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Spike and wave discharges (SWD), generated by the cortico-thalamo-cortical (CTC) network, are pathological, large amplitude oscillations and the hallmark of absence seizures. SWD begin in an initiation network in both humans and animal models, including the genetics absence epilepsy rats from Strasbourg (GAERS), where it is in the primary somatosensory cortex (S1). The mechanisms of how SWD rapidly spread across the brain is unknown. This thesis investigates mechanisms beyond the principal CTC network, i.e. in higher-order thalamic (HO) nuclei to explore their role in the generalisation of SWD in freely moving GAERS. Their diffuse connectivity, altered synaptic anatomy in GAERS and known interactions with the initiation network, make these nuclei serious potential candidates. Firstly, HO nuclei and cortical local field potentials revealed a novel feature of cortical SWD: synchrony in cortical regions far from S1 (such as primary visual cortex, V1) become transiently unsynchronised with the SWD, i.e. there are short breaks in SWD (named SWD-breaks). Additionally, SWD can occur in S1 only or in S1 and the neighbouring primary motor cortex (M1), but not elsewhere. These spontaneous events provided a unique insight since they represent unsuccessful maintenance and generalisation of SWD. Afterwards, the relevance of CTC relationships was verified by local inhibition of HO nuclei with muscimol. This increased the delay of SWD propagation and occurrence of SWD-breaks. Finally, neural dynamics of HO nuclei single units revealed three groups of putative excitatory neurons. All groups exhibited a switch from tonic to burst firing before onset of SWD, but they responded differently during V1 SWD-breaks and unsuccessfully generalised SWD. The results of these experiments converge on two main conclusions: that trans-thalamic cortical communication is utilised in the initial propagation of SWD and also has an active role in maintaining cortical synchrony throughout the paroxysmal activity.

Introduction: Skin and soft tissue infections (SSTIs) are considered a major public health problem in many countries. Pathogenic bacteria have evolved complex mechanisms for infection and colonization of numerous host cells. Despite the presence of various antibiotics for the treatment of infectious diseases, their random, inappropriate and excessive use has resulted in increasing incidence of several side effects and the development of antimicrobial resistance. Consequently, there is an urgent need to develop new and safe treatments for infections, especially those of the skin. Different new therapeutic approaches are currently being increased such as bacteriotherapy using probiotics. Dairy and non-dairy fermented foods have been used in traditional diets worldwide and are still to be broadly consumed, since they are considered as rich sources of probiotics, especially lactic acid bacteria. From commencement to cure, the progress and severity of infectious diseases are continuously affected by the interaction between the pathogen and the host. Therefore, investigations performed both in vitro and in vivo are important when determining the health- enhancing properties of probiotics. The greater wax moth larvae Galleria mellonella is a well-accepted model of infection by scientists, as an in vivo model for host-pathogen interactions due to several advantages, such as its low cost and no required ethical rights to be used in the experiments in vivo. Aim: This project aimed to investigate the antagonistic activity of Lactobacillus type strains and food isolates against the major causative agents of skin infections in vitro. It also aimed to explore the therapeutic potency of food-derived Lactobacillus species against skin pathogens, using both the injection method inside the Galleria mellonella larvae model, and the topical application on the larvae as a novel technique for infections and treatment which can assist in decreasing the occurrence of skin infections. Results: A total of twenty Lactobacillus species, including ten type strains obtained from different culture collections and ten food isolates derived from fermented yogurt and olives, all revealed significant inhibitory activity against the three skin pathogens used in this study; Staphylococcus aureus, Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis. Although the antagonistic effect was observed after the first day of incubation under both aerobic and anaerobic incubation conditions, the maximum antibacterial effect was detected following three days of anaerobic incubation. Furthermore, food-isolated Lactobacillus presented a higher inhibitory effectiveness than the type strains against the pathogens xx particularly under the anaerobic conditions. Characterisation of bacterial genomes by whole genome sequence (WGS) analysis showed that the sequenced genomes of food lactobacilli had a number of genes encoding for antimicrobial substances, in addition to other genes responsible for probiotic advantages. Multiple genes encoding for virulence factors were also detected in the pathogenic sequenced genomes. Diversity of the bacterial community of food samples by next generation sequencing (NGS) of 16S rRNA gene amplicons revealed that Lactobacillus delbrueckii isolated from yogurt by the culture-dependent procedure was found in low abundance as Lb. delbrueckii subsp. indicus using the culture-independent method. Despite the isolation of Lactobacillus plantarum from olives using culturing technique, no signal was observed for this species by NGS. Injection of Lb. delbrueckii and Lb. plantarum isolated from fermented foods in G. mellonella larvae presented a significant therapeutic activity of both species against pathogenic infections in a dose-dependent matter. Both the injected doses, 102 CFU/larva and 104 CFU/larva of Lb. delbrueckii washed cells, were effective in reducing S. aureus and S. pyogenes infections. Whereas the low injected dose of this species had more activity than the higher one to decrease the infection of S. dysgalactiae subsp. equisimilis in the larvae. Regarding Lb. plantarum, the doses of 103 CFU/larva and 104 CFU/larva injected in the larvae were both useful in reducing S. aureus infection, with more efficiency for the low dose. However, both doses had no therapeutic effect against S. pyogenes infection. The novel topical application technique of S. pyogenes cells on the dorsum of larvae followed by the application of Lb. plantarum washed cells and undiluted cell free supernatant (CFS) resulted in lower death rates of the treated larvae in comparison to the control group. Nevertheless, bacterial cells of Lb. plantarum revealed more activity than the CFS of the same species. Conclusions: The significant antagonistic effect in vitro and the significant therapeutic potency of food Lactobacillus species against skin pathogens, indicate that food-derived Lactobacillus isolates included in this study could act as potential candidates with promising probiotic advantages. This research delivers the basis for further investigation on the treatment of skin infections with food lactobacilli. Moreover, these food-based Lactobacillus species could be introduced as topical formulations to decrease the symptoms of skin infections or even in the context of skin infection treatments.

The firefly luciferase (Fluc) bioluminescence system has been widely studied and used by biomedical researchers for in vitro assays as well as a wide variety of optical imaging applications in small animals. However, Fluc is immunogenic to mice which limits its applications, especially in longitudinal experiments requiring time courses extending over more than four weeks. Murine acyl-CoA synthetases (ACSs) are structurally and functionally similar to Flucs and as native mouse proteins are inherently non-immunogenic. Both ACS and Fluc enzymes are composed of N and C-terminal domains (NTD and CTD). A strategy of hybridisation between Photinus pyralis (Ppy) Fluc and several medium-chain ACSs (ACSMs) was employed with the aim of creating hybrids with minimal immunogenicity whilst maintaining brightness when ultimately expressed in BALB/c mice. Several hybrid variants were created with regions of importance substituted into an ACSM backbone, but all the constructs tested failed to produce any bioluminescence when expressed in E. coli. Hybrids created containing the NTD of Ppy Fluc and the CTD of murine ACSMs 1, 2, and 3 were named PpA1, PpA2, and PpA3 respectively. Light emission of these hybrids was respectively ∼0.9%, ∼0.004%, and ∼0.2% that of WT Ppy Fluc when expressed in E. coli, but PpA1 was ∼10% and PpA3 was ∼0.2% as bright as when expressed in HEK cells. The PpA1 hybrid was selected as the best candidate and was expressed in immunocompromised NSG™ mice, producing ∼45% the light output of WT Ppy Fluc. Further modificationsto PpA1 included a construct which replaced a major epitope in the Ppy Fluc NTD (G160 - V168) of PpA1 with the equivalent sequence from a highly similar luciferase with lower predicted immunogenicity; when expressed in HEK cells this construct was marginally brighter than PpA1, emitting light with λmax 10 nm red-shifted.

