List of Projects 2017/18
Title: Bone marrow modelling for platelet production
Supervisor(s): Alessandra Balduini, Alessandro Malara
Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood. Under- or over-production of platelets has major clinical implications for many diseases, including thrombocytopenia and myeloproliferative neoplasms, where life-threatening side effects with incurable outcomes are common. The scientific and clinical communities are actively searching for new modes to generate functional platelets ex vivo to address clinical needs as well as for insight into fundamental studies of mechanisms. We hypothesize that engineering a 3D bone marrow mimic will propel mechanistic understanding of platelet shedding and determine future protocols for therapeutic inquiry. On the basis on our previous publications, the candidate will utilize non-thrombogenic silk protein biomaterial to perfect an ex vivo three dimensional (3D) tissue model of the bone marrow to study platelet release from megakaryocytes derived from human induced pluripotent stem cells. Megakaryocytes receive cues from the bone marrow environment including cell-cell contact, contact with extracellular matrix components, and physical characteristics of the tissue (topography and rigidity of the extracellular space) as well as shear stress generated by the blood flow in the vessels. By refining the environment in the 3D silk-based bone marrow system the aim will be to provide all the physical and biochemical characteristics necessary to improve ex vivo platelet release by megakaryocytes. The outcome of these studies is expected to be the design of new tools to mimic the bone marrow niches ex vivo gaining insight into the mechanisms that control platelet release in a physiological relevant manner.
1. Di Buduo CA, Wray LS, Tozzi L, Malara A, Chen Y, Ghezzi CE, Smoot D, Sfara C, Antonelli A, Spedden E, et al. Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies. Blood 2015;125:2254-2264.
2. Di Buduo CA, Currao M, Pecci A, Kaplan DL, Balduini CL, Balduini A. Revealing Eltrombopag’s promotion of human megakaryopoiesis through AKT/ERK-dependent pathway activation. Haematologica 2016;101:1479-1488.
3. Malara A, Gruppi C, Pallotta I, Spedden E, Tenni R, Raspanti M, Kaplan D, Tira ME, Staii C, Balduini A. Extracellular matrix structure and nano-mechanics determine megakaryocyte function. Blood 2011;118:4449-4453.
Title: Cellular and molecular mechanisms of skeletal muscle atrophy in ageing and disuse
Supervisor(s): Roberto Bottinelli
Skeletal muscle shows high structural and functional heterogeneity and plasticity, namely its structure and function can deeply adapt to physiologic and pathologic conditions such as exercise training, disuse, ageing, muscular dystrophy, chronic non-muscle diseases, drug administration. This is essential to enable the body to improve or maintain physical performance, and to cope with changes in energy and amino-acid supply such as those occurring in starvation or chronic diseases. Our research group has been working since the 80s on the cellular and molecular mechanisms underlying skeletal muscle plasticity in health and disease. Skeletal muscle wasting occurring in ageing and disuse is a major health concern for modern societies. It affects life span and quality of life, risk of chronic diseases and their prognosis. Despite much work performed, the role of single muscle fibres adaptations in the impairment of whole body exercise capacity and the mechanisms of single muscle fibres loss of mass and function are still debated. To address such issues, we study muscle structure and function in mice and human models at different levels: whole body; isolated muscles in vitro; individual muscle fibres; isolated myosin. Samples used for functional analysis are subjected to the following structural analyses: (i) muscle fibres size and type by immuno-histochemistry; (ii) concentration of myosin and myofibrillar proteins by quantitative electrophoresis; (iii) expression of myofibrillar proteins isoforms by high resolutions SDS-PAGE and Western blot; (iv) global protein pattern and post-translational modifications of proteins by proteomics; (v) intracellular signalling pathways controlling muscle mass and metabolism.
1. Brocca L, Toniolo L, Reggiani C, Bottinelli R, Sandri M, Pellegrino MA. FoxO-dependent atrogenes vary among catabolic conditions and play a key role in muscle atrophy induced by hindlimb suspension. J Physiol 2017;595:1143-1158.
2. Cannavino J, Brocca L, Sandri M, Grassi B, Bottinelli R, Pellegrino MA. The role of alterations in mitochondrial dynamics and PGC-1alpha over-expression in fast muscle atrophy following hindlimb unloading. J Physiol 2015;593:1981-1995.
3. Brocca L, Longa E, Cannavino J, Seynnes O, de Vito G, McPhee J, Narici M, Pellegrino MA, Bottinelli R. Human skeletal muscle fibre contractile properties and proteomic profile: adaptations to 3 weeks of unilateral lower limb suspension and active recovery. J Physiol 2015;593:5361-5385.
Title: Structural and functional adaptations of skeletal muscle to steroid administration and the underlying cellular and molecular mechanisms
Supervisor(s): Monica Canepari
Steroid myopathy is a consequence of glucocorticoid administration and excess occurring in Cushing’s syndrome. It is characterized by muscle weakness, which impairs patients’ capacity to perform everyday activities. The major pathogenetic mechanisms hypothesized are: decreased sarcolemmal excitability, imbalance between protein synthesis and degradation, impairment of mitochondrial function and redox imbalance. The triggers of the latter phenomena and muscle damage are still unknown. So far, analyses have focused on skeletal muscle at times when steroid myopathy had fully developed making it difficult to take apart the phenomena causing the myopathy from those being a consequence of it. The project aims to identify the molecular basis of steroid myopathy and to prevent it. To address the problem the early phases and the time course of muscle functional, structural and molecular adaptations will be investigated both in animals and humans. A potential therapy will also be tested. The project is supported by a grant from the Italian Ministry of Health.
1. Minetto MA, Qaisar R, Agoni V, Motta G, Longa E, Miotti D, Pellegrino MA, Bottinelli R. Quantitative and qualitative adaptations of muscle fibers to glucocorticoids. Muscle Nerve 2015;52:631-639.
2. Lossie J, Kohncke C, Mahmoodzadeh S, Steffen W, Canepari M, Maffei M, Taube M, Larcheveque O, Baumert P, Haase H, Bottinelli R, Regitz-Zagrosek V, Morano I. Molecular mechanism regulating myosin and cardiac functions by ELC. Biochem Biophys Res Commun 2014;450:464-469.
3. Canepari M, Maffei M, Longa E, Geeves M, Bottinelli R. Actomyosin kinetics of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach. Exp Physiol 2012;97:873-881.
