•    Responsabile Scientifico: Gandini Ilaria
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 20227RLEL8_002
•    CUP: J53D23011970006
•    Finaziamento MUR UniTS: 46.912,00 € 

 Abstract: Chimeric Antigen Receptor T-cells (CAR-T) therapy is an innovative therapeutic approach that have been recently approved for adult relapsed and/or refractory (R/R) Diffuse Large B Cell Lymphoma (DLBCL) and for acute B lymphoblastic leukemia (B-ALL) of the children and the young adults below 25 years on the basis of the long term survival advantage in comparison with conventional chemotherapy. For example, the rate of complete responders among R/R DLBCL was only 7% in the SCHOLAR-1 chemotherapy study compared to 30-40% in JULIET and ZUMA-1 CAR-T trials. Similar unprecedented clinical results had been achieved in other lymphoid setting, such as in Follicular and Mantle cell Lymphomas and Multiple Myeloma, pointing toward new approvations and timing of administration. However, CAR-T cells are associated with short and late severe adverse effects. Among the late-onset adverse effects, prolonged cytopenias are life-threatening complications that could impact patient survival not only because of the occurrence of infections but also because of the potential risk of myelodisplasia and subsequent secondary Myeloid Neoplasia (t-MN), of whom a few cases have been already reported in the clinical studies. Cytopenia risk factors include burden of previous treatments, age, residual poor bone marrow cellularity, severity of cytokine release syndrome and extent of the hematologic disease at infusion. Moreover, clonal hematopoiesis of indeterminate potential (CHIP), already known to be related with an increased risk of leukemic transformation and with an impaired peripheral blood stem cell (PBSC) collection in autologous stem cell transplant setting, was detected in the peripheral blood (PB) of a few patients before CAR-T administration and it seems to improve the efficacy of CAR-T therapy. The mechanisms by which CHIP influence CAR-T efficacy remain to be elucidated, because CHIP was never evaluated after CAR-T infusion nor in fractionated blood populations. In this project, we will study about 20 patients who will be treated with CAR-T cell therapy (tisa-cel and axi-cel) for the already approved indications in the FVG Network for Cellular Therapies (including Udine, Trieste and Aviano), before and after CAR-T administration. PB, bone marrow and lymphocytoapheresis products will be studied by Next Generation Sequencing, as targeted panel sequencing, by flow cytometry and by Wilms Tumor-1 (WT-1) expression as marker of leukemic transformation. We will analyse the mutational landscape of bone marrow hematopoiesis and separated T lymphocytes of patients who underwent CAR-T cell therapy before and after CAR-T administration. Our objective is searching any sign of myelodisplasia related abnormalities, thereby providing further insight into the mechanism of post CAR-T cytopenias, efficacy and risk of t-MN development.
 

•    Responsabile Scientifico: Sorrentino Giovanni
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022PWKZXE_001
•    CUP: J53D23012830001
•    Finaziamento MUR UniTS: 84.171€ 
 

 Abstract: Preliminary data generated in our laboratory demonstrate that non-alcoholic steatohepatitis (NASH) is associated with marked distortion of hepatocyte nuclei in both mice and humans. This distortion is caused by a mechanical stress generated by intracellular lipid vacuoles, a feature of hepatocytes with steatosis. This phenotype could be exacerbated in the context of mutations in epigenetic regulators capable of controlling the "stiffness" of the nucleus. The role of chronic mechanical stress in the progression of NASH is currently unknown. In order to study this phenomenon in complex systems in vitro, our laboratory has recently generated a model of macro-vesicular steatosis, using hepatic organoids, able to recapitulate both the presence of an intracellular lipid vacuole and the nuclear distortion associated with it. This model will allow us to study the role of aberrant nuclear mechanics in disease progression. In particular, our project is focused on dissecting the epigenetic alterations caused by nuclear deformation that may be at the basis of the mechanisms of hepatic inflammation.

•    Responsabile Scientifico: Mardirossian Mario
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022EKWRHB_001
•    CUP: J53D23001060001
•    Finaziamento MUR UniTS: 102.061€ 

Abstract: According to the World Health Organization, the antibiotic-resistant pathogens represent a medical emergency that will become in the next future a worrisome threat for human health. Even common infections may become incurable, with serious social but also economical. The need for new antimicrobial compounds is therefore urgent. In this scenario, the proline-rich antimicrobial peptides (PrAMPs) are in the spotlight for the development novel antibiotics. PrAMPs combine remarkable antimicrobial efficacy with good biocompatibility and are effective toward some concerning pathogens like multi-drug resistant Enterobacteriaceae. The selectivity of some PrAMPs for prokaryotic cells explains their tolerability by human cells. PrAMPs kill bacteria entering specifically their cytosol and inhibiting their protein synthesis after biding the prokaryotic ribosomes. PrAMPs therefore share the target with other common antibiotics blocking the protein synthesis, but overcome resistance mechanisms developed by bacteria against them.
Aim of this project is to optimize and exploit the antimicrobial potential of mammalian PrAMPs to develop new compounds for the treatment of multidrug-resistant Enterobacteriaceae.
The antimicrobial potential and mode of action of rationally modified mammalian PrAMPs will be described, starting from the effect on bacterial and human cells and descending to the atomic details of their interaction with the prokaryotic ribosome. Collected data will be combined to get hints for i) driving the synthesis of new PrAMPs to promote antimicrobial activity and subsequently ii) moving on from peptides to peptidomimetics, to develop compounds more robust compared to peptides. 
The biologic efficacy of the compounds will be tested in the clinical context of Enterobacteriaceae infections, i.e. blood stream infections and urinary tract infections by Escherichia coli and Klebsiella pneumoniae, to address a relevant clinical problem that will become dramatic in the future.

•    Responsabile Scientifico: Roman-Pognuz Erik
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022HASBMP_003
•    CUP: J53D23012160006
•    Finaziamento MUR UniTS: 13.040€ 

Abstract: Background. Early, successful restoration of myocardial perfusion after a ST-segment elevation myocardial infarction (STEMI) is the most effective way to reduce final infarct size and improve clinical outcome. However, experimental and clinical data have shown that reperfusion per se may have harmful effects, the “myocardial reperfusion injury” is identified as a target for cardioprotection. Molecular hydrogen (H2) is the most lightweight gas and abundant chemical element in the universe. Recent evidence has shown that H2 is a potent antioxidant, antiapoptotic and anti-inflammatory agent and so may have potential medical applications in cells, tissues and organs. The antioxidant advantages of H2 gas include: a high bio-membrane penetration and intracellular diffusion capability which enable it to reach subcellular compartments like mitochondria; selectively scavenging the deleterious hydroxyl radical while preserving other important reactive oxygen and nitrogen species for normal signaling regulation. The efficacy of molecular H2 for prevention and treatment of various diseases, with underlying pathological conditions of ischemia-reperfusion injury, has been reported by numerous non-clinical and clinical studies.
Preliminary data. H2 gas inhalation reduced infarct size and mitigated adverse left ventricular (LV) remodeling in a rat model. A recent phase I clinical study in Japan has shown that H2 inhalation during primary percutaneous coronary interventions (pPCI) is feasible and safe and may also promote LV reverse remodeling.
Aims. To evaluate the efficacy of a H2 inhalation on top of standard care when compared to standard care alone, on LV function and remodeling in reperfused STEMI.
Primary outcome. The primary efficacy endpoint is the global LV function assessed as ejection fraction (EF) changes from pPCI (day 1) to 6 months later. LV will be evaluated by echocardiography with imaging data independently analyzed in a central core echo lab.
Secondary outcomes. Concentration of cardiac troponins, CK-MB/CK, and natriuretic peptides after pPCI; incidence of ≥70% resolution of ST-segment elevation 1hr after pPCI; early LVEF change at day 4; LV end-diastolic volume at 3 months; MI size by cardiac magnetic resonance at 4 days after pPCI and at 6 months; rehospitalization for cardiovascular reasons; cardiovascular death, heart failure, and cardiogenic shock at 6 months.
Study design. Randomized, controlled, phase II trial. The study population comprises patients >18 years with a first STEMI. After informed consent, patients are randomized 1:1 to 1.3% H2 in 26% O2 or 26% O2 only. H2 inhalation will be achieved through a face mask upon arrival at the emergency room and continued during pPCI and for the first 2 hr after reperfusion. Considering a 15% LVEF improvement at 6 months after pPCI in the H2 group compared to standard of care, we plan to enroll a total of 120 STEMI patients (60 patients per group; 80% power, 5% 2-sided α).

•    Responsabile Scientifico: Pelin Marco
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022KZLJZH_002
•    CUP: J53D23006620006
•    Finaziamento MUR UniTS: 65.934€ 

Abstract: In the Mediterranean basin, harmful microalgal species and their toxins are detected in seawater and seafood with increasing frequency. The interest of scientific communities and the concern of Authorities (European Food Safety Authority, EFSA) are constantly rising due to detection of phycotoxins once found only in areas outside the European borders and whose regulatory limits have not been established, yet. These Emerging Marine Biotoxins (EMBs), occurring mainly in tropical areas, in recent years appeared in temperate latitudes, favored by global warming, causing great concerns for human and environmental health, tourism and fishing industry. Among them, palytoxins and ovatoxins are of particular concern for the Mediterranean regions while ciguatoxins, tetrodotoxin, azaspiracids, cyclic imines and cyanotoxins (freshwater phycotoxins that have been recently recorded also in the marine environment) are not monitored on a regular basis. Given the high toxic potential of these compounds, the associated hazard characterization and risk management need to be assessed. Despite the highly significant impacts related to these phycotoxins, several issues have not been elucidated so far: (i) the distribution of EMBs and of the producing microalgae along the Italian coasts; (ii) the ecological constraints affecting the harmful algal blooms; (iii) assessment of the distribution of EMBs in Mediterranean seafood chain; and (iv) the hazard characterization of some of these phycotoxins, including their mechanisms of toxicity. This hampers the establishment of regulations and makes difficult even to assess the actual risks posed by EMBs to humans, particularly to seafood consumers.
This project aims at elucidating the risks associated with the presence of emerging toxins in Italian seas through a multidisciplinary approach, including: (i) the identification of toxic microalgal species in coastal environment and seafood; (ii) the study of environmental factors affecting their growth, abundances and toxin production; (iii) their transfer to organisms at higher trophic levels, especially those used for human consumption; (iv) the implementation of chemical methods of analysis enabling the elucidation of the complex toxin profiles and their suitability to quantify the phycotoxins; and (v) toxicological studies to identify and characterize the hazard posed by EMBs to human health.
This project has the double purpose to assess the actual risk that ovatoxins, ciguatoxins, tetrodotoxins, azaspiracids, cyclic imines and cyanotoxins present for seafood consumers and to tackle the most urgent issues that currently hamper establishing regulation for the palytoxin-group of toxins - herein including ovatoxins, the major emerging toxins in the Mediterranean area.

