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Theses - Pathology

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  • ItemEmbargo
    Role of RNA methyltransferases in lung cancer
    Klimontova, Mariia
    In recent years, RNA-modifying enzymes have gained significant attention because their activities are relevant to cancer biology, and they are therefore potential targets for novel therapeutic intervention. Here, I target validated two RNA methyltransferases. I performed a late-stage study of METTL3, and I initiated and developed an early-stage analysis of THUMPD3. Advanced validation of METTL3, a well characterised m⁶A RNA methyltransferase, was performed based on the availability of a specific small molecule inhibitor, METTL3i. I explored whether differences in sensitivities of lung cancer cell lines to METTL3i might be due to METTL3 binding specific gene promoters in a cell-dependent manner. My findings indicate that this is not the case. I also performed a synthetic lethality screen to identify epigenetic-related inhibitors that sensitise a METTL3i-resistant lung cancer cell line to the inhibitor. This identified potential pathways that could be targeted to overcome resistance to METTL3i-based therapy. Independently of the above, an in-house bioinformatic analysis indicated a potential human RNA methyltransferase, THUMPD3, was linked to lung cancer biology. Furthermore, early in my project, THUMPD3 was reported to catalyse m²G in tRNAs. However, whether THUMPD3 also methylates non-tRNA substrates was not addressed. My project sought to address whether other RNAs are indeed m²G modified by THUMPD3 and to further investigate and validate the role of the enzyme in lung cancer biology. Consistent with our bioinformatic data, depletion of THUMPD3 from lung cancer cells induced several notable effects; it negatively impacted cellular proliferation, and migration. It also induced apoptosis and disturbances in RNA splicing. Importantly, exogenous expression of THUMPD3 in normal lung fibroblasts stimulated their proliferation rate. Furthermore, transcriptome analysis indicated that changes in expression of proteins on the cell surface and the extra cellular matrix (ECM) may occur. To identify potential RNA substrates containing THUMPD3-dependent m²G, I leveraged a newly invented method, PhOxi-seq, which is based upon blue light-induced photo-oxidation of certain modified nucleotides in RNA, including m²G. Initially, I further developed and optimised PhOxi-seq on tRNA, leading to the discovery of THUMPD3-dependent alterations in m²G6 tRNA modification. Subsequently, I broadened the scope of my screen by including purified human rRNA-depleted RNA from lung cancer cells as substrate in the photo- oxidation reaction. This approach unveiled a cohort of RNAs, mainly mRNAs, several of which are implicated in the ECM. This links in well with my phenotypic analysis on the same cells. Notably, this is the first time that m²G has been reported in human mRNAs, as well as other polyA+ RNAs. The implications of these findings are discussed. In summary, my work extends the application of PhOxi-seq to identifying THUMPD3- dependent m²G sites (and potentially other oxidisable modifications induced by different enzymes) in various RNA types. My works also identifies a potential oncogenic role for THUMPD3 in lung cells. Thus, my findings highlight THUMPD3 as a potential therapeutic target, especially in a lung cancer setting.
  • ItemOpen Access
    Using Tumour Evolution to Understand the Epigenetic and Transcriptional Adaptations of Cancer to Host Immunity
    Baird, Tarrion
    Immunoediting describes the process in tumour development whereby tumours evolve to avoid the immune system. The relevance of immunoediting in carcinogenesis, metastasis and immunotherapy resistance makes understanding the mechanisms of immunoediting essential in informing cancer therapy and prevention. Previous work on immunoediting has largely focussed on neoantigen loss, but this thesis aimed to expand the immunoediting paradigm to incorporate transcriptomic and epigenetic changes in tumour cells. This thesis describes the epigenetic and transcriptomic characterisation of carcinogen-induced immunoedited and unedited tumour lines after growth *in vitro*. The transcriptome and immune infiltrate of immunoedited tumours undergoing progressive growth, or unedited tumours undergoing immune-mediated regression in wild-type mice was investigated. The results suggested that expression of T cell inhibitory molecules, a reduction in inflammation, and reduced expression of L1 transposable elements may be involved in immune escape in immunoedited tumours. An over-expression screen was performed to attempt to identify key genes involved in immunoedited tumour escape *in vivo*. Finally, a collection of bioinformatic tools were developed to perform process-based analysis of The Cancer Genome Atlas. These tools can suggest hypotheses of gene function and regulatory mechanisms and explore the pathological significance of gene expression signatures in human cancers. These tools are publicly available as an online web-tool. Both the identification of potential mechanisms of immunoediting and the bioinformatics tools developed in this thesis can support and inform further research.
