VictorJuiz/thesis huggingface dataset:
Investigating glyphosate tolerance mechanisms in the liverwort Marchantia polymorpha

The evolution of herbicide resistance in weeds is a threat to global crop yields. The aim of this thesis is to increase our understanding of the evolution and mechanisms of herbicide resistance. To achieve this, I first determine the phylogenetic relationships of both the ATP-binding cassette (ABC) transporter and uridine diphosphate glycosyltransferase (UGT) protein superfamilies within the Archaeplastida lineage. Proteins from these families confer non-target site resistance (NTSR) in populations of weeds. I also determine that the liverwort Marchantia polymorpha is naturally tolerant to the non-selective herbicide glyphosate. Using enzyme activity assays, I demonstrate that glyphosate tolerance in M. polymorpha is not due to a resistant version of the glyphosate target EPSPS. Instead, I discover a novel form of glyphosate tolerance conferred by the peptidoglycan biosynthesis enzyme MurA, which is structurally similar to EPSPS. By expressing MurA in the angiosperm Arabidopsis thaliana and generating murA loss-of-function mutants in M. polymorpha, I determine that MurA confers glyphosate tolerance independently from its function in peptidoglycan biosynthesis. I show that MpMurA catalyses the same reaction as EPSPS, transferring the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P) forming 5-enolpyruvylshikimate-3-phosphate (EPSP) product. However, like EPSPS, it is also inhibited by glyphosate. I therefore propose that MurA confers glyphosate tolerance either through a mechanism analogous to target-site overexpression or by binding glyphosate, making it less available to inhibit EPSPS. Through a metabolomic analysis, I also show that the metabolic response to glyphosate treatment in M. polymorpha differs from that in A. thaliana. This may be a result of, or contribute to, glyphosate tolerance in M. polymorpha. These results therefore demonstrate a novel form of glyphosate tolerance and determine the physiological impacts of glyphosate treatment that may contribute to its mode of action.

Keywords: glyphosate
glyphosate tolerance
Marchantia polymorpha
herbicide resistance
Advisor: Dolan, L
Rhodium-catalysed regio- and enantioselective Suzuki-Miyaura cross-coupling reactions

In this thesis, we describe a range of rhodium-catalysed regio- and enantioselective Suzuki-Miyaura type cross-coupling reactions. Brief elaborations of products and modifications of drug molecules and natural products are demonstrated to show the utilities of these methodologies.
In Chapter 1, transition-metal-catalysed asymmetric Suzuki-Miyaura cross-coupling reactions are introduced in sequence, categorised by transition-metal type. Recent highlights in this area are discussed and the future of asymmetric Suzuki-Miyaura cross-coupling reactions is outlined.
In Chapter 2, we present the rhodium-catalysed regio- and enantioselective Suzuki-Miyaura cross-coupling reactions between vinylethylene carbonate and (hetero)arylboronic acids. We proposed that the chelation of substrate would help the regiocontrol. Combined experimental and computational mechanistic studies reveal the formation of a six-membered rhoda-1,3-dioxa-2-one intermediate after SN2’ oxidative addition during catalytic cycle, which is crucial for the regio- and enantiocontrol.
In Chapter 3, the method shown in Chapter 2 is elaborated by changing the ligand and solvent. The reaction scope now includes methyl-substituted vinylethylene carbonate. Notably, this represents the first examples of acyclic quaternary stereogenic centres formations via Rh-catalysed cross-coupling reactions between racemic tertiary electrophiles and achiral nucleophiles. The synthetic utilities of this method are demonstrated with formal syntheses of drug candidate and natural products (+)-epilaurene and (−)-cuparene.
In Chapter 4, we describe a series of general and modular regio- and enantioselective allylic arylation reactions. Unlike Chapter 2 and Chapter 3, the regioselectivities in this chapter are completely controlled by the rhodium/ligand complex. This method consistently produced arylation products in high levels of yield and selectivities (typically >90% yield, >95% ee and >20:1 r.r.). The synthetic applicability of these transformations is demonstrated with late-stage-functionalisations of naproxen, (−)-citronellal, (−)-perillaldehyde and estrone.

