Date of Award

1-1-2019

Document Type

Thesis

Degree Name

Master of Public Health (MPH)

Department

School of Public Health

First Advisor

Christian Tschudi

Abstract

Single cell RNA-sequencing (scRNA-seq) is a recent innovation that allows sequencing of transcriptomes from a vast number of single cells, thus providing an unbiased view of cell-to-cell variability in gene expression within a population of cells. The Tschudi laboratory has previously established an in vitro differentiation system based on the inducible expression of the RNA-binding protein 6 (RBP6) in cultured non-infective procyclics, which recapitulates Trypanosoma brucei developmental stages that have been previously described in tsetse fly vector. Thus, I aimed to utilize scRNA-seq in this system to describe the potential heterogeneity in transcript abundance of known and suspected T. brucei developmental genes, to determine if monoallelic expression of metacyclic variable surface glycoproteins (mVSGs) occurs from early onset of metacyclic development, and to establish if differential expression of RBP6 affects life-cycle progression capacity. Employment of 10x Genomics, Inc. scRNA-seq and downstream analysis with Partek®️ Flow®️ software revealed a baseline of heterogeneity present in uninduced cells. As differentiation starts, clusters emerged that are representative of induction of metacyclogenesis by RBP6, where T. brucei developmentally progress from procyclics to epimastigotes to metacyclics as it occurs in the tsetse fly vector. The three clusters of cells were marked by the surface protein genes of brucei alanine-rich proteins (BARP) and mVSG, or lack thereof. The presence of these clusters at both day two and day four of induction suggested that cells progress through metacyclogenesis at varying rates, with some cells unproductive in development. T. brucei cells appeared to have polyallelic early onset expression of mVSG prior to singular expression of an mVSG. The single cell polyallelic expression of mVSG happened at a distribution similar to population level expression, indicating either random chance selection of an mVSG or quorum sensing.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 08/28/2021

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