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The Northern Cape Province of South Africa is host to some of the largest deposits of iron and manganese metal in the world, contained in two separate districts, namely the Kalahari (KMF) and Postmasburg Manganese fields (PMF). The current thesis attempts to shed light on the chronicle of the complex epigenetic history of the two ore fields by focusing on the previously underexamined recurring theme of gangue alkali-rich assemblages, contained in and flanking a series of Fe and Mn orebodies. The principal outcomes of this study not only (a) set constrains on the characteristics, origin, sources, timing and relationship of alkali mineralization between the KMF and PMF, but also (b) challenge current ore genetic models by exploring the role of regional "alkali flooding" as a shared mechanism in the formation or at least hydrothermal upgrade of the deposits, (c) are beneficial for future exploration for new orebodies in the study and other prospective areas and (d) improve our knowledge of fluid circulation in sedimentary basins.

There are many ways to say "Hello," be it a wave, a nod, or a bow. We greet others not only with words, but also with our bodies. Embodied communication permeates our interactions. A fist bump, thumbs-up, or pat on the back can be even more meaningful than hearing "good job!" A friend crossing their arms with a scowl, turning away from you, or stiffening up can feel like a harsh rejection. Social communication is not exclusively linguistic, but is a multi-sensory affair. It's not that communication without these bodily cues is impossible, but it is impoverished. Embodiment is a fundamental human experience. Expressing ourselves through our bodies provides a powerful channel through which we express a plethora of meta-social information. And integral to communication, expression, and social engagement is our utilization of conversational gesture. We use gestures to express extra-linguistic information, to emphasize our point, and to embody mental and linguistic metaphors that add depth and color to social interaction. The gesture behaviour of virtual humans when compared to human-human conversation is limited, depending on the approach taken to automate performances of these characters. The generation of nonverbal behaviour for virtual humans can be approximately classified as either: 1) data-driven approaches that learn a mapping from aspects of the verbal channel, such as prosody, to gestures; or 2) rule bases approaches that are often tailored by designers for specific applications. This thesis is an interdisciplinary exploration that bridges these two approaches, and brings data-driven analyses to observational gesture research. By marrying a rich history of gesture research in behavioral psychology with data-driven techniques, this body of work brings rigorous computational methods to gesture classification, analysis, and generation. It addresses how researchers can exploit computational methods to make virtual humans gesture with the same richness, complexity, and apparent effortlessness as you and I. Throughout this work the central focus is on metaphoric gestures. These gestures are capable of conveying rich, nuanced, multi-dimensional meaning, and raise several challenges in their generation, including establishing and interpreting a gesture's communicative meaning, and selecting a performance to convey it. As such, effectively utilizing these gestures remains an open challenge in virtual agent research. This thesis explores how metaphoric gestures are interpreted by an observer, how one can generate such rich gestures using a mapping between utterance meaning and gesture, as well as how one can use data driven techniques to explore the mapping between utterance and metaphoric gestures. The thesis begins in Chapter 1 by outlining the interdisciplinary space of gesture research in psychology and generation in virtual agents. It then presents several studies that address presupposed assumptions raised about the need for rich, metaphoric gestures and the risk of false implicature when gestural meaning is ignored in gesture generation. In Chapter 2, two studies on metaphoric gestures that embody multiple metaphors argue three critical points that inform the rest of the thesis: that people form rich inferences from metaphoric gestures, these inferences are informed by cultural context and, more importantly, that any approach to analyzing the relation between utterance and metaphoric gesture needs to take into account that multiple metaphors may be conveyed by a single gesture. A third study presented in Chapter 3 highlights the risk of false implicature and discusses this in the context of current subjective evaluations of the qualitative influence of gesture on viewers. Chapters 4 and 5 then present a data-driven analysis approach to recovering an interpretable explicit mapping from utterance to metaphor. The approach described in detail in Chapter 4 clusters gestural motion and relates those clusters to the semantic analysis of associated utterance. Then, Chapter 5 demonstrates how this approach can be used both as a framework for data-driven techniques in the study of gesture as well as form the basis of a gesture generation approach for virtual humans. The framework used in the last two chapters ties together the main themes of this thesis: how we can use observational behavioral gesture research to inform data-driven analysis methods, how embodied metaphor relates to fine-grained gestural motion, and how to exploit this relationship to generate rich, communicatively nuanced gestures on virtual agents. While gestures show huge variation, the goal of this thesis is to start to characterize and codify that variation using modern data-driven techniques. The final chapter of this thesis reflects on the many challenges and obstacles the field of gesture generation continues to face. The potential for applications of Virtual Agents to have broad impacts on our daily lives increases with the growing pervasiveness of digital interfaces, technical breakthroughs, and collaborative interdisciplinary research efforts. It concludes with an optimistic vision of applications for virtual agents with deep models of non-verbal social behaviour and their potential to encourage multi-disciplinary collaboration.

This thesis presents the results of lattice Quantum Chromodynamics (QCD) calculations of hadronic matrix elements for pseudoscalar to pseudoscalar semileptonic decays involving c → s and b → s weak interaction transitions. We calculate scalar, vector and tensor form factors, and use them to test the Standard Model. Most prominently, we use D → Klv form factors, calculated on the lattice in the Highly Improved Staggered Quark formalism, to calculate a much more precise determination of the Cabbibo-Kobayashi-Maskawa (CKM) matrix element |Vcs|, allowing for more stringent tests of CKM unitarity. We also calculate branching fractions and other experimentally measurable quantities for the rare B → Kll decay, finding significant tension with experiment in this one-loop level process, which is sensitive to physics beyond the Standard Model.

Lung cancer is the most common form of cancer in the world, both in terms of diagnosis and number of deaths per annum. Approximately 85% of cases diagnosed are non-small cell lung cancer (NSCLC), of which adenocarcinoma is the most common histological subtype. Specific DNA mutations allow for molecular characterisation of the disease and activating mutations in KRAS are present in one third of cases. The oncogene MYC is a downstream target of KRAS and the two proteins have long been known to cooperate with one another to induce tumorigenesis. The gene editing enzyme APOBEC3B (A3B) is a cytidine deaminase, deaminating cytidine residues in the genome, resulting in C > T transversions. If these point mutations occur in the coding region of a gene this can result in a mutated protein with altered function. A3B has been shown to be involved in tumour evolution, invasion and metastasis, making it an attractive target for investigation. Additionally, upregulation of A3B in NSCLC has been correlated with an increase in mutations at GC base pairs and the A3B mutations signature has been noted in the tumours of both smokers and non-smokers. The mutations caused by A3B catalytic activity may result in the tumours being more immune visible, due to increased neoantigen production. This presents an opportunity for the tumour immune response to recognise tumour cells and destroy them before they can progress to a clinically relevant malignancy. Genetically engineered mouse models of cancer have contributed significantly to the field of cancer research. Tumours arising in advanced GEMMs closely mimic the features of their human counterparts and are able to spontaneously progress to metastatic disease. However, there are some limitations to currently available GEMMs, one such being that they are not truly reflective of the high tumour mutation burden seen in human non-small cell lung cancer. Therefore, there is an urgent need to develop models that reflect the mutational diversity seen in the human disease in order to better understand the challenges faced in the human disease, such as resistance to therapy. The work presented in this thesis describes the characterisation of a novel genetically engineered mouse model of NSCLC. This conditional model allows for the expression of the oncogenes Kras and Myc (KM) exclusively in the lung epithelium to drive tumour formation. A modest overexpression of A3B (KMA) is then used to drive increased tumour mutation burden.

Background: Small conductance potassium channels (SK) channels have been shown to participate specifically in atrial repolarisation, modulating action potential duration (APD). Atrial arrhythmias, including AF have been associated with overexpression of SK channels where they are thought to contribute to APD abnormalities. Their role in the ventricular electrophysiology remains controversial with limited evidence of their function. Purpose: This study aimed to reveal the role of the SK channel in the AP of atrial and ventricular muscle in healthy and AF susceptible hearts. Additionally, the sensitivity of the SK channel to [Ca2+]i was investigated using low extracellular [Ca2+] and intracellular [Ca2+]-buffers. Methods: Electrophysiological measurements of APs and [Ca2+] were made using optical techniques in enzymatically digested cardiomyocytes and isolated rabbit hearts. The specific SK channel blocker, ICAGEN was used to investigate the contribution of the current to the APD. [Ca2+]i was altered in two ways: (1) lowering extracellular [Ca2+] to 0.3mM and (2) chelating intracellular Ca2+ by incubating in 20µM BAPTA-AM. Results: Pharmacological block of SK channels in tissue preparations caused a significant increase in APD by ~30% and 40% prolongation of APD50 and APD75/90 respectively at 10min drug perfusion in atrial muscle. No response to ICAGEN was observed in the ventricle. Atrial APs in low extracellular [Ca2+] retained a significant ICAGEN-sensitivity causing comparable prolongation to that seen in 1.8mM [Ca2+]. However, buffering [Ca2+]i with BAPTA abolished the drug effect with no significant APD prolongation in either cells or tissue. No APD prolongation occurred in response to ICAGEN in both the atrium and ventricle of AF-susceptible hearts from a heart failure (HF) model. Conclusion: Using ICAGEN as a tool to probe SK channel activity, the data suggests that SK channels have an atrial-specific function in repolarisation in the normal rabbit. From the response to BAPTA loading, the channels appeared sensitive to local (possibly dyadic) [Ca2+] rather than global [Ca2+]i. The role of the channel in disease remains unclear with no evident contribution to AP repolarisation in an AF-susceptible rabbit model.

The Canadian Prairies rely heavily on ample amounts of freshwater, especially for their agricultural activities. Its position in the rain shadow of the Rocky Mountains and long-distance to major water bodies makes this area prone to re-occurring droughts and occasional floods. Especially the former creates costly natural and socio-economic disasters. Although, our understanding of climate drivers is fairly robust on a global scale, it is incredibly limited on a regional scale, which is indicated by our inability to fully explain the 2000s drought in the Canadian Prairie. Long-term regional droughts have the potential to occur abruptly but there is low confidence in current climate projections of their frequency, severity and timing (IPCC AR5, 2013). This uncertainty is to a large part caused by our limited understanding of climate drivers on regional and sub-centennial spatio-temporal scales. Here, the exceptionally low density of robust, (cross-) validated palaeoclimate records in the Northern Great Plains is particularly problematic. Dendrohydrological reconstructions are sparse, mostly short (<500 years) and of reduced fidelity in this region. Diatom-inferred salinity is based on the premise that water balance (P-ET) in a closed lake is traced by salinity but several in-lake processes exist that can decouple the salinity - water balance relationship. Pollen-inferred temperature/precipitation uses the vegetation's response to climate variability which limits its temporal resolution to 50-100 years. Moreover, training data sets in the Northern Great Plains that are used to find modern analogues are affected by anthropogenic impact on species distribution. The meta-problem for the Canadian Prairies hence is two-fold: we lack a) proxies that allow high spatio-temporal resolution and b) robust, site-validated palaeoclimate reconstructions of multi-millenial length. In this thesis, we substantially contribute to the understanding of the climate system in the Canadian Prairies and how novel proxies in terrestrial palaeoclimatology can offer us valuable insights into past climate variability. We advance comparatively novel proxies (in terrestrial palaeoclimatology) based on long-chain diols, ostracod trace metal geochemistry, X-Ray Fluorescence (XRF) and compound-specific isotope analysis (CSIA). Moreover, we apply long-chain alkenones, CSIA, n-alkanes, pigments, bulk isotope analysis (13C), XRF and grain size analysis to reconstruct past lake surface temperature, hydrology and organic matter composition. In the process, we use cutting-edge technology like Mass Spectrometry Imaging to gain sub-annual insights into temperature variability during the abrupt termination of the Holocene Climatic Optimum. Our ability to understand climate events, their causes and for the recent events (e.g. Dust Bowl Drought) our role in it, can be the cradle of desperately needed changes in how we manage the earth system. Climate reconstructions as the basis for projections offer a glimpse into a potential future and at the same time incentive to avoid that future.

Exploitation of the resources available in space is one of the key challenges for future space exploration. Many of these resources have been recognized as potentially low-cost alternatives to those launched from Earth. In particular, near-Earth asteroids are among the easiest objects to reach and could provide resources such as water, liquid propellants electrolysed form water, semiconductors, and metals. Several studies have shown that a useful quantity of accessible resources may be available to be transferred into Earth orbit with transfer energies lower than that required to exploit material from the Moon. To address this problem, different scenarios can be envisaged to transfer material to Earth orbit or Halo orbits, such as transport of the entire asteroid or transport of mined material, the optimal choice depending on the particular asteroid of interest. A further possibility is in-situ manufacturing using asteroid resources, for example to assemble space-structures directly nearby the asteroid or to process water for propellants or life support. Motivated by this growing interest in asteroid resource exploitation, this thesis investigates a novel strategy to deliver a fraction of the asteroid mass into orbit about the asteroid or to escape. The analysis has its roots in the idea of leveraging the rotational kinetic energy of a rotating body to lift material, for example with the concept of the space elevator. The elevator is envisaged as a tethered structure to connect a mass in synchronous (or higher) orbit and the surface of the body. The tether is in equilibrium by the balance of centripetal and gravitational forces acting on it; the payload, i.e. mass extracted from the asteroid, is then lifted to the desired altitude along the tether and, if synchronous orbit is reached, the payload could increase its altitude without further work required. A direct evolution of the space elevator is the orbital siphon concept which is the foundation of this thesis. In this case, rather than a single payload ascending along the tether, a chain of tether-connected masses is envisaged, where the centrifugal-induced pull due to the body's spin can overcome the gravitational force on the payloads, eventually allowing payloads to escape. A chain of payloads can therefore be envisaged to provide a continuous mass flow from the surface of a rotating asteroid into orbit (siphon effect): new payloads are connected to the chain while the top payloads are removed and released into orbit, without the need for external work to be done. The siphon, as with the space elevator, can in principle be used as a payload-raising mechanism on any rotating body. However, contrary to the space elevator, the siphon does not require external work to lift asteroid material below synchronous altitude. In support of mining operations, the siphon can be used to raise mined material to a collecting/processing station in orbit around the asteroid or directly connected to the siphon. Alternatively, the siphon can be used to release material to escape, without the need to use propellant-based methods. This thesis therefore will investigate the dynamics of an orbital siphon anchored at an asteroid and examine a range of applications in the context of asteroid manipulation and resource exploitation. Long-term effects of the siphon operation are discussed, showing that this device allows a significant quantity of mass to be raised to orbit or to escape. It is shown that an optimal siphon length can be chosen, such that the extracted mass is maximised. Key variables, such as achievable mass flow rates, tension on the tethers, timescales and anchor forces are discussed. It is demonstrated that the oscillations of this device resulting from Coriolis forces are damped and the siphon will eventually align with the local vertical if mass is released to a collecting spacecraft connected at the top of the siphon. Moreover, it is proposed that the siphon dynamics could be leveraged to deliver resource payloads to stable equilibria about the asteroid, with a smaller delta-v than direct transfer from the surface, which may be beneficial in a long-term mining scenario. Effects of an irregular gravity field on the siphon dynamics are also examined, using polyhedral shape models of two candidate asteroids. The siphon effect is still generated for the candidate asteroids analysed, even with motion of the anchoring system on the asteroid surface, thus allowing the mining location to be moved without interrupting the flow of material to the collecting spacecraft. If a large quantity of material is released to escape, the siphon effect may also be exploited to induce a small variation to the heliocentric velocity of a potentially hazardous asteroid for impact risk mitigation. It is shown that typical delta-v on the order of 1 cm/s can be achieved within a time window of a decade. Finally, use of the orbital siphon to generate artificial cavities for habitats or storage of mined material is discussed.

