Theses - Clinical Neurosciences

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Exploring Endoplasmic Reticulum as a Therapeutic Target Site to Antagonize Amyloid Production.
Gupta, Karnika
The excessive release of aggregation prone, toxic Amyloid-β (Aβ) from the Amyloid Precursor Protein (APP) by secretases has been genetically and biochemically established as central to Alzheimer’s Disease (AD) pathogenesis. Therapeutic strategies targeting extracellular deposits of Aβ, or inhibiting enzymes releasing Aβ from APP, have not been as successful as first envisioned and motivate alternative approaches, such as reducing the production of Aβ by modifying APP localisation. This requires a better understanding of the APP intracellular processing events which liberate Aβ peptide. In my thesis, I mapped the kinetics of APP cleavage to produce Aβ with subcellular resolution. To visualize APP’s processing with organellar resolution, I developed a dual-tagged APP reporter system combined with Retention Using Selective Hook (RUSH), which enabled the controlled localisation of the protein in the organelles of the secretory pathway whilst monitoring its cleavage status. Using pulse-chase long-term fluorescence imaging and analysing the label-signal decay patterns, I quantified APP cleavage within each of these compartments. The measurements demonstrate that APP undergoes cleavage in endosomes and Golgi, and to a lesser extent in the Endoplasmic Reticulum (ER) or plasma membrane. Biochemical analysis was performed using Single-Molecule Pulldown and Single Molecule Array to detect monomeric and aggregated Aβ, which demonstrated that retaining APP in the ER reduced amyloid production. Based on these insights, I explored a therapeutic strategy focused on redirecting APP to the ER, to reduce amyloidogenic processing. I developed an intrabody-based system to control localisation of endogenous (untagged) APP in the ER. This strategy proved effective in an immortalised cell line, human cortical neurons, and in vivo. A rationally designed ER-targeted intrabody that targets the APP luminal domain caused ER enrichment of endogenous APP in the mouse brain, leading to a substantial decrease in Aβ plaque formation and associated neuropathological features. The manipulation appeared inert in terms of viability and ER stress in cells and in vivo. These findings suggest that modulating APP early secretory pathway localisation leverages the organelle’s high proteostasis capacity and presents a promising therapeutic avenue that avoids the adverse effects associated with direct secretase inhibition or complete loss of APP expression.
Open Access
Specialist Healthcare Services for Concussion/Mild Traumatic Brain Injury
Karvandi, Elika
Mild traumatic brain injuries (TBIs) affect over a million people in England and Wales annually. While most individuals recover spontaneously within days to weeks, a significant proportion experience persistent symptoms that last months to years after injury. These symptoms are complex and heterogenous, affecting daily functioning, return to work or play, and quality of life. Patients with persistent symptoms have extended healthcare needs requiring ongoing care in outpatient services. However, guidance on mild TBI care in the outpatient setting is limited, and the standard of care in England remains unclear. This thesis aims to address how to plan care provision and service delivery for individuals with persistent symptoms after a mild TBI, focusing on their healthcare needs. This thesis presents exploratory research using a mixed-method approach, drawing on survey data. The first component evaluated the impact of head injuries on university student athletes to inform safety and return to play protocols and evaluate how to enhance clinical and university-based support services. Subsequently, current clinical practices and unmet healthcare needs were examined through quantitative and qualitative data. Solutions for planning care provision and service delivery based on healthcare needs were evaluated, and a consensus study was conducted to establish agreement on care provision and service delivery for specialist outpatient services. Additionally, the feasibility of using digital health interventions to identify individuals with persistent symptoms after a mild TBI and triage them to multidisciplinary clinics based on healthcare needs was assessed. These results highlight clear unmet healthcare needs in the mild TBI population with persistent symptoms. Improved safety and return to play protocols are necessary for student athletes, and students with persistent symptoms would benefit from improved support services, both clinical and educational. Current healthcare provision and service delivery are suboptimal, lacking capacity, and offering limited value to patient health. To address these needs and improve care quality, structured follow-up in multidisciplinary clinics should be made available and accessible to deliver appropriate and timely care. This requires adequate resource allocation and sufficient capacity in specialist outpatient services. Standardising multidisciplinary management is crucial to addressing the diverse needs within the population, but more comprehensive clinical practice guidelines are necessary to deliver high-value care. Services and guidelines should be integrated into a care pathway that extends from the acute phase through to achieving the desired outcome. A clear pathway to access these services is necessary, and this may feasibly be delivered using digital health tools. Further evidence is needed to evaluate the cost-effectiveness and clinical value of such services. This thesis provides the foundation for planning care provision and service delivery for specialist outpatient services based on healthcare needs in the mild TBI population. Future work should establish the definitions of concussion/mild TBI and persistent symptoms, planning the care pathway, and implementing evidence-based recommendations and effective interventions to improve the quality of care and patient outcome.
