PRIN 2022 - DISB - Dipartimento di Scienze Biomolecolari

FORGETFUL - the eFfects of cOrtisol excess on neuRoGenesis in AlzhEimer’s disease: a mandaTory Footpath throUgh stem celLs

Breve descrizione, finalità e risultati attesi: Alzheimer’s disease (AD) patients usually suffer from hypercortisolism. Consistently, certain patients with high cortisol levels progressively develop AD. Cortisol dysregulation is also responsible for a decrease in the volume of the hippocampus and memory impairment in patients with Cushing’s syndrome (CS). Memory impairment is distinctive in AD patients who show structural and/or functional disturbances in cognition-related brain regions, such as hippocampus. Adult hippocampal neurogenesis (AHN) is strongly associated with cognitive functions and its dysregulation has been found in animal model of AD and glucocorticoid alteration conditions. Therefore, AD and CS appear to be interconnected: both display high level of cortisol and progressive dementia. The common denominator is the excess of cortisol, but no one has really paid attention to their connection and to the possibility it offers to improve our knowledge about detrimental neurogenesis caused by excess of cortisol. Hence, the comprehension of this connection and the requirements for a productive AHN might help to develop a new understanding of AD and a causal strategy for prevention and therapeutic intervention. In this context, the project aims to evaluate the effects of cortisol excess as negative modulator of adult neurogenesis in AD but winking to CS. The questions inspiring this project have been:

- what does it happen to human mesenchymal stem cells (MSCs) (from adipose tissue of healthy controls) in vitro treated with high dose of cortisol and induced towards neuronal differentiation?

- is it the same for MSCs derived from CS patients (in vivo experienced)?

- and what does the concomitant treatment with glucocorticoid receptor antagonist (RA) cause on both types of MSCs?

- how does cortisol excess affect in vivo neurogenesis in AD model mice? And concomitant treatment with RA?

Our project is looking for answering these questions, using human and murine model, in vitro and in vivo approaches, and deep morphological and functional characterization of each step of neurogenesis. Not least, we will isolate circulating neuronal exosomes from human AD and CS patients and analyze miRNAs related to neurogenesis as predictive biomarkers, able to detect presymptomatic elderly subjects at high risk of AD, with a significant value for patient stratification in clinical trials and preventive interventions. In addition, the identified neurogenesis-associated miRNAs can be used as prognostic biomarkers to follow up the evolution of the disease under pharmacological treatment.

Importo totale UniUrb: € 84.000 di cui € 10.000 co-finanziati

Periodo: 17/10/2023 – 16/10/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Patrizia Ambrogini

Codice progetto: 2022XF7XZF

CUP: H53D23005520006 

Interactions of the white truffle Tuber magnatum with soil microbiome and plants

Breve descrizione, finalità e risultati attesi: Truffles are hypogeous ascomycetes, belonging to the Tuber genus (true truffles) that form ectomycorrhizal symbiosis with the roots of different plant species. The Italian white truffle (Tuber magnatum Pico) is the world's most expensive mushroom. Despite its economic value, it has yet to be successfully cultivated, due to knowledge gaps in its biology and ecology. The aim of the project is to highlight T. magnatum relationships with soil microbiome and plants. To reach these objectives, four work packages (WP) will be considered: WP1 will investigate the interaction between T. magnatum and soil microorganisms in vitro; WP2, will study the interaction between T. magnatum and soil microorganisms and viruses in the field; WP3 will investigate the interactions between T. magnatum and host/non-host plants and WP4 is dedicated to project management and dissemination. Researches will be carried out in lab, greenhouse and field. Taking advantage of the T. magnatum genome sequencing and of studies on other Tuber species carried out by the research groups involved in this project, transcriptomic studies will be carried out on T. magnatum pure cultures, in association with mycelium growth-promoting bacteria and plant roots. The role of microbial community towards T. magnatum development will be studied through Illumina MiSeq technology in productive and not productive patches of a truffle ground, as well as its answer towards the inoculum of bacteria promoting mycelium growth of T. magnatum. Viruses will be also considered as component of the complex soil microbiota for the first time. Lastly, the possible endophytism of T. magnatum with ectomycorrhizal host plants and herbaceous plants will be investigated by fluorescent in situ hybridization. The results of this project aim to expand knowledge on T. magnatum soil ecology, laying the foundation for its cultivation. They will also indicate the most suitable techniques for the management and conservation of natural truffle grounds. Moreover, this study will give new insights on how to improve the mycorrhization techniques with T. magnatum and how to evaluate the quality of mycorrhized plants. Besides the commercial value of T. magnatum ascomata, the presence of areas intended for truffle production is now more important than ever for an eco-friendly management of agroforestry environments and it can represent a promising socio-economic opportunity for marginal lands. The PI of the project has a long experience in coordinating truffle projects and the other three research groups involved in this project have been collaborating in truffle research for more than 20 years.