  • ItemEmbargo
    Characterisation of the Mechanism of Norovirus VPg-Nucleotidylylation
    Pinckert, Malte
    Noroviruses, which belong to the *Caliciviridae* family, have a substantial yearly impact on the global economy of about $60 billion and cause about 200,000 deaths per annum in children under the age of 5. Despite causing most cases of viral gastroenteritis, noroviruses remain poorly characterised compared to other viruses. Infectious norovirus RNA is covalently linked to VPg (viral protein genome-linked) at the 5’ end of the genome through VPg-mediated RNA priming of viral RNA synthesis. While poorly understood mechanistically, this process is reliant on the addition of a nucleotide to a highly conserved tyrosine residue within VPg - this process is referred to as VPg nucleotidylylation and is catalysed by the viral polymerase (NS7). Using murine norovirus (MNV) as the primary model, this project sought to better understand this key stage of the norovirus life cycle, providing insight into a previously ill-characterised crucial stage of the viral life cycle and may help to identify key points which could be therapeutically targeted. Mass spectrometric analysis of the 5’ end of infectious norovirus RNA purified from infected cells, confirmed that VPg is genome-linked via a GDP moiety at a highly conserved tyrosine residue. These results however are contradicted by the state of the art on calicivirus VPg nucleotidylylation and has, to date, not been replicated *in vitro*. Therefore, developing a specific assay to measure *in vitro* VPg nucleotidylylation producing physiologically relevant readouts laid at the core of this project. Subsequently, this, for the first time, brought previous *in vivo*, genomic and *in vitro* observations of VPg nucleotidylylation in agreement and enabled me to study this reaction. I identified negative sense subgenomic RNA (-sgRNA), magnesium ions (Mg2+) and the viral protease (NS6) as essential components that facilitate specific and physiologically relevant VPg nucleotidylylation. Further, I showed precursor forms of NS6, NS56 and NS67, to stimulate the reaction more efficiently than mature NS6. Mutations within potential RNA binding regions of NS6 affected VPg nucleotidylylation *in vitro*, suggesting a potential role in the reaction for it as stabilizing the nucleotidylylation complex, through binding the RNA template. I identified a region within the -sgRNA of MNV, spanning region in the 3’ end of open reading frame 3 (ORF3) and the untranslated region (UTR), to be both sufficient and necessary for stimulating VPg nucleotidylylation. Through subsequent serial truncations, internal deletions and point mutations, I was then able to refine this understanding. My findings indicate VPg nucleotidylylation to be reliant on three distinct structural elements within this region, one of them a small, predicted stem-loop in the UTR. Utilising a panel of MNV mutants within the 3’ region of the genome, previously found to affect virus viability, I observed a clear correlation between virus viability and the negative sense 3’ region’s ability to enhance VPg nucleotidylylation. Lastly, I demonstrated the ORF3 UTR region of HuNoV RNA to be able to template MNV VPg nucleotidylylation, suggesting template and positional conservation between MNV and HuNoV. In summary, this work provides new key insights into VPg nucleotidylylation - a previously poorly characterised, but vital, process of norovirus replication and may pave the way for further exploration of this process for therapeutic targeting.
  • ItemOpen Access
    The role of RNA structures in the evolution of respiratory RNA virus genomes.
    Rigby, Charlotte; Rigby, Charlotte [0009-0004-3533-6679]
    RNA secondary structures in RNA viral genomes play important roles in a range of functions, including packaging, protection from cellular nucleases, and regulating viral replication. Furthermore, RNA structures have been shown to be immunostimulatory. In the case of Influenza A virus (IAV), during infection aberrant RNAs are produced. One of these are mini-viral RNAs (mvRNAs) which are very potent RIG-I stimulators. However, our understanding of the mechanisms that underlie the role of RNA structures in the above processes and how RNA structures evolve is still limited. In my thesis I first investigate whether mvRNAs are produced in human patients. Clinical samples were obtained from three influenza seasons and presence of mvRNAs were analysed via RT-PCR. Due to both technical and sample issues, no conclusion was reached. I next examined how RNA structures, including those capable of inducing mvRNA synthesis and increasing their immunostimulatory potential, are changing during pandemic IAV H3N2 evolution. Using in silico analyses, I observed a decline in RNA structure stability in the polymerase segments over time. Control analyses showed that this change was independent to amino acid mutation and reduced in human-adapted viruses, but present in emerging H1N1 IAV. Using transfections, I subsequently identified that amino acid changes in the IAV RNA polymerase make the enzyme more sensitive to RNA structures, suggesting that the loss in RNA structure stability and change in enzyme activity are linked. To determine if the loss of RNA structure was present in other emerging viruses, I analysed sequences of SARS-CoV-2 and found a similar decline in thermodynamic stability over time. Having identified a pattern of RNA structure loss in both IAV and SARS-CoV-2 I repeated the analysis on Ebola Zaire and Lassa virus to determine if similar patterns are observed in zoonotic viruses. I found this not to be the case. Based on these observations, I propose a novel model for emerging and pandemic RNA virus adaptation and hypothesise that immune pressure may select for viruses with improved replication efficiency, leading to RNA structure loss and a reduction in detection by immune receptors over time.
  • ItemOpen Access
    TRIM5α is a poxvirus restriction factor: viral evasion strategies and a pathway to antivirals
    Zhao, Yiqi; Zhao, Yiqi [0000-0003-4348-0588]