Open Access
Management of Neurotrauma in Low-Resource Settings
Griswold, Dylan; Griswold, Dylan [0000-0003-0291-8360]
The first chapter presents an overview of the burden of disease of traumatic brain injury (TBI) - also referred to throughout the text as neurotrauma - a history of clinical practice guidelines, the Brain Trauma Foundation guidelines for the treatment of severe TBI (sTBI), the paucity of clinical trials carried out in low- and middle-income countries (LMICs) that make the development of treatment guidelines possible, the concept of resource-stratified guidelines (RSGs), the evolution of international TBI guidelines, and a proposed protocol for TBI management in low-resource settings. The introduction will be published by *Frontiers in Neurology* after incorporating the recommended changes from the reviewers. The second chapter systematically reviews the contribution of LMICs to neurosurgical randomised trials. In this chapter, we established that there is a substantial disparity between high-income countries (HICs) and LMICs in the number of published neurosurgical trials and call upon the global neurosurgery community to invest in research capacity building in LMICs as an essential step towards ensuring context- and resource-specific high-quality evidence is generated. A total of 73.3% of the 397 studies that met inclusion criteria were led by HICs, whereas 26.7% were led by LMICs. Of the 106 LMIC-led studies, 71 were led by China. Excluding China, only 8.8% were led by LMICs. HIC-led trials enrolled a median of 92 patients vs. a median of 65 patients in LMIC-led trials. HIC-led trials enrolled from 7.6 sites vs. 1.8 sites in LMIC-led studies. Over half of LMIC trials were institutionally funded. The results from this study are presented as a journal article, Neurosurgical randomised trials in low-and middle-income countries, published in the March 2020 issue of *NEUROSURGERY*. To access this publication, click [here](https://journals.lww.com/neurosurgery/fulltext/2020/09000/neurosurgical_randomized_trials_in_low__and.6.aspx). The third chapter reviews recent consensus-based algorithms, protocols, and guidelines proposed for the care of patients with sTBI in regions of limited resources (RLRs). Limitations of care for sTBI include suboptimal or non-existent prehospital care, overburdened emergency services, lack of trained human resources, and surgical and intensive care. Low-cost neuromonitoring systems are currently in testing, and formal TBI registries are forming to evaluate both long-term outcomes and best practices at every level of care, from hospital transport to the emergency department (ED) to the operating room (OR) and intensive care unit (ICU). Four multi-institutional collaborations between (HICs) and LMICs have developed evidence and consensus-based documents focused on capacity building for sTBI care to address this substantial burden of disease. The results from this study are presented as a journal article, Management of severe traumatic brain injury in regions with limited resources, published in the September 2021 issue of *Brain Injury*. To access this publication, click [here](https://www.tandfonline.com/doi/full/10.1080/02699052.2021.1972149). The fourth chapter provides a protocol and analysis of the first scoping review on the development, implementation, and validation of RSGs in LMICs. Nearly every field of medicine has some form of clinical practice guidelines. However, only within the past 5–10 years has the medical community acknowledged the need for well-developed guidelines tailored to the local healthcare needs and available resources. In most LMICs, healthcare workers depend on guidelines developed in HICs. Yet, many interventions validated in a HIC are ineffective when implemented in an LMIC. The variation in infrastructure, medical personnel, technology, and environmental conditions exhibited in LMICs relative to HICs necessitates a careful appraisal of the evidence base used in clinical guideline recommendations. This review aims to map the use of RSGs across all fields of medicine. The review seeks to answer three questions for the identified guidelines: (1) What was the method of development? (2) Have they been implemented? If so, (3) Have they been validated? The protocol was presented in the journal article, Development, implementation and validation of resource-stratified guidelines in low-and middle-income countries: a scoping review protocol, published in the September 2022 issue of *BMJ Open*. To access the publication, click [here](https://bmjopen.bmj.com/content/12/9/e059603.long). The results of the scoping review will be submitted to *BMJ Global Health*. The fifth chapter presents a protocol for a pilot study that aimed to identify the facilitators of, and barriers to, collecting data about patients with sTBI and to implement a stratified protocol across the treatment phases of prehospital, emergency department (ED), neurosurgery, and intensive care unit (ICU) in low-resource settings. A consensus process including experts from the four treatment phases took place in Colombia to develop a set of stratified protocols called BOOTStraP (Beyond One Option for the Treatment of Severe Traumatic Brain Injury: A Stratified Protocol), targeting resource-poor environments. However, it had not been systematically implemented and tested. The protocol is presented as a journal article, Protocol for a multicentre, prospective, observational pilot study on the implementation of resource-stratified algorithms for the treatment of severe traumatic brain injury across four treatment phases: prehospital, emergency department, neurosurgery, and intensive care unit in the August 2022 issue of *NEUROSURGERY*. To access this publication, click [here](https://journals.lww.com/neurosurgery/fulltext/2022/08000/protocol_for_a_multicenter,_prospective,.17.aspx). The sixth chapter presents the results of a Key Informant Survey, Bridging the gap: Investigating the treatment practices and guidelines for severe traumatic brain injury in Sub-Saharan African (SSA) urban referral centres and rural district hospitals. We sought to understand the current use of guidelines for the treatment of TBI in rural and urban regions of SSA and to discover and document the barriers to and facilitators of the utilisation of guidelines in this region to formulate a consensus statement and recommendations for improving the treatment of TBI patients in SSA centres. The results from this study will be submitted to *NEUROSURGERY*. A list of current publications can be accessed [here](https://scholar.google.com/citations?user=12sUiCEAAAAJ&hl=en).
Embargo
DNA Damage Response in Huntington’s Disease and Naked Mole Rat Brain Ageing
Ungerleider, Kyra
This thesis investigates the molecular mechanisms underlying healthy brain ageing in the exceptionally long-lived naked mole rat (NMR) and the role of the DNA damage response in the pathogenesis of Huntington's disease (HD). Multi-omic analyses of NMR brain ageing revealed a lack of a canonical ageing signature in the NMR cortex, with no observed increase in inflammation, DNA damage, or cellular senescence. Single-cell sequencing uncovered cell type-specific changes, with GABAergic neurons and astrocytes showing the most pronounced age-related alterations. Investigating the role of DNA damage in HD, the thesis demonstrated that HD cells display increased sensitivity to genotoxic stress and impairments in specific DNA repair pathways. CRISPR screens identified novel genes and pathways that modulate the cellular response to DNA damage in the context of expanded HTT. This thesis also investigated other DDR genes involved with repeat expansion in HD and identified potential modifiers of CAG expansion such as MBD4, PMS2, and RNASEH2A. Several hits from this arrayed CRISPR screen for modifiers of CAG repeat expansion converged on the regulation of R-loops, suggesting that these DNA-RNA hybrid structures may contribute to CAG repeat instability and genomic instability in HD. Integration of these findings highlights the central importance of genomic stability and cellular homeostasis in brain health and disease. The NMR's remarkable resistance to age- related brain pathology underscores the significance of maintaining an anti-inflammatory homeostasis and genomic integrity for healthy brain ageing. Conversely, the increased DNA damage sensitivity and repair deficits in HD cells demonstrate how disruption of these key cellular pathways can lead to neurodegeneration. The insights gained from this work could inform the development of interventions to promote healthy brain ageing and combat age- related neurodegeneration.
