Projects 2018 /19
List of Projects 2018/19
Project 1
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.
Project 2
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.
Project 3
Title: Novel therapeutic strategies against AL amyloidosis
Supervisor(s): Giampaolo Merlini
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.
Project 4
Title: Molecular, magnetic resonance, and echocardiographic imaging combined with biomarkers of cardiac and clonal disease to predict survival and assess response to therapy in cardiac AL amyloidosis –
Supervisor Giovanni Palladini
The severity of cardiac involvement is the main prognostic determinant in light chain (AL) amyloidosis. Currently, prognostic stratification and assessment of response is largely based on cardiac biomarkers N-terminal pro natriuretic peptide type B (NT-proBNP) and troponins (cTn). Evidence indicate that cardiac dysfunction in AL amyloidosis is mainly caused by direct cardiac toxicity exerted by the circulating precursor detected by NT-proBNP elevation. Thus, staging and response assessment are based on measurement of markers of clonal (free light chain, FLC) and cardiac (NT-proBNP) disease. While reduction of these markers improves survival, we lack a functional and morphological correlate of these changes. The availability of novel imaging techniques, such as cardiac magnetic resonance (CMR), echocardiographic assessment of global longitudinal strain (2D-GLS), and scintigraphy with amyloid-specific tracers can fill this gap.
Patients will undergo standard assessment of cardiac and clonal disease which includes: 1) measurement of FLC; 2) measurement of NT-proBNP and cTnI and staging; 3) echocardiography; 4) cardiac MRI. In addition, patients will undergo investigational Positron Emission Tomography CT scans with the amyloid specific tracer 18F-florbetapir (F-PETCT) to detect and quantify cardiac amyloid deposits. Assessments will be performed at baseline, and 6 months after chemotherapy initiation, during standard assessment of response evaluations. Patients will be followed for survival until the end of the project.
The correlation between echocardiographic, CMR, and F-PET data with cardiac staging and response to therapy will be evaluated, to detect functional and morphological (amyloid load, tissue characterization) correlates of disease severity. The ability of imaging data to predict early (within 12 months) deaths will be also assessed.
This study may shed light on the reason why 40% of patients do not reach cardiac response despite good quality hematologic response. This is particularly relevant at a time when novel treatments directly targeting the deposits are being evaluated in clinical trials.
References:
1. Palladini G, Merlini G. What is new in diagnosis and management of light chain amyloidosis? Blood. 2016;128(2):159-168.
2. Palladini G, Barassi A, Klersy C, et al. The combination of high-sensitivity cardiac troponin T (hs-cTnT) at presentation and changes in N-terminal natriuretic peptide type B (NT-proBNP) after chemotherapy best predicts survival in AL amyloidosis. Blood. 2010;116(18):3426-3430.
3. Palladini G, Dispenzieri A, Gertz MA, et al. New criteria for response to treatment in immunoglobulin light chain amyloidosis based on free light chain measurement and cardiac biomarkers: impact on survival outcomes. J Clin Oncol. 2012;30(36):4541-4549.
Project 5
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.
Project 6
Title: Skeletal muscle wasting in aging: molecular mechanisms and functional implications
Supervisor(s): Maria Antonietta Pellegrino
With extended life expectancy, quality of life of older people is a priority. Loss of mobility, increased morbidity and risks of falls have dramatic individual and societal impacts. In particular, impoverished neuromuscular control and sarcopenia are major factors of frailty in the elderly. Among the many factors involved, neurodegenerative changes, also associated with mitochondrial dysfunction [1], play a key role [2]. These changes involve (i) loss of motoneurons, (ii) alteration of the neuromuscular junction (NMJ), (iii) muscle deterioration: muscle atrophy, impairment of intrinsic muscle strength and of oxidative metabolism. In ageing skeletal muscle, several phenomena could also impair NMJ, potentially initiating denervation-reinnervation cycles: mitochondrial dysfunction, increased ROS production, decreased PGC-1a expression. Aim of the project is to understand the role of NMJ in aging. In fact, it is still unclear: (i) whether ageing actually causes alterations of NMJ in humans [3]; (ii) whether alterations of NMJ is followed by functional impairment; (iii) the mechanisms underlying NMJ alterations and muscle deterioration. This will be pursued through the following tasks: (I) define impairment of neuromuscular control and loss of motoneuron in older humans (extent and functional impact) (ii) characterize NMJ alterations in older humans (extent; relationship with functional impairment; molecular mechanisms) (iii) define the role of muscle fibres in causing NMJ alterations (molecular mechanisms; relative role of NMJ and muscle fibres alterations).
1] E. Masiero, L. Agatea, C. Mammucari, B. Blaauw, E. Loro et al 2009; Autophagy is required to maintain muscle mass, Cell Metab.; PMID: 19945408
[2] K.A. Rygiel, M. Picard, D.M. Turnbull, 2016; The ageing neuromuscular system and sarcopenia: a mitochondrial perspective, J Physiol; PMID: 26921061.