The pharmaceutical heparin is a complex polysaccharide of the glycosaminoglycan family, usually derived from porcine mucosa, and is a mainstay of anticoagulant therapy worldwide. Its activity can be modified, most notably, through depolymerisation. Yet, owing largely to the chain complexity, the progressive effects of environmental conditions on the heparin structure have not been fully described. A systematic study of the progressive effects of acidic and alkaline hydrolysis on heparin chain length and sulfate substitution has therefore been conducted. The initial analysis concerned the changes of weight-average molecular weight of heparin, induced by applied conditions. In acidic environments, the relation between the molecular weight loss and pH was inversely correlated, whilst the rate of degradation increased with temperature. In alkaline environments, the molecular weight loss was proportional to pH and temperature, although less effective. Under milder acidic conditions, desulfation was the major factor affecting the molecular weight of the polysaccharide, whilst in alkaline conditions, the hydrolytic sulfate scission was not as prominent. Glycosidic scission was observed only after the prolonged hydrolytic processing at pH 1, at 80 C. Stability studies confirmed the chain stability between pH 2 and pH 12. To understand desulfation order and further investigate possible rearrangements, selected acid and alkaline hydrolysates were monitored via 2D (HSQC) NMR. This study revealed that in acidic environments all sulfate groups of heparin were altered. It was observed that the sulfate groups were hydrolytically removed in the following order: N-sulfate (NS-), then 2-O-sulfate (2OS-), and finally 6-O-sulfate (6OS-), although the selectivity of last two was strongly dependent upon applied conditions. On the other hand, the NMR analysis of alkali treated heparin demonstrated that applied environments catalysed only iduronate desulfation (2OS-), followed by its exclusive rearrangements to a galacturonic residue. The research discussed in this thesis investigated the behaviour of heparin in aqueous systems altered by temperature and pH over time. The selected stressing factors closely resembled conditions applied over the industrial and/or pharmacological processing. Therefore, the contribution of presented findings reaches beyond theoretical knowledge and extends to possible practical applications, i.e., optimisation of manufacturing, storage and administration of pharmacologically active heparin products.

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.

Trace amines including β-Phenylethylamine (β-PEA) and tyramine are vasoactive monoamines closely related to the classical neurotransmitters, noradrenaline, serotonin (5-HT) and dopamine. As vasoactive substances, the trace amines are considered sympathomimetic, eliciting vasoconstrictor responses through noradrenaline release, although they can also induce vasodilator responses. The trace amines are well-known agonists of trace amine-associated receptors (TAARs), of which TAAR1 is expressed in rat aorta. Pharmacological characterisation of TAAR1 has proven difficult as TAAR1 is located in intracellular compartments and is poorly expressed at the cell surface. Previous studies have attributed both trace amine-induced vasoconstrictor and vasodilator responses to TAAR1. The aim of the current thesis was to pharmacologically characterise both the vasoconstrictor and vasodilator actions of the trace amines. β-PEA- and tyramine -induced vasoconstrictor response in rat aortic rings were both significantly potentiated by endothelium removal or inhibition of endothelial nitric oxide synthase (eNOS). Vasoconstrictor responses to β-PEA were found to be resistant to blockade of post-synaptic uptake-2 transporters, antagonists of 5-HT2 receptors, α1-adrenoceptors, D1 and D2-class dopamine receptors and the mouse-specific TAAR1 antagonist, EPPTB. This indicates that β-PEA-induced contractile responses are likely mediated by a currently unidentified cell surface receptor or receptors. As EPPTB, is a poor tool for the study of rat TAAR1, it is possible that TAAR1 located at the plasma membrane mediates these responses. β-PEA-induced vasodilator responses in rat aortic rings were partially attenuated by endothelium removal or inhibition of eNOS. Vasodilator responses to β-PEA were resistant to antagonists of muscarinic M3 receptors, β2-adrenoceptors and EPPTB. β-PEA-induced vasodilator responses were completely abolished by inhibition of uptake-2 transporters indicating that vasodilation is mediated by an intracellular receptor such as TAAR1.

Glioblastoma multiform (GBM) is a lethal brain tumour composed by many distinct cell types that are closely connected and dependent on their surrounding environment. Microglia are the brain immune cells, which are highly abundant in GBM and create an immunosuppressive microenvironment that promotes tumour progression. Caveolin-1 (Cav1) is the most important protein of caveolae and it is involved in cell signalling activity. In the GBM, Cav1 promotes the tumour invasion and it is correlated with a poor prognosis. In immunes cells, its role is not well explored, however it can be involved in immune response. Our hypothesis was that Cav1 could have an impact in the response of human microglia to the environment, influencing tumour progression. To test our hypothesis, a human microglia cell line and an iPSC cell line were used to generate Cav1 knockout clones using CRISPR-Cas9 technology. The iPSC was used to generated human microglia cells. Primary human microglia expressed low levels of Cav1, which could be regulated upon activation. The viral immortalized human microglia cells expressed strong Cav1 protein levels, possibly correlated with the immortalization procedure with SV40 large T antigen. This infection in combination with the culture conditions might lead to a constitutive pro-inflammatory phenotype, impacting the ability of microglia to react to other stimulus and to do phagocytosis. A slightly modified protocol to generate microglia from iPSC allowed the differentiated cells to be polarized towards pro-inflammatory and anti-inflammatory phenotype and to perform phagocytosis. In microglia, Cav1 was involved in the regulation of the inflammatory response, cell migration, phagocytosis, and sensitivity to temozolomide. The microglia cell line did not impact the tumour behaviour, likely due to the profile presented by the cells. However, the deletion of Cav1 in microglia derived from iPSC promoted the tumour invasion.

