Florey Department of Neuroscience and Mental Health - Theses
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ItemTowards biologics targeting vasopressin family receptors( 2022)G protein-coupled receptors (GPCRs) are integral membrane proteins that play a critical role in transducing information into a cell, and as such are important drug targets. Biophysical characterisation and drug discovery against GPCRs is challenging as they express at low levels in recombinant systems and demonstrate low stability when removed from the native lipid bilayer environment, which together, can mean that the routine generation of purified receptor protein is difficult. The inability to routinely generate purified GPCR protein is a bottleneck to the application of many biophysical techniques. In particular, this thesis focused on the application of anti-GPCR biologics discovery. The oxytocin receptor (OTR) and vasopressin 1A receptor (V1AR) are emerging therapeutic targets for social disorders such as schizophrenia and autism. The native ligands of these GPCRs are closely related nonapeptides oxytocin and arginine vasopressin. Despite therapeutic interest, there is a lack of selective compounds targeting OTR and V1AR, limitted understanding about how these peptides bind and activate their receptors, and a lack of delineation regarding their expression patterns in the brain. There is therefore a need for selective compounds for OTR and V1AR. I propose that OTR and V1AR binding antibodies, and nanobodies are an interesting modality to explore to these ends. Antibodies are an emerging method of targeting GPCRs, both therapeutically, and as tools for biomedical research. Theoretically, antibodies should bind to GPCR subtypes with improved selectivity compared to small molecules, while also providing benefits such as restricted biodistribution, an extended half-life, and additional functionality via the design of antibody-drug conjugates. However, the identification of antibodies against GPCRs has been hindered by the challenges associated with generating GPCR antigens that represent the 3D conformation of the receptor. The ability to generate a sufficient yield of stable, functional, purified GPCR protein for immunisation or panning is central to this challenge. Thus, the development of antibodies targeting GPCRs has lagged behind other protein families. Nanobodies are single VHH domain camelid antibodies, which are encoded by single gene. Domain antibodies such as nanobodies have emerged as a novel alternative for targeting GPCRs, as the smaller size, and extended complement determining region 3 (CDR3) can enable binding to smaller sites and can be advantageous over small molecule, or traditional antibody approaches. In this thesis I developed methodologies that aim to circumvent the main bottlenecks in receptor antibody and nanobody discovery, working towards the discovery of biologics targeting vasopressin family receptors. Firstly, I enhanced receptor protein expression of the OTR, using a novel method of lentivirus assisted mammalian cell directed evolution. Using this method, I discovered the variant OTR(3A) which has 8-fold enhanced expression compared to OTR(WT) by the introduction of only four amino acid point mutations. Then, I optimised the expression and purification of high expressing variants of V1AR and OTR from mammalian cells. Strategies for generating fluorescent receptor protein preparations were also explored. Subsequently, purified V1AR and OTR were used to generate an immune repertoire of anti V1AR and OTR nanobodies following alpaca immunisation. I developed a custom mammalian cell display panning methodology and demonstrated the feasibility of using a mammalian cell display approach for the identification of nanobodies, based on binding to a target antigen. Finally, in a pilot study, FPR1 and cognate antagonistic antibody Fpro0165 were used to demonstrate that a Fab expressed on the cell surface of a mammalian cell can bind to its cognate receptor expressed on the same cell and Fab-GPCR binding was detected using fluorescent ligand competition, and blockage of peptide induced signalling, laying the foundations for further development of this mammalian display method for anti-GPCR antibody discovery. Together, the work in this thesis has made significant contributions towards developing tools and methodologies that will aid in the discovery of selective binders to OTR and V1AR, and furthermore, will contribute to the generation of anti-GPCR biologics more generally.
