Projects 2019/20

Projects 2019/20


List of Projects 2019/20 


Research project proposed by: Antonio Di Sabatino supervisor  (Nicola Fazio internal tutor)

Title of the research project: Microbiota variations and effects in gastroesophageal cancer.

Description of the research project:
Microbiota plays an important role in human health, and an increasing number of studies have shown that changes in the composition of human microbiota may correlate with different diseases. There is growing evidence in oncology, that cancer therapy perturbs the microbiota and the host immune response, resulting in dysbiosis. On the other hand, existing evidence supports the hypothesis that gut microbiota can modulate the pharmacological effects and the toxicity profile of chemical therapy and novel targeted immunotherapy. Gastric adenocarcinoma is the third leading cause of cancer-related death worldwide, accounting for more than 720,000 deaths annually. Although H.pylori is the most common bacterial infection worldwide and colonizes greater more than 50% of the global population, only 1%–3% of infected individuals ever develop gastric cancer. However, studies in germ-free mice have shown that H. pylori alone is less likely to induce gastric cancer compared to germ-free mice co-colonized with complex intestinal flora and H. pylori synergistically in which invasive gastrointestinal intraepithelial neoplasia (GIN) has been seen in 80% of cases. These evidences suggest how the microbiota of the stomach, other than H.pylori, can both influence the development of gastric malignancies and drive the carcinogenesis sequence in H.pylori infected patients. The aims of this study are to characterize the microbiota composition in a gastric cancer patient population, its variations according to patient and tumor characteristics and subsequently if microbiota can modulate response and toxicity profile to cancer therapy.

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 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 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

the new project agreed with the student is:

Liver Extracellular Matrix Scaffolds Repopulated with Primary Liver Cancer Cells to Investigate the Role of the Tumor Microenvironment in Shaping Host Innate Immune Responses

Description of the research project:

Primary liver cancer is the 4th most common cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) accounts for approximately 90% of primary liver cancers and often develops in a background of chronic viral hepatitis, alcoholic liver disease, or non-alcoholic steatohepatitis (NASH), after a multistep process requiring chronic necroinflammation, eventually leading to liver fibrosis and cirrhosis. Beside HCC, cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical onset and a dismal prognosis. CCA is the second most common primary liver cancer after HCC, accounting for the remaining nearly 10% of all primary liver cancers.
Most solid organ cancers, including liver, harness several mechanisms of immune escape to evade anti-tumor immune responses, including modulation of the local tumor microenvironment to create an immunosuppressive milieu. Of note, recent findings from different laboratories, including our own, indicate that overexpression of inhibitory cytokines may profoundly influence innate immune responses, particularly natural killer (NK) cells. Moreover, a substantial proportion of liver cancers is surrounded by a reactive tumor infiltrate populated by host cells including cancer-associated immune cells. Cancer immunotherapyrescuing exhausted cytotoxic T cells with checkpoint inhibitors is among the biggest recent breakthroughs of cancer research allowing control of previously aggressive tumors.
The traditional platforms used for disease modeling and drug screening in primary liver cancer are 2-dimensional (2D) cell monolayers cultured on plastic surfaces, animal models, and human organ cultures. However, cells in monolayer cultures tend to de-differentiate because of the high stiffness of the artificial substrate. Thus cellular functions may be affected by the lack of signals from stromal cells and ECM proteins organized within a tissue-specific 3D architecture. Therefore, due to the high prevalence of liver cancer, there is a pressing demand to establish in vitro models of human normal and malignant liver able to more accurately recapitulate the complex pathophysiology of human primary liver cancers. Tissue engineering has provided new 3D platform technologies, such as organoids, spheroids, and scaffolds for the in vitro study of pathophysiological mechanisms underlying liver disorders. Organoids and spheroids derived from tissue obtained from biopsies or surgical sections have various limitations and high costs. In contrast, biological ECM scaffolds have successfully been obtained by decellularization of human and murine organs, including the liver. Previous studies, showed that a process of decellularization and recellularization technology of human liver 3D scaffolds is doable and constitutes a valuable platform for liver bioengineering through the repopulation of human liver ECM scaffolds with parenchymal and nonparenchymal liver cells. The aim of this project is to develop a liver cancer model using liver 3D scaffolds to recapitulate the tumor microenvironment and identify molecules controlling tumor-directed innate immune responses. The information retrieved from these studies will allow the design of innovative therapies for HCC and CCA in the expectation to improve the prognosis of these tumors.


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 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.