•    Responsabile Scientifico: Marchesi Giulio
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 20222YTC5A_001
•    CUP: J53D23006130006
•    Finaziamento MUR UniTS: 63.364 € 

Abstract: Molar incisor hypomineralization (MIH) is a phenomenon defined as a quality defect of enamel of systemic origin of one to four permanent first molars (PFMs), frequently associated with morphological changes of incisors. Children with MIH-affected teeth face a number of difficulties in their life such as hypersensitivity and pain even during brushing resulting in poor oral hygiene, rapid caries progression and consequent chewing difficulties. The low esthetics of the anterior teeth affected by MIH has a negative impact on child’s self-esteem, often preventing them from smiling. and, subsequently, leading to psychological problems. It has been universally proven that the physical and psychological problems consequent to this pathology result in an important social discomfort and in high therapeutic costs that affect in a not indifferent way the families and the national health service.
In the last decade, the incidence of patients with MIH-affected teeth increased exponentially, thus making this disease a worldwide burden. Among the different causes that have been hypothesized at the root of this disease, the most advanced theories include genetic and environmental influences (factors related to air pollution, water or food contamination). A possible correlation with other pathologies has been also proposed, suggesting the potential use of MIH diagnosis as meaningful tool for detecting such diseases (allergies, topical dermatitis etc.). Despite the increasing attention paid to this pathology at medical level, to date its etiopathogenesis remains unknown, recalling the need to perform studies that can fully explore the causes triggering MIH to be able, consequently, to implement appropriate therapy and standardized protocols. Understanding the distribution and prevalence of the disease in different territories is of fundamental importance to outline its trend. A literature search revealed that hardly any attention has been given to exploring the prevalence, regional distribution, and clinical characteristics of MIH-affected children in Italy. Consequently, this project will aim to provide useful and distinctive information on the epidemiological, clinical, genetic, and structural characteristics of MIH to furnish a clear background on MIH disease. As to do so, a complete perspective on regional trend and perspective of MIH in Italy through an epidemiological survey will be conducted considering all the pre-, peri-, and post-natal influences. The clinical characteristics of the MIH-affected teeth and their impact on the quality of child’s life will be assessed. Concurrently, the genetic evaluation of MIH-affected patient will be conducted and correlated with other disease of allergic or infective origins. Lastly, this study would report, for the first time, the expression of matrix metalloproteinases (MMPs) in saliva and dentin in children diagnosed with MIH, and it will also investigate novel therapeutic approaches in treating MIH-affected teeth.

•    Responsabile Scientifico: Stocco Gabriele
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 20228T28YC_001
•    CUP: J53D23006170006
•    Finaziamento MUR UniTS: 76.719 € 

Abstract: A curative pharmacological therapy for inflammatory bowel disease (IBD) does not exist and the therapeutic approach is mainly aimed at the treatment and control of inflammation. IBD is particularly severe in pediatric patients and therapy is more aggressive than in adults. While the cause of IBD is still unknown, accumulating evidence suggests that the intestinal epithelium plays an important role in its pathogenesis. Substances able to promote epithelial regeneration may have a therapeutic role, as recently demonstrated for IL22.
In this context, the development of a new category of drugs that are targeted to epithelial cell barrier functions and promote tissue repair may become one of the therapeutic choices to achieve mucosal healing in patients with IBD. Thanks to the recent development of intestinal organoids, it is now possible to culture the intestinal tissue of the individual patient, which reproduces the genetic and epigenetic features and the main functions of the organ of origin.
The aim of this study is to identify innovative drugs for pediatric IBD by applying intestinal organoid-based models.
Therefore, a focused drug repurposing screen will be performed using patient-derived intestinal organoids, evaluating the capability of the compounds to promote epithelial proliferation. The activity of selected compounds will be then validated by measuring the expression of candidate genes, involved in the epithelial injury and immune regulation, and the production of candidate inflammatory cytokines on pediatric IBD organoid lines.
From another perspective, preliminary data showed that the cytokine CD70, the ligand for CD27, is critical to sustain T cell-mediated intestinal inflammation by NF-kB activation and its expression is higher in IBD compared to non-IBD derived organoids, suggesting a potential role of intestinal epithelial cells in the regulation of immune response. Consequently, another significant outcome of this project will be the identification of selective binders for CD70, a new candidate therapeutic target for IBD, by a structure-based virtual screening. In this part of the project, the objective is to design, synthesize, and characterize a series of compounds capable of inhibiting the protein-protein interaction (PPI) between CD70 and CD27. In particular, the CD27 portion in complex with CD70 will be used as a template to guide the computational design of PPI inhibitors, relying on the CD70/CD27 X-ray crystal structure, which has been recently deposited in the Protein Data Bank. Such compounds will be then tested for their ability to block CD70/CD27 costimulatory pathway by coculturing IBD patient-derived intestinal organoids with CD27/NF-kB reporter-Jurkat recombinant cell line. The expected results could lead to identify intestinal epithelial cells-based therapies which may have regenerative or protective effects on the damaged epithelium, and thus promote mucosal healing.

•    Responsabile Scientifico: Manfioletti Guidalberto
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022MWE5JY_002
•    CUP: J53D23009090006
•    Finaziamento MUR UniTS: 72.850 € 

Abstract: The aim of the present study is to evaluate the efficacy of two new compounds, selected by in silico screening as acting on cell migration, in reducing glioma invasion in clinically relevant in vitro and in vivo models.

•    Responsabile Scientifico: Gardossi Lucia
•    Dipartimento: Scienze Chimiche e Farmaceutiche
•    Codice Progetto: 2022KL2ECA_002
•    CUP: J53D23010100006
•    Finaziamento MUR UniTS: 77.192 € 

Abstract: The BRAIN project aims at the design and implementation of a versatile platform of rationally designed materials applicable to the IN delivery of bioactive molecules.  The materials will be biosynthetized and functionalyzed thanks to microbial, biocatalytic and chemical methodologies already developed and consolidated by UNINA and UNITS within the frame of the CARDIGAN project (MIUR-PRIN 2017). Finally, the experimental validation of the IN-delivery platform will provide the frame for the exploration of the repurposing of the bioactive compounds towards still unexplored therapeutic fields.

•    Responsabile Scientifico: Del Sal Giannino
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022XBYNJP_001
•    CUP: J53D23009140006
•    Finaziamento MUR UniTS: 113.992 € 

Abstract: Biomechanical alterations are characteristic of most tumors and elicit cell responses contributing to tumor progression. Mechanical signals are transduced from the extracellular matrix to chromatin by connection of the cytoskeleton with Lamin nucleoskeleton across nuclear membrane. Nuclear mechano-transduction allows adaptation of nuclear envelope (NE) structure, chromatin organization, epigenetic modifications, and gene expression to mechanical cues. These processes are crucial in cancer cells, which experience mechanical challenges in all phases of tumor progression, including growth, invasion and metastasis. However, it is unclear how mechanotransduction is spatiotemporally regulated at the NE to control chromatin state at lamina-associated domains (LADs), and how these impacts on gene expression and genome integrity, unleashing aggressive tumor phenotypes. A key role in regulation of NE structure and function is played by Lamins, whose function is modulated by multiple phosphorylations. Our work indicates that in mechanically challenged normal and cancer cells, a crucial role in regulating NE structure, heterochromatin and DNA integrity is played by Pin1, a unique enzyme that isomerizes phospho-S/T-P sites in many cellular proteins, fine-tuning cell responses to multiple stimuli in a phosphorylation-dependent fashion. In tumors and metastases Pin1 upregulation is prevalent and amplifies tumorigenic pathways controlling proliferation and stem cell fate, while its depletion/inhibition curbs tumor growth and metastasis, sensitizing breast cancer (BC) cells to targeted- and chemo-therapies.
In this proposal, we plan to pursue our preliminary evidence that in BC cells Pin1 reacts to mechanical stress, contributing to preserve NE structure and heterochromatin. Based on this, we aim to investigate the interplay between NE, chromatin remodeling and tumorigenic gene expression in response to mechanical cues in BC, focusing on the role of Pin1 at LADs, and to assess the relevance of these mechanisms in tumor models and clinical samples. We expect that pharmacologic Pin1 inhibition may dampen pro-tumorigenic mechanosignaling and weaken the NE leading to heterochromatin relaxation, transposable elements activation and DNA damage, sensitizing tumor cells to anticancer therapy. To achieve our aims, we assembled an interdisciplinary team of investigators that already contributed, from different perspectives to relevant discoveries in this field. We generated cellular and animal BC models to study Pin1 function and will combine innovative omics methods to achieve high-resolution maps of chromatin organization with techniques to analyze the impact of mechanical cues on cultured cells.
We expect to clarify fundamental mechanisms of cell response to mechanical stimuli, linked to heterochromatin organization, whose deregulation contributes to cancer traits, and to identify factors that may be exploited to increase the efficacy of anticancer therapies.

•    Responsabile Scientifico: Cescutti Paola
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 20224T3X8K_002
•    CUP: J53D23001010006
•    Finaziamento MUR UniTS: 70.232 € 

Abstract: Antibiotic resistance has reached very high and worrisome levels, rendering antibiotic treatment of multi-drug resistant bacteria often inefficient. To tackle bacterial infections, a possible alternative to traditional antibiotics is the use of bacteriophages components. The aim of the present project is to prepare and test novel antimicrobials constituted of bacteriophages enzymes and antimicrobials which will be delivered by liposomes.