The oxygenated volatile organic compound, acetaldehyde, plays a fundamental role in atmospheric chemistry by altering the atmosphere's oxidative status or "self-cleaning capacity". Acetaldehyde flux from freshwater, estuarine, and marine environments is thought to be under significant microbial control, yet the identity and diversity of microbial acetaldehyde degraders, and the mechanisms of acetaldehyde degradation, are largely unknown. In this thesis, the key acetaldehyde-degrading microorganisms in the Colne Estuary, the River Colne and the River Gipping were identified using metagenetic sequencing and quantitative PCR. Bacteria were primarily responsible for acetaldehyde degradation, with members of the genera Pseudomonas and Arcobacter identified as key acetaldehyde degraders. Seventeen bacterial isolates were cultivated from the Colne Estuary and degraded acetaldehyde at a concentration of 2.27 mM, with Halobacillus strains A4 and A5, Rhodococcus strain A14, and Bacillus strain A17 identified as important acetaldehyde degraders. Fourteen previously identified bacteria, belonging to the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia, also degraded acetaldehyde, suggesting that the microbial acetaldehyde sink is highly diverse. Using genomics and proteomics, the pathway of acetaldehyde metabolism used by Rhodococcus strain A14 was proposed. Acetaldehyde is oxidised to acetate by aldehyde dehydrogenase enzymes, with acetate converted to acetyl-CoA via acetyl-CoA synthetase. These reactions are thought to be conserved amongst acetaldehyde-degrading bacteria. Under favourable growth conditions, acetaldehyde is dissimilated to CO2 for energy production via the oxidative decarboxylation steps of the tricarboxylic acid cycle. Acetaldehyde-derived carbon is assimilated into biomass by upregulating the glyoxylate shunt and gluconeogenesis pathway, ensuring carbon is conserved for growth. Using 14C-radiolabelling, rates of microbial dissimilation were shown to be significantly higher than assimilation rates in the freshwater rivers, estuaries, and coastal seas of south-west England, with summer rates significantly higher than winter. These findings suggest that microorganisms exert considerable control over acetaldehyde emissions and represent an important component of the global acetaldehyde cycle.

Heterozygous germline mutations in the GATA2 gene gives rise to an immunodeficiency syndrome characterised by cytopenias, severe infections and a predisposition towards haematological maligancies such as myeloidysplastic syndrome (MDS) and acute myeloid leukemia (AML). Clinical data from GATA2 mutated families reveal a variable age at presentation, a variable disease phenotype and a proportion of asymptomatic carriers, suggesting incomplete penetrance. Acquisition of secondary hits such as somatic mutations in the ASXL1 gene and cytogenetic abnormalities such as monosomy 7 and trisomy 8 are commonly found in patients that have progressed towards MDS/AML. Perturbation in the immune system of these patients leads to persistent inflammation. Indeed, scRNA-seq from patient samples showed immune and inflammatory pathway dysregulation at the HSC level, which has also been confirmed in our lab in HSCs from Gata2 heterozygous mice. Whilst there has been much research over the years investigating the loss of GATA2 in a variety of mouse and human models, many have failed to recapitulate key aspects of the disease. Using human iPSCs, primary cord blood CD34+ cells and transgenic mouse models, GATA2 immunodeficiency was modelled along with further investigations into inflammation as a potential driver of disease progression. In this thesis, CRISPR/Cas9 and CRISPR base editors were used to engineer specific GATA2 mutations - T354M and R361C - into human iPSC lines followed by truncating ASXL1 mutations to model GATA2 immunodeficiency and its progression towards myeloid malignancy. Unfortunately, due to technical and time limitations (including COVID19) this work was unable to be taken to fruition. Interestingly, in vitro bacterial stimulation selectively increased CFU output and CD14 expression in GATA2 knockdown cord blood CD34+ cells. In addition, chronic inflammation led to splenomegaly and granuloma formation along with a reduction of Gr1+ Mac1+ cells in the BM of mice with haematopoietic-specific deletion of a single Gata2 allele. Finally, in comparison with steady-state conditions where Gata2- heterozygous HSC levels were reduced by half, HSC numbers remained unchanged while control HSCs were reduced by half upon inflammation, indicating that Gata2 heterozygosity enabled HSCs mechanism to better adapt to inflammation. Taken together, this work highlights that a multi-model approach is desirable to study GATA2 familial MDS/AML and that inflammation is an important mediator for disease progression in GATA2 immunodeficiency syndromes thus opening new avenues for disease modelling and therapeutic intervention.

Fungal infections are a global issue affecting over 150 million people worldwide annually with 750,000 of these caused by invasive Candida infections. The outcomes of life-threatening systemic infections caused by Candida albicans are poor with mortality rates estimated to be between 46-75%. Azole drugs are the frontline treatment against fungal infections however resistance to current azole antifungals in C. albicans poses a threat to public health. Azole resistance can arise through several mechanisms with point mutations in sterol 14α-demethylase (CYP51) leading to amino acid substitutions a major contributor. The aim of this research is to design and synthesise novel azole inhibitors effective against wild type and fluconazole-resistance Candida strains. The development of potent and selective inhibitors from three azole series were investigated for CYP51 inhibitory activity, binding affinity, and minimum inhibitory concentration (MIC) against C. albicans strains biologically as well as computationally. The first series, short and extended novel imidazole/triazole derivatives were synthesised successfully. The short derivatives were more potent against the C. albicans strains (MIC 0.03 μg/mL) compared with the extended derivatives (MIC 1 μg/mL), while both series showed similar enzyme binding and inhibition (Kd low nM, IC50 submicromolar) and were comparable with the standards fluconazole and posaconazole. The short series had poor selectivity for CaCYP51 over the human homolog, while the selectivity of the extended series was higher (21.5-fold) than posaconazole (4.7-fold) based on Kd values, although posaconazole was more selective (615-fold) compared with the extended series (461-fold) based on IC50 values. Series two, 2-(arylphenyl)-N-(4-((4-arylphenyl)amido)benzyl)-3-(1H-1,2,4-triazol-1-yl)propanamides derivatives, were synthesised successfully. The novel inhibitors exhibited weak activity against C. albicans strains; however, a slight improvement in the IC50 was shown in chloro derivatives (IC50 4.6 -1.3 μM). A series of N-(2-(arylphenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)butanamide derivatives have been synthesised using an efficient synthetic route and shown to be potent against the C. albicans strains (MIC from <0.03 to 1 μg/mL) and potent inhibitors of CaCYP51 (IC50 0.78 to 1.6 μM) compared with the standard fluconazole. All series were studied computationally using CaCYP51 crystal structure (PDB 5FSA) for molecular modelling and molecular dynamic simulations to determine optimal fit in the active site and binding interactions. Leishmania was also of interest as it has been identified by the WHO as a disease with unmet needs with an estimated 700,000 to one million new cases each year in the endemic regions such as East Africa, North Africa and West Asia. CYP5122A1 an orphan enzyme has been identified as a CYP enzyme specific to leishmania, which could provide a novel target in the treatment of leishmania infections. A CYP5122A1 homology model was developed, as no crystal structure is available, using a combination of homology modelling, molecular dynamics simulations, and molecular docking to understand the active site and the binding interaction of CYP5122A1 and selected ligands complexes. Docking results for CYP5122A1 showed amino acids Glu365, Thr366, Val440 in the haem binding pocket and Tyr175, Phe178, Pro441, Asp584 in the access channel, which could have an important role in the binding interactions with designed ligands. Furthermore, some of the novel compounds synthesised in this research were also tested against Leishmania donovani to investigate the inhibitory potential.

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.

