VictorJuiz/thesis huggingface dataset:
Re-evaluating the basis of clonal selection in germinal centres

Effective humoral responses rely on the development of high-affinity antigen-specific B cells. However, the naïve repertoire of antibodies usually shows low affinity. B cells need to improve their affinity in secondary lymphoid structures known as germinal centres (GCs) during infections, a process known as affinity maturation. In GCs, B cells undergo iterative rounds of somatic hypermutation in the dark zones (DZs) to diversify BCRs then exit cell cycle and move back to light zones (LZs) for affinity-based selection. In the LZs, it has long been considered that only selected cells could subsequently reenter cell cycle and move back to DZs, a process known as cyclic re-entry. Affinity enhancements are thought to be conferred via T cells providing help to the highest affinity LZ B cells to initiate cyclic re-entry, however, this hypothesis was never directly tested. Using fluorescent cell cycle reporter mice, we found neither T cells depletion nor MHCII deletion abolished the cyclic re-entry initiation in LZ cells and this process was not detectably restricted by general intra-GC competition. Concurrently, using BATF as a marker for recent T-B interactions, we found B cells needed to compete in order to receive T cell mediated refuelling in an affinity dependent manner. However, this process was also limited by B cells’ intrinsic ability to acquire antigens. Consistent with the observations above, cyclic re-entry LZ cells were found to be heterogeneous at their BATF levels, further confirming cyclic re-entry initiation and receiving T cell mediated refuelling are two processes regulated independently. Our data is pointing towards a less binary selection mechanism in GCs, which confers proliferative advantage to higher affinity clones while allowing low-affinity cells to mature via more complex pathways so that antibody repertoires are matured for both affinity and clonal diversity.

Keywords: Germinal centre
Antibody affinity maturation
B cell
Advisor: Bannard, O
Delineating the causal role of the gut microbiota in experimental colitis

The gut microbiota is altered (dysbiosis) in patients with inflammatory bowel disease (IBD), although it is not currently understood whether this is causal of, or consequential to, disease development. Delineating the nature of these associations is imperative to inform microbiota-based IBD therapeutics. One such avenue is targeting of Enterobacteriaceae expansion, a dysbiotic signature common to murine and human colitis, through tungstate-mediated inhibition of Enterobacteriaceae growth. Although Enterobacteriaceae expansion is common to multiple animal models, tungstate inhibition of Enterobacteriaceae and amelioration of pathology has been predominantly shown in dextran sodium sulfate (DSS) colitis. Thus, in this thesis we aimed to replicate and expand on the therapeutic potential of tungstate in animal models, namely DSS, Helicobacter hepaticus + anti-interleukin-10 receptor (aIL-10R) and Citrobacter rodentium colitis. We found limited effects of tungstate on DSS-induced Enterobacteriaceae expansion and no effects on pathology development across all models. Thus, the ability of tungstate to ameliorate disease appears context-dependent. Although tungstate targets a feature of dysbiosis, it does not inform on the bacteria-bacteria and host-bacteria interactions which underpin disease pathogenesis. The H. hepaticus + aIL-10R model is poised to explore such interactions, as disease is dependent on the presence of other commensals, including the gnotobiotic 12-member Oligo-Mouse-Microbiota (OMM12). In this thesis, we characterise dysbiosis and intestinal metabolite alterations in the H. hepaticus + aIL-10R OMM12 model and show that E. clostridioformis YL32 is a key colitogenic member in this context. This is likely due to interactions between E. clostridioformis YL32 and H. hepaticus, as H. hepaticus upregulates putative virulence gene expression in E. clostridioformis YL32 mice, in-turn associated with changes in host inflammatory gene expression. Importantly, these interactions are therapeutically targetable as selective depletion of E. clostridioformis YL32 reverts established colitis. This work serves as proof of concept that microbiota inter-bacterial and host-bacterial interactions serve as novel therapeutic strategies for IBD.

