List of Projects 2019/20
L Arcaini (Haematology), M Paulli (Pathology)
Dissecting molecular heterogeneity of unclassifiable B-cell lymphoproliferative disorders
According to the WHO Classification1, discovery of new entities or subtypes within heterogeneous tumors must be based on a multiparameter approach encompassing tumor pathology, genetics, phenotype and clinical course. However, some cases do not present lymph node and/or extranodal involvement and bone marrow histology coupled with flow cytometry is not able to identify a recognized WHO entity (non Hodgkin’s lymphoma not otherwise specified, NHL NOS). Aim of the project is to molecularly classify these cases fully clinically annotated through a NGS approach. Analysis will be performed on DNA isolated from NHL NOS patients using a targeted sequencing strategy of candidate gene coding exons (a panel of 138 genes recurrently mutated genes in mature B-cell neoplasia). We will use the Nextera Flex for Enrichment Kit (Illumina) to generate DNA libraries. Bioinformatic analysis will be performed by an ad hoc pipeline. Machine learning approaches applied to multiple layers of “big” data led to the discovery of unexpected molecular clusters (MC). Scientific instrumentation to be used: Second generation sequencers: HiSeq output rapid run 90 Gb, Miseq 15 Gb. The project will be carried out in the research laboratories of Haematology Unit located at Fondazione IRCCS Policlinico San Matteo in collaboration with M Paulli (Pathology).
V Bellotti, S Giorgetti (Biochemistry), M Paulli, A Vanoli (Pathology)
Ruolo dell’ ipossia nel danno tissutale da deposizione amiloide
La deposizione amiloide nelle amiloidosi sistemiche e localizzate causa un sovvertimento della matrice extracellulare e una sua espansione che modifica le distanze tra le membrane cellulari e le pareti capillari. Abbiamo ipotizzato che tale alterazione possa modificare sostanzialmente il profilo del gradiente di pressione parziale di ossigeno causando una condizione di ipossia che potrà essere latente o conclamata a seconda della spinta metabolica al consumo di ossigeno mitocondriale. Nostri risultati preliminari, ottenuti in collaborazione con i colleghi A Vanoli e M Paulli e M Parola dell’Università di Torino, confermano questa ipotesi su un modello di amiloidosi da peptide IAPP. Questo tipo di amiloidosi è responsabile di diabete di tipo 2 in topi transgenici e ha ruolo patologico in via di definizione nel diabete di tipo 2 nell’uomo. La caratterizzazione molecolare e funzionale del ruolo dell’amiloide nel causare uno stato ipossico verrà condotta con tecniche di proteomica ed istologia molecolare per caratterizzare l’espressione dei fattori trascrizionali hypoxia-inducible factors (HIF-1) e (HIF-2). La pressione parziale di ossigeno verrà calcolata sia speimentalmente che attraverso modelli consolidati basati sulla distanza membrane cellulari-superficie endoteliale. Lo stato ipossico è responsabile dell’aumento di radicali dell’ossigeno che verranno opportu-namente quantificati e il loro valore correlato con PaO2 e l’espressione dei fattori trascrizionali. Il modello di studio dell’amiloide delle Isole di Langerhans verrà validato su un modello di amiloidosi cardiaca murino messo a punto in collaborazione con il National Amyloidosis Center di Londra e su tessuti bioptici cardiaci già a disposizione della nostra Unita e già coperti da permessi di utilizzo per scopi di ricerca ottenuti con consenso informato. La teoria che ci proponiamo di verificare è assolutamente originale e la verifica sperimentale potrà chiarire aspetti fondamentali, e ancora incerti, sul meccanismo di tossicita’ cellulare del materiale amiloide.