•    Responsabile Scientifico: Terlizzi Antonio
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022R5JJFC_001
•    CUP: J53D23006700006
•    Finaziamento MUR UniTS: 105.539 € 

Abstract: Marine invasive species are having huge environmental and economic impacts along Italian coasts (1). Some of them contain bioactive metabolites whose effects on the native fauna and flora are the subject of a growing literature (2–7). Among invasive benthic invertebrates, the calcareous sponge Paraleucilla magna commonly occurs as invasive fouler in Italian mussel farms and marinas. The sponge markedly selects mussel shells as its substrate, requiring constant efforts by aquaculture farmers to control its growth (8). The sponge material resulting from the cleaning of mussel rows is generally released at sea and accumulates on the seabed, with harmful effects on the environment. This huge waste material, however, has the potential to be exploited to obtain high added-value products of interest for industrial applications, within a knowledge-based strategy for biomass valorization. Actually, recent studies indicate that the crude ethanolic extract of P. magna could represent an important source of antifouling metabolites (9). This evidence is consistent with the evolution of chemical defense strategies in sessile invertebrates, which protect them from biofoulers. It is a natural solution to control fouling that could be effectively mimicked for engineering applications, towards the development of novel environmental-friendly antifouling paints (10). In this view, the harvesting and industrial exploitation of P. magna to obtain its antifouling components could both reduce its environmental impact in the Mediterranean, and bring economic benefits to local aquaculture farmers and fishermen. However, the active components of the crude extract of P. magna have not been identified yet, since the chemical composition of the sponge is still unknown. This lack of information prevents the selection of the active ingredients most suited for the development of novel antifouling products. A first thrust of the present research proposal is thus to fill the knowledge gap by analyzing the chemical composition of P. magna living along Italian coasts. This preparatory study phase will be conducted at the laboratories of the Institute of Biomolecular Chemistry (ICB) of the Italian National Research Council (CNR), with extensive experience in the study of marine natural products and their ecological role. Purified metabolites will be then tested for their inhibitory activity on the settlement of micro- and macrofoulers both at the Marine Biology Laboratory of the University of Trieste, and at the Stazione Zoologica “A. Dohrn” of Naples, taking advantage of the relevant expertise, infrastructure and instrumentation available at these two excellent institutions operating in the field of marine sciences. To select antifouling agents that could effectively substitute the extremely toxic ones that have been used for long time (11), the active components of P. magna will be also tested for their dose-dependent toxicity in vivo.

•    Responsabile Scientifico: Comar Manola
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022W9W4NL_002
•    CUP: J53D23001210006
•    Finaziamento MUR UniTS: 83.206 € 

Abstract: Candida is a commensal fungus that can colonizes the vaginal tract of healthy women. However, Candida can cause vulvovaginal candidiasis (VVC). Most of VVC are caused by Candida albicans. VVC is a very common pathology, affecting 70-75% of women of child-bearing age at least once in their lifetime. About 5-8% of these women will suffer from the chronic form (recurrent VVC). The symptoms include burning sensation, pain and reduced state of mental wellbeing. Nevertheless, the research funds dedicated to the study of this female-specific disease are incommensurate with their toll on public health. What induces C. albicans to switch from a harmless commensal to a virulent pathogen, is not yet clear. An epithelial tolerance threshold exists to Candida that varies between women. When this tolerance threshold is overcome, the host becomes susceptible to the fungus and triggers the symptoms of the disease. To date, it has not been possible to identify any disease-causing traits in Candida that correlate with human VVC. Our recent data suggest that C. albicans isolates from VVC can’t be distinguished from those obtained from colonized women based on clade, propensity to switch to hyphal morphology or candidalysin toxin allele. However, it has been possible to identify “vaginopathic” C. albicans strains based on their different ability to interact with vaginal epithelial cells in vitro. Indeed, VVC isolates grew more and were shed more from the epithelium than the colonizing isolates, in addition to a higher epithelial cell exfoliation. Our project will build on this striking difference and will determine the mechanistic basis of enhanced pathogenesis of strains associated to VVC. RNA-seq analysis comparing the responses of vaginal epithelial cells infected with either a selected VVC or colonizing strain has revealed many differentially regulated pathways, including type I interferon pathway, integrin-mediated signaling and ferroptosis.
Particularly, in this project we will focalize on studying the role of the type I interferon pathway. Indeed, our preliminary data suggest that VVC strains may be more pathogenic by dampening this signaling pathway. In our studies will use pathway-specific activation and inhibition systems by means of in vitro and in vivo models to define the role of this component in the mucosal response to Candida. We will collect a large-scale information from vaginal samples of VVC or healthy colonized women such as inflammatory parameters and composition of vaginal microbiome. This information will be cross analyzed with the data obtained from the in vitro and in vivo experiments in order to get a broader picture of the role of each player in the onset of the disease: Candida, immune response and microbiota. Our results will characterize new aspects of host response to define molecular mechanisms responsible for the VVC symptoms, potentially leading to more effective and long-lasting treatment for this poorly understood disease.

•    Responsabile Scientifico: Barbieri Pierluigi
•    Dipartimento: Scienze Chimiche e Farmaceutiche
•    Codice Progetto: 20229KTNRM_001
•    CUP: J53D23012060006
•    Finaziamento MUR UniTS: 88.105 € 

Abstract: Airborne transmission of respiratory diseases is a socially relevant interdisciplinary issue, as demonstrated in the COVID19 pandemic. The methods of studying the behavior of pathogens in the air are not standardized, and the relevance of the transmission of viral and bacterial infections through particles with dimensional modes of less than five micrometers (aerosol mode), as well as the role of aggregates between atmospheric particulate and carrier droplets viruses, are theorized but supported by limited experimental data bases. The role of airborne transmission is poorly defined: 1) the evolution of the primary particles of saliva carrying pathogens depends on the mode of emission (breathing, speech, singing, cough, sneezing), stage of the disease (asymptomatic, with symptoms initial, symptomatic, shedding tail), environmental conditions of evaporation, condensation, interaction with environmental particulate 2) absence of standardized methods for the determination of pathogen RNA and residual infectivity, in bioaerosol samples, in the face of a variety of instruments and procedures (filters, impactors, incorporation in liquid or gelatin, growth tube by condensation in moderate vapor flow, size selection, low, medium, high flow rates). 3) The variability of the microbiological composition in social scenarios relevant to the spread of infectious diseases (schools, public transport, shopping centers, hospital emergency rooms) is not defined. 4) The effectiveness of air sanitation technologies for relevant pathogens (eg SARS -CoV2 and variants) is not known. 5 WPs are defined.
WP1. Preparation of aerosol chamber consisting of instrumented glove boxes, which can be placed in the BSL3 laboratory to generate and mix abiotic aerosols and aerosols carrying microorganisms, and to study aggregation phenomena with optical particle counters, multistage impactors; assessment of the maintenance of infectivity in aerosol in the BSL3 laboratory. Testing of air treatment and infectivity mitigation technologies.
WP2. Comparative study of bioaerosol sampling / interaction between technologies (filter, impactor, impinger, MCGTs) and microorganisms, from an intense source of bioaerosol (aeration of wastewater treatment plant) with metagenomic detection.
WP3. Studies on the presence of antibiotic resistant bacteria in control points (e.g. wastewater treatment plant) and critical (e.g. health facilities) and on the transfer of antibiotic resistance genes between microorganisms
WP4. Application of technologies and optimized procedures to characterize the presence, distribution and variability of microorganisms in schools, public transport, shopping centers, emergency rooms, with metagenomic detection. Study of genetic indicators of infectivity in environmental samples.
WP5. production of optimized guidelines for risk mitigation and identification of criticalities for the transmission of pathogens by air

•    Responsabile Scientifico: Scocchi Marco
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022XFFTH5_003
•    CUP: J53D23001230006
•    Finaziamento MUR UniTS: 64.588 € 

Abstract: The bleakest outlook for a “post-antibiotic era” is one in which microbial infections can no longer be cured. The traditional antibiotic pipeline has been exhausted, while antimicrobial resistance has become a multifaceted crisis, imposing a serious threat to global health. There is thus an urgent and undeniable need for new antimicrobial treatments. Inspired by nature, antimicrobial peptides (AMPs) are rapidly gaining attention for their clinical translation, as they present distinct advantages compared to conventional antibiotics. AMPs have demonstrated a pivotal role in the innate immune system of living organisms; many of them are evolutionarily conserved with limited propensity to induce resistance. Research groups in our Consortium have identified short, naturally-occurring helical AMPs that are membrane-active (MA-AMPs) as well as several short proline-rich AMPs (PR-AMPs) that act intracellularly inhibiting protein synthesis.
The main goal of this project is to fully characterize and optimize existing MA-AMPs (Esc and Tmp peptides), PR-AMPs and a novel library of cyclic Tmp or their combination for the development of new anti-infective compounds to treat two types of relevant topic infections such as pulmonary and wound infections associated to the “priority pathogens” Pseudomonas aeruginosa and Staphylococcus aureus. Combined use of MA- and PR-AMPs with different modes of action is expected to increase activity and make the incidence of resistance even slower, while chemical modification to the peptide backbone can be used to improve biological activity and stability of the peptides and to reduce their potential cytotoxicity, if necessary.
We propose to pursue an experimental plan aimed at studying the potency of selected AMPs on a large panel of clinical isolates under conditions that better reflect physiology of the target districts as well as to assess their stability in biological fluids before testing their efficacy in a proper mouse model of infection. Furthermore, the project aims at exploring the effect of AMPs on the host cells that are present at the sites of infection in terms of metabolic and immunomodulatory activities which represent hitherto unexplored aspects. Another relevant objective of the project is the development of novel nanoparticulate systems tailoring the formulation for more efficient and quantitatively adequate AMP delivery to the target site, while improving AMPs stability to proteases and prolonging AMPs lifespan.
If successful our multidisciplinary approach will allow a deeper understanding of the effectiveness of AMPs-based therapeutic formulations and to identify the best candidates for the translation of novel drugs into real-life medicine to fight the current alarming antibiotic-resistant infectious diseases.