Title: Genomic profiling of myeloid malignancies and development of precision medicine strategies
Supervisor(s): Mario Cazzola, Elisa Rumi
Rare cancers are identified as those with an incidence of less than 6 per 100,000 persons per year: most myeloid malignancies belong to this category, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and myelodysplastic/ myeloproliferative neoplasms (MDS/MPN). All tumors are caused by somatic mutation, that is, the occurrence of abnormalities in DNA sequence of genes, and the identification of recurrent genetic events represents one of the most direct approaches to understand cancer biology. Precision medicine is a medical model that combines already established clinical-pathological parameters with advanced genomic profiling in order to create innovative diagnostic, prognostic and therapeutic strategies. In hematology, the paradigmatic example is chronic myeloid leukemia, in which the discovery of the BCR-ABL1 fusion gene and its oncogenic properties has translated into targeted therapy. We are currently combining genomic profiling with clinical studies to develop innovative precision medicine strategies for patients with MDS, MPN or MDS/MPN. State-of-art genomic profiling include mutational characterization of both germline and acquired gene mutations. We preferentially study patients enrolled into observational or interventional clinical trials. We analyze the interactions among driver mutations, gene expression, clinical variables and patient outcome to model natural history of disease and impact of treatment. We aim to identify genomic changes that can be used for classification, risk assignment, enrollment into clinical trials, and prediction of response. Phase I/II as well as innovative clinical trials, aimed to test safety and efficacy of new or repurposed drugs in molecularly characterized patients, are conducted in collaboration with pharmaceutical companies.
1. Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD, Them NC, Berg T, Elena C, Casetti IC, Milanesi C, Sant’antonio E, Bellini M, Fugazza E, Renna MC, Boveri E, Astori C, Pascutto C, Kralovics R, Cazzola M; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood 2014;123:1544-1551.
2. Elena C, Gallì A, Such E, Meggendorfer M, Germing U, Rizzo E, Cervera J, Molteni E, Fasan A, Schuler E, Ambaglio I, Lopez-Pavia M, Zibellini S, Kuendgen A, Travaglino E, Sancho-Tello R, Catricalà S, Vicente AI, Haferlach T, Haferlach C, Sanz GF, Malcovati L, Cazzola M. Integrating clinical features and genetic lesions in the risk assessment of patients with chronic myelomonocytic leukemia. Blood 2016;128:1408-1417.
3. Della Porta MG, Gallì A, Bacigalupo A, Zibellini S, Bernardi M, Rizzo E, Allione B, van Lint MT, Pioltelli P, Marenco P, Bosi A, Voso MT, Sica S, Cuzzola M, Angelucci E, Rossi M, Ubezio M, Malovini A, Limongelli I, Ferretti VV, Spinelli O, Tresoldi C, Pozzi S, Luchetti S, Pezzetti L, Catricalà S, Milanesi C, Riva A, Bruno B, Ciceri F, Bonifazi F, Bellazzi R, Papaemmanuil E, Santoro A, Alessandrino EP, Rambaldi A, Cazzola M. Clinical effects of driver somatic mutations on the outcomes of patients with myelodysplastic syndromes treated with allogeneic hematopoietic stem-cell transplantation. J Clin Oncol 2016;34:3627-3637.
Title: Pompe disease: genetic analysis of “extreme phenotypes”
Supervisor(s): Cesare Danesino, Paola De Filippi
Pompe disease (PD) (OMIM 232300) is a rare autosomal recessive disorder; GAA is the only disease causing gene. Late-onset phenotypes show a wide clinical variability for age of onset, rate of disease progression, pattern of muscle involvement. This extensive clinical variability, present both within and among families, suggest that some family members of patients with PD are carriers of two pathogenic mutations, show a very mild clinical phenotype and are not yet diagnosed as affected with PD. The efficacy of Enzyme Replacement Therapy (ERT) is well documented, but several reports show that some patients behave as “poor responders”. It is thus possible to identify “extreme phenotypes” (very mild or very severe) for most of the clinical features of the disease. The clinical variability of PD as well as the unpredictable response to ERT, clearly indicate that factors, other than GAA deficiency, may influence the mechanisms of muscle damage/repair, the severity of other clinical features and the response to ERT. The project is aimed to identify genetic factors, additional to GAA mutations, relevant for modulating the clinical presentation of the disease. We expect, within 1 year, thanks to ongoing nationwide collaborations, to collect 30 patients representative for “extreme phenotype” related to age of onset, muscle damage, respiratory impairment, and response to therapy. These patients will be studied by exome sequencing and genetic variants identified will be selected based on their frequency, possible pathogenic significance, belonging to pathways related to previously listed clinical features and validated. The project has been already funded
1. De Filippi P, Saeidi K, Ravaglia S, Dardis A, Angelini C, Mongini T, Morandi L, Moggio M, Di Muzio A, Filosto M, Bembi B, Giannini F, Marrosu G, Rigoldi M, Tonin P, Servidei S, Siciliano G, Carlucci A, Scotti C, Comelli M, Toscano A, Danesino C. Genotype-phenotype correlation in Pompe disease, a step forward. Orphanet J Rare Dis 2014;9:102.
2. Parenti G), Fecarotta S, la Marca G, Rossi B, Ascione S, Donati MA, Morandi LO, Ravaglia S, Pichiecchio A, Ombrone D, Sacchini M, Pasanisi MB, De Filippi P, Danesino C, Della Casa R, Romano A, Mollica C, Rosa M, Agovino T, Nusco E, Porto C, Andria G. A chaperone enhances blood α-glucosidase activity in Pompe disease patients treated with enzyme replacement therapy. Mol Ther 2014;22:2004-2012.
3. Musumeci O, la Marca G, Spada M, Mondello S, Danesino C, Comi GP, Pegoraro E, Antonini G, Marrosu G, Liguori R, Morandi L, Moggio M, Massa R, Ravaglia S, Di Muzio A, Filosto M, Tonin P, Di Iorio G, Servidei S, Siciliano G, Angelini C, Mongini T, Toscano A; Italian GSD II group. LOPED study: looking for an early diagnosis in a late-onset Pompe disease high-risk population. J Neurol Neurosurg Psychiatry 2016;87:5-11.
Title: Identifying pathogenic, prognostic and theragnostic factors in cancer-associated gastrointestinal inflammation
Supervisor(s): Antonio Di Sabatino
A higher risk for developing small bowel carcinomas (SBC) has been reported in celiac disease (CeD) and Crohn’s disease (CrD). However, most information concerning CeD or CrD-associated-SBC (CeD-SBC) is limited to few, retrospective series or case reports. It has been suggested that CeD-SBC show a survival advantage compared with sporadic SBC. Apart from SBC, CeD may predispose to enteropathy associated T-cell lymphoma (EATL), a rare but aggressive complication. Poor adherence to a gluten-free diet, HLA-DQ2 homozygosity, and late diagnosis of CD are recognized as risk factors for malignant evolution of CD. This tumor derives from the neoplastic transformation of aberrant intraepithelial T lymphocytes emerging in CeD patients unresponsive to a gluten-free diet. In a retrospective multicenter study we will assess more-in depth the features of EATL in CeD and CeD- or CrD-associated SBC. Aim of the project is to describe the spectrum of lymphoproliferations (of B-cell and T/NK cells) arising in CD by a histopathologic review and to assess the potential pathogenetic and prognostic role of the underlying gastrointestinal disorder. Molecular analysis is evaluated by microdissection technique the clonality of lesions as well as the clonal relationship between the inflammatory infiltrate, at different times of the disease evolution to overt lymphoma and, for B-cell lymphomas, the mutational status of immunoglobulin heavy chain genes. In the second part of the project we aim to investigate the immunophenotypic and molecular features of SBC associated with Crohn’s disease and celiac disease and explore possible clinical or molecular biomarkers of cancer evolution.
