Projects 2021/22
The PhD Programme in Translational Medicine has now reached its 5th year of activity. The programme has expanded year after year and now offers projects that span research in Biochemistry, Cellular and Human Physiology, Immunology, Cancer Biology, Gastroenterology, Hepatology, Cardiology and Haematology. A poster displaying the area of activity of the Programme is available at this page.
The PhD Programme in Translational Medicine has been greatly strengthened by the recent availability of core equipment (NMR spectroscopy, cryo-electron microscopy, super resolution microscopy, cell sorting, micro computed tomography, pre-clinical magnetic resonance imaging, mass spectrometry and a high performance computer cluster. This equipment is fully available to research students enrolling in the PhD Programme in Translational Medicine and much of is located in a central facility close to the main departmental buildings (Centro Grandi Strumenti).
The text below provides a list of the 13 projects available for the three year cycle starting on 1st October 2021. Interested students can contact Enza Maria Valente, coordinator of the PhD Programme in Translational Medicine for general questions concerning the admission procedure. They can also contact the supervisors of individual projects in order to seek additional information about each project.
List of research projects available for the three year cycle starting on 1st October 2021:
Project Title: Studying nuclear signalling events in muscle fibers
Supervisor: Roberto Bottinelli
Laboratory: Human Physiology
Research theme: Molecular and Cellular physiology / Muscle Physiology and signalling
Abstract
The skeletal muscle fiber is a highly organized cell that contains the minimal motility units (sarcomeres). A determining characteristic of muscle fibers is the presence of a large number of nuclei (50-300). Their gene expression was recently shown to be at the same time coordinately regulated, and also varying among nuclei in different portions of the fibers (e.g., neuromuscular junction, myotendinous junction, muscle fibre body). A high degree of functional independence exists among nuclei. While the variance in performance is likely linked to their specific tasks, the events that determine the functional singularity of each nucleus in a single fiber remain to be determined. The overarching goal of the proposed project is to investigate the molecular mechanisms that define how each nucleus perceives and elaborates cellular signals and how these signals are integrated in muscle physiology and pathophysiology. This project will study muscles in physiologic conditions and in conditions in which they profoundly adapt, e.g., ageing and unloading. In so doing the project will offer novel mechanistic insights and potential therapeutic routes.
Techniques
Primary mouse muscle fibers and immortalized cellular models (C2C12) will be used in live cell imaging FRET-based approaches and integrated with classic biochemistry and molecular biology techniques and with functional analysis
Project Title: SFibro-adipogenic precursors (FAPs): novel players in muscle plasticity.
Supervisor: Lorenza Brocca
Laboratory: Human Physiology
Research theme: Physiology, Cell biology, Regenerative medicine
Abstract
Skeletal muscle plasticity is of paramount importance in muscle and whole body homeostasis. Muscle mass can increase in response to contractions against high loads, shrink when loading or neuromuscular activity are decreased and regenerate after injury. Most studies on muscle plasticity focused on muscle fibers and satellite cells, i.e., muscle stem cells. Indeed, adaptations of muscle mass and function are modulated by a complex network of signals within muscle fibers. Satellite cells are necessary for hypertrophy, donating nuclei to muscle fibers, and for repairing or regenerating damaged or necrotic muscle fibers following injury or diseases. However, besides satellite cells, a relevant population of interstitial adult stem cells exists. Indeed, 19% of nuclei in a muscle belong to one of such populations, namely to fibro-adipogenic precursors (FAPs). Although FAPs do not directly regenerate muscle fibers, they are necessary for the regenerative function of satellite cells and play a key role in muscle integrity and function engaging in a complex network of cellular interactions through autocrine and paracrine signaling. They could be major players in orienting muscle adaptions towards muscle mass maintenance and regeneration or towards maladaptive phenomena such as muscle replacement by connective and adipose tissue. FAPS comprise many subpopulations with likely different roles. The project will address the role of FAPS and of their subpopulations in muscle adaptations to conditions such as ageing and disuse relating analyses of FAPS subpopulations, satellite cells, intracellular signaling pathways and of structural and functional features of skeletal muscle from human and mice models.
Techniques
High resolution microscopy, antibody staining, cell culture, cell sorting, RT-PCR, proteomics, analysis of muscle fibres contractile and metabolic function.