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ItemThe value of routine clinical imaging in predicting ischaemic stroke outcome( 2022)Research Objective To investigate the impact of ischaemic lesion characteristics and white matter changes – extracted from routine clinical scans – on outcome as a key biomarker for clinical prediction models. Methods Development of a stroke-population specific tool for semi-automated standardised assessment of lesion characteristics in routine clinical computed tomography (CT) and magnetic resonance imaging (MRI) scans; application of this tool to analyse the impact of lesion volume and lesion location together with white matter changes on functional outcome, gross motor outcome and language outcome; and analysis of the influence of very early mobilisation on functional outcome in the setting of large vessel occlusion (LVO). Results A stroke-population specific CT-MRI atlas with a validated robust and reliable CT normalisation pipeline was developed. Total lesion volume and white matter damage impacted functional and gross motor outcome the most. Aphasia, as a function-specific outcome was primarily impacted by infarction in core language areas. There was no evidence that very early mobilisation is especially harmful in patients with LVO. Implications The stroke-population specific CT-MRI atlas is a valuable tool to make routine clinical scans easily accessible for research in the context of lesion location and has the potential to standardise research in this field. Function-specific outcome parameters are necessary to further look into the lesion location – outcome relationship and systematic research using big data will be required to advance our understanding of the impact of lesion characteristics on stroke outcome. These points are crucial when aiming for the development and implementation of a clinical meaningful prediction model.
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ItemInvestigating mechanisms of mutant huntingtin toxicity by spatially mapping lipid metabolites in a mouse model of Huntington’s disease( 2022)Huntington’s disease (HD) features intraneuronal inclusion body formation by polyglutamine-containing fragments of the mutant huntingtin protein in many brain regions, including the hippocampus, neocortex and striatum. To better understand the molecular changes associated with inclusion body formation and associated pathogenesis, we examined the abundances and turnover rates of membrane lipids in the hippocampus, a region of pronounced inclusion formation associated with cognitive deficits, in a transgenic mouse model of HD (R6/1 line) using deuterium labelling in vivo. The R6/1 HD mice lacked inclusions in the hippocampus at six weeks of age, whereas inclusions were extensive by 16 weeks. We assessed one brain hemisphere collected at three timepoints (6, 12 and 16 weeks) by MALDI-mass spectrometry imaging (MALDI-MSI) and the other hemisphere for liquid-chromatography mass spectrometry (LC-MS) analysis. Hippocampal sub-fields (CA1, CA3 and DG) dense with inclusions showed a reduction in the relative abundance of neuronal-enriched lipids with roles in neurotransmission, synaptic plasticity, neurogenesis and ER-stress protection. Conversely, lipids in the phosphatidylinositol, phosphatidic acid and ganglioside class were increased in lipid synthesis in HD mice, relative to WT mice across all the age groups examined. The changes were also detectable in the HD mice at six weeks of age, indicating they arose prior to the formation of the inclusion bodies and disease symptoms. Since elevated synthesis of lipids in the PI, PA and ganglioside classes is a known adaptive response to Endoplasmic reticulum (ER) stress, our findings suggest this molecular mechanism serves as an early-stage adaptive response to ER stress in pre-symptomatic HD mice and may be targetable therapeutically. Additionally, our study has identified progressive changes in neuronal lipid abundances in the pre-symptomatic and symptomatic stages of HD that closely correlate with known hippocampal-dependent cognitive changes in HD, thus providing early lipid biomarkers that may be targeted therapeutically to slow down HD progression. Most importantly, we have spatially monitored disturbances in lipid metabolism at the primary site of inclusions in HD hippocampi, thus illuminating new insight into the cascade of molecular events in brain regions spanning the development of inclusions in HD mice. These findings required the development of a novel in-house bioinformatics software (KineticMSI), which is made available as an R package and will have broad neuroimaging applications.
