VictorJuiz/thesis huggingface dataset:
Photofragmentation studies of metal ion-molecule complexes and metal oxides

Gas phase metal-containing complexes provide suitable systems in which to study fundamental binding motifs between a metal ion and molecules in the absence of any solvent, support or competing charge effects. In this thesis, metal-containing species are explored experimentally using infrared resonance enhanced photodissociation (IR-REPD) spectroscopy and velocity map imaging (VMI). The experimental results are further interpreted with the aid of spectral simulations based on density functional theory (DFT). These are the first studies reported using a newly built IR-REPD spectrometer equipped with a purpose-built laser ablation source to allow for the study of single metal ion-molecule complexes. The laser ablation source is shown to efficiently produce various complexes including Rh+(CO2)n, VO2+(N2O)n and Au+(CH4)n and the IR-REPD spectrometer has been characterised against a well-studied system of V+(CO2)n complexes. In order to record the IR-REPD spectra for small metal ion-molecule complexes, an argon atom is employed as the inert messenger.
A combined IR-REPD spectroscopy and DFT investigation of M+(CO2)n complexes (where M = Co+, Rh+ and Ir+) reveals a common [M+(CO2)2] core structure for all three considered metal ions. Additional ligands, which are not directly bound to the central metal ion, experience lower perturbation as evident in the reduced blue-shift for the ligand in the outer coordination shells. A further IR-REPD/DFT study involving CO2 complexation around NbO2+ and TaO2+ ions reveals a strongly-bound core of four CO2 ligands around the MO2+ ion (M = Nb, Ta). A significant increase in the intermolecular bond distances for the second coordination sphere ligands coincides with a decrease in the calculated binding energies.
Velocity map imaging is employed to explore the rich photodissociation dynamics of VO in the vicinity of C4Σ- - X4Σ-(v',0) vibronic transitions in VO. The final quantum state distribution was observed to be strongly dependent on the intermediate vibronic state of VO via which the dissociation threshold is reached. This work provides a refined value for the VO dissociation energy of D0(VO) = 53190 ± 261 cm-1 in excellent agreement with available literature.

Keywords: Infrared spectroscopy
Metal clusters
Photodissociation
Velocity map imaging
Advisor: Mackenzie, S
Examining the influence of lineage relationships upon excitatory neocortical development

The mammalian neocortex comprises a diverse population of excitatory neurons that perform distinct roles in the neocortical circuit. These neurons are born from a heterogeneous population of progenitor cells during embryonic development and it is increasingly being recognised that individual progenitors can impart specific functional characteristics to their offspring. For example, clonally-related sister neurons in mouse neocortex are biased to form gap junctions with one another early in development, to form synaptic connections with one another as they mature, and to show similar response properties in the adult. This highlights a fundamental role for neuronal lineage in the formation of precise neocortical connectivity. However, the extent to which neuronal phenotype is determined by lineage, and the process by which this arises, is not fully understood. Nor is it known whether similar lineage relationships exist in the human neocortex. Deciphering neocortical lineage relationships has been limited by the techniques available to identify and manipulate clonally-related neurons.
In this thesis I have developed novel molecular tools for the identification and manipulation of clonally-related neurons and refined their use by in utero surgery in mice. I first developed a retrovirus encoding Cre recombinase and demonstrated that this can be successfully combined with reporter mice to capitalise on optogenetics for functional studies. In an effort to establish a reliable and unequivocal method of identifying clonally-related neurons, I then constructed a retrovirus encoding genetically-distinct RNA barcodes. I confirmed that the RNA barcode could be reliably retrieved from single neurons and used to determine clonal relationships in mouse neocortex and in an in vitro model of human neocortex derived from induced pluripotent stem cells (iPSCs). By comparing the dendritic morphology of barcoded mouse neocortical neurons, I was able to demonstrate that the dendritic arbor of clonally-related neurons is more similar than control neurons derived from different progenitors. This may contribute to specific patterns of synaptic connectivity amongst clonally-related neurons. Within the iPSC system, I demonstrate the utility of the retroviral RNA barcode and revealed that clonally-related human neocortical cells exhibit a higher probability of being gap-junction coupled. These studies advance our understanding of lineage relationships in neocortical excitatory neurons in mouse and provide the first evidence that human neocortical clones exhibit similar functional relationships to those observed in the rodent neocortex.