Type 2 immunity is activated in response to both allergens and helminths, but it can be detrimental or beneficial depending on the stimuli. These conflicting outcomes highlight the importance of understanding the regulation of this response. Th2 cells are key for the clearance of helminth infections in the SILP. IL-10 is a regulatory cytokine that is highly expressed during human and murine helminth infection, but its role in helminth infection remains unclear. The aim of this thesis is to assess if IL-10 acts directly or indirectly to promote the type 2 immune response while also suppressing counter-active Th1 cells which provide immune competition. We show that helminth infection results in increased IL-10 expression in both the SILP and the draining MLN in mice, with highest expression in the infected tissue. In vivo blockade of IL-10 led to a significant decrease in IL-13, IL-5 and GATA3 expression by Th2 cells in the intestine and not in the MLN. In vitro, IL-10 induced expression of GATA3 and production of IL-5 and IL-13 in purified CD4+ T cells. The impact of IL-10 was partially dependent on IFNγ signalling, and in vivo, surface IL-10 receptor expression was higher on Th1 cells than on Th2 cells and highest on Th1 cells in the SILP. Due to the nature of the H. polygyrus lifecycle, where at two-time points worms move through the wall of the intestine. We hypothesised that a possible indirect mechanism for IL-10 promotion of Th2 cells was via suppression of IFNγ responses as a result of bacteria stimuli. However, our data show no major disruption to the intestinal barrier during infection. Although, we do report IFNγ responses in the SILP, MLN and omentum over the course of H. polygyrus infection. Suggesting IFNγ may play multiple roles during infection. Furthermore, we found that MLN cells from H. polygyrus infected mice produced IFNγ when stimulated with HES, suggesting that IFNγ responses towards H. polygyrus itself may occur, although the function of this in the immune response to H. polygyrus remains unclear. Understanding immune competition during H. polygyrus infection contributes towards the understanding of the immune response to helminths and with further work may contribute towards treatment strategies. In addition, IL-10 optimisation of Th2 responses during helminth infection may be applied to the treatment of helminth infections and other type 2 mediated diseases.

Coalbed methane (CBM) is an important energy source in many countries. The origin and post-generation evolution of methane in coalbeds are rather poorly understood despite the importance of assessment of reserves and developing exploration strategies. Stable isotopes are the typical geochemical tools to analyse the gas origin and subsequent evolution. Noble gases are widely used for identifying the source of fluids in reservoirs and the impact of the tectonic and hydrologic activities on hydrocarbon generation and preservation. In this PhD, I have studied the composition of gases from the Southeast Qinshui Basin, the most CBM productive region in China, and the Liupanshui Coalfield, a CBM development target in southern China. Methane in these two regions was formed in the late Triassic during burial and heating driven by magmatic activity in the Late Jurassic to Early Cretaceous. Cenozoic basin inversion brought the coals to a shallow depth. This study aims to establish how the gas formed, to estimate the contribution of magmatic heating on methane generation and the influence of basin exhumation on gas storage. Nineteen gas samples were taken in April 2017 from methane-producing wellheads along an E-W transect perpendicular to the boundary fault in Southeast Qinshui Basin (SQB) in northern China. The stable isotope compositions (δ13C= -35.2 to -30.2‰, δD= -194 to -155‰) indicates a thermogenic origin with limited microbial input. They are, however, lighter than expected based on coal maturity, perhaps due to adsorption/desorption fractionation during commercial gas extraction. He-Ne-Ar isotopes are a mixture of crustal-radiogenic gas with air-derived noble gases from groundwater. 4He concentrations (0.52 to 33.25 ppmv) and 4He/40Ar* ratios (0.06-1.74) are unusually low. He-Ne-Ar concentrations are consistent with the open system Rayleigh fractionation of noble gases derived from air-saturated water (ASW) with 4He/40Ar*=1 during gas extraction. The low 4He/40Ar* compared with average crust (5) or local production (13) values, implies that radiogenic 4He produced in the coals was lost prior to equilibrium between gas and water phase in the reservoir. This likely occurred during rapid exhumation in Cenozoic. The event may have led to the loss of up to 44% of the methane from the coal seams. The depletion of Xe and Kr without significant enrichment of Ne suggests the strong retention of Kr and Xe in SQB coals. Ten CBM samples were collected in April 2018 from the four blocks containing different mature coals in Liupanshui Coalfield (LPC) in southern China. Two gas samples were collected from the same two wells in the Dahebian block in October 2018. This is the first gas geochemical work in LPC. Methane is mainly thermogenic in origin based on the C1/(C2+C3) ratio (16-971), δ13CCH4 (-42.9 to -34.9‰) and δDCH4 (-206 to -140‰). The difference in major gas and stable isotope compositions between the four blocks of LPC is mainly due to the different extent of maturation of coals in each block. Noble gases in LPC are a mixture of air-derived gases from ASW with crustal gases in the reservoir. The lower proportion of nucleogenic Ne and 4He/40Ar* ratios in gases from Qingshan block compared with other blocks implies an evident He and Ne loss. The gas loss is also inferred to be the result of basin inversion. 20Ne/36Ar ratios (0.23-0.70) are higher than local ASW (0.16), requiring a re-dissolution fractionation process similar to that proposed for CO2 gas reservoirs (Gilfillan et al., 2008). 84Kr/36Ar and 132Xe/36Ar ratios in LPC are mostly higher than the modelled fractionation lines. This is explained by desorption of initial trapped Xe and Kr in the coal matrix. The Qingshan block gases contain the same depleted Kr and Xe pattern as SQB, suggesting the stronger adsorption capacity of local coals to Kr and Xe. Over 93% of He and 9% of Ar budget of the coal basins is estimated to have been lost during the gas loss process. It explains the absence of mantle volatiles in the SQB and LPC CBM. This demonstrates the importance of basin inversion on gas preservation in shallow CBM and shows that the light noble gases are essential for tracing such a process.

Insulin regulates glucose homeostasis by stimulation of glucose transport into adipose and muscle tissues through the regulated trafficking of glucose transporter 4 (GLUT4). In response to insulin GLUT4 rapidly translocates from intracellular storage sites to the plasma membrane where it facilitates glucose uptake. Significant impairments in glucose transport and GLUT4 trafficking are a major hallmark of diabetes mellitus type II. Recent advances in light microscopy techniques enabled the study of GLUT4 dynamics in the plasma membrane and it was reported that the transporter was clustered in the basal state and insulin stimulation resulted in GLUT4 dispersal. The main aim of this study was to develop a microscopy-based assay to study and quantify insulin-stimulated GLUT4 dispersal dynamics in the plasma membrane. Insulin-stimulated GLUT4 dispersal has only been observed in adipocytes and therefore we have chosen this model as a starting point to investigate the molecular mechanisms behind GLUT4 clustering and dispersal. We explored a range of cluster analysis methods to find the most suitable way to quantify GLUT4 clustering dynamics. Furthermore, this project aimed to optimise super resolution imaging in a variety of cell culture models to determine whether insulin-stimulated GLUT4 dispersal operates in skeletal and cardiac muscle and whether this process is affected by disease. Using a range of approaches we showed that insulin results in GLUT4 translocation and dispersal within the plasma membrane of 3T3-L1 adipocytes. We found that AMPK activation attenuated insulin-stimulated glucose uptake in 3T3-L1 adipocytes and also GLUT4 dispersal. It was observed that cholesterol depletion resulted in increased glucose uptake rates and GLUT4 clustering. Knock down of the membrane-localised protein EFR3 that has previously been shown to be involved in glucose uptake resulted in disruption of GLUT4 dispersal in adipocytes. We also found that HeLa cells show similar insulin-stimulated GLUT4 dispersal as adipocytes and suggest that HeLa cells are a suitable experimental model for initial studies of GLUT4 trafficking and dispersal. Chronic insulin treatment was observed to induce a state of cellular insulin resistance in 3T3-L1 adipocytes and resulted in reduced GLUT4 translocation and a more clustered GLUT4 configuration for both basal and insulin-stimulated cells. This indicates that insulin resistance affects intracellular GLUT4 trafficking pathways as well as the organization of the transporter within the plasma membrane in adipocytes. Moreover, we found a negative correlation between adipocyte cell area and insulin-stimulated GLUT4 translocation. We also report that insulin did not stimulate the reorganisation of the transferrin receptor in the plasma membrane of HeLa cells suggesting that insulin-stimulated GLUT4 dispersal did not originate from endosomal compartments in HeLa cells and that this observed effect may be specific for GLUT4. Finally, we observed that insulin did not affect GLUT4 distribution in the membrane of a commercially available model of skeletal muscle from healthy and diabetic donors. Sortilin is a sorting receptor involved in the formation of GLUT4 containing vesicles and levels of this protein were found to be reduced in skeletal muscle myotubes derived from a diabetic donor. Finally, we discovered that insulin stimulated GLUT4 dispersal also operates in stem cell-derived cardiomyocytes and have investigated GLUT4 dispersal in a variety of in vitro models of cardiac muscle tissue. Taken together, this thesis has detailed several novel findings regarding the regulation of GLUT4 clustering in adipose and muscle tissues. A robust assay to measure GLUT4 dispersal has been established and molecular mechanisms behind the observed GLUT4 clustering dynamics have been described in adipocytes. Furthermore GLUT4 clustering was characterised in several cell culture models of skeletal and cardiac muscle for the first time.

Leishmania parasites are transmitted by the bite of a sandfly vector, and have the ability to subvert the host immune system and adopt sophisticated strategies to develop and survive within the mammalian host. Leishmania parasites survive within macrophages, inside a parasitophorous vacuole and cause leishmaniasis. The molecular communication between host and parasite decides the outcome of infection, but is incompletely understood. Additionally, there is no vaccine currently available against human leishmaniasis, existing chemotherapies are toxic, and drug resistance is prevalent. However, leishmaniasis is vaccinable, as evidenced by acquired immunity and the availability of canine vaccines. Live attenuated Leishmania parasites can be exploited as potential vaccines that deliver prolonged immune stimulation from the native parasite niche. We have compared genotype and gene expression of an attenuated Leishmania mexicana line with a virulent, isogenic wild-type precursor. The attenuated L. mexicana (H-line) was developed by in vitro culturing the parasites under gentamicin pressure. The H-line parasites of various Leishmania species have demonstrated potential as vaccines in rodents models and dogs, and a L. major H-line is currently undergoing phase 2 and phase 3 trials in humans. Therefore, we aim to identify key virulence factors and to explore the potential of the attenuated H-line as a vaccine candidate. This study was conducted on promastigotes of L. mexicana and involved comprehensive polyomics approaches to elucidate the molecular mechanism that underpins attenuation in Leishmania parasites. Log phase promastigotes of wildtype and the gentamicin-attenuated H-line were grown in parallel and harvested for comparative polyomics analyses. For genomics, DNA was extracted and converted into DNA library for next generation sequencing. For transcriptomics, RNA was isolated and converted into a library of cDNA molecules for cluster generation and RNA sequencing. For proteomics, protein extracts were labelled using 6-plex TMT and analyzed with LC-MS/MS. For metabolomics, finger-print and foot-print metabolites were extracted with chloroform/methanol/water (1:3:1) and analyzed with LC-MS. Integration of polyomics data was carried out based on the gene ontology of the modulated omics products. Selected genes were targeted by CRISPR-Cas9 to assess their individual role in attenuation. Genomics data of the attenuated H-line revealed the presence of many gene variants and an increase of copy number in chromosomes 16 and 30, which were triploid and tetraploid, respectively. We found 481 transcripts were differentially expressed, 18 proteins differentially expressed, and 26 identified metabolites were differentially represented in the attenuated H-line Leishmania. Statistical significance thresholds in these datasets were assigned for FC ≥1.5 and FDR ≤0.05. Most of the differentially expressed proteins and transcripts were metabolic enzymes, and the majority of the differentially expressed metabolites were substrates involved in nucleotide, carbohydrate and amino acid metabolism. Based on gene ontology, correlation of polyomics datasets revealed that nucleotide metabolism was significantly altered in gentamicin-attenuated Leishmania. Interestingly, several transmembrane nutrient transporters were modulated. Nucleoside (NT1) and nucleobase (NT3) transporters were significantly down regulated in proteomics analysis. Modulation of gene expression, observed through polyomics analyses, may relate to gentamicin selection, either directly through drug selection or as an adaptive consequence of drug selection. The H-line L. mexicana and LmexΔNT3 cells become more sensitive to allopurinol, a hypoxanthine analogue, at a lower concentration (EC50= 39.1 ± 6.35 and 14.1 ± 0.65 µM, respectively, comparing to the wild-type cells (EC50= 136 ± 4.23 µM). In contrast, LmexΔNT1 cells were less sensitive to tubercidin, an adenosine analogue, (EC50= 7.74 ± 0.21 µM) comparing to the wild-type cells (EC50= 0.76 ± 0.01 µM). This suggests that NT1 and NT3 transporters may contribute to Leishmania virulence. The attenuated H-line invaded but was unable to survive in vitro within THP-1 macrophage-like cells, whereas wild-type parasites were able to survive and multiply. Moreover, findings showed that the H-line, LmexΔNT1, and LmexΔNT3 L. mexicana harboured less than 2 parasites per an infected THP-1 cell compared to the wild-type parasites which harboured about 4 parasites per an infected THP-1 cell. Important concerns about the possibility of reversion to a virulent phenotype must be further addressed before live vaccines can be approved for use in the field.

Engagement is a key factor in understanding people's psychology and behaviours and is an understudied topic in children. The area of focus in this thesis is child engagement in the story-stems used in child Attachment evaluations such as the Manchester Child Attachment Task (MCAST). Due to the high cost and time required for conducting Attachment assessments, automated assessments are being developed. These present story-stems in a cost-effective way on a laptop screen to digitalise the interaction between the child and the story, without disrupting the storytelling. However, providing such tests via computer relies on the child being engaged in the digital story-stem. If they are not engaged, then the tests will not be successful and the collected data will be of poor-quality, which will not allow for successful detection of Attachment status. Therefore, the aim of this research is to investigate a range of aspects of child engagement to understand how to engage children in story-stems, and how to measure their engagement levels. This thesis focuses on measuring the levels of child engagement in digital story-stems and specifically on understanding the effect of multimedia digital story-stems on children's engagement levels to create a better and more engaging digital story-stem. Data sources used in this thesis include the observation of each child's facial behaviours and a questionnaire with Smiley-o-meter scale. Measurement tools are developed and validated through analyses of facial data from children when watching digital story-stems with different presentation and voice types. Results showed that facial data analysis, using eye-tracking measures and facial action units (AUs) recognition, can be used to measure children's engagement levels in the context of viewing digital story-stems. Using eye-tracking measures, engaged children have longer fixation durations in both mean and sum of fixation durations, which reflect that a child was deeply engaged in the story-stems. Facial AU recognition had better performance in a binary classification for discriminating engaged or disengaged children than eye-tracking measurements. The most frequently occurring facial action units taken from the engaged classes show that children's facial action units indicated signs of fear, which suggest that children felt anxiety and distress while watching the story-stems. These feeling of anxiety and distress show that children have a strong emotional engagement and can locate themselves in the story-stems, showing that they were strong engaged. A further contribution in this thesis was to investigate the best way of creating an engaging story-stem. Results showed that an animated video narrated by a female expressive voice was most engaging. Compared to the live-action MCAST video, data showed that children were more engaged in the animated videos. Voice gender and voice expressiveness were two factors of the quality of storytelling voice that were evaluated and both affected children's engagement levels. The distribution of child engagement across different voice types was compared to find the best storytelling voice type for story-stem design. A female expressive voice had a better performance for displaying the 'distress' in the story-stem than other voice types and engaged children more in the story-stems. The quality of the storytelling voice used to narrate story-stems and animated videos both significantly affected children's levels of engagement. Such digital story-stems make children more engaged in the digital MCAST test.