Importo totale UniUrb: € 73.711 di cui € 18.494 co-finanziati

Periodo: 11/10/2023 – 10/10/2024

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Antonella Amicucci

Codice progetto: 2022K272X8

CUP: H53D23005240006

NGLYNEU - Shared phenomics, genomics, and functional glycomics for better N-GLYcosylation evaluation and targeting in NEUrodevelopmental disorders

Breve descrizione, finalità e risultati attesi: Glycosylation is the enzyme-dependent process where oligosaccharides chains (glycans) are attached to proteins or lipids. Congenital disorders of glycosylation (CDG) are genetic disorders whose primary defect impacts one or more glycosylation pathways. CDG affect the central nervous system and lack clinical uniformity. Neurodevelopmental disorders (NDDs) are chronic, disabling diseases characterized by an atypical/delayed neuro-development. The genetic landscape of NDDs is complex and heterogeneous: almost 70% of patients with NDDs remain undiagnosed at the molecular level. Protein N-glycosylation (covalent linkage of glycans to protein Asn residues), plays an essential role in neural development and physiology via effects on synaptic proteins. Genetic defects of N-glycosylation are primarily autosomal recessive CDG causing global developmental delay/intellectual disability and other neurological symptoms. The rate of CDG in disease population with NDDs is unknown as yet. We aim to recognize the impact of pathogenic variants within the associated genes (glyco-genes) in NDDs while understanding their role on proper glycosylation functioning by coupling functional and omic analyses in cellular models and patients’ biological samples. We will analyse a population of 3000 patients with NDDs by targeting known and novel candidate glyco-genes in autozygous regions (ROHs) and in small, non-causative chromosomal deletions (Del), to enrich the detection of possible variants in a large set of glyco-genes. Patients with ROHs or Del including targeted glyco-genes, plus an additional sample of patients with NDDs selected by clinical criteria, accounting for more than 700 subjects, will be studied. Patient-derived fibroblast cultures with identified variants in Pathway Specific (PS) and nonPS glyco-genes and HEK293-derived cell models with genetic knockout, will be analysed. We will use established heavily glycosylated glyco-biomarkers analyses and as a novel approach, we will consider the signal transduction pathways of IGF-1R particularly sensitive to N-glycosylation defects, since not only the receptors but also their ligands depend on glycosylation in order to fold and function correctly. N-glycome profiling will be performed by a vast array of approaches, including mass spectrometry, on serum and on patient-derived fibroblasts and HEK293-derived cell lines, to outline possible occurrence of specific N-glycan signatures associated with PS and nonPS glyco-genes mutations. We expect to produce significant deliverables by: understanding the contribute of ROHs analyses for prioritizing candidate glyco-genes in NDDs patients; defining microphenotype features; developing novel cellular and secretory glyco-biomarkers related to the IGF-1/IGF-1R signaling components; releasing N-glycan signatures associated with PS and nonPS glyco-genes mutations. NGLYNEU development will be boosting molecular targets at the crossroad between rare diseases and NDDs.

