The first two departmental PhD Studentships
As part of a broad review of PhD studies in the Department and at the University of Pavia, the Department of Molecular Medicine aims to develop a new and strong PhD Programme in Molecular/Translational Medicine in years to come. On an interim basis the Department has funded two PhD studentships from its ‘Fondo Ricerca e Giovani’ for research projects to be awarded within the existing PhD Programme in Experimental Medicine. The awards will be made at the end of September 2016 for work to commence on the 1st of November 2016. Students in receipt of the two studentships will be able to choose among the following 9 research projects listed below.
Title: Towards the elucidation of pathophysiology of amyloid conversion of globular proteins
Supervisor(s): V Bellotti
Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. Despite much progress in understanding amyloid fibrillogenesis in vitro, the formation of amyloid fibrils in vivo is poorly understood. A comprehensive study to elucidate the amyloidogenic processes operating under physiological and pathophysiological conditions is critical to understand the natural history of the disease, its response to treatment and, crucially, the development of new and more effective therapies. The discovery of the first naturally occurring structural variant of human β2-microglobulin, D76N (1, 2) together with the characterization of most aggressive variant of transthyretin, S52P (3), and their propensity to form amyloid fibrils in vitro under physiological conditions offers two unique models to study the pathophysiology of amyloidosis (4). Supported by preliminary, but robust experimental evidence, we hypothesize that fibrillar conversion of globular proteins is based, “in vivo”, on the combination of an intrinsic instability of these proteins and their exposure to unfolding forces generated by the flow of the extracellular fluid and exposure to hydrophobic surfaces (1, 2, 3, 5). The candidate will study two prototypic globular amyloidogenic proteins, β2-microglobulin and transthyretin for which we have identified two natural pathogenic variants that represent two original models for our hypothesis. Expected results will contribute to promote a paradigm shift in theories so far available and only partially explaining the mechanism of protein amyloidogenesis in vivo. Elucidation of this mechanism is a crucial and unmet medical need for designing proper therapeutic strategies.
1. Mangione PP, Esposito G, Relini A, Raimindi S, Porcari R, Giorgetti S, Corazza A, Fogolari F, Penco A, Goto Y, Lee Y-H, Cecconi C, Naqvi MM, Gillmore JD, Hawkins PN, Chiti F, Rolandi R, Taylor GW, Pepys MB, Stoppini M, Bellotti V. (2013) Structure, folding dynamics, and amyloidogenesis of D76N beta2-microglobulin: roles of shear flow, hydrophobic surfaces, and alpha-crystallin. J Biol Chem 288:30917-30.
2.Natalello A, Mangione PP, Giorgetti S, Porcari R, Marchese L, Zorzoli I, Relini A, Ami D, Faravelli G, Valli M, Stoppini M, Doglia SM, Bellotti V, Raimondi S. (2016) Co-fibrillogenesis of wild-type and D76N β2-Microglobulin: The crucial role of fibrillar seeds. J Biol Chem. 291:9678-89.
3. Mangione PP, Porcari R, Gillmore JD, Pucci P, Monti M, Porcari M, Giorgetti S, Marchese L, Raimondi S, Serpell LC, Chen W, Relini A, Marcoux J, Clatworthy IR, Taylor GW, Tennent GA, Robinson CV, Hawkins PN, Stoppini M, Wood SP, Pepys MB, Bellotti V. (2014) Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis. Proc Natl Acad USA 111:1539-44.
4. Stoppini M, Bellotti V. (2015) Systemic amyloidosis: lessons from β2-microglobulin. J Biol Chem. 290:9951-8.
5. Marcoux J, Mangione PP, Porcari R, Degiacomi MT, Verona G, Taylor GW, Giorgetti S, Raimondi S, Sanglier-Cianférani S, Benesch JL, Cecconi C, Naqvi MM, Gillmore JD, Hawkins PN, Stoppini M, Robinson CV, Pepys MB, Bellotti V. (2015) A novel mechano-enzymatic cleavage mechanism underlies transthyretin amyloidogenesis. EMBO Mol Med. 7:1337-49.
6. Giorgetti S, Raimondi S, Pagano K, Relini A, Bucciantini M, Corazza A, Fogolari F, Codutti L, Salmona M, Mangione P, Colombo L, De Luigi A, Porcari R, Gliozzi A, Stefani M, Esposito G, Bellotti V, Stoppini M. (2011) Effect of tetracyclines on the dynamics of formation and destructuration of β2-microglobulin amyloid fibrils. J Biol Chem 286:2121-31.
7. Domanska K, Vanderhaegen S, Srinivasan V, Pardon E, Dupeux F, Marquez JA, Giorgetti S, Stoppini M, Wyns L, Bellotti V, and Jan Steyaert J. (2011) Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant. Proc Natl Acad USA 108: 1314-9.