R Bottinelli (Physiology)
YH10-dependent regulation of necroptosis a novel non-apoptotic programmed cell death type
Programmed cell death is a fundamental cellular process that has a central role in cancer. Indeed, deregulated cell death contributes to the progression of the disease, and, at the same time, cell death induction is one of the most promising therapeutic lines available. Until recently apoptosis was regarded as the sole programmed cell death, however the discovery of necroptosis, a form of programmed necrosis, forced us to re-evaluate the contribution of this death type in the processes that previously were attributed solely to apoptotic cell death. In relation to cancer, the role of necroptosis remains unclear with multiple studies supporting its anti-tumor function while increasing evidence suggests that necroptosis also promotes cancer progression. However, mechanistic studies on the role of necroptosis in cancer are in their infantry and it is unclear whether there are regulatory mechanisms that control a functional shift of and/or by necroptosis within the different stages of cancer progression. In a recent mass spectrometry screening, aiming to discover binding partners of the main regulator of necroptosis, Receptor Interacting Protein Kinase 3 (RIPK3), we identified myosin 10 (MHY10). Since conventional non-muscle myosins have been connected to apoptosis, we formulated the hypothesis that MYH10 may be a regulator of RIPK3 function and consequently necroptosis. Specific aims of the proposed project: The overall goal of the proposed project is to explore the mechanistic connection between MYH10, RIPK3 and necroptosis during cancer progression. The investigation will concentrate on 3 specific aims: (i) Determine the domains through which MYH10 interacts with RIPK3, (ii) Establish whether cellular stress induced by the cancerous microenvironment affects the binding of MYH10 and RIPK3 and whether this affects necroptosis and, (iii) Establish whether there is a functional connection between the MYH10/RIPK3 axis to metastasis. Impact, Significance, Novelty: This is a highly ambitious project and a pioneering effort that aims to explore a novel and previously unappreciated crosstalk between a structural component (MYH10) and a cell death-related kinase (RIPK3).
R Ciccone (Human Genetics), A Forlino (Biochemistry)
Functional characterization of ciliary genes causing neurodevelopmental and skeletal co-morbidities
A Postdoctoral Position is available at the Department of Molecular Medicine to investigate ciliopathies characterized by neurodevelopmental and skeletal co-morbidities. This is a collaborative project between the Medical Genetics Unit, researching the genetic basis of brain ciliopathies, and the Biochemistry Unit, with expertise in animal models of genetic skeletal diseases. Ciliopathies are clinically and genetically heterogeneous disorders caused by disfunction of the primary cilium. The project will focus on ciliopathy-related genes causing neurodevelopmental and/or skeletal abnormalities (e.g. CPLANE1 and CEP120), and aims at dissecting the molecular basis of such phenotypic variability using in vitro and in vivo approaches. Knock-out and knock-in zebrafish models will be generated by Crispr/Cas9 to reproduce mutations identified in patients with neurological and/or skeletal phenotypes, whose fibroblast-derived iPSCs are already available. High-resolution imaging (microCT, MRI, DLS, STED) and specific reporter zebrafish will be employed for deep in vivo characterization of neuronal and skeletal development. In parallel, patient-derived iPSCs will be differentiated towards neuronal and osteoblastic linages, to further characterize the developmental defects by monitoring developmental markers at the RNA and protein level and performing whole transcriptomic studies. The applicant will be placed in a stimulating, collaborative environment with excellent instrumental and intellectual resources for pursuing innovative research and career development. Successful candidates should have a PhD with training in Molecular and Cell Biology, Genetics, Developmental Biology, zebrafish work. The candidate must have documented peer-reviewed research productivity.
E Gherardi (Immunology and General Pathology), EM Valente (Human Genetics)
Structural and functional characterization of the interaction between PINK1 and Beclin 1
PINK1 is a protein mutated in autosomal recessive Parkinson Disease which plays a crucial role in mitophagy and was proven to interact with Beclin 1, a master regulator of autophagy, in the areas of contact between endoplasmic reticulum and mitochondria, a cellular region altered in diseases such as Parkinson’s. The aim of this project is the characterization of this interaction both from a structural and functional point of view, and the study could be possibly expanded to other binding partners. The structural work will take place in Prof. Ermanno Gherardi’s laboratory and will rely on techniques such as recombinant protein expression, purification, X-ray diffraction, NMR, cryo-EM. Functional studies will take place in Prof. Enza Maria Valente’s laboratory and will make use of integrated strategies of molecular biology, biochemistry, and imaging (real time qPCR, western blotting, co-immunoprecipitation, subcellular fractionation, ELISA assays, confocal microscopy, flow cytometry, SeaHorse technology etc). The strengths of this project are the integrated approach, which will benefit from the vicinity and ongoing collaboration between the two labs, and a constant look at the clinical significance of the work.