•    Responsabile Scientifico: Albert Umberto
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022C7AL7F_002
•    CUP: J53D23010910006
•    Finaziamento MUR UniTS: 28.177 € 

Abstract: Treatment-resistant depression (TRD) represents a serious morbid condition, with a prevalence of about 20-30% among subjects suffering from major depressive disorder. In recent years, several innovative pharmacological strategies, targeting new biological pathways other than those of traditional antidepressant drugs, have been proposed. The N-methyl-d-aspartate receptor (NMDAR) antagonist ketamine and its S(+) enantiomer esketamine have been identified among the most promising. In 2019, esketamine was approved in a nasal spray formulation as a supplemental drug for the management of TRD by both the US Food and Drug Administration and the European Medicines Agency. Several studies have proven the clinical efficacy and safety of intranasal esketamine. Yet, the mechanisms underlying the rapid decrease in depressive symptoms induced by esketamine and the illness-related biological differences between responders and non-responders remain unclear. Recent developments in metabolomics, testing a broad panel of biomarkers, could be useful to clarify the involved molecular pathways. Along with a more appropriate understanding of esketamine biological mechanisms, metabolomics may help to predict individual metabolic features associated with clinical response to esketamine, contributing to a personalised management of TRD. Nonetheless, there is a lack of studies exploring the predictive value of metabolomics. Thus, we designed the ReDREAM – Resistant Depression Response to Esketamine Assessing Metabolomics – research project, aimed at identifying metabolomics biosignatures that may represent novel, clinically meaningful, correlates of response to esketamine treatment. We plan to enrol 60 individuals with TRD from three different clinical sites. The subjects will be administered with esketamine nasal spray – as approved – twice a week for four weeks (‘induction phase’), then once a week for four additional weeks (‘maintenance phase’). We will test the correlations between baseline metabolomics and depressive symptoms improvement at the study endpoints (weeks 4 and 8), as measured by the Montgomery– Åsberg Depression Rating Scale (MADRS). In addition, we will complement our analytical approach exploring possible metabolome differences between responders (i.e., subjects with a MADRS score reduction >50% at week 4 and/or week 8) and non-responders. The profiling of clinical changes after esketamine treatment in TRD, potentially associated with unbiased data from untargeted metabolomics, will clarify specific biological pathways involved in treatment response. This research project may ultimately contribute to defining new paradigms for precision psychiatry-oriented, personalised care of TRD.

•    Responsabile Scientifico: Barazzoni Rocco
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 202229ET3S_002
•    CUP: J53D23011750006
•    Finaziamento MUR UniTS: 31.200 € 

Abstract: The decline of lean body mass and skeletal muscle functionality (namely, sarcopenia) is common and is exacerbated in individuals with obesity due to metabolic changes associated with a sedentary lifestyle, adipose tissue derangements, comorbidities (acute and chronic diseases), either extreme or repeated weight loss, and during the ageing process. Co-existence of excess adiposity and low muscle mass/function is referred to as sarcopenic obesity (SO), a condition increasingly recognized for its clinical and functional features that negatively influence important patient-centred outcomes. Effective prevention and treatment strategies for SO are urgently needed, but efforts are hampered by the lack of a universally established SO Definition and diagnostic criteria. Resulting inconsistencies in the literature also negatively affect the ability to define prevalence as well as clinical relevance of SO for negative health outcomes (1-5).
The European Society for Clinical Nutrition and Metabolism (ESPEN) and the European Association for the Study of Obesity (EASO) launched an initiative to reach expert consensus on a Definition and diagnostic criteria for SO (6). The jointly appointed international expert panel proposes that SO is defined as the co-existence of excess adiposity and low muscle mass/function. The diagnosis of SO should be considered in at-risk individuals who screen positive for a co-occurring elevated body mass index or waist circumference, and markers of low skeletal muscle mass and function (e.g. risk factors, clinical symptoms, or validated questionnaires). Diagnostic procedures should initially include assessment of skeletal muscle function, followed by assessment of body composition where presence of excess adiposity and low skeletal muscle mass or related body compartments confirm the diagnosis of SO. 
Individuals with SO should be further stratified into Stage I in the absence of clinical complications, or Stage II if cases are associated with complications linked to altered body composition or skeletal muscle dysfunction.
ESPEN and EASO represented by the expert panel advocate that the proposed SO Definition and diagnostic criteria be implemented in clinical practice and in interventional RCTs aimed in particular at exploring the impact of specific interventions on SO. In addition, they strongly encourage validation and prospective follow up studies as well as secondary analysis of existing cohorts with the aim to increase the scientific evidence needed to identify and treat SO patients.
Hence, the aim of the present study is, as suggested in the ESPEN-EASO consensus, to assess the validity of the entire diagnostic process, the need to include any other variables (e.g. calf circumference, visceral fat), the possibility to adopt a unified parameter (e.g. a body composition index) with both fat and skeletal muscle measurements related in a single criterion.
The implementation of the decision algorithm for the screening and diagnosis of SO proposed by ESPEN and EASO will contribute to "enhance the capacity for prevention" and "guarantee widespread access to care" as it is asked in the 6th mission of the PNRR (Piano Nazionale di Ripresa e Resilienza), by helping to the "creation of proximity networks and telemedicine structures" for use by local medicine based in particular on the "digitization of the SSN" (Servizio Sanitario Nazionale) with automatic processing of the collected data.

•    Responsabile Scientifico: Collavin Licio
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 202285XS52_001
•    CUP: J53D23006160006
•    Finaziamento MUR UniTS: 92.330 € 

Abstract: Cancer cells within tumor masses are chronically exposed to stress caused by nutrient deprivation, oxygen limitation, and high metabolic demand. They also accumulate hundreds of mutations, potentially generating aberrant proteins that cannot properly fold and cause proteotoxic stress. Finally, cancer cells are exposed to multiple damages during chemotherapy.
In a growing cancer, transformed cells eventually adapt to these conditions, eluding the death-inducing outcomes of multiple signaling pathways triggered by chronic stress. Indeed, activation of stress-related pathways may actually promote cancer progression, by inducing epithelial-mesenchymal transition (EMT), stimulating angiogenesis, and supporting tumor dormancy. Therefore, understanding the genetic determinants that enable adaptation of cancer cells to stress is critical to improve treatment efficacy.
Missense mutations in the TP53 gene are extremely frequent in human cancers and give rise to stable mutant p53 proteins (mutp53) that lose tumor suppressive activities. Various evidences suggest that cancer cells acquire selective advantages by retaining mutant forms of the protein; in fact, p53 mutant proteins can display powerful oncogenic activities, defined as Gain of Function (GOF), that promote tumor progression. Recent studies suggest that mutant p53 proteins play a peculiar role in supporting cancer cell homeostasis, by modulating inflammatory pathways, proteasome activity, folding of N-glycosylated proteins, and the response to ER stress.
Based on these premises, we propose to study the impact of cancer-related p53 mutations in the adaptation of cancer cells to stress; specifically focusing on the unfolded protein response (UPR) and cell death by ferroptosis. In fact, we have preliminary evidence that the response to ER stress and sensitivity to ferroptosis are genetically linked to each other, and may be related to crucial cancer phenotypes such as cell motility, invasiveness, and chemoresistance.
To this aim, we will analyze a panel of breast, prostate and pancreatic cancer cell lines with diverse missense p53 mutations, systematically testing their response to ER stress and ferroptotic stimuli to evaluate how expression of mutp53 – or specific p53 mutations – correlates with resistance or sensitivity. Next, we will investigate the specific role of mutp53 in this phenotype, by broad transcriptomic approaches and specific functional studies. Finally, we aim to define the mechanism by which mutp53 may affect the response to ER stress or ferroptosis by discovering molecular partners or target genes involved in the phenotype.
This study may provide important insights on the mechanisms that allow cancer cells to adapt and survive to chronic stress within a growing tumor, and in particular on the role played by specific TP53 mutations, hopefully providing novel potential targets for therapy and additional elements to guide the choice of available treatment options.

•    Responsabile Scientifico: Pauli Francesco
•    Dipartimento: DEAMS
•    Codice Progetto: 20227YCB5P_003
•    CUP: J53D23011990006
•    Finaziamento MUR UniTS: 46.155 € 

Abstract: BACKGROUND AND OBJECTIVES: Suboptimal diet has recently surpassed smoking as the leading risk factor for noncommunicable disease morbidity and mortality in the Global Burden of Disease Study. In particular, among the various risk factors for cancer, diet quality (e.g., Mediterranean diet) and quantity of food consumed have long been one of the main objectives of Public Health. Although diet may not inherently be as a strong risk factor as others, limitations in data collection and analysis paradigms contribute to a reduced ability to identify these effects accurately. Challenges include barriers to accurate and precise measurement of diet, appropriately modeling diet complexity, and residual confounding. 
The INDACO project is aimed at using novel statistical and machine learning (ML) approaches to tackle the previous challenges. The project will be divided in four main objectives:
1.Exploring nonlinearity and nonadditivity within the association between dietary patterns and cancer risk;
2.Evaluating the identified cancer risk prediction/classification models using newly and previously collected data from an Italian validation study and Swiss case-control studies, respectively;
3.Exploring the potential of machine learning approaches in support of novel image-based dietary assessment tools;
4. Validating results from machine-learning based image approach versus a newly collected pilot study of food images, food recipes, and nutrient intakes.
EXPECTED RESULTS AND IMPACT: With its team of competent experts, the INDACO project will define novel machine learning and statistical approaches to better leverage the full richness of diet in modeling health outcomes by incorporating more dietary variables and by accounting for nonlinear and nonadditive relations among them and with confounding factors. Furthermore, the INDACO project will validate ML approaches on an Italian dataset of food images and weights to provide opportunities in future development of technological dietary assessment tools. 
The INDACO project is expected to have a (long-term) impact on various levels: 1) on Italian and international research in nutritional epidemiology, statistics, and nutrition; 2) on the health of the Italian population by promoting effective public health messages, public awareness about diet and cancer risk factors, and the individual monitoring of these aspects; 3) on Italian economy by potentially reducing the preventable burden of a major chronic disease like cancer and providing background for future commercial exploitation of image-based dietary assessment tools.

