Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

First Advisor

Michael J. Kimber

Abstract

Since its first characterization in 1998 in the free-living nematode Caenorhabditis elegans, RNA interference has been considered a powerful reverse genetics tool to investigate nematode biology. But to date, current RNAi protocols for parasitic nematodes have proven unreliable and inconsistent.

We established an alternative RNAi protocol targeting the filarial nematode Brugia malayi in-host whereby the parasites are exposed to RNAi triggers as they develop within the intermediate host, the mosquito Aedes aegypti. Using this in vivo RNAi protocol, we successfully quantified the suppression of five B. malayi genes associated with known or putative drug targets. Administration of a random exogenous dsRNA resulted in no phenotypic abnormalities, demonstrating the specificity of the in vivo RNAi protocol.

In vivo RNAi experiments revealed that the cathepsin L-like cysteine protease Bm-cpl-1 plays a role in worm migration, survival and overall health. Suppression of Bm-cpl-1 resulted in inhibited worm motility and capacity to properly navigate to the head for transmission thus abolishing the transmission potential of the worm. Bm-cpl-1 suppression also affected worm development as evident by a reduction in worm length post Bm-cpl-1 suppression.

The potential of the in vivo RNAi protocol to aid drug development was further validated using four known or putative drug targets of interest: y-tubulin (Bm-tub-1), a glutamate-gated chloride channel alpha subunit (Bm-GluCl-y3A), a G protein-coupled acetylcholine receptor (Bm-gar-2), and a FMRFamide-like peptide (Bm-flp-21). Suppression of these genes resulted in a combination of decreased motility, worm survival, migration, and worm physiological abnormalities verifying or validating each of the four drug targets potential for anthelmintic drug development.

Copyright Owner

Chuanzhe Song

Language

en

Date Available

2012-04-28

File Format

application/pdf

File Size

162 pages

Share

COinS