Solar eruptive events are associated with radio emissions that appear as impulsive increases in intensity, known as solar radio bursts. Turbulence in the solar corona impacts the propagation of radio waves, obscuring the intrinsic emission properties. Here, anisotropic scattering on small-scale density fluctuations is investigated using novel 3D radio-wave propagation simulations. Several observed radio properties are simultaneously reproduced for the first time, verifying the necessity to consider anisotropic scattering. The sub-second evolution of fine radio burst properties at a single frequency is also investigated, enabled by conducting observations that utilise the unprecedented imaging capabilities of the LOw-Frequency ARray (LOFAR). The fundamental and harmonic sources of a Type IIIb burst are quantitatively compared, demonstrating that harmonic emissions arise from an intrinsic source with a finite size and finite emission duration. Drift-pair burst observations are successfully described by the radio echo hypothesis. It is shown that the radio echo, which produces the second Drift-pair component, is detected only when the anisotropy is strong. A dependence of the observed properties on the source's intrinsic location and on the assumed emission-to-plasma frequency ratio is inferred. Moreover, the subbands of a split-band Type II burst are simultaneously imaged for the first time. Despite the large separations observed between subband sources, it is shown that once scattering is quantitatively accounted for, the sources become co-spatial. Corrections on the observed source locations also allude to lower coronal densities. Additionally, the first observation of a Type II burst that transitions between a stationary and drifting state-termed as a transitioning Type II burst-is reported. The radio emissions are related to a jet eruption that drives a streamer-puff CME. Overall, state-of-the-art simulations and radio observations are combined and compared. The importance of accounting for radio-wave propagation effects-primarily anisotropic scattering-and the consequence of neglecting to do so on any subsequent interpretations is illustrated.

The topology of DNA is crucial to cellular processes such as replication, transcription, and recombination. The protein-DNA interactions of these processes can induce major topological changes into DNA molecules. The Xer site-specific DNA recombination system is a very well characterised topology-dependent mechanism. First discovered in Escherichia coli, Xer consists of a heterotetrametric tyrosine recombinase complex paired with several accessory proteins. Xer is involved in the resolution of chromosome dimers, and in the conversion of supercoiled plasmid multimers to monomeric states. Both Xer-dependent mechanisms are required for equal and faithful segregation of E. coli DNA at cell division. There has long been an aim to observe the topological complexity of DNA, e.g., the products of site-specific recombination, on a single molecule level. Existing techniques such as rotary shadowing electron microscopy have seen little recent development and have significant practical limitations. In this thesis I demonstrate the application of high-resolution Atomic Force Microscopy (AFM) in liquid to the topological determination of DNA knots and catenanes. The E. coli Xer synapse was used as a tool to generate knotted or catenated DNA in vitro. Several aspects of knot and catenane generation e.g., reaction conditions, substrate length modifications, repeatability, and DNA purification were probed in depth. Several putative DNA knots were classified from AFM micrographs by tracing their path and assigning chirality based on the topographical profile of the DNA-DNA crossovers. Every possible knotted configuration of three AFM-traced DNA molecules was determined by hand. These were most likely 4-node knot (4-1), 5-1* twist-knot, and an unknot. Subsequently, a 4-node catenated DNA with a homogenous topological nature was studied by AFM to focus solely on the discrimination of over-crossing DNA strands versus under-crossing strands. Individually, several AFM micrographs of 4-node catenanes were of extremely high image quality, where their crossovers could be readily determined and were consistent with prior biochemical evidence of chirality. However, the majority of catenanes adopted configurations where their crossings were clustered indecipherably. Only 13% of the traced 4-node catenanes were of conformations that might be conducive to the determination of crossover directionality. The work described in this thesis represents the first topological study of DNA knots and catenanes by AFM, and demonstrated the utility of this technique. Future studies of DNA knots and catenanes by AFM will require further optimisation of sample deposition conditions and imaging parameters to reduce the tendency of DNA molecules to cluster together, and to emphasise the directionality of knotted or catenated DNA crossovers.

Retinal imaging allows assessment of ocular health, and is important for the detection and monitoring of retinal diseases. One such device for imaging the retina is the ultra-wide field of view scanning laser ophthalmoscope (UWFoV-SLO), manufactured by Optos plc, which is capable of imaging up to 200 degrees of the retina in a single scan. This thesis details work in two areas, both relating to the image processing of UWFoV-SLO images. The first is the investigation of automated image quality assessment algorithms for UWFoV-SLO images. A novel image quality metric (referred to as the GVC metric) was developed, which is based on a textural measure of image patches that contain retinal vasculature. This metric is demonstrated to correlate with the assessment of image quality as graded by experts. The GVC metric was applied to images captured at a UWFoV-SLO manufacturing facility, and timeseries analysis of this data discovered a number of potential system modifications that could improve the quality of manufactured devices. These included the correction of a software bug that was impairing image quality, and to install all manufactured devices with a detector that captured higher quality images. These observations were further confirmed with forced-choice preference testing with expert assessors of image quality. The second investigation concerns the automated localisation of two retinal landmarks (the optic disc and fovea) in UWFoV-SLO images with convolutional neural networks. On a test set of 1485 images, optic disc localisation accuracy of 96.70% less than 1 optic disc radius from the ground truth was achieved, and 93.27% of fovea predictions were less than 1 optic disc radius from the ground truth. It is also shown that the laterality of the image (whether it is of the left or right eye) can be reliably inferred from the landmark coordinates. Finally, three methods to automatically detect unreliable landmark predictions are presented. These are based on prior knowledge of the spatial distribution of landmarks --- two methods employ Gaussian mixture models, and the third applies a threshold to the Euclidean distance between the predicted landmark coordinates.

Research into third party kinship recognition has been sparse even though kinship provides crucial insight into the biological underpinnings of pro-social and sexual behaviour. Furthermore, the studies that have been conducted are of varying quality and consistency, resulting in a myriad of different findings and conclusions. My doctoral research addressed the common issues in the literature by conducting studies using high quality stimuli, a consistent methodology and appropriate analyses. Study 1 investigated what facial information is used for making kinship judgments in 3D facial images, specifically the contribution of face shape and surface reflectance information (e.g., skin texture, tone, eye and eyebrow colour). Using binomial logistic mixed models, we found that participants were able to detect relatedness at levels above chance for all three stimulus versions. Overall, both individual shape and surface reflectance information contribute to kinship detection, and both cues are optimally combined when presented together. Study 2 investigated whether a smiling facial expression increases the accuracy of judging relatedness compared to a neutral facial expression in human raters. Contrary to expectations, smiling decreased the accuracy of relatedness judgments compared to a neutral facial expression. Study 3 aimed to replicate previous studies suggesting that birth order affects kinship detection ability. Our findings indicate that laterborns do not have an advantage in detecting child sibling pairs and that kinship judgment accuracy is therefore unaffected by rater birth order. Study 4 compared the performance of participants across three commonly used methods (i.e., kinship judgment, similarity rating, matching paradigm), using the same highly-controlled stimulus set. We found that while responses on all three tasks were correlated, performance varied significantly across the tasks. Furthermore, when looking at the effect sex and age of the portrayed individuals had on performance, we found that different results are found dependent on which method is used.

The rapid development of 3D imaging technology allows data to be collected directly in three-dimensional space. The high accuracy of the images requires further investigations on digitised objects, especially of local features. In the last decade, 3D Local features have played an important role in recognising and modelling real-world 3D objects. This thesis introduces a series of methods for 3D local features, including automatic keypoints detection, 3D model construction with curves, local region detection and statistical analysis of local features. Those methods are not only to build 3D local feature descriptors but also have a wide range of applications, such as shape comparison in medical facial treatments and evolutionary researches in biology. Conventional shape analysis, limited by the data-collection technology, project 3D objects into 2D space to analyse or focus on 3D discrete points which are not close to each other. Those points of anatomical meanings are called landmarks. Researchers used to manually place the landmarks on 2D or 3D images by eyes, but it generates the operator error which is not of interest but has a large influence on shape analysis. This thesis introduces a novel method to automatically estimate the landmarks on 3D models using Bayesian statistics. The Procrustes matching of the landmark sets shows that the variation of Bayesian placements is much smaller than the manual placements. Local shapes like 'ridges' and 'valleys', which are considered to contain rich geometric information, can be estimated based on landmarks. Existing methods rely heavily on landmarks, but in most cases, the number of landmarks is not enough and adding extra ones are time-and-labour consuming. A flexible and user-customisable method is introduced in this thesis to deal with complex surfaces marked with as few landmarks as possible. A simulation study is conducted, and the result shows that the method is stable and efficient in terms of local feature description. After the 3D curve is estimated, methods to analyse the local features using the curves are discussed. An algorithm to flexibly dissect the surface along the estimated curve is developed for extracting local pieces or divide the surface into pieces. The novelty of this method is that it applies directly on 3D shapes and dissects the shape along any 3D curves, such as the lip edge on a human facial model. Besides the novel method for 3D shapes, curvatures, which reflect the bending amount along the curves, are calculated. The curvatures of the same local feature on different individuals are aligned to analyse the average shape difference of groups, such as gender and age. A reconstruction procedure from the curvatures is discussed and the effect of noise on choosing the degree of freedom in smoothing is investigated. Another application of the estimated curves is in benchmarking the performance of different 3D camera systems. A new camera system developed by NCTech®, Edinburgh, is assessed using the evaluation outcome of facial deformity surgeries in Brazil. It is designed to be child-friendly, portable and low-cost. Validation studies are carried out at three stages of the development, and both landmarks and curves are used to evaluate the performance of the new camera system on estimating local features in comparison with mature products from DI4D® and Artec®.

As organizations collect petabytes of data, analysts spend most of their time trying to extract insights. Although data analytic systems have become extremely efficient and sophisticated, the data exploration phase is still a laborious task with high productivity, monetary and mental costs. This dissertation presents the Query-Driven learning methodology in which multiple systems/frameworks are introduced to address the need of more efficient methods to analyze large data sets. Countless queries are executed daily, in large deployments, and are often left unexploited but we believe they are of immense value. This work describes how Machine Learning can be used to expedite the data exploration process by (a) estimating the results of aggregate queries (b) explaining data spaces through interpretable Machine Learning models (c) identifying data space regions that could be of interest to the data analyst. Compared to related work in all the associated domains, the proposed solutions do not utilize any of the underlying data. Because of that, they are extremely efficient, decoupled from underlying infrastructure and can easily be adapted. This dissertation is a first account of how the Query-Driven methodology can be effectively used to expedite the data exploration process focusing solely on extracting knowledge from queries and not from data.

There is an emerging need for low-cost medical diagnostics for both high and middle to low-resource settings. Surface acoustic waves microfluidics are emerging lab-on-a-chip technologies which have the potential to provide all-in-one solution to actuate liquids and sense biomarkers, thus enabling point-of-care bioassays. DNA has become a key biomarker for a range of medical conditions, including infectious diseases, as it provides critical information on the pathogen or the response of the patients to particular treatment, on a personalized basis. This thesis will examine the effects of surface acoustic waves on DNA hybridization, with a view to integrate molecular diagnostic assays onto acousto-fluidic devices. The work used fluorescence to characterize the binding of DNA in a range of conditions, and revealed nucleobase-specific quenching (NB-S Quench) of fluorophores when attached to DNA as a double strand. This latter effect was examined as a replacement for common analytical markers used in standard techniques, such as melting curves, which typically rely on dyes which recognize DNA strands non-specifically (such as groove binders). The technique has been shown to be suitable for determine the concentration of DNA, performing DNA amplification and identifying the presence and melting temperature of target DNA. This work will have an impact on research into low-cost medical diagnostics, and improve the understanding of fluorescence of DNA modified with fluorophores, contributing to the understanding of future work in these areas.

Improvements in the diagnosis and management of preeclampsia (PE) have contributed to a reduction in maternal mortality in parts of the world. However, current treatments still carry safety and efficacy issues, and perinatal morbidity and mortality remains prevalent. Research into the pathology of PE and new treatment strategies are therefore warranted. Gene therapy represents a potential treatment strategy since PE is now recognised as a complex genetic disorder. Despite the clinical progress of gene therapy in foetal growth restriction, less invasive and non-viral delivery systems targeting the placenta are required to mitigate safety concerns and extend research to other pregnancy disorders, such as PE. Identifying a therapeutic gene target in PE remains a challenge since a lack of replication in gene association studies suggests rare variants with small effect sizes likely contribute to the development of PE. Alternatively, a growing body of evidence suggests placental microRNAs (miRNAs) play a key role in the pathology of PE and may therefore represent therapeutic targets. Preclinical studies however remain limited, and inconsistencies between clinical studies make it difficult to discern placental miRNA profiles characteristic of PE. Therefore, in addition to investigating non-invasive and non-viral delivery systems for gene therapy purposes in PE, there is a need for further preclinical and clinical studies to examine the role of placental miRNAs in PE and evaluate their therapeutic potential. Ultrasound-mediated gene delivery (UMGD) can be utilised for non-viral and non-invasive gene transfer, a method in which ultrasound (US) stimulates vector uptake through its mechanical effects following interaction with US contrast agents known as microbubbles. Although the technique has been investigated in a variety of tissues, research into targeting the placenta is limited, with a single UMGD study in baboons published to date. Chapter 3 sought to develop and optimise an in vivo UMGD protocol in rodents that stimulates targeted transfer of a reporter gene to a surrogate organ which could then be applied to the placenta, given the difficulty in targeting a specific placenta in litter-bearing species. These optimisation studies allowed issues to be identified and logical modifications to be made to the protocol, including the type and size of vector employed, the plasmid DNA and MB dose, and the organ targeted. Through these studies, a protocol was established showing evidence of gene transfer of a luciferase plasmid to mouse hearts, allowing progression to a proof-of-concept study. Chapter 4 sought to demonstrate proof-of-concept that UMGD can achieve tissue-specific transfer of an expression vector and is therefore suitable for targeting placenta. UMGD of a luciferase plasmid to mouse hearts showed luciferase activity was significantly greater in the ventricles compared to non-target organs (liver, lungs, and spleen) (p<0.05), providing evidence of tissue-specific transfer. Although there was a trend towards greater luciferase activity in the ventricles compared with the remaining non-target organs (skeletal muscle, left kidney, and right kidney), this was non-significant. Due to small n-numbers, significance testing was not performed comparing tissue from the negative control mice with UMGD treatment mice. The protocol was subsequently applied to pregnant mice to target the placenta. Low levels of luciferase gene transfer were evident in the placenta positioned closest to the cervix on the targeted left uterine horn. Non-target tissues were not evaluated for gene transfer due to time constraints, meaning this aspect of safety could not be confirmed. Placental-specific delivery of an expression vector with UMGD therefore remains to be demonstrated in order to support the clinical translatability of this technique. Identifying a placental miRNA dysregulated in preeclamptic patients or a subset of patients remains essential to identifying miRNAs that represent a clinically relevant therapeutic target. Animal models on the other hand provide a means of exploring the role and therapeutic potential of miRNAs in PE. In Chapter 5, a literature review was conducted to collate evidence from patient studies and identify clinically relevant miRNAs of interest for therapeutic targeting. Subsequently, the expression of candidate miRNAs was evaluated in whole placental tissue and individual placental layers of a rat model of superimposed PE (SPE) previously established by our group. The literature review identified placental miRNAs consistently detected as significantly differentially expressed by miRNA expression profiling studies in third trimester preeclamptic patients. Clinical studies were either in agreement or discordant in the direction of expression of the miRNAs in PE. Evaluation of candidate miRNAs in the placenta of the SPE rat model contributed to the growing body of evidence that placental miRNAs differ between PE subtypes and placental layers, also providing novel evidence of dysregulation of miRNAs within placental layers in rats, a finding previously only shown in humans. Furthermore, four miRNAs (miR-210-3p, miR-223-3p, miR-181a-5p, and miR-363-3p) were identified as potential therapeutic targets given their consistent dysregulation in PE. MiRNA expression profiling allows identification of differentially expressed placental miRNAs in a hypothesis free manner. In Chapter 6, miRNA sequencing was performed to identify differentially expressed placental miRNAs in the rat model of SPE, representing the first study to conduct miRNA sequencing in the placenta of an animal model of PE. Furthermore, the dysregulation of select miRNAs and predicted gene targets was examined by RT-qPCR in placental tissue from the SPE rat model as well as preeclamptic patients to evaluate the clinical relevance of the findings. Expression of miR-210-3p and its predicted gene target, fibroblast growth factor receptor-like 1 (FGFRL1), demonstrated an inverse relationship in the placentas of the SPE rat model and preeclamptic patients as well as in a BeWo trophoblast cell line. This is in agreement with published data that show miR-210-3p is upregulated in PE and provides novel evidence of altered FGFRL1 expression in the placentas of preeclamptic patients. This adds to the existing literature that suggests miR-210-3p plays a pathological role in PE and is a promising therapeutic target. This thesis has provided information on a non-viral and non-invasive gene therapy technique for targeting the placenta as well as miRNAs dysregulated in the placenta in PE and potential therapeutic targets. The development of safe and efficient delivery systems that target the placenta and the identification of suitable targets represent key aspects to establishing placental gene therapy as a treatment strategy in PE. The work in this thesis has provided means to further investigate UMGD for targeting the placenta in pregnant mice, important preclinical models in pregnancy research. Furthermore, the work in this thesis has identified aspects of placental miRNAs that should be considered in future work seeking to investigate the role of and/or target placental miRNAs. Finally, the work in this thesis has identified several miRNAs that are potentially therapeutic targets in PE.

