Science

Publication Search Results

Now showing 1 - 10 of 27
  • (2021) Peng, Wang
    Thesis
    The HIV-1 capsid interacts with mutiple host factors during its early life cycle to achieve succesfful infection. In this thesis we aim to expand our understanding of how interactions between the capsid and host cell factors regulate viral activities, with chapter 4 and 5 specifically focusing on the cytoplasmic transport process. Based on cellular and biochemical evidence, recent studies suggest that HIV-1 capsid hijacks both dynein and kinesin-1, via the adaptor proteins BICD2 and FEZ1 respectively, for active transport along microtubule network. We will present our work to reconstitute these complexes and demonstrate their motilities along microtubles in vitro, providing more direct evidence to support these models. To investigate the interactions between HIV-1 capsid and the motor adaptors, we applied a fluorescence fluctuation spectroscopy based binding assay and a total internal reflection microscopy (TIRFm) based binding assay. These approaches allowed us to generate quantitative descriptions of the interactions between HIV-1 capsid and the adaptor proteins. Subsequently, we reconstituted the motor-adaptor-cargo complexes (DDBC and KFC) using recombinant proteins as well as components isolated from native tissues. We have successfully demonstrated both the dynein- and kinesin-dependent transport of HIV-1 capsid along microtubules in vitro using a TIRFm based single molecule motility assay. We also further charaterized the motile behaviors and properties of the KFC complex. Our work validated the proposed models for the cytoplasmic transport of HIV-1 capsid and demonstrated the minimum requirement for this process. In general, this work has made solid contributions towards the understanding both HIV viology and motor-driven cargo transport, as well as opened oppotutnities to a range of exciting research questions in both fields.

  • (2021) Begik, Oguzhan
    Thesis
    RNA modifications, collectively referred to as the ‘epitranscriptome’, are not mere decorations of RNA molecules, but can be dynamically regulated upon environmental queues and changes in cellular conditions. This dynamic behaviour is achieved through the RNA modification machinery, which comprises “writer”, “reader” and “eraser” proteins that modify, recognize and remove the modification, respectively. Chapter1 presents a comprehensive analysis of the RNA modification machinery (readers, writers and erasers) across species, tissues and cancer types, revealing gene duplications during eukaryotic evolution, changes in substrate specificity and tissue- and cancer-specific expression patterns. Chapters 2 and 3 presents the exploration and development of novel methods to map and analyze RNA modifications transcriptome-wide. Nanopore direct-RNA sequencing technology was used to provide RNA modification maps in full-length native RNA molecules. Firstly, it is shown that RNA modifications can be detected in the form of base-calling ‘errors’, thus allowing us to train Support Vector Machine models that can distinguish m6A-modified from unmodified sites, both in vitro and in vivo. Secondly, it is demonstrated that distinct RNA modification types have unique base-calling ‘error’ signatures, allowing us to exploit these signatures to distinguish different RNA modification types. It is found that pseudouridine has one of the most distinct signatures, appearing in the form of C-to-U mismatches. Finally, this information was used to predict novel pseudouridine sites on ncRNAs and mRNAs transcriptome-wide, as well as to obtain quantitative measurements of the stoichiometry of modified sites. Chapter 4 presents the development of a novel nanopore-based method, which is termed ‘Nano3P-seq’, to simultaneously quantify RNA abundance and tail length dynamics in individual molecules in both the coding and non-coding transcriptome, from cDNA reads. It is demonstrated that Nano3P-seq offers a simple approach to study the coding and non-coding transcriptome at single molecule resolution regardless of the tail ends. Together, this work provides a comprehensive framework for the study of RNA modifications and polyA tail dynamics using third generation sequencing technologies, opening novel avenues for future works that aim to characterize their dynamics and biological roles both in health and in disease.