•    Responsabile Scientifico: Sblattero Daniele
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 20228MFALR_001
•    CUP: J53D23012030006
•    Finaziamento MUR UniTS: 96.000 €

Abstract: The aim of the project is to establish an in vitro selection "pipeline" of high throughput and high-quality recombinant antibodies.
These new antibodies will be produced against selected targets that are of diagnostic, clinical, and research interest but still lack a reliable detection reagent today.
The new detection reagents will be validated in two well-defined model disease models, liver carcinoma and glioblastoma, which so far lack the availability of specific detection reagents to perform multiplexing analyzes.
The reagents will be used in combination thanks to the use of mass cytometry which will allow advancing the state of the art in the field of multiplex imaging for diagnosis and precision medicine.

•    Responsabile Scientifico: Giacca Mauro
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022Z5PEHM_001
•    CUP: J53D23003340006
•    Finaziamento MUR UniTS: 73.037€ 

Abstract: This is an ambitious project aimed to develop CRISPR/Cas9 technologies for the precise correction of the genetic defects leading to inherited cardiomyopathies, including hypertrophic cardiomyopathy (HCM), which affects 1:500 individuals in the general population, and dilated cardiomyopathy (DCM) which has a prevalence of 1:2500.  The advent of the CRISPR/Cas9 technology has opened the unprecedented possibility to develop a curative therapy for these conditions. However, precise gene editing in the heart remains challenging, as genetic correction must occur in a sufficiently large number of cardiomyocytes to be effective. In addition, cardiomyocytes are post mitotic cells and thus refractory to homology directed repair (HDR) and large animal models in which to test the technology are largely missing. This project will try and overcome these limitations.
The project is divided into 3 main AIMS, which are correlated and bidirectional. AIM1 will fuel the project with technological tools. Besides standard saCas9 and spCas9, we will generate better gene editors with smaller size, improved efficacy and lower off-target effects, from metagenomic analysis of bacterial species and through in vitro direct evolution. AIM1 will also provide the project with cardiotropic AAV9 vectors and with lipid nanoparticles (LNPs) for cardiac delivery. 
The novel editors and delivery tools from AIM1 will be used in AIM2 to attempt correction of a mouse model of HCM, carrying a knock-in mutation in the MYBPC3 gene. AIM2 will also try and overcome the problem that cardiomyocytes are refractory to HDR. In one strategy, we will leverage on a series of pro-recombinogenic miRNAs we have identified. Alternatively, we will exploit Prime Editing, which allows HDR-independent gene correction. 
One of the challenges of cardiac gene editing is the efficiency of cardiac delivery. AIM3 will identify a most effective route of cardiac administration in pigs. We will explore LNP and/or AAV vector administration using catheterization of either the coronary arteries or the coronary sinus, which will be compared with intramyocardial injection. All these procedures are amenable to clinical translation. Finally, AIM3 will attempt gene editing in a large animal model of DCM, based on a knock-in mutation in the titin gene we developed. 
This is a highly integrated project among three groups with complementary expertise and converging interests, and with an extensive record of past interactions. There is a tremendous medical interest in developing therapies to prevent heart failure in inherited cardiac conditions, with relevant social and economic impact for patients and the national health system. This project has also important potential to generate IP and to attract interest from venture or pharma. The proponents have significant experience in dealing with the private sector to ensure continuity towards clinical application.

•    Responsabile Scientifico: Meroni Germana
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022J2WJC3_002
•    CUP: J53D23009020006
•    Finaziamento MUR UniTS: 46.000 € 

Abstract: A hallmark of eukaryotic cells is the evolution of subcellular compartments to ensure that specific biochemical reactions and cellular functions occur in a spatially restricted manner. Historically, only membrane-bound organelles, specified by their unique composition of phospholipids, were thought to confine a specialized array of molecules to different subcellular compartments. Recent studies have revolutionized this view: biomolecular condensation driven by liquid-liquid phase separation has emerged as an essential strategy to confine diverse cellular functions. With the rapid identification of different membraneless compartments comes a similar explosion of distinct domains between the Endoplasmic Reticulum (ER) and other organelles, known as membrane contact sites, which work as signaling hubs. Intriguingly, these two types of structures interact intimately and their coordinated actions ensure spatiotemporal control of various cellular functions. The dynamic nature of these compartments requires rapid and flexible adaptation. Ubiquitylation is an exciting and yet uncharacterized candidate for such regulation, and our published and unpublished data sustain this hypothesis. In this proposal we intend to elucidate how ubiquitylation of Ribonucleoproteins (RNPs) and ER contact sites governs neuronal homeostasis. We will focus on selective ubiquitin E3 ligases: HECW1, CRL3KLHL12 and TRIM18 that are the prototypic members of the main classes of E3s and we will investigate their ability to modify and modulate integral components of the two compartments.
The project is structured in work packages:
WP1 (Ubiquitin modulates RNPs) tackles the biochemical and biophysical characterization of the E3 enzymes and their substrates (FMRP, alpha4/PP2A) that are involved in RNP regulation.
WP2 (Ubiquitin modulates ER contact sites) aims at characterizing two key ubiquitylated ER-resident proteins (Lunapark and RTN3) by dissecting the mechanism and the function of their ubiquitylation. WP3 (Ubiquitin modulates local protein synthesis and autophagy in neurons) exploits the knowledge acquired in WP1 and WP2 to analyse the role of these ubiquitin networks in controlling local translation and degradation in neurons. We will focus on neurons as these highly polarized cells have an additional demand for rapid responses and thus, ready-to-use machineries and they are highly susceptible to alterations of the ubiquitylation process. By addressing these issues, we expect to provide advancement in: i) the role of ubiquitylation in dynamic cellular compartmentalization and ii) the role of ubiquitylation in neuronal peripheral protein synthesis.
Understanding the functional relationships between the ubiquitin machinery, the RNPs and ER contact sites, and probing the results obtained in neurons will allow us to contextualize their function in a physiologically meaningful manner and apply those principles to disease intervention.

•    Responsabile Scientifico: Muggia Lucia
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022Z7PXKW_001
•    CUP: J53D23010680006
•    Finaziamento MUR UniTS: 88.575 € 

Abstract: In the last decades, graphene has represented a groundbreaking nanomaterial which has been employed in many fields of innovative technologies. Although graphene-based materials (GBMs) bring several advantages, they will eventually degrade, break and be disposed of, potentially leading to an inadvertent and undesirable accumulation of graphene into the environment. Concerns about the presence of these new substances have arisen the need to study whether graphene potentially affect living organisms and human health. Despite this important issue and the general aim of predicting environmental the fate of graphene in terrestrial ecosystems, so far only few studies have tested whether natural degraders, such as saprotrophic fungi and bacteria, are able to degrade it. Being graphite the carbon allotrope from which graphene is obtained, graphite mining sites may be potential hotspots of microorganisms where these primary degraders could be found. Thus, this proposal will pursue a bottom-up approach starting from the in vivo analyses of graphite-associated microbial diversity to the in vitro evaluation of its capacities of biodegrading graphene. Three areas with graphite-rich soil and rock debris in Western Alps, and three neighboring graphite-free areas will be sampled. Here, soil core samples and rock debris will be collected and characterized for their chemical and physical properties, and will be used for extraction of environmental DNA (eDNA) and culture isolations of bacteria and fungi. eDNA will be processed for i) metabarcoding (amplicon sequencing) to characterize the taxonomic diversity of fungi and bacteria, and ii) metagenomic analyses to characterize the functional diversity (signatures of metabolic pathways) of the whole communities both graphite-associated and not. In order to translate the results from the omics studies into testable and reliable models, the isolation of single and pure bacteria and fungal strains will also be performed from the core samples. The isolated microorganisms will be identified and at least three bacteria and three fungi will be selected and tested in vitro (incubating them with few layers graphene (FLG), the material most similar to graphite and graphene) for their capacity of chemically altering or degrading graphene, thus potentially affecting its reactivity and environmental toxiciy. The taxa for which graphene-degrading capacities will be detected will also stand as suitable candidates for forthcoming researches aimed at testing their enzymatic activities and biodegradation capacities on diverse GBMs. Thus, the discovery of microorganisms which are naturally able to degrade graphene (because isolated directly from graphite-rich substrates) would advocate their use as natural biodegraders of graphene that will unavoidably accumulate in the environment due to its massive use.

•    Responsabile Scientifico: Generali Daniele
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022A7HJEM_003
•    CUP: J53D23003060006
•    Finaziamento MUR UniTS: 27.156 € 

Abstract: Breast cancer (BC) is one of the most frequently diagnosed malignancies in women, with a high mortality rate worldwide, due to or in the absence of estrogenic stimuli and in resistance to therapy. The results obtained in cell experimental models will be translated to human specimens to detect lncRNA molecules that might be potential prognostic factors and cancer/progression biomarkers. The final goal will be the identification of a small set of lncRNAs suitable as molecular targets for future clinical applications. molecular heterogeneity and the occurrence of therapy resistance phenomena causing disease recurrence. The estrogen receptor alpha (ERα) plays an important role in the clinical care of breast cancer patients, being the key factor in modulating estrogen signaling by its oncogenic effects, thus representing the main target of the endocrine therapy (ET) adopted in clinical practice.
Indeed, the first line management of hormone receptor positive BC both in pre- or postmenopausal women is represented by the ET. This therapy is based on functional inhibition of ERα activity by several approaches, such as estrogen biosynthesis blocks or use of antiestrogen molecules, determining the activation of anti-proliferative and apoptotic mechanisms in BC. Aromatase inhibitors (AI) such as letrozole, by eradicating estrogens, suppress both genomic and non-genomic action of ER and create harsh conditions for cancer cells; SERMs compete with estrogens for the ERα binding site determining the block of its activities, while SERDs cause selective ERα degradation.
Recently, a new role of ERα as RNA Binding Protein (RBP) has been defined, opening new clues over another class of ERα interacting partners and pointing to the receptor as a multifaceted gene expression modulator, at both transcriptional and post-transcriptional level, affecting BC progression and resistance to therapies. Among ERα-associated RNAs, lncRNAs have been also retrieved. These are a class of noncoding RNA molecules known to carry out a compartment–specific activity, mainly acting as oncogenic or tumor suppressive factors in several cancers, including BC, making them potential therapeutic targets. The proposed project aims at the investigation of the molecular mechanisms involving ERα-interacting lncRNAs in modulating ERα oncogenic activity in the presence or in the absence of estrogenic stimuli and in resistance to therapy. The results obtained in cell experimental models will be translated to human specimens to detect lncRNA molecules that might be potential prognostic factors and cancer/progression biomarkers. The final goal will be the identification of a small set of lncRNAs suitable as molecular targets for future clinical applications.