Keywords: colitis
microbiome
Advisor: Ilott, N
Kleanthous, K
Powrie, F
Investigating the molecular role of transcription factor MAFG in circadian rhythm regulation

The molecular mechanisms underlying circadian photoentrainment are complex, comprising several regulatory factors. There is evidence to suggest differential chromatin accessibility in the master clock, SCN, in response to light at different times of the day. Several genes identified close to differential regions of chromatin accessibility are already known to play an important role within the molecular clock and are recognized to be important for entrainment. Previous studies in the lab using ATAC-seq (Assay for Transposase Accessible Chromatin) showed that chromatin near the gene Mafg (MAF BZIP Transcription Factor G) closed following a nocturnal light pulse (LP), indicating repression of the gene. Additionally, the response element to which MAFG binds, which is the AP-1 motif, was highly over-represented in the light regulated accessible chromatin profile, leading us to hypothesise that MAFG, that is part of the AP-1 transcription factor family, regulates photoentrainment through the regulation of AP-1.
To investigate this, I conducted in vitro and in vivo analyses to evaluate MAFG function in circadian entrainment. Sections of the Mafg promoter that responded by closing in response to light were cloned into a luciferase reporter plasmid and transfected into U2OS cells. Forskolin (which simulates a LP via the AP-1 pathway) reduced reporter expression, and this was further reduced in cells with siRNA- mediated Mafg knockdown. Furthermore, knockdown of Mafg in BMAL1:Luc U2OS circadian reporter cells showed a reduction in amplitude. In vivo, the baseline circadian phenotype, and photic entrainment phenotype of Mafg knockout mice was studied by housing Mafg+/+ and Mafg-/- mice in strict light/dark cycles. Periodogram analysis showed reduced amplitude of circadian period in knockout mice, suggesting that they have weak & fragmented rhythms. Mafg knockouts also showed slower re- entrainment in a 6-hour phase-advancing jetlag study and reduced phase shifting in response to a nocturnal LP (CT 16), indicating reduced photic responses in Mafg -/- animals. However, qPCR analysis of gene expression in the SCN isolated from CT16 light pulsed Mafg+/+ and Mafg-/- mice shows very little difference in a subset of the light regulated genes assessed between the genotypes. In summary, a novel role for MAFG in regulating circadian photoentrainment was shown, however discrepancies in the mechanism of action remain to be addressed.

Keywords: luciferase reporter assay
photoentrainment
molecular neuroscience
circadian rhythms
chromatin accessibility
Advisor: Jagannath, A
Novel capabilities for gas-phase laser-induced gratings

Laser-Induced Grating Spectroscopy (LIGS) is a non-intrusive optical diagnostic primarily used for temperature measurements in the gas phase. The technique boasts excellent accuracy and precision, achieved at relatively low cost and complexity, compared to similar diagnostics.
A drawback of the technique is its temporal resolution, which has generally been limited to 10 Hz without compromising the accessibility of the technique (through the use of toxic tracer species or expensive lasers). The discovery of a new LIGS tracer species, biacetyl, which enables the use of accessible high repetition rate lasers is presented. Temperature measurements, obtained at a rate of 1 kHz, and the application of the technique to study thermoacoustic instabilities, have been demonstrated.
A significant problem for LIGS is an inherently poor spatial resolution due to an elongated (in the axial direction) measurement region. A novel approach enabling simultaneous 1-D spatially-resolved LIGS measurements in the axial direction is presented. This capability can be incorporated into standard LIGS experiments with a negligible increase in cost or complexity, thus preserving the accessibility of the technique. A computational model for the approach was developed and validated. A preliminary experiment demonstrated a 93% improvement in axial spatial resolution, as well as a three-fold increase in the number of single-shot measurements obtained, when compared to a standard LIGS configuration.
To exploit the significant advantages of the technique by enhancing its accessibility, a unique transportable LIGS system (named PILOT) was designed, manufactured and characterised. The mobile, self-contained system is capable of collecting tracer-free LIGS measurements at a rate of 400 Hz. An accuracy of less than 0.37% and precision of +/-0.7% was demonstrated for temperature measurements in room air. Characterisation experiments showed that the use of an avalanche photodetector for signal detection would permit the use of the system in scattering environments. A novel systematic investigation of tracer-free LIGS in different gases highlighted the polarisability of a gas as a key indicator for performance of the technique. These findings will inform experimental design for future applications. Finally, the deployment of PILOT in a research facility was demonstrated.