Land use classification is the process of characterising the land by the purposes of usage. It is significantly important since timely and accurate land use information is essential for land monitoring and management. Traditional classification relies heavily on labour-intensive land survey, which is time-consuming and expensive. The development of remote sensing (RS) technologies has greatly advanced the automation of land monitoring, since RS images can efficiently capture the physical attributes of the earth surface. However, it is challenging to use single RS images to recognise land use, especially for high-density cities with complex and mixed land-use patterns. There are several major challenges. Firstly, it is challenging to extract representative features from RS images that are highly related to land use semantics and invariant to complex landscapes. Secondly, RS images capture the physical information from nadir view, and thus miss out many useful ground-level details. Thirdly, RS images cannot directly capture information about human activities on the land, which is critical to determine land use. To address these issues, this thesis leverages deep learning techniques and exploits recently emerged geospatial big data to complement RS images for land use classification. In summary, the thesis has made following contributions: Firstly, a triplet deep metric learning network has been developed to enhance remotely sensed land-use scene retrieval and classification. The developed network can embed RS images into a semantic space where images from the same class are near each other while those of different classes are far apart through training with triplet loss. The produced features are discriminative and can be exploited for content-based RS image retrieval. Feature reduction methods have been investigated to reduce the redundancy of learned semantic features. Triplet selection strategies have also been examined to ensure effective and efficient training of the network. Furthermore, the extracted features have been leveraged for image scene classification. Comprehensive experiments have been conducted on popular land-use scene benchmarks, and the results demonstrated the effectiveness of the proposed methods. Secondly, a spatial-aware Siamese-like network has been proposed to learn distinguishable embeddings for ground-to-aerial geolocalisation, which can serve as a potential solution for the geotagging of ground-taken images without geotags. Spatial transformer layer was exploited to address the large view variation issue. A loss that combines the triplet loss with a simple and effective location identity loss has also been designed to train the proposed network to further enhance the geolocalisation performances. Extensive experiments were conducted on a publicly available dataset of cross-view image pairs, and the results demonstrated the effectiveness of the method. The proposed geolocalisation method can be used to infer the geolocation of ground-taken images, which makes it possible to further fuse them with RS images for land use classification. Thirdly, a deep learning-based approach has been developed to integrate RS images with geotagged ground-taken images for urban land use classification. Specifically, a deep neural network was used to extract semantic features from sparsely distributed street view images, and the features were further interpolated in the spatial domain to match the aerial images, which were then fused together through a deep fusion neural network for pixel-level land use classification. The proposed methods were tested on a large public dataset, and the results showed that the street view images contain useful information about land use and fusing them with aerial images can improve classification accuracy. Moreover, experimental studies have been presented to show that street view images add more values when the resolutions of the aerial images are lower, and case studies have also been presented to illustrate how street view images provide useful auxiliary information to aerial images to boost performances. Finally, a deep learning-based method has been proposed to fuse remote and social sensing data for urban land use classification. Two neural network based methods have been developed to automatically extract discriminative time-dependent social sensing signature features, which were fused with remote sensing image features extracted via a residual neural network. Besides, a deep learning-based strategy has also been developed to address the data asynchrony problem by enforcing cross-modal feature consistency and cross-modal triplet constraints during the training of the model in an end-to-end manner. Extensive experiments have been conducted on publicly available datasets to demonstrate the effectiveness of the proposed methods. The results showed that the physically sensed RS images and social activities sensed signatures can complement each other to help enhance the accuracy of urban land use classification.

The evaluation of how Bacteria respond to oil-contamination, and the application of dispersants and biosurfactants, in North Sea seawater microcosms is the focus of Chapter Two. Analysis revealed that dispersants and biosurfactants, which significantly reduced the interfacial tension between oil and water, significantly increased growth of obligate hydrocarbonoclastic bacteria (OHCB) in 24 hours, translating into significantly enhanced alkane-biodegradation. Early sampling of microcosms revealed how the OHCB Oleispira, hitherto considered a psychrophile, can dominate bacterial communities at the relatively high temperature of 16oC. Bacterial response to oil-pollution is examined further in Chapter Three, where an in situ oil-slick is compared to a chemically dispersed oil-slick in the North Sea. Results suggest a lack of hydrocarbon-degrading bacteria (HCB) growth, even in samples with measurable hydrocarbons, could potentially be attributed to phosphorous limitation. Whilst the Ecological Index of Hydrocarbon Exposure, which quantifies the proportion of a bacterial community with hydrocarbon-biodegradation potential, revealed an extremely low score, highlighting a limited capacity for the environment, at the time of sampling, to naturally attenuate oil. Analysis of sediments contaminated by the Agia Zoni II oil-spill (Greece, 2017), in Chapter Four, demonstrated significant growth of HCB five-days post-oil-spill. Whilst the relative abundance of HCB declined as oil was removed, a legacy effect was observed, with the OHCB Alcanivorax and Cycloclasticus persisting for several months after the oil-spill. Finally, analysis of sediments around a North Sea shipwreck (HMS Royal Oak), in Chapter Five, revealed low levels of pyrogenic polycyclic aromatic hydrocarbons and little evidence of HCB, indicating sediments showed no long-term impact by previous oil-pollution from the shipwreck. This thesis not only advances our understanding of microbial response to oil-spills, remediation strategies, and shipwrecks, in a range of marine environments, but also highlights the importance of harnessing such knowledge and data to advance post-incident monitoring guidelines and models.

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.

The global financialisation of urban space has led to challenges for low-income citizens to find affordable housing. The Indonesian government has attempted to solve the housing problems but has not been able to eradicate them. Moreover, the planning approach towards informal settlement upgrading is considered fragmented. Findings regarding the informal settlement relocation plan which violated spatial planning and flood mitigation control, raised the question of who will capitalise on this campaign of urban ecological security and whose resilience is enhanced. The thesis engages with the literature on state rescaling, how it exacerbates informality in Indonesia, where it has experienced a regime shift from centralised to decentralised governance. The thesis contributes to the exploration of collaborative planning processes in Indonesia, and how the hierarchical processes in existence tend to make informal settlement community not to have effective voice. It also attempts to expand concepts of collaborative governance and co-production adapted to developing countries by integrating the local culture of gotong-royong on the principle of collective action. By using Jakarta as a case study and conducting semi-structured interviews and focus group discussions, the thesis aims to critically assess the role of collaborative governance in addressing the problem of informal settlements in Indonesia.

The intricate linking of information processing and neural representations to the underlying hippocampal neural rhythms during episodic memory retrieval are yet to be fully explored in human subjects. In this doctoral thesis, the temporal order of these relationships was investigated, with emphasis on how the processes evolve and take place over time. Empirical evidence and neural network models suggest that hippocampus and the hippocampal theta rhythm play a central role in episodic memory. In the first two studies, different oscillatory dynamics in the hippocampal circuit thought to provide optimal states for encoding and retrieval were investigated. The third study investigated the role of the hippocampal theta oscillation as an adaptive mechanism in regulating competition between similar memories. And lastly, the fourth study investigated sharp-wave ripples in promoting successful episodic memory retrieval. Throughout the four chapters, memory content was decoded using multivariate pattern classification, and the timing of memory reactivation was linked to two prominent oscillatory brain signatures: the hippocampal theta rhythm on the one hand, and hippocampal sharp-wave ripples on the other. In sum, this doctoral thesis provides support for the powerful computations along the hippocampal theta oscillation, and the close interplay between hippocampus and neocortical areas, foremost at time of retrieval.