1. Vanoli A, Di Sabatino A, Biancone L, Martino M, et al. Small bowel Epstein-Barr virus-positive lympho-epithelioma-like carcinoma in Crohn’s disease. Histopathology 2017;70:837-839.
2. Vanoli A, Di Sabatino A, Furlan D, et al. Small Bowel Carcinomas in Coeliac or Crohn’s Disease: Clinico-pathological, Molecular and Prognostic Features. A Study from the Small Bowel Cancer Italian Consortium. J Crohns Colitis 2017 Feb 24. [Epub ahead of print] 3. Vanoli A, Di Sabatino A, Martino M, et al. Small bowel carcinomas in celiac or Crohn’s disease: distintictive histophenotypic, molecular and histogenetic patterns. Mod Pathol, in press
Title Structural characterization of new lectins with antineoplastic properties
Supervisor(s): Monica Galliano
Molecular imaging and targeted drug delivery have in common the use of biological (macro)molecules whose role is the recognition of other molecules (including carbohydrates and glycoproteins) that are indicative of a pathological state. Many cell surface proteins and lipids are glycosylated, and sugars can combine to produce a very large number of special carbohydrate structures. Different cell types can display on their surface very dissimilar glycans and, in particular, some neoplastic cells are known to expose on their membranes, carbohydrates that are not present in normal cells. A well-known example is the Thomsen-Friedenreich antigen which is a disaccharide linked to cell surface glycoproteins and hidden in healthy cells while exposed in a high percentage of human carcinomas and other neoplastic tissues. Lectins are proteins of non-immune origin devoid of any catalytic activity, that reversibly bind mono- and oligosaccharides with high specificity and are involved, through sugar binding, in many fundamental biological processes. Initially identified in the plant kingdom because of their hemagglutinating activity are now being widely used in basic and clinical research to develop new drugs for cancer therapy, to treat microbial and viral infections and to fractionate hemopoietic stem cells for transplantation. Our research project is aimed at the identification, purification and and structural characterization of new lectins that specifically recognize the T antigen and that show antineoplastic activity on different cancer cell lines.
1. Capaldi S, Faggion B, Carrizo ME, Destefanis L, Gonzalez MC, Perduca M, Bovi M, Galliano M, Monaco HL. Three-dimensional structure and ligand-binding site of carp fishelectin (FEL). Acta Crystallogr D Biol Crystallogr 2015;71:1123-1135.
2. Bovi M, Cenci L, Perduca M, Capaldi S, Carrizo ME, Civiero L, Chiarelli LR, Galliano M, Monaco HL. BEL β-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms. Glycobiology 2013;23:578-592.
3. Bovi M, Carrizo ME, Capaldi S, Perduca M, Chiarelli LR, Galliano M, Monaco HL. Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms. Glycobiology 2011;21:1000-1009.
Title: Adipose stem cells for cartilage regeneration
Supervisor(s): Giulia Gastaldi
Osteoarthritis (OA) is the most common chronic degenerative joint disease and affects 20 and 50 million of people in USA and EU respectively, causing pain and decreased functionality. Mesenchymal stem cells (MSCs) are emerging as minimally invasive solutions able to restore the joint homeostasis and to delay OA progression. MSCs derived from adipose tissue (ASCs) became an attractive stem cell type thanks to their abundance, the ease with which they can be harvested (with low donor-site morbidity), their rapid expansion and high proliferation potential. ASCs showed capability to inhibit OA progression, due to their immunoregulatory and anti-inflammatory properties and to their ability to produce paracrine factors, able to enhance tissue regeneration. Recently, cell microvesicles (MVs) have been shown to play a vital role in cell-cell communication and tissue regeneration. They are membranous small vesicles able to transfer proteins, lipids, messenger RNA and micro RNA into cells, invoking changes of the gene expression, proliferation and differentiation of the recipient cells. Aim of the project is to verify the effects of ASC on chondrocytes, comparing them with the effects induced by MVs obtained from ADSCs in OA treatment. Chondrocytes and ASCs will be co-cultured in the percentage of 50-50 in a micromass culture system. Chondrocytes alone (100) and hASC alone (100) will be used as references. To evaluate the in vitro effects of hASC-MVs on chondrocytes, the suspension of hASC-MVs will be added to the chondrocytes after the micromass formation. After 3 or 6 weeks of culture, the pellets will be collected and analyzed.
1. Benazzo F, Botta L, Scaffino MF, Caliogna L, Marullo M, Fusi S, Gastaldi G. Trabecular titanium can induce in vitro osteogenic differentiation of human adipose derived stem cells without osteogenic factors. J Biomed Mater Res A 2014;102:2061-2071.
2. Pers Y-M, Ruiz M, Noel D, Jorgensen C. Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives. Osteoarthritis and Cartilage 2015;23:2027-2035.
3. Gun-Il Im. Endogenous Cartilage Repair by Recruitment of Stem Cells. Tissue Engineering: Part B 2016;22:160.
Title: Role of HGF/SF in tissue regeneration
Supervisor: Ermanno Gherardi
Chronic degenerative diseases of internal organs and the brain constitute a major cause of morbidity and mortality. S Yamanaka’s demonstration that adult cells can be reprogrammed to a stem cell-like phenotype has opened interesting prospects for cell therapy but there are remain major technical, financial and regulatory challenges with cell-based therapies. This project outlines a different strategy for regenerative medicine, namely protein therapy. A number of post natal contain adult stem/progenitor cell populations and, while these cells often fail to achieve a substantial degree of tissue regeneration after damage, certain growth/motility factors can result in amplification and differentiation of adult stem cell populations in vivo yielding enhanced levels of regeneration. The project will focus specifically on the polypeptide growth and motility factor HGF/SF, a protein essential for the development of several major cell types and organs during embryonic life and with further, crucial physiological roles in tissue regeneration in post-natal life. The project will involve: (i) design of new HGF/SF constructs for expression in bacteria and mammalian cells, (ii) assessment of protein activity by physico-chemical techniques (SPR) and assays on cells in culture and, (iii) experiments in vivo in which therapeutic proteins are administered to several strains of transgenic mice in which chronic organ damaged is induced.
1. Sinha Roy R, Soni S, Harfouche R, Vasudevan PR, Holmes O, de Jonge H, Rowe A, Paraskar A, Hentschel DM, Chirgadze D, Blundell TL, Gherardi E, Mashelkar RA, Sengupta S. Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis. Proc Natl Acad Sci USA 2010;107:13608-13613.