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ItemEpileptic Encephalopathies: Identification and Characterization of Disease Mechanisms( 2022)Pathogenic mutations in the KCNQ2 gene, encoding the KV7.2 voltage - gated potassium channel, are known to cause neonatal seizure disorders, including severe Epileptic Encephalopathies. Epileptic Encephalopathies are characterised by pharmacoresistant seizures, developmental delay, and behavioural and cognitive deficits. Current therapies show limited efficacy in the treatment of seizures and fail to address the devastating comorbidities 1–5. KCNQ2 de novo variant K556E was identified in a patient with Epileptic Encephalopathies 6. In this research project we aimed to characterize the KCNQ2 K556E variant to better understand the underlying mechanisms of the disease and to set the stage for therapeutic screens that will help finding better treatments for this patient and for other patients like her. In order to assess the disease mechanisms of this variant, three disease models were used. Initially, the biophysical properties of the variant were investigated using in vitro expression in Xenopus oocytes followed by two - electrode Voltage - clamp Recordings. The data suggest a loss-of-function with no dominant negative effect caused by the variant. A loss of KV7 channel function indicate a significant reduction in the production of the potassium M - current. The M - current main biophysical role is setting the neuronal resting membrane potential and protecting against uncontrolled repetitive action potential firing. The loss-of-function might be the underlying cause of an excitable phenotype. We hypnotised that application of KV7 channels opener might rescue the significant reduction current seen in KCNQ2 K556E variant. However, retigabine, a KV7 opener, had no significant effect on the variant’s biophysical properties when the mutant channel was expressed as a homotetramer in Xenopus laevis oocytes. The second disease model we have generated and characterized is an in vivo disease model. A knock - in mouse model carrying the corresponding amino acid exchange in its KCNQ2 gene. Our observational studies revealed that both homozygous and heterozygous mice develop spontaneous seizures and present with increased mortality rates and premature death compared to wild-type littermate controls. The heterozygous mice are more susceptible to both heat induced seizures and chemically induced seizures. The heterozygous mice also expressed some behavioural changes when compared with wild-type littermate controls. The heterozygous mice bury less marbles in the marble burying test when compared with wild-type littermate controls. This might suggest a less anxious like behaviour and reduced cognitive abilities. Furthermore, the significant difference found between genotypes in this specific behavioural test could be used as an efficacy marker in a drug screening set of experiments in later stages. Lastly, we have generated and characterized patient specific in vitro iPSC-derived neuronal cultures. To this end we exploited a differentiation method based on the overexpression of Neurogenin 2 (NGN2) transcription factor to generate excitatory cortical neurons and investigated the electrophysiological characteristics of the neuronal cultures using whole cell patch clamping technique. Our findings suggest a more excitable phenotype of the patient-derived neurons in comparison with a control cell line from a healthy subject. In conclusion, the disease models indicate that a loss-of-function caused by the K556E variant likely leads to an increase in neuronal excitability which may be responsible for the increased susceptibility to epileptic seizures.
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ItemRelapse to alcohol-seeking: Examining VGluT1 vs VGluT2 ventral subiculum to nucleus accumbens shell projections( 2022)Alcohol use disorder imposes a substantial social and economic strain on society. Unfortunately, relapse is a major impediment to the successful treatment of alcohol use disorder. There are three main factors causing relapse-stress, drug re-exposure, and drug-related cues or contexts and the nucleus accumbens shell (NAc shell) is a key brain region in context-induced relapse. During my Ph.D., I investigated circuitry underlying context-induced relapse to alcohol-seeking, concentrating on glutamatergic (VGluT1 vs VGluT2) projections from the ventral subiculum (vSub) and basolateral amygdala (BLA) to the NAc shell. In chapter 3, using RNAscope, I found that the VGluT2 vSub-NAc shell projection was a subset of the VGluT1 vSub-NAc shell projection. However, the VGluT2 BLA-NAc shell projection was independent of the VGluT1 BLA-NAc shell projection. While the vSub-NAc shell projection was essentially entirely glutamatergic, the BLA-NAc shell projection was only ~70% glutamatergic, and ~30% non-glutamatergic BLA-NAc shell projections were GABAergic. In chapter 4, retrograde tracing was used to map VGluT1 vs VGluT2 projections to the NAc shell throughout the mouse brain. I found that VGluT1 projections to the NAc shell were mainly from the infralimbic cortex, dorsal peduncular cortex, prelimbic cortex, lateral entorhinal cortex, ventral subiculum, basolateral amygdala, and basomedial amygdaloid nucleus, anterior part. VGluT1 inputs to the NAc shell were greater in the ventral subiculum in both absolute cell number and density over VGluT2. VGluT2 inputs to the NAc shell were mostly from the infralimbic cortex, dorsal peduncular cortex, ventral subiculum, basolateral amygdala, basomedial