This work represents significant progress in the development of a Si3N4 waveguide platform for near infra-red wavelengths as required for 87Rb chip-scale atomic systems. The theoretical framework provided by ray optic and electromagnetic theory have formed a basis for understanding waveguide phenomena. Coupled mode theory has been used to determine the microring resonator coupling coefficient. An analytical model of a micro-ring resonator has been used to show that Q factor can be optimised by the decreasing the coupling coefficient. The Lacey-Payne scattering model suggests that increased waveguide width and reduced index contrast results in decreased loss due to sidewall scattering. Q factor has therefore been optimised by increasing waveguide width, developing a top cladding process, and operating in the undercoupled regime. By this approach, a record high Q factor value of (1.38*0.04)*10^6 has been demonstrated for buried waveguide micro-ring resonators operating around 780 nm corresponding to a propagation loss of 0.261*0.009 dB cm^−1. This high-Q micro-ring resonator could serve as a frequency reference for laser frequency stabilisation but a temperature dependent resonance wavelength shift of 7.34 pmK^−1 is expected and would require active temperature stabilisation to within 25mK. Passive athermalisation of a micro-ring resonator by optimising the thickness of polymer top cladding materials with a negative thermo-optic coeffcient has been investigated by simulation. A significant reduction of the temperature dependent wavelength shift of a micro-ring resonance peak has been found for PMMA and SU8 top cladding layers, 1.20 pmK^−1 and 0.14 pmK^−1 respectively corresponding to a required temperature stability of 167mK and 1.43K. Coupling of light from a Si3N4 waveguide to collimated free-space Gaussian beam has been considered within the context of developing an on-chip interferometer intended to measure the displacement of a MEMS gravimeter proof-mass. The divergence of a free-space coupled Gaussian beam has been determined. Taking into account the use of an SU8 spot-size converter, the Fresnel reflectance of the SU8 waveguide, and the measured reflectance of the silicon proof mass, the beam divergence is still found to be the dominant loss mechanism for the propagation distances involved in this application. Use of a micro-ball lens to collimate the beam was considered using various simulation techniques, determining that the vertical positioning of the lens would pose the greatest fabrication challenge. Processes were developed to produce SU8 waveguides, registration blocks, and micro-ball lens receptacles.

Glyphosate, N-(phosphonomethyl)glycine, is the active compound in glyphosate based herbicides. It is currently the most intensely applied herbicide across the globe. Likewise, the potential harmfulness of glyphosate has been heatedly debated as some studies have linked glyphosate exposure to cancer development and endocrine disruption in humans and other life forms. This work first aimed to investigate the effects of glyphosate and a glyphosate-based herbicide (Roundup) on the metabolism of Arabidopsis thaliana plants by utilising an untargeted metabolomics approach. Secondly, pure glyphosate toxicity was inspected upon exposure of HEK 293 cells for up to 24 h. Lastly, due to the prevalence of glyphosate in non-targeted environments and its controversial toxicological status, a novel quantification method for glyphosate was developed. The untargeted metabolomics approach in Arabidopsis thaliana plants treated with both pure glyphosate and Roundup for up to 48 h confirmed the previously established mode of action of the herbicide. The shikimate pathway was targeted which led to an accumulation of metabolites upstream the 5- enolpyruvylshikimate-3-phosphate synthase inhibition point. Levels of aromatic amino acids, however, accumulated over time despite the shikimate pathway being disrupted. Metabolites involved in plant immunity were also observed to accumulate alongside levels of amino acids. The observed metabolic disruptions indicated that nutrient recycling and proteolysis were engaged possibly to ensure plant survival. Next, the untargeted metabolomics approach was also employed to study the effects of pure glyphosate on the metabolism of HEK 293 cells. The cells were exposed to an agriculturally relevant concentration of glyphosate (21 mM) for up to 24 h in serum-free medium. Interestingly, the analysis revealed that the purine and pyrimidin pathways were disrupted which are involved in DNA and RNA synthesis. Also, the metabolites resulting from glyphosate metabolism (AMPA and methylphosphonate) were detected. Lastly, a sensitive and fast optical-based detection method for underivatized glyphosate in water was devised. A portable, in-house produced glyphosate specific-enzyme was utilised which was paired with a complementary metal oxide semiconductor (CMOS) photodiode array sensor. The powerful 16 x 16 photodiode array chip enabled the optical real-time detection of glyphosate. The sensitivity for glyphosate quantification (LOD= 0.47 μM and LOQ= 1.55 μM) met the set standards by the WHO and the EPA in the U.S. What is more, the enzymatic PD CMOS sensor was successful in quantifying glyphosate in spiked tap water samples and registered recoveries from 100-127%.

In recent years, peptides have emerged as potential therapeutic tools for the disruption of protein-protein interactions (PPIs). These interactions are twice as likely to contain a key α-helical interface as any other secondary structure. Incorporation of chemical constraints to pre-organise peptides into their α-helical bioactive conformation can increase binding affinity to protein targets, enhance stability to protease enzymes and allow cellular uptake. This thesis focusses on the design, synthesis and characterisation of peptides containing a novel diyne constraint as an improvement on the current state-of-the-art all-hydrocarbon linkage. Chapter 2 focusses on the synthesis of a series of truncated Omomyc peptides, constrained through the ring closing metathesis (RCM) of two unnatural terminal alkene amino acids. A staple scan was utilised to compare sequences for targeting the Myc/Max protein-protein interaction. Helicity and thermal melt analysis using circular dichroism (CD) upon incubating the peptides with the Myc protein allowed for determination of the optimal stapling position in the Omomyc sequence. An increase in protease resistance was observed for this stapled peptide, however no uptake into the cell line of interest was achieved. Chapter 3 describes the development of a new stapling strategy in which a diyne is formed through a Glaser coupling of two terminal alkynes. Alkyne containing amino acids were synthesised through a diastereoselective alkylation using a Ni(II) Schiff base complex. A model conotoxin peptide was synthesised and the Glaser reaction was optimised on this system, so it could be carried out on-resin to allow excess reagents to be washed away. The stability of the constrained mimetics was assessed in rat serum and the inhibition of the target voltage gated sodium channels (Nav1.4 and 1.6) was also studied. The peptides exhibited comparable binding to that of an all-hydrocarbon mimetic control. Chapter 4 details the production of alkyne stapled peptides to study the inhibition of Sam68 dimerisation. The Sam68 dimer is key in the process of alternative splicing which can lead to the generation of cancerous proteins. Initially, an alkene stapled control was synthesised and binding to Sam68 confirmed using FP. An in vitro splicing assay was developed to determine the effect of the alkene stapled mimetic on splicing function of Sam68, however no effect was observed using these conditions. A series of alkyne containing amino acids were synthesised to identify the optimal stereochemistry and chain length of the alkyne bridge required for the Glaser reaction. Peptides containing varying diyne bridges were synthesised with an S7S7 bridge shown to be the optimal configuration through both Glaser conversion and conferred helicity.

Coronary artery disease is the leading cause of death and disability worldwide and is typically caused by atherosclerotic narrowing of the coronary arteries, which impairs blood flow (ischaemia) to heart. In severe cases atherosclerotic plaques destabilise and rupture, resulting in complete occlusion of the coronary artery and progressive ischaemic necrosis (myocardial infarction, MI). The optimal treatment is to restore the patency of the coronary artery using primary percutaneous coronary intervention (PPCI) to reperfuse the ischaemic myocardium and limit necrosis. However, paradoxically, the full efficacy of PPCI is limited by additional injury initiated by reperfusion that causes cell death and contributes to contractile dysfunction. The collective injury caused by ischaemia and reperfusion, termed ischaemia-reperfusion (I/R) injury, leaves patients vulnerable to adverse cardiac remodelling and the development of heart failure, a hugely debilitating condition with high mortality rates. As such, there is a pressing, unmet need for novel therapeutic strategies that can be used as an adjunctive to reperfusion therapy to limit contractile dysfunction and adverse remodelling post-MI and prevent heart failure development. Previous work has highlighted RUNX1 as a promising therapeutic target for the management of I/R injury and adverse cardiac remodelling. RUNX1 belongs to the RUNX family of transcription factors and is upregulated in border zone cardiomyocytes following MI. Using mice with a cardiomyocyte specific deficiency in Runx1 (Runx1∆/∆ mice) it was previously shown that RUNX1 is detrimental to contractility and contributes to adverse cardiac remodelling in experimental models of MI and I/R injury. It was subsequently demonstrated that increased contractility of Runx1∆/∆ hearts at 2 weeks post-MI was explained by improved cardiomyocyte calcium handling, caused by a decrease in protein phosphatase 1 (PP1) expression, increased phosphorylation of phospholamban (PLB) and the relief of sarcoplasmic reticulum calcium ATPase (SERCA) inhibition. This thesis aimed to further investigate the role of RUNX1 in the context of myocardial I/R injury. Specifically, it aimed to study potential triggers for Runx1 mRNA that may arise in the infarct and border zone including I/R injury and stretch. It also aimed to explore whether Runx1 deficiency offers acute (<24 h) protection against contractile dysfunction in response to I/R injury and whether this was linked to altered calcium handling protein expression. To fulfil these aims, a mouse Langendorff model was established to allow evaluation of the acute responses of isolated hearts to I/R injury and myocardial stretch in the ex vivo setting. Two separate models of I/R injury were characterised: i) no-flow I/R injury where perfusion was completely halted during ischaemia and ii) low-flow I/R injury where residual perfusion was provided during ischaemia to mimic perfusion via collateral vessels. Isolated hearts demonstrated a robust and consistent response to these protocols that paralleled findings reported by others in the field. In addition, the results demonstrated that lengthening the duration of no-flow ischaemia increased the final infarct size, corroborating the work of others and further validating the I/R protocol used. Using the Langendorff set up, it was shown that perfusion was sufficient to induce rapid Runx1 mRNA upregulation, indicating that changes in Runx1 mRNA expression can occur within hours in the isolated heart, independently of neuroendocrine or systemic inflammatory interactions. No-flow I/R injury but not low-flow I/R injury, decreased Runx1 mRNA expression relative to time-matched continuously perfused control hearts. At the same time, the mRNA expression of Runx2 and Runx3 was also measured. Runx2 expression did not change in any of the conditions tested; however, Runx3 mRNA expression was decreased by perfusion, and this downregulation could be offset by no-flow I/R injury but not low-flow I/R injury. Using an intraventricular balloon to stretch the left ventricle of isolated hearts, we investigated whether stretch stimuli affected Runx1 mRNA. Interestingly, intermittent stretch (cycles of balloon inflation and deflation) but not sustained stretch was able to offset the upregulation of Runx1 mRNA by perfusion, indicating that stretch can regulate Runx1 expression when present as a dynamic stimulus. Using the Langendorff model of no-flow I/R injury, it was next demonstrated that Runx1∆/∆ hearts had improved post-ischaemic contractile recovery compared to control hearts, indicating, for the first time, that the protective effects of Runx1 deficiency manifest within an hour of I/R injury and occur in isolation from systemic stimuli. There was no significant difference between PLB, phosphorylated PLB at the serine 16 residue or PP1 expression between Runx1∆/∆ and control hearts either at the beginning or end of the reperfusion period. Taken together, this suggests that the effect of Runx1 deficiency on contractility post-Langendorff I/R injury occurs via a distinct mechanism from that at 2 weeks post-MI. In the final part of this project, the role of Runx1 in hypoxic responses in neonatal rat cardiomyocytes (NRCMs) was investigated. To explore whether Runx mRNAs are upregulated alongside a hypoxic gene profile, NRCMs were treated with hypoxic mimetics. Dimethyloxalyglycine (DMOG) did not affect the expression of Runx mRNAs, whereas cobalt chloride (CoCl2) and deferoxamine (DFO) selectively upregulated Runx1 and Runx3 mRNA, respectively. Further work is necessary to understand the different effects of these mimetics on the Runx family. In non-cardiac tissue, physical and functional interactions exist between hypoxia inducible factor 1α (HIF-1α) and RUNX1. As HIF1-α directs the transcription of a large set of genes (including VEGF and GLUT1) as part of the cellular response to ischaemia, we explored whether RUNX1 expression levels modulate expression of HIF-1α target transcripts in NRCMs. However, RUNX1 overexpression did not affect the mRNA expression of HIF-1α target genes VEGF or GLUT1 under baseline conditions or in the presence of CoCl2. Overall, the work in this thesis has confirmed that the beneficial effects of Runx1 deficiency previously found in vivo extend to the ex vivo setting and manifest acutely during reperfusion. It was demonstrated that a range of stimuli can alter Runx1 mRNA in the isolated heart, including intermittent stretch, which has not previously been linked to Runx1 mRNA regulation in the heart. Developing these findings further will be key to understanding myocardial RUNX1 function and the translational potential of targeting RUNX1 as a therapeutic strategy in the future.

An experimental investigation has been performed to measure the inflow of two and four-bladed propellers. This is conducted over a range of advance ratios (J = 0.36 to 1.54) and yaw angles (γ= 0 to 20◦) at a Reynolds number of Re≈111000, based on the chord length and advancing blade resultant velocity at the 70% spanwise position. Drawing on experimental results, three major qualitative trends are observed, that characterise the axial induced inflow of inclined propellers: the formation of an approximately sinusoidal inflow trend around the azimuth featuring broad regions of increased and reduced inflow, an azimuthal phase shift of the trend from the advancing and retreating blade positions, and the dependencies of the inflow maxima, minima and phase shift on advance ratio, yaw angle and radial position. These are subsequently compared against two widely used assumptions for calculating the aerodynamic environment of inclined propellers; firstly, that inflow at each radial and azimuthal elements calculated using an axial approximation of the whole propeller disc operating within the same condition as that element, and secondly, uniform induced inflow across the entire propeller plane. The comparison demonstrates the limitations of said assumptions by highlighting their inability to account for components of the trailed and shed vortical systems of the propeller wake. The results are also compared against CFD, demonstrating excellent consistency with the computational solution.