Title: Cardiac sympathetic denervation in patients with recurrent ventricular tachycardia/fibrillation and structural heart disease.
Supervisor(s): G De Ferrari
Antiarrhythmic drugs and catheter ablation are the standard of care in patients (pts) with recurrent ventricular arrhythmias (ventricular tachycardia/ventricular fibrillation, VT/VF) and implantable cardioverter-defibrillator (ICD) shocks. Both treatments have limitations and drawbacks, with suboptimal efficacy. Recently,neuromodulation has emerged as a clinical option for the prevention of ventricular tachyarrhythmias recurrence and the management of arrhythmic storms (1). Over the past 25 years we have strongly contributed to the experimental demonstration of the proarrhythmic effect of autonomic imbalance characterized by increased sympathetic activity and defective parasympathetic activity and we have pioneered the use of surgical left cardiac sympathetic denervation (LCSD) for the treatment of long QT syndrome and Catecholaminergic Polymorphic Ventricular Tachycardia (2,3). Recently bilateral cardiac sympathetic denervation (BCSD) has been proposed in a single centre study also for the treatment of patients with VT/VF in the setting of structural heart disease (ischemic and non) with very promising results (4). In addition of being retrospective, this study lacks any characterization of effects of the intervention, which deprives almost completely the heart of its adrenergic innervation, on cardiac contractility and haemodynamics, as well as on cardiac electrophysiology. The side effect profile and the quality of life have not been assessed and there is no analysis on the potential predictors of success/failure Finally, no histological and immunohistochemistral characterization of the removed bilateral sympathetic ganglia was provided, despite a recent human study in LQTS/CPVT patients suggests that T-cell–mediated cytotoxicity toward ganglion cells may boost adrenergic activity as to trigger or enhance electric instability (5). Therefore, goal of the present study will be to prospectively enroll pts with recurrent VT/VF after ablation (if indicated) assessing the effects of BCSD on a series of parameters including:
1. Standard and advanced echocardiographic indexes of myocardial contractility such as wall strain both at right and left ventricular level and data of electromechanical coupling (left and right ventricular total isovolumic time, electromechanical window and ventriculo-arterial coupling).
2. Haemodynamics (diastolic blood pressure, systolic blood pressure and heart rate), electrocardiographic parameters and bilateral palmar skin conductance at baseline, during 2 sympathetic stressors (exercise handgrip and mental stress test induced by Trier test), and at a final baseline (to assess recovery from sympathetic stress).
3. Myocardial refractoriness, measured through the implanted ICD (including atrial and both right and left ventricular sites if applicable and atrioventricular conduction)
4. 24-hour Holter recording with derived autonomic indexes of heart rate variability
5. Baroreflex sensitivity derived from spontaneous recording of RR intervals and blood pressure values before and after BCSD.
6. Sympathetic nerve ending and activity imaging with radiolabelled 123-iodine metaiodobenzylguanidine (123-I MIBG).
7. Quality of life (QOL) and evaluation of side effects and pain by means of specific questionnaires.
8. Additionally, the removed sympathetic ganglia will be analyzed evaluating neuronal size and number, neuronal phenotype (adrenergic vs cholinergic), neuropeptide Y immunoreactivity and the presence and the type of inflammatory cells. We will compare left and right-sided ganglia of the same patient and ganglia of pts with emergent vs. non-emergent indication for BCSD and of pts with different type of cardiomyopathies.
In pts with a previous myocardial infarction, we will assess whether left and right-sided ganglia neural remodeling is symmetric or asymmetric and if there is a relationship with the site and the size of the infarction. Finally, we will compare the type and the entity of ganglionic inflammatory response (if present) of these pts with structural heart disease with those observed in pts who had CSD in our centre for refractory VT/VF in the setting of purely electrical heart disease. The goal is to provide the first in vivo human inside into the potential specific role of ganglia hyperplasia and inflammation in triggering or enhancing electric instability in pts with SHD and refractory VT/VF. Sections will be immunostained with antibodies against T cells (CD3, CD4, CD8, CD20, Granzyme B), CD68 (macrophages), and HLA-DR (human leukocyte antigen-DR) antigens (activation marker). Immunopositive cells will be quantified as cells/mm2. Furthermore, polymerase chain reaction (PCR) and reverse transcription PCR will be performed to screen for herpes virus DNA (cytomegalovirus, HSV-1 and HSV-2, Epstein-Barr virus, human herpes virus 6 and varicella zoster virus) in order to determine the potential trigger for the immune cell infiltration. Neuronal phenotype will be quantified using antityrosine hydroxylase (-TH) antibody (adrenergic phenotype) and anti-choline acetyltransferease antibody (cholinergic phenotype) and express as absolute numer per slide and per mm2 and as percentage of the total number of neurons (per slide and per mm2). Finally, neuropeptide Y (NPY) immunoreactivity will bequantified using anti-NPY antibodies. The study will also assess efficacy of the intervention on the burden of VT/VF based on ICD interrogation and will evaluate the presence of possible predictors of efficacy including: baseline clinical variables, characteristics of ventricular arrhythmias, effects of the denervation on the parameters mentioned above. Finally, based on ICD interrogation and clinical data, we will also evaluate atrial tachyarrhythmias burden, ventricular response during atrial tachyarrhythmias and incidence of inappropriate discharges due to atrial tachyarrhythmias. We anticipate enrolling approximately 30 patients with ischemic or non-ischemic cardiomyopathy over the three-year program.