Project Title: Inflammation and inflammatory disorders – Type 2 inflammatory disorders and allergy
Supervisor: Antonio di Sabatino
Laboratory: Laboratory of Gastroenterology, Department of Internal Medicine
Research Theme: Allergology, Immunology, Internal Medicine
Abstract
Type 2 inflammation is a defining attribute of allergic disorders, such as asthma, food and drug allergy, anaphylaxis, and eosinophilic gastrointestinal disorders (EGID). These disorders have a significant impact in terms of morbidity and are rapidly increasing. The mechanisms leading to Type 2 inflammation are only partly known. In addition to classical IgE- mediated allergies, some allergic disorders display a predominant cellular-mediated component, which is only indirectly characterised through diagnostic techniques. This diagnostic gap applies particularly to EGID, that is the focus of this project, for which no functional tests are available. The main aim of the project is to characterise how the gastrointestinal mucosa contributes to the recruitment and activation of the different cellular players involved in EGID (), associated or not with asthma or food allergy. Specific focus will be given on the development of assays to detect in vitro and in vivo Th2 cellular activation, as well as on the characterization of mastocytes and eosinophils pathways. These data may allow a more precise phenotyping of patients and hence a better patients’ selection for biological therapies. This project particularly suits for candidates with a burning interest in mechanistic understanding of allergic inflammation with a translational research approach.
Techniques
Candidates will receive a careful training in molecular, cellular, and translational immunological techniques, including tissue immunohistochemistry, ELISA tests, immunoCAP, BAT tests.
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Project Title: Structural characterization of new lectins with antineoplastic properties
Supervisor: Monica Galliano
Laboratory: Biochemistry
Research Theme: Structural characterization of new isolated lectins to evaluate their antineoplastic activity against different types of tumour cells.
Abstract
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 structural characterization of new lectins that specifically recognize the T antigen and that show antineoplastic activity on different cancer cell lines. So, if you want to train in translational medicine and its tools in one of the oldest and most prestigious Italian Universities apply for the PhD course in Translational Medicine at the Department of Molecular Medicine #UniPV! Website: http://molecularmedicine.unipv.it/phd-programme/. (Graphycal abstract created with BioRender.com).
Project Title: Therapeutic antibodies directed to cancer antigens
Supervisor: Ermanno Gherardi
Laboratory: Immunology and General Pathology
Research theme: Cancer biology/cancer immunology/antibody engineering
Abstract
Receptor tyrosine kinases (RTK) control fundamental aspects of the development and adult life of multicellular organisms. They also play major roles in several types of cancer as a result of mutations or over- mis-expression. Accordingly, they constitute major target for cancer therapy. This project aims to develop several new antibodies directed against selected members of the RTK family and to proteins – such as CD47 – that associate with RTKs on cancer cells and promote cell survival and growth. The relevant antigens will be expressed in heterologous systems (bacterial or yeast cells) or in mammalian cells and, after purification, they will be used for selection of fragments of human antibodies (scFv) from phage antibody libraries. Antibody fragments binding cancer antigens with adequate affinity and specificity will be formatted as intact antibodies using protein engineering techniques and used in order to probe their biological in vitro and in vivo.
Techniques
Heterologous protein expression, protein purification, protein characterisation by size exclusion chromatography, surface plasmon resonance, X-ray crystallography
Project Title: Generation and characterization of clinical grade mesenchymal stromal cells and secretome to treat heart disease
Supervisor: Massimiliano Gnecchi
Laboratory: Molecular and Translational Cardiology
Research Theme: Translational Cardiology – Regenerative Medicine
Abstract
Heart failure (HF) and pulmonary arterial hypertension (PAH) are characterized by high morbidity and mortality. Currently available therapies do little to improve prognosis and organ transplantation is limited by a shortage of donors. Cell therapy may offer a novel therapeutic approach to PAH. For instance, mesenchymal stem/stromal cells (MSC), have been investigated in preclinical studies and showed therapeutic benefits. In particular, the administration of the MSC-derived conditioned medium (CM) or extracellular vesicles (EV) seems very promising since MSC-mediated beneficial effects are mainly related to their secretome. In this project we propose to develop a clinical grade protocol for the scale up of MSC of fetal origin isolated from human placenta (F-MSC) which in previous experimental settings have been shown to be more effective than adult MSC of bone marrow origin that, over the years, tend to lose therapeutic power due to aging and depletion of many paracrine factors. The therapeutic potential of CM and EV isolated from clinical grade F-MSC will be tested in vitro and in animal models. Considering the severity of HF and PAH, the discovery of new therapeutic strategies would greatly impact the prognosis and the quality of life of these patients.