Coronary heart disease is a leading cause of death worldwide despite advances in treatment and medical interventions. Cardiomyocyte death due to prolonged ischemia (myocardial infarction; MI) is accountable for the majority of deaths resulting from coronary heart disease. MI leads to adverse cardiac remodelling which clinically manifests as a change in size, shape and function of the heart- a process which is detrimental to cardiac function and can lead to heart failure (HF). At present, the main treatment for MI is to re-perfuse the myocardium in a process known as percutaneous coronary intervention but this does not fully prevent adverse cardiac remodelling. Novel therapeutic targets which can limit adverse cardiac remodelling and prevent the progression to heart failure are required. Runx1 is a master transcription factor which plays an essential role in definitive haematopoietic stem cell (HSC) development, HSC self-renewal and differentiation. In the cardiovascular system, Runx1 is minimally expressed in healthy adult cardiomyocytes but its expression is increased in humans and animal models following cardiac disease. The functional role of Runx1 in the heart remains unknown. Therefore, the main aims of this thesis were to investigate the role of Runx1 in a mouse model of MI. This was achieved by assessing Runx1 levels from 1-day post-MI in stock mice as well as in a tamoxifen-inducible cardiomyocyte-specific Runx1 deficient mouse model. The therapeutic potential of Runx1 deficiency was then assessed via a gene transfer approach using shRNA delivered directly to the heart in a mouse model of MI. Initially, Runx1 expression was assessed at between 1 and 7-days post-MI following MI in stock mice. In situ hybridisation using dual probes against Runx1 and pericentriolar material-1 (PCM-1), Runx1 was shown to be increased specifically within the border zone and infarct. PCM-1 detects cardiomyocyte-specific nuclei allowing the delineation of Runx1 in cardiomyocytes from non-cardiomyocytes. These results were further supported by qPCR data showing an increase in Runx1 in isolated cardiomyocytes from the left ventricle (LV). In a separate cohort of mice, LV contractile function, as measured by echocardiography, was reduced between 1 and 7-days post-MI. Next, it was hypothesized that the tamoxifen-inducible cardiomyocyte-specific Runx1 deficient mouse would have a preserved contractile function post-MI. Whilst LV fractional shortening in control mice decreased at 1- and 7-day post-MI, Runx1 deficient mice demonstrated a preservation of LV contractile function. This was also observed in the chronic stages of MI in mice with larger infarcts. The Runx1 deficient mice demonstrated a preserved LV contractile function up until the end of the study at 32 weeks post-MI. An infarct larger than 40% was induced in these mice in order to induce a greater degree of adverse cardiac remodelling. Finally, an adenovirus (Ad) expressing Runx1 shRNA, was utilised in a mouse model of MI and was delivered via an intracardiac injection in the border zone immediately after MI was induced. In situ hybridisation demonstrated reduced Runx1 within cardiomyocytes at the border zone at 7 days post-MI. Mice injected with Ad-Runx1-shRNA showed a preservation of LV contractile function compared to control group receiving scrambled virus between 1- and 7-days post-MI. Collectively, the data in this thesis demonstrates that Runx1 deficiency is cardioprotective in the setting of MI. Runx1 deficiency has potential as a novel translatable target to limit the progression of adverse cardiac remodelling in a mouse model of MI.

One of the most devastating processes of cancer is metastasis: i.e., the ability of primary tumour cells to migrate and form secondary tumours. Biochemical aspects of cancer are continuously investigated, whereas mechanical aspects of cancer invasion remain largely unexplored. Nonetheless, the importance of mechanics in pathological conditions has been increasingly recognized, underlining the need of novel advanced cell micro-rheology methods. Atomic force microscopy (AFM) is a powerful tool for measuring the mechanical properties of cells under physiological conditions with nanoscale precision. In this thesis, a novel AFM-microrheology method for educing the linear viscoelastic properties of complex materials and cells from stress-relaxation nanoindentation measurements, without the use of preconceived viscoelastic models, nor time consuming experimental procedures, was developed. Using this capability, an investigation into the chemotherapeutic treatment of breast cancer cells was undertaken, unveiling new, mechanical characteristic drug responses. The method is ready to be implemented in conventional AFM setups; providing a simple yet powerful tool to measure dynamic mechanical properties of living cells. Metastasis depends on the ability of cancer cells to migrate between tissue sites. How cells move and exert forces to achieve this is also an important contributor to how this disease spreads. A fast and user-friendly method for traction force microscopy (TFM) was established, and the correlation between cell to extracellular matrix (ECM) forces was studied and utilised as a complementary technique to the AFM-microrheology measurements. This technique uncovered a significant difference in the response of drug treated cancer cells, showing larger forces exerted across cell areas than non-treated cells. Mechanochemically tuneable ECM mimics allow a greater understanding of cell-ECM interactions. Therefore, peptide hydrogels were used as biomimetic substrates to corelate interactions between cancer cells and their environment. The mechanical properties of cells on soft substrates were interpreted by means of a novel, double-layer mathematical model, which found that the traditional Hertzian model underestimates the Young’s modulus of cells. The methods stated above were used to uncover new, quantitative, characteristic mechanical responses of drug-treated, invasive, cancer cells on environments with increasing biochemical and mechanical complexity.

Leukaemias, which include chronic and acute myeloid leukaemia (CML and AML), represent the tenth deadliest type of cancer, claiming over 300,000 lives each year worldwide. CML and AML are clonal haematological malignancies caused by mutations and chromosomal rearrangements. These occur in stem and progenitor haematopoietic cells, giving rise to leukemic stem cells (LSCs). While CML is characterised by the presence of the Philadelphia chromosome and the constitutively active BCR-ABL protein, AML can develop from a multitude of genomic aberrations. CML patients have greatly benefited from the introduction of BCR-ABL-targeting tyrosine kinase inhibitors (TKIs), which have brought the disease’s five-year survival rate above 80 %. TKIs however, are not a cure. Indeed, to avoid relapse most CML patients need to remain under treatment their whole lives, with associated toxicities and the risk of developing treatment resistance. In comparison to CML, the survival rate of AML patients has remained stubbornly low: below 40 % for patients under 60 years old, falling below 15 % for older ones. While a number of targeted therapies have been approved in recent years, the first line of treatment in AML remains aggressive chemotherapy and, for eligible patients, haematopoietic stem cell (HSC) transplantation. In addition, up to half of AML patients going through therapy will experience resistance and/or relapse. While therapies informed by genomic data have transformed the treatment of some cancers, they have also shown their limits. This is especially apparent in our current inability to drug certain targets, and the frequency of treatment resistance. Research into alternative vulnerabilities of cancer cells is therefore of great interest, including in myeloid leukaemia. Critically, a rewired cellular metabolism is now considered a hallmark of cancer. Early work in oncometabolism suggests that cancer cells are more glycolytic than their non-malignant counterparts, a mechanism known as the Warburg effect. In the past decade however, a more complex picture of cell metabolism in myeloid leukaemia has emerged. Indeed, oxidative phosphorylation (OXPHOS) now appears to be of importance in treatment-resistant cells and CML LSCs, while the metabolic profile of AML LSCs has yet to be fully characterised. Thus, in the present study, we aimed to determine whether the energy metabolism of AML LSCs differ from that of less primitive AML cells, and aimed to identify novel OXPHOS inhibitors for application in myeloid leukaemia. We therefore provided the first in-depth investigation of the energy metabolism of transcriptionally defined AML LSCs, performed in CD34- NPM1-mutated AML. This required the optimisation of media composition and cytokine supplementation for the metabolomic-based study of patient-derived AML samples. Analysis of RNA sequencing data, combined with stable isotope tracing and live-cell metabolic functional assays, revealed the distinct metabolic profile of patient-derived AML samples carrying a stem-like signature. Specifically, stem-like samples were less metabolically active than their differentiated-like counterparts and, noticeably, less glycolytic, potentially rendering them more sensitive to OXPHOS inhibition. In parallel, addressing our second aim, we performed a metabolism-specific drug repurposing screen. To identify safe and effective OXPHOS inhibitors, over 1,300 compounds were tested in CML and AML cell lines. Following initial validation of the screen’s top hits in cell lines, calcium channel inhibitor lomerizine, fatty acid amide hydrolase inhibitor PF-3845 and antifungal itraconazole were selected for further investigation. Metabolomic-based experiments in OCI-AML3 cells revealed that all three drugs inhibited OXPHOS, and that PF-3845 and itraconazole did so without significantly disrupting other central carbon metabolism pathways. In addition, all three compounds inhibited mitochondrial respiration in patient-derived AML cells, and presented minimal toxicity towards HSCs. Having shown activity against primitive AML cells in vitro, the efficacy of itraconazole was further tested in two in vivo models. In a patient-derived xenograft experiment, itraconazole in combination with cytarabine decreased the percentage of AML LSCs in the bone marrow (BM) of immunocompromised NRG-3GS mice. In a secondary transplant experiment, this combination treatment increased the survival of engrafted mice, although this did not reach statistical significance. In another xenograft model, itraconazole sensitised THP-1 cells to cytarabine treatment, decreasing their survival in mice BM. Overall, these results indicate that NMP1-mutated, transcriptionally defined CD34- AML LSCs are characterised by low metabolic activity and low glycolysis. This is of clinical interest, considering LSC signatures are associated with poor prognosis in AML patients. In addition, as HSCs are glycolytic, this could indicate a metabolic therapeutic window. Also translationally relevant, several safe and effective OXPHOS inhibitors were identified in a repurposing screen, highlighting the value of this drug development strategy to target oncometabolism. These compounds were then further validated in patient-derived AML samples. Finally, we showed that antifungal itraconazole, already used in routine infection prophylaxis, represents a clinically relevant candidate to target OXPHOS metabolism in chemo-resistant AML cells.

The brown trout (Salmo trutta L. 1758) is a widespread salmonid species that is well known for its multiple life history strategies. Some individuals remain in freshwater for the duration of their lives, and the life stages of these freshwater-resident fish have been well researched. But others implement an anadromous life history strategy where they migrate from their natal river into the marine environment before returning to their natal river to spawn. This life history strategy is beneficial to those individuals by providing them with access to additional food resources in the marine environment which ultimately leads to increased growth rates and fecundity. But with this shift in habitat comes additional risks such as increased predations and exposure to pathogens which can result in an increased level of mortality. Due to the geographic range of these anadromous fish in the marine environment, there are still several large gaps in our understanding of the movements of trout at sea, as well as the associated threats and subsequent impacts they might have on trout populations. In recent decades, it has been suggested that populations of anadromous trout (or sea trout) are experiencing a decline, however, little research has been done to quantify or explain this observed loss. This same decline was thought to have been observed in Scottish sea trout based on catch numbers, but no national trends have been reported beyond raw catch data reported by the Scottish Government on an annual basis. Using an Information Theoretic modelling approach, three measures of sea trout abundance and a variety of river, geographic and climatic variables were used to explain patterns of change in sea trout populations in Scotland. This study demonstrated overall sea trout numbers have declined 48% in the last 67 years, but that there were significant differences in the trends observed across coastal and regional spatial ranges, with some populations even showing increases in their numbers. Several river specific (river length, river gradient, geology and freshwater loch availability) and climatic variables (mean winter rainfall) were shown to act as consistent driving factors affecting population size change across this time period. One of the most consistently important drivers of sea trout populations was an interaction between river length and geographic region with longer rivers usually producing larger populations. However this relationship varied spatially and temporally and ultimately showed a significant decrease in the resilience of many sea trout populations across Scotland. The strength of the effect of some climatic variables, which vary spatially and temporally, changed with time and are predicted to continue changing given expected shifts in climate change pressures. One of the most well documented threats in the marine environment to sea trout populations is the presence of open-net pen Atlantic salmon aquaculture. This industry has been tied to numerous environmentally damaging impacts, including increased parasite levels and increased mortality in wild sea trout populations, in multiple countries in Europe and Scandinavia. The west coast of Scotland hosts one of the largest Atlantic salmon aquaculture industries that has expanded rapidly since the 1970’s. There has been little attempt to quantify the impacts of this industry on long term populations trends of sea trout on the west coast. Using an Information Theoretic modelling approach, environmental, climatic and aquaculture focused variables and three measures of abundance were used to identify drivers of change in sea trout populations on the west coast of Scotland over the last two decades. The results demonstrated that sea trout populations are reacting differently in areas with and without Atlantic salmon aquaculture but that these effects are relatively complex. Thus there is a negative effect of production biomass on sea trout populations that can be enhanced or lessened given changes in climatic variables. When sea temperatures rose above 11.0°C, sea trout populations declined with increasing net-pen biomass. During years of low winter rainfall, sea trout populations declined with increasing net-pen biomass. Given the high likelihood that these variables will change in the future due to climate change shifts, it is likely that they will impact sea trout more strongly in future years. Increased densities of Lepeophtheirus salmonis, the salmon louse, are frequently associated with areas of intensive Atlantic salmon aquaculture. At high levels, this ectoparasite can cause extensive physical damage to sea trout that can result in reduced osmoregulatory function and body condition and increased predation and mortality. To examine the range of increased lice densities on wild sea trout, sea trout populations were sampled in five sheltered coastal fjords on the Isle of Skye, Scotland at varying distances from active aquaculture sites (3 km – 48 km). While the likelihood of a sea trout having salmon lice present increased with distance from aquaculture facilities, the total lice burdens were found to be higher on individuals sampled within close proximity (within 13 km) to facility locations. The proportion of different life cycle stages of salmon lice on a sea trout was correlated with the proximity of the fish to aquaculture facilities. For example, the total lice count of a sea trout sampled near an aquaculture facility was primarily comprised of juvenile lice life stages, while sea trout sampled further away from a facility had a larger proportion of mobile adults and gravid female lice present. There is still a lack of understanding about the space use by sea trout in the marine environment in Scotland, however, it is suspected that trout populations spend part of their marine life stage in the same sheltered coastal areas that are increasingly occupied by Atlantic salmon aquaculture sites. Given the clear connection to increased salmon lice densities in the water column surrounding the aquaculture facilities in sheltered coastal areas, any overlap between habitat usage by wild sea trout and Atlantic salmon farming could have significant negative impacts on wild fish, particularly vulnerable post-smolts that have entered the marine environment for the first time. Acoustic telemetry techniques were used to gather data on the marine migration and spatial use of two different populations of sea trout post-smolts originating from two adjacent fjord systems located on the Isle of Skye, Scotland. One fjord system contained an active aquaculture facility and one did not. A total of 60 sea trout smolts were tagged and 46 of those individuals were detected on the receiver array. The study demonstrated that sea trout post-smolts maintain a strong fidelity to the coastal fjord system connected to their natal river during the first summer of their marine migration. A small percentage of the detected individuals (13 individuals, 28%) did migrate out of their natal fjord system but most (8 individuals, 17%) returned to that same fjord after a period of time foraging elsewhere. Survival and migration range were not significantly correlated to fish size. A small number of individuals (21% of detected individuals) were detected near the aquaculture facility but there was no significant difference between the amount of time spent near the facility and the amount of time spent elsewhere in the loch. The four studies presented in this thesis have combined modelling and empirical field study approaches to quantify the historical trends of sea trout populations in Scotland and identify current drivers of those trends. The results presented here can provide future insight to the predicted changes that sea trout populations will experience as their marine habitats undergo continued transformations brought on by both anthropogenic and climatic shifts.

Efficient folding of proteins frequently requires the introduction of disulfide bonds. The insertion of disulfide bonds could result in native disulfides, which can be found in the correctly folded and functional protein structure, and non-native disulfides which are not present in the native protein. The resolution of non-native disulfides is absolutely required for cells to fold secreted proteins correctly and to remove misfolded proteins to alleviate cell stress. The pathways for disulfide formation are well characterised. However, our understanding of the reduction of non-native disulfides is still limited. How the reducing equivalents are transferred across the ER membrane and whether there are additional components needed in the reduction pathway either in cytosol or ER lumen are the questions we need to address. Microsomes including redox-sensitive green fluorescence protein (roGFP) were used to address which membrane protein was involved in the ER reductive pathway. We demonstrated that a membrane protein is required for the reduction of microsomal proteins by the membrane impermeable reducing agent Tris(2-carboxyethyl)phosphine (TCEP) or a reconstituted thioredoxin/thioredoxin reductase (Trx/TrxR) pathway. Previous research in our group suggests that ERp57 has a specific role in the isomerisation of non-native disulfide bonds in specific glycoprotein. We optimised the determination of redox state of ERp57 in semi-permeabilised (SP) cells. It was found that the reduction of ERp57 in SP cells required reducing equivalents generated by the Trx/TrxR pathway and the reduction was dependent on catalytic cysteines in the Trx motif. It was also shown by proteinase experiments that different membrane proteins are required for the transduction of electrons from TCEP and the Trx/TrxR pathway. In addition, we took advantage of an in vitro translational system supplemented with SP cells. A post-translationally folding assay was developed to evaluate the ability of the cytosolic components to resolve non-native disulfides in nascent polypeptides proteins in the ER. We showed that the minimal components in the cytosolic Trx/TrxR pathway are sufficient for disulfide rearrangement in the ER and that the reduction process is Trx - dependent.