    This thesis describes cellular protein TRIM5α as a restriction factor for vaccinia virus (VACV) and two VACV countermeasures. This study followed up a proteomic analysis showing that VACV infection of TERT-immortalised human foetal foreskin fibroblasts (HFFF-TERTs) induced TRIM5 downregulation. This observation was validated in HFFF-TERT and HeLa cells, and TRIM5 degradation was shown to be proteasome-dependent. By using VACV mutants lacking blocks of genes near either genomic terminus, and thereafter mutants lacking individual genes, the gene encoding the viral protein that induces TRIM5α degradation was mapped to gene *C6L*. Furthermore, the inducible expression of C6 degraded endogenous TRIM5α outwith infection, showing that TRIM5α degradation does not require other viral proteins. Subsequent mechanistic studies showed that C6 interacts directly with the RING domain of TRIM5α. Finally, highly conserved orthologues of C6 in other orthopoxviruses such as cowpox virus (CPXV), rabbitpox virus (RPXV), camelpox virus (CMLV), elephantpox virus, monkeypox virus (MPXV) clades I and II and variola virus (VARV), were shown to co-precipitate and degrade TRIM5α outwith infection. A hypothesis to explain VACV-mediated TRIM5α degradation was that TRIM5α is an anti-VACV restriction factor, and therefore is removed by VACV to facilitate infection. To test this, cell lines lacking or over-expressing TRIM5 were generated. Whereas VACV replication and spread were enhanced in TRIM5-/- cell lines, over-expression of TRIM5α had the opposite phenotype. Reintroduction of wildtype (WT) TRIM5α into TRIM5-/- cells rescued TRIM5α-mediated restriction of VACV, but TRIM5α mutants that lacked E3 ubiquitin ligase activity, the ability to oligomerise or the C-terminal PRY/SPRY domain did not. Notably, during VACV infection, TRIM5α co-localised with virus replication factories, whereas in uninfected cells, TRIM5α was diffuse throughout the cytoplasm. In addition to C6-mediated degradation of TRIM5α, this thesis reports that VACV exploits the cellular protein, cyclophilin A (CypA), as an additional measure to counteract TRIM5α-mediated restriction. This work was based on prior observations that CypA is utilised by HIV-1 to evade restriction by human TRIM5α, CypA is incorporated into both VACV and HIV-1 virions and the inhibitor of CypA, cyclosporine A (CsA), has anti-viral activity. Whilst VACV replication and spread were reduced in CypA-/- cells, this phenotype was lost when TRIM5 was also knocked out, showing that CypA is proviral in a TRIM5-dependent manner. Furthermore, CsA restricted virus plaque size in WT but not in TRIM5-/- cells, showing that the antiviral activity of CsA is also TRIM5-dependent. To determine whether the peptidyl prolyl isomerase activity of CypA is required for its proviral function, two catalytic defective mutants were re-introduced into CypA-/- cells. Whilst WT CypA rescued virus replication and spread, the mutants did not, showing that CypA enzymatic activity is needed to antagonise TRIM5-mediated restriction. Next, the binding partner(s) of TRIM5α and CypA was identified by proteomic analysis of proteins that co-precipitated with TRIM5α and CypA during VACV infection. Of the several VACV structural proteins that co-precipitated with TRIM5α, only L3 was also precipitated by CypA, and this co-precipitation was lost in the presence of CsA. These interactions were validated during VACV infection and following ectopic expression of L3, and shown to be direct. The interaction between TRIM5α and L3 was mapped to the C-terminal domains of TRIM5α. During infection in the presence of L3, TRIM5α co-localised to virus factories, whereas when L3 expression was repressed, TRIM5α was elsewhere in the cell. L3 was shown to dimerise and dimerisation was enhanced by TRIM5α and this required its E3 ubiquitin ligase activity, and was antagonised by CypA. Furthermore, TRIM5α and L3 were shown to activate NF-κB synergistically, and this was antagonised by CypA but not a catalytically defective mutant. Like C6, L3 is highly conserved amongst orthopoxviruses and the L3 from MPXV and VARV co-precipitated with both TRIM5α and CypA, and the latter interaction was disrupted by CsA. Given the 2022 MPXV epidemic and the emergence of tecovirimat-resistant MPXV, the possibility of repurposing CsA as an anti-poxviral drug was tested. Since CsA is an immunosuppressant, two non-immunosuppressive derivatives, NIM811 and alisporivir, were also tested. All three drugs reduced VACV and MPXV replication and spread and disrupted the interaction between VACV and MPXV L3 and CypA. Therefore, NIM811 and alisporivir have anti-poxviral activity and potential clinical applications.
  • ItemEmbargo
    Algorithm development for RNA structure prediction in RNA viruses
    Tumescheit, Charlotte
    RNA structures play many different roles in the life cycles of RNA viruses, including in directing translational control, genome replication, subgenomic RNA synthesis, and encapsidation. However, predicting these structures can be challenging, especially when it comes to pseudoknots, long-range interactions, and mutually exclusive interactions or switches. Furthermore, separating biologically relevant structures from the vast number of interactions that could theoretically form can be problematic. Since the sequences of RNA viruses evolve rapidly and, for many species, there are a large number of sequenced isolates, comparative genomics can be used to predict structures that are conserved and therefore more likely to be biologically relevant. Here, a new program is presented that aims to fill the gaps among existing RNA structure prediction programs by looking for all possibly functionally relevant short-range and long-range interactions that are conserved within a multiple sequence alignment, thereby allowing for the detection of pseudoknots and mutually exclusive structures. An offset parameter makes it possible to find interactions that do not align perfectly, thereby allowing for the possibility to look at more divergent sequences. This can further improve the separation of functional structures from random structures. Furthermore, a phylogenetic weighting scheme aims to balance alignments that may mix an abundance of closely related sequences with a few distantly related sequences, besides mitigating potential problems introduced by sequencing errors. The free energy is taken into account to reduce the number of false positives. The program to predict potential secondary structures in RNA viruses is embedded in a pipeline that, based on a reference sequence, obtains related sequences, builds a sequence alignment and phylogenetic tree, and creates all the necessary files to perform the analysis. The pipeline is then applied to a selection of viruses. Also, a novel tool is presented for improving the quality of multiple sequence alignments by trimming poorly aligned regions, and for visualising alignments and alignment processing steps. The structure prediction pipeline presented identifies known functionally important structures and suggests new structures for potential experimental follow up. Different options and adjustable parameters allow for individual workflows. By filling an underrepresented niche, the software will hopefully help guide future molecular understanding of RNA viruses.