Importo totale UniUrb: € 63.658 di cui € 8.527 co-finanziati

Periodo: 04/10/2023 – 03/10/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Giosuè Annibalini

Codice progetto: 202255RLB4

CUP: H53D23004740006 

PhActHealth - Physical Activity and Healthy aging: fighting low grade chronic inflammation to put out the oncologic risk

Breve descrizione, finalità e risultati attesi: Breast Cancer (BC) is the most common neoplastic disease and among the leading cancer related causes of death among women worldwide. Many studies have shown how tumour progression was characterized by a sequence of dormancy and growth phases. The role of inflammation and host metabolism appears increasingly fundamental in tumour dormancy. Surgical removal of the primary tumor is believed to be able to perturb metastatic homeostasis, to induce both conversion to angiogenesis in a few a-vascular micrometastases, and to activate proliferation in dormant cells. We and others demonstrated the former effect will result in a first peak and the latter in a second peak of the hazard function for clinical metastatic recurrence. Tumours with high proliferation activity are expected to be highly sensible to the acute inflammatory stimulus from surgery which already proved to induce cancer cells proliferation. In the last years, several studies have established the relationships between impaired cardio-metabolism/fat accumulation and the associated low-grade chronic inflammation on the increased risk of developing BC and of distant disease recurrence. Every intervention able to diminished host systemic inflammation and boost immune system activation in BC patients can contribute to improve post-surgery outcome. Although the evidence of the benefits in the post-surgical context, there is still a major lack of knowledge on the role of physical exercise in the pre-surgery/neoadjuvant settings. In this perspectives, in BC patients with metabolic syndrome, physical activity prescription before surgery can represent an inexpensive treatment to effectively extinguish host systemic inflammation, impact on tumor features and ultimately impair surgery-induced growth of existing dormant tumor cells. The study hypothesis is that exercise may be able to prevent cancer recurrence by metabolic/immunological effects leading to the lowering of tumor proliferation activity levels as a marker of tumor distant spreading capability. The healthcare purpose of this study is to explore how an aerobic exercise program of pre-habilitation before surgery (cardiometabolic exercise that stimulates and strengthens the heart and lungs and improves the body's use of oxygen) can reduce tumor aggressiveness by improving immune response preventing metastatic spread and the following exit from tumor dormancy of the localized distant micro and nano metastases. The project is in a preventive Phase IV Health Technology Assessment perspective, exploiting the latest advancements in exercise biomedicine involving the role of autonomous nervous system in modulating the immune response in a homeostasis perspective to prevent cancer recurrence.

Importo totale UniUrb: € 74.836 di cui € 5.036 co-finanziati

Periodo: 27/09/2023 – 28/09/2024

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Elena Barbieri

Codice progetto: 2022FW5A5K

CUP: H53D23001780006

Genomic, proteomic, and metabolomic characterization of hypoxia-dependent Hematopoietic Stem and Progenitor Cells to improve their harvesting and downstream applications

