Pathological Aggregation of Cystatin C and α-Synuclein in Neurodegenerative Disease

Neurodegenerative diseases are chronic conditions that progressive dysfunction in cognition, muscle control, and speech, driven by neuronal death and malfunction due to protein misfolding and aggregation. Current treatments are limited, and understanding the aggregation pathways involved is key to developing more effective therapies. This thesis investigates two such aggregation systems with strong associations to disease. The first part focuses on cystatin C aggregation in Bunina bodies, observed in amyotrophic lateral sclerosis (ALS). Extensive attempts at optimising expression and purification of recombinant cystatin C from E. coli are shown, followed by in vitro aggregation assays. Cystatin C aggregation is induced in conditions similar to cytosol, where Bunina bodies form, by disrupting two structurally critical disulfide bonds. Kinetic analysis suggests a proline-isomerisation step during the aggregation process, as seen in other cystatins. Additionally, preliminary nuclear magnetic resonance (NMR) data hints at a potentially unreported binding interaction between cystatin C dimers and RNA. In vivo overexpression of fluorescently-tagged cystatin C in mammalian cells produces Bunina body-like inclusions, which appear to be actively transported toward the nucleus via the microtubule network. The second part explores α-synuclein aggregation on POPG lipid membranes, modelling Lewy body formation in Parkinson’s disease. This system has previously informed Parkinson’s drug development, including an ongoing phase II clinical trial, which heavily supports its disease relevance. This study shows that fully helical α-synuclein remodels POPG membranes into long cylindrical micelles, which form an inter-connected network, spaced 10-20 nm apart, consistent with a double-anchoring mechanism for α-synuclein reported in the literature. After prolonged incubation, a rapid structural transition from α-helix to β-sheet occurs, supporting a hypothesis of amyloid structural propagation along the micelle surface, providing a mechanism for accelerated aggregation and lipid sequestration by α-synuclein amyloids in Parkinson’s disease

Investigating the Mechanobiology of Macrophages: Implications for Inflammatory Bowel Disease

Macrophages are essential cells of the innate immune system, playing a key role in regulating inflammation, tissue repair, and homeostasis. Their behaviour is tightly controlled by various signalling pathways, including mechanical forces that influence their shape, movement, and function. This process, known as mechanotransduction, allows cells to sense and respond to mechanical signals from their environment, converting these signals into biochemical responses that regulate cellular behaviour. Dysregulation of macrophage functions can lead to chronic inflammatory diseases and cancer. Recent studies have shown that mechanical cues, such as extracellular matrix (ECM) stiffness, fluid flow, cell crowding, and topography, modulate macrophage behaviour in various physiological and pathological contexts. However, the effect of ECM stiffness at relevant physiological levels, particularly in inflammation and fibrosis, has not been fully understood. Previous studies have often relied on single or limited marker approaches, which may not capture the full complexity of macrophage polarization. To address this gap, we conducted a series of experiments aimed at characterizing THP-1 and bone marrow-derived macrophage (BMDM) protocols to ensure proper validation and reproducibility for our study. We then adapted ECM stiffness values, mimicking the conditions seen in inflammatory bowel disease (IBD), representing both normal and inflamed-fibrotic tissue. Experiments were conducted to assess macrophage polarization states in response to varying stiffness levels. Our results reveal that increasing ECM stiffness promotes the expression of YAP and IL-6 in M1 macrophages, driving a shift towards a pro-inflammatory phenotype. In contrast, M2 macrophages exhibited elevated levels of the anti-inflammatory markers CD163 and IL-10, reflecting an adaptive response to softer ECM conditions. Interestingly, M0 macrophages, which are considered to be non-polarized, adopted a hybrid phenotype, expressing both YAP and CD163, underscoring the inherent plasticity of macrophages when subjected to mechanical stress. In primary BMDMs, stiff ECM conditions induced also mixed phenotypes with favoured M1 polarization, as shown by a significant overlap with established M1 gene expression signatures, further emphasizing the role of ECM stiffness in driving pro-inflammatory responses. These findings challenge the traditional binary M1/M2 polarization model, suggesting that macrophage responses to mechanical cues are nuanced and context dependent. In the second part of this thesis, we investigated the mechanical regulation of the Poly(C)-binding protein 1 (PCBP1) in macrophages and its role in macrophage polarisation. PCBP1 is a multifunctional RNA-binding protein that plays a crucial role in regulating mRNA stability, splicing, and translation. It is also involved in iron metabolism, acting as an iron chaperone, and is involved in DNA damage repair. Our experiments demonstrate that ECM stiffness and cell density regulate PCBP1 subcellular localization in macrophages. In stiff ECM and low-density environments, PCBP1 localises mainly to the nucleus, while in soft ECM and high cell density, it remained cytoplasmic. PCBP1 knockdown increased CD163 expression, suggesting it modulates M2 polarization. Finally, we demonstrate a possible role of PCBP1 in ECM stiffness dependent DNA damage repair, suggesting a novel mechanism of mechanoprotection