This thesis aimed to address questions in two distinct areas of research in ageing and cognitive neuroscience. Firstly, given that the pre-stimulus state of cortical oscillations had been shown to predict behavioural and neural responses, we addressed the question of whether pre-stimulus oscillatory mechanisms change or remain consistent in the ageing brain. Secondly, previous research had shown that Audio-visual (AV) speech influences the amplitude and latency of evoked activity. Our research addressed the questions of whether/how AV enhancement and visual predictability of AV speech is represented in evoked activity in noisy listening conditions, and whether such Electroencephalographic (EEG) signatures remain stable with age. In Chapter 3 we investigated the consistency of how pre-stimulus activity influences auditory frequency discrimination performance in young and older participants. In both groups the power of pre-stimulus activity influenced the encoding of sensory evidence reflected by early evoked components, while the phase influenced choice formation in later-activated EEG components. Importantly, for the early EEG components we did not find evidence for a systematic difference in the time scales of the perceptually relevant pre-stimulus activity. In the later-activated EEG component we found a trend for perceptually relevant rhythmic activity to arise from slower frequencies in the ageing brain. At the same time our data replicate previous findings of a significant age-related slowing of Auditory Evoked Potential (AEP) latency, modulations of AEP amplitudes, and a flattening of the spectral profile of EEG activity. In Chapter 4, we investigated the consistency of behaviour and evoked activity underlying AV speech integration in a speech-in-noise discrimination task in younger and older adults. Behaviourally, younger and older adults performed comparably. Performance was greater for Audio-visually informative (AVinf) speech compared to Auditory-only informative (AOinf) speech across groups and noise levels, and was poorer at low noise levels. AV enhancement was greater in high noise levels, across all participants, and older adults derived greater AV enhancement compared to younger adults (an effect that was consistent across noise levels). In terms of visual predictability, we found that word discrimination performance was greater for target words with non-labial initial phonemes (assumed least visually predictive), compared to labial initial phonemes (assumed most visually predictive). Furthermore, we found that AV enhancement was greater for labial initial phonemes, compared to non-labial initial phonemes, and this was consistent across age groups. Neurally, we found that AV enhancement is represented by a centro-parietal P3-like activity in older adults and an N4-like fronto-central activity in younger adults, but found that this activity did not correlate with behavioural AV enhancement. Our results point to distinct patterns of late evoked activity underlying AV enhancement between younger and older adults, possibly representing distinct cognitive (memory) strategies in predicting upcoming target stimuli. At the same time our data replicate previous findings of a significant age-related slowing of AEP latency, modulations of AEP amplitudes, and a flattening of the spectral profile of EEG activity. In Chapter 5 we investigated the consistency of evoked activity underlying the visual predictability of AV speech. We found that visual predictability was reflected by late fronto-central negativity in older adults, but not in younger adults. However, we did not find evidence of an interaction between visual predictability and AV enhancement in terms of evoked activity, raising further questions about how visual predictability of speech is represented the brain’s electrophysiology. Our results point to distinct patterns of late evoked activity underlying visual predictability of visual speech, again possibly reflecting differential strategies in predictive coding. In summary, the results of this thesis demonstrate that pre-stimulus mechanisms in auditory pitch perception remain consistent in the younger and older adult brain, while spectral dynamics change with age. Our results also replicate previous work demonstrating age-related delays in peak latency, and changes in peak amplitude, of early auditory evoked activity. And lastly, we demonstrate that differences in the EEG signatures of AV enhancement between younger and older adults emerge in late evoked activity, and that visual predictability of speech is represented in late evoked activity only in older adults.

Introduction: Microvascular injury in acute ST-segment elevation myocardial infarction (STEMI) is an important predictor of adverse prognosis. However, persistent microvascular injury typically passes undetected after primary percutaneous coronary intervention (PCI), and is a problem of unmet therapeutic need. The aim of this work was to gain greater insight into the invasive assessment of acute microvascular injury following primary PCI, for risk-stratification, and to provide mechanistic insights into the effects of intracoronary alteplase on the microcirculation. Methods: The first part of the thesis sought to establish the influence of coronary flow on the effects of adjunctive intracoronary alteplase, and to investigate the effects of intracoronary alteplase on invasive physiology measures of microvascular function. Through a multi-centre, prospective, randomised controlled trial (T-TIME, NCT02257294), alteplase 10mg, and alteplase 20mg were compared to placebo, in patients undergoing primary PCI. Eligible participants presented within 6 hours from STEMI onset, and the study drug was administered before stent implantation. In 421 patients, TIMI (thrombolysis in myocardial infarction) flow grade was determined in the infarct-related artery immediately before study drug administration. In a subset of 144 patients, invasive physiology parameters were measured in the infarct-related artery at the end of the primary PCI procedure (the prespecified T-TIME physiology sub-study). These invasive physiology parameters included index of microcirculatory resistance (IMR), coronary flow reserve (CFR) and resistive reserve ratio (RRR). Microvascular obstruction (MVO) and myocardial haemorrhage were assessed on cardiovascular magnetic resonance (CMR) imaging at 2 to 7 days post-STEMI, and CMR imaging was repeated at 3 months. The second part of the thesis sought to prospectively compare IMR, CFR, RRR, myocardial perfusion grade (MPG) and TIMI frame count (TFC), for predicting MVO and myocardial haemorrhage, and clinical outcomes at 1 year, in the T-TIME physiology sub-study. The following adjudicated clinical outcomes were assessed: major adverse cardiac events, heart failure hospitalisations, and all-cause death/ heart failure hospitalisations. Furthermore, a retrospective analysis was performed in 271 acute STEMI patients from a single-centre observational study (MR-MI, NCT02072850), for the derivation of a newly conceived invasive physiology parameter termed temperature recovery time (TRT). The associations between TRT and MVO (on 2 to 7 day CMR imaging) and clinical outcomes, were assessed in the MR-MI cohort, and were prospectively validated in the T-TIME physiology sub-study population. Results: The main findings are summarised as follows: - Low-dose intracoronary alteplase given early during primary PCI, was associated with increased occurrence of MVO and myocardial haemorrhage in participants who had TIMI flow ≤2 immediately preceding drug administration. - In participants with TIMI 3 flow immediately preceding drug administration, there was no difference in MVO or myocardial haemorrhage with intracoronary alteplase compared to placebo. - There was overall no difference in microvascular function, measured by IMR, CFR and RRR, between intracoronary alteplase and placebo groups. - In patients with ischaemic time < 2 hours, CFR and RRR were higher with alteplase 20mg vs. placebo, whereas in patients with ischaemic times ≥4 hours, MVO extent was higher with alteplase 20mg vs. placebo. - In acute STEMI patients, lower RRR, IMR >40, and MPG ≤1 were associated with more MVO, myocardial haemorrhage presence and adverse clinical outcomes, whereas CFR ≤2 was not. - In acute STEMI patients, TFC >27 was associated with adverse clinical outcomes, but was not associated with MVO or myocardial haemorrhage. - Higher TRT independently predicted more MVO and adverse clinical outcomes, in two independent acute STEMI cohorts. Conclusions: The findings from this PhD are novel and clinically relevant. Invasive measures of microvascular injury during primary PCI allows potential for early administration of targeted adjunctive therapies to the highest risk patients. The data support IMR in conjunction with RRR instead of CFR, to select patients for adjunctive therapies. Moreover, TRT was found to detect failed microvascular perfusion and may have potential to refine risk stratification in acute STEMI. The findings raise a question as to the safety of intracoronary administration of alteplase in the context of STEMI when there is < TIMI 3 flow. Finally, the data suggest that future studies evaluating the effects of intracoronary fibrinolysis should limit recruitment to patients with short ischaemic time.

In this thesis, we present new algorithmic and complexity results for specific matching problems involving preferences. In particular we study the Stable Marriage problem (SM) and the Student-Project Allocation problem (SPA) and their variants. A matching in these scenarios is an allocation of men to women (SM) or students to projects (SPA). Primarily we are interested in finding matchings that are stable. A stable matching is a matching that admits no blocking pair, which is a pair of agents (not already allocated together) who would rather deviate from the given matching and become assigned to each other. In addition to stability, other objectives may be applied. We focus on finding either fair or large stable matchings in SM and SPA. In the Stable Marriage problem with Incomplete lists (SMI), the rank of a matched man or woman, with respect to a matching, is the position of their assigned partner on their preference list. The degree of the set of men in a matching is the rank of a worst-off man, over all matched men. A similar definition exists for the set of women. The cost of the set of men in a matching is sum of ranks of all matched men. Again a similar definition exists for the set of women. We introduce the following degree-based definitions of fairness in SMI. A stable matching is regret-equal if it minimises the difference in degree between the set of men and the set of women, over all stable matchings. Additionally, a stable matching is min-regret sum if it minimises the sum of the degree of men and the degree of women, over all stable matchings. We present polynomial-time algorithms to find these types of fair stable matchings, given an instance of SMI, and perform experiments to both test the performance of two algorithms to find a regret-equal stable matching, and to compare properties of several other types of fair stable matchings over both degree- and cost-based measures. Also in SMI, we investigate fairness in the form of profile-based stable matchings. The profile of a matching is a vector of integers, where the ith vector element indicates the number of agents assigned to their ith-choice partner. A stable matching is rank-maximal if its profile is lexicographically maximum taken over all stable matchings. A stable matching is generous if its reverse profile is lexicographically minimum taken over all stable matchings. A polynomial-time algorithm exists to find a rank-maximal stable matching, using weights that are exponential in the number of men or women [32]. We adapt this algorithm to work with polynomially-bounded weight vectors, and show using randomly-generated instances that our approach is significantly less costly in terms of space. Further experiments are carried out to compare these profile-based optimal matchings over several cost- and profile-based fairness properties. We additionally show that in the Stable Roommates problem, each of the problems of finding a rank-maximal stable matching or a generous stable matching is NP-hard. In the Student-Project Allocation problem with lecturer preferences over Students including Ties (SPA-ST) we study the problem of finding large stable matchings. A 3/2-approximation algorithm exists to find a maximum-sized stable matching in HRT, and we extend this to the SPA-ST case, developing a linear-time 3/2-approximation algorithm for the problem of finding a maximum-sized stable matching in SPA-ST. We test the performance of our approximation algorithm using the implementation of a new Integer Programming (IP) model that finds a maximum-sized stable matching, and show that in practice, our approximation algorithm produces stable matchings of size far closer to optimal than the 3/2 bound. Additionally, we give an example to show that this bound is tight. Finally, we look at fairness in the context of the Student-Project Allocation problem with lecturer preferences over Students including Ties and Lecturer targets (SPA-STL), an extension to SPA-ST in which each lecturer has an associated target, indicating their preferred number of allocations (or the number of allocations preferred by the matching scheme administrator). We first investigate load balancing without the presence of stability. A load-max-balanced matching is a matching in which the maximum difference between a lecturer’s target and their number of allocations is minimised. A load-sum-balanced matching is a matching in which the sum of differences between all lecturers’ targets and their number of allocations is minimised. Finally, a load-balanced matching is a matching that is both load-max-balanced and load-sum-balanced. We provide new polynomial-time algorithms to find matchings of these types. Additionally we show that in the presence of stability, each of the problems of finding a stable matching that is load-max-balanced, load-sum-balanced or load-balanced is NP-hard. Finally, we present new IP models for finding such types of optimal stable matching.

Tissue expansion is a technique used by plastic and restorative surgeons to cause the body to grow additional skin, bone or other tissues. Distraction osteogenesis (DO) is an example of tissue expansion which has been widely applied in lower limb surgery (trauma/congenital), and congenital upper limb reconstruction (e.g. radial dysplasia). This complex and tightly regulated expansion process has resulted in adverse effects such as severe soft-tissue contractures and loss of nerve function as well as microtrauma and micro-haematoma formation (Natu et al., 2014). Thus far, the procedure can only be optimised by long-term animal or human experimentation. This thesis explains the development of an in vitro model that will allow extension regimes (μm/h, continuous/ intermittent) and molecular pathways involved in soft tissue damage related to DO to be explored. Cells cultured onto polycaprolactone (PCL) polymer films can be stretched at very low, adjustable speeds, using a stepper motor and various 3D printed and laser cut designs. The idea here is that plastic flow of PCL can be utilised to enable the material to stay extended upon strain being released, to represent permanent stretching of soft tissue. PCL film for the purposes of this project was made using a solvent in conjunction with a spin-coating process; A semi-crystalline and amorphous derivative of the polymer was made (C-PCL and A-PCL respectively). Testing the two polymer sheets indicated that C-PCL is a more rigid material and that strain occurs in more localised regions when it is stretched in comparison to A-PCL. The profile of the stress-strain curve for both C-PCL and A-PCL closely resemble that of a typical soft tissue after it has passed its yield point (33% strain). Due to the known involvement of fibroblasts in mechanical loading of tissue (B. Hinz, 2004), they were used as an initial cell line to develop an in vitro model for growth by stretch. Both A-PCL and C-PCL were used as substrates and were stretched passed their yield point (33% strain) before cells were cultured on. Following fibroblast proliferation to confluency substrates were further stretched by 1mm (2.5% strain) over 24 hours (stepped stretching at 0.04mm per hour). Orientation analysis indicated that cells grown on C-PCL initially elongate and orient to the direction of pre-stretch (when substrates are initially stretched passed their yield point), then contract upon being further stretched by 1mm over 24 hours. Cells cultured on A-PCL, under the same stretching regime initially align to the direction of pre-stretch; after being further stretched by 1mm the majority of cells remain aligned, but also elongate in the direction of stretch. Initial alignment on both materials was deemed a result of tension in the material and/or or topographical features which formed during stretching of substrates before cells were cultured on. The alignment was more pronounced on the C-PCL substrate and cell nuclei were analysed to be more elongated indicating the topography caused the fibroblasts to reside in a stressed state. This aligned cell effect was lost on C-PCL during further 1mm stretching due to; stress relaxation after each step of stretching; and/or localised strain regions causing cells to round during the stepped 1mm stretch. A-PCL was further investigated as a substrate to model soft tissue expansion in relation to DO where MRTF-A nuclear translocation was shown to increase in response to stretch (by 3-fold). F-actin texture analysis further implied cytoskeletal involvement in the stretching regime utilised for this project. Based on the results obtained, it was concluded that A-PCL with the stretching regime detailed (where plastic flow is utilised), provides the basis for a representative in vitro model of stretching soft tissue in relation to DO. Future work outlined to build on this model would be to: further investigate the relation between strain and cell response at the cell level for both materials using live imaging (in conjunction with fiducial markers in the substrate) and atomic force microscopy methods; and to develop understanding of extracellular matrix (ECM) interactions with cells in response to the stretching in the plastic flow region by again using live imaging methods (fluorescently tagging ECM components).

Osteoporosis represents an ever increasing burden to health care services worldwide. Within the UK alone, over 500 000 fragility fractures secondary to osteoporosis occur each year, resulting in significant morbidity and mortality. Furthermore, the management of these injuries cost health care services billions of pounds. A revolution in the diagnosis and treatment of the disease is required to prevent significant suffering of future generations. MicroRNAs are known to regulate many key physiological processes, including mesenchymal stromal cell (MSC) differentiation. They have been demonstrated to be abnormally expressed in many musculoskeletal conditions, including osteoporosis. Patients who have suffered a neck of femur fracture are known to be at risk of, or have undiagnosed, osteoporosis. Dysregulated microRNAs have been identified in bone and serum samples of patients with this injury type. Great hope has been placed on the development of targeted therapies to manipulate microRNA expression and improve bone quality. This study has focussed on rigorously assessing the functionality of MSCs from patients who have suffered a neck of femur fracture. Extracellular MSC markers and differentiation capacity were shown to be significantly altered within this patient group. MicroRNA expression was analysed, and key microRNAs, microRNA-143 and microRNA-31, were manipulated using functionalised gold nanoparticles to further understand their role in MSC differentiation. Furthermore, the metabolomic effect of inhibiting microRNA-31 was explored to further understand its role in osteogenesis. To reduce the gap between in vitro and clinical research an optimal 3D, scaffold free, culture model was identified for bone marrow derived MSCs. These results provide further insight to the impaired MSC function of patients who have suffered a fragility fracture. Furthermore, the potential of microRNAs to be utilised as a biomarker or therapeutic target for osteoporosis has been reinforced.