Techniques
Stem cell isolation and culture, isolation of cell secretome and microvesicles, immunocytochemistry, microscopy, main molecular biology techniques, GMP procedures.
Project Title: Role of aquaporins in controlling the redox status of the cell in pathophysiological conditions: wound healing, sperm function, obesity, cancer
Supervisor: Umberto Laforenza
Laboratory: Membrane transport
Research Theme/Topic: Physiology
Abstract
ROS like H2O2 and 4-hydroxynonenal can exert a physiological effect at low concentrations, acting as intracellular second messengers (signaling molecules), or a cytotoxic effect at high concentrations that may trigger programmed cell death (apoptosis). The oxidative stress results from the imbalance occurring in the cells between ROS production and scavenging systems or detoxification. Great interest was aroused by the discovery that some aquaporins (AQPs) can facilitate the diffusion of H2O2 from the producing cells across the plasma membranes to the extracellular fluid and viceversa. AQPs are a family of water channel proteins present in mammals in thirteen isoforms (named from AQP0 to AQP12) with different permeability features and different localizations at cellular and subcellular levels. Some of them, AQP3, 5, 8, 9 and 11, were found to facilitate H2O2 diffusion and thanks to this characteristic, could constitute an important ROS scavenging mechanism that relieves normal cells from oxidative stress (Pellavio et al., 2017; 2020; Laforenza et al., 2016; Medraño-Fernandez et al., 2016). We surmise that (dis)regulation of H2O2 transport in cells could be a target in different pathophysiological conditions: wound healing, cancer, obesity, human sperm functions and dysfunction (infertility and HPV). Regulating redox signaling may open novel possibilities to control: cell proliferation and migration underlying tissue regeneration, cancer cell migration and apoptosis, inflammation in obesity and sperm infertility.
Techniques
Cell culture, gene silencing, qRT-PCR, immunoblotting, immunocytochemistry, stopped flow light scattering, real time H2O2 imaging with genetically encoded fluorescent probe (HyPer7).
Project Title: Targeting the Warburg effect to unlock cancer metabolism
Supervisor: Marco Lolicato
Laboratory: Immunology and General Pathology
Research Theme/Topic: Oncology, Structural Biology, Chemical Biology, Biophysics
Abstract
Tumor cells preferentially use glycolysis rather than mitochondrial respiration, even in aerobic conditions. This phenomenon, known as Warburg effect, is considered a hallmark of cancer cells and a promising drug target. Metabolic rewiring is, indeed, used by tumor cells to escape immunosurveillance leading to apoptosis evasion and uncontrolled proliferation. Switching the bioenergetics of malignant cells from OXPHOS to glycolysis leads to an increased lactate production which is exported to the microenvironment to feed neighboring cells by the so called “reversed Warburg effects”. Targeting cancer cell metabolism is, therefore, a powerful strategy to both slow down glycolysis and shut the metabolic coupling with neighboring cells, leading to the reduction of cancer cell survival. Alterations of cancer cell metabolism can be achieved by the inhibition of glycolytic enzymes or by modulating mitochondrial respiration. A key player in cancer cell survival is the association between the first glycolytic enzyme Hexokinase and the mitochondrial outer membrane channel VDAC. Both proteins are upregulated in many tumors and their association gives to the cancer cell both a metabolic advantage and a protection against mitochondria- mediated apoptosis. In the past decades, research efforts have been put in the identification of mechanisms allowing to dissociate the VDAC-HK complex by means of cell-penetrating peptides or small molecules. However, there was no progression to advanced clinical trials due to toxicity or cross reactivity. Furthermore, the lack of structural information on the VDAC-HK association limits the development of targeted drug therapies. With this project, given the urgent need of finding drugs capable of disrupting the VDAC-HK complex in cancer cells, we aim at the identification of new molecules specifically targeting the VDAC-HK interface without hampering functions of the two partners.
Techniques
X-ray crystallography, cryo-electron microscopy, electrophysiology, protein expression and purification, cell biology assays.