Embargo
Biochemical and Biophysical Studies of the UPR Kinase PERK and its Interactors in Health and Disease
Rieder, Lara-Sophie
The unfolded protein response (UPR) is a protective cellular mechanism that restores protein folding homeostasis under conditions of endoplasmic reticulum (ER) stress. The PERK-eIF2α branch of the UPR is activated by the accumulation of misfolded proteins in the ER lumen. PERK, upon activation through autophosphorylation, recruits its substrate eIF2α for phosphorylation, initiating a transcriptional and translational response. Overactivation of this signalling pathway is a major pathological feature in a range of neurodegenerative diseases. However, the mechanism by which the PERK pathway is activated in these conditions, where misfolded proteins are predominantly deposited in the cytoplasm rather than the ER, is still unknown. Recently, evidence for novel functions and modulation of PERK signalling has emerged. This has led to the working hypothesis that in disease, cytoplasmic disease-specific aggregates may directly interact with the cytoplasmic face of PERK, modifying its signalling in a non-conventional manner and resulting in the observed increase of PERK pathway activation. The overarching aim of this thesis is to further understand mechanisms of PERK pathway modulation and how these may potentially be altered in disease. This was addressed by developing a combination of biochemical techniques and biophysical methods, namely microfluidic diffusional sizing and Förster resonance energy transfer (FRET), to investigate interactions of PERK in solution and live cells. In-solution interaction studies confirmed recruitment of eIF2α by the unique insert loop situated within PERK’s kinase domain, but also indicated the presence of additional eIF2α binding sites in PERK, which may play a significant, but currently unknown, role in PERK signalling. Additionally, the interaction between PERK and its newly identified binding partner, the mitochondrial protein ATAD3A, was found to be direct, with ATAD3A’s tertiary and quaternary structure potentially being necessary for its modulatory effect on PERK signalling. Next, the question was posed whether disease-specific protein aggregates that accumulate in the cytoplasm, specifically α-synuclein oligomers, can act directly on the PERK pathway. In-solution techniques provided evidence to support the existence of a non-canonical mechanism of PERK signalling activation driven by these cytotoxic oligomers. Oligomeric α-synuclein interacted directly with PERK’s cytosolic domain and, strikingly, with PERK’s substrate eIF2α. This increased or stabilised the recruitment of eIF2α to PERK and resulted in elevated levels of phosphorylated eIF2α. Importantly, this was not observed with the monomeric α-synuclein protein. These findings could potentially explain the increased levels of PERK signalling and eIF2α phosphorylation observed in synucleinopathies and, possibly, other neurodegenerative diseases. To delve deeper into PERK:eIF2α interaction dynamics, FRET biosensors to monitor the interaction between the two proteins in live cells were developed. These successfully reported the specific, kinase insert loop-dependent, interaction of PERK with its substrate eIF2α. In the future, these biosensors hold potential for resolving local and temporal aspects of PERK:eIF2α binding dynamics under physiological and pathological conditions. Taken together, this work contributes to the understanding of PERK signalling modulation in health and disease. Whilst the proposed model for direct aggregate-mediated activation of the PERK pathway in neurodegenerative diseases requires further validation in cell-based and in vivo models, it may represent a unique therapeutic target which could enable specific inhibition of pathology-driven PERK pathway signalling.
Open Access
Metabolic control of neural stem cell function in a patient-derived model of multiple sclerosis
Ionescu, Rosana-Bristena
Multiple sclerosis (MS), the most prevalent inflammatory autoimmune disorder affecting the central nervous system (CNS), results in the deterioration of myelin sheaths and neuronal injury. For reasons that remain unclear, the majority of patients eventually enter a disease stage termed progressive MS (PMS), which is characterised by a substantial failure of the main endogenous brain repair mechanism seen in the early stages of the disease. During this period, patients experience a gradual accrual of neurological deficits, which are largely attributable to neuronal loss. There are limited treatment options for this phase of the disease. Notably, progression of the disease is associated with aging, with recent studies implicating cellular senescence (CS), particularly in the stem cell compartment of the PMS brain, as a probable contributor to disease progression by creating a hostile environment. Given the intricate interplay between cellular metabolism and CS, there is growing interest in exploring metabolic pathways as potential targets for therapeutic intervention in PMS to correct dysfunctional senescence-associated phenotypes that may be contributing to neuronal loss. My research aims to investigate the role of neural stem cell (NSC) metabolism in driving pathological changes in the PMS brain. Most MS models do not account for ageing, by either utilising middle-aged animals or employing reprogramming technologies which lead to cellular rejuvenation (e.g., induced pluripotent stem cells [iPSCs]). In the present study I am utilizing an innovative *in vitro* disease model employing human induced neural stem cells (iNSCs), which retain the epigenetic age of the somatic cells by bypassing the pluripotency stage in the reprogramming process. I conducted comprehensive phenotypic and multi-omics analyses of iNSCs generated from healthy controls and people with PMS. This investigation unveiled a distinct senescent gene expression profile in PMS iNSCs, primarily associated with inflammatory signalling, heightened metabolic activity, and the secretion of senescence-associated secretory phenotype (SASP) factors. PMS-derived iNSCs exhibited elevated synthesis of fatty acids and cholesterol, leading to the accumulation of cholesteryl ester-enriched lipid droplets. Furthermore, the lipogenic state mediated by HMG-CoA reductase activity triggered SASP release in PMS iNSCs via transcriptional regulation by cholesterol-dependent transcription factors. Notably, SASP from PMS iNSCs induced neurotoxicity, which could be mitigated through pharmacological intervention targeting HMG-CoA reductase using simvastatin (SV), a cholesterol-lowering drug. These findings highlight an intrinsic disease-linked, cholesterol-related, hypermetabolic phenotype in PMS iNSCs, culminating in neurotoxic signalling, which can be pharmacologically rescued, and establish iNSCs as a viable tool to study disease mechanisms in PMS.
Open Access
Exploring neurodegeneration in Ataxia-Telangiectasia
Tiet, May Yung
Ataxia-Telangiectasia (A-T) is a very rare autosomal recessive DNA repair disorder. The condition is characterised by a progressive neurodegenerative disorder. Cancer predisposition, immunodeficiency and respiratory disease can result in premature death. The life expectancy of a patient with A-T is 30 years old. The phenotype is highly heterogeneous when partially functioning ATM protein and kinase are present. The exact mechanism for disease causation and associated phenotype is unclear. It is also unknown why the cerebellum is so vulnerable in this condition. There are increasing reports of the association of mitochondrial dysfunction in A-T but how this is associated with DNA repair is unknown. The current lack of precise disease phenotyping, and metabolic and imaging biomarkers limits the progress of developing curative treatments and successful clinical trials for this disease. This study focuses on trial readiness and exploring the neurodegenerative process in A-T. I have focused on the development of a longitudinal study to effectively study the natural history of A-T and evaluated white matter hyperintensities, cerebral microbleeds and cognitive phenotype in adult A-T patients. I have also evaluated potential MRI and serum biomarkers. Finally, I have explored mitochondrial dysfunction and response to mitochondrial DNA depletion in human A-T fibroblasts. In this study, I have identified a cerebellar cognitive affective syndrome in A-T patients with executive and motor language dysfunction. Serum fibroblast growth factor 21 and dentate nuclei hypointensity could serve as potential molecular and imaging biomarkers. Finally, I have found a potential association of A-T with mitochondrial respiratory chain dysfunction and alterations of mitochondrial DNA replication.
