Projects 2019/20

Projects 2019/20


List of Projects 2019/20 

Project 1

Research project proposed by: Alessandro Malara, Alessandra Balduini

Title of the research project: Bone marrow modelling for platelet production

Description of the research project:
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.

Project 2

Research project proposed by: Roberto Bottinelli

Title of the research project: Evaluation of the therapeutic potential of PIN1 inhibition in muscular dystrophy.

Description of the research project:
One proposed strategy to prolong survival and increase quality of life of DMD patients is based on the observation that slower muscle fibers are more resistant to the dystrophic pathology relative to their faster counterparts [1]. Notably, the transcription factor MEF2C activates the transcription of the slow-fiber type gene program and of PGC-1a in skeletal muscles [2]. Furthermore, Pin1, a Peptydyl Prolyl cis/trans isomerase involved in post-phosphorylation control of protein function, interacts with the phosphorylated MEF2C?1 splice variant repressing MEF2-dependent activity [3]. We found that Pin1 loss results in a shift of skeletal muscle fiber type toward slow twitch fibers, suggesting that it might represent a good candidate target to promote slow fiber conversion and ultimately counteract muscle wasting of dystrophic muscle. Aims of the project are (i) to evaluate the mechanical and metabolic changes associated to the loss of Pin1 expression in skeletal muscles by investigating the phenotype of adult skeletal muscles lacking Pin1 (ii) to identify the molecular targets of Pin1 in adult skeletal muscle by Co-IP and Mass Spectrometry (iii) to establish whether the pharmacological inhibition of Pin1 has the potential to mitigate muscle damage in a murine model of Duchenne muscular dystrophy (mdx mice). The Pin1 inhibitor will be tested both in cultured myotubes and in mdx mice.

Project 3

Research project proposed by: Monica Canepari

Title of the research project: Molecular determinants of diaphragm muscle impairment in an intermediate mouse model of spinal muscular atrophy.

Description of the research project:
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease with an incidence in humans of approximately 1 in 10.000 births per year. SMA is caused by the deletion/mutation of the survival of motor neuron gene (SMN). The genetic defects occurring in SMA determine the degeneration of spinal motoneurons (MNs), leading to progressive muscular atrophy weakness and respiratory failure. Indeed increasing evidence suggest that SMA pathogenesis is more complex than expected: many authors have recently speculated that, even though MNs are the most affected cells in SMA, their loss might not only depend from the lack of SMN: retrograde signals coming from muscles and NMJs can be crucial players of the MN alteration.(1). A more extensive analysis is therefore needed to better clarify the molecular, functional and temporal defects affecting skeletal muscles and NMJs in SMA. In this context, diaphragm muscle is a muscle relatively poorly studied, notwithstanding is one of the earliest affected muscles. The central goal of this project is to assess intrinsic diaphragm muscle defects, the molecular causes underlying them and their pathogenic role in an intermediate SMA mouse model (SMN?7mice or Jackson Laboratory stock #005025) with median survival of 13 days. This goal is of primary importance for understanding the pathogenesis of SMA and for the development of appropriate pharmacological strategy. In this way, the project will have the potential to identify the causes and the factors influencing the course of the disease leading to respiratory failure and early death. To achieve its goal, the project will combine morphological, biochemical, functional and molecular analyses (2,3,4,5) on isolated diaphragm muscle. The project is supported by the BLUE SKY RESEARCH Grant.

Project 4

Research project proposed by: Roberto Ciccone

Title of the research project: Evaluating the contribution of rare, silent variants to cerebellar congenital malformations and other brain developmental disorders.