Project Title: FIBROmeltIN: Taregeting fibronectin in myelofibrosis
Supervisor: Alessandro Malara
Laboratory: Immunology and General Pathology
Research Theme/Topic: Haematology, Oncology
Abstract
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by an increased number of atypical megakaryocytes, progressive fibrosis in bone marrow and splenomegaly. Bone marrow fibrosis is the consequence of excessive deposition of extracellular matrix by stromal cells in response to secreted pro-inflammatory and pro-fibrotic cytokines from proliferating megakaryocytes. In patients with PMF, high fibrosis grade is associated with worse prognosis. To date, therapeutic attempts of bone marrow fibrosis reversal have demonstrated only limited success. Fibronectin (FN) is a glycoprotein that mediates the assembly of other extracellular matrix proteins and regulation of growth factor activity to direct myofibroblast differentiation and neo-angiogenesis. FN homeostasis is significantly dysregulated in myelofibrosis and FN fibres represent a major component of the fibrotic scar. Thus, in this project we propose an integrated targeting strategy to reduce FN deposition and polymerization in vivo in mouse models of myelofibrosis. The primary outcomes of this project will be to assess whether preventing FN fibril assembly, ablation of its expression, and combination thereof will result in the reversal of the altered bone marrow microenvironment and fibrosis in mouse models of myelofibrosis. Successful completion of the proposed translational study will provide new targeting strategies to treat and/or manage myelofibrosis effectively.
Techniques
Bone Marrow isolation, Cell cultures, Immunofluorescence, Western blot, Flow Cytometry, Cell Sorting, ELISA, Immunohistochemistry, Animal Models, qPCR, Gene Silencing.
Project Title: Inflammation and Cancer: histologic and molecular studies of precancerous changes in coeliac and Crohn’s disease
Supervisor: Antonio di Sabatino
Laboratory: Laboratory of Gastroenterology, Department of Internal Medicine
Research Theme: Pathological Anatomy, Gastroenterology, Oncology, Immunology.
Abstract
Inflammation is a recognized driving factor in carcinogenesis in several sites, including the small bowel. Particularly, coeliac (CD) and Crohn’s disease (CrD), two common inflammatory disorders, have a significantly higher risk of developing small intestine adenocarcinoma (SBA) compared to the general population. Nevertheless, both in CD and CrD, very scarce knowledge is available in regard to either molecular mechanisms underlying mucosal changes, metaplastic or dysplastic, and, with particular reference to CrD, the histologic and immunophenotypic features of non-canonical epithelial lesions. Furthermore, to date, the microenvironment surrounding small bowel dysplastic and neoplastic growths in such cancer-predisposing inflammatory disorders has been poorly characterized, although its pivotal role as both negative and positive regulator of cancer development has been long established. The aims of this project are: i) to characterise precancerous lesions, histologically and molecularly, in CD and CrD and, thus, to define CD- and CrD-associated SBA’s natural history; ii) to study the interplay between the microenvironment and dysplasia/neoplasia in CD and CrD patients. These data will help better understand the molecular pathways leading inflamed and remodelled small bowel mucosa to adenocarcinoma, as well as the role of inflammation in both early and late carcinogenetic processes in CD and CrD.
Techniques
Molecular, histologic and translational, including immunohistochemistry, light microscopy, RT-PCR, NGS, flow cytometry.
Project Title: Neurogenesis and neuroimmunity in tinnitus
Supervisors: Roberto Pizzala, Paola Perin
Laboratory: Immunology and General Pathology
Research Theme: Neurogenesis, neuroinflammation, auditory disease,
Abstract
Tinnitus is an auditory disease characterized by the perception of phantom sounds. Although several risk factors are identified, no clear causative hypothesis for tinnitus exists yet, although chronicization appears to require a subcortical onset and a subsequent cortical rearrangement. Several studies have identified the dorsal cochlear nucleus as a necessary station for tinnitus onset after peripheral trauma, similarly to spinal cord dorsal horn in chronic pain. Animals exposed to tinnitus-inducing stimuli but remaining free from tinnitus display differences compared to animals developing tinnitus in the activating ratio between dorsal cochlear nucleus and cerebellar floccular node. Moreover, a similar switch between the two structures has been observed in neurogenesis, and neuroinflammatory stimuli are able to affect the onset and development of tinnitus. Our works focuses on the auditory system responses in a rat model of tinnitus. In particular, we are interested in understanding the role of the 4th ventricle choroid plexus in tinnitus-related plasticity and neurogenesis. In the lateral ventricle, choroid plexus is essential in regulating adult neurogenesis, but quite little is known about the 4th ventricle. We have developed a novel clarified brain-temporal bone preparation to investigate choroid plexus contacts with the auditory system (which appear to be mainly directed to the dorsal cochlear nucleus, where we have observed macrophages shared between the surface of the plexus and of the nucleus, especially after cochlear damage) and are starting to dissect the factors involved in dorsal cochlear nucleus cellular plasticity.