Breve descrizione, finalità e risultati attesi: Hematopoietic Stem and Progenitor Cell (HSPC) transplantation efficacy relies on threshold quantitative and qualitative levels of HSPCs to reconstitute hematopoiesis. However, current mobilization, harvesting and manipulation procedures, carried out at present in non-physiologic ambient air, can be improved and optimized. It has been established that HSPC processing in conditions that more closely resemble their native environment in the bone marrow, such as hypoxia or three-dimensional (3D) bioreactors, or the ex vivo treatment with nicotinamide (the vitamin precursor of the coenzyme NAD), improve HSPC function and engraftment outcomes. The proposed project intends to dissect the role of hypoxia in modulating HSPC metabolism, stemness and differentiation potential with the final aim of increasing the recovery of transplantable high-quality HSPCs. HSPCs will be isolated and cultured under hypoxia conditions, in combination with modulators of NAD levels and/or NAD-dependent pathways, in 2D or in 3D scaffolds to identify optimized conditions for the ex vivo processing. In depth metabolomic, proteomic and transcriptomic analyses will be performed on: HSPCs harvested from cord blood using a modified cell sorter to maintain a controlled anaerobic environment; HSPCs cultured in a hypoxia workstation that allows accurate control of oxygen at 0.1% increments; HSPCs cultured in hypoxia using a 3D bone marrow model developed by our group using silk derived biomaterials; HSPCs cultured with a combination of NAD-pathway modulators and hypoxia. Modulation of the main metabolic pathways will be compared among the different conditions. Automated image analysis algorithms applied to two-photon excited fluorescence, already in use in our laboratories, will allow label-free real-time monitoring of HSPC metabolism in the 3D scaffolds at single-cell resolution. The results of this study should help to define the impact of low oxygen levels (hypoxia) on the outcome of collection and ex vivo manipulation of HSPCs and allow for functional characterization of the molecular bases of stemness, thus advancing research on HSPC-based cell therapies. The three Research Units involved in the project have complementary scientific and technical skills: (i) leadership in HSPC biology and bone marrow modeling (University of Pavia); (ii) expertise in molecular biology and biochemistry with a “next-generation” mass spectrometry facility, access to an advanced computing platform and collaboration with the Italian Node of ELIXIR for transcriptomic analysis (University of Urbino); (iii) solid and internationally-recognized background in the role of NAD precursors and NAD-dependent pathways in different cell systems (University of Genova).

Importo totale UniUrb: € 88.868 di cui € 24.843 co-finanziati

Periodo: 27/09/2023 – 28/09/2024

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Marzia Bianchi

Codice progetto: 2022P9RM9M

CUP: H53D23003100006

3D-printed antibiotic oral dosage forms for pediatric use. [p3Diatrics]

Breve descrizione, finalità e risultati attesi: The development of age-appropriate and personalized medicines is still a necessity. Indeed, nowadays it is widely accepted that the pediatric population has different needs from adults. For example, the swallowing difficulty is typical of most types of medicinal treatments among children. Specifically to antibiotic therapy, additional limitations can be mentioned such as inappropriate biopharmaceutical properties, low stability in the gastrointestinal tract and bitter taste. Therefore, there is an urgent need for developing age-tailored dosage forms for antibiotic administration in pediatric patients. This project will explore and combine innovative strategies to obtain suitable oral antibiotic formulations, specifically designed for various pediatric age groups, through the advancement of new manufacturing approaches. By using a highly efficient and iterative research plan among the four research units involved in the p3Diatrics project, new medicines will be designed and developed starting from the preformulation stage to the formulation and assay studies, using a bottom-up experimental approach. The goal is to improve safety, efficacy and acceptability, in order to fulfill specific pharmaceutical requirements such as bioavailability, dose uniformity, easy and safe administration. In the preformulation stage, the project will explore the cocrystallization to improve physico-chemical properties, absorption characteristics, and palatability of selected antibiotic molecules. The final oral formulations will be manufactured using different 3D printing methodologies. Among them, direct powder extrusion (DPE) will be employed to develop solid dosage form, while pressure-assisted microsyringes (PAM) will be considered to process soft dosage forms. The formulations will be developed by mixing the preformed cocrystals with selected excipients regarded as safe for children. The obtained dosage forms will be characterized as regards the physico-chemical, technological and functional properties. The main advantages with a 3D printing manufacturing approach are the possibility to personalize the shape and dosage based on the patient needs, change flavor and drug release profile by changing the formulation matrix all with the aim to improve the patient adherence to the therapy. With the development of this approach, 3D printers could be present in both hospitals and local pharmacies to directly prepare personalized formulations. p3Diatrics will impact current clinical scenarios by providing personalized therapies combining both materials science and formulation technology fields with an innovative approach and represent an important step forward for the improvement of pharmacological compliance in pediatric antibiotic treatments. Finally, the positive impact on the environment is also an important objective of p3Diatrics; the possibility of personalizing the therapy in terms of doses and dose-units can contribute to drug waste minimization.