  • ItemOpen Access
    Role of the Innate Immune Response to DNA During Therapy with Oncolytic Viruses
    Wagner, Emma
    Oncolytic viruses (OVs) are viruses which selectively replicate within and directly lyse cancer cells whilst sparing host tissue. This selectivity not only helps their safety profiles and minimise toxicities but also allows OVs to potentially increase their therapeutic dose over time as they replicate and spread throughout tumours. Whilst tumour lysis helps reduce tumour burden, a secondary purpose of OVs as immunotherapies is to stimulate the host's immune system culminating in a systemic anti-tumour response. To do so relies on OVs ability to trigger to two distinct forms of immunity simultaneously: anti-tumour and anti-viral. OV-induced anti-viral immunity typically refers to release of virions, cytokines and damage- or pathogen-associated molecular patterns (DAMPs and PAMPs) to the tumour microenvironment (TME) upon tumour lysis. Doing so helps to recruit innate and adaptive immune cells to the TME, helping to overcome its naturally immunosuppressive state and stimulate the generation, recruitment and activation of tumour antigen-specific T cell responses. In theory, this combination of anti-viral and anti-tumour immunities has the potential to build to a systemic anti-tumour T cell response that targets distant, secondary tumour sites. In reality, although effective in reducing local tumour burden in the short-term, OVs struggle to produce sustainable anti-tumour responses in the long-term as monotherapies. Recent research into improving OV efficacy has focused on enhancing anti- tumour immunity by combining OVs with other immunotherapies such as immune checkpoint inhibitors (ICI). However, to ultimately achieve a sustainable systemic anti-tumour response against future tumour relapse, a greater understanding of OV-induced anti-viral immunity is needed. In particular, little is known about cancer cell’s intrinsic ability to sense and respond to OV infection and how this may impact OV efficacy. The purpose of this study was to gain a mechanistic understanding of how tumour cells may sense infection by a vaccinia (VACV)-derived OV, superior killing virus (SKV), through cytoplasmic DNA sensing pathways and how manipulation of this pathway may impact OV efficacy. Here, I screened several syngeneic murine cancer cell lines for their expression of cytoplasmic DNA sensing proteins and susceptibility to OV infection. After establishing the BRAF-mutant melanoma cell line 4434 as our model system, I found that deletion of cytoplasmic DNA sensor cyclic GMP-AMP Synthase (cGAS) resulted in loss of STING-dependent signalling in response to DNA stimulation and SKV infection whilst deletion of DNA-dependent protein kinase (DNA-PK) complex protein Ku80 did not. Despite these differences in STING-dependent signalling, loss of either cGAS or DNA-PK resulted in loss of pro-inflammatory signalling and significantly increased SKV replication *in vitro*. This was also true *in vivo*, where cGAS-/- tumours had significantly increased SKV load in comparison to WT tumours. Interestingly, deleting cGAS also had an impact on tumour growth and immune cell infiltration to the TME prior to SKV treatment. Altogether, this work highlights the importance of investigating the underlying activity of cytoplasmic DNA sensing pathways in tumours prior to OV therapy. In particular, these results indicate that VACV-derived OV treatment efficacy of tumours expressing cGAS and DNA-PK may be enhanced by incorporating cGAS and DNA-PK inhibitors to their design.
  • ItemEmbargo
    Comparative analysis of African and Asian/American Zika virus strains at the transcriptional and translational level
    Lefèvre, Charlotte
    Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. Initially, ZIKV was considered of low importance as infection was usually asymptomatic or caused mild illness. However, the emergence of the Asian/American lineage, in contrast to the African lineage, was considered a public health threat. The Asian/American lineage has been associated with neurological complications such as Guillain-Barré syndrome in adults and congenital ZIKV syndrome (e.g., microcephaly) in newborns. Currently, it is not understood why this newly emerged Asian/American ZIKV results in a more severe disease than the African ZIKV. This project focused on understanding the molecular mechanisms underlying the differences in virulence and pathogenicity between these two ZIKV lineages in human brain cells. In order to identify the molecular determinants that influence viral replication and subsequent ZIKV virulence, the differences between an African and an American ZIKV strain were studied at different levels. Firstly, RNA-Seq and ribosome profiling (Ribo-Seq) was performed on astrocytoma-glioblastoma cells (U251 cells) infected with an African and an American ZIKV strain to determine the transcriptional and translational profile of each virus. Two previously overlooked upstream open reading frames (uORFs) in the 5′ untranslated region (UTR) of the American ZIKV genome were identified, initiating at non-canonical CUG and UUG codons; and a single uORF in the 5′UTR of the African ZIKV, initiating at a CUG start codon. The uORFs in the American ZIKV are referred to as ‘uORF1’ and ‘uORF2’, and the one in the African ZIKV as the ‘African uORF’. Secondly, considering their overlap with the main ORF, the viral polyprotein, it was hypothesised that these uORFs could play a role in the translational regulation of the main ORF. To investigate this, luciferase reporter assays were used, which showed that when the ZIKV 5′UTR harboured an African uORF or when uORF1 was not expressed, the main ORF was better translated compared to the wild-type, in contrast to the knock-out of uORF2. Thirdly, the presence of these uORFs was shown to modulate virus growth and replication by using mutant viruses that modulate the expression of the different uORFs and comparing these to the wild-type American ZIKV strain by performing viral growth curves and competition assays. Interestingly, the uORF2 knock-out virus showed a similar infectivity as the wild-type American virus, whereas the uORF1 knock-out virus and the African-like virus reached higher fitness than the wild-type virus. Additionally, transient overexpression of the uORF1 peptide and its subcellular localisation within the cytoskeletal fraction suggests a contribution to the dysregulation of the cytoskeleton, which has been associated with neurodegenerative diseases. Finally, a differential gene expression analysis was performed in which cellular genes that were differentially transcribed by using the RNA-Seq datasets, but also the genes that had a different translational efficiency by using the Ribo-Seq datasets in response to infection with different ZIKV strains were thoroughly analysed. Infection of U251 cells with an African ZIKV strain led to an early upregulation of innate immune response genes, but infection with an American ZIKV strain induced the expression of a broader range of genes related to the antiviral response. With this first analysis of neurotropic flavivirus gene expression using ribosome profiling, the functional characterisation of novel uORFs and the comparative analysis of differentially up- or down-regulated cellular genes, novel insights were provided into important differences between the African and the Asian/American ZIKV lineages, two viruses that result in different pathologies.