Title: Induced pluripotent stem cells to model inherited cardiomyopathies
Supervisor(s): Massimiliano Gnecchi
Recent advances in 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 such genetic variations cause the phenotypic alterations in pathobiology and treatment response still remains challenging. Human induced pluripotent stem cell (iPSC) technologies are emerging as promising strategies to fill the knowledge gaps between genetic association studies and underlying molecular mechanisms. Breakthroughs in genome editing technologies and continuous improvement in iPSC differentiation techniques are making this research direction more realistic and practical. 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 long QT syndrome (LQTS) and other forms of inherited cardiomyopathies. In our laboratory, since few years we use the iPSC technology in the context of LQTS and other rare diseases. The PhD candidate will be involved in projects aiming at: 1) identification factors able to modify the clinical penetrance of inherited cardiomyopathies; 2) identification of variants of unknown significance (VUS) and its mechanism of action. The candidate will deal with the latest technologies in genetics, molecular biology and cell biology and he/she will learn how to independently design experiments and interpret the results.
1. Mura M, Mehta A, Ramachandra CJ, Zappatore R, Pisano F, Ciuffreda MC, Barbaccia V, Crotti L, Schwartz PJ, Shim W, Gnecchi M. 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. Int J Cardiol 2017 Apr 12 (epub ahead of print) doi: 10.1016/j.ijcard.2017.04.038.
2. Gnecchi M, Stefanello M, Mura M. Induced pluripotent stem cell technology: Toward the future of cardiac arrhythmias. Int J Cardiol 2017 Mar 21. (epub ahead of print) doi: 10.1016/j.ijcard.2017.03.085.
3. Rocchetti M, Sala L, Dreizehnter L, Crotti L, Sinnecker D, Mura M, Pane LS, Altomare C, Torre E, Mostacciuolo G, Severi S, Porta A, De Ferrari GM, George AL Jr, Schwartz PJ, Gnecchi M, Moretti A, Zaza A. Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes. Cardiovasc Res 2017;113:531-541.
Title: H2O2 membrane crossing by aquaporins may control oxidative eustress and distress
Supervisor(s): Umberto Laforenza
In all cells, H2O2, the most abundant and stable reactive oxygen species (ROS), can have a different effect depending on concentration. Recently, the diffusion of H2O2 from producing cells across plasma membranes to the extracellular fluid has been considered an important ROS scavenging possibility, mediated by some water channel protein – aquaporins (AQPs), isoforms. The proposed project has a dual purpose: – to further understand the mechanisms of AQPs as peroxiporins in mediating H2O2 diffusion through cellular and subcellular compartments. The study will investigate the contribution of different AQPs expressed in HeLa cells by selective silencing of single isoforms and functional experiments of water and H2O2 permeability. Moreover, the gating of AQPs in presence/absence of oxidative stress will be analyzed by testing known antioxidant and novel plant extract compounds. – to investigate whether AQPs expression and functioning is affected by HPV infection in human sperm cells, in which four AQPs were recently identified, involved in volume regulation and ROS elimination. A relationship between sperm number/motility and AQPs functioning has been also demonstrated. HPV infection is closely related to decrease in sperm motility and morphology. Therefore, ejaculated sperm cells from normospermic and sub-fertile subjects screened for HPV infection will be investigated for AQPs expression and functioning. In particular, water and H2O2 permeability as well as the ROS basal quantification in sperm cells from health and HPV infected subjects, both normal and sub-fertile will be also investigated. The results could indicate a correlation between AQPs, HPV infection and male fertility.
1. Medraño-Fernandez I, Bestetti S, Bertolotti M, Bienert GP, Bottino C, Laforenza U, Rubartelli A, Sitia R. Stress Regulates Aquaporin-8 Permeability to Impact Cell Growth and Survival. Antioxid Redox Signal 2016;24:1031-1044.
2. Laforenza U, Pellavio G, Marchetti AL, Omes C, Todaro F, Gastaldi G. Aquaporin-Mediated Water and Hydrogen Peroxide Transport Is Involved in Normal Human Spermatozoa Functioning. Int J Mol Sci 2016;18:E66.
3. Bienert GP, Chaumont F. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. Biochim Biophys Acta 2014;1840:1596-1604.
Title: Novel therapeutic strategies against AL amyloidosis
Supervisor(s): Giampaolo Merlini – Giovanni Palladini
Systemic immunoglobulin light chain (AL) amyloidosis is a severe protein conformational disease caused by misfolding and deposition of patients-specific monoclonal immunoglobulin light chains produced by a small and otherwise indolent bone marrow plasma cell clone. If diagnosed late and not responsive to therapy, AL amyloidosis is rapidly fatal, mainly due to cardiac dysfunction. Therapeutic interventions specifically designed for AL amyloidosis are still lacking, possibly due to the limited knowledge of the biology of the disease-causing plasma cell clone. Aim of this PhD project is to study the sensitivity of the disease-causing plasma cell clone to novel small molecule drugs targeting the ubiquitin proteasome system (UPS) and possibly identify candidate drugs or combinatorial therapies for future clinical studies. Primary plasma cells from diagnostic leftovers of bone marrow aspirations from patients with AL amyloidosis, evaluated at the Italian Referral Center for Systemic Amyloidoses in Policlinico San Matteo, will be sorted using magnetic beads and characterized by flow cytometry. Primary plasma cells will be exposed to different doses of the investigational drugs in vitro and efficacy will be assessed with cell viability assays. For drugs showing anti-plasma cell effects, biochemical and genetic analyses (including RNA interference or genome editing) will be performed to confirm the mechanism of action, and the possible synergistic effect with other anti-plasma cell drugs will be investigated. The most promising molecule(s) will be finally validated in vivo using a recently established xenoengraftment murine model.
1. Palladini G, Merlini G. What is new in diagnosis and management of light chain amyloidosis? Blood 2016;128:159-168.
2. Nuvolone M, Sorce S, Pelczar P, Rushing E, Lavatelli F, Rognoni P, Valentini V, Palladini G, Merlini G, Aguzzi A. Regulated expression of amyloidogenic immunoglobulin light chains in mice. Amyloid 2017;24(sup1):52-53.
3. Oliva L, Orfanelli U, Resnati M, Raimondi A, Orsi A, Milan E, Palladini G, Milani P, Cerruti F, Cascio P, Casarini S, Rognoni P, Touvier T, Marcatti M, Ciceri F, Mangiacavalli S, Corso A, Merlini G, Cenci S. The amyloidogenic light chain is a stressor that sensitizes plasma cells to proteasome inhibitor toxicity. Blood 2017;129:2132-2142.