Early diagnosis of Alzheimer's disease is important because current treatments only provide symptom relief or slow the disease and therefore rely on early diagnosis to be effective. Neuropsychological tests are important in early diagnosis because there is currently no reliable diagnostic test using biomarkers. Previous research has suggested that binding processes in memory might be particularly vulnerable in the early stages of the disease, and therefore useful in diagnosis. The thesis critically reviews this research, and highlights a number of methodological problems and gaps. The thesis then describes a study designed to address some of these issues. Groups of people with Alzheimer's disease, age-matched healthy controls, and younger people (n=26 in each group) completed six binding tests, along with a battery of other measures. The results suggested that binding tests do provide a promising method for early diagnosis, but that they vary in their effectiveness. The Face-Name and the Paired Associates Learning tests performed best. More research is needed to establish the characteristics of binding tasks that are most vulnerable to the early disease processes. The results provided no evidence to support the claim made in previous literature that spatial binding may be more vulnerable than non-spatial binding.

Inflammatory bowel disease (IBD) has progressively become a global epidemic and now affects nearly 0.5% of the Western population. The aetiological factors that initiate and drive mechanisms associated with IBD remain unclear. A cure has been even more elusive. Changes in the gut microbial diversity and profiles in individuals with this disease is a characteristic feature, however a causal relationship has yet to be proven. In my PhD I have attempted to explore host-microbiota interactions and its influence on mechanisms of ulcerative colitis (UC) and primary sclerosing cholangitis associated inflammatory bowel disease (PSC-IBD). Patients with UC have a greater abundance of Clostridiaceae at inflamed compared to non-inflamed sites. Immunophenotyping demonstrated significantly higher proportions of colonic mucosal Th17 and IL-17 producing CD4 cells in patients with UC and PSC-IBD compared to healthy controls. Through an open label study (STOP-Colitis pilot phase), I demonstrated that faecal microbiota transplantation (FMT) resulted in a clinical response in 47% of patients (8/17; intention to treat). This response was associated with a significant increase in colonic mucosal regulatory T cells (Treg), effector memory Tregs, gut homing Tregs and IL-10 producing CD4 T cells population along with a concurrent decrease in Th17, IL-17 producing CD4 T cells and CD8 populations. Colonic mucosal transcriptomics revealed that responders to FMT had significant downregulation of antimicrobial defence and proinflammatory immunological pathways and an increase in butanoate metabolic pathways compared to both baseline and non-responders. Finally, through a multi-omic exploration of colonic mucosal biology, I demonstrated that the gene expression profiles in patients with PSC-IBD was significantly different to UC and was associated with dysregulation of bile acid homeostasis and signalling in association with colonic dysbiosis.

The LIM-Only protein 2 (LMO2) is a transcriptional co-regulator that forms a multi-protein complex with TAL1, GATA, and LDB1. This complex regulates transcription from the onset of haematopoietic development and differentiation. However, LMO2 is normally downregulated during T-cell development. Genetic abnormalities that trigger the aberrant expression of LMO2 and other members of the complex lead to T-cell acute lymphoblastic leukaemia (T-ALL). The components of the LMO2 complex are not widely explored in T-ALL and its functions are not fully characterised in this type of leukaemia. Through proteomic analysis we identified the Plant homeodomain zinc finger protein 6 (PHF6) as a novel interactor of LMO2. Mutations of PHF6 have been found to occur in several types of leukaemia including T-ALL. We show a functional interaction between PHF6 and the LMO2 complex members by genome-wide approaches in T-ALL. These outcomes indicated that the LMO2 complex recruits PHF6 to the DNA and regulates gene expression. Importantly, we identified many genes of transcription factors, which are involved in haematopoietic stem cell (HSC) self-renewal and development of early T-cell progenitors, as putative targets of LMO2/PHF6 complex, providing compelling indications of an oncogenic mechanism of the complex. Additionally, Phf6 gene knockdown in myeloid cells suggested a role for PHF6 in the differentiation of this lineage. Finally, loss of PHF6 and LMO2 caused chromosomal instability and signs of DNA damage in these cells, implying a possible function in chromosome and genome stability.

T cell metabolism is differentially regulated in order to support their activation, replication and effector functions. For example, T cells preferentially upregulate glycolysis following activation and this is a crucial determining factor at the Th17/Treg axis. Productive T cell activation requires several signals namely 1) antigen, 2) co-stimulation, and 3) cytokines but if, and how, these influence the metabolic changes associated T cell activation is largely unknown. To investigate this we assessed the metabolic phenotypes of CD4+ T cells following activation with; 1) variable strengths of stimulation through antigen-T cell receptor (TCR) engagement, 2) incubation with CHO cells transfected with individual costimulatory molecules CD80 or CD86, 3) exposure to the inflammatory cytokine IL-6. Metabolism was assessed using NMR spectroscopy-based metabolomics and metabolic flux analysis to assess the balance between the major pathways of glycolysis and oxidative phosphorylation. We optimised the techniques for determining human CD4+ T cell metabolic phenotype and have observed quantitative and qualitative differences associated with variations in the activating signals 1 to 3. In particular, we found that exposure to IL-6 prior to TCR stimulation leads to an increase in glycolysis, a response which might prepare T cells for the metabolic burden following activation. Subsequent flow cytometry analysis demonstrates that IL-6 exposed cells have an increased proliferative phenotype, greater mitochondrial mass and markedly upregulate GLUT1, however, these changes were only observed in naïve cells and not memory. Abnormal T cell responses and systemically measurable dysregulated metabolism are hallmarks of autoimmune and inflammatory diseases such as rheumatoid arthritis. An improved understanding of T cell metabolism has implications for the treatment of these diseases and the mechanisms of action of biological therapies such as abatacept and tocilizumab which target T cell differentiation and function.

The maintenance of intramuscular creatine (Cr) is important for sustainability of skeletal muscle mass across the life-course. Creatine transporter (CreaT) is the only known specific Cr transporter which embedded in the cell membrane as a gate-keeper that transports Cr from blood into skeletal muscle. The impairments of structure and functions in CreaT will lead to insufficient intracellular Cr concentration, resulting in the disturbance of cellular energy homeostasis, metabolic disorders, muscle atrophy and neurological disorders. Impaired energy homeostasis could also have implications for dysregulated proteostasis, and muscle atrophy. Maintenance of proteostasis is essential to sustain skeletal muscle mass and therefore vital to health across the life-course. This is especially important to populations in the context of musculoskeletal ageing and scenarios of disease/injury. In this regard, detections of tissue CreaT content may aid the clinical diagnosis of diseases. As such, to understand the CreaT regulation mechanisms and its role in Cr metabolism can help the development of therapeutic interventions to improve intramuscular Cr levels and support muscle mass in health and disease. However, the role of CreaT in modulating Cr metabolism in skeletal muscle remains largely unexplored. The existing measurement techniques lack precision to fully explain the regulatory role of CreaT in different physiological scenarios of muscle deterioration. Accordingly, Chapter 2 of this thesis validated and optimized an immunofluorescence microscopy technique and provided a more precise approach to identify CreaT protein expression and distribution in human skeletal muscle. A new method of calculation was designed and developed to identify CreaT protein intensity in specific sub-cellular regions. By using these developed methods, Chapter 3 of this thesis investigated whether CreaT distribution and sub-cellular expression is altered in older age. A sub-set of quadriceps muscle biopsy samples from 6 young and 6 older healthy individuals were involved in study to identify CreaT content and distribution, Creatine kinase (CK) and high-energy phosphagens. No significantly difference of CreaT content was observed between young and older individuals but with a declined concentration of phosphocreatine (PCr) (P<0.05) in older muscles. CreaT protein localisation was determined to predominantly distribute at the cell peripheral in both young and old individuals (P<0.05) but with fibre type specific difference in old individuals. These results demonstrated that Cr metabolism did alter by ageing, however, CreaT content remains relatively unaffected. Chapter 4 of this thesis investigated the effect of immobilization on CreaT protein expression and distribution in human skeletal muscle. The 7-days leg immobilization trial was undertaken in which 15 healthy young individuals. The results elucidated that 7 days immobilization lead to a declined muscle isometric strength and fat free mass (P<0.01), in addition with a reduced type II muscle fibre cross-section area (CSA) (P<0.05). CreaT protein content was greater in immobilised leg compared control leg (P<0.01). CreaT was mainly distribute at the cell peripheral in both immobilised and control legs with fibre type specific difference (P<0.01). These outcomes determined that the greater muscle fibre CreaT expression with immobilization may have important implications for skeletal muscle energy production during disuse-induced atrophy and in the recovery of muscle mass upon re-loading. In conclusion, this thesis provides a novel immunofluorescence approach to investigate CreaT activities in human skeletal muscle. This developed method provides valuable insight into the role of CreaT in alteration in intramuscular Cr metabolism across the spectrum of health and disease. In addition, this thesis enhances our understanding of CreaT protein expression and sub-cellular localisation in the role of Cr in skeletal muscle physiology.