Keywords: rhodium
regioselective
asymmetric catalysis
Suzuki-Miyaura coupling
Advisor: Fletcher, S
Investigating a novel catecholamine-associated cardiomyocyte subpopulation

Excessive sympathetic stimulation of the heart, via catecholamines, is involved in a range of cardiac disease, from takotsubo cardiomyopathy to heart failure. Canonically, there are two main sources of catecholamines for sympathoexcitation. 1) Adrenaline, secreted by chromaffin cells in the adrenal medulla and carried by the bloodstream to the heart. 2) Noradrenaline, released by sympathetic nerve terminals within the heart. However, there is a growing body of literature highlighting a non-neuronal cellular source of catecholamines within the heart. This project aimed to characterise a cellular cardiac subpopulation associated with the gene expression of a catecholamine-synthesising enzyme. Mouse embryos and hearts from embryonic day 8.5 to neonates were single-cell RNA-sequenced. RNA velocity, a form of in-silico lineage inference, in combination with differential gene expression analysis and pseudotime inference suggested first heart field splanchnic mesoderm as the developmental origin for this subpopulation. An optogenetic lineage tracing mouse line for the gene encoding this catecholamine-synthesising enzyme was generated. Langendorff-perfused ex-vivo hearts from this mouse line were subjected to pulsed light stimulation. In addition, fluorescent microscopy of immunostained heart sections and genetic fluorescence was performed. These methods showed that the subpopulation was spatially distributed in the ventricular endocardium, subendocardium, atria and interventricular septum. The refractory periods of the cells were not significantly heterogeneous across the heart. The cells of the subpopulation had the size, morphology, striations and protein expression of cardiomyocytes. The subpopulation was shown to be proximal to sympathetic nerve terminals. Characterising subpopulations associated with intrinsic cardiac catecholaminergic signalling could lead to a better understanding of, and potential therapeutics for, cardiac pathologies where excessive catecholaminergic signalling plays a key role.

Keywords: sympathoexcitation
catecholamines
RNA sequencing
lineage inference
heart
electrophysiology
optogenetics
RNA velocity
Advisor: Lei, M
Paterson, D
Seifert fibered spaces: triangulation and recognition

In this thesis, we study 3-manifolds from an algorithmic point of view. Our motivation is the conjecture that 3-manifold homeomorphism is in NP. The hope is that if one can prove this conjecture in the special case of geometric 3-manifolds, then one can use geometrisation to prove the general statement. This thesis predominantly concerns itself with the Seifert fibered case.
The idea is to show that we can bound the size of characteristic topological structures in an arbitrary triangulation. To that end, we first study the size of minimal triangulations of Seifert fibered spaces. We show that we can realise certain important curves, the singular fibres, as subcomplexes by increasing the size of an arbitrary triangulation by a linear factor. We use this result to give bounds on the triangulation complexity of Seifert fibered spaces with non-empty boundary, which is the minimum number of tetrahedra in a triangulation of a manifold. We give a formula for the triangulation complexity which is correct up to a multiplicative factor. We also give an optimal bound for the triangulation complexity of a connect sum.
We then turn our attention to the 3-manifold homeomorphism problem. We first show that recognising (closed) Euclidean and Nil 3-manifolds, and deciding if a given set of Seifert data is correct for them, is in NP. We then consider the case of recognising Seifert fibered spaces with non-empty boundary. We prove that the recognition problem for Seifert fibered spaces with non-empty boundary is in NP, and that deciding whether a given Seifert fibered space with non-empty boundary admits certain Seifert data is in NP ∩ co-NP. To do this, we give bounds on the size of a complete collection of normal annuli in such a Seifert fibered space. The technique we use for this bound, which is the theory of normal surfaces in split handle structures, comprises half this chapter and can be used generally to give a bound of the form c|T|² on the weight of a complete collection of (disjoint) incompressible normal surfaces in an arbitrary triangulation T.