amygdaloid nucleus, anterior part, central medial thalamic nucleus, mediodorsal paraventricular thalamic nucleus, and thalamic nucleus. VGluT2 inputs to the NAc shell were significantly greater in the paraventricular thalamic nucleus in both absolute cell number and density over VGluT1. In chapter 5, retro-TRAP was validated and using this technique the VGluT1 and VGluT2 containing projections from the vSub-NAc shell and BLA-NAc shell were isolated and sequenced. Chapter 6 highlighted the molecular differences between VGluT1 and VGluT2 BLA and vSub to the NAc shell projections. With bioinformatics and ddPCR, my study showed that Pfkl was higher in the VGluT1 vSub-NAc shell IP sample compared to its input sample; Fuz was higher, whereas Dlg1 and Sparcl1 were lower in the VGluT2 vSub-NAc shell IP sample compared to its input sample. Overall, my research uncovered the detailed arrangement of VGluT1 and VGluT2 projections to the NAc shell. My research also uncovered differentially expressed genes in the vSub-NAc shell projections of VGluT1 and VGluT2 cells. My study provides the foundation for the molecular mechanism for context-induced relapse to alcohol-seeking.
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ItemPhysical Activity, Skeletal Muscle and Clinical Outcomes after Ischaemic Stroke( 2022)Stroke is a leading cause of adult disability. Understanding modifiable risk factors associated with ischaemic stroke may inform strategies to reduce burden after stroke. Pre-stroke physical activity and skeletal muscle contribute to stroke prevention and may contribute to favourable outcomes after stroke. However, there remains key knowledge gaps regarding their benefits to stroke survivors. This thesis aims to address knowledge gaps for the benefits of physical activity and skeletal muscle towards key factors associated with clinical outcomes after stroke. I addressed the following questions: What is the current evidence for the association between pre-stroke physical activity and acute hospital admission stroke severity (i.e., the clinical severity of the stroke when the person admits to the hospital)? What is the association between pre-stroke physical activity and cerebral collateral circulation in acute ischaemic stroke patients? What is the association between pre-stroke skeletal muscle mass and acute hospital admission stroke severity in acute ischaemic stroke patients? What is the association between post-stroke physical activity and white matter hyperintensities (WMHs)? In Study 1, a systematic review revealed that higher pre-stroke physical activity levels may be associated with lower admission stroke severity. Pre-stroke physical activity may be associated with decreased likelihood of risk factors for severe strokes. However, among the risk factors identified, the included studies did not examine the association between pre-stroke physical activity and cerebral collateral circulation. This knowledge gap motivated the aims of Study 2. Furthermore, no authors had examined the associations between pre-stroke skeletal muscle mass and strength, sarcopaenia, and admission stroke severity. This knowledge gap motivated the aims of Study 3. Study 2 was a cross-sectional study recruiting acute ischaemic stroke survivors within 7 days of hospital admission. In Study 2, no association between pre-stroke physical activity and cerebral collateral circulation was observed. However, participants with anterior circulation occlusion strokes who engaged in pre-stroke leisure walking, sports, and recreational activities had lower median hypoperfusion intensity ratio (lower values indicate favourable collateral circulation) compared to those who did not engage in these activities. These results suggests that certain physical activity types are potentially associated with favourable cerebral collateral circulation. While inconclusive, these results provide direction for future research. Study 3 was a cross-sectional study recruiting acute ischaemic stroke survivors within 7 days of hospital admission. In Study 3, no association between pre-stroke skeletal muscle mass and admission stroke severity was observed. However, participants with sarcopaenia (combined low skeletal muscle mass and strength) had higher median National Institutes of Stroke Scale scores compared to those without sarcopaenia. Furthermore, I demonstrated that specific components of European sarcopaenia assessments guidelines were feasible in an acute ischaemic stroke setting. These results provide direction for future research examining sarcopaenia in acute ischaemic stroke survivors. Study 4 was a study using neuroimaging and physical activity data from the Cognition And Neocortical Volume After Stroke cohort, involving both cross-sectional and longitudinal analyses. In Study 4, ischaemic stroke survivors who engaged in at least 30 minutes moderate-to-vigorous physical activity per day were associated with lower WMH volumes at 12-months post-stroke. However, these associations were not observed in the multivariable analysis, where only older age remained associated with higher WMH volumes. These results revealed a complex relationship between physical activity, age, and WMH in ischaemic stroke survivors. Overall, I provide preliminary evidence suggesting that physical activity, before and after stroke, and skeletal muscle may contribute to clinical outcomes in ischaemic stroke survivors. These findings provide directions for future research, and further highlights physical activity as a strategy to improve clinical care and reduce burden for ischaemic stroke survivors.