Protein function is inherently linked to amino acid sequence; however, this vocabulary is innately limited. One natural method to supplement protein chemistry is the application of PTM. This modification has significant implications for both the structure and function of protein and can now be emulated with non-natural chemistry. This thesis examines the influence PTM has, on protein stability (Chapter 3), function (Chapter 4), structure (Chapter 5), and how nnPTM can be applied to induce functional communication (Chapters 6 and 7). This thesis first explores the expression of HRP in E. coli (Chapter 3), wherein it is demonstrated that soluble HRP expression, is achieved by the inclusion of the full-length proto-HRP sequence. These proto-HRP regions are then shown to cleave, and additionally, it is also shown that the removal of unoccupied N-linked glycosylation sites from the surface of HRP decreases the rate of precipitation observed in recombinant HRP. Next, recombinant apo-HRP is investigated as a corrective agent in a commercial immunoassay (Chapter 4), to establish if the corrective function of glycosylated apo-HRP can be achieved without glycosylation. Evidence generated indicates that glycosylation of apo-HRP is essential for the removal of false-positive rogue signalling. Subsequently, Chapter 5 explores the influence of PTM on HRP's structural rigidity. Using fluorescence emission to assess the red edge excitation shift of W117, it is observed that both haem binding and glycosylation increase protein structural rigidity, and this is confirmed by CD spectra. The latter two chapters of this thesis explore the potential of chromophore communication which can be induced by nnPTM. Together, both in silico interface modelling, and nnAA incorporation, is utilised for the formation of two fluorescent dimers. Firstly, this system was applied to the structurally similar combination of sfGFP and mCherry (Chapter 6). In which the enhanced chromophore proximity resulted in a significant decrease in function of mCherry, contrary to expectation. In Chapter 7 sfGFP and cytochrome b562 were dimerised to generate dimeric protein which could transfer energy by way of FRET (Chapter 7). Lastly, both nnPTM and natural PTM were combined in the formation of trimeric protein which was linked by SPAAC and disulphide linkage.

The particulate nature of nanoparticles (NPs) dictates a preferential interaction with cells of the immune system assigned to recognition and elimination of foreign particulates. Probing safety of nano-objects by defining immune responses of environmental organisms is therefore key to environmental nanosafety. Earthworms represent major immunosafety models representing keystone ecosystem engineers and being in intimate contact with soils ensuring exposure to terrestrial NPs. Innate immunity represents the first line of defence against pathogens in invertebrates and despite extensive description of cell populations involved a dearth of information about the associated molecular components exists. This thesis aimed to use genomics approaches to generate a comprehensive systems immunity description of earthworm prior to exploiting transcriptomics to explore the interaction of NPs and earthworms. A tissue-specific transcriptomic atlas has been established for Eisenia fetida and Eisenia andrei representing six tissues from each species. The resultant comparative transcriptomic resource represented an innate immunity database containing immune-related genes from major immune signalling pathways. To refine the tissue-specific database we generated a de novo genome for E. fetida with high contiguity and completeness. Finally, to enhance insight into the different immune functions of the individual types of coelomocytes we generated transcriptomic datasets from eleocytes, hyaline and granular amoebocytes. The interaction between NPs and the earthworm immune system was then explored by combining the direct introduction of copper oxide NPs with bacterial challenge and following the transcript changes within individual coelomocytes cell populations. This resolved the spatial-temporal impact on the immune system into three distinct phases: direct, systemic and differentiation responses. A complementary soil-based exposure using a range of CuNPs, Silicon-CuNPs and copper ion doses explored the comparative response after soil-based biotransformation. This revealed Si-CuNPs to elicit a negligible response whilst differentially regulated genes under high CuNPs were distinct from equivalent copper ion exposure the pathways impacted intersected substantially.

Lymphoid Tissue inducer (LTi) cells are part of the Innate Lymphoid Cell (ILC) family and have been implicated in the development of thymic microenvironments and support their recovery following damage. However, a detailed characterisation of other ILC subsets within the thymus is lacking. This study aimed to characterise the ILC composition of the thymus across ontogeny and investigate their function. Inhibitor of DNA-binding 2 (Id2) is a transcriptional regulator required for the development of ILC subsets. Several in vivo models, including Id2 fate mapping and Id2 reporter mice, were used to dissect ILC populations within the thymus. LTi cells, a subset of ILC3, were prominent in the embryo, but numbers reduced in the neonate and continued to decline in the adult, where ILC2 became the dominant population. ILC2 were capable of producing IL-5 and IL-13 ex vivo, and through confocal imaging we discovered ILC2 within the thymic medulla. Surprisingly, the majority of Id2 expressing cells in the thymus were not ILC but developing thymocytes, which are present in much greater numbers compared to the number of ILC2. Collectively, we have revealed changes in the ILC compartment of the thymus across ontogeny and started to characterise Id2 expression amongst other lymphocytes.

Lateral roots (LR) branch out from the primary root in order to provide essential stability and allow water and nutrient uptake. The threat of climate change and an increasing global population represent crucial challenges to plant biodiversity and food security, and the manipulation of plant root system architecture could offer the potential to produce new and more resilient crop varieties. The transcription factor AtMYB93 is known to inhibit the initiation of new LR primordia in the model organism Arabidopsis, preventing resource expenditure on unnecessary root growth. It is hypothesised that the transcription factor AtMYB93 manipulates the suberin barrier layer in the endodermis, however the molecular mechanism through which AtMYB93 acts is poorly characterised. Here we begin to answer questions about AtMYB93, its regulatory network, and the phenotypic responses of AtMYB93 and its S24 clade members - AtMYB92 and AtMYB53 - under stress, and have attempted to apply these findings on MYB93 homologues to genetic manipulation of crop root systems.