Advisor: Akerman, C
Newey, S
Streamlined synthesis of taxol analogues

This thesis centres on the synthesis of taxol analogues via late-stage hydroxylation with P450 enzymes. To accomplish this, the taxane core, specifically taxa-4(5),11(12)-dien-2-one, was synthesised by classical synthetic methods, and subsequently oxidised using P450BM3 mutants.
Chapter 1 introduces enzymatic catalysis, and the advantages and disadvantages of its application to organic synthesis. Additionally, an overview of taxol, including its discovery, mode of action, biosynthesis and large-scale production, and a summary of the previously reported approaches to the taxane core are described.
Chapter 2 details the problems encountered and solutions implemented when reproducing Baran’s route to taxa-4(5),11(12)-dien-2-one. Furthermore, approaches to some of its intermediates and an alternative route to taxa-4(5),11(12)-dien-2-one, which is based on Baran’s, are discussed.
Chapter 3 describes the development of a new, practical and short synthetic route to taxa-4(5),11(12)-dien-2-one which, ultimately, led to 1,3-di-epi-taxa-4(5),11(12)-dien-2-one. Additionally, the application of this route to the synthesis of a model compound and attempts to convert this racemic synthesis into an enantioselective route are reported.
Finally, the enzymatic oxidation of taxa-4(5),11(12)-dien-2-one and related molecules using P450BM3 mutants is explored in Chapter 4. A preliminary study to determine the substrate enantioselectivity of the mutants is also described, along with the biological assays of the oxidised compounds produced during the study.

Keywords: Organic chemistry
enzymatic catalysis
biocatalyisis
Late stage C-H functionalisation
oxidation with P450 enzymes
synthesis
Advisor: Robertson, J
Developing optogenetics for use in vascular research

Optogenetics is a recently established experimental technique that involves the heterologous expression of light sensitive proteins (opsins) in mammalian cells to modulate cell function. One of the most commonly used opsin is the blue light gated depolarising channel, channelrhodopsin-2 (ChR2). Optogenetics involving ChR2 has revolutionised the field of neuroscience by enabling the definition of novel brain circuitries. Optogenetic control of the electromechanical coupling in vascular smooth muscle cells (SMCs) is now emerging as a powerful research tool with potential applications in drug discovery and therapeutics. However, the exact ionic mechanisms involved in this control remain unclear. The overall aim of this thesis was to establish ChR2 for use in vascular optogenetics. The main findings of this thesis are:
1) Blue light activation of ChR2 and the ChR2 variant ChR2(H134R) led to long-lasting and non-inactivating depolarising currents.
2) Transgenic mice expressing ChR2(H134R) selectively in SMCs were generated. Isolated SMCs obtained from these mice demonstrated blue light induced depolarising whole-cell currents. Fine control of artery tone was attained by varying the intensity of the blue light stimulus. This arterial response was sufficient to overcome the melanopsin-mediated light-depended arterial relaxation observed in the presence of contraction-eliciting agonists.
3) Pharmacological analyses revealed that Ca2+ entry through voltage-gated Ca2+ channels, and recruitment of plasmalemmal depolarising channels (TMEM16A and TRPM) and intracellular IP3 receptors were mandatory for the ChR2(H134R)-mediated arterial response to blue light at intensities &LT;~0.1 mW/mm2. Light stimuli of greater power evoked a significant Ca2+ influx directly through ChR2(H134R) and produced dramatic intracellular alkalinisation of the SMCs. The light intensity range that enables optical control of arterial tone primarily through the recruitment of endogenous channels and without substantial alteration of intracellular pH, was identified. Within this range, mice expressing ChR2(H134R) in SMCs are a powerful experimental model for achieving accurate and tuneable optical voltage-clamp of SMCs and finely-graded control of arterial tone, offering new avenues to the discovery of vasorelaxing drugs.
4) The Cl--selective ChR2 mutant iChloC mediates depolarising currents while also preventing H+ or Ca2+ fluxes. This mutant was also found to have enhanced sensitivity to blue light. This new optogenetic tool could be a good candidate for an improved mouse model of vascular optogenetics.
To conclude, the work presented in this thesis represents an in depth analysis of the use of ChR2(H134R) in vascular optogenetics. The cellular mechanism linking ChR2(H134R) opening with contraction of vascular SMCs was defined. Additionally, the limitations of the current optogenetic mouse model have been identified, and iChloC was shown to resolve these limitations. This study established vascular optogenetics as novel research tool vascular physiology and pharmacology.

