Riassunti dei progetti del corso di dottorato di ricerca in biologia molecolare e cellulare
Project leader: STEFANO RICAGNO (stefano.ricagno@unimi.it)
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- Project leader: PAOLA RIVA (paola.riva@unimi.it)
- Project leader: GIULIA SOLDA’ (giulia.solda@unimi.it)
- Project leader: CHIARA TONELLI (chiara.tonelli@unimi.it); (lucio.conti@unimi.it)
- Location: Department of Biosciences, University of Milan, Italy
Project leader: STEFANO RICAGNO (stefano.ricagno@unimi.it)Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYStructural-Based Drug Discovery against RNA-based VIRUSES The number of fatalities that occur worldwide as the result of infections by RNA viruses is in the order of many millions annually. Increasing human and animal population density combined with increasing mobility, commercial transport, land exploitation and climate change, all have an impact on virus emergence and epidemiology. Over the past 3 decades, many RNA virus threats were identified or “re-discovered”, including a variety of pathogenic flaviviruses. The flavivirus group includes several pathogens of global medical importance, i.e. dengue viruses (DENV); Infections by these neurotropic viruses may result in life-threatening aseptic encephalitis, with high risk of life-long debilitating neurologic sequels. About 500,000 people with severe dengue require hospitalization each year, a large proportion of which are children. WHO currently estimates there may be 50-100 million dengue infections worldwide every year. There is an increasing number of cases as the disease spreads to new areas, causing severe outbreaks. The threat of a possible outbreak of dengue fever now exists in Europe and local transmission of dengue, in addition to several imported cases, was reported for the first time in 2010. Vaccine development has proven particularly challenging because of the existence of 4 dengue serotypes and antibody dependent enhancement of infection. Hence neither vaccines nor antiviral therapy (or prophylaxis) are available today. We urgently need advanced levels of preparedness with which to confront and ultimately control these viral pathogens. The infective enveloped flavivirus contains a (+) single-stranded RNA genome encoding 7 non-structural proteins involved in viral replication. The non-structural proteins NS3 and NS5 play key roles in the replication cycle, therefore are ideal antiviral targets. For discovering small-molecule inhibitors of viral replication, our approach relies on the three-dimensional structure of target proteins (“structure-based drug discovery”). The key outcomes of the project will be discovery and optimization of novel inhibitors that bind viral replicative enzyme domains and/or viral replication complexes. Inhibitors active against dengue may possibly also be active against other flaviviruses, including West Nile virus, Yellow fever and Japanese encephalitis virus. This provides a unique opportunity to fully address the "Societal challenges" issue of the Horizon 2020 program, with a clear emphasis on world population health and well-being. Project leader: PAOLA RIVA (paola.riva@unimi.it)Location: Department of Medical Biotechnology and Translational Medicine, via Viotti 3/5, 20133 Milano RESEARCH PROJECT SUMMARYCompetitive endogenous RNA (ceRNA) Cross-Talk in Neurofibromatosis type 1 phenotype expression variability Neurofibromatosis type 1 (NF1 [OMIM + 162200]) is a common autosomal dominant disorder affecting 1/3500 individuals and caused by the point mutations or deletion of NF1, a tumor-suppressor gene with a Ras-GTPase activity, encoding neurofibromin. NF1 is characterized by a highly variable expressivity with multisystemic symptoms that may manifest at birth and evolve during lifetime. The clinical signs, including café au lait spots, axillary and inguinal freckling, dermal or plexiform neurofibromas, iris Lisch nodules, but also an increased risk of other benign and malignant tumors, highlight altered developmental pathways and provide insights into the close relationship between development and cancer. Central to these interconnected aspects is neurofibromin defective deregulation and the resulting hyperactivation of the Ras signal transduction machinery. The heterogeneous clinical expression is hardly conceivable on the basis of a great majority of NF1 deletions or truncating mutations. We will focus our interest on the identification of mechanisms leading to dermal (DNF) rather than plexiform neurofibromas (PNF) in patients carrying the same lesions of NF1 gene. Given that the NF1 constitutional mutation is a necessary condition to develop the two above kinds of benign tumors and that no tumor-associated mutations have been till now demonstrated, the involvement of modifier genes and/or further pathogenetic mechanisms have been proposed, even if the basis of this variable expressivity is currently unknown. Considering that a new emerging layer of gene regulation, based on competitive endogenous RNA (ceRNA) activity, plays important roles in the development of different diseases including cancer, we hypothesize that this mechanism may be involved in the expression regulation of the functional copy of NF1 gene and have a consequence on the variability of NF1 clinical