Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas disease representing the highest parasitic economic burden. The therapeutic drugs currently used to treat the disease have low efficacy and are highly toxic for the patient, so the development of new drugs with improved efficacy, higher specificity for the parasite and less toxicity for the host is imperative. Although little is known about the global mechanisms governing proliferation and differentiation of trypanosomatids, genomic studies have revealed that they greatly diverge from those of the human host, which makes them particulate suitable targets for the development of parasite specific pharmacological therapies. Our project aims to uncover new molecules responsible for the control and progression of the proliferative cycle of T. cruzi through the high throughput, comprehensive and quantitative identification of genes differentially expressed in the various parasite stages. We will perform RNA-seq studies from total RNA (transcriptome) and Ribo-seq studies of actively translated mRNA (translatome). The latter is obtained from polysomes after a nuclease digestion that generates ribosome footprints (Ingolia et al., 2009), whose abundance represents the rate of the mRNA translation for each gene. Since trypanosomatids exert a poor control of gene expression at the level of transcription, mRNA stability and translatability are proposed to represent major regulatory points. There is in fact mounting evidence of the global regulation at RNA stability obtained by microarrays or RNA-seq in trypanosomatids, but we still lack any assessment of the importance of translation regulation in these organisms. “Ribo-seq” or global ribosome footprint analysis has emerged as a new tool to address this question. It also has the advantage to provide a more realistic picture of the gene expression profiles in the cell than the obtained by the transcriptome. In order to obtain purified T. cruzi populations, representative of the different stages of the parasite cycle, we plan to perform a synchronization of epimastigotes (rendering G1, S and G2 phases of the cell cycle. We will isolate total mRNA and ribosome footprints from each stage to generate adapted libraries to be sequenced with high throughput sequencers. The data obtained will be analyzed bioinformatically to identify genes differentially expressed during the proliferative and developmental cycle. We will also study the patterns of gene regulation at the level of stability and translatability of the mRNAs (degradation and translation elements, regulons, signaling pathways, molecular networks). We this data we will be able to assess the characteristics of the global translation regulation of the mRNA for the first time in trypanosomatids. We will also study the differentially regulated proteins specific for the parasite, with no ortologous in the human host, in order to select those which meet the criteria for a desirable drug target. Overall, our study would help to further understand the mechanisms that determine parasite replication and differentiation and to discover new targets for the design of more specific drug therapy.