•    Responsabile Scientifico: Stampalija Tamara
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022SSFN2E_001
•    CUP: J53D23012400006
•    Finaziamento MUR UniTS: 82.074€ 

Abstract: State of the art: blood pressure is an easily measurable parameter but reflects a delicate hemodynamic balance between cardiac output (CO), effective circulating plasma volume (PV) and peripheral vascular resistance (PVR). In uncomplicated pregnancy, important haemodynamic changes occur which include increased CO, accompanied by cardiac remodeling, a decrease in PVR and an increase in PV. Several studies have shown the existence of two different phenotypes of hypertensive disorders in pregnancy from a hemodynamic point of view. The first characterized by high PVR and low CO. The second characterized by high CO and low systemic TVR with clear implications from a therapeutic point of view. However, maternal haemodynamics assessment is not currently evaluated for personalized therapeutic management.
Objective: the aim of the study is to evaluate whether maternal haemodynamic assessment and personalized calibration of antihypertensive therapy can improve the blood pressure control and improve the perinatal and maternal outcomes.
Methods: this is an Italian multicenter open lable randomized controlled trial. The goal is to enroll pregnant patients at the time of the diagnosis of mild gestational hypertension or mild preeclampsia. Patients will be randomized into two arms with a 1: 1 ratio. In the intervention group, an assessment of maternal hemodynamics will be performed using a USCOM device. For patients with high CO the initial treatment will consist in the use of beta blocking drugs while for patients with low CO the initial treatment will be the calcium antagonist drug. In the control group, the patients will be treated according to the internal protocols, without maternal hemodynamics assessment. The primary outcome will be the development of severe arterial hypertension while in the secondary outcomes will consist in adverse perinatal and maternal outcomes.

•    Responsabile Scientifico: Malfatti Francesca
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022JKAFHB_001
•    CUP: J53D23006600006
•    Finaziamento MUR UniTS: 75.487 € 

Abstract: Marine bacteria (and Archaea) play major roles in the oceanic carbon biogeochemical cycle thus structuring our planet and promoting Earth habitability. Within the marine biogeochemical cycle of carbon, microbes determine the fate of the fixed carbon: respiration, nutrient regeneration, biomass for higher trophic levels, sinking detrital particles for carbon storage. One layer of complexity within the microbial functioning that hasn't been explored yet, is the associated mobilome focused on antimicrobial resistance genes (ARGs), the resistome, within the microbial biogeochemical role in ocean-atmosphere as interconnected environments. Now more than ever, we understand the importance of the human-microbe nexus via air-water environment and that we urge to shift gears in managing the antimicrobial resistance crisis. Mobilome can be defined as specific information embedded in microbial genomes and can be exchanged among microbes. Swapping genes allow microbes to develop novel strategies for thriving in the environment. Within the mobilome, resistome refers to all genes that directly or indirectly contribute to antibiotic resistance. Furthermore, due to technical difficulties to recreate the “atmosphere as a microbial environment” there is a general lack of fundamental knowledge on bacteria behavior, physiology and ultimately gene expression in the air. FEMTO combines biogeochemical measurements with targeted resistome-mobilome metagenomics, and innovative culture-based experiments in an atmospheric simulation chamber. This research will map focused resistome-mobilome in the sea spray aerosol (SSA) and water column and estimate the potential transfer ARGs linked to specific mobile elements thus predicting the resistome-mobilome dynamics along a gradient of anthropogenic and climate-induced stressors. UniTS, UniGE and OGS form the FEMTO teams who are experts in environmental microbiology, aerosol physics and marine biogeochemistry. As a tangible product, we will create a risk map based on resistome-mobilome dynamics and expression focusing on ARGs within the carbon biogeochemical framework. Success in our goals should yield a mechanistic understanding of the marine biogeochemical significance of microbial ARG pools and their expressions. Our findings will provide data for supporting the management of the antimicrobial resistance issue within the One Health approach where human, animal and environmental health must be managed in synchrony across environments. Bimonthly group meetings will ensure high-flow communication in the FEMTO team and prompt risk management. We will rapidly communicate our findings in wide circulation open-access journals, at conferences and in open-day events, and via social media tools. FEMTO could shift the current paradigm on microbial carbon cycling and marine ecosystem functioning thus revealing a novel resistome-mobilome-derived context for carbon biogeochemistry across the air-ocean continuum.

•    Responsabile Scientifico: Zacchigna Serena
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 20225NA287_001
•    CUP: J53D23012580006
•    Finaziamento MUR UniTS: 74.758 € 

Abstract: The heart is a muscular pump that constantly contracts throughout our life. Thus, cardiac cells are exposed to high mechanical stimulation, linked to both cardiac contraction and pressure-volume load inside heart chambers. 
Mechanical forces suddenly increase in the heart early after birth, exactly at the time by which cardiomyocytes permanently exit from the cell cycle and heart loses its regenerative potential. Whether mechanical forces in the adult heart inhibit the proliferation of cardiac cells is an intriguing, yet largely unexplored question. In addition, to what extent the low proliferative capacity of cardiac cells is linked to the low incidence of cancer in the heart also remains elusive. This is due, at least in part, to the lack of simple models by which to study the effect of multiple types of mechanical stimulation on various cell types.
McHEART wishes to develop innovative tools to apply mechanical stimuli on cells ex vivo, identify the mechano-transduction pathways activated by these stimuli and eventually define their effect on the proliferation of both cardiac and cancer cells. On the one hand, these data will provide novel insights on the mechanisms that block cardiac cell proliferation and thus offer novel targets to induce cardiac regeneration. On the other, they will explain the low incidence of cancer in the heart and pave the way to a transformative way of fighting cancer growth, based on mechanical stimulation.

•    Responsabile Scientifico: Cesca Fabrizia
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 20224YX5ZX_001
•    CUP: J53D23010800006
•    Finaziamento MUR UniTS: 150.500 €

Abstract: As of today, 46 mutations in the KIDINS220 gene have been linked to HSP, and their number is constantly increasing. In vitro and in vivo studies, several of which authored by the proponent, provided a thorough knowledge of the physiological functions of Kidins220 in the nervous system. However, nothing is yet known about the mechanisms by which mutations cause spastic paraplegia, and patients face a complete lack of knowledge about their pathology. By combining in vitro studies on primary murine neural cells and on patient-derived stem cells, this proposal aims at addressing this gap in knowledge. The proposed project will be possible thanks to the collaboration with distinguished scientists, leaders in their fields and crucially, with the families of affected children. Importantly, we expect that our findings will be relevant also for other genetic forms of paraplegia, thus expanding their potential impact to wider cohorts of patients.

•    Responsabile Scientifico: Bulla Roberta
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 202275A8KE_001
•    CUP: J53D23001020006
•    Finaziamento MUR UniTS: 95.033 € 

Abstract: For pre-pubertal girls or women who require immediate chemotherapy, cryopreservation of oocytes and/or embryos is not an option. As an alternative, some patients opt to cryopreserve ovarian tissue and undergo auto-transplantation once in remission and ready to start a family. The rate of successful outcomes of auto-transplantation is increasing, but graft survival and follicular output remain relatively low and graft ischemia in the 5 to 7-day window post-transplant remains a significant obstacle to maintaining tissue viability. Our main aim is to improve the knowledge about the ovarian immunological milieu before and after cryo-preservation and create 3D cellularized scaffold, in order to the better the success rate of ovarian autologous transplantation and identify a novel approach of ovarian tissue or follicles auto- transplantation

•    Responsabile Scientifico: Gerdol Marco
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022ZRZBZ7_001
•    CUP: J53D23006830001
•    Finaziamento MUR UniTS: 106.266 € 

Abstract: A pan-genome include core genes, which are invariably shared by all the individuals belonging to a given species, and dispensable genes, which may be missing in some individuals or populations. Although pan-genomes have been long thought to be a prerogative of prokaryotes, large-scale genomic investigations have recently revealed the important functional implications of dispensable genes in several species of plants and fungi. Despite the growing interest on this topic, to date just a handful of studies, mostly focused on vertebrates, have investigated the presence of pan-genomic architectures in the animal kingdom. The mussel Mytilus galloprovincialis is a commercially important bivalve mollusc species abundant in marine coastal areas, native to the Mediterranean Sea but highly invasive in temperate waters worldwide. Although mussels are able to thrive in highly polluted and anthropized areas, being poorly susceptible to pathogenic infections causing massive mortality on other bivalves, the factors underpinning this unique adaptation potential are still poorly understood.
The study of the mussel genome has recently revealed the widespread occurrence of gene presence-absence variation (PAV), which is linked with the massive presence of hemizygous genomic regions, associated with nearly one third of all annotated protein-coding genes. The significant enrichment of the dispensable genome in functions linked with immune recognition and survival strongly suggests that accessory genomic regions might play a critical role in providing mussels an improved toolbox to adapt to challenging environments and colonize new niches. This project will investigate the impact of this highly unusual pan-genomic organization on fertilization and developmental competence up to the D-veliger stage, verifying whether the structural variations associated with widespread hemizygosity may be implicated in establishing intraspecific reproductive barriers. In parallel, the project will assess whether the inheritance of dispensable genes confers improved survival to larvae in response to heat, Vibrio and bisphenol A exposure during the first 72 hours post-fertilization.

