VictorJuiz/thesis huggingface dataset:
Understanding the disease mechanisms of cerebellar ataxia through induced pluripotent stem cell models

The cerebellar ataxias are a heterogeneous group of incurable disorders that are characterised by progressive dysfunction and degeneration of Purkinje cells. Although numerous causative genes have been identified, the underlying pathogenic mechanisms remain incompletely understood, due to the limited availability of human brain tissue and the lack of reliable disease models. Accumulating evidence has suggested that subtypes of autosomal dominant ataxias (SCAs) are likely to be caused by shared pathogenic pathways. Focused on spinocerebellar ataxia type 14 (SCA14), a subtype of SCAs, this work aimed to understand the aetiology of SCA14 and related SCAs using novel disease models derived from human induced pluripotent stem cells (iPSCs). SCA14 is caused by mutations in protein kinase C gamma (PKCγ), a central molecule in signalling pathways that are important for Purkinje cell development and function. Using iPSCs derived from patients carrying two distinct SCA14 mutations (H36R and H101Q), this work has revealed three novel interconnected disease mechanisms. (1) SCA14 mutations promoted formation of PKCγ foci, which accumulate in the cytoplasm. (2) Mutant PKCγ showed increased kinase activity resulting in increased phosphorylation of substrates. (3) The mislocalisation of hyperactive mutant PKCγ is likely to affect the phosphorylation of its targets at the membrane and the cytoplasm. Importantly, results obtained in SCA14 iPSCs faithfully recapitulated the pathological findings observed in the patient post-mortem cerebellum. Finally, to determine the developmental changes in Purkinje cells, human Purkinje cells were generated from control and SCA14 iPSCs using a novel simplified protocol. Notably, the dendritic development and survival of human SCA14 Purkinje cells were significantly compromised in long-term culture. CRISPR/Cas9-mediated genome editing was also employed to correct the mutations and produce isogenic controls of SCA14 iPSCs. Together, this work highlighted the importance of a faithful disease model and the value of a patient material-driven approach for understanding the aetiology of neurological disorders. In the future, these patient iPSCs would be an ideal platform for further investigation of molecular pathways and therapeutic development for SCA14 and other cerebellar ataxias.

Keywords: protein kinase C gamma
PKCg
Purkinje cells
cerebellum
SCA14
neurodegeneration
induced pluripotent stem cells
cerebellar ataxia
postmortem brain
Advisor: Becker, E
Talbot, K
Insights from primary immunodeficiencies: investigating the role of GIMAP6 in the immune system

While molecular, cellular, and animal models play a vital role in immunological research, the study of patients with inborn errors of immunity provides a unique opportunity to elucidate the direct importance of specific genes in the human immune system. In this thesis, I have investigated the disease etiology of four sets of patients believed to have novel primary immunodeficiencies (PIDs) using whole exome sequencing coupled with in vitro and in vivo assays of both patient cells and model systems. In one patient, I confirmed a mutation in DNMT3B, a DNA methyltransferase known to cause a previously identified PID. I next investigated whether a homozygous missense mutation in TICAM2, a toll-like receptor signaling adaptor, might underlie the disease of a patient with recurrent pneumonia. In collaborative project, I then examined the potential role of a newly described PID-associated gene, GTPase of the immunity-associated protein 5 (GIMAP5), in autophagy and metabolism. GIMAP5 is a member of a family of conserved GTPases with a poorly understood role in leukocyte development and survival. Stemming from the work on the GIMAP5 cohort, I identified a patient presenting with splenomegaly and autoimmunity who has a novel homozygous missense mutation in GIMAP6. The majority of this thesis focuses on verification of GIMAP6 as a PID-associated gene and investigation of its molecular function. Using both patient cells and knockout mouse models, I demonstrated an autophagic defect in GIMAP6-deficient T cells, implicating GIMAP6 as a regulator of autophagy. Furthermore, I determined that Gimap6 knockout mice have early mortality due to microangiopathic kidney disease. I demonstrated that this renal disease was not immune-mediated therefore implying that GIMAP6 has a novel role in endothelial cell function. The work presented in this thesis thus highlights both the challenges of studying PIDs and the promise of these diseases to help elucidate the mechanisms of human immunity and therapies for PID patients.