1. 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 Jul 15;594(14):3911-54.
2. Wilde AAM, Bhuiyan ZA, Crotti L, Facchini M, De Ferrari GM, Paul T, Ferrandi C, Koolbergen DR, Odero A, Schwartz PJ. Left cardiac sympathetic denervation for catecholaminergic polymorphic ventricular tachycardia. N Engl J Med. 2008;358:2024–2029.
3.De Ferrari GM, Dusi V, Spazzolini C, Bos JM, Abrams DJ, Berul CI, Crotti L, Davis AM, Eldar M, Kharlap M, Khoury A, Krahn AD, Leenhardt A, Moir CR, Odero A, Olde Nordkamp L, Paul T, Rosés I Noguer F, Shkolnikova M, Till J, Wilde AA, Ackerman MJ, Schwartz PJ.Clinical Management of Catecholaminergic Polymorphic Ventricular Tachycardia: The Role of Left Cardiac Sympathetic Denervation. Circulation. 2015;131(25):2185-93.
4. Vaseghi M, Gima J, Kanaan C, Ajijola OA, Marmureanu A, Mahajan A, Shivkumar K. Cardiac sympathetic denervation in patients with refractory ventricular arrhythmias or electrical storm: intermediate and long-term follow-up. Heart Rhythm. 2014;11:360-6.
5. Rizzo S, Basso C, Troost D, Aronica E, Frigo AC, Driessen AH, Thiene G, Wilde AA, Van der Wal AC. T-cell-mediated inflammatory activity in the stellate ganglia of patients with ion-channel disease and severe ventricular arrhythmias. Circ Arrhythm Electrophysiol. 2014 7 (2):224-9.
Title: Structural characterization of a new group of lectins with antitumoral properties and molecular genetics of analbuminaemia
Supervisor(s): M Galliano, L Minchiotti
The proteins that selectively recognize and bind carbohydrates without modifying them enzymatically are called lectins. Their sugar selectivity can be very useful and they are widely used in both basic and applied science for example for the purification of glycoproteins, stem cell fractionation and in targeted drug delivery. In collaboration with Prof. Monaco’s group in Verona we have identified and structurally characterized several new lectins. Among these, i) FEL, a fish egg lectin (1,2) that appears to plays a role in the innate defence system and, ii) BEL and BEL betatrefoil (3) from the popular edible wild mushroom Boletus edulis, which show different X-ray structures. Howeve, both interact with the T-antigen disaccharide, and possess anti–proliferative activity on several different human cancer cell lines. We intend to evaluate the potential biomedical applications of these lectins. In particular, lectins have been proposed as potentially useful candidates to coat nanoparticles in targeted drug delivery toward cancer cells but an obstacle to this use is the likely generation of antibodies against the foreign molecule. Our strategy involves the use of protein engineering to render them as similar as possible as a human counterpart and therefore nonrecognizable by the immune system. BEL β-trefoil is a potential candidate for this type of application because its cDNA sequence is available and we have found that the protein is expressed readily in a soluble form by E. coli. In addition, a human acidic fibroblast growth factor is found to be structurally highly similar to BEL β-trefoil, it is therefore conceivable to modify the latter to attempt to evade the immune response.
Human serum albumin, the major blood protein, is encoded by a single autosomal gene, which lies at position 4q13.3, near the centromere of chromosome 4. Mutations in this gene may result in the presence of two circulating forms of the protein (bisalbuminaemia or allo- albuminaemia) or in the virtual absence of the protein from the blood (congenital analbuminaemia, CAA)(4). We intend to continue the study of the molecular defects causing CAA in humans (5,6). Our key goal is to shed light on the molecular basis underlying the analbuminaemic trait. In order to avoid sequencing the whole gene, we will use the following experimental approach for a rapid localization of the mutation, which has been developed in our laboratory (5). The 14 coding exons of the albumin gene and their intron–exon junctions will be PCR-amplified from genomic DNA using specific primer pairs and screened for mutations by single-strand conformational polymorphism and heteroduplex analysis (5). In most cases this strategy allows a rapid identification of the mutated fragment which will then subjected to direct DNA sequencing (5,6).
