VictorJuiz/thesis huggingface dataset:
Protein modulation of ion channels

Ion channels lie at the heart of electrical excitability. They represent attractive targets for treatments of a range of pathophysiological conditions, including pain, ischemia and epilepsy. To date, small molecule approaches have dominated ion channel drug discovery, but small molecules often lack the appropriate safety and specificity, ultimately leading to their rejection in the clinic. One way in which to overcome this is to use proteins which use large molecular scaffolds to very precisely place a few key residues in order to modulate ion channel function. To this end, we characterise α-scorpion toxin binding to Nav channels, showing α-scorpion toxin AahII binds to VSDIV and inhibits upward voltage sensor movement. We interrogate the pharmacology of these naturally occurring toxins and probe electrochemical coupling involved in the fast-inactivation process of Nav channels which was uncovered in our work, in order to gain insights into the gating cycle of the channels. Better understanding of this process may allow development of novel therapeutics targeting inactivated Nav channels. Additionally, we characterise VHH domain antibody binding to K2P channels, showing they are capable of both activation and inhibition of channel activity and show high selectivity towards their target. We postulate a mechanism of action through the external pH-sensing pathway and show their activity on rodent channels, uncovering analgesic potential. Additionally, we develop the VHH antibodies as tools to allow visualisation of TREK-2 in native cells, showing selective binding against their target over closely related channels and provide some preliminary data to show localisation of TREK-2 within neurons of rat sympathetic cervical ganglia but not in the glial cells, consistent with RNA-seq data.

Keywords: protein modulation
structure-function
electrophysiology
ion channels
Advisor: Tucker, S
Carpenter, E
Regulation and function of innate-like T cells

KLRB1, encoding for CD161, is one of the most favourable prognostic pan-cancer genes. It is expressed by NK- and T-cell subsets, particularly by innate-like T cells such as MAIT and γδT cells. CD161-expressing T-cell subsets share a transcriptional and functional phenotype, including the ability to be activated by cytokines in a T-cell receptor-independent manner, similar to NK cells. The regulation and function of CD161-expressing cells as well as their role in cancer is not fully understood, however. In this study, an imaging-based high-content cytometry method, called ChipCytometry, was developed as a truly viable technique to identify and deeply phenotype CD161-positive cell types in blood and tissue. Novel mechanisms for the activation of CD161-expressing T cells were discovered by stimulation with the relatively underexplored cytokine IL-32. The ability of CD161-expressing T cells to also express IL-32 upon activation was demonstrated in vitro. To elucidate the role of IL-32 in disease, a pan-cancer IL32 gene signature was generated from The Cancer Genome Atlas (TCGA) RNA-seq data. The IL32 signature genes were associated with immune response- and T-cell activation-related pathways. Single-cell RNA-seq data from breast cancer-derived immune cells showed that IL32 gene expression and the expression of the most significant IL32 signature genes were strongly associated with innate-like T cells, including MAIT and γδT cells, in addition to memory T cells and Tregs. Finally, estimated immune cell fractions within TCGA data revealed that the favourable prognostic association of CD161 across cancers was attributable to multiple CD161-expressing cell types. These include NK cells, γδT cells, tissue-resident memory T cells, cytotoxic T cells and the still poorly studied T follicular helper cells in cancer. In summary, the findings of this study expand the knowledge of the regulation and function of CD161-expressing immune subsets and their role in cancer, setting the stage for their use as a clinical biomarker or in cancer immunotherapy.