  • (2021) Chilver, Miranda
    Thesis
    Mental wellbeing, a state of positive subjective experience and psychological functioning, is a key component of mental health. Despite this, little is known about how mental wellbeing is manifested in the brain, or how such neural associations covary with depression and anxiety symptoms. Thus, the aim of this thesis was to explore potential electrophysiological markers of wellbeing using electroencephalography (EEG) and event-related potentials (ERPs). To facilitate further investigation into the association between electrophysiology and wellbeing, a positive psychology intervention was also developed. Following an introduction to the key topics, Chapter 2 examined the relationship between mental wellbeing and resting EEG power. This study identified a specific profile of resting EEG power that is associated with wellbeing, independent from depression and anxiety symptoms. Twin modelling clarified that this EEG profile shares a genetic correlation with mental wellbeing. Chapters 3 and 4 shift towards using ERPs to investigate how wellbeing and depression and anxiety symptoms are associated with emotional and cognitive processing. Chapter 3 reported that wellbeing was not significantly associated with emotion processing after accounting for depression and anxiety symptoms, which were significantly associated with alterations in emotion processing. In Chapter 4, no evidence was found for an association between wellbeing or depression and anxiety symptoms with cognitive ERPs, although associations with behavioural performance reported in previous studies were replicated. Finally, Chapter 5 reports on the effectiveness of an online positive psychology intervention which was found to significantly improve wellbeing, particularly subjective wellbeing. Although COVID-19 restrictions prevented the evaluation of causal links between wellbeing and EEG using this intervention, it is discussed for purposes of future research. Together, this thesis provides one of the first investigations into the electrophysiological correlates of mental wellbeing. Resting EEG power was identified as the most promising avenue for future research aiming to establish endophenotype markers of mental wellbeing, with the task-related measures assessed here were not associated with wellbeing. A short and effective online intervention was developed that could be used to facilitate future investigations into the use of resting EEG as a predictor and marker of mental wellbeing.

  • (2022) Jamshidi, Javad
    Thesis
    Wellbeing, a key aspect of mental health, is defined as a state of positive subjective experience and optimal psychological functioning. This thesis presents a series of studies devised to comprehensively explore phenotypic, genetic, and neural correlates of wellbeing. The first study (Chapter 2) aimed to compare the heritability and stability of different wellbeing measures in the TWIN-E dataset (N~1600) to discern the most suitable approach for measuring wellbeing for subsequent gene discovery efforts. This twin-based study concluded that multi-item measures of wellbeing such as the COMPAS-W scale, were more heritable and stable than single-item measures. Wellbeing-associated variants were identified via genome-wide association studies (GWAS) and highlighted the need for larger sample size. The subsequent studies were conducted using population-scale data from the UK Biobank comprising ~130,000 participants with phenotypic and genetic data. Thus, in Chapter 3, I constructed a multi-item “wellbeing index” measure using UK Biobank data to investigate its relationship phenotypically and genetically (using GWAS, polygenic scores and LD score regression) with negative mental health indicators (e.g., neuroticism and loneliness), childhood maltreatment and psychiatric illness. I confirmed that SNP-heritability of wellbeing index was higher than both single-item measures and estimates previously reported (SNP-h2 = 8.6%). Moreover, I provide an overview of phenotypic and genetic correlations between wellbeing index and negative mental health indicators. In addition, childhood maltreatment and psychiatric illnesses were associated with reduced wellbeing, with evidence that genetic factors may influence their correlations. In Chapter 4, I investigated the genetic and phenotypic associations between wellbeing index and brain structure, using magnetic resonance image-derived phenotypes from the UK Biobank. This study found associations between wellbeing and volumes of brainstem, cerebellum and subcortical regions, and structural morphology of various cortical regions. Thus, wellbeing is associated with complex structural variations, each with a small effect. Together, this thesis explores the multifaceted nature of wellbeing, elucidating its phenotypic and genetic relationships with related phenotypes, childhood maltreatment, and psychiatric outcomes, and provides novel insights into the associations between wellbeing, its genetic signatures and brain structure.

  • (2022) Cao, Jun
    Thesis
    This thesis focuses on the development and applications of magnetic resonance electrical properties tomography (MREPT), which is an emerging imaging modality to noninvasively obtain the electrical properties of tissues, such as conductivity and permittivity. Chapter 2 describes the general information about human research ethics, MRI scanner, MR sequence and the method of phase-based MREPT implemented in this thesis. Chapter 3 examines the repeatability of phase-based MREPT in the brain conductivity measurement using balanced fast field echo (bFFE) and turbo spin echo (TSE) sequences, and investigate the effects of compressed SENSE, whole-head B_1 shimming and video watching during scan on the measurement precision. Chapter 4 investigates the conductivity signal in response to short-duration visual stimulus, compares the signal and functional activation pathway with that of BOLD, and tests the consistency of functional conductivity imaging (funCI) with visual stimulation across participants. Chapter 5 extends the use of functional conductivity imaging to somatosensory stimulation and trigeminal nerve stimulation to evaluate the consistency of functional conductivity activation across different types of stimuli. In addition, visual adaptation experiment is performed to test if the repetition suppression effect can be observed using funCI. Chapter 6 explores if resting state conductivity networks can be reliably constructed using resting state funCI, evaluates the consistency of persistent homology architectures, and compares the links between nodes in the whole brain. Chapter 7 investigates the feasibility of prostate conductivity imaging using MREPT, and distinctive features in the conductivity distribution between healthy participants and participants with suspected abnormalities.