•    Responsabile Scientifico: Martellos Stefano
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022JBP5F8_005
•    CUP: J53D23006580006
•    Finaziamento MUR UniTS: 38.557 € 

Abstract: The overarching aim of this proposal is to fill gaps in the knowledge on the main drivers of invasion by the Invasive Alien Plants of (European) Union Concern (IAPUC) in the Italian Protected Areas (PAs), applying, testing, and delivering tailored and replicable novel methodologies. Innovatively, three main categories of drivers will be taken into account for modelling, prevention, and early warning: phenotypic traits of IAPUC, landscape characteristics, and social activities. The acquired knowledge will give a contribution to Biodiversity conservation and to Nature’s Contribution to People (NCP) in the Italian PAs. To this aim, 4 main objectives (OB) and 6 work-packages (WP) are planned as follows:
OB1: Identifying the main biotic and abiotic drivers and genetic/physiological traits promoting introduction of IAPUC in the Italian PAs, their establishment, spread, and invasion success in current and in future scenarios; OB2: Assessing the vulnerability of the Italian PAs to be invaded and negatively impacted by IAPUC, identifying invasion risk hotspots, habitats at risk, and priority IAPUC; OB3: Designing, testing, and delivering novel methods for the prevention and the early detection of IAPUC in Italian PAs in current and future scenarios, and with specific insights for a selected number of representative case studies; OB4: Raising public awareness on the risk of invasion in Italian PAs and identifying priority PAs that could take advantage from Citizen Science for prevention and early detection.
WP1 will establish a geo-database with all available information on IAPUC in Italian PAs, supporting all WPs. WP2 will produce suitability models for IAPUC at different scales, for current and future scenarios, accounting for both climate and land-use change as well as plant traits information variation within and among populations of IAPUC. WP5 outputs (mobility of people and goods) will be integrated in both the species distribution models (WP2) and in the risk model (WP4). WP4 is a central WP within this proposal: besides landscape and plant-trait data, model will take into account social factors relevant to invasion (WP6). In WP4 the distribution models of WP2 will be integrated also with information from selected case studies (WP3), i.e., specific combinations of PAs and IAPUC, evaluating the risk of negative impacts to biodiversity, sustainable development, and human health. In addition, WP6 will demonstrate the effectiveness of volunteers’ involvement in the framework of prevention and early detection. Dissemination will be supported by all the Research Units and by a dedicated plan and will occur e.g., during national and international conferences, and among the members of the European Citizen Science Association.

•    Responsabile Scientifico: Crocè Saveria
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 2022Y8FZCP_002
•    CUP: J53D23001240001
•    Finaziamento MUR UniTS: 71.197 €

Abstract: Colorectal cancer (CRC) is a disease caused by multi-etiological factors, both environmental and genetic, affecting more than 1.8 million people worldwide. CRC causes are continually debated, and a recent thrust in the scientific community was given by the discovery of mucosa-associated microbiota (MAM) members able to trigger and drive host DNA lesions and a prolonged cancer-related cellular inflammation. It was also recently proposed that CRC genomic instability would be driven by human endogenous retroviruses (HERVs), ancient viruses embedded into our genome, able to exert a strong oncogenic potential due to their (re)activation and translocation properties. MAM bacterial members are also able to: i) develop and fine-tune the gut-associated immune system, which acts a role in defining the CRC-related immune tonus and inflammation; ii) modify gene expression of underlying intestinal epithelial cells (IECs) through epigenetic modifications such as histone deacetylase and DNA methyl-transferase inhibition. Once stimulated, IECs within a CRC lesion (re)activate HERVs through hypomethylation and shed lot of extracellular vesicles (EVs) which contain HERVs. EVs could act both locally (intestine) and systemically (blood) by triggering cell transformation through horizontal gene transfer, thus spreading the HERV oncogenic potential.
The HERMIA project thus aims at characterizing biopsy HERVs methylation and expression, the MAM-immune system axis, the IECs-derived EVs shredded into the bloodstream, ultimately finding a definite bacterial species or consortium (named Species Interacting Group, SIG) able to counteract the oncogenic potential of HERVs (re)activation.
To this aim(s), HERMIA will employ state-of-the-art high throughput sequencing for MAM and immune system characterization, along with innovative bioinformatic analysis (social networks, strain profiling, gene/pathways) and culturomics approaches to select and cultivate distinctive species/consortia harboring the function of HERVs up- and down-regulation. The project HERMIA will thus produce not only deliverables (datasets and bioinformatic pipelines), but also a user-friendly tool for individual prediction of the (re)activation of a definite HERV of interest. This integrated approach of bioinformatic and microbiological analysis techniques, simulating the interrelationships between the different datasets and the theranostic power of SIGs or distinct cultivated species, could then be eventually applicable in clinics in order to downregulate HERVs expression in CRC patients, thus facilitating the translation from bench to bedside.

•    Responsabile Scientifico: Schoeftner Stefan
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022KWLYAL_001
•    CUP: J53D23012780006
•    Finaziamento MUR UniTS: 108.322 € 

Abstract: Epithelial ovarian cancer (EOC) is the most lethal gynaecological cancer with > 60.000 new cases per year in the US and EU and single therapy that is beneficial for all EOC patients is currently not available. We found that self-renewal circuits based on the self-renewal transcription factor OCT4 enhance ovarian cancer aggressiveness by programming a tumor promoting microenvironment. In this context OCT4 is tightly regulated by a derivative pseudogene lncRNA that deposits epigenetic silencing complexes to the OCT4 promoter using an unprecedented epigenetic mechanism, impinging on ovarian cancer aggressiveness. This highlights that relevance of tight regulation OCT4 dependent cancer pathways to program a tumor microenvironment (TME) in ovarian cancer. With this this proposal we want to understand how OCT4 dependent gene expression signatures program the an tumor-promoting TME and unravel the detailed mechanism by which the human OCT4 pseudogene 3 lncRNA (hOCT4P3) targets silencing complexes to the OCT4 promotor and identify alternative target regions across that ovarian cancer genome. 
In particular, we want to reach the following goals:
1.    Build a EOC specimen collection stratified for hOCT4P3 and OCT4 and define cellular TME components by IHC. 
2.    Define OCT4 dependent gene expression signatures and identify compounds that revert OCT4 driven pathways of TME programming and EOC aggressiveness
3.    Unravel central mechanisms of OCT4 driven EOC cell – TME communication involving the cGAS-STING pathway
4.    Define sequence-dependent mechanism underlying hOCT4P3 lncRNA directed deposition of silencing complexes
5.    Map hOCT4P3 lncRNA target sites in the EOC genome and integrate with gene expression data to understand a role of hOCT4P3 in modulating EOC aggressiveness
The collaborative effort will provide new insights into the evolution of novel, clinically relevant epigenetic mechanisms and provide new inroads in disrupting a tumor promoting TME in ovarian cancer.

•    Responsabile Scientifico: Pastore Stefano 
•    Dipartimento: Ingegneria e Informatica
•    Codice Progetto: 20224CACME_001
•    CUP: J53D23011820006
•    Finaziamento MUR UniTS: 90.159 € 

Abstract: Alzheimer’s disease and dementia are recognised as a growing societal health and care problem due to the increasing survival of older people in society. They are complex neurodegenerative diseases with multiple cognitive and behavioral symptoms. The heterogeneity of AD and related dementias makes these diseases difficult to diagnose, manage, and treat, leading to calls for better methods to early detect, forecast and monitor disease progression, especially for designing appropriate treatments. On the other hand, early diagnosis and intervention increases the quality of life and care planning for people with dementia and their caregivers, which promote independence, and it is a top priority in recent policies for dementia care.
Current assessments and their associated ‘markers’ for dementia can comprise several types, from clinical history, biological (e.g. blood- or brain-based) assessment, to neuropsychological and functional assessments. The role of digital data in supporting AD and dementia diagnosis is increasingly recognized, with major focus on advanced digital imaging and biomarkers, analyzed with machine learning methodologies. However, large screening cannot be achieved with clinical snapshots or high-cost procedures. Conversely, digital technologies have the potential to enable reliable, high quality, continuous data collection from large patient populations in their naturalistic settings. In turn, this could help improve our currently limited understanding of natural disease course, patients’ own experiences of the illness, symptom manifestation, and individualized treatment response. Several systems and applications for low-cost and low-impact large scale assessments were proposed, but they lack the multimodal approach needed to provide as personal and personalized view.
We therefore propose to implement an integrated infrastructure for low-impact data collection that will (1) provide a tool for early detection of the disease; and (2) represent the basis for a longitudinal digital twin able to follow the disease progression and the patient in later stages. To do so, we will:
-    Implement a multimodal system including apps, wearables, and cognitive tests and exercises, based on the current evidence of relevant factors and predictors of the disease;
-    Implement the digital infrastructure that integrates the data collected through the multimodal system and the classical clinical assessments used for diagnosis and disease progression evaluation (clinical scales and biomarkers);
-    Test and validate the multimodal system and the digital infrastructure in preclinical, early, and advanced stages of dementia and AD, and in healthy controls. 
The aim of this project cannot be the validation of a long-term digital twin, nor the validation of an early detection or prognostic tool for dementia and AD. However it represents the necessary technological and infrastructural first step for these future developments.

•    Responsabile Scientifico: Di Bella Stefano
•    Dipartimento: Scienze Mediche, Chirurgiche e della Salute
•    Codice Progetto: 202229FKJF_001
•    CUP: J53D23000960006
•    Finaziamento MUR UniTS: 51.616 € 

Abstract: Staphylococcus aureus is one the most important bacteria responsible for bloodstream infections (BSIs) attributable mortality. The high mortality observed is due to several factors: antibiotic resistance properties, invasivity of S. aureus (adhesins, etc.), strong cytokine release induction and bacterial pathogenicity in terms of toxins’ production, both in a qualitative (which toxins) and quantitative (amount) manner. 
S. aureus carries more than 30 toxin genes. The different toxins have different potentials in determining tissue and systemic damage. Their pathogenic activity involve antiphagocytosis, hemolysis, coagulation system alterations, etc. 
S. aureus bacteremia is a stable problem both in community and hospital settings, and the 30-day all-cause mortality of S. aureus bacteremia is 20% and has not changed since the 1990s (van Hal et al. 2012). 
The standard of care toward S. aureus BSIs is still antibiotic therapy alone (± source control). However, similarly to what we have witnessed with bezlotoxumab (monoclonal antibody toward toxin B) for Clostridioides difficile, the field of bacterial infections is preparing to be investigated for a targeted therapy with monoclonal antibodies (usually administered as adjuvants to antibiotics) (Sakari et al. 2022).  
In support of this, Liu et al. recently (2021) identified a human monoclonal antibody vs S. aureus alpha-toxin (Liu et al. 2021). From this perspective, in the near future, it will become significantly important to know “which toxins” are produced and “how much” they are produced. This would lead to choosing who will benefit from monoclonal antibodies adjuvant therapy.
Existing human studies on the role of S. aureus toxins and mortality focused on the presence or absence of toxins (yes/no) and the outcome (survivor/non-survivor). These studies analyzed infections caused by strains with disrupted toxin genes. It is likely that the quantitative assessment of toxins (detection power improvement) will overcome the “qualitative” studies, in order to identify those at higher risk of complicated infections/death.
Prof. Alessandra di Masi already studied the relationship between toxemia levels and clinical outcome in C. difficile human infection, demonstrating that high serum levels of toxin A correlate with disease severity in patients with C. difficile infection (Granata et al. 2021).
A similar investigation for S. aureus in  humans is lacking. The main outcome of our proposal would be: to investigate the relationship between the amount of S. aureus toxins produced and the clinical outcomes of patients with S. aureus BSI.
Additionally, we set out other 2 objectives: to set up a zebrafish model of S. aureus toxin-associated damage in order to provide a cheap and more simple animal model to the scientific community to deeply investigate these infections to assess if human serum albumin could prevent the toxin-associated damage both  in cell culture and zebrafish animal study.
This second additional objective relies on previous experiments from our group demonstrating that human serum albumin retains binding properties against toxins produced by Gram positive bacteria (Vita et al. 2020; di Masi et al. 2018).