Defective in cullin neddylation-like (DCNL) proteins are known to coordinate the addition of NEDD8 to the cullin subunit of the largest family of ubiquitin E3 ligases, the Cullin-RING ligases (CRLs), in a process known as neddylation. The human genome encodes five DCNL proteins which are thought to exhibit a large degree of overlap in function, with only a few neddylation processes having been definitively ascribed to a single DCNL homologue. It currently remains unclear whether these DCNL proteins have functions that extend beyond their roles in cullin neddylation. In the present study we now describe a novel role for one of the family members, DCNL5, in the programmed cell death response known as apoptosis. We have shown that cells lacking DCNL5 function fail to promote caspase 8 cleavage – an important early activation step - in response to various inducers of the extrinsic apoptosis pathway. Caspase 8 cleavage and activation requires polyubiquitination which is known to be mediated by cullin 3 in coordination with the dual ubiquitin and NEDD8 ligase RBX1. This process is thought to occur in lipid rafts at the plasma membrane and in the cytosol. In the present work, we provide the first indication that DCNL5 is able to translocate out of the nucleus where it was previously thought to be exclusively located, and this occurs in response to TNFα-related apoptosis-inducing ligand (TRAIL) stimulation. In addition, we present evidence for the first known interaction between DCNL5 and cullin 3 in U2OS cells under endogenous conditions. The DCNL5 KO cells demonstrated a lack of a polyubiquitination event that occurs in WT cells; unmodified caspase 8 was shown to associate with a polyubiquitinated protein (the identity of which we were unable to determine) in response to TRAIL and this interaction was absent in KO cells, perhaps representing the key mechanism underlying DCNL5 involvement. This emerging function for DCNL5 in promoting caspase 8 cleavage was confirmed in multiple cancer cell lines including U2OS, H460 and HeLa cells. Importantly, we demonstrated that siRNA-mediated silencing of DCNL5 prevented CASP8 cleavage. A lot of our work suggested that DCNL5’s role in CASP8 activation is mediated by the cullin CUL3. However, treatment with the neddylation inhibitor MLN4924 caused a reduction, but not a total loss of caspase 8 cleavage, suggesting that if cullin 3 is involved, it may be independent of its neddylation status. This hints at the surprising possibility that a CRL complex exists that that does not require neddylation for some of its function. Furthermore, while our data suggests that DCNL5 may regulate apoptosis via cullin 3, we were unable to exclude a cullin neddylation-independent role for DCNL5 in this process. Future work will need to answer this question by identifying and characterising the molecular target of DCNL5 in apoptosis signalling to ascertain the precise mechanism underlying DCNL5 regulation of this clinically important signalling event.

Acute myeloid leukaemia (AML) is one of the most common haematological malignancies, characterised by the clonal expansion of abnormal or poorly differentiated myeloid cells, infiltrating the bone marrow (BM), blood or extramedullary tissues. Although treatment of AML has progressed over the past few decades, in some patients, particularly elderly patients, usually defined as older than 60-65 years of age, the outcome is dismal due to the remarkable genetic complexity, epigenetic alterations, and the dynamics of the disease. In this study, a newly developed CDK2/9 inhibitor CYC065 was investigated as a treatment option for AML.

The Trypanosomatidae are a family of parasitic protozoa that infect various animals and plants. Several species within the Trypanosoma and Leishmania genera also pose a major threat to human health. Among these are Trypanosoma cruzi and Leishmania infantum, aetiological agents of the highly debilitating and often deadly vector-borne zoonoses Chagas disease and American visceral leishmaniasis. Current treatment options are far from safe, only partially effective and rarely available in the impoverished regions of Latin America where these ‘neglected tropical diseases’ prevail. Wider-reaching, sustainable protection against T. cruzi and L. infantum might best be achieved by intercepting key routes of zoonotic transmission, but this prophylactic approach requires a better understanding of how these parasites disperse and evolve at various spatiotemporal scales. This dissertation addresses key questions around trypanosomatid parasite biology and spatial epidemiology based on high-resolution, geo-referenced DNA sequence datasets constructed from disease foci throughout Latin America: Which forms of genetic exchange occur in T. cruzi, and are exchange events frequent enough to significantly alter the distribution of important epidemiological traits? How do demographic histories, for example, the recent invasive expansion of L. infantum into the Americas, impact parasite population structure, and do structural changes pose a threat to public health? Can environmental variables predict parasite dispersal patterns at the landscape scale? Following the first chapter’s review of population genetic and genomic approaches in the study of trypanosomatid diseases in Latin America, Chapter 2 describes how reproductive polymorphism segregates T. cruzi populations in southern Ecuador. The study is the first to clearly demonstrate meiotic sex in this species, for decades thought to exchange genetic material only very rarely, and only by non-Mendelian means. T. cruzi subpopulations from the Ecuadorian study site exhibit all major hallmarks of sexual reproduction, including genome-wide Hardy-Weinberg allele frequencies, rapid decay of linkage disequilibrium with map distance and genealogies that fluctuate among chromosomes. The presence of sex promotes the transfer and transformation of genotypes underlying important epidemiological traits, posing great challenges to disease surveillance and the development of diagnostics and drugs. Chapter 3 demonstrates that mating events are also pivotal to L. infantum population structure in Brazil, where introduction bottlenecks have led to striking genetic discontinuities between sympatric strains. Genetic hybridization occurs genome-wide, including at a recently identified ‘miltefosine sensitivity locus’ that appears to be deleted from the majority of Brazilian L. infantum genomes. The study combines an array of genomic and phenotypic analyses to determine whether rapid population expansion or strong purifying selection has driven this prominent > 12 kb deletion to high abundance across Brazil. Results expose deletion size differences that covary with phylogenetic structure and suggest that deletion-carrying strains do not form a private monophyletic clade. These observations are inconsistent with the hypothesis that the deletion genotype rose to high prevalence simply as the result of a founder effect. Enzymatic assays show that loss of ecto-3’-nucleotidase gene function within the deleted locus is coupled to increased ecto-ATPase activity, raising the possibility that alternative metabolic strategies enhance L. infantum fitness in its introduced range. The study also uses demographic simulation modelling to determine whether L. infantum populations in the Americas have expanded from just one or multiple introduction events. Comparison of observed vs. simulated summary statistics using random forests suggests a single introduction from the Old World, but better spatial sampling coverage is required to rule out other demographic scenarios in a pattern-process modelling approach. Further sampling is also necessary to substantiate signs of convergent selection introduced above. Chapter 4 therefore develops a ‘genome-wide locus sequence typing’ (GLST) tool to summarize parasite genetic polymorphism at a fraction of genomic sequencing cost. Applied directly to the infection source (e.g., vector or host tissue), the method also avoids bias from cell purification and culturing steps typically involved prior to sequencing of trypanosomatid and other obligate parasite genomes. GLST scans genomic pilot data for hundreds of polymorphic sequence fragments whose thermodynamic properties permit simultaneous PCR amplification in a single reaction tube. For proof of principle, GLST is applied to metagenomic DNA extracts from various Chagas disease vector species collected in Colombia, Venezuela, and Ecuador. Epimastigote DNA from several T. cruzi reference clones is also analyzed. The method distinguishes 387 single-nucleotide polymorphisms (SNPs) in T. cruzi sub-lineage TcI and an additional 393 SNPs in non-TcI clones. Genetic distances calculated from these SNPs correlate with geographic distances among samples but also distinguish parasites from triatomines collected at common collection sites. The method thereby appears suitable for agent-based spatio-genetic (simulation) analyses left wanted by Chapter 3 – and further formulated in Chapter 5. The potential to survey parasite genetic diversity abundantly across landscapes compels deeper, more systematic exploration of how environmental variables influence the spread of disease. As environmental context is only marginally considered in the population genetic analyses of Chapters 2 – 4, Chapter 5 proposes a new, spatially explicit modelling framework to predict vector-borne parasite gene flow through heterogeneous environment. In this framework, remotely sensed environmental raster values are re-coded and merged into a composite ‘resistance surface’ that summarizes hypothesized effects of landscape features on parasite transmission among vectors and hosts. Parasite population genetic differentiation is then simulated on this surface and fitted to observed diversity patterns in order to evaluate original hypotheses on how environmental variables modulate parasite gene flow. The chapter thereby makes a maiden step from standard population genetic to ‘landscape genomic’ approaches in understanding the ecology and evolution of vector-borne disease. In summary, this dissertation first demonstrates the power of population genetics and genomics to understand fundamental biological properties of important protist parasites, then identifies areas where analytical tools are missing and creates new technical and conceptual frameworks to help fill these gaps. The general discussion (Chapter 6) also outlines several follow-up projects on the key finding of meiotic genetic signatures in T. cruzi. Exploiting recently developed T. cruzi genome-editing systems for the detection of meiotic gene expression and heterozygosis will help understand why and in which life cycle stage some parasite populations use sex and others do not. Long-read sequencing of parental and recombinant genomes will help understand the extent to which sex is diversifying T. cruzi phenotypes, especially virulence and drug resistance properties conferred by surface molecules with repetitive genetic bases intractable to short-read analysis. Chapter 6 also provides follow-up plans for all other research chapters. Emphasis is placed on advancing the complementarity, transferability and public health benefit of the many different methods and concepts employed in this work.

Brucellosis is a widespread neglected zoonotic disease. It can cause severe and prolonged illness in people, as well as impacting on animal health and productivity. Brucellosis is endemic in much of sub-Saharan Africa (SSA). The global burden of brucellosis is suspected to be highest in SSA, where there are many livestock-keeping communities. Cattle, sheep and goats are common maintenance hosts of zoonotic Brucella spp. Pastoralist communities in frequent contact with these livestock species are at increased risk of infection. This study was performed to improve our understanding of the epidemiology of brucellosis in Tanzania through: a risk factor analysis for human acute brucellosis cases; trialling an active surveillance approach to identify additional cases through household screening in a high-risk population; and latent class analyses to evaluate diagnostic test performance in different animal hosts. In Chapter 2, questionnaire data were collected from febrile patients attending a rural hospital in the Ngorongoro Conservation Area (NCA), Tanzania. Risk factors associated with acute brucellosis were: having herded cattle, sheep and/or goats in the past 12 months; and decreasing age in years. In Chapter 3, active surveillance in the form of screening household members of febrile patients for exposure to Brucella spp. was implemented in the NCA. Screening household members of febrile patients with acute brucellosis led to identification of additional acute cases. However, the study did not find a significant association between the Brucella spp. exposure of household members and the household member who sought care at hospital. In Chapter 4, Bayesian latent class analyses were used to evaluate the Rose Bengal plate test (RBT) and the competitive enzyme-linked immunosorbent assay (cELISA) for the diagnosis of livestock brucellosis in northern Tanzania. Sensitivity was variable across livestock models, RBT sensitivity was comparable to cELISA in the bovine model and greater than cELISA in ovine and caprine models. RBT and cELISA specificity was essentially comparable in all livestock models. Conducting parallel RBT and cELISA testing optimised diagnostic test performance in all livestock models. These novel findings can inform the development and implementation of effective, evidence-based brucellosis prevention and control measures in SSA. Improved knowledge of acute human brucellosis risk factors is important in understanding temporally relevant risks associated with active infection and is a vital tool in developing interventions that prevent transmission. Active surveillance by screening household members requires further study but may prove too resource-intensive for routine implementation in Tanzania. However, it can provide valuable data on disease burden for the population that do not reach a healthcare facility, as well as assist in targeting prevention and control measures towards high-risk populations. In livestock, a parallel RBT and cELISA diagnostic testing approach, potentially implemented at the herd/flock level, would be more effective than using either test alone or serial approaches. Using these data, identification of a national sampling and testing approach can guide the development of a surveillance strategy which is a crucial step towards improving our understanding of brucellosis burden across livestock-keeping settings in Tanzania and wider SSA.

Background: The complex shape of the pleural cavity and the close proximity of normal radiosensitive structures render the delivery of radical radiotherapy in malignant pleural mesothelioma (MPM) challenging. However, the advent of conformal, intensity modulated radiotherapy (IMRT), where dose is selectively delivered to the tumour whilst sparing normal tissues, can facilitate safe dose escalation. SYSTEMS-2 is the only randomised controlled trial of radiotherapy dose escalation to be attempted in MPM and is comparing the palliative efficacy of two hypofractionated radiotherapy regimes to sites of pain using conformal techniques. Although traditionally associated with unacceptable late normal tissue toxicity, the success of stereotactic radiotherapy (SABR) and the discovery that two common malignancies exhibit low α/β ratios, has enhanced the popularity of hypofractionated regimes. While the radiobiology of MPM is not well understood, its slow growth and apparent radioresistance suggests that it may exhibit a low α/β ratio and therefore that it may respond more favourably to dose hypofractionation. Aims of thesis: To investigate the possibility of further radiotherapy dose escalation in MPM, beyond that delivered in the SYSTEMS-2 study. Methods: I. Novel radiotherapy dose constraints were generated for use in the SYSTEMS-2 study and tested on five patients from the SYSTEMS study. II. Multi criteria optimisation (MCO) software was used to assess whether the original dose escalated radiotherapy plans for the Glasgow cohort of SYSTEMS-2 could be improved, without compromising target volume coverage. III. A clinically relevant 3D in vitro spheroid model was used to investigate the radiobiology of two independent MPM cell lines (H2052 and 211H). Spheroids were established and exposed to the same total dose of ionising radiation (IR) delivered in different doses per fraction. Data was used to investigate response to dose fractionation and to estimate the α/β ratio of this tumour. IV. The response of H2052 and 211H spheroids to two radiosensiting agents was investigated in combination with fractionated radiotherapy. Spheroids were incubated with increasing concentrations of either NU7441 (a DNA-PKcs inhibitor) or A1331852 (a BH3 mimetic) before being exposed to fractionated IR. The immunohistochemical (IHC) expression of DNA-PKcs and Bcl-xL was explored in diagnostic biopsies obtained from MPM patients to investigate clinical validity of the targets. V. IHC expression of nine proteins, selected for their potential to impact on radioresponse, was analysed in diagnostic tumour tissue collected from SYSTEMS and SYSTEMS-2 patients. Expression data was correlated with baseline clinical trial data in all patients, and with clinical trial outcome data from SYSTEMS patients. Results: I. Initial planning studies showed that none of the five SYSTEMS patients met all of the SYSTEMS-2 dose constraints, but the plans were deemed to be potentially clinically acceptable and the constraints were taken forward in the trial. The value of familiarity with a planning technique was evidenced by the fact that all constraints were achieved when the cases were re-planned by the same staff member in April 2019. II. MCO re-planning of dose escalated SYSTEMS-2 plans achieved clinically significant dose reductions to organs at risk (OAR) without compromising target volume coverage in 13/20 cases. Plans which did not meet OAR constraints or conform to the prescribed target volume coverage may still have been clinically acceptable. III. In vitro studies confirmed that growth of MPM spheroids can be delayed by IR. Spheroids demonstrated sensitivity to changes in dose per fraction, with the greatest volume reductions observed in hypofractionated radiotherapy regimes. This data implies that these MPM cell lines may exhibit a low α/β ratio, a suggestion which was further supported by in vitro multi-fraction IR studies. IV. Data suggest that NU7441 and A1331852 are potent radiosensitisers of MPM spheroids and that both are valid clinical targets in MPM. The supposition that a BH3 mimetic may offer tumour specific radiosensitisation, combined with the observation that A1331852 demonstrated greatest efficacy with hypofractionated IR, suggests that this agent may be clinically valuable in the radiosensitisation of MPM. V. No statistically significant correlations were found between baseline clinical characteristics and expression of the proteins of interest and no potential biomarkers of radiosensitisation were identified in the SYSTEMS cohort. Conclusions: Novel dose constraints are being used to facilitate the delivery of hypofractionated, dose escalated palliative radiotherapy in the SYSTEMS-2 study. Results from this trial may guide future dose escalation in this disease and data from MCO planning studies suggest that further dose escalation to the target volume may be feasible without breaching OAR limits. In vitro studies suggest that MPM is sensitive to IR, responds more effectively to dose hypofractionation and may have a low α/β ratio. This data may be helpful in determining dose and fractionation regimes in future MPM radiotherapy trials. Combination of BH3 mimetics with IR may provide MPM specific radiosensitisation, achieving greatest efficacy with dose hypofractionation. Ongoing IHC analysis of tumour samples from the SYSTEMS-2 study may identify a biomarker of radiotherapy response which would be helpful in guiding radiotherapy treatment decisions for future patients. In summary, this thesis has investigated ways in which radiotherapy could be delivered with radical intent in MPM. Practical aspects of radiotherapy planning and delivery have been considered and are presented in conjunction with laboratory data to demonstrate how technical advances can be combined with an appreciation of disease radiobiology to facilitate radical treatment.