  • (2022) Young, Clara
    Thesis
    A breakdown in B cell self-tolerance can lead to antibody-mediated autoimmune disease. This thesis aims to explore how B cell tolerance can be broken in two distinct, but complementary projects within the context of the Goodnow somatic mutation hypothesis. In both scenarios, B cells that escape self-tolerance and generate autoantibodies are referred to as “rogue” B cells. First, this thesis aimed to elucidate the precise steps undertaken by expanded rogue B cell clones in patients with chronic Hepatitis C virus (HCV)-associated cryoglobulinemic vasculitis, an autoimmune disease characterised by the production of a rheumatoid factor cryoglobulin autoantibody. The rogue B cell clones in the HCV cryoglobulinemic vasculitis patients were confirmed to be the source of the autoantibody. The rogue B cell clone precursor antibodies failed to bind the HCV envelope glycoprotein E2, yet bound multimerised self-antigen IgG relative to membrane IgM density. These findings disfavour a molecular mimicry hypothesis, and instead indicate IgG immune complexes may be sufficient to drive recruitment of the rogue B cell clone precursors. Finally, the rogue B cells clones were found to carry somatic lymphoma-associated, non-immunoglobulin gene mutations and chromosomal aberrations, predicted to cause hyperactivation of the NF-kB signalling pathway and escape of B cell tolerance. This finding provides additional evidence in support of the Goodnow somatic mutation hypothesis. Second, this thesis examined rogue germinal centre (GC) B cells that arise in the absence of the receptor FAS. Rogue GC B cells loose specificity for the foreign antigen and incidentally generate autoantibodies. However, the accumulation of rogue GC B cells cannot be explained by our current understanding of affinity-based selection in the GC. This work revealed rogue GC B cells, unlike “conventional” GC B cells undergoing affinity maturation to the foreign antigen, can be identified by low expression of CD21 and high expression of B220 (CD21loB220hi). Moreover, rogue GC B cells were found to be rapidly entering cell cycle, enriched for a dark zone phenotype and T-cell dependent, reminiscent of positively selected GC B cells. Thus, rogue GC B cells typically removed by FAS, likely persist in the competitive GC microenvironment despite their loss of BCR specificity to foreign antigen, because they retain the capacity to undergo T-cell dependent positive GC selection.

  • (2022) Farrell, Megan
    Thesis
    T cells are critical in the body's defence against viruses and cancerous cells. They specifically recognise viral or tumour antigens presented on antigen presenting cells using their T cell receptor (TCR). Antigen binding triggers the TCR, transmitting signal intracellularly and resulting in the recruitment of a plethora of signalling proteins to the membrane. The signal is transmitted by post-translational modifications, such as the phosphorylation of tyrosine residues in intracellular tails of receptors, resulting in the recruitment of signalling proteins via their interaction domains. The spatial organisation of signalling proteins at the membrane determines the effector response of the T cell and is therefore critical for understanding the complex array of T cell responses. In this thesis, I develop a novel microscopy technique that reports on the nanoscale locations of signalling proteins and their binding kinetics to receptors at the T cell membrane. This technique utilizes the SH2 interaction domains of various signalling proteins which selectively and transiently bind to phosphorylated tyrosines on receptors. This transient binding results in the stochastic blinking necessary for super-resolution microscopy. Using this technique, termed protein point accumulation in nanoscale topography (pPAINT), I investigate the binding of multiple signalling proteins, achieving multiplexed imaging both simultaneously and sequentially with a combined microfluidic and microscopy approach. In the second half of this thesis, I apply pPAINT to study how chimeric antigen receptors (CARs) signal in T cells. In CAR-T therapy, patient cytotoxic T cells are isolated and transduced with a CAR construct that recognises tumour antigens, and are then reintroduced into the patient where they find and eliminate cells expressing the CAR target antigen. CAR constructs are made up of an antigen recognition domain fused to various intracellular signalling motifs from the TCR complex and co-stimulatory receptors, such as CD28, which are crucial for T cell activation. The first-generation of CARs contained an intracellular tail of the TCR, the CD3ζ chain, but it has been the second-generation CARs, with the addition of co-stimulatory receptor signalling domains, that have proven clinically effective. However, CAR therapy is not successful in all patients; limitations reducing their efficacy include inefficient recognition of low antigen densities, finite persistence in the body and off-target side effects in patients. It follows that a detailed knowledge and understanding of CAR activation and signalling is needed to optimise CAR design. In this thesis I use pPAINT to gain a unique perspective on how different generations of CARs signal upon activation, identifying key similarities and differences to signalling from the standard TCR. Signalling was investigated in CAR-T cells generated in a similar way to clinically used CAR-T therapies. In doing so, unique signalling mechanisms utilized by CARs were identified that will be valuable for the development of more effective chimeric antigen receptors. The results demonstrate that although CARs utilise the signalling domains of the TCR and co-stimulatory receptors, the pattern of adaptor protein recruitment is different from that of T cells stimulated through the TCR and co- stimulatory receptors. Specifically, I found that whilst hubs of signalling proteins spatially diverged from clusters of activated TCR, they were instead closely colocalised with activated CARs. The incorporation of the CD28 coreceptor in CAR design improves signalling protein recruitment patterns, however, the patterns of protein binding were still vastly different to co-stimulated T cells. Collectively, the results indicate that CARs utilize a signalling pathway unique to that of costimulated T cells, in a mechanism that may have ramifications in the functional responses exhibited by cells used in CAR-T therapy.