 Schubert AF, et al. Nature 2017; 552(7683):51-56. PMID: 29160309
 Gelmetti V et al. Autophagy 2017; 13:654-669. PMID: 28368777
M Gnecchi (Cardiology)
Modeling inherited cardiomyopathies and arrhythmias with induced pluripotent stem cells-‐derived cardiomyocytes
Recent advances in the study of epigenetic and DNA sequencing revealed how human genetic variations associate with differential health risks, disease susceptibilities, and drug responses. Such information is now expected to help evaluate individual health risks and design personalized treatments. However, understanding how genetic and epigenetic variations cause the phenotypic alterations in pathobiology and treatment response still remains challenging. Human induced pluripotent stem cell (iPSC) technology emerged as promising strategies to fill the knowledge gaps between genetic/epigenetic association studies and underlying molecular mechanisms (1). Breakthroughs in genome editing technologies and continuous improvement in iPSC differentiation techniques are paving the way for a clinically useful translation of this approach. Pioneering studies have shown that iPSCs derived from a variety of monogenic diseases can faithfully recapitulate disease phenotypes in vitro when differentiated into disease-‐relevant cell types. For instance, iPSC-‐derived cardiomyocytes (iPSC-‐CMs) have been used to study inherited arrhytmias, such as long QT syndrome (LQTS), or other forms of inherited cardiomyopathies, such as laminopathies. Since few years, we use the iPSC technology to model LQTS and other rare cardiomyopathies (2,3). Here, we propose a project aiming at: 1) identification of factors able to modify the clinical penetrance of inherited cardiomyopathies; 2) identification of novel putative therapeutic targets. The candidate will deal with the latest technologies in genetics, molecular biology, microscopy, cell sorting and cell biology. Taking advantage of the recent acquisition by the Department of a state of the art nuclear magnetic risonance (NMR) spectroscopy platform, we propose to use this technique as a mean to predict the impact of lamin mutations on SCN5A mediated peak sodium current (INa) based on the molecular multidimentional interaction between the 2 proteints. Patient specific iPSC will be than used to verify in the impact of different LMNA mutation on peak sodium current (INa) and to assess the predictive capability of NMR spectroscopy on the cellular and clinical phenotype. The candidate will work in the group led by Prof. Gnecchi at the Unit of Cardiology of the Department, at the Cardiology Division (patient recruitment) and at the Laboratory of experimental cardiology (experimental part) of the Fondazione IRCCS San Matteo. The project will be carried out in collaboration with the Unit of Human Genetics of the DMM. Briefly, the Laboratory of experimental cardiology is fully equipped with laminar and chemical hoods, CO2 incubators, refrigerated centrifuges, freezers (-80°C and -30°C), refrigerators, multimode imaging plate reader (time resolved fluorescence, fluorescence polarization, flash luminescence, glow luminescence, dual color luminescence and absorbance) and gradient and real-‐time thermocyclers. In the laboratory there is also all the required equipment for cloning (microbiological incubator for prokaryotic dishes and shaking incubators for prokaryotic cultures) and molecular analysis (electrophoresis and western blotting apparatus associated with the Odyssey infrared imaging system for quantitative analysis). Moreover an inverted fluorescent microscope (Zeiss Axio Observer Z1 equipped with ApoTome system) with a wide array of filters is available in the laboratory.
 From patient-‐specific induced pluripotent stem cells to clinical translation in long QT syndrome Type 2. Schwartz PJ, Gnecchi M, Dagradi F, Castelletti S, Parati G, Spazzolini C, Sala L, Crotti L. Eur Heart J. 2019 Feb 6. doi: 10.1093/eurheartj/ehz023.