Advisor: Klenerman, P
Buffa, F
Willberg, C
The role of non-state actors in foreign policy decision-making processes in the non-western world

This thesis investigates the involvement of non-state actors (NSAs) in foreign policy decision-making processes in the non-Western world in order to find answers to why, how, and under what conditions such entities become engaged in foreign policy-making and influence state behaviour. Based largely on primary sources, it offers an exclusive and original data set, organised around a set of hypotheses and according to an organically developed conceptual framework. This allows for an unprecedented insight into the modus operandi of different non-state actors and contributes to an analytical diversification away from a prevailing Western-centrism in the scholarly literature.
The thesis demonstrates that non-state actors, assuming a variety of different operational roles, are able to become informally but nonetheless meaningfully involved. Thus, by adopting a networked analysis of international relations, the dissertation is able to shed analytical light on a grey area, existing alongside the formal, state-driven initiatives. This ultimately reveals a hybrid form of foreign policy at work, blurring the boundaries between the actions of states and non-state actors.
Hence, whilst informality in foreign policy might raise questions of legitimacy, the thesis suggests that hybridity should be acknowledged and perhaps welcomed – especially when it proves to be more efficient than more formal and supposedly legitimate forms of diplomacy. The thesis offers a conceptualisation of informal networks and adopts a methodology able to illustrate their inner functioning. It concludes that coordinated policies of state and non-state actors, capable of recognising and utilising the formal and informal networks they are embedded in, are already prevalent in distinct regional settings, determining the international rules of the game.

Keywords: foreign policy
Advisor: Zielonka, J
The origins and development of professional legal attorneys by reference to thirteenth-century eyre proceedings

In exploring the origins and development of professional legal attorneys by reference to thirteenth-century eyre proceedings, it is firstly argued that attorneyship, as it existed circa 1200, originated in the coalescence of three separate traditions based, respectively, on putting in place, attornment and procuration. Evidence for this proposition lies in the remarkably diverse vocabulary employed by contemporary scribes, clerks and commentators in describing the activity of attorneyship.
Simple attorneyship, the basic core function of which was physical substitution, is then shown to have accommodated over the course of some 60 years legal attorneyship with its numerous duties and capacity for professionalisation. This process is charted by reference to the activities of attorneys at eight selected eyres held between 1218-19 and 1281. Themes and issues discussed include the circumstances surrounding the recorded presence of attorneys in court; outcomes achieved; the several motivations of litigants in appointing attorneys in increasing numbers; and the emergence of men displaying lawyerly qualities and characteristics including those of competence, articulacy, literacy, administrative skill and legal expertise.
The activities of professional legal attorneys at a series of eyres in the 1280s are next examined. Issues addressed include how many of them there were; their identities; where they practised; for whom they acted; what they did; how they organised themselves; and the extent to which they associated.
Finally, it is argued that the reason for professionalisation and the means of its emergence lie in the concept of supply and demand. The respective contributions of secular men and women, husbands and wives litigating together, and of ecclesiasts in the creation, firstly, of a critical mass of attorneys able to accommodate a minority of legal professionals and, secondly, of a demand for expert services supplied by those professionals, are analysed, as are the sources of the expertise demanded.

Keywords: history of the English legal profession
Advisor: Brand, P
A corpus study of formulaic variation and linguistic productivity in early Greek epic

This thesis addresses the general issue of formulaic variation in early Greek epic – not formulaic change, in that I do not adopt a diachronic perspective or seek to restore the original form of any formulae, but simply to describe how different variants of the same formulae coexist, and what their patterns of variation can tell us about formulaic language. I introduce the concepts of formula and formulaic variation in part I, which is functionally a two-part introduction (chapters I and II). Part II addresses the issue of syntactic variation, focusing on formulae composed by an adjective and a (common) noun; the behaviour of these formulae is analysed in a corpus made of Homer, Hesiod, and the major Homeric Hymns, and compared to the behaviour of similar structures in a non-formulaic 5th-century baseline corpus. The final chapter in part II (chapter V) analyses the behaviour of the same Adj + N pairs in a Hellenistic poem, the Argonautica of Apollonius of Rhodes. Part III focuses on semantic variation, and applies Distributional Semantics in order to study the range of meaning that objects of transitive verbs can take in Homer and Hesiod. Both Parts II and III have an introductory chapter of their own (chapters III and VI, respectively), in which the linguistic framework that will be used in the whole section is introduced and applied to a smaller-scale study.