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ItemHarnessing addiction neuroscience to understand compulsive overeating( 2022)With >2.8 million deaths/year attributable to overweight/obesity, it is now the fifth leading cause of global deaths and is rapidly surpassing smoking as the number one killer in the industrialized world. Difficulty in managing food intake, especially ultra-processed highly palatable food, is thought to be the main factor leading to non-genetic types of obesity (i.e., diet-induced obesity). A cardinal feature of pathological overeating is that although individuals want to lose weight and are aware of adverse and serious negative consequences of their behaviour, they have great difficulty changing it. Clinical studies show that people struggle to maintain healthy eating routines and usually re-gain all the lost weight and some more after a period of abstinence. Therefore, it is crucial to understand why people overeat and why it is so difficult to resist the urge to eat “junk food”. Many people with obesity describe their overeating as compulsive and addiction-like. A growing body of research has identified striking similarities between attributes of addiction and overeating in obesity. This emerging evidence supports the hypothesis that the brain’s reward circuitry may be dysregulated in people who suffer from disordered overeating behaviour such as binge eating, emotional eating and loss-of-control eating, typically co-morbid with obesity. During my PhD, I have used animal models of diet-induced obesity and binge-like eating to investigate the parallels between drug addiction and disordered overeating. I aimed to investigate 1) changes in eating behaviour induced by chronic overeating of high-fat high-sugar food in diet-induced obese rats; 2) compulsive-like eating in rats given sporadic intermittent access to high-fat high-sugar food; 3) changes in markers of synaptic plasticity at glutamatergic synapses in the dorsal and ventral striatum induced by chronic overeating or binge eating; 4) conduct a preclinical trial of N-acetylcysteine, a compound known to restore glutamate homeostasis. The results presented in this thesis demonstrate that diet-induced obesity in outbred Sprague-Dawley rats is associated with glutamatergic dysfunction in the nucleus accumbens and addiction-like behaviour towards high-fat high-sugar food. Critically, I showed that a compound that restores glutamatergic homeostasis, N-acetylcysteine, ameliorates addiction-like behaviour observed in diet-induced obese rats. Separately, this thesis has also demonstrated that the pattern of access to junk food has an important role in the development of compulsive-like behaviour towards junk food. Occasional access to junk food, for only one hour, three times per week, despite not causing significant body weight gain over eight weeks, is sufficient to induce compulsive-like behaviour towards this food, as well as changes in expression of glutamate receptor subunits (GluA1 and GluN2B) in the dorsal striatum. Collectively, the findings of this thesis indicate that both extended and occasional access to the obesogenic diet has the potential to lead to compulsive-like behaviour towards junk food in vulnerable individuals and induce glutamatergic dysfunction in addiction-related circuits in the brain. Furthermore, these findings support the concept of addiction-like glutamatergic dysfunction underlying disordered overeating behaviour and provide promise in the form of a potential new pharmacotherapy. Lastly, these findings have possible implications for how we view diet-induced obesity as a disease and around the regulation of high-fat high-sugar foods in society.