 The KCNH2-‐IVS9-‐28A/G mutation causes aberrant isoform expression and hERG trafficking defect in cardiomyocytes derived from patients affected by Long QT Syndrome type 2. Mura M, Mehta A, Ramachandra CJ, Zappatore R, Pisano F, Ciuffreda MC, Barbaccia V, Crotti L, Schwartz PJ, Shim W, Gnecchi M. Int J Cardiol. 2017 Apr 12. pii: S0167-‐5273(17)30298-‐X. doi: 10.1016/j.ijcard.2017.04.038.
M Lolicato (Applied Biology)
Imaging of the human Voltage-Dependent Anion Channel (VDAC) in complex with its regulatory partner Hexokinase in a native-like environment
The Voltage-Dependent Anion Channel (VDAC) is a transmembrane mitochondrial protein crucial for the cell energetics. VDAC is implicated in apoptosis and cancer cell survival. Hexokinase (HK), the first enzyme of glycolysis, binds VDAC on mitochondria. HK is overexpressed in many cancer types and involved in the Warburg effect. HK association to VDAC inhibits apoptosis: such mechanism is thus exploited by tumor cells to escape apoptosis. The molecular mechanism of VDAC/HK interaction is still largely unknown.The proposed project aims at the understanding at a molecular level the interaction between VDAC and HK. The project will involve the generation of the three-dimensional structure of VDAC in complex with the full-length HK in a bilayer setting. High-resolution crystal structure of VDAC in complex with a HK peptide able to interfere with the channel activity (Patent #WO2017158502A1), instead, will be used as a starting point to identify new drugs able to disrupt the VDAC/HK interaction. Electrophysiological recordings will be performed to validate the structural findings. The project will involve protein expression and purification, reconstitution of the complex in lipid bilayers, biophysical characterization of the sample using cryo-electron microscopy, X-ray crystallography and electrophysiology.
C Olivieri (Applied Biology)
Quantitative and functional analysis of circulating miRNAs in a rare disease
The project aims to verify the hypothesis that, beyond the disease-causing mutation, a circulating microRNAs (miRNAs) dysregulation is present in patients affected with Hereditary Haemorrhagic Telangiectasia (HHT). Very few data are present in literature about circulating miRNAs and HHT. Our preliminary results let to a list of differentially expressed miRNAs in plasma, including many “angiomiRs”, miRNAs involved in angiogenesis. As HHT is an autosomal dominant disease leading to abnormal vessel structures, these results are very encouraging. The project includes two steps: 1) validation of the circulating miRNAs profile already identified, using real time PCR and Taqman™ probes on RNA from a large number of HHT Patients; 2) investigating the functional role of circulating miRNAs in HHT pathogenesis by in silico and in vitro analyses on selected target genes (i.e. western blotting, ELISA, luciferase and wound healing assays). The patients are enrolled by the ENT Unit of IRCCS Policlinico “San Matteo” in Pavia, which is our main clinical partner. For both aims we will take advantage of facilities already present in the Dept of Molecular Medicine (real time, cell culture, image analyzer, functional study lab) ; in particular, the research will be performed in the General Biology and Medical Genetics Unit.
A Rossi (Biochemistry)
Molecular basis of heritable skeletal disorders
In the Rossi laboratory at the Department of Molecular Medicine Unit of Biochemistry the overarching goal is the study of the molecular basis of heritable skeletal disorders caused by defects in proteoglycan metabolism. Using animal and cellular models, the objectives of our research are: (i) the understanding of the molecular basis of heritable skeletal diseases to gain a deep knowledge of the cellular, molecular and genetic aspects contributing to the initiation and progression of the disorders; (ii) the development of effective approaches for the diagnosis, prevention and/or treatment of the disorder. We are looking for a highly motivated and enthusiastic post-doctoral fellow working at the molecular basis of Desbuquois dysplasia, a skeletal disorder we have recently studied (Matrix Biol 2018 Nov 12. doi: 10.1016/j.matbio.2018.11.002) caused by defects in a calcium activated nucleotidase of the Golgi. Using animal and cellular models the main goal of the project is the study of the alterations in organelle homeostasis due to a lack of function of the nucleotidase. The project will involve biochemical, molecular biology and confocal/electron microscopy techniques. Successful candidates should be well trained in mouse work, mammalian cell culture, biochemistry and cell biology.