1. Galliano M, et al. (2003) Structural and biochemical characterization of a new type of lectin isolated from carp eggs. Biochem J. 376:433-40.
2. Capaldi, S. et al. (2015) Three-dimensional structure and ligand-binding site of carp fishelectin (FEL) Acta Crystallogr D Biol Crystallogr. 1123-35
3. Bovi M, et al .(2013) BEL β-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms. Glycobiology. 578-92
4. Minchiotti L, et al. Hum Mutat 29:1007-16 (2008).
5. Minchiotti L, Biochim Biophys Acta. Congenital analbuminaemia: molecular defects and biochemical and clinical aspects.2013 Dec;1830
(12):5494-502. doi: 10.1016/j.bbagen.2013.04.019. Epub 2013 Apr 21.
6. Caridi G, et al. A nucleotide deletion and frame-shift cause analbuminemia in a Turkish family. Biochem Med (Zagreb). 2016;26(2):264-71. doi:10.11613/BM.2016.031.
Title: Engineering growth factors and antibodies for tissue/organ regeneration
Supervisor(s): E Gherardi, H de Jonge, L Iamele
Chronic degenerative diseases of internal organs and the brain constitute a major cause of morbidity and mortality. Organ transplantation has had a significant impact on survival and life expectancy of patients with certain degenerative diseases (chronic renal failure, for example) but had a far more limited impact on control of chronic diseases of other internal organs, such as lung and liver, for example) and is inapplicable to chronic degenerative diseases of the central nervous system. In recent years cell therapy has emerged as a powerful alternative to organ transplantation in regenerative medicine. S Yamanaka’s demonstration that adult cells can be reprogrammed to a stem cell-like phenotype through forced expression of a limited number of transcription factors has overcome the controversies and limitations of the use of embryonic stem cells and there is a real prospect that HLA-defined banks of induced, pluripotent stem cells may be produced within a few years. There remain problems nevertheless with cell therapy of both time (the need to expand and differentiate cell populations prior to their use) and resources as the cost involved in propagating and differentiating cells for treatment of individual patients are very considerable.
Synopsis. The project proposed here pursues a different strategy for regenerative medicine, namely protein therapy. The rationale for this approach is threefold: (i) a number of post natal contain adult stem/progenitor cell populations, (ii) these cells often fail to achieve substantial tissue regeneration after damage but, (iii) exposure of these cells to specific growth factors can result in amplification and differentiation of these adult stem cell populations in vivo and induce a much enhanced level 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 and essential physiological roles in tissue regeneration in post-natal life as demonstrated with tissue-specific gene targeting experiments. HGF/SF works on target cells via the receptor tyrosine kinase MET and previous work in the laboratory has generated both engineered fragments of HGF/SF as well as MET specific antibodies with potent mitogenic and motogenic activity on target cells. Throughout the project the activity of the engineered fragments of HGF/SF and the MET antibodies will be explored in mouse models of chronic degenerative diseases of internal organs (lung and liver) and the central nervous system (motor neuron disease).
Methods and Technologies. The project involves: (i) protein 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 mice – transgenic and non – in which chronic organ damaged is induced. Protein activity in these animals will be assessed by functional parameters and by histological analysis. The project may also involve further rounds of structure-based protein engineering depending on initial results from the invivo experiments.
Roy SR et al. Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis. Proc Natl Acad Sci USA 107:13608-13 (2010) doi: 10.1073/pnas.1006007107.
Ross J et al. Protein engineered variants of hepatocyte growth factor/scatter factor promote proliferation of primary human hepatocytes and in rodent liver. Gastroenterology 142:897-906 (2012) doi: 10.1053/j.gastro.2011.12.006.
