Human embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos. Once explanted and culture in vitro, human ES cells retain their pluripotent potential (i.e capacity to differentiate into all somatic cells) and acquire the ability to self-renew indefinitely. These two properties of human ES cells make them an invaluable resource for developmental biology, cell replacement therapies, drug development and toxicology screening. However, it has been widely reported that human ES cells frequently acquire karyotypic changes throughout culture in vitro, termed 'culture-adaptation'. These changes occur sporadically in cultures but appear to be more common in high passage cell lines. The changes also appear to be non-random, frequently involving the gain of chromosomes 1, 12, 17 and 20 suggesting that these chromosomes harbour genes that provide the cells with a selective advantage. These karyotypic abnormalities are also commonly found in human embryonal carcinoma cells and primary human tumours, a concern for the potential use of human ES cells in therapeutic applications. A recent large-scale study by the International Stem Cell Initiative identified a small copy number variant (CNV) on chromosomal region 20q11.21, which was amplified in >20% of 125 karyotypically normal human ES cell lines. Here we show that the high prevalence of 20q11.21 amplification in human ES cells can be attributed to a strong selective advantage provided by BCL-XL. Cell lines containing the CNV show increased protection against apoptosis resulting in increased population growth rates allowing variant cells to rapidly out-compete normal diploid cells. Cell lines containing the 20q11.21 amplification also show altered differentiation patterns and increased survival of polyploidy. We also describe a method for the rapid detection of the 20q11.21 amplification in human ES cell cultures which is sensitive, cost-effective and applicable for stem cell laboratories worldwide.

Human embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos. Once explanted and cultured in vitro under appropriate conditions, human ES cells retain pluripotency (i.e. capacity to differentiate into all somatic cell types) and acquire the ability to self-renew indefinitely. These two properties of human ES cells make them an invaluable resource for developmental biology, cell replacement therapies, drug development and toxicology screening. In order to exploit these unique cells and to better understand human development, it is imperative to understand the mechanisms behind the specification of somatic cell types. Much work has been conducted on monolayer formats to delineate signalling and gene expression networks responsible for lineage specific differentiation, however less focus has been on the use of embryoid bodies as a more representative model of in vivo differentiation. In this study we develop a differentiation assay to better recapitulate embryonic development, which we also show as a useful model for predictive toxicology. Within the assay, however, we found persistently heterogeneous differentiation. To better understand how hESCs make lineage decisions, we went back to interrogate heterogeneity within the stem cell compartment, and show that discreet, but functional heterogeneity biases cells to particular fates. Finally, we shed light onto a potential mechanism through which heterogeneity arises, which could offer a platform for future work to homogenise stem cells thus resulting in uniform, controlled differentiation.

Aneurysmal subarachnoid hemorrhage (SAH) carries a high morbidity and mortality. The current protocols used to treat the unruptured Intracranial Aneurysms (IAs) are inadequate underscoring the need of finding new descriptors. As demonstrated by the studies performed in this manuscript, haemodynamics plays an important role in the aetiopathogenesis of IAs. An evaluation of haemodynamic indices can provide a useful alternative to predict the behavior of an unruptured IA at an early stage. Studies performed by me demonstrate that Computational Fluid Dynamics (CFD) can be used successfully to predict haemodynamic indices where detailed in vivo measurement of haemodynamic flow variables is not possible owing to technical limitations. European Commission funded Project @neurIST was the first project of it’s kind that brought together a number of multidisciplinary professionals from 32 European institutions and made possible development of state-of-the-art tools for personalised risk assessment and treatment IAs using CFD. These tools have been constantly improved and amended in the light of feedback gathered from their controlled exposures conducted world over, as described in the manuscript. However, need of a well-designed Randomized Controlled Trial in this context cannot be overemphasized, before these tools can be accepted by clinicians and patients. In my study on the validation of different concepts used in CFD, I demonstrated that there is no added advantage of complex Womersley-flow-profile over the much simpler plug-flow profile. One of my studies on initiation and rupture of IAs showed that the haemodynamic patterns of IAs during these two phases are significantly different with values of supra-physiological Wall Shear Stress (WSS) being higher in initiation while lower in rupture phase. I also investigated the effects of pharmacological agents on the aetiopathogenesis of IAs and found that heparin induces significant derangements in the haemodynamics of both, pre-aneurysmal as well as ruptured IA. I propose that heparin (and its derivatives) can, on the one hand may facilitate the rupture of existing IAs, on the other hand they may suppress the formation of new IAs. I have also found significant differences in the results using patient-specific vs. Modeled Boundary Conditions and showed that the 1D circulation model adopted by @neurIST performs better than other approaches found in the literature. I also proposed a novel mechanism of increase in Blood Viscosity leading to high WSS as one of the important underlying mechanisms responsible for the increased incidence of IA formation in smokers and hypertensive patients. In my study on patients with pre-existing Coarctation of Aorta (CoA) and Intracranial Aneurysms, I demonstrated that the cerebral flow-rates in CoA patients were significantly higher when compared to average flow-rates in healthy population. It was also seen that the values and the area affected by supraphysiological WSS (>15Pa) were exponentially higher in patients with CoA indicating the possible role of increased haemodynamic WSS secondary to the increased flow-rates playing an important role in the pathogenesis and rupture of IAs in CoA patients.