Perin P, Marino F, Varela-Nieto I, Szczepek AJ. Editorial: Neuroimmunology of the Inner Ear. Front Neurol. 2021 Feb 9;12:635359. doi: 10.3389/fneur.2021.635359. PMID: 33633679; PMCID: PMC7899967.
Voigt FF, Kirschenbaum D, Platonova E, et al. The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue. Nat Methods. 2019 Nov;16(11):1105-1108. doi: 10.1038/s41592-019-0554-0. Epub 2019 Sep 16. PMID: 31527839; PMCID: PMC6824906.
Perin P, Voigt FF, Bethge P, Helmchen F, Pizzala R. iDISCO+ for the Study of Neuroimmune Architecture of the Rat Auditory Brainstem. Front Neuroanat. 2019 Feb 13;13:15. doi: 10.3389/fnana.2019.00015. PMID: 30814937; PMCID: PMC6381022.
Project Title: Nanotechnology applications for innovative therapeutic approaches
Project Title: Inactivity-induced neuromuscularimpairment: from whole body phenomena to molecular determinants
Supervisor: Simone Porcelli
Laboratory: Human Physiology
Research Theme: Physiology, Neurology, Sport medicine
Abstract
Inactivity is a major risk factor of chronic diseases and death, as relevant as smoke, hypertension, and high cholesterol. A simple decrease in the number of daily steps has a dramatic impact on metabolic health parameters, leading to anabolic resistance, peripheral insulin resistance and dyslipidaemia. Concomitantly, inactivity induces a reduction in cardiorespiratory fitness, muscle mass and strength. Previous studies investigating the negative effects of inactivity on human body by the step reduction model disregarded adaptations at the level of nervous system, neuromuscular junction and skeletal muscle. Moreover, it is still unclear which is the minimum level of activity required for preventing neuromuscular and metabolic alterations. We will address such open issues using the step reduction model from 7,000-9,000 to 3,500 and 1,500 steps/day, in young and middle age healthy humans. Time-course of changes at neuromuscular and metabolic levels will be evaluated by measurements at 7 and 15 days. In-vivo evaluations will include: (i) cardiorespiratory and metabolic responses to exercise; (ii) muscle strength and power; (iii) neuromuscular control. We will also perform ex-vivo and in-vitro analyses on muscle biopsy samples to assess: (i) mitochondrial function; (ii) mitochondrial dynamics; (ii) NMJ integrity. The results of this project will extend current understanding of the interplay, time-course and molecular mechanisms of neural, muscular and metabolic alterations induced by a “real-world” approach of inactivity.
Techniques
-in vivo: superficial electromyography, nerve and muscle electrical stimulation, maximal oxygen consumption, VO2 kinetics, transthoracic bioimpedance to estimate cardiac output, near infra-red spectroscopy to evaluate fractional O2 extraction at skeletal muscle level, muscle strength, muscle power.
-ex vivo: mitochondrial function by high-resolution respirometry; in vitro: confocal microscopic analyses, Western blot and RT-PCR
Project Title: Unveiling the germline predisposition to myeloproliferative neoplasms
Supervisor: Elisa Rumi
Laboratory: Haematology, Fondazione IRCCS Policlinico San Matteo Pavia
Research Theme: Haematology
Abstract
There are several preliminary lines of evidence that support the role of germline genetic factors in the pathogenesis of myeloproliferative neoplasms (MPN): familial clustering of MPN, biclonal haematopoiesis in MPN and common polymorphisms predisposing to MPN. We hypothesize that MPN with germline predisposition tend to present at a younger age than their sporadic counterpart. Then screening younger subjects (<27 years-old) with MPN might help in identifying germline mutations. We aim to identify a germline predisposition to MPN through the application of a Next Generation Sequencing (NGS)-based panel test in young patients. We will interrogate our MPN database to identify all patients with age below 27 years (corresponding to the 5 percentile of our cohort) with available DNA. Germline screening panel includes 71 genes identified from literature as potentially involved in germline predisposition to myeloid neoplasms. Correlation between inherited predisposition, somatic driver and subclonal mutations, clinical phenotype and disease evolution will be performed. Combining data on somatic driver and subclonal mutations with germline variants could clarify the mechanism driving progression from inherited predisposition to overt malignancy. An in-depth understanding of the genetic pathogenesis of oncogenesis would allow to tailor treatment and follow-up.