The new project agreed with the student Brunelli is:

PINK1: fine tuning the balance between cell proliferation and degeneration

Description of the research project:

PINK1 is a mitochondrial serine/threonine kinase involved in the pathogenesis of familial forms of Parkinson’s Disease (PD). It is a master regulator of mitophagy, the autophagic degradation of damaged mitochondria, but there is evidence that this protein is crucial in other cellular processes, including autophagy at large, inhibition of apoptosis, promotion of growth and proliferation, regulation of oxidative metabolism.
The project involves the study of PINK1 and its interactome from a combined approach of structural biology and cell biology.
Structural studies will focus on the expression of whole-length proteins or single domains of PINK1 and its interactors, such as Beclin 1, in different expression systems, including bacteria, yeasts, and mammalian cells.
Cell biology studies will be primarily aimed at understanding the effects of these interactions and the consequences of mutations in the contact sites. They will entail cultures of immortalized cells silenced for PINK1 and/or other proteins or expressing mutated variants, as well as iPSCs derived from fibroblasts of PD patients carrying a PINK1 mutation.

The new project agreed with the student Lidia Pollara, with supervisor Virginie Sottile, is: Innovative strategies for in vitro modelling of rare human neurogenetic diseases

Description of the research project:

Over the past decade, the routine advent of next generation sequencing techniques has led to impressive progress in the identification of genes causative of rare genetic diseases, even in small families or sporadic cases. These genetic discoveries often require validation through functional experiments, to understand the biological function of proteins encoded by candidate genes, and how this function is disrupted by mutations. While in vivo animal models are not always available or physiologically accurate, the use of human cell lines may represent a valid alternative but, especially in neurogenetic disorders, this may present limitations related, for instance, to the differences with the actual tissues affected by the disease.
My PhD project will concern the generation of innovative in vitro models to investigate the molecular basis of selected neurogenetic disorders such as Joubert syndrome (JS), based on the use of new patient-derived induced pluripotent stem cells (iPSCs), which can be differentiated in any cell type, while recapitulating the full genetic background of the patient.
JS is a congenital cerebellar ataxia with autosomal recessive or X-linked inheritance, characterized by a peculiar cerebellar and brainstem malformation, the molar tooth sign. It is a genetically heterogeneous disease and over 40 associated genes have been identified, overall accounting for about 60% of cases. All known JS-linked genes encode proteins of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues, enabling the cataloguing of JS as a ciliopathy. There is clinical and genetic overlap among ciliopathies because the same gene may be responsible for distinct phenotypes and the same clinical manifestation can be caused by many genes.
In the first part of my project, I will focus on the development and optimization of differentiation protocols to obtain specific neuronal types, to characterize genetic mutations identified in JS patients. Isogenic controls will be made by CRISPR/Cas9 technology.
In the second part of the project, I will attempt to further grow selected iPSCs into 3D organoid structures (“minibrains”), which will allow a deeper characterization of disease-specific changes in mechanisms such as neuronal migration defects or neuron-glia interaction.
The final goal is to look for common end-route pathways that could be similarly affected in JS patients with mutations in distinct genes and/or understand how the same gene can produce different phenotypes (for example pure neurological, neurological+retinal, neurological+retinal+renal phenotypes), through transcriptome 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).

Research project proposed by: Giovanni Palladini

Title of the research project: Evaluating Diagnostic and Prognostic Markers of Cardiotoxicity in the setting of Hematopoietic stem cell Transplantation and cart-cells

Description of the research project: Allogeneic and autologous hematopoietic stem cell transplantation are well-established therapeutic procedure for different haematological diseases. They can be the only curative approach, but their toxicity profile is stillchallenging, mostly for allogeneic transplantation.
CART-cell therapy is an innovative approach in haematological neoplasia with a specific and known toxicity, but it still needs an accurate evaluation in real-life practice.
Cardiotoxicity is linked to drugs used during the conditioning regimen in transplant setting or to CART-induced cytokine-release syndrome and can significantly affect the survival outcomes. Some pathological conditions (like AL amyloidosis with cardiac involvement), age and a history of cardiac events represent risk factors for thistoxicity. Evaluating the diagnostic and prognostic significance of some biological markers of cardiac damage and dysfunction, like BNP and troponin, can be a useful tool to stratify patient’s risk, make an early diagnosis and therapeutic project and monitor therapy efficacy. Predictive value of biological markers has still to be validated and could stimulate patient-adapted strategy. Clinical significance of an integrated approach of biological markers and imaging must be evaluated. Improving knowledge about drug- and CART-related cardiotoxicity and its clinical management can lead to a reduced therapy-related morbidity and mortality.

























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