  • (2023) Green, Nicole
    Thesis
    The onset of psychiatric disorders, such as autism spectrum disorder, schizophrenia, anxiety, and major depressive disorder, can be associated with both genetic and environmental factors. This thesis examines transcriptional changes caused by either genetic or environmental factors linked to psychiatric disorders. We investigated regulatory regions which harbour mutations associated with psychiatric disorders, and the effects of early life stress on the developing brain and its consequent adverse mental health outcomes. Here, we assessed the function of active enhancers in the brain to understand their regulatory roles, and their link to mutations associated with psychiatric disorders. This was achieved using a CRISPR interference (CRISPRi) screen to repress almost one thousand enhancers in primary human astrocyte cells. The effect of enhancer repression on transcription was measured using single cell mRNA sequencing (scRNA-seq), a which measures gene expression from individual cells. This thesis describes the first CRISPRi screen to be performed in brain cells, and the first to be performed in primary cells. Active enhancers targeted in this screen were characterised based on their chromatin state and through analysing enhancer marks from relevant published data. Regions of open chromatin are a marker for enhancers and were determined in primary human astrocytes using ATAC-seq, a method which transposes DNA in an open chromatin state, before using sequencing to determine regions of open chromatin. Candidate enhancers were filtered based on the ability to detect potential target genes of the enhancer by scRNA-seq, to which gene expression was required to be above an expression threshold. The expression of potential target genes was determined by mRNA-seq, and candidate enhancers were required to be in the same topologically associated domain as a detectable target gene expressed above the threshold. The CRISPRi screen was optimised with a pilot screen to determine the degree of silencing due to CRISPRi and the detectability of the transcriptional effect of silencing enhancers by CRISPRi using scRNA-seq. A large-scale CRISPRi screen was then performed to investigate and validate candidate enhancers in primary human astrocytes. This identified almost one hundred enhancers active in the human brain that have disease-associated variants, and 140 enhancer target genes associated with disease. We also investigated the transcriptional effect of early life stress using murine models. A limited nesting (LN) model was used, as this emulates human maternal neglect and has been previously shown to increase measures of anxiety like-behaviours in rats. We performed RNA sequencing on the prefrontal cortex (PFC) of rats that underwent an LN paradigm as a model for early life stress. Our results indicated that this primarily led to transcriptional repression in the PFC. We further identified a molecular signature of LN in rat PFC that, when compared with other studies, is observed across ELS protocols and replicable in other murine models.