Title: MicroRNA and acute myocardial infarction
Supervisor(s): M Gnecchi
Background and rationale: MicroRNAs (miRNA) are small, non-coding, RNA molecules approximately 22 nucleotides in length that act as post-transcriptional regulators of gene expression and are key regulators of complex cell biological processes. It has been described that miRNA are present also in the circulation and that plasma levels of some circulating miRNA are altered following AMI, probably because of the ongoing ischemic process (1). While there is little convincing evidence that miRNA can outperform traditional biomarkers, such as cardiac troponins, in the diagnosis of AMI, there is potential for miRNA to complement existing risk prediction models and act as valuable biomarkers of post-AMI prognosis (2). A recent work demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNA responsive to antiplatelet therapy (3). This is a relevant issue to bear in mind in the interventional cardiology setting and it might raise the hypothesis that miRNA are involved in platelet aggregation. The exciting world of miRNA-based research and its potential clinical application is developing rapidly: from a clinical perspective, miRNA may represent valuable diagnostic and prognostic biomarkers and may provide promising therapeutic targets, given their key function in gene regulation. In our Laboratory we work on miRNA since few years now. For instance, we have shown that the overexression of miRNA499 and miRNA133 increases the differentiation capacity of cardiac progenitors toward differentiated cardiomyocytes (4). More recently, we performed a study to identify specific miRNA whose levels are altered following AMI and we subsequently searched for a correlation between these biomarkers and left ventricular function. Our results show that miRNA1233, 423-5p, 345, 483-3p and 52 are up-regulated at one or more time points following AMI and that two of them correlate and can predict cardiac dysfunction measured at 6 months. Based on these preliminary but solid results, we wish to offer a PhD project with the following aims: I) Evaluate the diagnostic/prognostic role of up-regulated circulating miRNA after AMI (1233, 423-5p, 345, 483-3p e 52) in an independent and more extended population and examine if their expression level correlates with clinical and echo parameters; II) Evaluate the role of these and other miRNA in platelet aggregation and in the pathogenesis of intracoronary thrombosis; III) Study the role of these and other miRNA in the destabilization of the atherosclerotic plaque and identify their putative targets in a mouse model.
Experimental approaches: Aim I. To validate our preliminary findings, we will enroll 200 consecutive AMI patients to quantify the expression of miRNA1233, 423-5p, 345, 483-3p e 52 at different time points. Plasma will be isolated from blood samples and stored in a bio-bank at -80 °C. Platelets will also be isolated from blood samples and stored at -80 °C. MiRNA will be isolated from plasma and platelets using dedicated kits. Quantification will be done by real time PCR. During enrollment, the candidate will also need to fill the clinical record files (CRF) and keep track of the clinical follow-up. The results obtained after qPCR analysis will be statistically analyzed and correlated with clinical, laboratory and echo data in order to establish the prognostic role of our candidate miRNA. The sensitivity and specificity of miRNA will be computed by performing ROC curve analysis. Aim II. Thrombi aspirated during the primary coronary intervention will be collected in some of the 200 patients enrolled in the study and their miRNA content quantified. Moreover, immunoistochemistry will be performed on thrombi fixed and placed in paraffin. In particular, in situ hybridization assays will be performed to identify the miRNA of interest inside the thrombi. Then, to verify if miRNA1233, 423-5p, 345, 483-3p and 52 or other miRNA play a role in thrombus formation, platelets will be transfected with miRNA precursors and platelet aggregation evaluated by aggregometry studies following epinephrine, ADP agonists, collagen, arachidonic acid, thrombin and ristocetin addition (5). At the contrary, assay of miRNA inhibition on the platelets isolated from AMI patients or on platelets overexpressing the miRNA of interest will confirm the putative role of the miRNA of interest. Aim III. To accomplish this goal, the candidate will need to learn and use different molecular biology and immunohistochemistry techniques. Most importantly, he/she will be required to handle small rodents. Indeed, to evaluate the role of candidate miRNA in the pathophysiology of atherosclerotic plaque instability, miRNA precursors will be administered to Apo-/- mice fed with a cholesterol-rich diet for 6-12 weeks and the atherosclerotic lesions analyzed at the level of the aorta. Bioinformatic analysis will be used to identify the targets of the miRNA. Last, chemically engineered oligonucleotides, such as antagomiRs, will be employed both in vitro and in vivo in order to confirm targets and pathways previously identified.