Keywords: Primary immunodeficiencies
GIMAP6
Advisor: McMichael, A
Simon, K
Lenardo, M
Evaluating selfish spermatogonial selection and its role in human disease

The majority of de novo mutations originate in the paternal germline. Prior evidence has shown that a subset of activating mutations arise spontaneously within the spermatogonial stem cell (SSC) and can expand clonally along the length of a seminiferous tubule. This process, Selfish Spermatogonial Selection (SSS), explains the high birth prevalence, and increase in frequency with paternal age, of several rare congenital disorders (e.g. Apert syndrome, achondroplasia).
Quantification of these low-level mutations represents a significant technical challenge. Mutation-specific assays have been developed to assess mutation levels directly in sperm and testis samples; to date, however, only 16 loci in six genes, all of which encode members of the RAS-Mitogen-Activated Protein Kinase (RAS-MAPK) pathway, have been assessed.
The aim of this thesis is to evaluate whether other mutations, genes and pathways are subject to SSS by developing scalable screening methods that allow distinction of true variation from confounding errors.
My initial work focused on an existing discovery screen which utilised massively parallel droplet PCR combined with ultra-deep sequencing to assess a candidate gene panel. I undertook manual curation and targeted high-fidelity PCR to resolve a long-list of candidate variants. The validated variants display a striking overlap with clinically relevant mutations and double the number of SSS implicated genes in the RAS-MAPK pathway.
Distinguishing signal from noise remains a significant challenge with PCR- based methods. To improve sensitivity, two different capture-based tech- nologies incorporating unique molecular identifiers (UMIs) were investigated. Target panels were developed to assess hotspots in candidate genes, including genes involved in cancer, intellectual disability and clonal haematopoiesis. The first mutations potentially subject to SSS outside the RAS-MAPK path- way is described, with the identification of mutations at hotspots in SMAD4, a key mediator of the TGF-β BMP signalling pathway.
In summary, my work has strengthened the association of the RAS-MAPK pathway with SSS. Using refined methods to increase sensitivity and scalability, I also report the first hints that SSS may impact additional signalling processes in the testis.

Advisor: Goriely, A
Wilkie, A
Application of ultrasound for enhanced delivery of biological therapeutics into solid tumours

Drug development for cancer is heading towards increased use of highly-targeted and complex biological therapeutics. As the complexity and costs of drug development increase, the importance of ensuring good tumoural delivery and distribution of these drugs is paramount.
The tumour microenvironment, which possesses many characteristics distinguishing it from healthy tissue, presents both challenges to and opportunities for drug delivery. Countless novel nanoscale formulations or reformulations of existing drugs have been created to harness the potential of the ‘enhanced permeability and retention’ of tumours. The scale of this effect has, however, come under recent scrutiny, and a clear role for alternative delivery mechanisms has emerged.
Ultrasound-mediated cavitation has been evaluated as a non-invasive external stimulus to improve delivery of drugs to solid tumours. Cavitation nuclei can be used to seed cavitation at relatively low pressures to achieve cavitation levels capable of enabling enhanced drug transport, without safety concerns, suggesting a path for clinical translation.
In this thesis, the effects of ultrasound-mediated cavitation on the delivery of a range of therapeutic drug classes to solid tumours was investigated: a cancer-targeting antibody (cetuximab), an actively targeted nanoparticle (cetuximab-GNP), and an oncolytic virus (ColoAd). Two classes of cavitation nuclei were employed: a microbubble ultrasound contrast agent (SV) and sonosensitive gas-entrapping nanoparticles (SSPs).
Ultrasound-mediated cavitation was shown to enhance the delivery of all therapeutics tested here, though the scale of the effect varied. Cavitation activity achieved with SV or SSPs in vivo showed different characteristics, with SSPs demonstrating more sustained cavitation. Delivery of a long-circulating antibody was significantly more enhanced with SSPs than with SV, but the two cavitation nuclei provided a comparable benefit to the shorter-circulating targeted nanoparticle and oncolytic virus. Furthermore, site-specific density-modification of ColoAd was investigated, in an attempt to further enhance its cavitation-mediated transport, though the conclusion of this work was that the proposed modification strategy was not viable.
The work presented in this thesis highlights the challenge of achieving effective tumoural delivery of systemically administered biological therapeutics, and provides compelling evidence for the further investigation of ultrasound-mediated cavitation as an effective tool for enhancing the delivery of biological therapeutics into tumours.

Advisor: Carlisle, R
St Ethernan: a philological and historical case-study of a Pictish saint

The persistant endurance of local saints’ cults is an attribute of the ecclesiastical culture of the medieval Celtic societies. Many of these early saints are historically obscure, and this is equally true of the Pictish regions where evidence of early Christian figures is scanty and poorly documented, although much of the material of the later medieval saints’ cults in Scotland is derived from the eighth through tenth centuries. We find that the majority of the saints’ cults in Scotland are to Irish-born ecclesiastics associated with the Columban Church. Most of these saints flourished between years 563 (the year in which St Colum Cille founded the monastery of Iona), and c. 710 when the Pictish king Nechton mac Derili (c. 709-724) converted to the Roman tradition by means of Northumbrian influences.
[Introduction continues in thesis]
Clinical prediction modelling to guide decision making for essential neonatal services in Kenyan hospitals