With the emergent role of protein glycosylation as a potential biomarker of disease, new technologies are required to selectively recognise unique glycan structures symptomatically expressed in disease states. Currently, glycan recognition is achieved through methods lacking high affinity and selectivity or through complex analytical methods and are poorly suited to clinical laboratories. In this thesis we propose an alternative glycan sensing strategy based on molecular self-assembly and imprinting of glycan ligands with phenyl boronic acid (PBA), imbuing selective oligosaccharide recognition to a surface. This strategy employs extensive use of the copper catalysed click reaction providing a simple highly biocompatible and adaptable self-assembled monolayer intended to form a foundation for future modifications. The synthesis and characterisation of various SAM monomers has been described here alongside azide functionalised-PBA CuAAC ligands and PBA terminated SAM monomers. SAMs formed with these were characterised for surface organisation and the CuAAC reaction yield established thus confirming reaction utility in functionalisation of alkyne terminated SAMs. Saccharide binding of PBA functional SAMs was then examined accounting for surface organisation between SAM monomer designs. Having determined a suitable SAM for functionalisation, we then produced a novel oligosaccharide-PBA imprinted surface demonstrating high selectivity between structurally different oligosaccharides.

An investigation of cognitive task engagement and subsequent muscular endurance performance and brain endurance training (BET) - increased cognitive demand during physical training. Chapter two utilised a triple task (cognitive-physical-cognitive) to investigate the dose-response of a response inhibition task on submaximal handgrip. Handgrip was impaired following cognitive tasks of 10 minutes but not after 5 and 20 minutes. Learning effects occurred during the 20 minute cognitive task. A prior response inhibition task improved performance in a novel response inhibition task. These results can be explained by cognitive control theory. Chapter three demonstrated that engagement in a mentally demanding cognitive task, without response inhibition, for a period of 20 minutes did not impair submaximal isometric handgrip exercise. Chapter four investigated temporal effect of mental fatigue with 4 blocks of 10 minutes cognitive task followed by 5 minutes of rhythmic handgrip. Over time indices of mental fatigue diverge, with self-report measures accentuating and physiological measures attenuating. After 20 minutes this state of mental fatigue impaired physical performance. Chapter five demonstrated that 6 weeks BET alongside submaximal handgrip training, increased endurance performance by 32%, which occurred with a higher prefrontal cortex oxygenation, relative to 12% improvements in control.

This thesis covers studies on nano-photonic bandgap structures and devices inspired from nature, with the potentials to be used as filters and smart sensors such as hydrogel based photonic bandgap sensors for detection in various industries including medical applications, food and cosmetics. Research was conducted on two samples of feathers that exist in nature namely Nicobar pigeon and Monal pheasant bird species in order to study the structural colouration originating from the internal barbule nanostructures. The structural colouration of these species have not been studied or reported anywhere else before. Optical microscopy and spectroscopy measurements were performed showing a variety of colours observed with change in illumination and viewing angle. The variation in colour observed is reflected by red, green and blue photonic bandgap structures present in the internal structure of the feathers. Hydrophilic and Hydrophobic properties of the optical material were also investigated. Contact angle measurements were performed resulting in ~ 156° in the case of Nicobar pigeons demonstrating it to be super hydrophobic whereas a contact angle of ~ 120° was recorded in case of Monal pheasant. Feathers were also immersed in different liquids and the spectral shifts were recorded and analysed which were correlated with the refractive indices of these liquids. The structural colouration in Nicobar pigeon and Monal pheasants can be mimicked for applications such as colour filters, non-reflecting coating, bio sensors, photonic bandgap hydrogel sensors and sensors based on refractive indices used in food, medical, health monitoring and cosmetic industries. To support the theory, a simple silica thin film grating structure was designed using silicon and silicon dioxide having different refractive indices. The structure was simulated with variation of various parameters in order to get the best colour change. Two samples were successfully fabricated for testing and confirmation purposes. Both concepts of thin film and diffraction grating were in combination and the mechanism was studied in detail. Titanium nanostructures were also fabricated with varied dimensions in size using laser interference ablation method in order to study the interaction of a liquid droplet with nanostructures. The change in hydrophobicity with respect to change in nanostructure size was captured, analysed and studied in detail. A surface profile with large spacing and size in nanostructures prevents the absorption on the surface with availability of wider channels for the liquid's excessive flow allowing very low contact angles whereas very narrow and smaller size of nanostructures allow no wettability with ability of nanostructures to hold the liquid drop resulting higher contact angles. The main focus was to explore more on photonic bandgap structures that exist in nature and have not been investigated and suggest their possible use in applications such as integration in hydrogel based photonic bandgap sensors to enhance fast and facile fabrication with simple detection methods using change in physiological conditions of the photonic bandgap structure integrated in the hydrogel.

Asteroseismology - the study of stellar oscillations - is one of the key tools used to study stars in modern astronomy. This thesis focuses on asteroseismology of solar-like oscillators, which exhibit variability driven by the same mechanism as the Sun. By studying variations in the brightness of stars on their surface, asteroseismology probes the full stellar interior, giving insight into stars' internal processes and fundamental properties. In this thesis, I provide an introduction to asteroseismology and the state of the field. This is followed by a description of asteroseismic analysis tools I developed for the open source Python package 'Lightkurve', and a presentation of two studies that use asteroseismology to probe different aspects of astrophysics. The first study uses an asteroseismic ensemble of 5576 evolved Red Clump stars to calibrate data from the Gaia mission, and quantify systematic differences in asteroseismic modelling techniques. The second focuses on 91 main sequence stars like the Sun, using asteroseismology to measure their rotation rates. Along with asteroseismic ages, these new rotation rates are used to quantitatively state that stars experience a change in how they lose angular momentum half-way through their main sequence lifetimes. A common thread throughout both these studies is the use of Bayesian statistics, which allows us to leverage large asteroseismic ensembles to make inferences about adjacent fields of astronomy.

This thesis examines the use and value of fuzzy sets, fuzzy logic and fuzzy inference in wireless positioning systems and solutions. Various fuzzy-related techniques and methodologies are reviewed and investigated, including a comprehensive review of fuzzy-based positioning and localisation systems. The thesis is aimed at the development of a novel positioning technique which enhances well-known multi-nearest-neighbour (kNN) and fingerprinting algorithms with received signal strength (RSS) measurements. A fuzzy inference system is put forward for the generation of weightings for selected nearest-neighbours and the elimination of outliers. In this study, Monte Carlo simulations of a proposed multivariable fuzzy localisation (MVFL) system showed a significant improvement in the root mean square error (RMSE) in position estimation, compared with well-known localisation algorithms. The simulation outcomes were confirmed empirically in laboratory tests under various scenarios. The proposed technique uses available indoor wireless local area network (WLAN) infrastructure and requires no additional hardware or modification to the network, nor any active user participation. The thesis aims to benefit practitioners and academic researchers of system positioning.