A Balduini (Clinical Biochemistry), L Stivala (Immunology and General Pathology)
Multiple roles of DDB2 protein in cancer progression
DDB2, known for its function in DNA repair, was recently shown to exert multiple roles, being involved in several biological processes, including tumorigenesis and cancer progression. We have previously demonstrated that it directly interacts with PCNA, a master regulator of DNA replication and repair. The mutant form (DDB2PCNA-) abolishing this interaction, delays DNA repair and promotes cell proliferation of HEK293 cells, through a defective DNA damage checkpoint activation (1). Aim of this project will be to understand whether the altered expression or specific mutations (e.g. those affecting PCNA association) of DDB2 influence the onset of the metastatic phenotype. Since cellular adhesion, migration and invasion are key events associated with the epithelial to mesenchymal transition, particular attention will be paid to the role of DDB2 in these processes. Using DDB2Wt and DDB2PCNA- HEK293 clones, it will be investigated: i) their adhesion on glass, plastic and extracellular matrix components, ii) their anchorage-independent growth, iii) their migration and invasion in 3D scaffolds, and iv) their nanomechanical properties by atomic force microscopy.
L Visai (Biochemistry)
Immunotherapy in Staphylococcal INfectious DisEase – Displacing Antibodies (IMMUNE)
Since the discovery of penicillin, antibiotics have provided efficacious treatments for bacterial infections. The growing numbers of antimicrobial strains among different pathogens challenge this strategy and underscore the threat that bacterial infections still pose to world health. In this context, we must consider novel strategies to combat infections such as immunotherapy. We have studied for a long time staphylococcal virulence factors in terms of their ligand interactions and the antibody responses they can trigger. We have focused on surface proteins of the MSCRAMM family identifying four types of antibodies (Abs) that differ in terms of the effects that these have on the interactions of a bacterial antigen with its host targets: i) Abs that bind to the virulence factor but do not affect the virulence factor’s activity. ii) Abs that inhibit the virulence factors activity. iii) Abs that specifically recognize the complex of the virulence factor bound to its host ligand but does not inhibit the activity of the virulence factor and iv) Abs that can displace the ligand from the virulence factor. These latter Abs are especially interesting in immunotherapeutic strategies since they can reverse an interaction already established. So far, we have conclusive data showing the presence of displacing murine mAbs against the S. aureus collagen binding MSCRAMM, CNA. Furthermore, we have been able to isolate human anti-CNA Abs from a library of single chain Fragment variable (scFv) antibodies. CNA is the prototype of a large family of structurally related collagen-binding surface adhesins, present on most Gram-positive pathogens but also on some Gram-negative bacteria. Then, although, CNA is present in a subset of clinical isolates of S. aureus, the MSCRAMM is an important prototype for testing our hypothesis that displacing Abs can be effective immunotherapeutic. The main aim to pursue is the generation and characterization of the mechanism(s) of action of displacing anti CNA antibodies. To fulfill this aim, we intend to complete the characterization of human anti-CNA scFv selected by antibody phage display and pursue the crystallographic studies on human scFv::CNA complex. Methodology: Production and characterization of the human anti-CNA scFv-Fc (IgG like), antigen affinity through Surface Plasmon Resonance (SPR), protein stability through Differential Scanning Fluorimetry (DSF) to determine protein Tm, epitope mapping and assay of inhibition and displacing activity using clinical isolate of S.aureus strains; flow cytometry analysis and structural analysis of dynamic biological macromolecules with Cryo-EM. Equipment: Surface Plasmon Resonance (SPR), Differential Scanning Fluorimetry (DSF), ELISA assay, SEM, flow cytometry, X-ray crystallography analysis and Cryo-EM. The project is a collaborative effort between the laboratories of L Visai in Pavia, M Hust in Germany and M Hook in USA.