Description of the research project:
NGS-based investigations such as Whole Exome Sequencing (WES) represent a primary tool for identifying causal variants in rare diseases, although the interpretation of sequencing data still remains a challenging task. This is particularly true for silent variants (i.e: intergenic, intronic, and synonymous variants) which are much harder to interpret and usually discarded in sequencing analyses. However, a growing body of evidence indicates that silent variants can potentially determine a deleterious effect by altering the gene transcription and/or the splicing process, possibly explaining a proportion of the “missing diagnoses” in many rare diseases.
Aim of this PhD project is to apply novel strategies to identify potentially deleterious silent variants in patients in whom genetic testing failed to identify pathogenic variants related to their condition. The student will focus on a large cohort of pediatric patients with a range of cerebellar malformation disorders (Joubert syndrome – JS, ponto-cerebellar hypoplasia etc), which has been recruited and genetically tested in the lab over the past years. For instance, despite over 40 causative genes are known, a genetic diagnosis is not reached in 35-40% of JS patients, but a subset of them actually shows a single pathogenic variant, suggesting that the second one can reside outside the coding regions.
To pursue this aim, a combined strategy based on in-silico analyses, gene expression and genomic investigations will be used. The PhD student will become confident with different approaches including NGS, gene expression assays and in-silico prioritization of coding and silent variants.

Project 5

Research project proposed by: Lia Crotti

Title of the research project: Combined molecular and therapeutic approach to investigate neonatal life-threatening calmodulin mutations

Description of the research project:
Calmodulin is a small ubiquitous Ca2+-binding protein, encoded by 3 CALM genes (CALM1-3) and pivotal for the correct functioning of many organs, including the heart. Mutations in CALM genes have been linked to severe neonatal cardiac disorders that can manifest phenotypically as Long QT Syndrome (LQTS), Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), Idiopathic Ventricular Fibrillation (IVF) or overlapping syndromes and this has led to the definition of a new clinical entity called “Calmodulinopathy”. Current pharmacological therapies for calmodulinopathies are largely ineffective, while implantable cardioverter defibrillators (ICDs), designed for older subjects, are burdened with important side effects (sometimes-lethal), thereby challenging physicians and exposing these subjects to high risk of death. Indeed, data from the International Calmodulinopathy Registry which our group has established and leading, show that  around one third of the cases enrolled have died either suddenly or, more rarely, due to   ICD-related complications.
The aim of this project is to identify novel therapeutic strategies for the treatment of calmodulinopathies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from our patients with CALM mutations. This project will harness multiple state-of-the-art technologies in genetics, electrophysiology and molecular biology to investigate the phenotypic effects of disease causing mutations and to identify potential pharmacological therapies to rescue the severe phenotype. The outcome of this project will also generate new knowledge on calmodulin mutations, their mechanism of action and new information for the diagnosis and treatment of neonatal life-threatening Calmodulinopathies.

Project 6

Research project proposed by: Antonio Di Sabatino

Title of the research project: Targeting intestinal fibrosis in inflammatory bowel disease

Description of the research project:
Extensive tissue fibrosis is the end-stage process of a number of chronic conditions affecting the gastrointestinal tract, including inflammatory bowel disease (Crohn’s disease, ulcerative colitis). Fibrogenesis is a physiological, reparative process that may become harmful as a consequence of the persistence of a noxious agent, after an excessive duration of the healing process. In this case, after replacement of dead or injured cells, fibrogenesis continues to substitute normal parenchymal tissue with fibrous connective tissue, leading to uncontrolled scar formation and, ultimately, permanent organ damage, loss of function, and/or strictures. Several mechanisms have been implicated in sustaining the fibrogenic process, including inflammatory and non-inflammatory pathways that are partially known. The only available therapy is the surgical removal of the affected gastrointestinal tract. Through this PhD project, the candidate will focus on the development of experimental 3D intestinal models (scaffolds) of fibrosis, ex-vivo expression of pro-fibrogenic cytokines, and clinical characteristics of patients displaying a high rate of progression to intestinal fibrosis. Also, aim of this project is to investigate possible novel biomarkers of intestinal fibrosis that may be useful in identifying patients who are more likely to develop this complication, and to identify potential therapeutic targets to conteract the fibrogenic process and the consequent intestinal stricture development in patients with Crohn’s disease.

Project 7

Research project proposed by: Monica Galliano

Title of the research project: Structural characterization of new lectins with antineoplastic properties

Description of the research project:
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.