  • ItemEmbargo
    Investigating the ubiquitin conjugating machinery in *Plasmodium falciparum*
    Smith, Cameron
    *Plasmodium falciparum* is the causative agent of the most lethal form of human malaria. In the absence of a vaccine and the emergence of drug-resistant parasites, novel targets against malaria are required. The ubiquitin-proteasome system (UPS), which involves the attachment of the small protein ubiquitin to target proteins, is essential to all eukaryotes and represents a promising target for antimalarial therapies. However, the components of this system are largely uncharacterised. The work undertaken in this thesis explores the ubiquitin landscape of *P.falciparum* with a focus on E3 ubiquitin ligases as the most numerous and diverse component of this pathway. Activity based probes (ABPs) were used to identify ubiquitin pathway enzymes expressed and active during the asexual stage of *P.falciparum*. Two ABPs Ub-Dha and Ub-PA were used co-operatively in concert with immunoprecipitation and mass spectrometry (IP-MS/MS) to identify several families of ubiquitin enzymes as well as proteins of unknown function. In vitro ubiquitination assays validated the activity of identified E2 ubiquitin conjugating enzymes, and a HECT E3 ligase, PfHEUL. The catalytic residue of PfHEUL was found to be C8558 by site-directed mutagenesis, however, attempts to genetically modify the PfHEUL locus in vivo were unsuccessful indicating gene immutability and emphasising the importance of the gene function to asexual stage parasites. Two families of Cullin-RING ligases (CRLs) were identified in *P.falciparum*, and a transgenic approach was used to tag endogenous PfCullin1 and PfCullin2 to facilitate localisation and co-immunoprecipitation studies. Associated proteins identified through this approach indicated a role for PfCullin1 in DNA replication and PfCullin2 in protein homeostasis. PfFBXO6, an F-box domain containing protein found to be associated with PfCullin1, was shown to interact with PfSkp1 and to be involved in DNA replication through interactions with the MCM complex in the nucleus. Conditional knockdown of PfFBXO6, in conjunction with ubiquitin-derived diGly remnant enrichment, revealed potential ubiquitination substrates of PfFBXO6 containing CRL1 (PfSCFFBXO6) involved in DNA replication and gametocytogenesis. The utilisation of data-independent acquisition (DIA) mass spectrometry generated the largest Plasmodium ubiquitome to date. These findings contribute to a deeper understanding of the ubiquitin pathway in *P.falciparum* and provide potential targets for the development of antimalarial therapies.
  • ItemEmbargo
    Mechanisms of cell adhesion regulation by herpes simplex virus
    Barrow, Henry
    Herpes simplex virus (HSV)-1 is a highly prevalent human pathogen that establishes a life-long infection. HSV-1 promotes its replication and spread by expressing multi-functional proteins that extensively remodel the host cell. Three such proteins are pUL21, pUL7 and pUL51. pUL21 is a viral phosphatase adapter. pUL7 and pUL51 form a complex that localises to juxtanuclear membranes and sites of cell-matrix adhesion termed focal adhesions. All three proteins are required for efficient virus assembly, egress and cell-to-cell spread. However, the viral or cellular binding partners required for these functions have not yet been fully identified. Furthermore, very little is known about how the interactions of pUL7:pUL51 at focal adhesions promote virus replication, spread or survival. Biotin-proximity ligation (BioID) was used to identify novel viral and cellular protein interaction partners for pUL21 and pUL7:pUL51. The IPP complex, which consists of integrin-linked kinase (ILK), PINCH and parvin, was identified as a potential interaction partner for pUL7:pUL51 at focal adhesions. Colocalisation and a direct interaction was confirmed using immunofluorescence microscopy and biochemical approaches respectively, with binding between pUL51 and ILK identified as primarily responsible for the interaction. Expression of pUL7:pUL51 was shown to alter focal adhesion morphology and be important for preventing infected cell rounding and detachment. Cell lines recombinantly expressing pUL7:pUL51 showed that the complex directly alters cell adhesion dynamics by likely preventing focal adhesion disassembly. pUL7:pUL51 could not localise to focal adhesions and prevent cell rounding in the absence of ILK, confirming the importance of this interaction for function. However, no large defect in cell-to-cell spread was observed in the absence of ILK, suggesting this is not a mechanism by which pUL7:pUL51 promotes virus spread between cells. When performing the BioID experiments it was observed that fusion of the biotin ligase to pUL7 had a dominant negative effect on viral replication and cell-to-cell spread. This selective pressure was exploited to perform *in vitro* evolution experiments in which the virus adapted to this attenuation. Whole genome sequencing of the adapted virus population identified several HSV-1 genes that may promote virus replication and cell-to-cell spread through interactions or functional relationships with pUL7:pUL51. Focal adhesions are highly dynamic cellular platforms that mediate bidirectional signalling between the interior of the cell and the extracellular environment. This study characterises the only known example of a virus directly manipulating focal adhesion dynamics to prevent cell detachment, identifying a direct interaction between pUL7:pUL51 and ILK. The findings provide a molecular framework to understand the regulation of focal adhesions by HSV-1, accelerating future molecular and functional studies.
