Riu, Federica (2010) Identificazione e caratterizzazione di una putativa pathway di RNA interference nel Protozoo a trasmissione sessuale Trichomonas Vaginalis. Doctoral Thesis.
RNA interference (RNAi) is a conserved mechanism triggered by dsRNA that leads to the sequence-specific cleavage of homologous mRNA. The biological functions of this phenomen are the resistence to viral and endogenous dsRNAs, and the regulation of the expression of protein-coding genes. Among pathogenic protists, RNAi was described in African Trypanosomes, E. hystolytica, and T. gondii, showing to be an unvaluable tool to manipulate gene expression. RNAi in T. vaginalis hasn’t been observed yet, nor have genes putatively involved in RNAi processes identified. The genome sequencing allowed the detection of the RNAi genetic hallmarks in T. vaginalis: a Dicer-like, 2 AGO and 41 DEAD/DEAH-box helicase genes, that were all shown to be transcribed. In order to assess the presence of a functional RNAi pathway, T. vaginalis was transfected with a synthetic dsRNA homolog to alfa-actinin. We obtained contradictory and difficult to reproduce results, wich in a first attempt showed a gene silencing. Since this experimental system do not prove to be reliable and reproducible, we constructed a plasmid system for selectable and inducible expression of dsRNA in vivo. This will hopefully lead us to show a possible RNAi activity in T. vaginalis, thus providing a new tool for the genetic dissection of this microorganism, allowing the clarification of several aspects of its biology, including pathogenesis and identification of possible novel drug targets. Furthermore, the demonstration of a functional RNAi pathway in an early diverging eukaryote such as T. vaginalis can also lead to new insights into the evolutionary origin of this mechanism in the eukaryotic lineage.
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