Title: Alloalbuminaemia and Analbuminaemia
Supervisor(s): Lorenzo Minchiotti
Human serum albumin, the major blood protein, is encoded by a single gene at position 4q13.3, near chromosome 4 centromere. Mutations in this gene may result in the presence of two circulating forms of the protein (bisalbuminaemia or alloalbuminaemia) or in the virtual absence of the protein from the blood (congenital analbuminaemia, CAA). Alloalbumins are markers of migration and population genetics, and can provide an useful tool for the study of the unique binding properties of the protein. Usually, they do not seem to be associated with disease, except for the mutations causing familial dysalbuminemic hyperthyroxinemia and familial dysalbuminemic hypertriiodothyroninemia. CAA is an extremely rare autosomal recessive disorder, characterised by a relatively mild phenotype, because of the compensatory increase of other serum proteins. In contrast to the benign presentation of CAA after birth, the pre- and peri-natal course appears less favourable, supporting the hypothesis that the rarity of the trait may be attributed to the fact that only a few analbuminaemic individuals survive past the neonatal state. As part of a nearly thirty-years research project, we are continuing to study the molecular defects causing alloalbuminaemia and CAA in humans. Our research is aimed at improving the knowledge of the structure-function relationship for albumin and is addressed to shed light on the molecular basis underlying the analbuminaemic trait. Major information on this research can be found at the web-site www.albumin.org, which is presently managed by Lorenzo Minchiotti, Monica Campagnoli and by Dr. Ulrich Kragh-Hansen of the Aarhus University (Denmark).
1. Minchiotti L, Galliano M, Caridi G, Kragh-Hansen U, Peters T Jr. Congenital analbuminaemia: Molecular defects and biochemical and clinical aspects. Biochim Biophys Acta 2013;1830:5494-5502.
2. Kragh-Hansen U, Minchiotti L, Galliano M, Peters T Jr. Human serum albumin isoforms: genetic and molecular aspects and functional consequences. Biochim Biophys Acta 2013;1830:5405-5417.
3. Minchiotti L, Galliano M, Kragh-Hansen U, Peters T Jr. Mutations and Polymorphisms of the Gene of the Major Human Blood Protein, Serum Albumin. Hum Mutat 2008;29:1007-1016.
Title: Genetic and clinical heterogeneity in Shwachman Syndrome
Supervisor(s): Antonella Minelli
Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive disease (OMIM #260400) characterized by pancreatic exocrine insufficiency, haematological abnormalities, bone marrow failure (with increased risk for myelodysplasia and acute myeloid leukaemia) and skeletal alterations. The clinical picture shows a wide variability for all clinical signs listed, and patients with mild form of the disease and late diagnosis are common. The disease causing gene is SBDS localized at 7q11.21. Detection of biallelic pathogenic variants in SBDS confirms the clinical diagnosis, but about 10% of patients with clinical features strongly suggesting SDS remains negative for mutations in SBDS. Recently, genetic heterogeneity was proven: exome analysis revealed biallelic mutations in DNAJC21, the second gene now associated to SDS, and mutations in EFL1, an SBDS partner, were also found in patients with SDS-like phenotype. The project will cover two topics: i) to perform exome analysis on SBDS negative patients collected thanks to the ongoing national collaboration on SDS, searching for additional genetic heterogeneity; we expect to analyse 10 new cases in two years; ii) to analyse the exome data already available for 16 SDS patients with SBDS mutation, searching and validating damaging variants in genes other than SBDS, which might be relevant for developing the haematological alterations.
1. Nacci L, Valli R, Pinto RM, Zecca M, Cipolli M, Morini J, Cesaro S, Boveri E, Rosti V, Corti P, Ambroni M, Pasquali F, Danesino C, Maserati E, Minelli A. Parental Origin of the deletion del(20q) in Shwachman-Diamond patients and loss of the paternally derived allele of the imprinted L3MBTL1 gene. Genes, Chromosomes & Cancer 2017;56:51-58.
2. Minelli A, Nacci L, Valli R, Pietrocola G, Ramenghi U, Locatelli F, Brescia L, Nicolis E, Cipolli M, Danesino C. Structural variation in SBDS gene, with loss of exon 3, in two Shwachman-Diamond patients. Blood Cells Mol Dis 2016;60:33-35.
3. Minelli A, Maserati E, Nicolis E, Zecca M, Sainati L, Longoni D, Lo Curto F, Menna G, Poli F, De Paoli E, Cipolli M, Locatelli F, Pasquali F, Danesino C. The isochromosome i(7)(q10) carrying c.258+2t>c mutation of the SBDS gene does not promote development of myeloid malignancies in patients with Shwachman syndrome. Leukemia 2009;23:708-711.
Title: Sialic-acid-binding immunoglobulin-like lectin-7 (Siglec-7) as a biomarker of fibrosis and efficiency of NK cell responses in chronic viral hepatitis.
Supervisor(s): Mario Mondelli
Sialic acid-binding immunoglobulin-like lectins (Siglecs) are transmembrane proteins containing an amino-terminal V-set immunoglobulin domain mediating sialic acid recognition. Siglec-7 is expressed in highly functional NK cells whereas a dysfunctional subset lacking Siglec-7 is overrepresented in chronic viral infections, including HIV-1, HBV, and HCV. In the latter, we have recently shown significant correlations between soluble serum Siglec-7, and liver inflammation/fibrosis, suggesting it could be a biomarker of advanced liver disease and increase discrimination of existing scores predicting mortality in cirrhosis. Serum Siglec-7 was reduced after sustained virological response in patients with hepatitis C treated with interferon-α/ribavirin or after stable HBV suppression using nucleos(t)ide analogues (NUC), suggesting diverse clinical applications. Here we plan to validate Siglec-7 as biomarker of liver fibrosis in larger cohorts of patients with chronic viral hepatitis. Furthermore, we shall establish whether serum Siglec-7 can improve sensitivity and specificity as part of an algorithm to predict fibrosis and clinical and virological relapse in patients with chronic hepatitis C treated with DAAs. We also plan to investigate the basic mechanisms regulating expression of Siglec-7 on NK cells, the proteomic profile of Siglec-7 negative and positive NK cells, and the possible role of Siglec-7 negative dysfunctional NK cells in reduced surveillance against cancer. Finally, we shall determine whether soluble Siglec-7 can be used to predict relapse after structured NUC treatment interruption in patients with chronic hepatitis B achieving complete viral suppression, to avoid life-long treatment.
1. Oliviero B, Varchetta, Paudice E, Cerino A, Michelone G, Zaramella M, Mavilio D, De Filippi F, Bruno S, Mondelli MU. Natural killer cell functional dichotomy in chronic hepatitis B and chronic hepatitis C virus infections. Gastroenterology 2009;137:1151-1160.
2. Varchetta S, Mele D, Mantovani S, Oliviero B, Cremonesi E, Ludovisi S, Michelone G, Alessiani M, Rosati R, Montorsi M, Mondelli MU. Impaired intrahepatic natural killer cell function in chronic hepatitis C virus infection. Hepatology 2012;56:841-849.