Introduction
More than 98% of neonatal deaths occur in low and middle income countries(LMICs) and hospitals in these regions are expected to play a key role toward achievement of SDG 3.2 “reduce neonatal mortality to 12 per 1000 live births and below. One approach in efforts to achieve this is to use data for decision making to improve quality of hospital care for neonates.
Methods
Using the largest neonatal unit data set in Kenya (11,850 observations): (1) I conducted the first analyses of in-hospital mortality in Kenya using Kaplan-Meier and Competing Risks analysis, (2) developed, validated and explored the potential utility of two novel and simple prognostic models using the Prognosis Research Strategy (PROGRESS) recommendations. Model development was informed by a systematic review guided by the Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modelling Studies (CHARMS) recommendations.
Results
A tenth of the neonates studied died (839/9115, 9.2% [95% C. I 8.7 to 9.8%]). The overall median length of stay was 2 days (range of 0-98 days, IQR 1-5days). Competing risk analysis showed different cumulative incidence of death for the 1.5 to 1.99kg and 2 to 2.49kg weight categories that are often grouped together (1.5 to 2.49). In addition, the competing risk method provides a more clinically meaningful estimate of in-hospital death compared to Kaplan-Meier. Two simple prognostic models that use treatments (NETS) and signs and symptoms (SENSS) as predictors provide accurate predictions of in-hospital death. Discrimination by c-statistic is 0.89 for each model with calibration slope 0.90 for SENSS and 0.76 for NETS.
Conclusion
Prognostic data on in-hospital mortality may inform decisions on treatment, demand and capacity planning as well as balancing of groups in clinical trials. Scale up of such data collection and analyses is required in LMICs to support efforts to improve neonatal survival.

Advisor: English, M
Collins, G
Berkley, J
Establishing a detailed tephrochronological framework for Japan to aid the synchronisation of palaeoenvironmental records across the Asian/Pacific region

Tephra (volcanic ash) layers have the potential to synchronise palaeoenvironmental archives across regional to hemispheric scales. Highly productive arc regions, like those in East Asia, offer a considerable number of widespread isochrons, but before records can be confidently correlated using these layers, a refined and integrated tephrochronological framework is required. The Lake Suigetsu sequence, obtained from a small tectonic lake in central Honshu, is the most precisely dated sedimentary record for Japan. Here, the sediments are examined for the presence of thin (sub-millimetre in thickness) and ‘non-visible’ (cryptotephra) layers in order to provide a comprehensive record of East Asian volcanism, and a linchpin site for correlating sequences.
On average, cryptotephra layers are identified four times more frequently than visible markers in the studied sediments of Lake Suigetsu, providing an unprecedented record of ash fall events that reached the densely populated regions of central Honshu. Major and trace element glass geochemistry obtained from these tephra layers is compared to new and published datasets to pinpoint their volcanic source. The cryptotephra were erupted from sources that span the length of Japan, and volcanoes further west, including Ulleungdo (South Korea) and Changbaishan (North Korea/China).
Constrained eruption ages are modelled for the tephra layers identified in the sequence, which can be transferred into other sedimentary records that contain these markers. Cryptotephra identifications show that many eruptions dispersed ash more widely than previously anticipated, and are able to synchronise palaeoclimate records over hemispheric scales. For example, the archive provides the most southerly tephra occurrence of the B-Tm tephra, synchronising this mid-latitude record to the Greenland ice cores. The augmented Lake Suigetsu eruption archive is established as a key node within the tephrostratigraphic framework for Japan, and provides an important resource for volcanological research, with constraints on the frequency, reoccurrence intervals and dispersal of numerous eruption events.

Keywords: Cryptotephra
Tephrostratigraphy
B-Tm tephra
Japan
Quaternary Science
Changbaishan volcano
Lake Suigetsu
Age-modelling
Lake Sediments
East Asia
Tephrochronology
Varve Sediments
Ulleungdo volcano
Tephra framework
Advisor: Smith, V
Albert, P
Malaria transmission in Cambodia: Are plantations a hotspot?

Background: Malaria is a leading cause of morbidity in Cambodia with an estimated 55,000 cases in 2014. Malaria prevalence is highest in forested rural areas, where a large proportion of the population live and work. Plantations are hypothesised to be “hotspots” for malaria transmission due to their proximity to forests and the vectors therein.
Methods: An analysis of malaria incidence data collected on plantations and in surrounding local villages in north-eastern Cambodia, as well as epidemiological analysis of risk factors for malaria prevalence on plantations. A deterministic compartmental model was developed to simulate malaria transmission dynamics on plantations.
Findings and conclusions: Malaria incidence in both villages and plantations follows a seasonal pattern with a peak in cases toward the end of the wet season in October to November. There was no statistical difference in incidence between settings. Mondulkiri province has a second peak at the start of the wet season which may be attributed to the type of work conducted on the plantations at that time or population migration. Permanent non-vector permeable housing in the wet season (OR 0.30; 95% CI: 0.11–0.86, p <0.05) and use of bed nets in the dry season (OR 0.15; 95% CI: 0.03–0.62, p <0.001) were the only factors found to have a significantly reduced odds of malaria prevalence. The plantation malaria model demonstrates that importation of cases by migrant workers is currently not a factor in malaria transmission on plantations. Plantation workers with immunity and asymptomatic malaria act as a reservoir enhancing malaria transmission and need to be targeted in elimination efforts. The malaria model also shows that as the plantations approach elimination, the transmission pattern will shift from a stable endemic to unstable epidemic pattern due to declining immunity which is likely to result in more clinical and severe malaria.