In this thesis we study the Student-Project Allocation problem (SPA), which is a matching problem based on the allocation of students to projects and lecturers. Students have preferences over projects, where each project is offered by one lecturer; whilst lecturers have preferences over students, or over the projects that they offer. We seek stable matchings of students to projects, which guarantee that no student and lecturer have an incentive to deviate from the matching by forming a private arrangement involving some project. We present new structural and algorithmic results for four problems related to SPA . We begin by characterising the stable matchings in an instance of the Student-Project Allocation problem with Lecturer preferences over Students (SPA-S) where the preferences are strictly ordered, in the special case that for each student in the instance, all of the projects in her preference list are offered by different lecturers. We achieve this characterisation by showing that, under this restriction, the set of stable matchings in an instance of SPA-S is a distributive lattice with respect to a natural dominance relation. Next, we study a variant of SPA - S where the preferences may involve ties — the Student- Project Allocation problem with Lecturer preferences over Students with Ties (SPA-ST). The presence of ties in the preference lists gives rise to three different concepts of stability, namely, weak stability, strong stability, and super-stability. We investigate stable matchings under the super-stability (respectively strong stability) concept. We present the first polynomial-time algorithm to find a super-stable (respectively strongly stable) matching or to report that no such matching exists, given an instance of SPA-ST . We also prove some structural results concerning the set of super-stable (respectively strongly stable) matchings in a given instance of SPA - ST . Further, we present results obtained from an empirical evaluation of our algorithms based on randomly-generated SPA-ST instances. Moving away from variants of SPA with lecturer preferences over students, we study the Student-Project Allocation problem with lecturer preferences over Projects (SPA-P). In this context it is known that stable matchings can have different sizes and the problem of finding a maximum size stable matching, denoted MAX-SPA-P , is NP-hard. There are two known approximation algorithms for MAX-SPA-P , with performance guarantees 2 and 3/2 . We show that MAX-SPA-P is polynomial-time solvable if there is only one lecturer involved, and NP-hard to approximate within some constant c > 1 if there are two lecturers involved. We also show that this problem remains NP-hard if each preference list is of length at most 3, with an arbitrary number of lecturers. We then describe an Integer Programming (IP) model to enable MAX-SPA-P to be solved optimally in the general case. Following this, we present results arising from an empirical evaluation that investigates how the solutions produced by the approximation algorithms compare to optimal solutions obtained from the IP model, with respect to the size of the stable matchings constructed, on instances that are both randomly-generated and derived from real datasets.

Despite significant improvements in surgical implant manufacturing there remains a clinically unmet need for implantable bone regenerating scaffolds. Current clinical solutions such as the use of pelvic autologous bone graft give rise to additional symptoms and are unpredictable. An engineered customisable osteoinductive coating has been developed within this research group, however the translatable aspects have not yet been explored. As an orthopaedic surgeon with a clinical interest in complex trauma and limb reconstruction, an invested interest in the practical aspects in the application of this technology is maintained. In this work, an engineered osteoinductive coating consisting of poly (ethyl acrylate) with fibronectin and bone morphogenetic protein 2 (BMP-2) is evaluated as a potential coating for surgical implantation. Firstly, it was found that the use of ethylene oxide to sterilise the system (on a variety of base materials) could be employed without significant detriment to the fibronectin network, however BMP-2 was not resilient enough to the process to warrant addition prior to sterilisation. The use of decellularised bone chips commercially available as a transport medium for the osteoinductive coating was explored using an in vivo critical defect model which demonstrated evidence of mature bone formation with BMP-2. In vivo experiments were carried out in the University of Glasgow Veterinary Research Facility and performed under personal (n°I44130F69) and project license (n°70/8638) issued by the Home Office under the Animals (Scientific Procedures) ACT 1986. A fully synthetic transport medium by way of a polymer (poly caprolactone) scaffold was investigated with mechanical compression testing and in vitro cell biocompatibility. Scaffold materials were found to accommodate cell adhesion and differentiation. Despite changes in scaffold pore size, density and addition of hydroxy apatite as a composite filler, mechanical properties did not reach those of current commercial products, specifically poly (methyl methacrylate) bone cement. The findings of this work demonstrate potential translatable aspects of this osteoinductive scaffold. However further rigorous in vivo assessment of retained osteoinductive function following sterilisation is required, followed by analysis of safety and efficacy. Further work on the improvement on mechanical properties of a bioresorbable polymer scaffold are warranted.

In our centre, we had developed nanovibrational bioreactor generating nanoscale vibration by piezo actuator for bone tissue engineering. Recently, nanovibrational stimulation (NS; 30 nm at 1000 Hz) showed the success of osteogenic induction in mesenchymal stem cells (MSCs) seeded collagen hydrogel without chemical supplement. However, culturing MSCs in the collagen hydrogel for long term NS stimulation in NS bioreactor is challenging due to its mechanical properties. The principle aim of this thesis is to develop the scaffold for nanovibrational bioreactor which is suitable for surgical application. Three strategies including ingel scaffolds, collagen concentration optimization and genipin crosslinking were trialled which aimed to improve hydrogel stiffness and handleability, possess biocompatibility and allow NS force transmission. The role of high amplitude stimulation (90 nm at 1000 Hz) on 3D osteogenesis was also studied. Interestingly, increasing NS amplitude successfully enhanced 3D osteogenesis through multiple pathways and it was biologically safe. Metabolomics during NS revealed the evidence of low level of reactive oxygen species production and inflammation which was controlled in physiological level through multiple intracellular signals such as redox balancing, NFkB and MAPK pathways. To propose the technique how to use NS induced MSCs for clinics, MSCs seeded biphasic scaffolds compositing collagen hydrogels and freeze dried collagen sponges were developed. Cell-hydrogel-sponge composite (CHSC) was reproducible, handleable and biologically safe. CHSC allowed a good fidelity of NS. NS with high amplitude stimulation successfully induced 3D osteogenesis. NS protocol in CHSC was optimized in order to identify a stimulating duration which can induce osteogenesis without phenotypic reversibility. Interestingly, two-week stimulation possibly committed MSCs in the preosteoblast stage.

AMP-activated protein kinase (AMPK) has a central role in maintaining cellular energy homeostasis. AMPK is activated allosterically by AMP and signals to inhibit ATP consuming pathways while simultaneously promoting ATP producing pathways. As such AMPK exerts an anti-proliferative effect to induce cell cycle arrest when nutrient resources are limited. Although many of the anti-proliferative effects of AMPK have been well documented during interphase, less is known about how AMPK signalling may influence mitosis. A few publications have reported that levels of active phospho-T172 AMPK increase as mitosis progresses and a link has also been reported between AMPK and myosin regulatory light chain (MRLC) in mitosis. In this Thesis, a combination of cellular, genetic and biochemical techniques were used to further characterise the role of AMPK in mitosis. A novel localisation of the AMPKα1 subunit was described, where AMPKα1 was detected at the microtubule organising centres (MTOCs) during mitosis in both primary human cells and in HeLa cells. AMPKα2 localisation to the midbody was also demonstrated in primary cells and in cell lines. Modulation of AMPK activity by either pharmacological or genetic means caused cell cycle arrest and influenced polyploidy in a number of cell lines. A yeast genetic approach was utilised to investigate the link between the AMPK pathway and a number of key mitotic regulates. Genetic interactions were detected between the AMPK pathway and S. pombe aurora kinase (ark1), polo-like kinase (plo1), anillin (mid1) and components of the endosomal sorting complex required for transport (ESCRT) machinery. Furthermore, a network of cross phosphorylation events were described in vitro between the mammalian protein orthologues. This included the phosphorylation of aurora A, aurora B and PLK1 by AMPK and the phosphorylation of aurora A and aurora B by Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Considering the localisation of AMPK to mitotic structures and the numerous interactions documented with mitotic proteins, a role for AMPK in the regulation of mitosis is clear although the precise cellular effects remain difficult to discern.

This thesis presents and evaluates software for simultaneous, high-resolution time-frequency discrimination. Whilst this is a problem that arises in many areas of engineering, the software here is developed to assist musicological investigations. In order to analyse musical performances, we must first know what is happening and when; that is, at what time each note begins to sound (the note onset) and what frequencies are present (the pitch). The work presented here focusses on onset detection, although the representation of data used for this task could also be used to track the pitch. A potential method of determining pitch on a sample-to-sample basis is given in the final chapter. Extant software for onset detection uses standard signal processing techniques to search for changes in features like the spectrum or phase. These methods struggle somewhat, as they are constrained by the uncertainty principle, which states that, as time resolution is increased, frequency resolution must decrease and vice versa. However, we can hear changes in frequency to a far greater time resolution than the uncertainty principle would suggest is possible. There is an active process in the inner ear which adds energy and enables this perceptual acuity. The mathematical expression which describes this system is known as the Hopf bifurcation. By building a bank of tuned resonators in software, each of which operates at a Hopf bifurcation, and driving it with audio, changes in frequency can be detected in times that defy the uncertainty relation, as we are not seeking to directly measure the time-frequency features of a system, rather it is used to drive a system. Time and frequency information is then available from the internal state variables of the system. The characteristics of this bank of resonators - called a 'DetectorBank' - are investigated thoroughly. The bandwidth of each resonator ('detector') can be as narrow as 0.922Hz and the system bandwidth is extended to the Nyquist frequency. A nonlinear system may be expected to respond poorly when presented with multiple simultaneous input frequencies; however, the DetectorBank performs well under these circumstances. The data generated by the DetectorBank is then analysed by an OnsetDetector. Both the development and testing of this OnsetDetector are detailed. It is tested using a repository of recordings of individual notes played on a variety of instruments, with promising results. These results are discussed, problems with the current implementation are identified and potential solutions presented. This OnsetDetector can then be combined with a PitchTracker to create a NoteDetector, capable of detecting not only a single note onset time and pitch, but information about changes that occur within a note. Musical notes are not static entities: they contain much variation. Both the performer's intonation and the characteristics of the instrument itself have an effect on the frequency present, as well as features like vibrato. Knowledge of these frequency components, and how they appear or disappear over the course of the note, is valuable information and the software presented here enables the collection of this data.

According to the IPCC Fifth Assessment Report (IPCC, 2014), climate change is projected to reduce surface and groundwater resources and significantly undermine water quality and availability. Modelling studies have demonstrated that for each degree of global warming, approximately 7% of the global population is expected to be exposed to a decrease of replenishable water resources of at least 20% (IPCC, 2014). The Canadian Great Plains, encompassing large portions of the provinces of Alberta, Saskatchewan and Manitoba, is one of the regions where IPCC hydrological and air temperature models show the lowest agreement (<60%); future drought scenarios appear to be particularly complex as, although dry spells are projected to last longer, winter rainfall is also predicted to increase. Climate modelling for future scenarios cannot be achieved by modern observations alone, the potential changes are too vast and complex, and the timescale of occurrence is not yet fully understood. Understanding modern climate change is therefore reliant on our ability to characterise the Earth’s climate system at multiple timescales and over a variety of time periods. Particular focus should be given to atmospheric circulation, such as El Niño Southern Oscillation (ENSO), since simulations of droughts at global scale during the 20th century have shown strong correlations between ENSO and hydrological droughts. A multi-proxy approach is presented in this study, combining biomarkers (Long Chain Alkenones – LCAs, Long Chain Diols – LCDs, and Glycerol Dialkyl Glycerol tetraethers – GDGTs), X-ray fluorescence (XRF) and sedimentary organic analysis (Total Organic Carbon –TOC, carbon/nitrogen ratio – C/N). Biomarkers were evaluated for quantitative paleo-climate and -environmental reconstruction with focus on GDGTs. Two types of GDGTs (branched – br, and isoprenoidal – iso) were studied in 96 lakes from the Saskatchewan province, within the Canadian Great Plains. It was observed that the iso-GDGT based TEX86 index could not be applied to the studied lakes due to the large relative proportion of soil derived and methanogenic components. Similarly, br-GDGT derived indices, such as MBT/CBT, were not applicable as the predicted temperatures were considerably lower or higher than those measured. Statistical analysis conducted on the GDGT dataset showed that temperature and salinity accounted for a large and statistically independent fraction of the variation in the dataset. Based on these results, two novel regression models were proposed for salinity using br-GDGTs: Brsalinity (R2 = 0.76, RMSE = 4.9 g/L) and Brsalinity.b (R2 = 0.84, RMSE = 3.47 g/L). This is the first study that presents a GDGT-based calibration for salinity. This thesis presents the first study of lacustrine downcore application of biomarkers in the Canadian Great Plains to determine past climatic and hydrologic variability in this region over the past 2000 years. Two long lacustrine sediment cores from the northern (Humboldt Lake) and southern (Lac Pelletier) Great Plains, were retrieved to investigate shifts in air and water temperature, hydrology and lake productivity over the past 2000 years within the two lakes and their catchment. For the Humboldt Lake sediment core all three biomarkers were analysed for air and water temperature reconstruction. LCA proxies including R3b, UK37 and UK'37 were successfully applied downcore and they provided the first quantitative May Water Temperature (WT), MAAT and spring WT records specific to the Saskatchewan prairies. Additionally, LCA results, suggest that RIK37 and fGII can be qualitatively applied to infer past salinity but also might have the potential to be applied quantitatively as paleo-salinity proxy. For Humboldt Lake, GDGTs had to be excluded due to sample contamination. Finally, statistical analysis between LCDs and recorded MAAT and WT did not produce reliable models and were therefore not used for paleoclimate reconstruction. For the Lac Pelletier sediment core, GDGTs and LCD were successfully applied as palaeo-thermometers for MAAT and autumn WT, respectively. GDGT-based Brsalinity.b calibration developed in this thesis was also applied downcore. However, as instrumental record for salinity was not available, Brsalinity.b was qualitatively used to infer salinity changes. By combining the data generated from XRF, biomarkers, TOC and C/N, this study provided novel quantitative information on the climate and hydrological history of the Canadian Great Plains. Specifically, this thesis suggests that the Great Plains are not an uniform hydrological region, and distinct factors affect water availability and quality in different part of the plains. It was also inferred that the north to south divide seen today as a result of the mid-tropospheric pattern has been present consistently in the past 2000 years. It is evident from the results presented in this thesis that solar forcing and large-scale climate events are key drivers of shifts of air temperature in the Canadian prairies. However, and perhaps more importantly, negative phase of ENSO (La Niña) and Pacific Decadal Oscillation in conjunction with increased wind activity and glacier fluctuations were identified as the fundamental drivers of the extent and severity of wet and dry periods in the Canadian Great Plains.