3. Varchetta S, Mele D, Lombardi A, Oliviero B, Mantovani S, Tinelli C, Spreafico M, Prati D, Ludovisi S, Ferraioli G, Filice C, Aghemo A, Lampertico P, Facchetti F, Bernuzzi F, Invernizzi P, Mondelli MU. Lack of Siglec-7 expression identifies a dysfunctional natural killer cell subset associated with liver inflammation and fibrosis in chronic HCV infection. Gut 2016;65:1998-2006.
Title: Circulating angiomiRs involvement in Hereditary Hemorrhagic Telangiectasia (HHT).
Supervisor(s): Carla Olivieri
HHT is an autosomal dominant vascular dysplasia affecting about 1:5000 subjects worldwide. It is a rare disease, caused by mutation in at least 4 different genes but 85% of Patients carry a mutation in either ENG or ACVRL1; both genes belong to the BMPs pathway. The pathogenetic model is haploinsufficiency, however contrasting results are present when the precise mechanism underlying the disease is investigated. Moreover, large phenotypic variability can be observed also within the same family. miRNAs are short single-stranded non coding RNAs, involved in gene expression regulation. Circulating miRNAs have been identified in body fluids since 2008. Recent works (Zhang et al.; Tabruyn et al. 2013) suggest a role for circulating miRNAs in HHT. We already obtained additional evidence of a misregulated expression of miRNAs in 10 HHT patients versus 5 controls, the largest cohort studied in HHT up to now. We plan to validate these results on a sizeable number of Patients in order to identify miRNAs to further investigate from a biological point of view, focusing at first on angiomiRs identified in the preliminary step.The PhD student will be involved in this validation step and subsequent functional analyses (i.e. miRNAs over expression in HUVEC, Western Blotting, Luciferase assay, ELISA and wound Healing assay). The project focuses on two/three angiomiRs, significantly dysregulated in Patients, to identify their targets and understand if they play a role in the pathogenesis of the disease. A particular attention will be paid to genotype/phenotype correlations.
1. Invernizzi R, Quaglia F, Klersy C, Pagella F, Ornati F, Chu F, Matti E, Spinozzi G, Plumitallo S, Grignani P, Olivieri C, Bastia R, Bellistri F, Danesino C, Benazzo M, Balduini CL. Efficacy and safety of thalidomide for the treatment of severe recurrent epistaxis in hereditary haemorrhagic telangiectasia: results of a non-randomised, single-centre, phase 2 study. Lancet Haematol 2015;2:e465-473.
2. Lesca G, Olivieri C, Burnichon N, Pagella F, Carette MF, Gilbert-Dussardier B, Goizet C, Roume J, Rabilloud M, Saurin JC, Cottin V, Honnorat J, Coulet F, Giraud S, Calender A, Danesino C, Buscarini E, Plauchu H. Genotype-phenotype correlations in hereditary hemorrhagic telangiectasia: Data from the French-Italian HHT network. Genet Med 2007;9:14-22.
3. Olivieri C, Pagella F, Semino L, Lanzarini L, Valacca C, Pilotto A, Corno S, Scappaticci S, Manfredi G, Buscarini E, Danesino C. Analysis of ENG and ACVRL1 genes in 137 HHT Italian families identifies 76 different mutations (24 novel). Comparison with other European studies. J Hum Genet 2007;52:820-829.
Title: Understanding disease development in Friedreich’s ataxia in a time-resolved way
Supervisor(s): Annalisa Pastore
Friedreich’s ataxia (FRDA) is a recessive autosomal ataxia caused by reduced levels of frataxin, an essential highly conserved mitochondrial protein. The exact role of frataxin and its primary function remain unclear although this information is important for designing new therapeutic approaches. A main difficulty is that of establishing a temporal relationship between the different observations that could allow a distinction between causes and secondary effects and provide a link between aging and disease development. Most studies based on cellular and animal models, however, did not effectively investigate the time-course of the disease: the phenotype was tested only at few time points that were often temporally spaced. When the phenotype becomes observable most of the events that lead to it have already taken place. We want to approach the problem by developing a cellular model in which we can switch off/on in a time-controlled way the frataxin gene partially mimicking what happens in the disease. This system will allow us to follow disease progression and understand the early stages in the FRDA development. The knowledge acquired will help to identify novel markers that can be used in diagnostic analysis.
1. Adinolfi S, Iannuzzi C, Prischi F, Pastore C, Iametti S, Martin S, Bonomi F, Pastore A. Bacterial frataxin CyaY is the gate keeper of iron sulfur formation catalysed by IscS. Nat Struct Mol Biol 2009;16:390-396.
2. Prischi F, Konarev PV, Iannuzzi C, Pastore C, Adinolfi S, Martin SR, Svergun DI, Pastore A. Structural bases for the interaction of frataxin with the central components of iron-sulfur cluster assembly. Nat Commun 2009;1:95.
3. Yan R, Konarev PV, Iannuzzi C, Adinolfi A, Roche B, Kelly G, Simon L, Martin SR, Py B, Barras F, Svergun DI, Pastore A. Ferredoxin competes with bacterial frataxin in binding to the desulfurase IscS. JBC 2013;288:24777-24787.
Title: Nemaline myopathy: structural and functional adapations, molecular mechanisms and therapy
Supervisor(s): Maria Antonietta Pellegrino
Nemaline myopathy (NM) is a rare and fatal neuromuscular disorder with an estimated prevalence of 1 in 50.000 live births. The main clinical feature of NM is muscle weakness, which impairs swallowing and causes severe respiratory problems. In children suffering from NM, the diaphragm is often severely affected, leading to suffocation. There is currently no treatment for NM, and efforts are directed at alleviating symptoms and compensating for disabilities as far as possible. Previous studies have yielded important results on (1) the pathophysiology of muscle weakness in NM, on (2) new NM biomarkers, and on (3) in vitro effects of troponin activators on muscle fibres of NM patients. However, the pathogenesis of nemaline myopathy is still unsettled and no therapy still exists.
In this study, we will study the intracellular signaling pathways underlying the structural and functional adaptations of skeletal muscle to the myopathy and the efficacy of tirasemtiv, a fast skeletal troponin activator, on structure and function of skeletal muscle in four NM mouse models. Muscle function will be studied at whole muscle and single fibre level, energy metabolism will be studied at whole muscle level and moelcualr adaptions will be studied on samples of several muscle in vitro. The project is supported by a E-Rare EU grant and will be performed through the collaboration of three groups: Free University Amsterdam (The Netherlands), Univeristy of Marseille (France), Univeristy of Pavia.
1. Hvid LG, Brocca L, Ortenblad N, Suetta C, Aagaard P, Kjaer M, Bottinelli R, Pellegrino MA. Myosin content of single muscle fibers following short-term disuse and active recovery in young and old healthy men. Exp Gerontol 2016;87:100-107.
2. Cannavino J, Brocca L, Sandri M, Bottinelli R, Pellegrino MA. PGC1-alpha over-expression prevents metabolic alterations and soleus muscle atrophy in hindlimb unloaded mice. J Physiol 2014;592:4575-4589.
