Applications of scRNA-seq to capture Trypanosomatid life cycle stage transitions and heterogeneity

Laidlaw, Ross (2024) Applications of scRNA-seq to capture Trypanosomatid life cycle stage transitions and heterogeneity. PhD thesis, University of Glasgow.

Abstract

Single cell RNA-sequencing has transformed transcriptomic analysis, giving researchers unparalleled access to minute changes in gene expression at the level of single cells. This resolution is especially useful when analysing biological processes, as cells (and their transcriptome) from various points in the process can be captured. The applications of scRNA-seq to parasite life cycles is thus clear. In this thesis, I have applied scRNA-seq and a variety of analysis methods to interrogate the transitions that occur in the Trypanosomatid group of parasites, specifically Trypanosoma brucei and Trypanosoma cruzi. Firstly, a tool was created that aligns and compares biological processes across time between two conditions. This tool returns more accurate alignments of asymmetrical processes than current tools and allows extraction of meaningful genes that define the differences between the two conditions. Next, we confirmed the effectiveness of scRNA-seq to profile the transcriptome of T. cruzi, in the process capturing marker genes of the four major life cycle stages of the parasite and creating the first scRNAseq atlas of T. cruzi. From this data the process of metacyclogenesis was captured at the single cell level, characterising various differentially expressed genes by their expression peak across the process and identifying several differentially expressed RNA binding proteins which had not previously been associated with metacyclogenesis. The transcriptomic profile of trypomastigote subsets was investigated, possibly identifying functional trypomastigote subsets in the context of infection. Finally, we utilised scRNA-seq on the RBP6 overexpression in vitro model of the insect stage life cycle of T. brucei to identify genes which drive the transition of life cycle stages. These results revealed an unexpected lack of transcriptomic signature epimastigotes being generated by the model but did capture the generation of few metacyclic-like cells who express mVSG but have many differences in transcriptome when compared to the in vivo forms, including metabolism associated genes. PCF-like cells, high in expression of PAG, were also detected in the data, with the transcriptome of these cells being highly expressed towards the end of the RBP6 overexpression process in published bulk RNA-seq data of the model.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Otto, Professor Thomas, McCulloch, Professor Richard, Briggs, Dr. Emma and Matthews, Professor Keith
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84743
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 06 Dec 2024 16:53
• Last Modified: 06 Dec 2024 16:54
• Thesis DOI: 10.5525/gla.thesis.84743
• URI: https://theses.gla.ac.uk/id/eprint/84743