Open Access
Mathematical Modelling of Brain Haemodynamics and Pressure-Volume Compensation
Chu, Ka Hing
Traumatic brain injury (TBI) is a major global health issue with diverse demographics and a dynamic clinical profile. Modern care of a pathology with such variabilities involves individualised treatment, which requires an accurate understanding of pressure-volume interactions inside the cranium. This thesis used a hydrodynamic model representing the circulation of cerebral fluids to investigate the relationships between intracranial pressure (ICP) and cerebral blood volume (CBV) changes. The effects of these relationships on the performance of indices describing vascular dynamics were also examined. The background of this project is discussed in Chapters 1 to 4 of this thesis. Chapter 1 provides a description of the anatomical and physiological aspects of the cerebrospinal space, including the circulation of cerebral fluids and the pressure-volume interactions among different compartments within the cranium. Chapter 2 introduces the pathophysiology of cerebral hydrodynamics in patients with TBI, and the important role of neuro-monitoring in its management. Chapter 3 specifies the aims and hypotheses of this project, while Chapter 4 includes a literature review of previous modelling work in cerebral blood flow (CBF) and cerebrospinal fluid (CSF) dynamics. In this project, a computer programme was written to create an implementation of the electrical equivalence of the hydrodynamic model. With a set of model parameters and imported data, the programme was used to simulate the pressures and flows of fluids in different intracranial compartments. Details of the programme were specified in Chapter 5, while experiments with the model were included in Chapters 6 to 9. In Chapter 6, the validity of an existing model was tested with a series of experiments simulating various clinical phenomena. The simulations include pathophysiological features caused by dynamic variations in cerebrovascular properties, as well as prolonged changes in CBV. In Chapter 7, the existing model was modified to include a compartment representing the bulk flow of cerebral interstitial fluid (ISF), coupled with the cerebrovascular compartment. The new model was tested using the same sets of experiments in the previous chapter, with its results compared to the original model. In Chapter 8, the modified model was used to assess the performance of common autoregulation indices in response to changing strength of cerebral autoregulation (CA), thus identifying the physiological factors determining the reliability of CA assessments. In Chapter 9, the modified model was used to explore the interplay between brain compliances and vascular reactivity, and its influence on pressure reactivity indices. The ability of the model to replicate clinical features observed in TBI patients was also investigated. In conclusion, the model has proved to be a robust tool to study the pressure-volume interactions among various intracranial compartments, as well as the performance of CA and pressure reactivity indices in different scenarios. The work in this thesis has laid the foundation of creating a ‘digital twin’ of cerebral hydrodynamics, with the potential of improving the individualisation of treatments in neuro-intensive care, particularly in dynamic pathologies such as TBI.
Open Access
Fatigue in Cerebral Small Vessel Disease and its Relationship to Cognitive Behavioural Symptoms
Jolly, Amy A; Jolly, Amy A [0000-0003-2669-4998]
Introduction
Cerebral Small Vessel Disease (SVD) is a disease affecting the small blood vessels in the brain. SVD is a leading cause of both stroke and vascular dementia, whilst cognitive behavioural symptoms such as fatigue are also frequent. Fatigue has a major impact on quality of life, yet little is known about its pathogenesis. This thesis aimed to assess the prevalence of, and possible mechanisms driving, fatigue whilst also assessing the role of other cognitive behavioural symptoms. Methods
Both monogenic SVD (*n*=251) and sporadic (*n*=36) SVD cohorts were utilised. Neuroimaging features and inflammation (both central nervous system (CNS) as assessed by ¹¹C-PK11195 PET and peripheral as assessed by circulating blood biomarkers) were investigated as possible mechanisms associated with fatigue, before moving on to assess the role of cognitive behavioural symptoms in the prevalence and severity of fatigue. Results
Fatigue was present in around half of both the sporadic SVD and CADASIL groups. There were no clear associations between neuroimaging features and fatigue, although meta-analysis highlighted some preliminary associations with network disconnection. There was no association between fatigue and CNS inflammation as measured using ¹¹C-PK11195 PET. There was an association of elevated C-reactive protein, a marker of systemic inflammation, with some fatigue measures in the sporadic SVD cohort, however, there was no association with a wide variety of blood biomarkers on the Olink panel after adjustment for multiple comparisons. The strongest finding throughout was the association of fatigue prevalence with both depression and cognitive impairment, in both sporadic SVD and CADASIL cohorts. Cognitive behavioural symptoms did manifest alone but were frequently comorbid. Pathanalysis confirmed that these symptoms seemed to exacerbate both presence and severity of one another. This was most prominently seen for fatigue and depression. Conclusion
Fatigue was prominent in both sporadic SVD and CADASIL cohorts. This thesis helps to further the research surrounding fatigue, a symptom which is poorly understood, and inform where future research may be best placed. Although there were no clear associations with neuroimaging or CNS inflammation seen in the projects, two areas that may show promise include network analysis and systemic inflammation. Further, the role of cognitive behavioural symptoms in the prevalence of fatigue cannot be understated and requires more research to disentangle their comorbid nature.
Open Access
Investigating immunosuppression as a disease modifying strategy in Parkinson's disease
Greenland, Julia
Increasingly, the immune system is implicated in the aetiology and progression of Parkinson’s disease (PD). Epidemiological studies have shown that the risk of developing PD is linked to polymorphisms in immune-related genes, increased by the presence of inflammatory conditions and reduced by the use of immunosuppressant and anti-inflammatory drugs. The immune also system shows an abnormal profile in PD patients. Microglia, the immune cells of the brain, are activated in PD. Abnormalities are also present in the peripheral immune system, with T-lymphocytes primed to respond to alpha-synuclein and a higher proportion of pro-inflammatory subsets, impaired regulatory function and a lack of immunosenescence normally seen with aging. T lymphocytes infiltrate the brain in PD, where they are found in higher numbers than in controls. Inflammatory cytokine levels are also elevated in the cerebrospinal fluid in PD[1], and a pro-inflammatory cytokine profile in the blood has been linked to faster disease progression. There is currently no treatment to slow the progression of PD and given the substantial evidence supporting the role of the immune system, we are conducting a phase 2 double-blind randomised controlled trial of azathioprine (“AZA-PD”), an immunosuppressant medication, to investigate whether suppressing the peripheral immune system has a disease-modifying effect in PD. At the time of writing, I am still blinded to treatment allocation, therefore this thesis does not include any data on the clinical efficacy of azathioprine. Chapter 1 explores the background and rationale for this treatment strategy, including a review of published trials targeting the immune system in PD. Chapter 2 describes the design of the AZA-PD clinical trial, with a summary of the trial protocol and a justification of the eligibility criteria and selection of the outcome measures. This chapter also covers the progress of the trial, including recruitment, adverse events and treatment withdrawals. Chapter 3 discusses the optimisation of the immunophenotyping analysis using flow cytometry which is being used as an exploratory outcome measure in AZA-PD, and reports the baseline immune profile of the trial cohort compared to a matched control group, as well as the profile of a group of patients with REM sleep behaviour disorder (RBD). This analysis illustrates the potential utility of FBC-derived variables as biomarkers in PD, and positions innate immune cell populations, particularly classical monocytes and natural killer (NK) cells, as variables of interest in the final trial analysis. Chapter 4 reports the imaging data from the sub-group of AZA-PD trial participants who had [11C]- PK11195 PET prior to starting treatment, which assesses microglia activation. The [11C]- PK11195 binding potential in regions of interest is compared to that of a previously collected control cohort. This analysis suggests the pallidum, putamen and lateral orbital gyrus as areas to take forward to the investigation of the potential effect of peripheral immunosuppression with azathioprine on neuroinflammation. This chapter also shows a correlation between peripheral NK populations and whole brain [11C]- PK11195 signal. Chapter 5 uses multiple regression analysis to explore the contribution of these markers of peripheral and central immune to the clinical phenotype of the AZA-PD trial participants prior to starting treatment. Chapter 6 analyses the treatment effect of azathioprine on both the peripheral and central immune profiles, using flow cytometry immunophenotyping analysis and [11C]- PK11195 binding potentials. Chapter 7 presents the key conclusions from this thesis and plans for future work.