  • ItemEmbargo
    Applying phenotypic phage selection to Chlamydia trachomatis infection
    Adams, Simone
    *Chlamydia trachomatis* (*Ctr*) is an obligate intracellular pathogen that causes the most common bacterial sexually transmitted infection worldwide and is the agent of trachoma, an ocular infection that is the leading cause of preventable blindness. *Ctr* has a biphasic lifecycle dependent on an infectious extracellular form, the elementary body (EB), and a non-infectious replicative form, the reticulate body (RB). EBs induce entry into host cells and reside within a specialised vacuole termed an inclusion where EBs differentiate into RBs. Following replication and redifferentiation from RB to EB, *Ctr* bacteria exit the host cell by stimulating host cell lysis or extrusion of the inclusion. *Ctr* virulence proteins are translocated into the host cell by EBs and RBs, and subvert host cell functions from the inclusion membrane, host cytosol or the inclusion lumen to propagate this cycle. The detection of *Ctr* infection remains challenging, and no vaccine is available. As the obligate intracellular lifecycle of *Ctr* is more reminiscent of viral than most other bacterial infections, bacterial entry and exit might present opportunities for intervention. Monoclonal antibodies (mAb) have recently been identified that enable the detection and treatment of viral infectious diseases but have yet to be comprehensively applied to study bacterial infections. Such mAbs can be identified by target based or phenotypic phage display. Here, phenotypic phage display was performed in two ways: (1) on a population of cells infected with *Ctr* or (2) on EB-enriched material derived from lysed, infected cells. In (1), panning for antigens was performed on the surface of infected cells to understand how antigen exposure at the plasma membrane may be altered by intracellular infection. Antibodies identified by this screen provided insights into the challenges of selection and revealed two differentially expressed surface antigens that signify *Ctr* infection. In selection (2), panning was performed on material released from infected cells including intact, asynchronous bacteria, and host and chlamydial antigens. Antibodies derived from this screen specifically recognised *Ctr*-infected cells when probed by immunofluorescence. Three distinct binding patterns (diffuse, inclusion-recognising, or inclusion-recognising with filaments) were evident. By immunofluorescence, four of the antibodies recognized a punctate structure within the inclusion lumen at 24 – 48 hours post infection (hpi) which then expanded to form a novel vesicular structure, primarily by 56 hpi. The vesicles are constant in diameter and most abundant in inclusions nearly devoid of bacteria. These previously unrecognised sparsely populated inclusions appear to develop during infection as while 7% of inclusions are sparse at 24 hpi this increases to 30% at 56 hpi. Characterization of the vesicles by microscopy indicates that they are membrane-enveloped structures with distinct composition to the inclusion membrane that sometimes colocalize with canonical bacterial membrane markers. Co-immunoprecipitation and mass spectrometry were used to identify the protein target of one antibody. Interestingly, binding to a phospholipid array revealed the antibody also strongly recognized phosphatidylinositol (5) phosphate. This study has demonstrated that the application of phage display to *Ctr* infection can be a valuable tool to study intracellular infections. The antibodies derived from the screen have provided insights to the *Ctr* lifecycle and host-pathogen interactions. The identification of a novel structure within the inclusion reveals how little is known during the late stage of the lifecycle, and the findings raise questions about the fate of the inclusion after infection when lysis does not occur. In the future, this technology could be applied to other intracellular pathogens to better understand their functions and to potentially create therapeutics.
  • ItemOpen Access
    Investigating the function of Citron kinase and its regulation by other mitotic kinases
    Halcrow, Ella
    Cytokinesis is the final stage of cell division, resulting in the generation of two separate daughter cells. This stage encompasses the physical segregation of the cytoplasm between the nascent daughter cells and is driven by the constriction of the actomyosin contractile ring that bisects the mother cell and the segregated genomic material. The contractile ring progressively compacts the central spindle microtubules to form an intercellular bridge that contains a small organelle at its centre, known as the midbody. The midbody is essential for the final abscission events, acting as a platform to recruit the essential proteins. Failure in cytokinesis is associated with many human diseases, including cancer, microcephaly, infertility and blood disorders, thus understanding the mechanisms underpinning this process is crucial for the development of treatments for these pathologies. In this thesis I investigated the function and regulation of the contractile ring component Citron-kinase (CIT-K). CIT-K is a conserved serine/threonine kinases with an evolutionary conserved role in the formation and organisation of the midbody in late cytokinesis. In the absence of CIT-K, the midbody matrix becomes scarce and detached from the equatorial cortex, highlighting the importance of CIT-K in maintaining the proper architecture of the midbody. Recent research from the host laboratory has indicated that CIT-K is regulated by other mitotic kinases, including Cdk1 and Aurora B, suggesting that its functions must be tightly controlled. Furthermore, although several lines of evidence indicate that CIT-K kinase activity is important (the most significant being the identification of mutations in the CIT-K kinase domain in microcephaly patients), only one known CIT-K substrate, INCENP, has been identified so far. To assess the role and function of CIT-K kinase activity I employed the chemical genetic approach of generating an analogue sensitive (AS) variant capable of accepting a “bulky” ATP analogue that would selective inhibit this kinase. Through a series of *in vitro* phosphorylation assays of four AS variants, I identified that the AS-CIT-K M174G variant retained the highest levels of kinase activity relative to the WT levels. I then tested four inhibitors for their ability to inactivate AS-CIT-K M174G kinase and identified 3MB-PP1 as the most efficient one. Unfortunately, expression of the CIT-K M174G mutant in cultured cells failed to rescue the functions of CIT-K, most likely because this mutant did not retain sufficient kinase activity. To investigate the regulation of CIT-K by other mitotic kinases, I focused on the phosphorylation of two serines, S440 and S699, by Cdk1 and Aurora B. I utilised phospho-specific antibodies and phospho-mimetic and non-phosphorylatable mutants to investigate the role of these two phosphorylation events. My experiments indicated that CIT-K is phosphorylated at both S440 and S699 in early mitotic stages, but whilst S440 is de-phosphorylated right after anaphase onset, S699 remains phosphorylated into anaphase and telophase. Furthermore, perturbing both phosphorylation events led to incorrect localisation of CIT-K, and in the case of S440 to abnormal midbody formation, and accumulation of midbody remnants. *In vivo* pull downs indicated that both phosphorylation events significantly reduced the interaction of CIT-K with its midbody partners Aurora B, KIF14 and KIF23/MKPL1. Together, these findings indicate that the coordinated regulation of CIT-K by Cdk1 and Aurora B temporally regulates the association of CIT-K with its partners in order to finely control midbody formation.