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ItemExploring how the indoor hospital physical environment may impact physical and social behaviour, and affective responses of people with brain injury( 2022)The physical environment of the interior hospital environment is an aspect of the environment of care of the person with brain injury that has received little attention in studies. Although several physical design attributes (e.g. daylight, patient room) have been studied in diverse healthcare settings (neonatal, cancer, ICU) few studies have involved people with brain injury. In addition, this field of research in the design of the physical environment is complex, requiring a multi-pronged approach to examine how and to what extent the physical environment might impact on people with brain injury in hospital. The overall objective of my thesis research was to explore how the physical environment of hospitals, specifically the indoor physical environment, might impact the physical and social behaviour, and affective responses of people with brain injury. Methods A series of sequentially-conducted studies were done over the course of this thesis. Firstly, I utilised a behavioural mapping approach and an adapted environmental checklist to compare the physical, and social activity of people in hospital for neurological conditions (including stroke) during a ‘before and after’ hospital re-location study. Then I conducted a systematic review where I aimed to differentiate the physical design attributes in a ‘single patient room’ from a ‘multi-patient room’, and to establish the strength of empirical evidence supporting one patient room type over the other. The systematic review motivated me to embark on a scoping review of the theories utilised by others in the field to attempt to explain or to test how the physical environment impacts on adults in health facilities. With the findings of studies 1-3, I was positioned to be able to design and conduct a factorial experimental design, using a virtual reality technology, to explore the impact of the physical design attributes on people with stroke, using numerical, categorical, and interviewing data collection. Results Firstly, the observational study showed that people with neurological conditions (>50% with stroke) spend most of the daytime in the patient room (social activity, physical activity), in both the old and new hospital ward environments. Further, these people spent time in close proximity to the window outlook and window seat (new ward), and not in communal areas. The systematic review revealed the paucity of diverse physical design attributes, beyond occupancy, used to examine the content of patient rooms, and highlighted the heterogeneity of outcomes (mostly hospital-acquired infection and falls rates) with which to compare the room types. Although a variety of theories were identified through the scoping review, few of them had been utilised consistently during a health research design study. One theory (the Theory of Supportive Design) was found to be consistently applied through a research study of the hospital ward environment. Finally, putative physical design characteristics (including ‘single’ versus ‘multi-patient’ room), led to the final thesis study (RiSE-VR). Identification of the magnitude and direction of stroke participant choice-preference and affective responses during exposures to a number of physical design characteristics were primarily sought. Quantitative findings were elaborated using interviews in the qualitative study. Feasibility of use of Virtual Reality in stroke was shown. Conclusions/ Implications New insights have been provided about how to study the impact of the interior hospital physical environment and people with brain injury. A novel study approach using virtual reality was developed and utilised to evaluate selected typical hospital physical design attributes in people after stroke. Feasibility and safety of this approach was shown. Regression analyses revealed the impact of different physical design attributes when co-existing with other physical attributes, in daytime and night-time. Thematic analysis provided triangulation and explanation of the quantitative data findings
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ItemUnravelling the role of microglial Mertk in myelination( 2022)Multiple sclerosis (MS) is a neurological disease characterised by chronic inflammation and demyelination of the central nervous system (CNS). Of the therapies currently available to treat MS, none are effective in promoting myelin repair, particularly in more progressive forms of the disease. Microglia are the resident innate immune cells of the CNS. In recent years, it has become apparent that microglia play key roles in mediating oligodendrogenesis and myelination. Broadly speaking, microglia can assume pro- or anti-inflammatory phenotypes, which influence their morphology and functions. While pro-inflammatory microglia can be drivers of demyelination, anti-inflammatory microglia can promote oligodendrocyte repopulation, especially