Embargo
Investigating the protein disaggregation machinery in the early secretory pathway
Skov, Lise
Protein misfolding and subsequent aggregation represent key pathological features in various forms of dementia, notably Alzheimer’s disease (AD). AD is characterised by the presence of extracellular plaques, comprised of aggregated amyloid beta (Aβ) peptides, and intracellular neurofibrillary tangles, consisting of hyperphosphorylated tau protein aggregates within brain tissue. Therefore, a comprehensive investigation into the molecular mechanisms driving and preventing protein aggregation is important for gaining molecular understanding of the development and progression of dementia. A primary contributor to AD pathogenesis, Aβ, originates from a membrane protein and traverses the secretory pathway. While tau is also implicated in AD, particularly affecting the ER-Golgi secretory pathway, recent research highlights the significance of Aβ due to its initial extracellular processing and subsequent re-entry into neurons. Additionally, emerging evidence suggests that compromised intracellular protein folding quality control mechanisms may be part of facilitating the accumulation of toxic protein aggregates. The scientific evidence underlines the importance of exploring the intricate system responsible for managing aggregation-prone proteins. Recent findings from our laboratory have illuminated the inherent capability of the early secretory pathway, specifically the endoplasmic reticulum (ER), in resolving preformed protein aggregates, particularly under conditions of pharmacologically-induced ER stress. These discoveries have led us to postulate that the induction of this system could potentially mitigate the pathology associated with protein aggregation. Conversely, any dysfunction within this system may pose an increased risk for the development of AD-related pathology. Hence, the primary objective of this research was to investigate the molecular mechanisms and precise interactors comprising the intrinsic disaggregation system within the ER. This aim was achieved through a series of proteomics investigations involving the isolation of surrogate metastable proteins and its interaction partners under disaggregation conditions. Additionally, a parallel mission to engineer an aggregation biosensor, which will facilitate the detection and characterisation of aggregation-prone proteins in their various molecular states was undertaken. The outcomes of the proteomics investigation identified various chaperones important to ER folding processes. Consequently, this discovery prompted a more in-depth exploration of their potential involvement in the disassembly of ER aggregates. Additionally, success in the construction of closed and open conformation versions of a FRET-FLIM paired biosensor, which demonstrated its efficacy upon detection through Fluorescence Lifetime Imaging Microscopy (FLIM).
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Low-dose IL-2 for T-regulatory cell expansion after alemtuzumab therapy in relapsing-remitting multiple sclerosis
Georgieva, Zoya; Georgieva, Zoya [0000-0002-9531-8884]
T-regulatory cells (Tregs) are vital for the prevention of autoimmunity, and they require interleukin-2 to function optimally. Their expression of the high-affinity IL-2 receptor, containing the CD25 subunit, renders them 10-fold more sensitive to IL-2 than effector cells (Teffs). Low-dose IL-2 preferentially expands Tregs in patients with a variety of autoimmune disease, correlating with clinical improvement. Autoimmunity is a frequent complication of treatment with the lymphocyte-depleting therapy alemtuzumab in patients with multiple sclerosis (RRMS)- and Treg insufficiency may be involved. In this work I: 1) tested whether low-dose IL-2 could selectively expand Tregs in this novel context; and 2) comprehensively characterised the reconstituting Treg compartment following alemtuzumab therapy. Using quantitative flow cytometry and PhosFlow techniques, I showed that following alemtuzumab therapy, naïve CD4 Teff cells gained higher CD25 expression and became more sensitive to IL-2 *in vitro*, potentially narrowing the therapeutic gap. In mice treated with alemtuzumab the timing of IL-2 treatment proved critical. Treg expansion predominated in animals whose immune system had partially reconstituted after alemtuzumab. However, early post-alemtuzumab, IL-2 clearly caused Teff proliferation, while not expanding Tregs. Gene expression analysis and spectral flow cytometry confirmed that human reconstituting Tregs early after alemtuzumab are highly activated, and remain responsive to endogenous IL-2, though they later acquired exhaustion markers. Given the limitations of my animal model to reflect human pharmacokinetics and pharmacodynamics, I performed a mechanistic study of low-dose IL-2 treatment in 6 RRMS patients following alemtuzumab therapy. There was unprecedented lack of Treg expansion with doses of IL-2 that ordinarily expand this population. I discuss the postulated mechanisms and clinical implications behind this unexpected outcome.
Embargo
Mother-newborn EEG connectivity during affectionate touch and impact of hypoxic-ischaemic encephalopathy
Kromm, Grace
Mothers are the first, natural communication partners of their newborn babies, who are born wired for social connection. One of the main ways that mothers and their newborn babies communicate is through affectionate physical touch—skin-to-skin care and gentle stroking—which is crucial for the mother-infant relationship and for infant neurodevelopment across the lifespan. Very little is known, however, about how mothers’ and newborn babies’ brains respond to and engage during different modes of affectionate touch in healthy or vulnerable populations. I designed the Brain Activation in Mother and BabY (BAMBY) study to assess mothers’ and babies’ neural responses to affectionate touch in the days after birth. I recruited and studied 37 mother-newborn dyads, recording hyperscanning electroencephalography across social (mother and baby skin-to-skin) and nonsocial (mother and baby separate) contexts and across socially encoded (static touch and slow stroking) and non-socially encoded (fast stroking) touch types. I present the first evidence of event-related desynchronisation and of increased functional connectivity in the human newborn brain in social compared to nonsocial contexts and during socially compared to non-socially encoded touch types, and I show that these responses are sleep state-dependent. Mothers’ brains also showed neonatal state-matching, where mother functional connectivity was adjusted to newborns’ sleep states. Additionally, I present the first evidence of a mother-newborn inter- brain neural network measurable just after birth that is responsive to these social and touch cues and dependent on the baby’s sleep state. Finally, I present preliminary evidence that neural connectivity may be associated with cortisol changes during skin-to-skin care and with newborn neurophysiological responses. Hypoxic-ischaemic encephalopathy (HIE), a brain injury caused by lack of oxygen and blood flow to the brain, disrupts early neurodevelopment. I conducted a systematic review of HIE outcomes, and I found that HIE newborns are at increased risk of disrupted socioemotional, behavioural, and psychological development—the foundation of which is laid at the start of life. I piloted the BAMBY study in a cohort of four HIE mother-newborn dyads and present preliminary evidence of enhanced functional neural connectivity during gentle, slow stroking maternal touch. Together, these findings highlight the rich complexity of mother-newborn interaction at the neural level across salient neonatal cues, demonstrate the importance and efficacy of characterising these dynamics following HIE, and provide support for maternal touch interventions in healthy and HIE populations.