  • ItemControlled Access
    Targeting PfUCHL3 Using Chemically Constrained Peptides
    King, Harry
    PfUCHL3 is a highly conserved, dual specificity deubiquitinating and deNeddylating enzyme acting within the ubiquitin proteasome system (UPS) of *Plasmodium falciparum*, the deadliest and most prominent *Plasmodium* species responsible for the transmission of malaria to humans. Malaria kills on average 450,000 people per year across 90 different countries, and there is at present no broadly effective vaccine against the disease. PfUCHL3 is essential to *P. falciparum* cell viability, and through the use of the RaPID peptide discovery system (Yamagishi Y et al, 2011), 10 novel, non-natural, cyclic peptides were selected for against PfUCHL3. The peptides all display tight dissociation constants for PfUCHL3 in the low nanomolar range (6-35 nM) and 4 of the 10 peptides inhibit the activity of PfUCHL3 in cleaving its ubiquitin substrate *in vitro*. The peptides all have selectivity for PfUCHL3 over HsUCHL3, the human homologue of the enzyme, and through NMR analysis it was shown that all 4 of the inhibitory peptides bind to the substrate recognition region of PfUCHL3. Thus, the peptides act by interfering with ubiquitin’s ability to bind to PfUCHL3 and represent the first reported inhibitory peptides for PfUCHL3. Through the use of thermal shift assays the inhibitory peptide’s were shown to stabilise the proteins structure upon binding and increase the melting temperature. Work is currently underway to determine the cell permeability properties of the peptides and their ability to kill *Plasmodium* parasites *in vivo* with the long-term aim of taking these peptides forward for the generation of a novel class of anti-malarial therapeutics. (Yamagishi Y et al, 2011) - Yamagishi Y, Shoji I, Miyagawa S, Kawakami T, Katoh T, Goto Y, Suga H. Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem Biol. Dec 23;18(12):1562-70. 2011
  • ItemEmbargo
    The Role of Ribosomal Maturation Proteins WBSCR22 and EMG1 Methyltransferase Activity in Cancer
    Benedetti, Anna Mary
    Internal RNA modifications are found both in coding and noncoding RNAs. In the ribosome, two highly conserved proteins catalyse the methylation of guanosine 1639 and pseudouridine 1248 residue of the 18S rRNA and they are known as WBSCR22 and EMG1. Both proteins are key factors for ribosome biogenesis and previous studies have demonstrated that in this context, they both act as scaffolds and that their catalytic activity is dispensable for 18S rRNA maturation. Moreover, high expression of both methyltransferases has been reported in acute myeloid leukaemia (AML) and uveal melanoma (UVM), where it strongly correlates with poor prognosis. However, the specific role of these enzymes in cancer is still unknown. I identified both WBSCR22 and EMG1 catalytic activity as essential for the growth of AML and UVM cells with a model in which the endogenous proteins are knocked down through a stable shRNA-inducible system and rescued with a wild-type or a catalytically dead variant. In AML, inducible depletion of WBSCR22 and EMG1 through both shRNA knock-down and CRISPR-Cas9 targeting, causes cell cycle arrest which cannot be rescued by catalytically inactive protein variants and is independent from ribosome maturation. Similarly, inactivation of WBSCR22 in uveal melanoma results in proliferation arrest, decreased migration, and reduced anchorage-independent growth, all while preserving ribosome integrity and function. These data show that, WBSCR22 has an additional function extrinsic to ribosome biogenesis as a regulator of oncogenic protein translation. By using polysome profiling coupled with next generation sequencing I characterized genes that showed no differences in total mRNA expression levels but are less translated when the m7G modification is removed from the 18S rRNA. Among these genes, I identified essential drivers of metastatic uveal melanoma such as cMET. Finally, the therapeutic potential of WBSCR22 catalytic inactivation was explored for the first time as a putative anti-metastatic combination therapy with crizotinib. Preliminary data show that sub lethal concentrations of crizotinib are sufficient to reduce uveal melanoma WBSCR22 catalytic mutant cell metastatic potential. Together, these data define WBSCR22 as a regulator of migration and endothelial-mesenchymal transition pathways that drive metastasis formation in uveal melanoma and identify this enzyme as a potential therapeutic target.
  • ItemOpen Access
    Molecular Mechanism of MDA5 RNA Sensing in Innate Immunity and Disease Pathogenesis
    Singh, Rahul
    The innate immune response plays a crucial role in recognising and combating viral infections. Double-stranded RNA (dsRNA) is a potent proinflammatory signal, and its recognition by MDA5 and its coregulatory factor, LGP2, is essential for initiating antiviral responses. This study aimed to investigate the structure and function of MDA5 and LGP2. I showed the cooperative nature of ATP hydrolysis in MDA5-dsRNA filaments. I discovered that adjacent MDA5 subunits in a filament hydrolyse ATP cooperatively, leading to coupled filament disassembly which allows MDA5 to displace tightly bound proteins from dsRNA. Unlike MDA5, LGP2 exhibits noncooperative ATP hydrolysis and ATP dependent internal binding to dsRNA which promotes the assembly of shorter MDA5 filaments, potentially enhancing filament stability or expanding the range of dsRNA ligands that MDA5 can interact with. The M854K MDA5 variant is associated with interferonopathy. I revealed that the M854K mutation abolishes ATPase activity, stabilises MDA5-dsRNA complexes, increases IFN-β transcription, and disrupts signal transduction. Cryo-electron microscopy structures of wild-type (WT) and M854K MDA5-dsRNA filaments provided detailed insights into how the M854K mutation hinders conformational changes necessary for ATP hydrolysis and dissociation from endogenous RNAs, resulting in constitutive activation of signalling pathways. In contrast, the partial loss-of-function I923V variant of MDA5 is type 1 diabetes (T1D) protective but increases vulnerability to viral infections. I showed how the I923V mutation causes hyperactive ATP hydrolysis, resulting in shorter and less stable filaments that have a relaxed grip on dsRNA. The I923V mutation disrupts the molecular brake function of residue 923, leading to accelerated ATP hydrolysis and expansion of the RNA binding footprint. This work provides structural and functional data of MDA5 variants and its regulatory factors during the innate immune response. Understanding the mechanisms of MDA5 variants has the potential to inform the development of novel therapeutics and enhance personalised medicine approaches.