Keywords: 3-manifold algorithms
triangulation complexity
normal surfaces
Seifert fibered spaces
Advisor: Lackenby, M
Estimation of NMR signals in the time domain: methodology, applications and software

Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique employed in many scientific disciplines that is able to provide insights into the structures and dynamics of chemical species. To maximise the utility of NMR, appropriate data treatment and analysis is necessary. The conventional route to extracting quantitative information from the raw experimental data — the free induction decay (FID) — is to convert it to an NMR spectrum, through application of the Fourier transform (FT). NMR spectra provide a human-interpretable representation of data; trained practitioners are able to rationalise the appearance of a given spectrum by mapping its component peaks to chemical environments in the sample from which the dataset was acquired. However, the FT suffers from poor resolution, with peaks of similar frequencies exhibiting over- lap. Disentangling the information associated with such peaks is not feasible using typical methods such as integrating user-defined regions of the spectrum. As an alternative approach, parametric estimation techniques aim to provide a detailed description of each signal which contributes to the FID. These methods have been shown to perform effectively even in scenarios where significant spectral peak overlap exists.
This thesis focusses on the development of a parametric estimation method for the analysis of FIDs derived from solution-state NMR experiments. The guiding principle behind the method is that it should require as little user input as possible, while being able to provide accurate and reliable signal estimates. Beyond simply providing a breakdown of individual signal components, many useful applications may be realised when estimation techniques are employed. The initial motivation for this work was to develop a procedure for the generation of broadband homode- coupled (pure shift) NMR spectra with desirable properties from 2DJ datasets. Furthermore, a means of analysing datasets such as those from inversion recovery (Τ₁), Carr-Purcell-Meiboom- Gill (CPMG) (T₂), and diffusion experiments, in which each FID exhibits a variation in its amplitude, is presented. The last application described is a means of producing phased, ultra-broadband NMR spectra from an experiment comprising a single frequency-swept (chirp) excitation pulse.
The methods presented in this thesis are incorporated into a software package written in the Python programming language, called NMR estimation in Python (NMR-EsPy), of which more information can be found at https://github.com/foroozandehgroup/NMR-EsPy.

Keywords: signal processing
python programming
chirp pulse
NMR
numerical optimisation
diffusion NMR
2D J-resolved NMR
matrix pencil method
Advisor: Probert, F
Claridge, T
Foroozandeh, M
Hellenistic mouldmade relief bowls in Thessaly

This thesis offers the first comprehensive and comparative study of Hellenistic mouldmade relief bowls discovered in Thessaly. The material under examination originates from old (1906-1916) and more recent excavations (1970s – to this day) across 23 Thessalian sites, conducted under the auspices of the Archaeological Society of Athens (under A. Arvanitopoulos) and the local Ephorates of Antiquities. Out of the 616 complete and fragmentary bowls examined here (which are recorded in 568 catalogue entries), only a few groups have been partially published to this day.
The aim of this study is to offer a regional perspective regarding the production, distribution and consumption of a very popular category of fine-ware, dispersed across the Hellenistic world. The examination of relief bowls within their excavation contexts (chapter.II) enables the identification of production centres and local workshops across the region. At the same time, it sheds light to the offering and use of such vessels in funerary assemblages. The interpretation of the bowls’ iconography (chapter.III) explains how literacy, education, theatricality, myths, cults, and everyday life were expressed in aspects of Thessalian material culture during the Hellenistic period. Emphasis is placed on the relationship of mouldmade bowls with other contemporary and earlier forms of art. Recording and discussing various types of inscriptions occurring on the bowls (chapter.IV) enhances our understanding of their relationship with manuscripts, of local dialects and paleographical trends. The classification of the bowls into typological groups and comparisons with other regional productions, mostly of mainland Greece and Asia Minor, allows the establishment of a typological sequence for the Thessalian production (chapter.V). Identifying imported specimens from other major production centres showcases the place of Thessaly within a network of Aegean pottery workshops, and the significance of its cosmopolitan harbours and land-routes as transit centres (chapter.VI). Ultimately, this research improves our understanding of material culture in Thessaly during the last three centuries BC and comprises an original contribution to Hellenistic pottery studies.