Open Access
Ultra-high field (7T) magnetic resonance fingerprinting
Shirvani, Saba
Ultra-high field (7T+) magnetic resonance imaging (MRI) improves image quality substantially compared to normal clinical 1.5T or 3T MRI. However, 7T MRI brings new challenges pertaining to B1+ inhomogeneity, B0 inhomogeneity, increased spectral bandwidth, and RF heating. Quantitative parametric mapping produces maps of tissue T1, T2 and other parameters in standard units rather than the weighted images normally produced in MRI. This has advantages by facilitating multi-site studies, long-term investigations, and making it easier to detect diffuse disease. At 3T, the Multi-Parametric Mapping (MPM) approach of Weiskopf [Weiskopf et al, Frontiers in Neuroscience, 2013] has been used in many neuroimaging studies, but it has been difficult to adapt for 7T. Magnetic Resonance Fingerprinting (MRF) is an alternative model-based technique to quantify multiple tissue properties after a single scan. MRF was initially created for clinical (1.5T and 3T) MRI scanners, where it has been widely applied to map T1, T2 and other parameters. MRF is more challenging at 7T due to B0 and B1+ inhomogeneity, limited peak B1+ and specific absorption rate (SAR) limits. My thesis aims to develop the methodology for 7T MRF on the first clinically-approved model of 7T MRI: the Siemens “7T Terra”. Chapter 1 and Chapter 2 introduce my thesis and summarise key background theory. Chapter 3 reviews the theory of MRF. Chapter 4 introduces an interleaved Cartesian MRF acquisition and analysis algorithm. To my knowledge, this is the first MRF implementation on the 7T Terra platform. MRF estimates of T1, T2, PD, and B1+ for brain and abdomen applications are evaluated, emphasizing the sensitivity of the MRF technique to B1+, B0, and slice profile effects. In Chapter 5 and Chapter 6, I address the challenge of generating a model that accurately reflects experimental magnetization evolution at 7T, which is more difficult than for 3T MRF. Chapter 7 compares the challenges of MRF at 3T versus 7T. In Chapter 8 and Chapter 9, I implement two strategies to accelerate the 7T MRF sequence: GRAPPA and sliding-window radial undersampling. These are steps towards a clinically acceptable scan time for 7T MRF. Chapter 10 describes my work to understand the parallel transmit framework on our new 7T Terra scanner, culminating in a successful human in vivo liver scan utilizing an 8-channel body transmit array coil. Overall, I have implemented key steps towards a viable 7T MRF sequence for human in vivo studies. This work will pave the way to a robust quantitative parametric mapping approach at 7T.
Embargo
The distinct contributions of cardiovascular risk factors to cognitive ageing
King, Deborah
This thesis aims to understand how individuals within an ageing global population can maintain cognitive health and independence throughout life. It is generally accepted that what is good for the heart is also good for the brain. However, it is not clear whether different aspects of cardiovascular health – such as blood pressure or heart rate – affect the brain through shared or independent pathways, partly because they are often condensed into a single composite score or examined in isolation. The research reported in the following chapters asks: do distinct elements of cardiovascular health have independent effects on cognition? And, if so, do they act by impairing brain structure and or brain function? The research builds a framework mapping unique pathways from multiple vascular factors to cognition. Understanding these pathways could inform the selection of specific vascular factors as targets for developing interventions to preserve cognition into old age. Chapter 1 is the General Introduction and describes the importance of cardiovascular health for cognitive performance. It also reviews the evidence for individual vascular risk factors, such as blood pressure, and how these might affect the brain’s structure and function. Chapter 2 is the General Methods and briefly summarises approaches shared across the three subsequent empirical chapters. These chapters use interdisciplinary and multivariate statistical analyses, applied to a large cohort of healthy adults (n=708, 18-88 years). Chapter 3 investigates whether multiple discrete vascular factors make distinct contributions to cognition. It shows unique and additive contributions from multiple vascular factors, including that high pulse pressure (systolic – diastolic) predicts a marker of cognitive decline. Pulse pressure typically increases with age and indicates increasing stiffness and decreasing elasticity of the major arteries, which then shunt the pulsatile wave of blood flow towards the brain, where it causes damage. In my analysis, the effects of pulse pressure on cognition are stronger for older adults. Importantly, the effects of pulse pressure are independent to other vascular risk factors, education and medications. Chapter 4 hypothesizes that pulse pressure impairs cognition by disrupting white matter microstructure. It shows a significant and substantial effect where high pulse pressure disrupts white matter, which then slows processing speed. It also indicates that pulse pressure driven declines in processing speed impair higher cognitive abilities such as problem solving, which is termed fluid intelligence. Overall, this chapter highlights the broad importance of pulse pressure in cognitive ageing. Chapter 5 investigates whether distinct vascular factors have unique associations with patterns of connectivity in large-scale structural and functional brain networks. It shows significant effects for heart rate variability, which represents the variation in the time interval between heart beats, where high variability is healthy. Lower variability associated with changes in the connectivity of functional brain networks, and lower fluid intelligence. This chapter also builds on the previous findings, to show that functional connectivity links with the now established effects of pulse pressure on white matter and processing speed. Altogether, this provides further evidence for the multivariate nature of vascular ageing. The results are discussed in the context of distinct aspects of cardiovascular health contributing to cumulative differences between individuals in the maintenance of brain tissues, and in the development of reserves in cognitive function, which both become important in old age. Chapter 6 brings together the findings of the three previous empirical chapters to draw a hypothetical framework of the multivariate contributions of cardiovascular health to cognitive ageing. The framework is hypothetical at this stage because it is informed by findings in cross-sectional data. I outline ideas to validate the framework in longitudinal datasets. Overall, I propose that managing pulse pressure may help to preserve cognition, particularly in older adults.