Project 8

Research project proposed by: Giulia Gastaldi

Title of the research project: Adipose stem cells for cartilage regeneration

Description of the research project:
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.

Project 9

Research project proposed by: Umberto Laforenza

Title of the research project: Aquaporins and malignant pleural mesothelioma: possible prognostic markers and new potential therapeutic targets

Description of the research project:
Malignant pleural mesothelioma (MPM) has a poor prognosis due to the poor response to common medical treatments. Unfortunately, until now no reliable prognostic clinical markers have been developed.
In the last decade, convincing evidence has emerged of a key role of aquaporins (AQPs) in cancer biology. For this reason, they have been indicated as possible therapeutic targets for cancer. Our study aims to investigate the three different histotypes (epithelioid, sarcomatous, and biphasic) of MPM in terms of expression, location, and function of the different AQPs.
Materials and methods
MPM cell lines and tissue sections will be obtained from the Biological Bank of Malignant Mesothelioma (BB-MM, Pathological Anatomy, Hospital of Alexandria). This study aims to investigate the three different histotypes (epithelioid, sarcomatous and biphasic) of MPM in terms of: 1) mRNA and protein expression of AQPs in MPM cell lines; 2) cellular localization by immunohistochemistry; 3) contribution of the different AQPs expressed in MPM cells by selective gene silencing of single isoforms using siRNA and pharmacological blockers such as AqB050; 4) proliferation, migration, invasion, apoptosis resistance and angiogenesis in single AQP-null cells; 5) functional evaluation of AQPs present in MPM cells by: stopped-flow light scattering for the measurement of osmotic water permeability and use of the CM-H2DCFDA fluorescent probe for the measurement of cell permeability to hydrogen peroxide.
Expected results
The treatment of MPM is complex and the survival results as well as the overall survival data are, to date, disappointingly discouraging. Our research project shows a real translation potential with beneficial effects for patients with MPM, presenting the AQPs with a dual role: as a possible new biomarker, providing an improvement in terms of early diagnosis, and as a therapeutic target, expanding therefore the possibilities of approach.

Project 10

Research project proposed by: Marco Antonio Lolicato

Title of the research project: Structural and functional characterization of the Voltage-gated proton channel (Hv1) in complex with drugs

Description of the research project :
Ion channels are molecular machines important in regulating membrane potential and ion homeostasis. They, also, play a key role in cell proliferation and apoptosis. Uncontrolled cell growth, resistance to apoptosis and a dysregulated chemical environment are all cancer hallmarks. Therefore, ion channels are considered excellent drug targets.
In metastatic breast cancer cell, the voltage gated proton channel Hv1 is involved in cell migration and in the maintenance of intracellular pH. To date, only one class of specific Hv1 inhibitor is known; mostly because only one structure of Hv1 channel is available. However, no mechanism of action has been, yet, described.
The goal of the project is to understand the pharmacology of Hv1 channel from a structural perspective. The project involves the generation of multiple structures in complex with small molecule modulators; validation of the structural findings with electrophysiological experiments and the screening of compound libraries to identify new potential drugs.
The PhD candidate will be actively involved in all the project steps, form molecular cloning to data collection, structure determination and functional analysis. He/She will learn the state-of-the-art methodologies in molecular biology, biochemistry, cell biology and electrophysiology. I will guide the PhD candidate to his/her own independence by learning how to design experiments, interpret the results and present the data to an audience.

Project 11

Research project proposed by: Antonella Minelli

Title of the research project: Genetic and clinical heterogeneity in Shwachman-Diamond Syndrome