Keywords: distributional semantics
hexameter poetry
quantitative linguistics
construction grammar
early Greek epic poetry
formulaic language
formulae
Advisor: Probert, P
The impact of cytomegalovirus infection and viral vector vaccination on human and mouse immune systems

Viral vectors are a next-generation vaccine platform that elicit a robust response from the humoral and cellular arms of the immune system. The importance of this vaccine platform was demonstrated during this PhD with the emergence of SARS-CoV-2 and the development of the chimpanzee adenovirus-vectored vaccine ChAdOx1 nCoV-19. Although this vaccine is immunogenic, pre-existing infections such as cytomegalovirus have been shown to reduce antigen-specific vaccine immunogenicity in other vaccines. Cytomegalovirus is a chronic herpesvirus that is prevalent in ~40-100% adults worldwide, and although infection is often asymptomatic it can increase the frequency of terminally differentiated immune cells in healthy young adults.
This thesis aims to characterise the effect of viral vectored vaccines including ChAdOx1 on immune populations not traditionally studied in vaccinology, including NK cells. It also investigates how cytomegalovirus impacts the immune phenotype and vaccine immunogenicity in humans and mice. This thesis demonstrates that viral vector vaccination activates NK cells and induces robust cytokine secretion in both mouse and human studies, although can affect NK cell phenotype following vaccination with MVA. Furthermore, CMV seropositivity elicits a terminally differentiated phenotype on the immune system, but does not affect viral vector vaccine immunogenicity following a single dose or homologous boost of ChAdOx1. Overall, these findings which are especially important for the ongoing vaccination regimen against SARS-CoV-2, and provide further insight into the interplay of viral vectored vaccines and viral infection on the immune system.

Keywords: NK cells
viral vector
ChAdOx1
vaccines
SARS-CoV-2
Cytomegalovirus
immunology
Advisor: Lambe, T
Gilbert, S
Klenerman, P
Targeting the 5' untranslated region of SMN2 as a therapeutic strategy for spinal muscular atrophy

Nucleic acid therapeutics allow sequence-based targeting of disease genes, such as the genes involved in spinal muscular atrophy (SMA) pathogenesis. SMA is a neuromuscular disorder caused by mutations in the survival motor neuron 1 gene (SMN1). All patients have at least one copy of a paralog, SMN2, but a C-to-T transition in this gene results in exon 7 skipping in a majority of transcripts. As a result, only 10 to 20 percent of SMN2 transcripts encode the fully functional SMN protein. Nusinersen, an FDA-approved therapeutic for SMA, is an antisense oligonucleotide (ASO) that promotes exon 7 inclusion in the SMN2 transcript. The ceiling effect associated with splice-switching approaches makes complementary therapeutic strategies necessary. Increasing the total pool of SMN2 transcripts and increasing the translational efficiency of these transcripts are strategies that can be used in combination with splice correction. I sought to determine whether the 5’ untranslated region (5’UTR) of SMN2 contains an element that reduces its expression that can be targeted as a means of increasing SMN levels. I found that ASOs targeting the 5’ end of SMN2 increase SMN mRNA and protein levels, and that this effect is due to ASO-mediated inhibition of SMN2 mRNA decay. Moreover, using the 5’UTR ASO in combination with a splice-switching oligonucleotide (SSO) increases SMN levels above those attained with the SSO alone. I show that the effect of this 5’UTR ASO is human-specific. These results add to the current understanding of SMN regulation and point toward a new therapeutic target for SMA.