Techniques
Experience in recruiting patients with MPN, experience in NGS, experience in translational research.
Project Title: Structural basis of antibody affinity maturation
Supervisor: Claudia Scotti
Laboratory: Protein engineering in physiology and disease
Research Theme: Immunology / Antibody structure and function
Abstract
Affinity maturation is a process that leads to the emergence of more efficient antibodies following initial antigen encounter and represents a key strategy of the adaptive immunity of vertebrate organisms. A model system to investigate this feature of the immune system is represented by the mouse response to the hapten 2-phenyl-5-oxazolone (phOx). Earlier and detailed sequence studies of the antibody response to phOx performed by the Nobel Prize Cesar Milstein defined three different classes of antibodies. Class I antibodies use the VHOx1/VκOx1 gene pair and dominate the early stages of the anti-phOx response, class II antibodies use the VκOx1 gene but a different VH segment and are common in the intermediate stages, and class III antibodies use the TEPC15/Vκ45.1 gene combination and play the greatest role in the late stages. Only the crystal structures of some anti-phOx antibodies are available so far and more are needed to have a full picture and to be able to translate this knowledge to “real-life” systems. The aim of the project is to determine the structure of at least 4 anti-phOx antibodies of the anti-phOx model system. The main structural techniques used will be X-ray crystallography, NMR and cryo-EM. Both natural and recombinant sources of the proteins will be used.
Techniques
Antibody fragments will be produced in recombinant form in bacterial, yeast and mammalian cell systems, purified, characterized from the biochemical point of view (Surface Plasmon Resonance) and crystallized for X-ray studies to be performed at European synchrotrons (ESRF, Diamond, EMBL). Alternative techniques will involve NMR or the study of antibody-antigen complexes by cryoEM. Site directed mutagenesis and structural modelling will also be applied to investigate specific features of the molecules involved in affinity maturation.
Project Title: Therapeutic approach against the novel molecular target SerpinB3 for hepatocellular carcinoma
Supervisor: Cristian Turato
Laboratory: Immunology and General Pathology
Research Theme: Cellular and Molecular Mechanisms of Liver Injury
Abstract
Hepatocellular carcinoma (HCC) represents a leading cause of cancer mortality worldwide. Growing evidence indicates that non-alcoholic fatty liver disease (NAFLD) is becoming a dominant cause of HCC, but mechanisms of NAFLD progression are largely unknown and biomarkers to predict individual at risk as well as validated therapeutic strategies are still lacking. The serine protease inhibitor SerpinB3 is mechanistically involved in experimental progressive NAFLD, acting as pro-inflammatory and pro-fibrogenic stimulus. SerpinB3 may lead to HCC through different methods, including inhibition of apoptosis and upregulation of pro-oncogenic pathways. Moreover, SerpinB3 is overexpressed in the subset of the most aggressive forms of HCC, characterized by early tumour recurrence after surgical resection. Aim of this PhD project is to investigate the biological role and prognostic significance of SerpinB3 in HCC, NAFLD progression towards HCC and preclinical therapeutic strategies. This will be pursued through the following tasks: 1) A comprehensive molecular characterization in terms of tumorigenic potential and drug-response, with simultaneous profiling of the transcriptome and epigenome, will be performed by using human cell lines and HCC human organoids. 2) Validation of the new preclinical therapeutic strategies to interfere with potential pro-carcinogenic role of SerpinB3; 3) An investigation of the in vivo tumorigenic potential of organoids by subcutaneous serial transplantation in xenograft mouse models; 4) Disclosure of the biological role (mechanisms and signalling pathways involved) of SerpinB3 in the progression of experimental NAFLD/NASH towards HCC development.
Techniques
Liver cell isolation, human and mouse primary liver cancer-derived organoid cultures, disease modeling and drug screening, transcriptome and epigenome analysis, gene silencing, microscopy analysis.