Corruption, Deprivation and Economic Development in sub-Saharan Africa

This thesis broadly examines the impact of lived corruption experiences on healthcare deprivation and tax evasion in sub-Saharan Africa (SSA). While Chapter 1 introduces the thesis, Chapter 2 examines how corruption causes healthcare deprivation in 29 SSA countries. Employing the fifth, sixth and seventh waves of the Afrobarometer survey spanning 2011-2018, I find that corruption in the form of bribe payments within the healthcare sector increases healthcare deprivation. Additionally, corruption experienced in sectors outside health such as education, police, public utilities and identification authorities, have adverse spillovers on healthcare deprivation. Furthermore, I show that corruption impacts healthcare deprivation through two key channels: Loss of income and loss of trust in public institutions. Chapter 3 utilises individual-level datasets to explore the effect of people’s lived corruption experiences on their propensity to evade taxes. I show that the likelihood of tax evasion rises by 19–39.5 percentage points for individuals who have paid bribes to government officials in various sectors–health, education, police, public utilities and identification authorities, compared to their counterparts who have never been extorted. Chapter 4 documents the spillover effects of firm-level corruption (unrelated to taxation) on tax evasion decisions, with evidence from 17 SSA economies. Chapter 4 also examines (a) whether and how corruption in tax agencies impacts tax evasion and (b) the key mediating mechanisms through which corruption impact tax evasion among firms. In contrast to the extant literature, I demonstrate that while corruption involving tax authorities rises tax evasion, corruption outside tax authorities (i.e., bribes paid to obtain operating licenses and secure government contracts) has adverse spillovers on tax evasion. The thesis concludes in Chapter 5 outlining key policy implications

Trans and non-binary people’s experiences of accessing and attending cervical screening in the North of England

Transgender people face discrimination and inequality in various areas of life, including within healthcare. A key health disparity is the limited uptake of cervical screening compared to cisgender women. A scoping review highlighted the paucity of research in this area. Therefore, the aim of this thesis was to undertake an in-depth, qualitative study to explore transgender men and non-binary peoples (trans+) experiences of cervical screening to develop recommendations to improve experiences of this service. 15 trans+ people shared their experiences of cervical screening in a semi-structured interview (10), or an online, asynchronous focus group (5) conducted between November 2022-March 2023. Participants were recruited through purposive and snowball sampling using various social media and LGBTQ+ newsletters. Inclusion criteria were being trans+, aged 25 or over, having or previously having a cervix, and living in the North of England. Participants were aged between 25-45, and the majority (13/15) had a disability, and were mostly (13/15) white British. Data were analysed using reflexive thematic analysis, through which four themes were developed that explore various barriers and facilitators to attending cervical screening. For example, in cases where health professionals were not sensitive and respectful, trans+ participants reported more negative experiences, such as heightened physical pain and gender dysphoria, compared with then health professionals were knowledgeable and reassuring. Further, trans+ people shared complexities of deciding whether to attend cervical screening. These decisions were shaped by previous experiences of health services, concerns about heightened gender dysphoria or pain, and the potential emotional impacts of cervical screening. The findings had multiple implications for practice, policy and future research to improve cervical screening for trans+ people. For example, trans+ people value being given choice in cervical screening, and shared what they would like health professionals to know about their identities and health needs