3. Pellegrino MA, Desaphy JF, Brocca L, Pierno S, Camerino DC, Bottinelli R. Redox homeostasis, oxidative stress and disuse muscle atrophy. J Physiol 2011;589:2147-2160.
Title: Neuroimmune interactions in the auditory brainstem
Supervisor(s): Roberto Pizzala
Our research project aims at the comprehensive evaluation of immune modulation in brainstem auditory nuclei and in particular of the modulation of microglia and macrophages in the dorsal cochlear nucleus, which is located in a privileged position to take part to neuroimmune interactions.Work will be developed along three distinct and complementary directions: (1) Microglia/macrophages’ characterization in the cochlear nuclei of young and old rodents: localization and density of cell subsets (epiplexus cells, meningeal and perivascular macrophages, blood-derived monocytes, true microglia), colocalization with neurons and other elements in the brain parenchyma and activation state. Assays will be performed in control subjects, and in animals receiving noise trauma or unilateral cochlear ablation; correlations will be sought with cochlear damage as well as with functional impairments (tinnitus, hyperacusis, hearing loss) induced by treatment. (2) We will investigate whether monocyte inflammatory status may be considered as a clinical sign of tinnitus/presbycusis, potentially employable in clinical routine. Density and inflammatory phenotype of circulating monocytes will be characterized in the blood of rodents and tinnitus/presbycusis patients with gender and age-matched controls. In humans, blood results will be correlated with age and severity of tinnitus or hearing loss. (3) Functional tests for hearing loss, central processing dysfunctions and tinnitus/hyperacusis will be performed on rodents after modulation or selective ablation of specific microglia/macrophage/monocyte cell populations. In particular, we will investigate the role of resident microglia, perivascular and meningeal macrophages, and circulating monocytes, and the functional effect of modulating their activation and density in the brain parenchyma.
1. Venturino A, Colombo G, Sanchini G, Vitale V, Bertone V, Oda A, Pizzala R, Perin P. Does blocking microglial activation prevent tinnitus onset? J Neuroimm Pharmacol 2016;11:#16.
2. Spaccapelo L, Galantucci M, Neri L, Contri M, Pizzala R, D’Amico R, Ottani A, Sandrini M, Zaffe D, Giuliani D, Guarini S. Up-regulation of the canonical Wnt-3A and Sonic hedgehog signaling underlies melanocortin-induced neurogenesis after cerebral ischemia. Eur J Pharmacol 2013;707:78-86.
3. Giuliani D, Mioni C, Altavilla D, Leone S, Bazzani C, Minutoli L, Bitto A, Cainazzo M-M, Marini H, Zaffe D, Botticelli AR, Pizzala R, Savio M, Necchi D, Schiöth HB, Bertolini A, Squadrito F, Guarini S. Both early and delayed treatment with melanocortin 4 receptor-stimulating melanocortins produces neuroprotection in cerebral ischemia. Endocrinology 2006;147:1126-1135.
Title: Targeting miR-21 to investigate its Role in Chronic Lung Allograft Dysfunction (CLAD) post Lung Transplantation
Supervisor(s): Elena Rossi
This project aims to investigate the role of miR-21 in cellular pathways involved in BOS onset, together with the possibility to target miR-21 or one of its downstream targets, in a selective and unique manner, in order to block or reverse the fibrogenic process. Bronchiolitis Obliterans Syndrome (BOS) represents the major clinical phenotype (70%) of chronic lung allograft dysfunction (CLAD) and is responsible of the poorer long term survival after lung transplantation (LTx). Although the exact pathogenesis of BOS is still unknown, and this represents the main obstacle to BOS prevention and therapy, aberrantly active mesenchymal cells seem to be the main actors in the fibrotic processes that bring to CLAD. These cells seem to origin through a conversion from an epithelial bronchiolar phenotype to a mesenchymal one leading to an over-proliferation with production of extracellular matrix and lately fibrosis. MicroRNAs, in particular pro-fibrotic miR-21, have been shown to play a relevant role in the epigenetic regulation of fibrosis in several organs and might play a role also in BOS through the induction of Epithelial-to-Mesenchymal Transition (EMT). Once started, this process cannot be halted and actually no effective pharmacological treatment is available. Based on these assumptions, this project aims to: 1) lighten the role of miR-21 in BOS onset and in the transition of epithelial to mesenchymal cells; 2) gain insights into relevant pathogenetic factors driving BOS onset 3) block or reverse the fibrogenic process in vitro and in vivo (animal models) through inhibition of miR-21 expression.
1. Di Carlo S, Rossi E, Politano G, Inghilleri S, Morbini P, Calabrese F, Benso A, Savino A, Cova E, Zampieri D, Meloni F. Identification of miRNAs Potentially Involved in Bronchiolitis Obliterans Syndrome: A Computational Study. PLoS One 2016;11:e0161771.
2. Cova E, Colombo M, Inghilleri S, Morosini M, Miserere S, Peñaranda-Avila J, Santini B, Piloni D, Magni S, Gramatica F, Prosperi D, Meloni F. Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction. Nanomedicine (Lond) 2015;10:9-23.
3. Meyer KC, Raghu G, Verleden GM, Corris PA, Aurora P, Wilson KC, Brozek J, Glanville AR; ISHLT/ATS/ERS BOS Task Force Committee; ISHLT/ATS/ERS BOS Task Force Committee. An international ISHLT/ATS/ERS clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome. Eur Respir J 2014;44:1479-1503.
Title: Asparaginase-based Antibody-Drug conjugates
Supervisor(s): Claudia Scotti
E. coli Asparaginase (ASNase) is an enzyme able to catalyse cleaveage of asparagine and glutamine. It has been a key element of the multi-drug approach to treat pediatric Acute Lymphoblastic Leukemia (ALL) for more that 40 years, thanks to the sensitivity of these cells to the deprivation of these two amino acids. Thanks to this therapy, more than 90% cases of ALL are successfully cured. However, several limitations remain, especially for the high-risk forms of LLA and for the side effects ASNase generates. The latter mainly depend on its sistemic effects and on the production of anti-ASNase antibodies by the host. With our research, we are dissecting this enzyme in order to clarify both its mechanism of action and the basis of its problematic features, and to generate improved versions of the molecule. Recent evidence both from our group and others suggest that targeting of ASNase onto cancer cells could be a solution to the systemic effects of the enzyme. In this project, we propose to build on our previous knowledge of the enzyme and on our experience in antibody engineering in order to generate targeted forms of ASNase able to specifically reach cancer cells. scFv-ASNase fusions will be generated selecting specific cell-surface receptors, like CD19 and CD20, present on cancer cells, as targeting antigens. The students will be guided to determine enzymatic and structural features of each fusion and its cytotoxicity on selected leukaemia cell lines.
1. Parmentier JH, Maggi M, Tarasco E, Scotti C, Avramis VI, Mittelman SD. Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines. Leuk Res 2015;39:757-762.