(1) D’ALESSANDRA Y, DEVANNA P, LIMANA F, STRAINO S, DI CARLO A, BRAMBILLA PG, RUBINO M, CARENA MC, SPAZZAFUMO L, DE SIMONE M, MICHELI B, BIGLIOLI P, ACHILLI F, MARTELLI F, MAGGIOLINI S, MARENZI G, POMPILIO G, CAPOGROSSI MC, 2010. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J., 31(22), 2765-73
(2) ROMAINE SP, TOMASZEWSKI M, CONDORELLI G, SAMANI NJ, 2015. MicroRNAs in cardiovascular disease: an introduction for clinicians. Heart , 101(12), 921-8
(3) WILLEIT P, ZAMPETAKI A, DUDEK K, KAUDEWITZ D, KING A, KIRKBY NS, CROSBY-NWAOBI R, PROKOPI M, DROZDOV I, LANGLEY SR, SIVAPRASAD S, MARKUS HS, MITCHELL JA, WARNER TD, KIECHL S, MAYR M., 2013. Circulating microRNAs as novel biomarkers for platelet activation. Circ Res., 112(4), 595-600
(4) PISANO F, ALTOMARE C, CERVIO E, BARILE L, ROCCHETTI M, CIUFFREDA MC, MALPASSO G, COPES F, MURA M, DANIELI P, VIARENGO G, ZAZA A, GNECCHI M, 2015. Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation. Stem Cells., 33(4):1187-99
(5) ZHOU L, SCHMAIER AH, 2005. Platelet aggregation testing in platelet-rich plasma: description of procedures with the aim to develop standards in the field. Am J Clin Pathol., 123(2), 172-83
Title: Possible role of aquaporins in the pathogenesis of ROS mediated inflammatory bowel disease
Supervisor(s): U Laforenza
This study aims to understand the role aquaporins (AQPs) in the pathogenesis of inflammatory bowel disease. In particular, the function of AQP as peroxiporins in mediating the diffusion of H2O2 through the cellular and subcellular compartments that promotes the H2O2 scavenging will be studied in normal and pathological conditions. To this purpose this study will investigate the expression of AQPs in a cellular model of human colonic epithelial cells, Caco-2, at mRNA and protein level by RT-PCR and western blotting. Then immunohistochemistry techniques will be used to define their localization in the plasma membrane and in the intracellular organelles. Successively, functional experiments will be performed to measure the water permeability by using stopped flow light scattering and the H2O2 permeability by using the CM-H2DCFDA probe in the presence and in the absence of oxidative stress. This latter is a chloromethyl derivative of H2DCFDA, useful as an indicator for reactive oxygen species (ROS) in cells fluorescence techniques (microplate reader). These experiments will be carried out to confirm what has already been shown on HeLa cells. Recently, in fact it has recently been shown that diverse cellular stress conditions, including heat, hypoxia and endoplasmic reticulum stress, reversibly inhibit the permeability of AQP8 to both water and H2O2 (Medraño-Fernandez et al., Antioxid Redox Signal. 2016 Apr 19). Gating of AQP8 has been speculated to impede the bidirectional transport of H2O2, with two important consequences; 1) NOX-derived H2O2 cannot exert its growth-promoting effects, 2) the excess of H2O2 produced intracellularly upon stress cannot be extruded through AQP8, causing growth arrest and apoptosis. Finally, oxidative stress does not impair H2O2 and water flow in cells expressing a mutant AQP8 in which cysteine 53 had been replaced by serine. This make them resistant to stress-, drug- and radiation-induced growth arrest and death. Therefore, AQP8 could be considered a new possible therapeutic target for IBD treatment. Two different approaches will be considered. In the first, the effectiveness of antioxidants known to protect AQP8 permeability will be tested, since the beneficial effect of antioxidants in the treatment of IBD has been suggested (Moura et al., Redox Biology 2015. 6; 617–639). Secondly, in the animal or cell model of colitis the mutant non gated AQP8 C53S will be delivered in the hypothesis that the maintaining of H2O2 scavenging could maintain mucosal integrity and functioning.
Stress Regulates Aquaporin-8 Permeability to Impact Cell Growth and Survival. Medraño-Fernandez I, Bestetti S, Bertolotti M, Bienert GP, Bottino C, Laforenza U, Rubartelli A, Sitia R. Antioxid Redox Signal. 2016 Jun 20;24(18):1031-44. doi: 10.1089/ars.2016.6636.
Antioxidant therapy for treatment of inflammatory bowel disease: Does it work? Moura FA1, de Andrade KQ2, dos Santos JC3, Araújo OR4, Goulart MO5. Redox Biol. 2015 Dec;6:617-39. doi: 10.1016/j.redox.2015.10.006.