  • ItemOpen Access
    HPV E6 inhibits E6AP to regulate epithelial homeostasis by modulating keratinocyte differentiation commitment and YAP1 activation
    Yin, Wen
    Human papillomaviruses (HPV) typically cause chronic infections by modulating homeostasis of infected basal cell to ensure persistence. Using FUCCI and cell-cell competition assays, we established the role of two common viral targets of low-risk and high-risk E6 proteins, E6AP and NHERF1, on the key components of epithelial homeostasis: cell density, proliferation, commitment to differentiation and basal layer delamination. Importantly, we demonstrate that deletion of E6AP in keratinocytes delayed the onset of differentiation and the abundance of E6AP is reduced in HPV-infected tissue. RNA sequencing revealed similar transcriptional profiles of E6-expressing cells and E6AP-/- cells. Amongst the pathways identified, YAP target genes were activated by either E6 expression or E6AP depletion. This is confirmed by the analysis of YAP expression pattern in both monolayer cell culture and HPV-infected lesions. As the conserved binding partner of Alpha group HPV E6 proteins, the precise role of E6AP in modulating keratinocyte phenotype and associated signalling pathways have not been defined. Our study suggests a model which explains the preserved functions of low-risk and high-risk Alpha E6 in epithelial homeostasis by inhibiting E6AP’s activity, hence leading to alteration of multiple downstream pathways including YAP activation. Potential treatments can thus be developed.
  • ItemEmbargo
    Post-translational control of mitochondria by Fbxo7 in Parkinson's disease
    Simpson, Lorna
    Parkinson’s disease is a progressive, neurodegenerative disorder which is characterised by the loss of dopaminergic neurons within the substantia nigra. At a cellular level, mitochondrial dysfunction, oxidative stress and proteasomal dysfunction are all implicated in the pathogenesis of the disease. The majority of PD cases are late onset and arise sporadically, with age being the primary risk factor. Approximately 5-10% of cases however are early onset, occurring in individuals under the age of 50. These early forms of PD are typically caused by mutations in one or more Parkinson’s-related genes. In 2008, mutations in the gene FBXO7 were identified as causing early-onset Parkinsonian pyramidal syndrome. Fbxo7’s canonical function is as the substrate recruiting domain of an E3 SCF ubiquitin ligase complex. Mitochondrial defects have previously been detected in cells lacking Fbxo7 expression, although the precise mechanisms through which Fbxo7 protects cells from mitochondrial dysfunction and neuronal cell death are not currently fully understood. In this thesis I characterised two patient fibroblast cell lines with novel mutations in FBXO7. A leucine to proline mutation at amino acid 250 was found to be destabilising and to disrupt the interaction between Fbxo7 and its binding partner PI31. Fibroblasts carrying this mutation were also found to have reduced levels of mitochondrial respiration, increased ROS and increased sensitivity to oxidative and proteasomal stress. Fibroblasts with an N-terminal truncating S93X mutation were also found to have higher levels of ROS, increased mitochondrial fragmentation and increased mitochondrial membrane depolarisation, indicative of elevated mitochondrial dysfunction. In addition, I validated the mitochondrial fission adaptors MiD49 and MiD51 as novel binding partners of Fbxo7. MiD49 and MiD51 protein levels were found to be stabilised in the presence of Fbxo7, independently of Fbxo7’s activity as part of an SCF ligase complex. MiD49 and MiD51 were also found to interact with PI31 with initial evidence suggesting that PI31 may compete with the fission adaptors for binding to Fbxo7 through the ligase’s FP domain. Overall, in this thesis I provide further evidence for the role of Fbxo7 in the development of PPS and describe a novel mitochondrial function for Fbxo7 in the stabilisation of the fission adaptors MiD49 and MiD51.
  • ItemEmbargo
    Exploring antiviral effector cells in vaccinia virus-infected tissues
    Shannon, John
    Vaccinia virus (VACV) is a large, double-stranded DNA virus belonging to the Poxviridae family that was used as the live vaccine leading to the eradication of smallpox. Despite eradication through global vaccination, the precise immune mechanisms underlying recovery from infection are incompletely understood. VACV is commonly studied after infection of the lung to model human smallpox vaccination; however, most current VACV infection models lack information on the anatomical organisation of immune cells interacting with virus-infected cells in tissues. To understand antiviral immunity in critical barrier tissues, this study investigated the spatial organisation of both leukocytes and VACV-infected cells in two different models: epicutaneous infection of the skin and a newly developed model of mouse lip (labial mucosal) infection. After epicutaneous infection of the skin, T cells traffic into the dermis and eliminate VACV- infected inflammatory monocytes. During the course of infection, VACV replication leads to a marked increase in the permeability of local blood vessels that occurs prior to the entry of CD8+ T cells. Pharmacological Inhibition of VEGF receptor (VEGFR)-signalling abrogated VACV-induced vascular leakage. Furthermore, CD8+ T cell-mediated clearance of infected monocytes also reduced vascular permeability. After mucosal infection in the lip, VACV replicates to high titres and both group 1 innate lymphoid cells (ILCs) and T cells are recruited to infected tissue. Intravital imaging revealed that group 1 ILCs continuously patrol the epithelium in both naïve and infected tissues and are the major immune force limiting oral mucosal viral infection at early timepoints. Unexpectedly, group 1 ILCs produce interferon gamma (IFN-γ) irrespective of contact with virus-infected cells, even in the uninfected epithelium, invoking an antiviral state independent of infection. Elimination of group 1 ILCs or neutralisation of IFN-γ decreased the expression of several key antiviral genes in the uninfected oral mucosa. Although ILC1s play an early critical role in restricting VACV spread in the labial mucosa, these cells are not sufficient alone to clear labial VACV infection as *Rag1-/-* mice (which lack T and B cells, but retain ILCs) have prolonged viral replication which can be reduced by adoptively transferred T cells. Together, these findings illustrate the complexities of immune-cell-mediated interactions and protection in the tissue, from the kinetics of leukocyte entry and elimination of virus-infected cells to the amplification of antiviral signals.