SACM - United Kingdom
Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/9667
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Item Restricted Dissecting the structural requirements for Notch/ligand interactions with mutations derived from cancer genome sequencing(The University of Manchester, 2025) Alanazi, Areej; Baron, MartinNotch is a cell surface receptor with critical roles in development and cellular differentiation, and its altered activity is frequently associated with cancer. Despite extensive sequencing efforts identifying numerous cancer-associated mutations in Notch, the functional consequences of many of these mutations remain poorly understood. Notch activation is mediated by its interaction with cell surface ligands, Delta and Serrate/Jagged, triggering proteolytic cleavage events that release the Notch intracellular domain (NICD). The NICD translocates to the nucleus to regulate transcription, and Notch can also be activated through ligand-independent mechanisms following endocytosis. This thesis exploits the high sequence conservation between human and Drosophila Notch to dissect how cancer-associated mutations alter receptor functionality, using an approach comprising in vitro and in vivo analyses. This work focuses on mutations within the ligand-binding region that are associated with cancers where Notch acts as a tumour suppressor. Through cell culture assays, I categorised Notch mutants based on their ligand-binding properties, signalling efficiencies, and ligand-independent activities. Using CRISPR/Cas9, these mutations were introduced into the Drosophila genome, enabling a comprehensive study of their phenotypic consequences. My analyses revealed diverse mutant classes, including those that completely eliminate signalling or retain partial functionality. For example, I identified mutants that discriminated between different Notch ligands, removed ligand-dependent signalling while retaining ligand-independent activity, and one mutant that retained ligand-dependent activation but removed ligand-independent activity. I also found that different cell-based assays could distinguish between levels of ligand associations required for cell adhesion and cell signalling. In vivo genetic interaction studies further refined our understanding of mutant classifications, revealing Notch mutant-specific interactions with a panel of genetic modifiers and uncovering phenotypes that deviated significantly from null-like behaviour. Additionally, I demonstrated that several mutations compromised cis-inhibitory interactions, with heterogeneous impacts on this regulatory mechanism across different developmental contexts. These findings provide valuable insights into the regulatory dynamics of Notch signalling, paving the way for advancing our understanding of the pathway’s role in development and disease.23 0Item Restricted Using a high-throughput sequencing approach to investigate the effect of ribavirin treatment on the human respiratory syncytial virus genome and its impact on host cells(University of Liverpool, 2023-12) Shawli, Ghada; Hiscox, JulianHuman respiratory syncytial virus (HRSV) infection is the most common cause of lower respiratory tract infections in infants and children worldwide. It can affect individuals of all ages, particularly those with compromised or weakened immune systems. By the age of two, nearly everyone has been infected with HRSV, with symptoms ranging from mild cold-like illness to severe bronchiolitis or pneumonia. Unfortunately, there is currently no safe and effective antiviral treatment available for HRSV infection. Ribavirin (RBV), a guanosine analogue, is the only drug approved for the treatment of severe HRSV lower respiratory tract infections. However, its use is associated with concerns due to its toxicity. RBV can act as a mutagen because its triazole carboxamide pseudobase can base pair with both uracil and cytosine, depending on the orientation of the amide group, leading to increased mutation rates in the viral genome. Understanding both the direct effects of RBV on HRSV infection and its indirect effects on host cells could therefore be valuable for advancing therapeutic strategies. This thesis focused on investigating the antiviral effects of RBV on both HRSV infected and uninfected A549 cells using high-throughput sequencing methods, including both short-read and long-read sequencing platforms. The first approach quantified the increased mutation rates between HRSV infected and RBV treated HRSV conditions using short-read Illumina sequencing. To date, the mutagenic effect of RBV on the HRSV genome in A549 cells has not been tested. A significant increase in transition mutations was observed in the presence of RBV treatment. Complementing the Illumina sequencing results, long-read direct RNA sequencing was employed to detect ribavirin triphosphate as a single molecule incorporated into the viral genome. Using xPore software, the differences in signal intensity between synthetic and natural nucleotides was calculated. Only kmers with NNGNN or NNANN were included in this analysis; eleven positions were identified where ribavirin triphosphate may have been incorporated into the viral genome during RNA synthesis. Interestingly, the counts of NNANN kmers were four-fold higher than those of NNGNN kmers, suggesting that RBV preferentially mimics adenine (A) nucleotides. Next, the incorporation of RBV during the polyadenylation process was investigated by examining the difference in the poly(A) tail length between HRSV and HRSV_RBV conditions using Nanopolish software. A significant reduction in poly(A) tail of some HRSV transcripts treated with ribavirin was found at 9 h and 24 h post infection. This long-read sequencing data provides further insight into ribavirin triphosphate incorporation and its potential use as a substrate by viral polymerase during polyadenylation. Finally, the effect of RBV on the host cellular processes during infection was examined through differential gene expression analysis to identify up and down regulated genes in infected cells or uninfected cells in the presence or absence of the RBV treatment. Overall, these findings improve our understanding of ribavirin’s antiviral mechanism against HRSV in A549 cells, particularly its direct inhibitory effect by increasing the mutation rate within the viral genome and its indirect effects on host cells, which might be crucial for inhibiting the viral replication.22 0