Description of the research project :
Shwachman-Diamond Syndrome (SDS) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. It is inherited as autosomal recessive disease (OMIM #260400) and its clinical aspects include also skeletal alterations and an increased risk for myelodysplasia and acute myeloid leukaemia.
All clinical signs listed show a wide variability and patients with mild form of the disease and late diagnosis are common. The major disease gene is SBDS localized at 7q11.21. Detection of biallelic pathogenic variants in SBDS confirms the clinical diagnosis, but about 10% of patient, with clinical features strongly suggesting SDS, remains negative for mutations in SBDS.
Recently, genetic heterogeneity was proven: exome analysis revealed biallelic mutations in DNAJC21 and EFL1, while heterozygous dominant negative mutations in SRP54 were also found in patients with SDS-like phenotype.
Nevertheless, the number of causal SDS genes reported could be very limited even now if compared to other IBMFSs for which the situation is very different (>20 loci in Fanconi anemia or >10 loci in Diamond-Blackfan anemia, and several loci in severe congenital neutropenia).
Consequently, our project will cover two topics:
i) to obtain an etiological diagnosis in patients lacking mutations in SBDS gene but in whom a clinical diagnosis of SDS is suggested. In these patients we plan to set up western blot analysis for the identified genes and to perform exome sequencing;
ii) to search for damaging variants by an in silico analysis of the exome data already available for 16 SDS patients with SBDS mutation, focusing for which variants might be relevant for promoting clonal progression to MDS and leukaemia.

Project 12

Research project proposed by: Mario Mondelli

Title of the research project: NKG2D and NKp30 ligand expression on hepatocellular carcinoma cells: implications for immunotherapy of liver cancer

Description of the research project:
Hepatocellular carcinoma (HCC) is a common cancer accounting for a significant proportion of all cancers worldwide. The tumour is ubiquitous and one of the most challenging complications of advanced liver disease. HCC develops deviously and there is no robust opportunity for early detection. Treatment of HCC has considerably improved over the last few decades from mere surgical resection in a minority of patients with good hepatic function and small tumours to orthotopic liver transplantation in selected patients, to locoregional ablation procedures, transarterial chemoembolization, and more recently antiangiogenetic drugs that increase survival in patients with advanced HCC for whom no therapy was hitherto available. Cholangiocarcinoma (CCA) is the second most common primary liver cancer after HCC. Infestation with liver flukes, primary sclerosing cholangitis, hepatitis B (HBV), hepatitis C (HCV), biliary stones and cirrhosis are recognized risk factors for CCA; however, the 5-year survival rate is still disturbingly low at 10% and less than 1/3 of the patients with CCA are amenable to treatment at diagnosis. Natural killer (NK) cells are an essential component of innate immunity being instrumental in anti-tumour immune responses. Interestingly, recurrence-free survival correlated with sustained functional NK cell activation, suggesting a role for these cells in the control of liver cancer. Recent data have shown that the level of expression of ligands of a major NK cell activating receptor, natural-killer group 2, member D (NKG2D), correlates with liver cancer progression and recurrence after surgical or ablative procedures. Poor NKG2D ligand expression was associated with poor differentiation and progression, whereas dysplastic nodules and well-differentiated tumours expressed large amounts of the ligand. Interestingly, NKG2D ligand expression on cancer cells is generally associated with indolent disease progression and prolonged survival in several types of tumour. It should be possible to manipulate NK cell ligand interaction in vitro and in vivo to provide the necessary basic information to design future immunotherapeutic trials in patients with advanced HCC treated with infusion of activated or resting allogeneic NK cells in the expectation that this approach will contribute to the arrest of cancer progression

Project 13

Research project proposed by: Mario Ulisse Nuvolone

Title of the research project: Novel therapeutic strategies against AL amyloidosis

Description of the research project:
Systemic immunoglobulin light chain (AL) amyloidosis is a life-threatening plasma cell tumor. A significant proportion of AL amyloidosis patients do not respond to anti-plasma cell drugs and die before being offered second-line therapies. Hence, predicting which patients are more likely to resist frontline drugs and identifying novel small molecules overcoming drug resistance has the potential to change the natural history of this fatal disease. By combining pharmacologic, immunophenotypic, genetic and proteomic approaches on primary, patients-derived bone marrow cells (plasma cells and mesenchymal stromal cells) and clinical monitoring of a prospectively enrolled cohort of AL patients, we plan to identify biomarkers predictive of response to frontline regimens and study mechanisms of drug resistance.
Primary plasma cells and mesenchymal stromal 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 isolated with established protocols. Primary cells will be exposed to different doses of the different drugs (bortezomib, melphalan and investigational drugs) in vitro and efficacy will be assessed with cell viability assays. A thorough biochemical and genetic characterization of primary plasma cells will be performed. Clinical data will be collected at baseline and at each subsequent follow up visit and response to frontline therapy will be assessed. By combining the data collected in the course of this study, we aim at identifying the best predictors of response to frontline therapies for AL patients. The planned research activities should also shed light on the molecular mechanisms of drug resistance and pave the way for the identification of novel promising drugs to treat AL amyloidosis.