Project Title: Genetic and functional characterization of Joubert syndrome
Supervisor: Enza Maria Valente
Laboratory: Human Genetics
Research Theme: Genetics of rare diseases
Abstract
Joubert syndrome (JS) is a clinically heterogeneous condition characterized by a unique mid- hindbrain malformation, so called “molar tooth sign”. To date, over 40 JS-causative genes have been identified, all encoding proteins of the primary cilium. Despite the high number of known genes, a genetic diagnosis is reached only in 60-70% JS patients. While biallelic loss of function (LOF) variants usually lead to a severe phenotype, the presence of at least one non-LOF variant results in a widely variable phenotypic spectrum, indicating a possible different impact on the protein function. Among negative cases, 45% carried a heterozygous variant in a JS gene.
Aim of this PhD project is to improve diagnostic yield and gene- phenotype correlates in JS. This will be pursued through the following tasks: (a) perform Whole Genome Sequencing in 29 heterozygous patients to possibly identify “cryptic” variants (CNV, non-coding variants, structural variants) or hypomorphic variants previously filtered out; (b) perform functional studies in vitro to evaluate the effect of novel variants; (c) characterize the impact of 20 missense variants on primary cilium structure or protein interaction or localization, by overexpressing the mutant protein in HTERT-RPE1 cells previously knocked-out using CRISPR-Cas9 technology.
Techniques
MINI/MIDI gene assay, cDNA fragments analysis and qtRT-PCR, firefly/renilla luciferase reporter, 4C/Hi-C experiments, cell lines and bacteria maintenance, CRISPR-Cas9 assay, immunofluorescence and co- immunoprecipitation.
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Project Title: Genetic and functional studies on Parkinson disease
Supervisor: Enza Maria Valente
Laboratory: Human Genetics
Research theme: Neurogenetics, neurodegeneration
Abstract
Parkinson’s Disease (PD) is a common neurodegenerative motor disorder. Its etiology is complex and multifactorial, with approximately 10% of patients showing a genetic predisposition. Several genes have been implicated in monogenic familial forms of PD and a growing number of studies is highlighting non-Mendelian genetic factors contributing to the disease. The first aim of this project is the screening of selected patients and families via classical and innovative sequencing techniques (Sanger, MLPA, NGS).
A definitive causal link between gene mutations and the main pathological feature of PD – the degeneration of dopaminergic neurons in the substantia nigra pars compacta – is so far elusive, but strong evidence points towards misfolded protein accumulation, defects in autophagy- lysosomal pathway, and mitochondrial dysfunction as suspects. The second aim of the project is the functional characterization of pathways dysregulated in PD with state-of-the-art cellular models, including iPSC-derived dopaminergic neurons. A particular attention will be devoted to proteins responsible for monogenic forms of PD (such as PINK1, Parkin) and to relevant risk factors (GBA), with the goal to analyze genotype-phenotype correlation at the cellular level, potentially identifying novel targets for pharmacological treatment.
Techniques
PCR, Sanger sequencing, MLPA, next-generation sequencing, cell culture, immunofluorescence & other microscopy techniques, Western Blot
Project Title: Nanotechnology applications for innovative therapeutic approaches
Supervisor: Livia Visai
Laboratory: Cells and biomaterials interactions
Research Theme: Biochemistry, chemistry, molecular biology, biology, microbiology, stem cell differentiation, histology, bioengineering and nanotechnology.
Abstract
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 the applications of nanotechnology for innovative therapeutic approaches in 4 different topics: Osteoporosis treatment in simulated microgravity and on International Space Station (ISS). In this research line we are interested in the development of preventive counter-measures for osteoporosis treatment in simulated microgravity and on ISS by using nanotechnology, bio- chemical, biomolecular and biological techniques. Biological characterization of nano-scaffolds for regenerative medicine. This study aims to investigate by biochemical, molecular and biological methods the interaction of human mesenchymal stem cells (hMSCs) with nano/microelectrospun fibers and their differentiation to osteoblasts in different experimental conditions (exposure to physical, mechanical or chemical stimuli). Biological characterization of 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 types of nanosystems have been developed and characterized in in vitro cell model using Trastuzumab/Herceptin. Biological characterization of nanoparticles/nano-antibodies for treatment of bacterial/viral infections. In this study, different nanotechnological approaches including phage display, biochemical and biomolecular techniques will be performed to reduce bacterial adhesion and biofilm formation in order to improve biofilm dispersal. So, if you want to train in translational medicine and its different tools in one of the oldest and most prestigious Italian Universities apply for the PhD course in Translational Medicine at the Department of Molecular Medicine #UniPV! Website: http://molecularmedicine.unipv.it/phd-programme/. (Graphycal abstract created with BioRender.com).