Importo totale UniUrb: € 73.229 di cui € 19.790 co-finanziati

Periodo: 15/10/2023 – 14/10/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Luca Casettari

Codice progetto: 2022FRNFMT

CUP: H53D23004550006

HECT E3 Ligases as therapeutic targets for SARS-CoV-2 and Emerging Respiratory(HECORES) Crisis

Breve descrizione, finalità e risultati attesi: The aim of the project is to demonstrate the antiviral properties of indole-3-carbinol (I3C) specifically in the treatment of SARS-CoV-2 infection and other RNA viruses such as metapneumovirus (hMPV). I3C is made up of a substance called glucobrassicin which is found in vegetables such as broccoli, Brussels sprouts, cabbage, etc. The results in our laboratory demonstrated that this compound exerts an anti-SARS-CoV-2 direct replication activity. Furthermore, we have established that this is a direct interaction between NEDD4 and WWP1 enzymes (HECT-E3 members) with SARS-CoV-2 most likely favoring their exit through ubiquitination. It is therefore conceivable that members of the HECT family may influence the outcome and natural history of COVID-19 and similarly hMPV infection at multiple levels, also affecting non-cellular autonomous antiviral defense mechanisms.

Importo totale UniUrb: € 88.600 di cui € 22.000 co-finanziati

Periodo: 27/09/2023 – 28/09/2024

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Andrea Duranti

Codice progetto: 2022ZSLRPT

CUP: H53D23000750006

The role of small RNA in human and canine Leishmania infantum infection: a one-health approach

Breve descrizione, finalità e risultati attesi: Leishmaniasis is a neglected disease affecting humans and other mammals, caused by the protozoa Leishmania, an intracellular parasite infecting mainly macrophages. Many host cell gene expression and signaling pathways are targeted by Leishmania allowing parasite survival/replication, even though the underlying molecular mechanisms are not fully elucidated. Recently, miRNAs have emerged as regulatory molecules with an important role in inflammation and immune responses, as evidenced by studies in which the dysregulation of miRNAs in the host was related to infectious diseases and associated with the eradication or susceptibility to the infection. In leishmaniasis, the role of miRNA has recently been evaluated mainly in human or murine models. Focusing on Leishmania infantum (endemic in Mediterranean region), the causative agent of human cutaneous and visceral leishmaniasis and of canine leishmaniasis, only few reports regarding host miRNA dysregulation have been published and only two studies have evaluated the expression of selected miRNA in serum/plasma of infected dogs. This project aims to: 1) identify the miRNA(s) dysregulated during L. infantum infection and characterize their regulatory role in the immune response in a one-health approach (human and canine); 2) evaluate/identify miRNA(s) as diagnostic/prognostic markers in plasma of infected dogs at different clinical stages. The objective 1) will be pursued in in vitro models of infection using human and canine cell lines (e.g. THP1 and DH82 cells, respectively). Infections will be performed with L. infantum MHOM/TN/80/IPT1 reference strain and/or other canine isolates available. Total RNA, including small RNA will be extracted from infected cells harvested at different time from infection and from culture medium/purified exosomes. Small RNA sequencing will be performed using the Ion Torrent S5 instrument. After identification of dysregulated miRNAs, sequencing results will be validated by qPCR and the role of selected miRNA(s) in infection will be first evaluated in silico and then experimentally using miRNA mimics and/or inhibitors. The objective 2) will be pursued by testing a number of miRNA selected from objective 1) in plasma samples from dogs at different clinical stages (healthy; infected healthy; infected unhealthy at different stages based on renal involvement). Initially, pooled plasma samples will be used to identify dysregulated miRNA that can be considered as diagnostic/prognostic markers. Once informative miRNA(s) will be identified, at least 25 dogs for each condition can be tested to validate the results obtained, using individual miRNA assays. The identification of dysregulated miRNA in both human and canine cellular models and plasma of infected dogs, as well as the characterization of their function in the context of the immune response, will allow to use them as diagnostic/prognostic biomarkers or as new drugs/drug targets to help to control this neglected disease.