Advisor: Tammaro, P
Cybercrime: the industry of anonymity

The central theme of this thesis is that cybercrime has matured into a large profitdriven industry. Hobby hackers still exist and hacktivists have attracted some attention in recent years, but a very significant component of contemporary cybercrime is now financially motivated. While its goods and services are usually illicit, the cybercrime industry operates according to the same broad principles of industrial organisation observed across numerous other contexts. But the development of this industry is somewhat puzzling. Ostensibly much cybercriminal cooperation takes place online. As a result, cybercriminals are often partnering with online criminals whose true identities are unknown to them. They also have no means of physical enforcement should deals go awry. Partnering with conventional criminals, who operate outside the protection of the state, already appears to present significant challenges for cooperation. The challenge of anonymity makes cooperation even more difficult and suggests that cybercriminals would often act alone, or in small groups. Instead a large successful industry has formed. This thesis first addresses the industrialisation of cybercrime. It then addresses this puzzle of how cybercriminals have overcome the challenges to cooperation to build an industry on such a scale.

Advisor: Varese, F
The role of DOT1L in MLL-AF4 leukaemia

DOT1L is a methyltransferase which has been shown to methylate H3K79 (Ng et al. 2002; Feng et al. 2002; Lacoste et al. 2002). DOT1L and H3K79me have been shown to be involved in active transcription and in particular has been shown to be important for MLL-AF4 leukaemia, where high levels of H3K79me are found at MLL-AF4 gene targets (Krivtsov et al. 2008; Bernt et al 2011). The exact mechanism of how DOT1L is recruited to MLL-AF4 gene targets leading to high levels of H3K79me is currently unknown, especially as it has been demonstrated that AF4 and DOT1L exist in mutually exclusive complexes (Leach et al. 2013; Yokoyama et al. 2004; Biswas et al. 2011). In addition to the recruitment mechanism of DOT1L, the function of H3K79me remains unclear. In this thesis, the recruitment mechanism of DOT1L at MLL-AF4 gene targets was investigated using the in vivo TetR-recruitment system (Blackledge et al. 2014). Using this, it was found that DOT1L complex members AF9, ENL and AF10 were sufficient for DOT1L recruitment, in addition to PAF1, a member of the PAF1 elongation complex. To investigate the function of H3K79me at MLL-AF4 gene targets, it was necessary to identify a set of gene targets which were dependent upon H3K79me for transcription. To do this, the DOT1L inhibitor, EPZ-5676, was employed to treat an MLL-AF4 leukaemia cell line (SEM) followed by Nascent RNA seq and ChIP rx seq. From this a set of hypersensitive MLL-AF4 gene targets were identified. Importantly, Capture-C, ATAC seq and ChIP seq revealed a subset of hypersensitive targets with a putative intragenic enhancer. Following EPZ-5676 treatment, Capture-C revealed a disruption in the interaction between the putative intragenic enhancer and promoter of some MLL-AF4 hypersensitive genes targets. This provides evidence for a novel, context dependent role of H3K79me which may be involved in enhancer-promoter interactions and function at a subset of hypersensitive MLL-AF4 gene targets.