Keywords: laser-induced grating spectroscopy
LIGS
temperature
optical diagnostics
Advisor: Williams, B
Computational approaches to molecular fragment elaboration

Recent advances in machine learning have led to considerable interest in their application to drug discovery, in the development of accurate predictive algorithms which can rapidly predict whether a given compound is likely to bind to a target protein, and in generative algorithms that propose novel high affinity binders in silico.
In this thesis, we propose a deep generative model for fragment elaboration for use in fragment-to-lead campaigns. We consider the problem of generating elaborations which are highly similar to a ground truth elaboration, first via reinforcement learning, where we propose a novel curriculum-based reward function, and also via the imposition of physically meaningful pharmacophoric constraints. We describe the development of a simple-to-use web application, allowing users to generate molecules with no prior programming experience and without installing anything.
Next, we describe a novel method for extracting useful and interpretable information from a target protein and providing it to a deep generative model. By computing a fragment hotspot map of the protein, which describes regions of the binding pocket that are likely to make a disproportionate contribution to binding affinity, we derive a set of pharmacophoric constraints which can be passed to our deep generative model, helping it generate functional groups which are likely to interact with the protein.
Finally, we consider the problem of virtual screening. In addition to being able to accurately identify high affinity binders, it is desirable that virtual screening models are able to identify the functional groups most responsible for binding. Assessing the ability of models to accurately attribute binding to specific functional groups is challenging due to the difficulty in specifying a ground truth which model attributions can be compared to. To address this, we propose a novel synthetic data generation process with a deterministic binding rule. First, we show that a recently proposed deep learning-based model is better able to identify important functional groups than a fingerprint-based model. Second, we demonstrate that training models on datasets which exhibit ligand-specific bias degrades the ability of the model to identify important functional groups, underscoring the importance of curating high quality datasets for virtual screening.

Advisor: Deane, C
Morris, G
New cation-mediated enantioselective transformations and catalytic enantioselective photocyclization

This thesis describes the development of novel catalytic enantioselective reactions. Chapter 1 describes the development of transformations mediated by chiral ammonium salts. The historical context of enantioselective phase transfer catalysis is provided and the key fundamental advances are detailed. The development of a cation-mediated kinetic resolution of atropisomeric biaryls is described as well as the synthesis and resolution of a range of substituted BINOLs.
The extension of the chiral cation mediated approach to the dearomatization of 2-naphthols by electrophilic amination and of indoles by C3 alkylation is also described. In both cases, good reactivity and moderate enantioselectivity were achieved.
Chapter 2 focuses on the development of a Lewis acid mediated approach to the catalytic enantioselective [6π] photocyclization of acrylanilides. The work is contextualized by a discussion of the previous approaches to enantioselective photochemical reactions covering direct irradiation, triplet energy transfer and photoredox strategies. The elucidation of competing triplet energy transfer and photoredox pathways to the same product is described alongside an account of the design and development of a photocatalyst that engages predominantly in the photoredox pathway. The reaction was tested on a range of substrates to determine the scope and limitations of the method.

Keywords: dearomatisation
atropisomerism
photocyclisation
catalysis
photochemistry
phase transfer catalysis
Advisor: Smith, M
Characterising the molecular basis of WDR82 binding in multiple contexts