Project 14

Research project proposed by: Carla Olivieri

Title of the research project: Functional characterization of variants of unknown significance in rare diseases

Description of the research project:
In the era of NGS-based genetic analysis, it is now the rule to identify a large number of novel genetic variants whose pathogenic impact is difficult to establish (the so called “variants of unknown significance”, which pose major problems in term of interpretation of NGS data and confirmation of a genetic diagnosis. an unknown effect. Although different in silico tools are available to predict the possible pathogenetic impact of a novel variant (based on conservation of the mutated residue, type of aminoacid change, impairment of key functional domains of the protein etc), they often generate conflicting results and none of them can be considered resolutive. Very often, the decision regarding the classification of a variant remains challenging, and functional studies represent the only strategy to solve this conundrum.
The present PhD project will focus on the functional characterization of selected VUS by applying functional in vitro studies and bioinformatic modeling strategies. The project builds upon a long tradition of our lab in the genetic screening of rare disorders; in particular, we will focus on two disorders, Hereditary Hemorrhagic Telangiectasia (HHT) and developmental brain ciliopathies. For these disorders, we have already screened large cohorts of patients and, for a subset of them, we were able to define some VUS as pathogenic, for instance by demonstrating that they resulted in aberrant splicing or reduced transcription levels of the involved gene.
The PhD student will perform a range of functional studies using site-directed mutagenesis and cell transfection to test whether the VUS will impact, for instance, on protein stability, subcellular localization, enzymatic activity or interaction with specific substrates. Whenever possible, we will also adopt patient-derived cell models, such as fibroblasts or induced pluripotent stem cells, which can be generated in the lab. The student will also learn to apply bioinformatic modeling techniques to predict the impact of the VUS on the protein folding, function and stability.

Project 15

Research project proposed by: Roberto Pizzala

Title of the research project: Neuroimmune interactions in the auditory brainstem

Description of the research project:
The auditory system can be damaged by several acute and chronic insults (noise, ototoxic drugs, endocrine and immune imbalances, aging), resulting in several hearing impairments. Although cochlear inflammation is central to most of these problems, central auditory pathways are able to modulate pathological cochlear signals, leading to perceptual normalization (compensation) or hearing impairment worsening (maladaptive plasticity). This is especially important in tinnitus and age-related hearing loss.
Neuroinflammation plays a major role in CNS plasticity; however, neuroinflammatory responses of the auditory system are still incompletely characterized.

Our research focuses on neuroinflammatory responses of the dorsal cochlear nucleus (DCN), which forms the earliest “noise filter” in the brain and is involved in tinnitus onset. This nucleus, together with its ventral counterpart, represents the first auditory station in the CNS, but also receives non-auditory information (e.g. trigeminal, vestibular) which contributes to sound filtering and perception modulation.
The DCN is surrounded by the 4th ventricle choroid plexus (CP), and CP-associated macrophages contact its surface and change their distribution upon cochlear damage, potentially affecting its inflammatory status.