Open Access
The Role of Neuronal Activity in Adult Myelin Plasticity
Jia, Wanyi; Jia, Wanyi [0000-0003-2177-3654]
In the central nervous system, oligodendrocytes wrap membranes around axons to produce myelin. Their functional roles include rapid neurotransmission, synchronization, and functional maintenance. In the adult brain, 5% or more of the total cellular population consists of oligodendrocyte precursor cells, stem cells that proliferate and differentiate to produce new oligodendrocytes. While myelination is largely a postnatal process that occurs rapidly during development, it has been recently shown that new oligodendrocytes form throughout adulthood in both the grey and white matter; this *de novo* adult oligodendrogenesis plays a role in memory and learning. Research has also demonstrated that neuronal activity plays a central role in early myelin development and promotes oligodendrogenesis and adaptive myelination. Thus, it becomes important to understand how neuronal activity affects myelin. In this thesis, the role of neuronal activity in myelin plasticity during adulthood was investigated. Designer receptors exclusively activated by designer drugs were used to alter the activity of neurons bidirectionally to observe the activity dependence of myelin. Long-term neuronal activity changes were applied to the optic nerve, a tract in the central nervous system that is fully myelinated during adulthood, to determine how new and existing oligodendrocyte lineage cells respond. With sustained, increased neuronal activity over an 8-week period, it was observed that both existing and newly formed oligodendrocytes recapitulated the morphology of development by forming longer and fewer sheaths. With persistent, decreased neuronal activity, newly formed oligodendrocytes were fewer in numbers and formed shorter and fewer sheaths when compared to control, with existing oligodendrocytes following the same morphological pattern. Data presented in this thesis demonstrated that bidirectional neuronal activity manipulation yielded retraction, lengthening, and shortening of myelin sheaths in existing cells in a fully myelinated tract, which has not been demonstrated in the myelin remodelling field. Furthermore, on the circuitry level, it was demonstrated that with 8 weeks of sustained, increased neuronal activity, the signal conduction velocity from the retina to the visual cortex was significantly higher. This increase in signal transmission could be attributed to the myelin remodelling occurring during the activity modulation, yielding a functional change to the circuit. The work in this thesis shed light on how myelin remains plastic during adulthood and contributes to a greater understanding of how myelin is remodelled after development. Elucidating the role of neuronal activity on the white matter may lead to the discovery and development of more effective therapeutics for myelin-related diseases.
Open Access
Investigating Synapse Function In Organotypic Hippocampal Slice Culture Models of Neurodegeneration
Humphrey, Robert
Organotypic hippocampal slice cultures (OHSCs) are a useful in vitro model for researching synapse dysfunction; they provide easy access to the tissue for biomarker analysis, whilst retaining much of the neuronal architecture and endogenous cell types, representing a reasonable compromise between the advantages of *in vivo* and *in vitro* preparations. OHSCs prepared from TgCRND8 mice develop phenotypic changes that are relevant to early changes in human Alzheimer’s disease (AD), including increased amyloid beta and progressive loss of the presynaptic protein synaptophysin; however, direct analysis of functional changes in this model has not yet been achieved. Additionally, wild type mice exposed to the inflammatory molecule Lipopolysaccharide (LPS) experience similar loss of presynaptic protein, indicating the possible loss of presynaptic terminals. Synapse dysfunction and loss are some of the earliest changes in AD and other neurodegenerative conditions; by understanding how changes in neuronal and synaptic activity are related to the mechanisms of synapse loss in OHSCs we have the best chances of identifying possible therapeutic targets for combatting cognitive decline in neurodegenerative diseases. In this thesis we develop an electrophysiological method for investigating synapse function in OHSCs, establishing a robust experiment and analysis pipeline using whole cell patch clamping to measure spontaneous and miniature synaptic events in CA1 pyramidal neurones. Applying this method to long term (8 weeks) TgCRND8 OHSCs, we found that activity within OHSCs underwent significant changes throughout the culture period, but there were no strong genotype related effects, despite decreases in the presynaptic protein synaptophysin. We go on to investigate function in an LPS induced neuroinflammation model in OHSCs, and found significant impairment of excitatory and inhibitory synaptic function, accompanied by significant decreases in membrane capacitance. Ablation of microglia in these cultures partially recovered LPS induced synaptophysin loss, but did not recover functional deficits. These results provide insight into how best OHSCs should be used as a tool to explore neurodegeneration, indicating that they may be best suited for research with acute models rather than long term chronic models. Additionally, they underscore the need for critical evaluation of synaptophysin as a marker of presynaptic health and function.
Open Access
From big data to personal narratives: a supervised learning framework for decoding the course of traumatic brain injury in intensive care
Bhattacharyay, Shubhayu; Bhattacharyay, Shubhayu [0000-0001-7428-5588]
The management of traumatic brain injury (TBI) in the intensive care unit (ICU) generates vast clinical data, much of which is never analysed or interpreted. At the same time, the dynamic, complex disease course of TBI is not sufficiently characterised for truly patient-tailored treatment. This thesis capitalises on an opportunity to widen the context of information considered by individualised, dynamic models of functional outcome and therapeutic intensity after TBI. This opportunity is jointly afforded by the large-scale data collection of the Collaborative European NeuroTrauma Effectiveness Research in TBI (CENTER-TBI) study and recent advances in machine learning (ML) for time series modelling. This thesis combines a range of neural network (NN) architectures to propose a methodological framework by which all of the CENTER-TBI data collected before and during a patient's ICU stay can be dynamically mapped to ordinal endpoints. All of the CENTER-TBI variables are tokenised and embedded into lower-dimensional vectors which are then fed into gated recurrent neural networks (RNNs) to identify time-varying, informative patterns from the full dataset. The RNN outputs are then decoded by an ordinal output layer to return probability estimates, calibrated on validation sets, at each threshold of the endpoint. Regularised model weights are trained through supervised learning, and the reliability and information content of the modelling strategy are evaluated with repeated k-fold cross validation. Finally, the contribution of recorded clinical events to trained model outputs is estimated with a temporal extension of the SHapley Additive exPlanations (TimeSHAP) algorithm. The first endpoint of my supervised learning framework is functional outcome at six months according to the Glasgow Outcome Scale – Extended (GOSE). For ordinal GOSE prediction, expanding the predictor set with the tokenisation-embedding encoder (i.e., making models ‘wider’) significantly improves prediction performance whilst adding hidden layers does not (i.e., making models ‘deeper’). Functional outcome prediction is more difficult at higher GOSE thresholds and for patients with longer ICU stays. The full set of CENTER-TBI variables accounts for approximately half of the ordinal variation in GOSE, and static (pre-ICU and admission) variables account for the vast majority of this prognostic information. Variables with the greatest contribution to prognosis include physician-based impressions, imaging features, protein biomarkers, and neurological assessments. Then, I perform a clinimetric validation of the Therapy Intensity Level (TIL) scale and its five-category summary (TIL(Basic)) to measure the overall intensity of intracranial pressure (ICP) management. With TIL(Basic) as the second endpoint of the modelling framework, the full range of CENTER-TBI variables again explain approximately half of the ordinal variation in next-day transitions of ICP management after the second day of ICU stay. A patient's prior treatments, age, brain lesions, ICP, metabolic derangements, serial protein biomarkers, and neurological function are most predictive of future changes in TIL. However, a considerable proportion of these variations remain unaccounted for, suggesting the significant influence of a physician's preferences or unmeasured factors in contemporary ICP management. Supervised ML with NN-based architectures proves useful for improving the detail of model inputs (over time) and outputs but requires thorough assessment of potential overfitting. Insights from this thesis can inform the design of dynamic causal inference models and future data-collection or informatics projects for TBI.