Nano-scale Iron-Oxide ferrofluids exhibit a special property, ‘superparamagnetism’, that induces an attractive force toward an external magnetic field. The aim of this project is to investigate the use of ferrofluids for tissue retraction during Minimally Access Surgery (MAS). In the in-vivo porcine experiments, 0.3 ml of ferrofluid (200 mg/ml concentration) containing 10 nm particles is injected subserosally into the small bowel, respectively. A 0.6 T magnetic field is created using a combination of 10 mm and 20 mm diameter Neodymium Iron Boron magnets. The vertical retraction distance is measured up to 80 mm and video-recorded. The results demonstrate the capacity of ferrofluid to facilitate the tissue manipulation and analysis of the migration of the particles within the tissue using micro computed tomography (CT). A theoretical model developed to validate the experimental results is also beneficial for predicting retraction force. In conclusion, this feasibility study provides a protocol for systematically using small volumes of ferrofluid, without the need to mechanically grasp the tissue.

The main objective of this work was to determine the feasibility of using indirect selective laser sintering (SLS) to produce parts for bone replacement applications from glass-ceramic materials. A castable glass based on the system SiO2-Al2O3"P2O5-CaO-CaFth2a t crystallises to a glass-ceramic with apatite and mullite phases was produced, ground to a powder and blended with an acrylic binder at various ratios by mass. An experimental sinterstation with a 250W CO2 laser was used to determine the viability of indirectly sintering the glass ceramic across a range of processing parameters. Green parts with good structural integrity were produced using a wide range of processing conditions, allowing both monolayer and multilayer components to be constructed. The effect of powder properties, such as glass particle size and binder content, on the surface qualities and structural integrity of the parts was also examined. Following SLS the parts were post-processed to remove the binder which may otherwise have rendered them unsuitable for biological use, and fully crystallise the material, evolving the apatite and mullite phases to improve both biological and mechanical properties. The parts were heated to 1200°C using a number of different time-temperature profiles, following which the processed material was analysed by DTA, XRD, SEM, gCT, and tested for its flexural strength. An increase in strength was achieved by infiltrating the brown parts with PMMA and a resorbable phosphate glass, although the latter altered the crystal phases present in the material. In vitro cytotoxicity and bioactivity tests were carried out to assess the biological properties of the produced parts. The laser sintered material was found to be non-toxic by both contact and extract methods. There was no evidence of an apatite layer forming on the surface of the material when soaked in a simulated body fluid suggesting that the material was unlikely to exhibit bioactive behaviour in vivo. However, following implantation in rabbit tibiae for 4 weeks, bone was seen to have grown into the porous structure of the laser sintered parts, and appeared to form a close bond with the material surface.

This thesis describes the first Text Analytics approach to predicting Causes of Death (CoD) from Verbal Autopsies (VA). VA is an alternative technique recommended by the World Health Organisation for ascertaining CoD in low and middle-income countries (LMIC). CoD information is vitally important in the provision of healthcare. CoD information from VA can be obtained via two main approaches: manual, also referred to as the physician-review and automatic. The automatic-based approach is an active research area due to its efficiency and cost effectiveness over the manual approach. VA contains both closed responses and open narrative text. However, the open narrative text has been ignored by the state-of-art automatic approaches and this remains a challenge and an important research issue. We hypothesise that it is feasible to predict CoD from the narratives of VA. We further contend that an automatic approach that could utilise the information contained in both narrative and closed response text of VA could lead to an improved prediction accuracy of CoD. This research has been formulated as a Text Classification problem, which employs Corpus and Computational Linguistics, Natural Language Processing and Machine Learning techniques to automatically classify VA documents according to CoD. Firstly, the research uses a VA corpus built from a sample collection of over 11,400 VA documents collected during a 10 year period in Ghana, West Africa. About 80 per cent of these documents have been annotated with CoD by medical experts. Secondly, we design experiments to identify Machine Learning techniques (algorithm, feature representation scheme, and feature reduction strategy) suitable for classifying VA open narratives (VAModel1). Thirdly, we propose novel methods of extracting features to build a model that predicts CoD from VA narratives using the annotated VA corpus as training and testing set. Furthermore, we develop two additional models: only closed responses based (VAModel2); and a hybrid of closed and open narrative based model (VAModel3). Our VAModel1 performs reasonably better than our baseline model, suggesting the feasibility of predicting the CoD from the VA open narratives. Overall, VAModel3 performance was observed to achieve better performance than VAModel1 but not significantly better than VAModel2. Also, in terms of reliability, VAModel1 obtained a moderate agreement (kappa score = 0.4) when compared with the gold standard– medical experts (average annotation agreement between medical experts, kappa score= 0.64). Furthermore, an acceptable agreement was obtained for VAModel2 (kappa score =0.71) and VAModel3 (kappa score =0.75), suggesting the reliability of these two models is better than medical experts. Also, a detailed analysis suggested that combining information from narratives and closed responses leads to an increase in performance for some CoD categories whereas information obtained from the closed responses part is enough for other CoD categories. Our research provides an alternative automatic approach to predicting CoD from VA, which is essential for LMIC. Therefore, further research into various aspects of the modelling process could improve the current performance of automatically predicting CoD from VAs.

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Mitochondria continuously undergo changes in their morphology by two dynamic processes called mitochondrial fusion and mitochondrial fission. Mitochondrial fusion results in longer mitochondria and is important for the complementation of mtDNA and intermixing of essential mitochondrial proteins and nutrients to maintain healthy mitochondrial population. Mfn1 and Mfn2 are involved in outer mitochondrial membrane fusion while OPA1 mediates inner mitochondrial membrane fusion. On the other hand, mitochondria are broken into smaller units for easy transport and removal of damaged mitochondria by Drp1. Defective mitochondrial dynamics has been linked with various common neuropathies and neurodegenerative diseases. Charcot Marie Tooth Type 2A (CMT2A) and its subtype Hereditary motor and sensory neuropathy type VI (HMSNVI) are caused by mutations in Mfn2 and result in progressive loss of distal motor and sensory neurons of peripheral nervous system. However, the pathomechanism of Mfn2 mutations and specific degeneration of peripheral motor and sensory neurons is still unclear.