Title: Molecular methods for the characterization of HPV-mediated oncogenesis on bioptic and cytological samples
Supervisor(s): P Morbini
Background: Human papillomavirus (HPV) infection is responsible for the development of squamous cell cancers in the genital area and in the oropharyngeal region. Ongoing research projects on the role of host immunological responses, the definition of HPV-specific cellular immunity, the disregulation of miRNA induced by HPV infection, and the design of innovative therapeutic approaches (as virus-specific T-cell therapy, immunomodulatory therapy, etc) require a precise definition of the oncogenic action of the virus in every single patient in particular with oropharyngeal cancer, to correctly classify the tumors that are actually HPV-driven from those in which the infection is not present or is not oncogenic (passenger), that can be used as negative controls. So far the gold standard for identifying oncogenic HPV infections is the PCR amplification of the mRNA of the 2 viral oncogenes E6 and E7, which is standardized only for high risk HPV16, requires fresh tumor tissues and dedicated facilities and is time-consuming. These constraints strongly limit the selection of patients that can be enrolled in prospective and retrospective studies to those for whom fresh tissue samples are available, and always require an invasive procedure. Aims: to implement and validate a method(s) to identify oncogenic/transcriptionally active HPV infection on archival (formalin-fixed paraffin-embedded) and cytologycal (cervicovaginal and oral washing) samples to support ongoing and future multidisciplinary research. Experimental approach: we are currently collecting a series of paired fresh, fixed, and cytological samples from oropharyngeal cancer patients enrolled in an ethically approved study (enrollment rate 12-15 pts/year), characterizing HPV DNA-positive cases and the specific viral genotype with a patented diagnostic platform (SPF10 LiPA, Innogenetics) based on viral DNA PCR amplification and reverse line blot, and assessing and quantifying (1) genotype-specific E6 mRNA, and (2) the proportion of integrated HPV genomes, in fresh samples by real-time PCR with a set of primers that we designed targeting genotype-specific E6/7 gene mRNAs and E2 and E6 gene DNAs. After completing the mRNA and integration study (gold standard) in 30 HR HPV-positive cases we will implement: 1. genotype-specific mRNA in situ hybridization on fixed samples from the case series, 2. mRNA extraction and E6/7 gene rt-PCR from the fixed samples, 3. mRNA extraction and E6/7 gene rt-PCR from cytological oral washing samples. The results obtained with the 3 above methods will be compared for each study case with the results of the gold standard test in order to validate their accuracy in identifying transcriptionally active HPV infection and identify a test can be used for patient selection that is most time- and cost-effective and less disturbing for the patients (ideally based on cytology). Finally, to allow the application of the validated methods to the classification of patients with cervical or other genital cancers we will translate the experimental approach to a case series from the gynecology department.
Rautava J, Syrjänen S. Biology of human papillomavirus infections in head and neck carcinogenesis. Head Neck Pathol 2012;6:S3-15
Holzinger D, Schmitt M, Dyckhoff G, Benner A, Pawlita M, Bosch FX. Viral RNA patterns and high viral load reliably define oropharynx
carcinomas with active HPV16 involvement. Cancer Res 2012;72:4993-5003. Morbini P, Dal Bello B, Alberizzi P, et al. Oral HPV infection and persistence in patients with head and neck cancer. Oral Surg Oral Med Oral Pathol Oral Radiol 2013;116:474-8.
Morbini P, Alberizzi P, Tinelli C, et al. Identification of transcriptionally active HPV infection in formalin-fixed, paraffin-embedded biopsies of oropharyngeal carcinoma. Hum Pathol 2015;46:681-9
Title: Understanding the molecular mechanism of Friedreich’s ataxia
Supervisor(s): A Pastore
Friedreich’s ataxia (FRDA) is a recessive autosomal neurodegenerative disorder caused by partial silencing of the frataxin gene (FXN) that encodes for an essential mitochondrial protein. It is one of the most common ataxias, it has an incidence of 1 every 50,000 individuals and the onset is around 20 years of age. The disease is characterized by progressive neurodegeneration, limb gait and cardiomyopathy . Patients derived cells present mitochondrial iron accumulation, increased oxidative stress and abnormalities in iron-sulphur (Fe-S) cluster biogenesis . Although frataxin has been extensively studied, its primary function and the early stages of the disease are still unclear. It was linked to the iron-sulfur cluster (isc) machine which is essential for life. Iron–sulfur (FeS) clusters are essential acid-labile prosthetic groups containing iron and sulfur which act as electron transfer agents thanks to their versatile electron-donor/acceptor properties . In nature, they are widely distributed in most organisms, including anaerobic, aerobic and photosynthetic bacteria, fungi, plants and animals . Enzymatic FeS cluster biosynthesis is a complex process which is under the control of the complex isc machine which is highly conserved from bacteria to primates. Determining the functions of isc proteins is important for understanding the molecular mechanism of Friedreich’s ataxia and, more in general, providing insights into FeS cluster assembly in eukaryotes.
