguration of the local political economy, and impinged upon structural transformations in southern Africa.

Keywords: Indirect Rule
History of the British Empire in Bechuanaland
Family History
Silas Molema
Advisor: Beinart, W
Cavity ringdown spectroscopy for broadband and microfluidics applications

Cavity Ringdown Spectroscopy (CRDS) is a highly sensitive, absorption spectroscopy technique. From the measurement of the decay of light within an optical cavity, spectroscopic parameters of an absorbing sample can be determined. Thus far the technique has mostly been applied to gas-phase samples. The work described in this thesis attempts to extend the technique to the condensed phase. A supercontinuum source and streak camera detector were used to enable broadband measurements (~ 500 – 600 nm) of the absorption spectra of gaseous NO2 and I2 samples. The setup had a detection sensitivity of 20 ± 7 ppm, and was capable of making an accurate measurement of the wavelength-dependent reflectivity of the cavity mirrors. Microfluidic devices have been employed in a range of fields for purposes including small volume organic synthesis, environmental monitoring and medical diagnosis. They enabled us to make measurements on condensed phase samples by integrating a ‘test’ microfluidic chip into a single wavelength CRDS setup and flowing liquid through its channels. Using a series of rhodamine 6G solutions the detection sensitivity of our setup was determined to be (4.2 ± 0.3) x 10-6 M, with a sample volume of 12.3 nL. The technique was also used to measure the titration curve of phenolphthalein prepared in a series of buffer solutions with pHs ranging from 7.7 to 11.0, and was capable of determining the pH of nanolitre samples to an accuracy of ± 0.028. In the final set of measurements reported here, the technique was used to follow the kinetics of the iodine clock reaction. The results showed good agreement with bulk measurements made using a single-pass spectroscopic technique.

Keywords: condensed phase
detection
cavity enhanced
pH
kinetics
spectroscopy
gas phase
microfluidics
broadband
supercontinuum
cavity ringdown
Advisor: Vallance, C
'Le parfait Ambassadeur'. The theory and practice of diplomacy in the century following the Peace of Westphalia

This thesis examines the theory of diplomacy in the formative phase of the European states-system. From the viewpoint of the discourse on the 'ideal ambassador', it explores early modern diplomacy as cultural history encompassing ideas, discourses, perceptions and 'codes'. The scope of study is the century following the Peace of Westphalia (1648), and three states and regions (France, Britain, the Holy Roman Empire) serve as case studies for a comparative approach of diplomatic theory and practice. In five parts, the adaptation of the theory and practice of diplomacy to the new demands of international relations after 1648 are considered. The first section sets the stage by illustrating that the mid-seventeenth century was regarded as a turning point in the practice of diplomacy. Part II examines diplomatic theory as a particular 'language' in its intellectual and socio-professional contexts. While published treatises on the 'ideal ambassador' build the core of this study, related genres of international law theory, ceremonial theory and political and state science are also taken into account. From the viewpoint of this diplomatic theory, the following section examines the ways in which the instruments and practices of diplomacy were aligned to the new framework. These ranged from changes in the structural framework of diplomacy to the evolution of norms and procedures of negotiation, international law and ceremonial. Part IV reconsiders the issue of 'professionalism' in diplomatic theory with regard to the preparation and training of diplomats. Special attention is given to proposals for diplomatic 'academies', which are for the first time examined in comparison. Finally, section V recasts the findings of this thesis in a comparative perspective. It underlines that, with the emergence of a states-system, the techniques of diplomacy became formalised and uniform, constituting a common European diplomatic practice. Against the background of the different regional and structural conditions, the alleged model role of France in the evolution of diplomatic theory and practice is re-evaluated.

Keywords: diplomacy
diplomats
Peace of Westphalia
international relations
political science
Advisor: Parrott, D
Development of catalytic methods to exploit sulfur dioxide in organic synthesis

In the following thesis, new methodologies towards the synthesis of a range of sulfonyl (-SO2-) containing functional groups are documented. These methods utilise easy-to-handle sulfur dioxide surrogates, such as DABSO (vide infra), and exploit palladium catalysis as a new mechanistic protocol for the incorporation of the -SO2- unit.
Chapter 1 is a literature review surveying sulfur dioxide in organic synthesis, the established uses of SO2 surrogates and the importance of the sulfonyl moiety in chemistry. Palladium-catalysed (carbonylative) cross-couplings are also broadly discussed as they provide inspiration for, and mechanistic similarities with, the proposed chemistry.
Chapter 2 describes a de novo synthesis of the sulfonamide functional group; a three-component and convergent methodology coupling (hetero)aryl and alkenyl halides with sulfur dioxide (provided by easy-to-handle surrogates such as DABSO) and hydrazine nucleophiles, is documented. This is achieved through the action of a readily available palladium catalytic system and is the first example of a metal-catalysed sulfonylative cross-coupling of halide based electrophiles.
Chapter 3 presents a new method of generating (hetero)aryl and alkenyl sulfones. The ability of organometallic reagents to add to sulfur dioxide (supplied via DABSO) is applied to deliver the corresponding metal sulfinate salt. This in situ derived sulfinate is coupled with an (hetero)aryl or alkenyl (pseudo)halide using palladium catalysis to form the desired sulfone. An electronically modified XantPhos-type ligand was designed for the reaction in order to suppress unwanted aryl-aryl exchange.
Chapter 4 documents the generation of (hetero)aryl and alkenyl sulfinates from the corresponding halide and DABSO through a palladium-catalysed sulfination protocol, obviating the need for organometallic reagents. A mild set of conditions using IPA as both a solvent and reductant together with a low loading of palladium catalyst offers an attractive route to sulfonyl compounds thanks to the in situ derived sulfinates being converted into a broad variety of functional groups via established onwards reactivity.
Chapter 5 discusses the conclusion of the research and the potential for future work.
Chapter 6 presents the experimental data.