  • (2022) Mc Guinness, Conall
    Thesis
    Perfringolysin O (PFO) is a cholesterol dependent cytolysin (CDC) secreted by Clostridium perfringens, which forms pores in cholesterol containing membranes. CDCs are part of the larger Membrane attack complex-Perforin/CDC (MACPF/CDC) superfamily, containing pore formers responsible for controlling infectious disease and cancer in humans. CDCs are secreted as soluble hydrophilic monomers which oligomerise on lipid bilayers, ultimately forming bilayer spanning closed ring or open arc-shaped β-barrel pores. Perfringolysin O (PFO) was the first CDC to have its crystallographic structure resolved in its soluble monomeric form and has since become the prototypical CDC for investigating pore-forming mechanism. Previous studies on PFO have revealed a general outline of the steps involved in CDC pore formation; recognition of cholesterol and membrane binding, oligomerisation and ultimately membrane insertion to form large amphipathic pores. These steps have been elucidated using bulk assays and static imaging techniques such as electron microscopy or atomic force microscopy, however key mechanistic details remain uncharacterised due to the lack of time resolved data at a single pore level. Here we present a novel assay using microfluidics and total internal reflection microscopy to track PFO pore formation dynamics. Fluorescently labelled PFO and dye encapsulating liposomes were employed in conjunction to measure the kinetics of PFO binding from solution, nucleation, and oligomerisation on the surface of cholesterol containing liposomes. By visualising fluorescent dye release from our liposomes, we were able to determine the number of molecules necessary for an oligomer to insert and form a bilayer spanning pore. This information was collected from a large number of liposomes and used to build a mathematical model to quantify parameters controlling PFO accumulation and insertion kinetics on liposomes. We have also demonstrated how a different membrane composition affects these parameters by performing the assay on virus-like particles derived from HIV. This also displayed the adaptability of the assay which can be applied to different membranes and other pore forming proteins.

  • (2023) Seyedzadeh, Hadi
    Thesis
    Cytotoxic T lymphocytes (CTLs) can target and induce apoptosis in cancer cells during the anti-tumour immune response. However, the cytotoxicity (or killing) function of CTLs can be perturbed directly by cancer cells or via the tumour microenvironment (TME). Among the various factors in TME that can influence T cell function, the effect that mechanical properties of the extracellular matrix (ECM) have on CTL responses is unclear. Research into CTL-mediated cytotoxicity is typically performed in either two-dimensional (2D) matrix-free culture or in complex in vivo animal models. In vitro, 2D studies are limited in recapitulating the CTL response in vivo, whereas it is very difficult to manipulate the TME and perform high-throughput experiments using in vivo models. Recently 3D culture models have been introduced to fill the gap between 2D and in vivo studies. In this study, we used an automated 3D bioprinter to incorporate OT-I T cells and cognate and non-cognate target cells in a polyethylene glycol (PEG)-based hydrogel and studied the killing efficiency in comparison with 2D culture and manually-prepared gels. Here, we showed that the 3D bioprinter embeds both CTLs and target cells in the hydrogel and enables control over the dimensions of the embedding matrix as well as the number and spatial organisation of cells. Moreover, the ability to digest the gel and release the cells allowed us to perform killing efficiency comparisons and downstream high-throughput CTL functional analyses using flow cytometry. This novel 3D cell culture system allowed us to investigate the effects of tunable ECM mechanical properties in a reproducible cytotoxicity model of matrix-embedded CTL and target cells. Our results demonstrate that in matrices with higher density, CTL killing efficacy was compromised. This demonstrates that matrix stiffness, independent of matrix porosity or other variable characteristics, has a large impact on CTL function. From another perspective, cancer cells can directly induce dysfunctional programming in CTLs. Repeated stimulation of the T cell receptor (TCR) on CTLs with the tumour-associated antigen leads to overexpression of inhibitory receptors such as programmed cell death (PD)-1 on the surface of T cells, leading to aberrant response and eventually tumour escape. TCR signalling machinery can be affected by the expression of inhibitory receptors, but it is not clear whether inhibitory receptors alone are responsible for the dysfunction of exhausted T cells or to what degree other mechanisms contribute. To address this, we used a mouse model of T cell dysfunction, finding that T cells could exhibit a dysfunctional phenotype with minimal upregulation of inhibitory receptors and without downregulation of TCR. Instead, we found a decrease in the proximal signalling kinases Lck and ZAP70, specifically in dysfunctional cells. To confirm these results, we developed a human primary in vitro CD8+ T cell dysfunction model, which allowed us to study the effect of repeated antigen stimulation on the inhibitory receptors expression and expression of Lck and ZAP70 in human T cells. In this model, we again found that dysfunctional T cells had lower expression of Lck and ZAP70, confirming the results from the mouse model. Future experiments could be performed in which Lck and/or ZAP70 expression is enhanced in dysfunctional T cells. If this restores the functional phenotype, it may confirm that a low level of Lck and ZAP70 protein expression is a cause of T cell dysfunction. This research sheds light on how the external (matrix stiffness) and internal factors (TCR stimulation) affect the CTL response.