Molecular Responses of Giardia lamblia to Gamma-Irradiation
Type of DegreeDissertation
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Giardia lamblia (syn intestinalis, syn duodenalis) is an important human pathogen and the etiological agent of giardiasis, the most common gastrointestinal disease in the world. Since its discovery in 1681, Giardia has been extensively studied; however, much of the basic biology of this organism still remains a mystery. The goal of this dissertation was to determine the molecular responses of Giardia lamblia trophozoites to gamma (?)-irradiation, a means of disinfection. Dose-response curves were determined for the Giardia lamblia WB isolate trophozoites after ?-irradiation using a Cobalt-60 source. Microscopic observation of trophozoites, along with direct observation of subcultures was used to determine the dosing levels that gave 10, 37 and 90% killing. Dose/Response curves for 4 additional isolates from assemblages A and B were also generated in order to determine if differing sensitivities to ?-irradiation existed among isolates of importance to human disease. In addition, dose/response curves were generated using methyl methane sulfonate (MMS), an alkylating agent that mimics damage induced by radiation. MMS was used to determine whether it induced a similar alteration in gene expression to ?-irradiation. A high-throughput method for quantitating trophozoite killing by both MMS and ?-irradiation using 96 well-plates was tested against the laborious method of manually counting viable cells with a hemacytometer. In order to evaluate changes in gene expression for genes commonly involved in DNA damage repair due to ?-irradiation and MMS, real-time (RT) PCR using primers designed to amplify ~60 known repair genes was conducted. Profiles generated indicate whether or not the gene of interest is upregulated, downregulated, or remains the same as the untreated control cells. Comparisons between MMS and ?-irradiation were then made to determine whether there were differences between expression profiles between the two treatments. The results of this study indicated that there were statistically different responses between isolates of Giardia lamblia. Different sensitivities also existed between isolates when MMS and ?-irradiation treatments were compared. The lethal dose of ?-irradiation required to inactivate Giardia lamblia, isolate WB, trophozoites was found to be 10.0 kGy, while the recovery doses ranged from 0.25-1 kGy. Doses below 0.25 kGy were ineffective at inactivating any of the trophozoites. It was determined decreased use of media, time, and allowed for a higher-throughput screening of toxic agents.