Date of Award

Summer 2013

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Program/Concentration

Biology

Committee Director

Roland A. Cooper

Committee Member

Wayne Hynes

Committee Member

David Gauthier

Call Number for Print

Special Collections LD4331.B46 D35 2013

Abstract

Malaria is among the most devastating human diseases, and a majority of lethal cases are caused by the protozoan parasite, Plasmodiumfalciparum. The emergence of multi-drug resistant P.falciparum is a major obstacle to malaria control and is highlighted by the abandonment of chloroquine (CQ) as a first-line treatment of P.falciparum infections worldwide. Chloroquine resistance (CQR) is associated primarily with mutations in the transmembrane digestive vacuole protein, PfCRT. However, CQR P.falciparum parasites harboring the same mutant pfcrt allele vary in their CQ response, suggesting the CQ response is multigenic in nature. No gene outside of pfcrt is completely associated with CQR. Thus, our understanding of the complex nature of CQ resistance remains inadequate. To study the evolutionary pathway and accompanying genetic changes associated with CQR, we employed a long-term in vitro on/off CQ selection strategy to select P.falciparum parasites capable of recovering from extended, high-dose CQ exposures. We posit the use of clinically relevant CQ doses for gradually longer durations will bottleneck the CQS parasite populations and select only those parasites harboring genetic backgrounds that provide a selective advantage. Also, the gradual increase in CQ selection pressure provides a timeline of the putative genetic and molecular changes that confer elevated CQ responses in natural populations. Here, we show selected and control parasite lines could survive a 48 h exposure to a CQ dose up to 400-fold greater than the estimated IC50• There was no change in CQ response between GC03 sequentially selected (GC03-CQ) and GC03 parasites. We did select a CQ-tolerant line (HB3-24 h) that showed a 1.7-fold higher 24 h LD50 and identified genetic changes located on chromosomes 10 and 12. We provide evidence that CQS P. falciparum parasites can survive high doses of CQ through a ring stage quiescence mechanism. Also, P. falciparum strains with different genetic backgrounds vary in their capacity to recrudesce. Our study implies P. falciparum recrudescence following high dose CQ exposure occurs readily in laboratory-treated parasites and may be a critical factor in malaria treatment failures.

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DOI

10.25777/067k-pd86

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