Advisor: Milne, T
Roberts, I
Ethical business: an ethnography of ethics and multiplicity in commercial settings

This thesis is a study of ethics and multiplicity as found within contemporary commercial settings. Drawing on Science and Technology Studies (STS) sensibilities and ethnographic-style research, the thesis proposes that current ethical phenomena should be understood as a user-enacted chimerical object: an object that is multiple in its ontology and as much enacted by what it is, as what it is not.
This research is particularly pertinent now because the term 'ethical' has become commonplace in modern Western life, including crucially within commercial activities. In certain uses, doing ethics becomes synonymous with doing business. Despite the increasing prevalence of what is considered 'ethical business', the exploration of how the term is appropriated and enacted remains largely under-examined.
Through examination of research material gathered during extensive ethnographic studies in three self-avowedly 'ethical organisations' - an ethical start-up, an ethical confectionery company, and an ethical consultancy - the thesis addresses this research gap. By focusing on the users of ethical business, the investigation questions traditional market assumptions of homogeneity within producing organisations, the supposed linear transfer of ethical knowledge, what we can know about 'users', and the genesis of novel ethical realities. Through this questioning the thesis provides new insights on the ethical object. The thesis additionally builds upon questions of how far we can push the boundaries of what we can know about knowledge, and whether it is possible to bring the mess of investigation back into the reporting. Developing previous applications of constitutive reflexivity, the research symmetrically investigates the appropriateness of my application of STS sensibilities to ethical business as a new research area, and interrogates my thesis as an ethical object in order to address the underlying question(s) of whether 'STS means ethical business?'

Keywords: Management Studies
Ethnography
Ethical Trading
Multiple Realities
Science and Technology Studies
Ethical Business
Chimera Realities
Multiplicity
Anthropology of Business
Chimera Object
Business Studies
Modern Commercial Setting
Advisor: Woolgar, S
Analysis of two pore channel proteins in Dictyostelium development

Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular responses including development and proliferation. Calcium (Ca2+) is stored in both neutral and acidic stores and its release through gated channels has been implicated in regulating development in Dictyostelium discoideum. This thesis aims to understand the roles of the calcium channel proteins, in particular the two-pore channel proteins (TPCs), found on acidic stores in Ca2+ signalling during Dictyostelium development.
Bioinformatic analysis indicates conservation of a gene encoding an orthologue of TPC2 in Dictyostelia and, similar to plant TPCs, a Ca2+ sensing domain is predicted along with a novel potential calmodulin binding site. To investigate the role of intracellular Ca2+ channels, a series of strains was generated, disrupted in one or more of genes encoding the channels TPC2 and mucolipin (TRP-ML), predicted to be located on acidic stores, and IplA, located on neutral stores. All disrupted strains, including one lacking all three channels, are able to complete development. However, strains lacking TPC2 show a pronounced delay in early development, correlating with reduced expression of some early developmental genes. Vesicles derived from tpc2-null cells show normal Ca2+ release compared to parental cells but an increased rate of Ca2+ uptake. During early development, the pH of acidic vesicles is increased in the absence of TPC2. However development of tpc2-null cells showed increased sensitivity to weak bases in producing fewer aggregates but resistance to sodium chloride and weak bases in later development suggesting a complex role for TPC during development.
In vivo cytosolic Ca2+ responses were analysed in strains expressing an ultra-sensitive Ca2+ indicator YC-Nano 15. Growing tpc2- and iplA- cells have lower basal cytosolic Ca2+ than parental Ax2 cells. Intercellular Ca2+ waves were observed in aggregates from Ax2, mcln- and tpc2- cells but were greatly reduced in iplA- aggregates, as was the increase in cytosolic Ca2+ in response to extracellular cAMP. In tpc2- aggregates, wave frequencies were reduced and the response to cAMP addition abolished after treatment with caffeine, a known adenylyl cyclase inhibitor in Dictyostelium.
This work demonstrates that TPC2 plays a role in the early stages of Dictyostelium development. TPC2 is important for pH regulation in acidic vesicles and cytosolic Ca2+ levels, either or both of which could influence development either directly or via changes in early developmental gene expression.