Keywords: malaria transmission
plantation
Cambodia
malaria modelling
migrant workers
rubber plantation
south east Asia
hotspot
Malaria
Advisor: White, L
Maude, R
Merson, L
Development and characterisation of human monoclonal antibodies against the Ebola virus glycoprotein

The current Ebola virus outbreak in West Africa has infected over 27,000 people and claimed over 11,000 lives, making it the deadliest and most widespread Ebola epidemic in history. The high caseload and rapid geographic spread has been due in part to crumbling healthcare infrastructure, critical shortages of healthcare workers, paucity of resources, and local traditional practices. In addition, although supportive care is available at treatment centres, there are no effective therapies available in the affected countries. A number of potential Ebola virus therapeutic agents are currently being tested in accelerated clinical trials, but there is still no drug with proven efficacy in humans.
In this dissertation I describe the isolation of the first fully human Ebola-specific monoclonal antibodies (mAb) from vaccinees during phase 1 of the Oxford Ebola vaccine trial. This was achieved through single cell sorting of antibody secreting cells (ASC) at day seven post boost, isolation of the immunoglobulin (Ig) heavy and light chain genes, expression vector cloning and transfection of human cell lines to produce mAbs. I also attempt to characterise the Ebola-specific mAb’s binding affinity, binding site, and cross-reactivity with other historic Ebola virus strains by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assays (IFA). I have also tested their neutralising ability with an in vitro pseudotyped influenza microneutralisation (MN) assay. I present a diverse panel of twelve mAbs with a range of binding characteristics and neutralising abilities, four of which are cross-reactive with both the Zaire and Sudan Ebola viruses. Once fully characterised, these antibodies may potentially be used as part of a mAb cocktail for pre- or post-exposure prophylaxis and cure for Ebola virus disease.

Keywords: antibodies
Ebola virus
human monoclonal antibodies
monoclonal
immunotherapy
therapeutic antibodies
Ebola virus glycoprotein
Advisor: Elias, S
Draper, S
Substituted indenyl complexes for ethylene polymerisation

The principle aims of this thesis have been to synthesise novel substituted indenyl complexes inspired by the very active Me2SBI*ZrCl2. By looking to improve on the ethylene polymerisation activity and the racemo-selectivity, the Me2SBI* ligand framework has been modified. The synthesised complexes have been tested for homogeneous and heterogeneous ethylene polymerisation activity and evaluated for their potential industrial applications.
Chapter One provides a background to the chemistry of cyclopentadienyl and indenyl ligands, group 4 ansa-metallocenes and their ethylene polymerisation activity. The mechanism of ethylene polymerisation is reviewed. A summary of the development of homogeneous and heterogeneous ethylene polymerisation is discussed including the use of solid supports; silica, solid polymethylaluminoxane (sMAO) and layered double hydroxides.
Chapter Two describes modification of the Me2SBI* ligand by introducing different alkyl and aryl groups on the silyl bridge and at the 3position of the indenyl rings. Et2SBI*H2, nPr,MeSBI*H2, All,MeSBI*H2 and Me2SB(3EtI*)H2 pro-ligands have been synthesised. All pro-ligands and their synthetic intermediates have been fully characterised by NMR spectroscopy.
Chapter Three details the synthesis of substituted indenyl group 4 complexes. All complexes have been fully characterised by single crystal X-ray diffraction and NMR spectroscopy. Comparisons of the crystal structure metrical parameters are made.
Chapter Four investigates the homogeneous and heterogeneous ethylene polymerisation activity of the synthesised substituted indenyl complexes. The complexes have been immobilised onto sMAO and characterised by solid state NMR spectroscopy and SEM. The resulting polyethylenes have been characterised by GPC, XRD and SEM.
Chapter Five studies the solid support comparison between sMAO, SSMAO and LDHMAO. The optimisation of polymerisation conditions for the sMAO-[Me2SB(3-EtI*)ZrCl2] catalyst has been carried out. Potential industrial applications including polyethylene waxes and LLDPE have been explored.
Chapter Six provides experimental details and characterising data for the preceding chapters. Electronic appendix contains the CIF, CheckCIF and PLATON files of all single crystal structures.

Advisor: O'Hare, D