[3] R.A. Jones, C. Harrison, S.L. Eaton, M. Llavero Hurtado, L.C. Graham et al 2017; Cellular and Molecular Anatomy of the Human Neuromuscular Junction, Cell Rep.; PMID: 29186674cle fibres alterations).
Project (supported by dedicated, extarnal funds)
Title: Ex vivo bone marrow models for platet production
Supervisor: Alessandra Balduini
Mechanisms that trigger platelet release from human megakaryocytes (MKs) are not fully understood. In 2008 two new drugs that mimic the effect of thrombopoietin (TPO) became available to treat thrombocytopenia. Despite current clinical practice, the mechanisms governing TPO mimetic’s impact on human hematopoiesis are largely unknown, in part due to the impossibility of using traditional in vivo models. Understanding the efficacy and safety of TPO mimetic on pathologic samples before treating patients in vivo would represent an important step towards creating more personalized and effective therapies. Therefore, further improvement of the current knowledge strongly depends on the possibility to create laboratory assays in order to understand the effects of thrombopoietic drugs on human cells, both in physiologic and pathologic conditions. To investigate their impact on megakaryocyte functions, the candidate will establish a translational study made up of two complementary approaches. The first will be based on the development of a culture system for the study of the basic mechanisms of the action of drugs on human hematopoietic stem cells, focusing on the evaluation of their ability to promote megakaryopoiesis and platelet formation. The second will be dedicated to the implementation of this knowledge into our recently established silk-based bone marrow model, which integrates important physical and physiological elements of the hematopoietic niche. This research will clarify the underlying mechanisms that govern the action of drugs on MK functions and their relevance in clinical practice.
1. Kuter DJ. Thrombopoietin and thrombopoietin mimetics in the treatment of thrombocytopenia. Annu Rev Med. 2009;60:193-206.
2. 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.
3. 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. Haematolo gica 2016;101:1479-1488.
Project (supported by dedicated, external funds)
Title:Out of hospital cardiac arrest: strategies for the identification of subjects at risk and for improving management
Supervisor: Gaetano De Ferrari
Out of hospital cardiac arrest (OHCA) affects about 1 person per 1000 inhabitants every year and it is one of the leading causes of death in the industrialized countries. The chance of survival after an OHCA is presently only around 5%. This figure can be potentially improved acting on several aspects including the identification of subjects at higher risk of cardiac arrest in the general population to reduce the incidence of this type of event, an improvement in the management of cardiac arrest by the lay bystander as well as an improvement in the treatment of the patient in hospital and after discharge. The general aim of the project is to increase the survival after an OHCA trying to improve all related aspects related.
Based on these assumptions, the goals of the research project will be:
1) to develop and validate both the prediction models for sudden cardiac arrest occurrence,
2) to improve the involvement of lay bystanders in cardiac arrest response system and to assess the added benefit of these approaches
3) to improve pre-hospital treatment and survival prediction of cardiac arrest victims
4) to assess and to implement novel approaches to improve in-hospital and after discharge treatment and prognosis of cardiac arrest patients.
1. G Gräsner JT, Lefering R, Koster RW, Masterson S, Böttiger BW, Herlitz J, Wnent J, Tjelmeland IB, Ortiz FR, Maurer H, Baubin M, Mols P, Hadžibegovi? I, Ioannides M, Škulec R, Wissenberg M, Salo A, Hubert H, Nikolaou NI, Lóczi G, Svavarsdóttir H, Semeraro F, Wright PJ, Clarens C, Pijls R, Cebula G, Correia VG, Cimpoesu D, Raffay V, Trenkler S, Markota A, Strömsöe A, Burkart R, Perkins GD, Bossaert LL; EuReCa ONE Collaborators. EuReCa ONE-27 Nations, ONE Europe, ONE Registry: A prospective one-month analysis of out-of-hospital cardiac arrest outcomes in 27 countries in Europe. Resuscitation. 2016;105:188-95. Erratum in: Resuscitation. 2016;109:145-146.
2. Wissenberg M, Lippert FK, Folke F, Weeke P, Hansen CM, Christensen EF, Jans H, Hansen PA, Lang-Jensen T, Olesen JB, Lindhardsen J, Fosbol EL, Nielsen SL, Gislason GH, Kober L, Torp-Pedersen C. Association of national initiatives to improve cardiac arrest management with rates of bystander intervention and patient survival after out-of-hospital cardiac arrest. JAMA. 2013;310:1377-84.
3. Mauri R, Burkart R, Benvenuti C, Caputo ML, Moccetti T, Del Bufalo A, Gallino A, Casso C, Anselmi L, Cassina T, Klersy C, Auricchio A. Better management of out-of-hospital cardiac arrest increases survival rate and improves neurological outcome in the Swiss Canton Ticino. Europace. 2016;18:398-404.