2. Maggi M, Chiarelli LR, Valentini G, Scotti C. Engineering of Helicobacter pylori L-asparaginase: Characterization of two functionally distinct groups of mutants. PloS one 2015;10:e0117025.
3. Covini D, Tardito S, Bussolati O, Chiarelli LR, Pasquetto MV, Digilio R, Valentini G, Scotti C. Expanding targets for a metabolic therapy of cancer: L-asparaginase, Recent patents on anti-cancer drug discovery. 2012;7:4-13.
Title: Identification of novel genes and novel pathogenetic mechanisms in congenital ataxias
Supervisor(s): Enza Maria Valente
Congenital ataxias (CAs) are heterogeneous disorders of brain development, due to malformations of the cerebellum and often of the brainstem and other brain areas. CAs are characterized by neonatal or paediatric onset of a spectrum of neurological features including hypotonia, cerebellar signs (ataxia, dysarthria), oculomotor anomalies, psychomotor delay and intellectual impairment. While for some CAs the diagnosis is suggested by the presence of peculiar clinical or neuroimaging signs, in other forms the presentation remains unspecific and does not permit to discriminate among distinct disorders with similar onset ages. Recent progresses in genetic technologies have led to an impressive acceleration in the discovery of causative genes yet many patients still remain undiagnosed. Aim of this PhD project is to apply Next Generation Sequencing (NGS) techniques to identify novel CA genes and improve gene-phenotype correlates in over 200 uncharacterized CA patients. This will be pursued through the following tasks: i) optimization of NGS-based panels for simultaneous sequencing of known CA genes; ii) whole-exome sequencing in selected patients negative to panel analysis and functional studies in cellular models (mainly patients’ fibroblasts) to confirm the pathogenicity of identified variants; iii) deep integration of clinical, imaging and genetic data. The PhD student will become confident with a range of NGS strategies, tools for bioinformatic data analysis as well as a range of cell biology and biochemistry techniques. The project is supported by a grant from the Italian Ministry of Health and will be carried on in collaboration with several research groups in Italy and abroad.
1. Roosing S, Romani M, Isrie M, Rosti RO, Micalizzi A, Musaev D, Mazza T, Al-gazali L, Altunoglu U, Boltshauser E, D’Arrigo S, De Keersmaecker B, Kayserili H, Brandenberger S, Kraoua I, Mark PR, McKanna T, Van Keirsbilck J, Moerman P, Poretti A, Puri R, Van Esch H, Gleeson JG, Valente EM. Mutations in CEP120 cause Joubert syndrome as well as complex ciliopathy phenotypes. J Med Genet 2016;53:608-15.
2. Micalizzi A, Moroni I, Ginevrino M, Biagini T, Mazza T, Romani M, Valente EM. Very mild features of dysequilibrium syndrome associated with a novel VLDLR missense mutation. Neurogenetics. 2016 Jul;17(3):191-5.
3. Lee JE, Silhavy JL, Zaki MS, Schroth J, Bielas SL, Marsh SE, Olvera J, Brancati F, Iannicelli M, Ikegami K, Schlossman AM, Merriman B, Attié-Bitach T, Logan CV, Glass IA, Cluckey A, Louie CM, Lee JH, Raynes HR, Rapin I, Castroviejo IP, Setou M, Barbot C, Boltshauser E, Nelson SF, Hildebrandt F, Johnson CA, Doherty DA, Valente EM, Gleeson JG. CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium. Nat Genet 2012;44:193-199.
Title: Nanotechnology applications for innovative therapeutic approaches
Supervisor(s): Livia Visai
This study aims to investigate by using biochemical/molecular and biological methods, the applications of nanotechnology for innovative therapeutic approaches as follows: a. gold nanoparticles conjugated with drugs for treatment of breast cancer; b. nanoparticles/nano-antibodies for treatment of bacterial infections; c. nanoparticles as a countermeasure for treatment of osteoporosis.
1. Risi G, Bloise N, Merli D, Icaro-Cornaglia A, Profumo A, Fagnoni M, Quartarone E, Imbriani M, Visai L. In vitro study of multiwall carbon nanotubes (MWCNTs) with adsorbed mitoxantrone (MTO) as a drug delivery system to treat breast cancer. RSC Adv 2014;4:18683.
2. Frasnelli M, Cristofaro F, Sglavo VM, Dirè S, Callone E, Ceccato R, Bruni G, Cornaglia AI, Visai L. Synthesis and characterization of strontium-substituted hydroxyapatite nanoparticles for bone regeneration. Mater Sci Eng C Mater Biol Appl 2017;71:653-662.
3. Liang X, Garcia BL, Visai L, Prabhakaran S, Meenan NA, Potts JR, Humphries MJ, Höök M. Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs. PLoS One 2016;11:e0159118.
Project 24 (supported by dedicated, external funds)
Title: Determinants of ventricular fibrillation during myocardial infarction
Supervisor: Gaetano De Ferrari
Sudden cardiac death is one of the leading causes of death and is caused in most cases by the occurrence of a malignant ventricular arrhythmia. This can occur due to the presence of a predisposing condition such as an arrhythmogenic disorder or an altered myocardial substrate, such as a previous myocardial infarction or a cardiomyopathy. Very often, the malignant arrhythmia is triggered by acute myocardial ischemia in the setting of an acute myocardial infarction. As a matter of fact, sudden cardiac death is often the first manifestation of coronary artery disease. Goal of the present research project will be to search the epidemiologic, clinical and genetic determinants of sudden cardiac death and aborted sudden cardiac death, looking specifically at patients with acute myocardial infarction complicated by an episode of ventricular fibrillation and comparing them with a cohort of patients without ventricular fibrillation.
1. Rocchetti M, Sala L, Dreizehnter L, Crotti L, Sinnecker D, Mura M, Pane LS, Altomare C, Torre E, Mostacciuolo G, Severi S, Porta A, De Ferrari GM, George AL Jr, Schwartz PJ, Gnecchi M, Moretti A, Zaza A. Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes. Cardiovasc Res 2017;113:531-541.
2. Crotti L, Hu D, Barajas-Martinez H, De Ferrari GM, Oliva A, Insolia R, Pollevick GD, Dagradi F, Guerchicoff A, Greco F, Schwartz PJ, Viskin S, Antzelevitch C. Torsades de pointes following acute myocardial infarction: evidence for a deadly link with a common genetic variant. Heart Rhythm 2012;9:1104-1112.
3. Shivkumar K, Ajijola OA, Anand I, Armour JA, Chen PS, Esler M, De Ferrari GM, Fishbein MC, Goldberger JJ, Harper RM, Joyner MJ, Khalsa SS, Kumar R, Lane R, Mahajan A, Po S, Schwartz PJ, Somers VK, Valderrabano M, Vaseghi M, Zipes DP. Clinical neurocardiology defining the value of neuroscience-based cardiovascular therapeutics. J Physiol 2016;594:3911-3954.