Precise regulation of gene expression is essential for the development of multicellular organisms. The progression of RNAPII transcription through initiation, elongation and termination is accompanied by mRNA processing events such as capping and splicing, and requires numerous regulatory factors. The extended C-terminal domain (CTD) of the largest RNAPII subunit, RBP1, consists of YSPTSPS heptapeptide repeats that can be post-translationally modified in a number of ways, most notably by phosphorylation. These different phosphorylation states occur in distinctive patterns across genes and have been implicated in regulating various aspects of transcription and mRNA processing. WDR82 is a small WD40-repeat protein which has been shown to bind the RNAPII CTD when it is phosphorylated on serine 5 (S5P), a modification associated with transcription initiation. Removal of WDR82 from cells has profound effects on transcription genome-wide. However, interpreting these results is complicated by the inclusion of WDR82 in three independent complexes that regulate transcription in different ways; the SET1 complexes act at promoters to support gene transcription, the PNUTS-PP1 complex promotes cleavage and polyadenylation-coupled transcription termination, and the ZC3H4 complex mediates premature transcription termination. Whilst the subunit compositions of these complexes have been characterised to varying extents, the molecular basis of WDR82 incorporation into each complex is unknown. Furthermore, the molecular function of WDR82 in these different contexts has not been well characterised. It has been proposed that WDR82 mediates SET1A and ZC3H4 binding to S5P CTD, whilst its role in the PNUTS complex is less clear.
To characterise WDR82 incorporation into these different complexes, I use endogenously tagged cell lines to purify SET1A, ZC3H4, PNUTS, and WDR82 from mouse embryonic stem cells and identify their interactors by mass spectrometry. I define the constitutive components of each complex and identify additional interactors which could provide functional specificity. I then use protein structure prediction combined with in vivo validation to characterise the molecular basis of SET1A, ZC3H4, and PNUTS binding to WDR82. I discover that SET1A and ZC3H4 bind WDR82 via remarkably similar interfaces, whereas PNUTS employs a distinct binding mode which nevertheless shares some features with SET1A and ZC3H4. Finally, I begin to characterise the molecular function of WDR82 in each complex. I propose that WDR82 provides an RNAPII CTD binding adapter function to the SET1A and ZC3H4 complexes and contributes to PP1 phosphatase regulation in the PNUTS complex. Together these results characterise the different binding activities of WDR82 and its function in multiple contexts, and provide key tools for further dissection of its role in regulating transcription.

Advisor: Klose, R
Mechanistic studies of H2 oxidation and evolution catalysed by NiFe hydrogenase enzymes

The complexity of metalloenzyme catalysis has led to the development of a range of techniques, broadly separated into those that probe activity, and those that provide structural information. However, reconciling data from each of these approaches is challenging due to the differences in the physical form of samples, and that most methods cannot simultaneously provide both structural insight and information on catalytic activity.
Two IR spectroelectrochemical techniques are utilised in this Thesis to provide insight into the catalytic mechanism of [NiFe]-hydrogenases.
In situ protein film infra electrochemistry bridges the gap between activity- and spectroscopic-based measurements and has revealed that the rate determining step in H+/H2 catalysis is different for the O2-tolerant Hyd-1 and O2-sensitive Hyd-2.
A technique is developed which combines single crystal IR spectroscopy with electrochemical control to unify structural and spectroscopic information of metalloenzymes. With this technique, a complete redox characterisation of single crystals of E. coli Hyd-1 is described, demonstrating that proton transfer equilibria and electron transfer redox equilibria are maintained in crystallo. Thus the crystalline state can be thought of as a dynamic system, much like in solution.
Studies on single crystals of E. coli Hyd-2 provide evidence of the Nia-L species which was not conclusively identified before for Hyd-2. The observation of the Nia-C species in frozen solution converting into Nia-L upon illumination confirms the assignment of the Nia-L species in Hyd-2.
Overall this work highlights the necessity for utilising a variety of techniques when investigating metalloenzyme catalysis. The single crystal IR microspectroscopicelectrochemical technique developed in this Thesis will provide valuable opportunities for unifying the structural and spectroscopic insights on the catalytic mechanisms of hydrogenases and other metalloenzymes in the future.

Keywords: hydrogenase
IR spectroscopy
spectroelectrochemistry
Numerical methods for unsteady film cooling in gas turbine