Open Access
Gastrointestinal dysfunction in Parkinson’s disease: Interactions between the gut, immune markers and disease outcome
Camacho Goncalves, Marta
Although Parkinson’s disease (PD) is commonly characterized as a movement disorder, it is also associated with important non-motor symptoms. Gastrointestinal dysfunction in PD (GID-PD) may include weight loss, dysphagia and sialorrhoea. Constipation, the most prominent feature, is often present before the onset of motor symptoms and has a significant impact on quality of life. Accumulation of alpha-synuclein aggregates in the enteric nervous system in early stages of the disease has led to the hypothesis that, in a subset of cases, PD pathology may begin in the gut before spreading to connected areas of the nervous system. These gut changes may trigger an immune activation response which accelerates disease progression. Despite an exponential growth of research efforts on the mechanisms of the gut-brain axis’ role in the pathophysiology of PD, a comprehensive characterization of GID-PD and an understanding of its prognostic and mechanistic significance was lacking. I addressed this issue through 3 inter-related studies. I conducted a retrospective analysis of the impact of constipation severity at PD diagnosis and its relationship with long-term disease progression in a large longitudinal dataset (Chapter 2). I found a significant association between severe constipation in early PD and faster progression to dementia at an 8-year follow-up. To further explore the relationship between gut dysfunction and disease progression in PD, I developed the Gastrointestinal Dysfunction Scale – Parkinson’s Disease (GIDS-PD) and validated it against other GID assessment instruments in a large PD cohort, and proved that the GIDS-PD has good psychometric properties (Chapter 3). In Chapter 4, I used the GIDS-PD to characterize GID across PD stages. Using cross-sectional and longitudinal psychometric and objective transit time assessments, I observed that constipation is not a universal feature of PD but only affects a subtype who are likely to experience it through the disease course. I also conducted a prospective longitudinal study comparing a newly diagnosed PD cohort and household healthy controls as well as a prodromal PD cohort and collected data on gut symptoms, motor and cognitive features, and blood samples at baseline and 1 year follow-up (Chapter 5). I identified circulating peptidoglycan as a potential endotoxin of interest in the gut-brain axis and total ghrelin as potential protective agent. My project has clarified the presence of GID in the earliest stages of the disease, and investigated links with immune activation. This may pave the way for future intervention studies targeting GI function and associated immune changes in PD, with the goal of delaying disease progression.
Open Access
The Neurocognitive Sequelae of Chronic Traumatic Brain Injury and the Utility of Treatment with Methylphenidate
Peattie, Alexander; Peattie, Alexander [0000-0003-2115-7640]
Traumatic Brain Injury (TBI) is a serious world-wide public health concern with a pressing need for adequate, effective long-term treatment. Cognitive deficits are a common complaint after TBI and persistent disabilities endure well into the chronic phases of the injury. Few recognised standard treatments exist for treating cognitive deficits associated with TBI. Advances in Magnetic Resonance Imaging (MRI) technology using structural, functional, and diffusion imaging have shown utility in revealing subtle neurobiological abnormalities in patients with TBI which were previously undetectable with routine brain imaging. These have been employed here to study the behavioural, cognitive, and functional consequences of TBI alongside the neurobiological correlates of improved cognition with pharmacological treatment i.e., Methylphenidate (MPh). This work aims to further our understanding of the detrimental functional consequences of TBI and inform targeted neurorehabilitation options. In the first experimental chapter, I use task-based functional MRI data acquired during a Tower of London (TOL) task that gauges working memory, visuospatial planning, and executive function i.e., executive planning. I explore the detrimental effects of TBI and the usefulness of MPh treatment on behavioural performance, activity, regional connectivity, and MPh-driven behavioural-neurofunctional relationships. I found that patients with TBI have deficits in accuracy and reaction time, together with differences in activity and connectivity, and some of these deficits are ameliorated with a single dose of MPh. These were in the insula, striatum, thalamus, parietal, and cerebellar regions. Moreover, differences in connectivity between the cerebellum, thalamus, and parietal cortex indicate MPh-driven behavioural improvements. In the second experimental chapter, I use task-based functional MRI data collected during a Rapid Visual Information Processing (RVIP) task that evaluates attention, and more specifically, one particular domain, namely sustained attention. I found that patients have deficits in accuracy, sensitivity, and response bias. Patients demonstrate hyperactivity in frontal, parietal, and subcortical structures and exhibit connectivity deficits within a network dedicated to sustained attention. A single dose of MPh normalised aspects of regional hyperactivity and resulted in striatal areas connecting to additional frontoparietal regions to support sustained attention. Furthermore, MPh-induced changes in connectivity of the cerebellum and striatal regions related to behavioural improvements with MPh administration. In the third experimental chapter, I investigate the effects of MPh on resting-state functional connectivity to ascertain the relationship between resting-state neurofunctional differences and changes in the in-scanner behavioural results with MPh treatment that demonstrated significance from previous findings and work. I found that resting-state functional connectivity from the right insula, inferior frontal, and opercula regions were impaired whether patients took MPh or not. However, MPh appeared to upregulate the resting-state connectivity of parts of the left insula and IFG to parts of the cerebellum. These functional connectivity changes are linked to changes in performance in multiple cognitive domains including working memory, sustained attention, and executive planning. In the fourth and final experimental chapter, I use diffusion MRI to examine the regional structural deficits following TBI, how residual structural connectivity contributes to enhanced cognitive function in cognitive domains from in-scanner behavioural results that demonstrated significance from previous findings and work, and how structural connectivity and integrity might inform more successful responsiveness to MPh treatment. Patients display reduced white matter integrity in regions relevant for specific cognitive domains, disrupted fibre tractography, and compromised axonal projections. These were in the corona radiata, cingulum, and external capsule which connect frontoparietal and subcortical regions. Nonetheless, patients display some increased structural connectivity which could be attributed to compensatory neuroplasticity following TBI. Furthermore, differences in residual structural integrity between patients are related to improvements in numerous cognitive domains including working memory, executive planning, sustained attention, and response inhibition with MPh treatment. Through these investigations, this thesis aims to further the existing understanding of the neurocognitive consequences of brain injury, to carve out a place for functional and diffusion imaging in understanding these neurocognitive consequences and treating them in patients with chronic TBI, as well as explore the utility of MPh in treating this complex and heterogenous condition.