Keywords: antisense oligonucleotides
SMN2
5′ UTR
spinal muscular atrophy
Advisor: Fischbeck, K
Wood, M
A medicine of constitutions: Sasang medicine in contemporary South Korea

This thesis investigates the process of standardisation, scientisation, and diversification of Sasang medicine in contemporary South Korea, through the lens of its central concept ch’ejil (constitution 體質). Sasang medicine (sasang ŭihak 四象醫學) is a current of medical tradition which emerged from the scholarly physician Yi Chema (李濟馬) in late 19th century Chosŏn. Sasang medicine is known as ch'ejil ŭihak (constitutional medicine 體質醫學) because it assumes that human beings have different constitutions with different physiologies and pathologies as well as different psychological traits. Sasang medicine is a unique example in [traditional] Korean medicine because it has constantly been practiced for over a century and is ingrained into health practices of modern Koreans as well as the contemporary health care system in South Korea.
Based on multi-sited ethnographic fieldwork in a school of Korean medicine and its teaching hospital, in a national research institute, and in private clinics, this thesis meticulously explores how an established form of Sasang medicine has been situated in the institutionalised system of Korean medicine. Ethnographies from different field sites provide microscopic details which are interspersed with various issues of collaboration, confrontation, and negotiation of different values and epistemologies. Furthermore, this thesis scrutinizes ‘how practitioners of Sasang medicine know what they know’ in such different social settings. These attempts are fulfilled by exploring how the central concept of Sasang medicine, ch’ejil, is understood in each field site. In so doing, this thesis aims to elucidate to what extent the practice of Sasang medicine in historical and social formation diversifies concepts of ch’ejil, which have played a role as a fluid medium to raise legitimacy or “efficacy” of Sasang medicine.

Keywords: East Asian medicine
chejil
Sasang medicine
Constitutional medicine
(post-)modernity
habitus
Korean medicine
constitution
Advisor: Hsu, E
Pulsed squeezed light: characterization and applications

Squeezed light is expected to enable new quantum metrology and quantum computation applications, but the very high degree of experimental control required in practice places severe demands on the physical devices used to generate it. As ultrafast squeezed light sources are reaching an unprecedented degree of brightness and modal control, there is a strong interest in developing methods for their experimental characterization and theoretical understanding. In this thesis, we demonstrate both a new application and a new characterization technique for pulsed squeezed light.
In the first part of the thesis, we present a new metrological application of pulsed squeezed light: quantum-enhanced label-free nonlinear microscopy. Stimulated emission microscopy (SEM) is a state-of-the-art pump-probe microscopy technique whose sensitivity is ultimately limited by shot noise in the probe beam. We propose to overcome this fundamental limitation by using a sub-Poissonian probe generated via optical parametric amplification. We demonstrate an intensity-squeezed sub-picosecond pulsed probe, and a simple stimulated emission microscope. Using this squeezed probe, sub-Poissonian statistics are observed in the SEM signal. After this proof-of-principle experiment, we aim to provide an improved design for a source of intensity-squeezed light. First, we develop a theoretical framework to simulate the photon statistics of a general sub-Poissonian source, considering pulsed and focused pump and seed fields in a unified spatio-temporal description. Next, we use this framework to suggest possible improvements to our previous squeezed probe.
In the second part of this thesis, we provide new theoretical and experimental tools to analyze the high gain regime of pulsed spontaneous parametric down-conversion (SPDC). We study a waveguided source capable of generating twin-beams with tens of photons in a single spatio-temporal mode. We develop a framework to efficiently simulate SPDC for an arbitrary nonlinear gain, including the effect of parasitic nonlinearities such as self-phase modulation (SPM) of the pump and cross-phase modulation (XPM) between pump and twin-beams. We experimentally characterize our source using a new technique based on stimulated difference-frequency generation (DFG). In a type-II down-conversion process, we measure the frequency generation produced not only in the orthogonal polarization to the seeding beam, but also in its same polarization, which we interpret as cascaded difference-frequency generation. We argue that the additional information obtained in this measurement allows for a self-referenced estimation of the squeezing gain. Using these measurements, we accurately fit our theoretical model, revealing that, in the high gain regime, parasitic nonlinearities strongly affect the spectral structure of the twin-beams. We propose a simple strategy to reduce the impact of these nonlinearities on the modal structure of SPDC.