by clearing myelin debris. One receptor tyrosine kinase expressed by these anti-inflammatory microglia is Mertk, which mediates immunoregulatory processes such as phagocytosis of apoptotic cells and debris. Given the importance of microglia in myelin repair, and Mertk in microglial function, this thesis aimed to investigate the microglial-specific role of Mertk in myelination. Using a Mertk conditional knockout (cKO) mouse model, I assessed the consequences of microglial Mertk deletion on glial cell production and myelination during development and in a mouse model of demyelination. From these experiments, Mertk cKO mice were found to have impaired oligodendroglia production and abnormal myelin ultrastructure during development. These developmental deficits were exacerbated during demyelination, whereby Mertk cKO mice showed signs of reduced mature oligodendrocyte production and thinner myelin. In collaboration with colleagues at the Paris Brain Institute, I expanded my work in mice to a transgenic tadpole model of demyelination, to determine if Mertk signalling was also beneficial in these animals. Not only was the promyelinating effect of TAM receptor signalling conserved Xenopus laevis, but pharmacological inhibition of Mertk signalling had detrimental effects on oligodendrocyte production. I then returned to mouse models and transplanted Mertk-expressing microglia into the brains of demyelinated animals to assess their ability to enhance remyelination. However, in these animals, I did not observe any beneficial effect of microglia transplantation in promoting remyelination above the natural level of recovery. This work is the first to describe microglial Mertk as a mediator of oligodendrocyte production and myelin ultrastructure during both development and disease, and highlights Mertk as a potential therapeutic target for promoting myelin repair in demyelinating diseases such as MS.
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ItemChronic Upper Limb Pain in Stroke: Beliefs and Perceptions, Factors Impacting, and Clinical Implications( 2021)Chronic pain is commonly experienced following stroke, with post-stroke shoulder pain (22-47%) and complex regional pain syndrome of the hand (21-31%) being among the most frequently reported pains by stroke survivors. Individuals with chronic pain post-stroke experience higher rates of depression, fatigue and anxiety in addition to restricted mobility and cognitive functioning compared to those without pain. Pain is frequently identified as an unmet need by survivors of stroke, and there is a lack of evidence for effective interventions. Many interventions used in clinical practice are based on outdated models of thinking related to pain, and do not consider the multifactorial nature of chronic pain. However, there is currently a lack of knowledge informing the mixed nature of pain post-stroke, and as such, development of treatment strategies has been limited. The primary aim of this thesis was to characterise the nature of chronic pain for individuals with stroke, and identify factors that may impact on the pain experience. A further aim was to investigate the impact of a learning-based approach to sensory rehabilitation (SENSe Therapy) on pain outcomes in people with stroke who experience somatosensory loss and chronic pain. To achieve these aims, two main studies were conducted. The first (RECOGNISE) was an online observational study developed for this thesis. RECOGNISE utilised a survey questionnaire and a series of interactive tasks to characterise the nature and experience of chronic pain post-stroke from the perspective of the stroke survivor (N=533), and investigate if relationships existed between somatosensory ability, body perception and chronic pain. The second study was part of a larger randomised trial (CoNNECT) in which stroke survivors with somatosensory loss and chronic pain (N=29), undertook a program of SENSe Therapy over a six-week period, with assessments performed at pre-post intervention and follow up at six-months. Findings from RECOGNISE revealed that there is not a common chronic pain experience post-stroke, as individuals with stroke report a mix of symptoms (spontaneous, evoked and paroxysmal). Further, chronic pain post-stroke was often associated with negative pain beliefs, somatosensory impairment and altered body image. Review of existing literature showed that these identified factors are modifiable using targeted interventions in other complex pain conditions, resulting in a reduction in pain. Findings from the exploratory study targeting somatosensory function of the upper limb in stroke survivors with chronic pain found that somatosensory retraining is a viable treatment option that had a positive effect in reducing pain in stroke survivors. In conclusion, this thesis advances knowledge about the mixed nature of pain experienced by individuals with stroke, and identifies several opportunities for development of intervention strategies that can be tailored to the individual with stroke based on their symptoms and presenting features.