Keywords: Hellenistic pottery
mouldmade relief bowls
Thessaly
Advisor: Stamatopoulou, M
Is native speakerism a privilege in TESOL employment? A comparative study of self-reported hiring discrimination in native and non-native ESL teachers across Spain and the UK

A quarter of a century has passed since the launch of The NNEST Movement, a consequential movement deriving from years of discrimination towards NNESTs, yet it appears very little has changed in the world of TESOL. This study examines whether TESOL employment and hiring practices are still subjected to native speakerism ideologies in Spain and the UK. This comparative mixed methods study examines self-reported discrimination of NESTs and NNESTs in Spain and the UK to temperature check experiences in 2023. A questionnaire was administered to n=210 ESL teachers across Spain and UK (n=134 NEST, n=76 NNEST), with 17 Likert-scaled statements and one optional qualitative question. Then, four follow-up semi structured interviews were conducted to gather further qualitative data from NEST and NNESTs. A principal component analysis was conducted, followed by independent samples t-tests, showing that participating NNESTs suffer significantly more (p<.001) self-reported discrimination when hired as an ELT than NESTs. Findings further show that NNESTs in Spain suffered significantly more (p<.001) discrimination than NNESTs in the UK. Thematic analysis was conducted on the qualitative data, which further triangulated this data and showed parallel findings. Suggestions to shape the future of hiring for TESOL teachers are made with calls to action for teachers, recruiters, and policymakers.

Keywords: NNEST
the NNEST movement
TESOL employment
native speakerism
Advisor: Rose, H
Targeting PDK and PARP to improve radiation response in preclinical models of non-small cell lung cancer

Approximately 50 % of all NSCLC patients receive radiation therapy during the course of their disease, and the quest to enhance the radiation response of these patients is still ongoing. Here, we investigate the potential to increase the radiation response of NSCLC by targeting the PI3K signalling pathway with PDK1 inhibitors and/or targeting DNA repair with the PARP inhibitor olaparib. The in vitro radiosensitising effect of the PARP inhibitor olaparib and the PDK1 inhibitors BX912 and GSK2334470 was determined using clonogenic survival assays. The combination of PDK1 inhibitors with radiation did not increase the radiosensitivity of NSCLC cell lines, irrespective of the TP53 status. However, a significant radiosensitisation was observed by addition of olaparib to radiation treatment (SER50 1.6 to 2.2). Immunofluorescent quantification of γH2AX foci formation indicated that the radiosensitisation effect of olaparib could be explained by inhibition of DNA repair. Subsequently, we demonstrated that olaparib enhances the radiosensitivity of a poorly vascularised NSCLC xenograft model by decreasing DNA repair and increasing apoptosis. In contrast, a well vascularised NSCLC model was not sensitised by addition of olaparib to radiation treatment. The effect of olaparib on in vivo vascular perfusion was assessed using dynamic contrast enhanced-magnetic resonance imaging. No significant differences were observed between olaparib and vehicle treated xenografts, suggesting that the radiosensitising effect can be mainly attributed to inhibition of DNA repair. We demonstrated that olaparib is a potent radiosensitiser in a poorly vascularised lung cancer model but further in vivo experiments are warranted to identify the subset of patients that would benefit from olaparib addition to radiation therapy.

