Riassunti dei progetti del corso di dottorato di ricerca in biologia molecolare e cellulare
Project leader: GIOVANNA BERRUTI (giovanna.berruti@unimi.it)
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Project leader: GIOVANNA BERRUTI (giovanna.berruti@unimi.it)Location: Department of Biosciences, University of Milan, Italy. RESEARCH PROJECT SUMMARYAn organ culture-approach to study the fertility gene product USP8/UBPy USP8/UBPy, recently found to be a candidate to 'male-fertility gene' [1], is a deubiquitinase preferentially expressed in the testis and the central nervous system, which acts as a regulator of the endocytic vesicle trafficking [2]. USP8 is involved in the biogenesis of the acrosome, an organelle essential to fertilization. USP8-KO is lethal, consequently functional studies in vivo are not feasible [3]. The purpose of this research is to investigate the role and importance of USP8 in spermiogenesis, using a system of organ culture developed recently that could allow us to recapitulate 'ex vivo' what happens in vivo [4]. As organ donors there will be used male mice Acr-GFP [5] that accumulate EGFP in the acrosome during its biogenesis, thus making it detectable at fluorescence microscopy. The initial phase of the research will be devoted to the development of organ culture conditions optimal for obtaining, from highly immature cells, spermatids that have achieved development stages corresponding to each of the 4 acrosomogenic phases (acrosomogenesis ‘in vivo’ requires about 2 weeks in the mouse). The cultured Acr-GFP testicles will assist to identify the phases in non-invasive manner. Analysis of confocal double-immunolabeling to highlight USP8 and classical markers of the biosynthetic and endocytic traffic will be performed to identify the type/s of traffic that carry the cargo protein to the acrosome in development. Acrosomogenesis is microtubule (MT)-dependent and USP8 has a MIT (microtubule interacting and trafficking) domain [2]; however, the MT-arrays involved in acrosomogenesis are transient structures devoid of any visible centrosomal foci. It will be investigated in the organ cultured cells whether the possible association USP8-MT is preferential with dynamic MTs (tyrosinataed tubulin) or more stable MTs (detyrosinated, acetylated tubulin, etc.); this will be performed by using confocal double-immunolabeling and in vitro protein-protein interaction assays (GST pull down). To silence the activity of USP8, we propose to produce a non-viral vector that replicates episomally and carries the expression cassette for a USP8-siRNA, in addition to the cassette for RFP as a reporter protein [6]. In this way we could see what effect the silencing of USP8 has on the development of Acr-GFP acrosome in the 'red' cells. Comparisons will be with the respective not silenced controls. As an alternative to silencing, the activity of USP8 could be blocked through transfection experiments by expression of a catalytically inactive variant of USP8, USP8C748A[7], fused with RFP (the recombinant construct has to be generated). In conclusion, this research combines two innovative approaches, i.e., the ‘organ culture’ and ‘non-viral vector-mediated’ silencing/inhibition of the USP8/UBPy enzymatic activity. If we succeed in both, it could be provided an experimental platform for studying not only USP8, but other key fertility gene products, for which the homologous deletion results in embryonic lethality, in a system that recapitulates the ‘in vivo’ situation. Kosova et al, Amer J Hum Gen 90, 950-61, 2012 Berruti et al, Biol Reprod 82, 930-39. 2010 Nierdof et al., Mol Cell Biol 27, 5029-39, 2007 Sato et al, Nature 471, 504-7, 2011 Nakanishi et al. FEBS Lett 449, 277-83, 1999 Jenke et al, Hum Gen Ther 16, 533-9, 2005 Alwan & van Leeuwen, JBC 282, 1658-69, 2007 Project leader: GIOVANNI BERTONI (1) (giovanni.bertoni@unimi.it)Location: Department of Biosciences, University of Milan, Italy. RESEARCH PROJECT SUMMARYDetailing of small RNA-based regulatory networks by parallel transcriptomic-proteomic profiling Pseudomonas aeruginosa is an important opportunistic pathogen in immune-compromised and cystic fibrosis patients responsible for numerous acute and chronic infections. Crucial traits contributing to pathogenicity of P. aeruginosa are the production of a large assortment of virulence factors, biofilm formation and the ability to rapidly develop resistance to multiple classes of antibiotics. Expression of these traits is fine-tuned by a dynamic and intricate regulatory network [1], in which more than 50 regulatory proteins play key roles as transcription regulators. On the contrary, the involvement in this context of small RNAs (sRNAs), important regulatory molecules acting post-transcriptionally on target mRNAs and/or via interactions with proteins, has been studied to a lesser extent. Preliminary results suggest that four novel P. aeruginosa sRNAs, which were identified in the proponent lab [2], are involved in the regulation of virulence traits in response to infection-relevant stimuli. In actual fact, overexpression of these sRNAs was shown to influence the expression of P. aeruginosa virulence descriptors such as motility, biofilm formation, secretion of proteases, toxic secondary metabolites and siderophores. In addition, it was observed that the expression of these sRNAs can be responsive to temperature shift from room to body temperature, oxygen availability, iron limitation, envelope stressors and quorum sensing, a system of stimulus and response correlated to bacterial cell density that coordinate the expression of several virulence genes. Focusing on these four sRNAs, the main aim of this research project is to unravel the cognate regulons, i.e. the set of genes which can be both direct and indirect targets of the sRNA-mediated regulation. This aim will be accomplished combining quantitative proteomics with transcriptomics. For virulence-relevant direct targets, the sRNA/target mRNA interaction will be characterized. 1. Balasubramanian D, Schneper L, Kumari H & Mathee K (2013) A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res, 41:1-20.
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