In order to characterize DCN neuroinflammatory and neuroimmune responses, current work is being carried out on the following topics:
(1) Age-related differences in microglia and macrophage populations in healthy rodents and after ototoxic insults (noise trauma, ototoxic drug exposure).
(2) Macrophage distribution and trafficking in the cochlear nerve (CN) and CP.
(3) CP mechanical and biochemical relations with the DCN surface

Since both CN and CP are very easily damaged and/or distorted upon mechanical sectioning, we have developed a novel decalcified-clarified preparation where the whole auditory system (including the cochlea) can be imaged by immunofluorescence without the need for bone removal or physical sectioning. Using this preparation we are reconstructing the geometry of the intact CP components (blood vessels, stroma, epithelium, associated immune cells) in order to model diffusional or non-diffusional (e.g. convective, contact) pathways for cell and substance transfer at the CP and DCN surface.(4) Characterization of inflammatory markers in the CSF and blood in relation to hearing impairment. In particular, this line of research aims at finding useful inflammatory markers in the diagnosis of tinnitus/presbyacusis, potentially employable in clinical routine.

Project 16

Research project proposed by: Elena Rossi

Title of the research project: Characterization of the genetic basis of intellectual developmental disorders in a cohort of Italian patients.

Description of the research project:
Intellectual Developmental Disorders (IDD) are characterized by significant limitations in both intellectual functioning and adaptive behavior with childhood onset and can be isolated or syndromic. Their predicted prevalence is ~1%, with estimated socio-economic burden up to ~25.000€/patient/year. A genetic basis for IDD is well established and highly heterogeneous, encompassing both chromosomal defects and monogenic forms (mostly de novo heterozygous variants). Over 700 causative genes are known, each accounting for <0.5% cases. However, diagnosis of IDD is impaired by scarce access to testing and difficulties in variant interpretation, and it is often descriptive, leading to uncertain prognosis. Understanding the genetic mechanisms implicated in the disease is challenging, requiring the application of different approaches and techniques. However, the increase of diagnostic yield is an essential prerequisite towards the development of personalized treatments of IDD patients.
Aim of this PhD project is to identify the underlying genetic causes of IDD in a cohort of pediatric patients which is being recruited in our lab, through a combined approach of CGH-array and whole exome sequencing of the trios (proband + parents). The student will become acquainted both with array-CGH techniques and data analysis, as well as with next generation sequencing protocols, bioinformatic analysis of data and validation of identified variants with molecular biology techniques (Sanger sequencing, real time PCR, minigene assay, bioinformatic modelling etc).

Project 17

Research project proposed by: Elisa Rumi

Title of the research project:
Unraveling the molecular basis of triple negative patients and the genetic predisposition to familial cases of myeloproliferative neoplasms

Description of the research project:
To date around 10% of patients with myeloproliferative neoplasms (MPN) remain negative for any of the known MPN associated mutations (JAK2, CALR and MPL).
Most of the MPN are sporadic, however familial clustering has also been observed. JAK2, CALR and MPL mutations are somatically acquired events also in familial cases, as in sporadic patients.
The aim of this project is to clarify through whole genome sequencing (WGS) the pathogenesis of JAK2/CALR/MPL negative MPN patients and to identify the germline genetic factors that underlie familial clustering of MPN.
Identification of new somatic drivers or of inherited predisposing genes would lead to improvement in the diagnosis of these diseases and perhaps in their management, providing new targets for specific therapies.

Project 18

Research project proposed by: Claudia Scotti

Title of the research project: Structural basis of apolipoprotein (a) function

Description of the research project:
Lipoprotein (a) [Lp(a)] is a novel independent cardiovascular risk factor. This particle is similar to an LDL, but it includes, beyond apolipoprotein B100, apolipoprotein (a) (apo(a)). Apo(a) contains multiple kringle domains and a catalytically inactive serine protease domain. The apo(a) gene has evolved from the plasminogen gene by gene duplication and it comprises 14-37 K4-like repeats, which are classified into 10 subtypes denoted as KIV-1 – KIV10. Apo(a) molecular weight is dependent on the number of genetically encoded kringle IV type 2 repeats and inversely related to Lp(a) plasma concentration. Risk thresholds for molecular weights have been proposed, but there is not a full consensus, and the role of the different isoforms in pathogenesis has not yet been clarified. It is expected that structural information will be relevant to clarify these open issues. At the moment, the atomic structure of this lipoprotein has not yet been fully elucidated. Only 5 of the kringle structures have been determined (KIV-6, KIV-7, KIV-8 and KIV-10 and KV) and, recently, one by our laboratory (KIV-2, manuscript in preparation).
The aim of the project is to determine the atomic structure of the missing kringles and of the protease domain of apo(a), and to use them as probes to solve the complete structure of apo(a) by cryoEM. Both natural and recombinant sources of the protein will be used to isolate and produce the full-length apo(a) protein with different numbers of KIV-2 tandem repeats. Interactions with known binders (apoH, fibulin 5 and anti-Lp(a) antibodies) will be explored at the structural and functional level and potential practical applications of the results evaluated. Partnership with companies is already ongoing and will be further developed along the project to explore the possibility to design a novel antibody-based diagnostic tool.