Keywords: femtosecond lasers
quantum optics
squeezed light
Advisor: Walmsley, I
Characterisation of endometrial B cells and lymphocytes in the peri-implantation window

Immunological regulation at the implantation site is a complex process, dysfunction may contribute to infertility, recurrent implantation failure (RIF) or recurrent pregnancy loss (RPL). Apart from endometrial NK and T cells, whose phenotypic profile and functional involvement have been recognised, evidence suggests that endometrial B cells may also have a potential role in reproductive pathologies. This thesis aimed to investigate endometrial immune profiles during human implantation, with a specific focus on the role of endometrial B cells.
Both healthy women and infertility, RIF or RPL patients were recruited for endometrial immune profiling, focusing on cell proportions (NK, T and B cells) and activation status. Additionally, peripheral blood samples were collected and analysed in parallel. Significant differences were observed between peripheral and endometrial immune profiles, in minor cell populations like CD56bright CD16dim NK cells and CD4 CD8 double positive T cells, such differences were not previously reported. A standard operating protocol (SOP) was established so that comparison between controls and infertility, RIF or RPL patients could be made with more patients being enrolled in the near future.
The role of B cells in the human endometrium was investigated. A systematic review was performed to understand the B cell involvement in reproductive pathologies. A number of immunohistochemistry studies were retrieved that identified endometrial B cells in association with T cells in ‘lymphoid aggregates (LAs)’, however, neither have been extensively characterised. RNA-sequencing and flow cytometric analysis were performed on CD19+ endometrial B cells. B cells comprised 1-5% of endometrial immune cells, the majority were naïve or memory B cells, with few plasma cells. Compared with circulating B cells, endometrial B cells had an activated phenotype, with increased expression of CD69, HLA-DR, CD74 and CD83, and IL-10 production capacities. PD1+CXCR5+ICOS+ T follicular helper-like cells and FAS+IgD􀀀 germinal centre-like B cells were present in the endometrium. Together with previously reported LAs, endometrial B cells may be an active player in shaping the endometrial immune environment.

Keywords: flow cytometry
recurrent implantation failure
recurrent pregnancy loss
T follicular helper cells
RNA-seq
B cells
endometrium
Advisor: Granne, I
Southcombe, J
Establishing an antibody mimetic applicable to targeting in the gastrointestinal tract

Proteins are a valuable class of therapeutics because proteins, like antibodies or non-antibody scaffolds, can bind their targets with high selectivity and affinity. The upper gastrointestinal tract, however, rapidly inactivates protein-based targeting reagents. Compared to injections, oral administration of protein therapies is favourable for treating gastrointestinal disease. In this thesis, I have developed a protein scaffold termed a gastrobody, that is suited to applications in the gastrointestinal tract. Gastrobodies are derived from Kunitz-type soybean trypsin inhibitor (SBTI). SBTI is resilient to digestion with stomach-like concentrations of pepsin and pH 2. Furthermore, SBTI is stable in the presence of intestinal proteases and bile acids, as well as being resilient to high temperatures. I used the protein modelling software Rosetta to assist design of a binding surface on SBTI. I also established phage display of SBTI to enable directed evolution. As proof of concept, gastrobody binders to domains of two toxins of the enteric pathogen Clostridium difficile were developed. After optimisation of the binding surface and affinity maturation, I identified gastrobodies that bound their target with nanomolar affinity. Gastrobodies bind their target after incubation with physiological concentrations of gastrointestinal proteases, including pepsin at pH 2. Lastly, I explored alternative gastrobody backbones using structural homologues of SBTI. The work presented in this thesis may provide a foundation for generating binding reagents for therapeutic and diagnostic use in the gastrointestinal tract.

Keywords: synthetic biology
phage display
gastroenterology
nutrition
protein engineering
Advisor: Howarth, M