signs, comprising the development of DNF or PNF. The recent discovery of competitive endogenous RNAs (ceRNA), natural decoys that compete for a common pool of miRNAs, provides a framework explaining the activity of (miRNA) response elements (MRE)-harboring non-coding RNAs, small or long non coding RNAs and transcribed pseudogenes, in relation to the target protein-coding mRNA regulation. Functional interactions in ceRNA networks aid in coordinating a number of biologic processes and, when perturbed, contribute to disease pathogenesis. If little is known about the NF1 gene post-transcriptional regulation, only a few targeting miRNAs have been validated, the possible involvement of NF1 mRNA in ceRNA cross talk has never been investigated. Knowing that several NF1 pseudogenes are differently expressed in different tissues, the aims of the proposed project are 1) to investigate the possible involvement of NF1 mRNA in ceRNA cross talk identifying possible interferences of specific NF1 pseudogenes with the interactions between NF1 mRNA and the validated targeting miRNAs, 2) to study the expression profile of miRNA targeting NF1 mRNA, NF1 transcript and specific NF1 pseudogenes by a comparison of their levels in both constitutional and tumor mRNAs from patients with DNFs and PNFs. The first goal will be pursued first by identifying NF1 pseudogenes sharing the same binding sites of miRNA targeting the NF1-coding mRNA by bioinformatic analysis. The expression profile of the selected pseudogenes and NF1-coding mRNA will be defined by interfering with specific miRNAs and pseudogenes in cell lines from different tissues. The detection of both NF1-coding and non coding mRNA levels consistent with the interference by the same miRNA, will indicate that the NF1-coding mRNA is involved in ceRNA cross talk. The data obtained will address the expression studies of specific NF1 pseudogenes and miRNAs in a current casuistry of 8 DNFs and 9 PNFs patients that will be increased. This project will provide new insights on NF1 post-transcriptional regulation and might open new perspectives on identification of mechanisms leading to the characteristic variable NF1 phenotype. Project leader: GIULIA SOLDA’ (giulia.solda@unimi.it)Location: Department of Medical Biotechnologies and Translational Medicine, via Viotti 3/5, 20133 Milano RESEARCH PROJECT SUMMARYIdentification of genetics and molecular bases of inherited sensorineural hearing loss by whole-exome sequencing Inherited nonsyndromic sensorineural hearing loss (NSHL) shows an extremely high genetic heterogeneity, with more than 70 genes already associated with NSHL, and many others still to be discovered (http://hereditaryhearingloss.org). We are currently applying whole-exome sequencing (WES) as a cost- and time-effective strategy to search for pathogenic variants underlying deafness. Indeed, this technique has already proven to be helpful for the discovery of novel genes/mutations responsible for recessive NSHL (DFNB82 and DFNX4 loci) [1-3]. Seven NSHL families, with a clear recessive (autosomal or X-linked) inheritance pattern and at least two affected individuals, have been already selected for WES; additional families are being recruited. The WES of the first 16 patients has already been performed through external NGS services at BGI (Beijing Genomics Institute, China), and Yale Genome Center (New Haven, USA). The proposed PhD project will involve a combination of both in-silico and wet-lab approaches in order to:
Develop data analysis pipelines to efficiently detect and prioritize candidate variants; Functionally characterize by in-vitro and in-vivo studies novel genes/mutations. The data analysis will include, among others, the implementation of the detection of splicing mutations, indels, and structural variants. Putative pathogenic mutations identified by WES will be tested to evaluate their segregation with the disease within the probands’ families and their recurrence in sporadic and familial NSHL cases. In this frame, the availability of a large non-syndromic deafness series (about 1300 individuals) of patients/families will be a key resource in the validation step, to screen for the identified mutations and to search for additional genetic defects in the candidate genes pointed out by WES. A in-house database of all variants identified by WES in a wide (>3000) cohort of Italian subjects is also available to the study, to identify population-specific polymorphisms. We are currently analyzing the putative pathogenic role of a novel variation in a candidate NSHL-causing gene both at the mRNA and at the protein level, by expression experiments in eukaryotic cell lines. In addition, the function of this newly identified gene in the auditory system is being tested in zebrafish, by adopting the CRISPR-Cas system cutting-edge technology to selectively and stably inactivate the gene of interest [4], thus allowing a life-long analysis of its roles in ear development and homeostasis. Similar approaches will be adopted for the characterization of additional genes/mutations derived by WES data analysis. References Walsh T et al. 2010. Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 87:90-4. Schraders M, et al. 2011. Next-generation sequencing identifies mutations of SMPX, which encodes the small muscle protein, X-linked, as a cause of progressive hearing impairment. Am J Hum Genet 88:628-34. Huebner AK, et al. 2011. Nonsense mutations in SMPX, encoding a protein responsive to physical force, result in X-chromosomal hearing loss. Am J Hum Genet 88:621-7. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227-9. Project leader: CHIARA TONELLI (chiara.tonelli@unimi.it); (lucio.conti@unimi.it)Tutor: LUCIO CONTI Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARYThe role of ABA in the floral transition: site and mechanism of action Drought stress triggers an increase in abscisic acid (ABA) levels, leading to an acceleration of the floral transition, a phenomenon known as drought escape response (DE). Our current understanding of the DE response posits that ABA promotes the upregulation of the florigen genes FT and TSF under long day phototoperiodic conditions. Conversely, under a short day photoperiod ABA negatively regulates flowering, independently of the florigen genes. The overarching questions of the proposed project are: how does ABA participate in the transcriptional upregulation of florigen genes? which is the site of ABA action in flowering? Two main avenues of research will be pursued. First, we will study the distribution of plant ABA by analysing available transgenic plants harbouring an ABA–responsive promoter fused to a reporter gene. We will next examine the spatial regulation of ABA signalling in flowering by expressing dominant ABA signalling components under the control of tissue–specific promoters. Alterations in flowering time of these transgenic plants will inform about where ABA signalling is required to stimulate or inhibit flowering. We will then address the physiological significance of the reported interaction between the ABA –downstream transcription factor ABI3 and master regulator of the photoperiodic response CONSTANS. Knowledge obtained through these experiments will provide concepts that help to understand how enormously variable water–dependent signals are translated into developmental information in plants. The project will also elaborate novel mechanisms underlying gene expression regulation by addressing the mechanistic basis of the interaction between ABA and photoperiod. Project leader: CHIARA ZUCCATO (chiara.zuccato@unimi.it) Location: Department of Biosciences, University of Milan, ItalyRESEARCH PROJECT SUMMARY An in vivo study of the impact of ADAM10 dysfunction in Huntington’s Disease Huntington's disease (HD) is a genetically dominant, neurodegenerative disorder caused by an elongated polyglutamine (polyQ) segment in the huntingtin (Htt) protein. Mounting evidence indicates that mutant Htt disrupts normal synaptic function (contributing to HD behavioral, cognitive, and motor symptoms), and that alteration of the cortico-striatal excitatory circuit occurs early in HD progression (Zuccato and Cattaneo, Progress in Neurobiology 2007; Milnerwood and Raymond, TINS 2010; Zuccato et al., Physiological Review 2010). We believe that the search for effective strategies to help restore the activity of the cortico-striatal synapse (regarding structure, function, and plasticity) is an important area in HD research that requires further study. This project focuses on the role of metalloprotease disintegrin ADAM10 in HD. ADAM10 is critical both for the developing and adult brain. ADAM10 has only recently emerged in the HD field on the basis of our demonstration that normal Htt inhibits its activity during brain development (Lo Sardo and Zuccato et al., Nature Neuroscience 2012). Recent works show that ADAM10 exerts a critical role in the adult brain by controlling the structure and function of excitatory synaptic circuitries (Malinverno et al., The Journal of Neuroscience 2010; Marcello et al., The Journal of Clinical Investigation 2013; Saftig and Reiss, Eur J Cell Biol. 2011). Therefore, ADAM10 represents a good and novel target of investigation in HD due to its inherent structural and functional role in excitatory synaptic circuitries. Our preliminary data show that ADAM10 activity is significantly increased in the brain of HD mice and in post-mortem HD caudate. We also show increased proteolytic processing of ADAM10 synaptic substrates in the brain of HD mice. Notably, administration of GI254023X, a compound that specifically targets the catalytic domain of ADAM10 with no effects on other ADAMs, rescues the severe brain defects observed in zebrafish embryos expressing human mutant Htt. Finally, we found that treatment of organotypic slices from symptomatic HD mice with ADAM10 blockers restores pro-survival pathways in the HD brain. The goal of this project is to evaluate whether the increased activity of ADAM10 is a relevant component in the dysfunction of the cortico-striatal glutamatergic circuitry in HD. We will normalize ADAM10 activity in the brain of HD mice by crossing HD mice with the new line of CAMKIIalpha-cre Adam10Fl/+ heterozygous knock-out mice produced by P. Saftig at the University of Kiel (Prox et al., The Journal of Neuroscience 2013). By combining biochemical and behavioural studies we will test whether the inhibition of ADAM10 normalizes synaptic defects and is beneficial to HD mice. Download 336.57 Kb. Share with your friends:
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