Project 19

Research project proposed by: Enza maria Valente

Title of the research project: PINK1 neuroprotective roles in Parkinson’s disease

Description of the research project :
Parkinson disease (PD) is a common neurodegenerative disorder, due to loss of aminergic neurons of the substantia nigra. Upon stress conditions, different pathways are activated to maintain neuronal health, such as mitophagy (a type of autophagy aimed at clearing dysfunctional mitochondria while new ones are generated), the unfolded protein response (which counteracts endoplasmatic reticulum (ER) stress), and metabolic pathways. Among these, the serine/glycine metabolism ensures cellular replication and the production of the antioxidant molecule glutathione, at the same time regulating levels of D-serine, a regulator of synaptic plasticity whose deprivation leads to oxidative stress.
Aim of this PhD project is to investigate new neuroprotective roles of PINK1, a mitochondrial kinase whose mutations cause autosomal recessive PD. PINK1 is a known sensor which responds to cellular stress signals by accumulating on mitochondria to promote mitophagy, inhibit apoptosis and regulate chaperons and calcium homeostasis. The hypothesis, based on preliminary results obtained in the lab, is that PINK1 could regulate neuronal survival via maintaining their energetic reservoir by a possible interaction with the ER and the regulation of metabolic pathways, such as serine/glycine biosynthesis. The project will be pursued through the following tasks: i) to assess the role of PINK1 in regulating enzymes of L-serine synthesis and metabolism, and to measure levels of L-serine and metabolites (glutathione, D-serine, glycine) in cellular models lacking functional PINK1; ii) to evaluate whether serine starvation or supplementation can modulate bioenergetics, oxidative stress and apoptosis in PINK1-deficient models; iii) to explore the interaction of PINK1 with BECN1, a protein localized in the ER, which regulates cell fate decision balancing autophagy and apoptosis. The project will be carried out in collaboration with the unit of Immunology and Molecular Pathology, led by Prof. E. Gherardi, experienced in protein crystallization and protein-protein interaction analysis.

Project 20

Research project proposed by: Livia Visai

Title of the research project: Nanotechnology applications for innovative therapeutic approaches

Description of the research project:
The use of nanotechnology in medicine – referred to as nanomedicine- offers some exciting possibilities. It could revolutionize the way we detect and treat damage to the human body and disease in the future, and many techniques only imagined a few years ago are making remarkable progress towards becoming realities. In view of this, the proposed studies aim to investigate by using various biochemical, molecular and biological methods, the applications of nanotechnology for innovative therapeutic approaches in 3 different topics:
a. nanosystems conjugated with drugs for treatment of cancer. The basic idea is the characterization of polymer coated gold nanoparticles and the biological evaluation of their efficacy on in vitro cells models. So far, different type of nanosystems have been developed and characterized in in vitro cell model using antineoplastic mitoxantrone (1).
b. nanoparticles/nano-antibodies for treatment of bacterial infections. In this study, different nanotechnological approaches will be performed to reduce bacterial adhesion and biofilm formation in order to improve biofilm dispersal (2).
c. nanoscaffolds for regenerative medicine. This study aims to investigate by biochemical/molecular and biological methods the interaction of hMSCs with hydroxyapatite/strontium doped nanoparticles or nano/microelectrospun fibers and their differentiation to osteoblasts in different experimental conditions (exposure to physical, mechanical or chemical stimuli or in simulated microgravity) (3).


























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