Infectious diseases that spread from person-to-person and continent-to-continent are a cause for concern for any health entity. One such disease is malaria, a mosquito-borne infection instigated by the protozoan parasite, Plasmodium falciparum. Hundreds of millions of people are affected annually and it is responsible for nearly 1 million deaths. It is the most fatal species causing malaria and proliferates in human red blood cells with a life cycle occurring every 48 hours. At this time, the parasite’s late stage form or schizont bursts from the erythrocyte releasing immune-inducing particles and infective forms (merozoites) into the bloodstream. The merozoites go on to infect other red blood cells as human immunity leads to fever. Fever is a hallmark symptom of malaria and effectively inhibits the growth of late stage parasites. Plasmodium still manages to complete its life cycle as early stages or rings are not affected by febrile temperatures. It is this facet of parasite biology that prompts our research into identifying genetic factors associated with fever.
The parasite’s response under elevated body temperature may offer further insight into its adaptive mechanism. A heat shock assay was developed in order to simulate fever in vitro. Mutant parasite cultures were subjected to 41°C for 8 hours and returned to normal body temperature or 37°C for the remainder of the life cycle. The piggyBac mutagenesis system allows for the evaluation of phenotypes associated with a particular genotype as the transposon inserts randomly into the gene. This often leads to changes in function that may cause delays in invasion or attenuation of growth. Determining the genes responsible for these phenotypes would be a great advantage to the field of drug discovery.
Collaborative efforts to develop vaccines and new antimalarial drugs are underway as resistance to current methods of treatment is on the rise. Such circumstances require new technologies for detecting novel drug targets or pathways in the parasite that can be significantly affected by these therapeutics. QISeq is a next generation sequencing tool that identifies genes with a particular phenotype that may alter intraerythrocytic development of P. falciparum. This technique was utilized in our study to confirm the heat shock phenotype with a high-throughput approach. The genomic DNA of pooled parasite cultures was sequenced to reveal those mutants sensitive and/or resistant to febrile temperature exposure. Through bioinformatics analyses, functional associations between genes can be made that lead to biological pathways of interest for therapeutic research.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-6983 |
| Date | 16 September 2015 |
| Creators | Thomas, Phaedra J. |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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