Platelets are crucial players in many (patho)physiological processes including haemostasis, thrombosis and inflammation. The immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor glycoprotein VI (GPVI) is the major platelet signalling receptor for collagen, which is exposed upon vascular injury. Once bound to collagen, multiple GPVI dimers cluster at the platelet surface increasing the avidity for collagen and regulating the signalling. In suspension, GPVI is cleaved by the metalloproteinases ADAM10/17 upon collagen activation. This thesis aimed to elucidate the signalling dynamics underlying GPVI clustering in platelets spread on collagen. Super-resolution microscopy and two-layer cluster analysis revealed that immobilised collagen induces long-term GPVI clustering and signalling in tandem with minimal shedding and low levels of GPVI/ADAM10 colocalisation. Conversely, GPVI is massively shed and the signalling rapidly downregulated in platelet suspensions stimulated with collagen. Syk, but not Src, kinase-dependent signalling is required to maintain clustering of the collagen integrin receptor α2β1, whilst neither is required for GPVI. This thesis proposes that GPVI clustering on immobilised collagen protects GPVI from shedding, thereby enabling longer-lasting Src and Syk-dependent signalling, integrin α2β1 activation and stable adhesion to collagen. Nanobodies targeting human GPVI have potential uses in many different biological applications from anti-platelet approaches to super-resolution imaging.

Bacterial cellulose (BC) was biosynthesised by Gluconacetobacter xylinus. The aim of this work was to study the effect of surface treatment with 5, 7 and 9 wt.% NaOH on the properties and structure of BC. The effect of BC (2, 4, 6 8 and 10 wt.%) as a filler on the mechanical properties and biocompatibility of reinforced polycaprolactone (PCL) composites processed by electrospinning and twin extrusion followed by injection moulding was also studied. FTIR showed an increase in crystallinity and cellulose II/I ratio with increasing NaOH concentration and treatment time. Thermogravimetric analysis (TGA) showed improved thermal stability of BC with increasing NaOH solution concentration and time. There was a significant difference between the viability of bone marrow cells on days 6 and 10 for the samples treated with higher NaOH concentration, leading to the conclusion that mercerisation did have a significant effect on the biocompatibility, studied by MTT assay. Cryogenic SEM studies showed that the morphology of BC changed with NaOH treatments from a dense fibre network to randomly distributed fibres with increased porosity. PCL-BC composites did not show significant improvement in the thermal stability studied by DSC with increasing BC content. An increase in the melting temperature of PCL-BC composites with increasing BC content was observed. A decrease in the ultimate tensile strength was observed with increasing BC content, whereas the yield strength did not show much change. The Young's modulus increased significantly with increasing BC content. The electrospun PCL-BC composites showed a decrease in the degree of crystallinity studied by DSC as the BC content increased. Cell studies showed successful cell proliferation and viability.

Aqueous foams are dispersions of gas bubbles in a liquid continuous phase and they are important for various applications from biological systems to Enhanced Oil Recovery. Foam stability relies on the use of surfactants/nanoparticles and on the rheological properties of the foaming liquid. One of nowadays challenges is to solve key foam industrial issues while considering their environmental impact. This duality is addressed by employing microfluidics to study static and dynamic foam stability. In this thesis the foam stability is first studied in static conditions by investigating the use of highly stable bubbles termed bulk nanobubbles as potential surface-active agent on non-ionic and anionic surfactant-stabilised foams. This work has shown that the presence of nanobubbles in solution impacted significantly the foam stability and foamability. These findings suggest that nanobubbles attracted surfactant molecules at their interface and that nanobubbles adsorbed at the foam gas-liquid interface. At low surfactant concentration, nanobubbles attraction removed surfactants molecules from the gas-liquid interface. This effect increased destabilisation of non- ionic surfactant foam. Oppositely, for anionic surfactant foams, the presence of nanobubbles enhanced foam stability. Indeed, nanobubbles enhanced the disjoining pressure within the thin liquid film due to an increase in the electrostatic repulsive forces between the two contiguous interfaces forming the film. Subsequently, the rheological properties of the foaming liquid are considered. In particular, the influence on the "dynamic" foam stability or the property of the foam to resist and/or recover after the deformation was evaluated in a microchannel. This work proposed a microfluidics approach to determine qualitatively the foam hysteretic behaviour after an induced deformation. This study has found that the viscosity and the bulk elasticity reduced the foam recovery. The result of this investigation showed that two mechanisms led to foam structural hysteresis after deformation: a retardation effect linked to the increase in viscosity and a tension thickening effect arising from the bulk elasticity.

BET proteins function as epigenetic readers, and BET inhibition has been shown to have activity against many different cancers. BET inhibitors have been fast-tracked into first clinical trials, however the effects of these inhibitors are still poorly understood. A more detailed understanding about the physiological effects of BET inhibition is important and may lead to improved clinical applications as well as allowing safer use of BET inhibitor drugs. In this thesis we have been able to identify that BET inhibition unexpectedly leads to an increase in RNA synthesis that is associated with conflicts between transcription and replication, leading to replication fork slowing, a sign of replication stress. We have identified BRD4 as the main target of BET inhibition in this process which is needed for normal fork progression. Interestingly, our data suggest that increased RNA synthesis requires free P-TEFb to be released from its inhibitor complex HEXIM1 to allow increased RNA polymerase II phosphorylation. HEXIM1 is required for BET inhibitor-induced replication-transcription conflicts. We have shown that BET inhibitor-induced fork slowing does not activate the canonical ATR-Chk1 replication stress response pathway. However, it activates the homologous recombination factor RAD51, which is recruited into nuclear foci in response to BET inhibitor treatment. RAD51 depletion followed by BET inhibition prevents replication fork slowing but activates the replication stress response. HEXIM1 depletion has the same effect, preventing fork slowing and activating the replication stress response. These data suggest that i) replication fork slowing is required to prevent DNA damage formation in presence of BET inhibitors and ii) this depends on HEXIM1, which is upstream of transcription-replication conflicts, and RAD51, which acts downstream of transcription-replication conflicts. Our data shed light on the initial stress response during the first 8 hours of BET inhibition. They implicate HEXIM1 and RAD51, which both play potential roles in BET inhibitor resistance, in the BET inhibitor-induced replication stress pathway.

The automotive industry is undergoing a major transformation process in which nearly every part of the vehicle is becoming digital and connected. Modern vehicles are often connected to the internet, feature several wireless interfaces and will soon communicate directly with surrounding vehicles and roadside infrastructure using V2X technology. However, this transformation has not yet been paralleled by the development of techniques or standards which address the cyber security challenges posed by these systems. The automotive industry has historically failed to use secure cryptography or appropriate key management techniques and there is no sign that things have improved. In this thesis, we present several new cryptographic and key management flaws in an existing automotive immobiliser system and we develop two new V2X architectures for improving the safety and privacy of tomorrow's connected and autonomous vehicles. Specifically, we study the AUT64 automotive block cipher and its associated authentication protocol in a real-world immobiliser system. Despite having a 120~bit key, we find a number of flaws in the system which we combine to present several practical key-recovery attacks. Our first new V2X architecture, IFAL, provides a practical and secure improvement to the leading European standard for V2X. IFAL introduces a new certificate issuance mechanism that eliminates the trade-off between pseudonym duration and bandwidth. Our second architecture, VDAA, addresses the need for efficient techniques that preserve vehicle privacy despite dishonest or colluding certificate authorities.