Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology, Neurobiology and Anatomy

Abstract

Inflammation is central to the pathogenesis of numerous human diseases. in many contexts, inflammation drives immune responses which are critical to protection against many pathogens. However, uncontrolled inflammation can drive the pathology of numerous diseases, including neurodegenerative diseases, intestinal disorders, cancer, and autoimmune diseases. a primary mediator of inflammation is the inflammasome complex, which drives the release of inflammatory mediators through caspase-1 activation. Our goal was to develop a tool that would allow us to monitor caspase-1 activation in vitro and ultimately in vivo. to this end, we employed a circularly permuted form of luciferase in which the N- and C-terminal domains necessary for bioluminescence are physically separated by a linker region. Caspase-1 target sequences were cloned into this linker region, so that upon caspase-1 activation, these sequences are cleaved allowing the two domains of luciferase to come together and luminescence can be quantified. in pilot experiments, we identified putative sensors that exhibited a significant increase in bioluminescence following caspase-1 activation in vitro. We generated transgenic mice expressing our top biosensor and identified lines that exhibited biosensor expression in all tissues. to determine if the caspase-1 reporter was functioning in vivo, mice were challenged with various inflammatory stimuli and bioluminescence was quantified in living animals and tissues ex vivo. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. Our results suggest that caspase-1 biosensors may serve as an outstanding tool to monitor and quantify inflammation in living animals.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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