•    Responsabile Scientifico: Baj Gabriele
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022FSPCH2_002
•    CUP: J53D23012750001
•    Finaziamento MUR UniTS: 77.200 € 

Abstract: Rett syndrome (RTT) is a genetic, progressive neurodevelopmental disorder mainly caused by sporadic mutations in the MECP2 gene, located in the X chromosome (Amir 2000, Kyle 2017). Neuronal MeCP2 ablation causes synaptic and morphological dysfunctions that are the primary pathogenic cause in RTT (Pohodich 2015). Even prior to the recognition of the genetic causes of RTT, clinical findings suggested the presence of metabolic abnormalities and more recently mitochondrial dysfunctions and the presence of a subclinical inflammatory status were identified as important contributors to the pathophysiology of RTT (Eeg-Olofsson 1988, 1990; Shulyakova 2017).
In fact, it has been reported that mitochondria of muscle and frontal lobe biopsies from RTT patients present abnormal ultrastructure as determined by electron microscopy (Armstrong 1992, Bauman 1995). Furthermore, these observations were corroborated by gene expression profile of RTT patients and Mecp2-knockout mice samples showing impaired expression of genes required for mitochondrial structure, function and redox homeostasis (Filosa 2015, Pecorelli 2016). These defects were proposed to further contribute to the disease progression (Grosser 2012, Sbardella 2017). 
Besides the increased oxidative damage-related markers and reduced activity of the main antioxidant enzymes (Pecorelli et al., 2011; Cervellati et al., 2015), RTT patients show chronic subclinical inflammatory condition characterized by deregulation of acute phase response proteins and perturbed cytokine/chemokine profile (Cortelazzo et al., 2014; Leoncini et al., 2015; Pecorelli et al., 2016). Furthermore, RTT PBMCs present mitochondrial morphological abnormalities associated with up-regulation of arachidonate 15-lipoxygenase (ALOX15) mRNA, an enzyme implicated in the oxidation of polyunsaturated fatty acids including linoleic acid and generation of bioactive lipid metabolites such as hydroxyoctadecadienoic acids (HODEs) (Pecorelli et al., 2016; Pecorelli et al., 2019). ALOX15 transcript levels are correlated to augmented serum levels of 13-HODE in RTT patients (Pecorelli et al., 2019). Even macrophage populations, including microglia, are  dysregulated in Mecp2-null mice (Cronk et al., 2015; Schafer et al., 2015). A transcriptomic study on microglia from Mecp2-null female mice at pre-phenotypic and phenotypic stages revealed an impaired expression of genes associated with innate immunity and cellular stress responses, suggesting an early potential role of microglia in the later onset of neurological symptoms (Zhao et al., 2017).
In this context, mitochondria can play a key role in fueling this positive  feed-back cross-talk between altered redox homeostasis and increase inflammatory status (OxInflammation) RTT (Pecorelli et al., 2016). 
For instance, mitochondrial dysfunction is considered crucial for the activation of NLRP3 inflammasome, a multiprotein complex that mediates proinflammatory responses (Sandhir et al., 2017). As part of the innate immune defense, inflammasomes may play a crucial role in this chronic inflammatory condition. Inflammasomes are a group of intracellular multi-protein complexes assembled in response to different stimuli (both pathogens and sterile stressors) and characterized by the activation of inflammatory caspases (caspase-1, -4, -5, and -11), able to activate the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. A variety of distinct inflammasome complexes has been identified, however the best-characterized is NLRP3 complex, which comprises in its canonical form the sensor protein ‘NLR family pyrin domain containing 3’ (NLRP3), the adaptor protein ‘apoptosis-associated speck-like protein containing a caspase activation and recruitment domain’ (ASC), and the effector protein pro-caspase-1 (Liu et al., 2020). NLRP3 inflammasome is generally activated through a two-step process. During the priming step (signal 1), upon the activation of different receptors (e.g., TLR4) induced by pathogen- or damage-associated molecular patterns (PAMPs and DMAPs, respectively), NF-κB translocates to the nucleus, where it can transcribe for several genes including NLRP3, pro-IL-1β, and pro-IL-18. The activation of NLRP3 can occur through a multitude of signals (PAMPs/DAMPs, potassium efflux, lysosomal-damaging environmental factors, and endogenous factors, like excessive ROS production and mitochondrial damage), leading to the assembly of the machinery and the autocatalytic cleavage of pro-caspase-1 into its active form (signal 2). Caspase-1 not only is responsible for the activation and release of IL-1β and IL-18, it can also cleave gasdermin D protein and its N-terminal fragment that can create pores into the plasma membrane and eventually trig a pro-inflammatory type of cell death known as pyroptosis (Liu et al., 2020). The assembly of NLRP3 inflammasome system leads to a cascade of events able to promote a multitude of finely regulated immune responses useful for restoring the physiological conditions. However, an uncontrolled and prolonged inflammasome activation can participate in the onset and progression of chronic inflammatory states associated with several human diseases (such as cancer, gout, type II diabetes, neurodegenerative diseases, and autism) (Saresella et al., 2016; Moossavi et al., 2018; Song et al., 2017). 
We have recently found a dysregulated inflammasome activation in primary cells obtained from RTT patients (Pecorelli et al., 2020; Cordone et al., 2022). Interestingly, increased plasma levels of IL-1β and IL-18 showed, respectively, positive correlation with MECP2 mutation severity and negative correlation with the stage of the pathology, suggesting a role for an aberrant inflammasome response in RTT pathophysiology, even in the early stages of disease (Cordone et al., 2022).
Based on literature and the above-described findings, we hypothesize that altered mitochondrial dynamics and a constitutive hyperactivated status of NLRP3 inflammasome may play a critical role in the ‘OxInflammatory’ condition typical of RTT. In particular, in this project, we want demonstrated their contributing role in the shift from the pre-symptomatic to symptomatic stage characteristic of RTT. 
In this context, future investigations on therapeutics that address mitochondrial dysfunction have a potentially broad clinical impact on RTT since altered mitochondrial dynamics and inflammasome activation are causatively related events. In fact, defective mitochondrial dynamics promotes mitochondrial localization of NLRP3 and activation of the inflammasome (Mishra et al., 2021). Therefore, treatments that restore the mitochondrial homeostasis, and the subclinical inflammatory status could be key targets to improve patient outcome.

•    Responsabile Scientifico: Cingolani Lorenzo Angelo
•    Dipartimento: Scienze della Vita
•    Codice Progetto: 2022PREBHB_001
•    CUP: J53D23011040006
•    Finaziamento MUR UniTS: 89.138 € 

Abstract: Objective: We aim to develop a mechanogenetic toolkit to regulate synaptic connectivity of neural circuits, with the ultimate goal of advancing therapy for treatment-resistant brain disorders. Mechanical stimulation of synapses is a completely novel strategy to regulate brain activity.
Background/Rationale: The emerging field of mechanogenetics attempts to control neural networks by combining the advantages of optogenetics with those of magnetic stimulations; like optogenetics, it relies on targeted actuators to achieve circuit specificity while exploiting magnetic fields, which penetrate tissue, to stimulate remotely the brain. Although there exist some mechanogenetic approaches to regulate neuronal firing, we lack non-invasive means to control remotely synapses, which are defective in many brain disorders. Because recent developments indicate that the synaptic adhesion molecules integrins function as biomechanical receptors, we reasoned that targeting them would give us the possibility to gain remote control of synaptic connectivity.
Approach: The ‘mechano’ technology of the mechanogenetic toolkit is based on magnetic nanoparticles functionalized with ligands for the perisynaptic β3 integrin. The ‘genetic’ technology relies on CRISPR-mediated activation of transcription to promote expression of β3 integrin, thus lowering the energy threshold for mechanical stimulation of synapses. The magnetic nanoparticles will be delivered to primary neurons, human brain organoids and the mouse cortex in vivo, where they will bind to β3 integrin and function as receptor actuators of locally applied magnetic fields. We will bring this toolkit through biological testing in human and mouse models of one neurodevelopmental disorder (fragile X syndrome). By hijacking the signaling pathways of synaptic mechanoreceptors, we aim to promot a lasting change in functional connectivity and network activity of the targeted circuits.
Anticipated output: Our approach, based on magnetic fields that penetrate freely into tissue and are not hindered by the size of the human brain, is predicted to have great medical potential. Importantly, we do not attempt to repair a specific genetic mutation but to gain therapeutic control of synaptic function. The technology we propose is therefore not limited to a specific disease but in principle applicable to any brain disorder arising from defective functional connectivity.
Expertise: To achieve this ambitious goal, we have gathered an interdisciplinary consortium interfacing scientists having cutting-edge know-how in bio-nanotechnology and genome-editing with renowned experts in neuronal cell differentiation and signaling of synaptic mechanoreceptors. By exploiting this complementary expertise, a novel, versatile technology for remote control of brain connectivity is envisaged, which will be a breakthrough for normalizing network activity within the following decade.