RIASSUNTI DEI PROGETTI DEL CORSO DI DOTTORATO DI RICERCA IN BIOLOGIA MOLECOLARE E CELLULARE

A.A. 2013 (XXIX CICLO)

1.Beltrame Monica: Deciphering the molecular network involving the transcription factor Sox18 in blood vascular and lymphatic development

2.Berruti Giovanna: An organ culture-approach to study the fertility gene product USP8/UBPy

3.Bertoni Giovanni (1): Detailing of small RNA-based regulatory networks by parallel transcriptomic-proteomic profiling

4.Bertoni Giovanni (2): Characterization of novel essential functions in the opportunistic pathogen Pseudomonas aeruginosa

5.Bolognesi Martino: Structure-based epitope discovery from B. pseudomallei antigens for vaccine

Development

6.Briani Federica: Looking for novel Pseudomonas aeruginosa inhibitors: S1 ribosomal protein as an unexploited target for new antibacterial molecules

7.Caporali Elisabetta: Key cell wall modelling factors to design fruit shapes in Arabidopsis

8.Cappelletti Graziella: Dissecting microtubule dynamics at the synapse

9. Cappelletti Graziella: Molecular architecture of microtubules: unravelling the contribution of - and tubulin to neuronal identity

10.Caretti Giuseppina: Epigenetic mechanisms underlying skeletal muscle atrophy.

11.Cattaneo Elena: hES cells differentiation into striatal neurons by conditional over-expression of

transcription factors

12.Colombo Lucia: Genetic and epigenetic control of seed number in Arabidopsis

13.Costa Alex: Functional characterization of Arabidopsis thaliana iGluRs (glutamate receptors)

channels

14.Cotelli Franco: Characterization of the role played by numblike during vascular development of the brain and Cerebral Cavernous Malformations onset using zebrafish as model system

14.Duga Stefano: Whole-exome sequencing applied to the study of the genetic basis of Parkinson

disease

16. Fornara Fabio: Molecular control of flowering in rice

17. Gissi Carmela: Evolutionary dynamics of nuclear genes involved in replication and repair of the mitochondrial genome in fast-evolving chordates

18.Gnesutta Nerina: NF-Y partners in the regulation of CCAAT promoters

19.Guerrini Luisa: Role of the p300 acetylase in regulating the activities of the p63 transcription factor

20.Kater Martin: Mining the Molecular Pathways Controlling Rice Reproductive Development

21.Lazzaro Federico: Determine the biological significance of the rNMPs incorporated in genomic

DNA

22.Mantovani Roberto: NF-YA isoforms in ES cells and cancer

23.Marini Federica: Unravelling the role of the Fanconia anemia protein P/Slx4 and the DNA damage checkpoint factor 53BP1/Rad9 in responding to double strand DNA lesions

24.Messina Graziella: Identification of the mechanism(s) regulating Nfix expression during fetal

Myogenesis

25.Messina Graziella: Genome-wide mapping of Nfix binding sites in skeletal muscle

26.Moroni Anna: The structural mechanism of KAT1 channel regulation by the cytoplasmic domain CNBHD

27.Muzi Falconi Marco: Haspin kinase: a new regulator of cell polarity and cell division

28.Nardini Marco: Structural analysis of transcription factor/DNA complexes

29.Pavesi Giulio: Development and Application of Bioinformatic Tools for the Analysis of ChIP-Seq Data

30. Petroni Katia: Role of anthocyanin-enriched diet on cardioprotection

31.Plevani Paolo/Giacomo Buscemi: DAXX protein and the human DNA damage response:chromatin remodeling, gene expression, genome stability and tumorigenesis

32.Pesaresi Paolo: Chromatin Remodeling Enzymes are at the basis of SVP transcription factor

function

33.Ricagno Stefano: Structural-Based Drug Discovery against RNA-based VIRUSES

34.Riva Paola: Competitive endogenous RNA (ceRNA) Cross-Talk in Neurofibromatosis type 1 phenotype expression variability

35.Sold Giulia: Identification of genetics and molecular bases of inherited sensorineural hearing loss by whole-exome sequencing

36.Tonelli Chiara/Conti Lucio: The role of ABA in the floral transition: site and mechanism of

action

37.Zuccato Chiara: An in vivo study of the impact of ADAM10 dysfunction in Huntingtons Disease

Project leader: MONICA BELTRAME (monica.beltrame@unimi.it)

Location: Department of Biosciences, University of Milan, Italy

RESEARCH PROJECT SUMMARY

Deciphering the molecular network involving the transcription factor Sox18 in blood vascular and lymphatic development

Our group is interested in gene expression regulation during embryonic development in vertebrates. We are studying a family of transcription factors, the Sox (Sry-related HMG box) proteins, which are found throughout the animal kingdom and play key roles in embryonic development. Mutations in several Sox genes have been shown to result in developmental anomalies, from fly to mammals. The emerging picture of SOX transcription factors is one of tissue-specific switches that induce changes in gene expression required for cell-type specification or differentiation.

Our interest is currently centered on SOX18, which is mutated in patients affected by the Hypotrichosis-Lymphedema-Telangiectasia syndrome. SOX18 is transiently expressed in the endothelial component of nascent blood and lymphatic vessels during embryonic development and in adult life, when neovascularization occurs.

Despite its relevance, relatively little is known about upstream factors and downstream targets involving SOX18 in endothelial cell differentiation and vascular development. We are addressing these questions using the zebrafish model system, as it provides several advantages over other vertebrate model organisms for the in vivo imaging of the vascular system and for the genetic or experimental manipulation of vascular development. We have shown that Sox18 and the closely related Sox7 protein (both belonging to the SoxF group) play redundant roles in arterio-venous differentiation of endothelial cells in zebrafish, while only Sox18 plays a conserved role in lymphatic development (Cermenati et al., 2008, Blood; Cermenati et al. 2013, ATVB). Gene expression profiling, at key developmental stages, under conditions of perturbed SoxF protein expression will serve as a basis to shed light on the molecular networks controlling blood vascular and lymphatic development. Loss-of-function and gain-of-function approaches in specific transgenic lines will be used to assess the functional relevance of interesting genes, whose expression is modified when SoxF proteins are perturbed. Given the pathological relevance of angiogenesis/lymphangiogenesis and lymphatic dysfunction in humans, the identification of new players might open up new therapeutic perspectives.

Project leader: GIOVANNA BERRUTI (giovanna.berruti@unimi.it)

Location: Department of Biosciences, University of Milan, Italy.

RESEARCH PROJECT SUMMARY

An 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 approa