Understanding contemporary political division in Western Europe: a multidimensional approach

Recent years have seen an entrenchment of partisan and ideological division across Western European democracies, with pernicious consequences. As cultural conflicts over questions of immigration divide electorates, urgent political challenges like climate change go unresolved in the face of political disagreement, and inter-group hostility undermines efforts to achieve democratic consensus, we appear to be entering a new age of political conflict with worrying implications for democracy. Yet, whilst these processes of polarization have been the subject of a prolific stream of research in recent years, there remains scope for additional contributions. Research on social division and political polarization often approaches the phenomenon from a macro-level perspective that emphasises the impact of socio-economic transformation, or from a micro-level perspective that emphasises the importance of individual-level psychological mechanisms in driving polarization. This thesis instead adopts a multidimensional approach to explain political polarization and division, examining how context – encompassing both immediate factors in the individual’s local environment and broader characteristics – influences and interacts with these individual-level processes. This approach, synthesizing micro- and macro-level explanations, offers new avenues to better understand an increasingly prescient threat to democratic health. Using data from Britain and Norway, this thesis focuses on three different sources of partisan and attitudinal division: first, authoritarianism and conflicts over cultural issues; second, climate change and responses to exogenous shocks an; third, the emotion of anger. Moving beyond the individual dimension, however, each study examines how these micro-level processes are influenced by contextual factors, encompassing the political information environment, partisan-ideological sorting and political discussion networks. Taken together, findings demonstrate the importance of context in shaping processes of polarization. In doing so, they highlight the characteristics of contemporary Western European democracies that are exacerbating these attitudinal divides - including increasingly sorted and geographically polarized electorates - and point to potential avenues for mitigating polarization

Fundamentals of self-acceleration and morphological evolution of premixed hydrogen flames

