Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Arjun Raj

Abstract

Single-molecule RNA FISH is a robust method for visualizing individual molecules of RNA within intact cells that has been used extensively for describing single-cell hetero- geneity in gene expression. In this thesis, we leverage RNA FISH-based technologies for two major problems in biology and medicine: rapid detection of viral infections and understanding mechanisms of resistance to targeted therapy in cancer. Until recently, RNA FISH was not a viable technology for rapid diagnostics, as the hybridization process required a minimum of 6 hours. We start by presenting a modification to the RNA FISH protocol developed by Raj et al. 2008, that enables hybridization in only 5 minutes, and then use these improvements in hybridization time to develop RNA FISH for detection of respiratory viruses. We demonstrate that RNA FISH is capable of de- tecting influenza, rhinovirus, and adenovirus, and propose two probe design strategies with clinical value for discriminating viral strains and detecting many strains at once. Ultimately, we extend these techniques to discriminate single-base differences in the viral sequences, which is clinically useful as single-base mutations can render viruses resistant to our best antiviral medications. In the next section of this thesis, we use RNA FISH for another application: examining single-cell heterogeneity in cancer cells treated with targeted therapy. We first show that melanoma cells can display profound transcriptional variability at the single cell level that predicts which cells will ultimately resist drug. This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces an epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming is a progressive process consisting of a loss of SOX10-mediated differentiation followed by activation of new signaling pathways, partially mediated by activity of Jun-AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics. Taken together, these two applications of RNA FISH show its generalizability for exploring many different questions in biology and clinical medicine.

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