Keywords: Calcium channels
Dictyostelium
Cell signaling
Advisor: Pears, C
Galione, A
Observing bright stars and their planets from the Earth and from space

The discovery and characterization of extrasolar planets is a leading frontier of science, which is limited by our ability to extract real astrophysical signals masked by systematic noise. In this Thesis I develop techniques for self-calibration in optical astronomy, in both imaging and photometry, applicable both to observations from the ground and for space telescopes, and apply these to searching for exoplanets.
Kernel phase interferometry is a method for improving high angular resolution astronomical imaging by cancelling out the effects of the turbulent atmosphere. I derive a generalization, kernel amplitude, to correct also for the effects of scintillation, or twinkling. I go on to demonstrate kernel phase for the first time from the ground using the Palomar Hale 200-Inch Telescope, as a test case for the extreme adaptive optics instruments SPHERE and GPI.
The Kepler satellite and its successor, the K2 mission, have been crucial to our understanding both of exoplanets, and via asteroseismology, of stellar physics. The success of K2 depends on correcting for its unstable pointing. Using semi-parametric Bayesian statistical models to overcome this issue, I report the discovery of 145 new transiting planet candidates in data from Campaigns 5 and 6 of the K2 mission. Furthermore, with two novel techniques, 'smear' and 'halo' photometry, which dramatically extend the dynamic range of Kepler and K2, I recover light curves of bright stars that were previously too saturated to study, and definitively detect variability in the seven naked-eye stars in the Pleiades cluster. These new data analysis approaches enable the K2 and TESS space missions to discover planets transiting the nearest and brightest stars, which will be ideal targets for the coming era of exoplanet characterization.

Advisor: Aigrain, S
The in-vivo study of pain in neuromyelitis optica

A thesis exploring the aetiology of pain in neuromyelitis optica (NMO).

Neuromyelitis optica is a severe autoimmune neuroinflammatory disorder characterised by longitudinally extensive myelitis and severe optic neuritis. An oft-neglected symptom of the condition is severe, intractable chronic pain that can be detrimental to quality of life and is marked out by its severity and prevalence in comparison to the related disorder, multiple sclerosis.
The experiments within this thesis make use of both conventional and advanced MRI techniques applied to the spinal cord and brain, and 1H NMR spectroscopy of blood plasma to explore the mechanisms driving chronic pain in NMO. The principle findings are:

i. Thoracic lesions are associated with greater pain, irrespective of lesion length or cervical lesion volume. They are associated with spinothalamic tract damage in the cervical cord that correlates with the severity of pain. A possible autonomic aetiology is proposed.
ii. Periaqueductal grey (PAG) to dorsolateral prefrontal cortex and to pregenual anterior cingulate cortex functional connectivity is correlated with pain severity, conversely PAG to rostroventromedial medulla connectivity is negatively associated with pain severity. Disruption of descending pain modulatory circuits is considered.
iii. PAG glutamate concentration is negatively correlated with pain scores and is higher in low pain patients compared to controls and high pain patients. The discussion includes consideration of pain vulnerability.

Chronic pain in NMO can devastate lives. The studies undertaken in this thesis are some of the earliest MRI imaging studies directed at understanding pain in NMO and the association of pain with thoracic lesions and the aberrations in the descending pain modulatory network are novel findings. There is more to be done and my hope is that this body of work will serve as a stepping-stone to a better understanding of chronic pain in NMO and the future development of effective treatments.

Keywords: neuroinflammation
MRI
pain
Devic's
neuromyelitis optica
Advisor: Palace, J
Tracey, I
Kong, Y