Global warming, primarily driven by CO2 emissions from fossil fuel combustion, necessitates a shift to sustainable energy sources. Hydrogen, being carbon-free and renewable, is essential for achieving net-zero emissions and addressing the global energy crisis. The combustion of hydrogen, particularly in a lean premixed condition, offers significant benefits: controlling flame speeds, reducing exhaust gas temperatures, and lowering nitrogen oxide (NOx) emissions. However, these flames are susceptible to thermodiffusive and hydrodynamic instabilities, which may induce self-acceleration and significantly impact the turbulent burning velocity across various combustion systems, thereby elevating fire and explosion risks. Identifying the regimes of cellular instability and self-acceleration could enhance combustion modelling, a critical tool in the design of combustion systems and in assessing fire and explosion hazards. To address these challenges, a full investigation and understanding on self-acceleration characteristics of hydrogen-air flames under various conditions was conducted, with a particular focus on flame speed and flame surface area. This research employed advanced experimental techniques, including Schlieren imaging, Particle Image Velocimetry (PIV), a 3D swinging laser sheet system, and Direct Numerical Simulation (DNS) supported by the Advanced Flow Simulator for Turbulence Research (ASTR). Using Schlieren imaging system, the onset of instability was identified by the critical stretch rate, where the flame speed deviates rapidly from its previous response to stretch. Notably, the critical Peclet number (Pecl) increased with higher equivalence ratios and temperatures, indicating a more stable flame. Conversely, Pecl decreased with increased initial pressure due to the associated decrease in the flame speed Markstein number (Mab). Correlations of Pecl and Kacl were developed as a function of Mab with increasing pressure, facilitating the estimation of the severity of large-scale atmospheric hydrogen flames. Comprehensive quantitative data on the self-acceleration of unstable laminar hydrogen-air flames was obtained, revealing self-similarity after instability onset. The previously assumed acceleration exponent α = 1.5 was found to be invalid, with derived α values ranging from 1.125 to 1.39. Higher acceleration exponents were observed in the lean condition, while lower exponents were found in the rich condition. A modified theoretical expression for the constant (A) was proposed and validated against experimentally derived results, highlighting a global pulsating acceleration pattern during the acceleration phase after flame instability. Particle Image Velocimetry (PIV) was utilized to explore the disturbance of unstable laminar hydrogen-air outwardly propagating spherical flames. It was found that self-acceleration of gas velocities ahead of the front, and shared the same acceleration exponents as the flame front. The power spectral density (PSD) displayed by the flow ahead of the flame front exhibited similarity to flame front fluctuations, attributed to wrinkled flame front-driven gas disturbance. Higher local gas velocities were observed just ahead of the tips of the cellular structures, compared to other regions along the flame front, particularly for the extra lean conditions. 3D laser sheet measurements were employed to quantify the flame surface area of hydrogen flames. For planar flames, the parameters ϵ, representing the deviation of the Lewis number from a critical value, and ω2, derived from classical linear stability analysis to represent thermal-diffusive effects, both exhibit a distinct linear correlation with the enhancement in flame surface area observed in planar flames. This suggests that the 3D swinging laser sheet system is an effective method for investigating flame surface area. For spherical flames, the stretch factor I_0 (the ratio of the increase in flame burning velocity to the enhancement in flame surface area) exceeds 1 when the equivalence ratio (ϕ) is 0.3 (lean condition), particularly under high-pressure conditions. The morphological characteristics and acceleration behaviour of cellular flames were investigated using Direct Numerical Simulation (DNS) with simplified chemical kinetics. The DNS results indicate that the self-acceleration capacity of thermodiffusively unstable flames (ϕ = 0.4) is significantly higher compared to thermodiffusively stable cases (ϕ = 0.6, 0.8). The stretch factor I_0 for thermodiffusively unstable flames exceeds unity, whereas for thermodiffusively stable flames, it remains approximately equal to one. This suggests that, in thermodiffusively stable flames, the primary contribution to acceleration arises from increased flame surface area. In contrast, thermodiffusively unstable flames exhibit additional mechanisms contributing to self-acceleration. Further analysis of local normal strain rates revealed elevated values at the tips of the finger-like protrusions within the cellular flame structures, indicative of rapid expansion at these points. The significant variations in normal strain rates in thermodiffusively unstable flames are attributed to enhanced flame instabilities or localised effects. Species distribution analysis showed that active radicals, such as OH, O, and H, are highly concentrated at these protrusion tips, referred to as ‘leading points’. In thermodiffusively unstable flames, the high mass diffusivity of hydrogen results in the formation of more ‘leading points’, altering the dynamics of flame expansion. This leads to elevated local normal strain rates and increased concentrations of active species like hydroxide radical (OH) , oxygen radical (O) , hydrogen radical (H) at these locations. Consumption rates of hydrogen and production rates of water are strongly influenced by curvature, with positive curvature regions enhancing localised combustion due to hydrogen's high diffusivity under lean conditions

Advancing Space Weather Prediction: Machine Learning and Bayesian Modelling for CMEs and Coronal Jets

This thesis investigates the use of Machine Learning (ML) and Bayesian inference to improve the prediction and understanding of Coronal Mass Ejection (CME), a critical aspect of space weather forecasting. Several ML techniques, including supervised learning methods such as support vector machines, decision trees, and ensemble methods, are used to develop predictive models based on CME data, aiming to enhance the accuracy of CME arrival time forecasts. A key focus is placed on model interpretability, achieved through Shapley Additive exPlanation (SHAP) values, which provide insights into the feature space and allow for a better understanding of how different variables influence model outputs. Additionally, the thesis applies Bayesian inference and Monte Carlo Markov Chain (MCMC) techniques to refine probabilistic models of CME propagation using drag-based models, further improving the robustness and reliability of the predictions. The work also extends ML applications to the study of other solar phenomena, specifically coronal jets, by augmenting the dataset for jet identification. This leads to increased dataset diversity, improved detection of rare events, and a better understanding of solar dynamics. Overall, this thesis presents advancements in the application of ML and Bayesian techniques to space weather forecasting and the study of solar phenomena. The tools and methods developed in this research hold considerable potential for future applications, with the capacity to improve prediction accuracy and mitigate the impacts of space weather on technological systems