Large eddy simulation (LES) of film cooling flows are focused on, not only because the complexity of film cooling flows brings challenges to accurate numerical simulations, but also because to improve the RANS model which is the main source of inaccuracies of RANS, a systematic set of Reynolds stress and turbulent heat flux data are needed. Achieving high accuracy of prediction for the film cooling flows mandates the exploitation of all the available elements of numerical modelling and computation methods. These methods as a whole should be able to result in reliable budgets of Reynolds-stress tensor and heat flux tensor in scale-resolving simulations. The LES framework for this flowfield is designed by including key ingredients of accuracy -- i) a meshing system (automatic hexahedral mesh generator) resolving small scales and representing complex geometries, ii)a high-resolution scheme (a thrid-order limiter), handling unwanted oscillations while retaining high order accuracy; iii)an efficient inflow generation (synthetic generator based on eigen-decoposition from realistic approximations of the covariance tensor ) minimizing the required inlet development length. Results reveal that the set of modelling methodologies is valid for calculating film-cooling performance in terms of velocity, turbulence, heat flux profiles, and discharge coefficient for fan-shaped and cylindrical holes. The energy-carrying turbulence structures in and near the cooling holes are illustrated from a standpoint of turbulent Reynolds stresses. The mechanisms by which energy is extracted from the mean flow and distributed among the normal Reynolds stresses are highlighted. A qualitative summary of the structure of the film in terms of jet edge, potential core, the lower edge of the jet, and confined region is proposed on the basis of their positions and relative magnitudes of the mean temperature, turbulent heat fluxes, and temperature variance in each region. A scaling law for the outer part of a three-dimensional wall jet has been proposed. Similarity patterns of velocity, turbulent heat fluxes, non-dimensional temperature, Reynolds stresses, and budget profiles are discussed. Far downstream of the cooling films, the Reynolds stress budgets near the wall present similarities with the Reynolds stress budgets in a boundary layer, while the Reynolds stress budgets further away from the wall resemble budgets in a free-shear flow. Results show that the dynamic balance of all budget terms is significantly influenced by the cooling hole shape, while each budget term may scale with the velocity defect, temperature deficit, and the normalized streamwise distance to the power of an exponent dependent on the hole shape.

Keywords: gas turbine
scaling law
LES
Reynolds stress budgets
high order scheme
discharge coefficient
Film cooling
turbulence
self-similarity
turbulent heat flux
eigenmode decomposition
inflow generator
conjugate heat transfer
Reynolds stress
turbulent heat flux budgets
fan-shaped
boundary layer
hexahedral mesh
Advisor: Di Mare, L
Endothelial colony-forming cells as a platform for early cardiovascular risk assessment

The human endothelial colony-forming cells (ECFCs), a subpopulation of endothelial progenitor cells (EPCs), offer a potential platform for cellular assessment due to their remarkable angiogenic expansion, regenerative, and self-renewable potentials in vitro. This thesis investigated how ECFC function and proteome profiles are altered in young adults with elevated cardiovascular disease (CVD) risk factors, including increased blood pressure (BP). It also demonstrated the effect of an exercise intervention on ECFC function and the potential to use ECFC to predict response to exercise intervention.
The main investigations of this thesis were based on a cohort of young adult participants aged 18 to 35 years, recruited through the Trial of Exercise to Prevent HypeRtension in young Adults (TEPHRA). Detailed clinical measurements, including BP measurements, retinal micro-vascular imaging, pulse wave velocity, blood biochemistry and lipid profiles, as well as ECFC culture and cellular assessments, were performed at baseline pre-exercise intervention. Measurements were repeated following a 16-week exercise intervention training period in a subgroup of participants. Our data presented a distinct proteomic signature with altered angiogenic mechanisms in dysfunctional ECFCs. An increase in CVD risks, including higher BP, body mass index and impaired lipid profile, were associated with a reduction in expansion and vasculogenic capacity in dysfunctional ECFCs. Further analysis revealed that the young adults with elevated BP had significantly reduced ECFC functionality and presented a potentially adverse retinal micro-vascular structure and greater macro-vascular resistance. Although no significant changes were observed in the ECFCs after exercise intervention, we demonstrated that baseline ECFC functional characteristics might positively affect aerobic training response. In addition, baseline ECFC functional characteristics may predict response to exercise, suggesting that mechanisms or substances modulating ECFC colony formation and function may be essential factors that affect an individual’s response to exercise.

Keywords: exercise
proteomics
pulse wave velocity
retinal microvasculature
endothelial colony-forming cells
hypertension
Advisor: Lewandowski, A
Leeson, C
Bertagnolli, M