PhD Project: Aims/Description. The aim of this project is to understand the relationship between frataxin and the other components of the isc machine. Pastore’s group has been working on frataxin for 20 years and accumulated a considerable know-how on this protein and its relationship with other cellular partners. The student will have to study in particular the relationship and the mode of interactions between frataxin and the two chaperonines which assist cluster assembly using the bacterial proteins as model systems and then expanding the knowledge to the eukaryotic orthologues. Plasmides for the proteins are already available in the group. We plan to use an integrated multidisciplinary approach  that exploits hybrid structural methods based on state-of-the-art NMR, small angle X-ray scattering and computational techniques to study how the various components of the iron sulfur cluster biogenesis machine talk to frataxin . The student will have ample space to establish new approaches and use different techniques. The project will provide training both in advanced computational methodologies and bioinformatics and in structural methods allowing the development of new hybrid methodologies based on the combined use of NMR and SAXS. Pandolfo, M., Pastore, A. (2008) The pathogenesis of Friedreich ataxia and the structure and function of frataxin. J. of Neurol. 256 Suppl 1, 9-17
 Pastore, A. and Puccio, H. (2013) Frataxin: a protein in search for a function. J. of Neurodegeneration 126, 43-52. Adinolfi, S. and Pastore, A. (2014) Chronochemistry in neurodegeneration. Frontiers in Neurosciences Mar 31; 7:20. doi: 10.3389/fnmol.2014.00020. eCollection 2014.
 Prischi, F., Konarev, P.V., Iannuzzi, C., Pastore, C., Adinolfi, S., Martin, S.R., Svergun, D.I., Pastore, A. (2010) Structural bases for the interaction of frataxin with the central components of iron-sulfur cluster assembly. Nature Communications 1, 95.
 Pastore, A. and Temussi, P.A. (2012) Hybrid approaches in Structural Biology. Edt. by Guagliardi, A. and Masciocchi, N., Insubria
Title: Understanding the antiproliferative molecular mechanisms of resveratrol-derived compounds and strategies for enhancing their targeted delivery
Supervisor(s): M Savio
Considerable epidemiological, preclinical and clinic studies have suggested that plant-derived dietary agents, nutraceuticals, have the potential to prevent and treat various cancers due to their broad chemical diversity, multi-targeting action and safety profile. Some nutraceuticals are currently in clinical trials and others have already been approved for human use. An ideal therapeutic agent should be non toxic, potent, inexpensive and easy available. One of the most promising compounds extensively studied in the last decades is resveratrol (3,5-4’tri-hydroxy-trans-stilbene), a phytoalexin found in several vegetables and fruits. Our research has been focused on the biological properties of some resveratrol-derivatives obtained linking structural component and specific activity, mainly against cancer cell proliferation (1). Among them, 4,4’-dihydroxy-trans-stilbene (DHS) has shown a preventive effect on cancer invasion and metastasis in a mouse model (2). We are interested in understanding its detailed molecular mechanism, not fully understood yet, together with an improvement of its bioavailability in the tumour site. The development of innovative formulation strategies, able to overcome physicochemical and pharmacokinetic limitations of resveratrol or its analogues, could be beneficial. To this end, the key goals will be to clarify the molecular mechanisms involved in cell proliferation inhibition by using different approaches. Notably, cellular enzymes (DNA replicative polymerases), transcriptional factors (STAT3 and its activators), and estrogen receptors, whose activities are closely involved in cellular proliferation, will be studied by cellular (proliferation, adesion, migration and invasion assays, immunofluorescence and western blot studies, flow cytometry and confocal analyses) and molecular biology techniques, including RNA sequencing. The second part of the project will be identifying a specific biomarker of mouse lung cancer cells to make strategies improving the delivering of DHS in tumour masses. There are three categories of promising biomarkers (3) including tumour immune and microenvironmental factors, epithelial-to-mesenchymal transition associated proteins or independent factors such as aquaporin-1 (4). Mouse cancer cell lines and mouse tumor tissue treated with resveratrol or its analogue, will be used to identify specific and promising biomarkers both in vitro and in mouse experiments. Using siRNA technique, immunofluorescence analysis, western blotting and immunohistochemistry, the specificity of the identified factor will be ascertained before starting with the design and the encapsulation of the compounds in polymeric nanoparticles (in collaboration with Dr. Sechi, University of Sassari). Functionalized nanoparticles will be used as vehicle for DHS in lung cancer cells and in mice to verify the targeted delivering of the studied compounds.
1. Stivala LA, Savio M et al. Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. JBC, 2001; 276:22586-94.
2. Savio M, Ferraro D et al. Resveratrol analogue 4,4’-dihydroxy-trans-stilbene potently inhibits cancer invasion and metastasis. Scientific
Reports 2016; 1-6:19973. doi10.1038/srep19973.
3. Vargas AJ and Harris CC. Biomarker development in the precision medicine era: lung cancer as a case study. Nature Rev Cancer 2016 Jul
8, doi: 10.1038/nrc.2015.56.
4. Yun S, Sun PL et al. Aquaporin1 is an independent marker of poor prognosis in lung adenocarcinoma. J Path and Trans Medicine 2016; 50: 251-257