Keywords: cross-coupling
palladium catalysis
DABSO
sulfur dioxide
Advisor: Willis, M
Needle-free vaccination: formulation and dermal delivery of diphtheria toxin CRM197 mutant

The unsafe use of needles propagates cross infections with bloodborne pathogens and reduces the positive impact of vaccinations on global health. While a plethora of needle-free injection devices exist, the reformulation of protein-based vaccines is largely empirical and costly, which presents a barrier to their widespread clinical application.
This thesis contributes to the identification of approaches that facilitate rapid vaccine reformulation and enhance the immunogenicity of needle-free dry-powder vaccines with the help of novel antigen delivery platforms.
We hypothesised that the thermodynamic stabilisation of diphtheria toxin mutant 197 (CRM197), a glycoconjugate vaccine carrier protein, may enhance its structural preservation during spray-freeze-drying (SFD), and that its formulation in either soluble, surface-adsorbed, or nanoparticle form impacts the elicited immune response. Differential scanning fluorimetry was used to study the effect of excipients on the thermal stability of CRM197. Dry-powder formulation of CRM197 used i) encapsulation into a thermodynamically stabilising excipient matrix by SFD, ii) surface-immobilisation via physisorption onto a novel potassium-doped hydroxyapatite (kHA) carrier microparticle formed by molten salt synthesis, and iii) chemical conjugation and surface presentation on amphiphilic block copolymer nanoparticles that were incorporated into SFD-powders (SFD-NP). The structural integrity of CRM197 was assessed by size separation in addition to various spectral and thermal analysis methods. The immunogenicity of dry-powder CRM197 formulations was subsequently tested in vivo.
The results suggest that the thermodynamic stability of CRM197 in solution does not ensure its structural stability during SFD. While needle-free dermal vaccination with kHA-adsorbed CRM197 induced comparable antibody titres to conventional IM injection of alum-adjuvanted CRM197, needle-free SFD and SFD-NP powders were less immunogenic. The highest mean IgG titre and most balanced Th1/Th2 response was achieved with nanoparticle-conjugated CRM197 by IM, which outperformed the current clinical standard.
Therefore, future vaccine design should combine thermodynamic and kinetic stability screening, and place special emphasis on the delivery and structural presentation of the antigen to the immune system.

Keywords: Spray freeze drying
CRM197
Needle-free
Vaccination
Hib
Hydroxyapatite
Nanoparticles
Advisor: Pollard, A
Schiffter, H
Nineteenth century English oratorio festivals

Oratorio festivals were an important cultural feature of nineteenth-century English society. These massive musical events lasted for three or four days and some involved up to 4,000 musicians and 83,000 in the audience. This dissertation advances the hypothesis that the oratorio festivals, and the grand new buildings in which they were staged, coalesced to create a musical monumentalism in a society steeped in the (mainly Protestant) Christian sentiments of the day. In particular, the dissertation contends that a central premise of nineteenth-century musical thought was that the musical value of a performance was directly in proportion to the size of the performing forces and the audience.
A framework devised mainly from Stephen Little's definition of monumental art (2004) is used as a critical tool to examine from a new perspective aspects of nineteenth-century oratorios such as 'physical scale', 'breadth of subject matter', and 'ambition to be of lasting significance'. Furthermore, this dissertation argues that a complex ideology of an English musical monumentalism underpinned the concatenation of circumstances that allowed oratorio festivals to flourish at this time.
The spectacle of the Crystal Palace in London and the Great Handel Triennial Festivals it housed are contrasted with the provincial festivals, such as those of Bristol, Manchester, Liverpool, Birmingham, and Leeds. The analyses of the latter rely on substantial original material uncovered from rich primary source documents about the provincial oratorio festivals and the buildings in which they were held. Musical scores themselves, including some of Sir Michael Costa's orchestral manuscripts, are also examined as monuments. A comprehensive study of these festivals is well overdue and this study will aim to understand why these events grew to such a mammoth size at this time.

Advisor: Allen, R