Keywords: PDK inhibitor
NSCLC
radiotherapy
Olaparib
Advisor: Ryan, A
The application of nuclear magnetic resonance to investigate biological processes


To understand disease, we need to understand the behaviour of individual biomolecules, how they interact, and how this complex ’dance’ gives rise to organism level phenotypes. Proteins are an important class of molecules on which to focus, the ’machinery’ of the cell. It is clear that to understand how they perform their various functions, we need a detailed description of not only their ’static’ or average structure, but also the dynamical motions that govern how they move. Biological NMR, as a solution state, atomic resolution technique, is very well suited to this characterisation. Despite their power, biological NMR techniques are not widely applied, mostly because the barrier to entry is very high when compared to alternative techniques such as cryo-EM. In this thesis, a deconvolution algorithm, UnidecNMR, is developed to automate ’peak picking’, an aspect identified as a rate limiting step in all NMR analysis. This step is manual on all but the simplest targets. UnidecNMR is demonstrated to be either as good, or better, than a human for real experimental data (chapter 2). It is then embedded into multiple pipelines to address experiment-specific problems, and by doing so, we develop novel methodology. Initially, application to 3D triple resonance data leads to near-automatic backbone and side chain assignment, together with a visualisation that lets a user clearly follow how the data maps onto the assignment (chapter 3). An application to 4D Methyl-TROSY NOE spectra and pseudo-3D CPMG relaxation dispersion data from a dynamical 396kDa oligomer unveils previously undocumented dynamics likely important for function (chapter 4). To further improve the sensitivity of triple resonance NMR data, ’Seedless’ band selective GRAPE pulses are developed, where we experimentally note that specific improvements of B1 field homogeneity compensation during NMR experiments can provide dramatic sensitivity enhancements. For example, the sensitivity of an NHSQC at 950 MHz is increased by 45%, and an HNCACO at 600 MHz by 55% (chapter 5). Finally, it formed the core of a new saturation transfer analysis pipeline for studying protein - ligand interactions. This led to atomic resolution, kinetic and structural information about a very relevant interaction between the SARS-CoV-2 spike protein and N-linked glycans found on cell surfaces (chapter 6). These applications are rendered feasible through the capabilities of UnidecNMR. By providing this software to the community, and demonstrating what can be achieved using these approaches, I hope this thesis will lower the barrier for entry to bring biological research into the field to enable a greater understanding of protein dynamics and hence deepen our understanding of disease.

Advisor: Baldwin, A
Investigating centriole orientation in the Drosophila embryo

Centrioles are barrel shaped organelles that are known to nucleate microtubules (MT) contributing to the mitotic spindle and act as basal bodies during ciliogenesis. Mammalian centrioles possess distal appendage proteins (DAPs) which allow them to anchor to the cell cortex via their distal end. This distal ended orientation often plays a functional role within the cell. In many other cell types, centrioles are specifically positioned close to the cell cortex. However, we do not yet understand how centrioles may be oriented in these systems. Our lab has long observed that in Drosophila embryos, which do not possess DAPs or make cilia, that most centrioles appear upright. Using a fixed assay and differential fluorescence labelling, I show that most centrioles have a preference to be upright with their distal end pointing towards the cell cortex, much like in mammalian cell types where centriole orientation is known. In collaboration with a statistician, we develop an assay to image embryos in real-time using super-resolution microscopy and analyse spatiotemporal aspects of their orientation using a neural network. Initial analysis further showed that centrioles have a preferential upright, “Ring” oriented state that is mostly stable over S-phase. Using a combination of drug- and candidate-based approaches, my data suggests that centrioles may indirectly be pushed and pulled into different orientations gradually by a balance of surrounding forces from microtubules and the centriole’s surrounding pericentriolar material. However, I also uncover a possible role for scaffold protein Centrosomin (Cnn) and distal cap proteins, most strikingly distal cap protein Cep97, which may be instrumental in maintaining the distal ended orientation of centrioles. My data strongly suggests that distally oriented centrioles are the default state in Drosophila embryos and there is an active mechanism maintaining this orientation.

Keywords: centrioles
ciliogenesis
drosophila
cep97
orientation
centrosomes
cnn
machine learning
microtubules
centrosomin
drosophila embryo
cp110
neural network
distal appendage proteins
Advisor: Raff, J