Importo totale UniUrb: € 153.429 di cui € 19.137 co-finanziati

Periodo: 27/09/2023 – 26/09/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Luca Galluzzi

Codice progetto: 2022KH5MBK

CUP: H53D23000700006

Unveiling new molecular players involved in myogenic stem cells homeostasis and skeletal muscle regeneration

Importo totale UniUrb: € 55.000 di cui € 13.000 co-finanziati

Periodo: 15/10/2023 – 14/10/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Pietro Ghezzi

Codice progetto: 2022YR9W2P

CUP: H53D23006330006 

The burden of persistent pain after total knee arthroplasty: novel strategy of prevention and adjunct management based on selected nutraceuticals from Mediterranean edible plants.

Importo totale UniUrb: € 111.790 di cui € 16.883 co-finanziati

Periodo: 15/10/2023 – 14/10/2024

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Matteo Micucci

Codice progetto: 2022FHF5BE

CUP: H53D23006150006 

GLUTAMATE TRANSPORTER GLT-1 IN CEREBRAL CORTEX: A NOVEL LOCALIZATION FOR A NOVEL FUNCTION

Breve descrizione, finalità e risultati attesi: Glutamate (Glu) mediates point-to-point synaptic transmission, but also spills out of the cleft to enter that of neighboring synapses, exerting effects at farther sites. Astrocytic processes capable of removing Glu are crucial in determining the extent of spillover and the crosstalk among synapses. We showed that in neocortex a fraction of synaptic GLT-1, the main Glu transporter, overlaps with VGAT, a marker of GABAergic synapses, and that numerous VGLUT1+ terminals are contiguous to terminals forming inhibitory synapses. We hypothesize that, in neocortex, GLT-1 located at GABAergic synapses prevents Glu escaped from adjacent excitatory synapses from binding to Glu receptors located on GABAergic terminals, and modulates interneuron (IN)-pyramidal cell (PYR) transmission. We will therefore set up to detail the organization of GLT-1 at cortical GABAergic synapses (Aim 1); to unravel the functional role of GLT-1 expressed at inhibitory synapses and the underlying mechanism(s) (Aim 2); and to verify whether a GLT-1-mediated modulation of GABAergic inhibition occurs at PV and SST subtypes of cortical IN (Aim 3).

AIM 1 - The objective of Aim 1 is to demonstrate the presence of GLT-1 at perisynaptic astrocytic processes (PAP) membranes associated to GABAergic inhibitory synapses. We will estimate the amount of VGAT spatially related to GLT-1 by confocal microscopy, and whether a fraction of GABAergic synapses is associated to GLT-1 by pre-embedding EM. Next, we will define the spatial organization of GLT-1 in PAPs located near GABAergic terminals by immunogold EM. These studies will demonstrate that GLT-1 is localized to PAPs surrounding terminals forming symmetric synapses and its extrasynaptic localization is strategic to regulate Glu spill-in from excitatory synapses.

AIM 2 - To test the hypothesis that GLT-1 localized at PAPs surrounding inhibitory terminals modulates inhibition at postsynaptic targets, we will analyze the effects of GLT-1 blockade on IPSCs recorded from PYR neurons, verify whether mGluRs are involved, and unveil the molecular mechanism mediating the effect of Glu receptors activation at GABAergic synapse, using electrophysiological, pharmacological and EM techniques. The studies will reconstruct the cascade of events activated by Glu spill-in at inhibitory synapses and leading to reduced efficacy of IN-PYR synapses.

AIM 3 - In order to provide the entire picture (GLT-1, mGluIIIR, axon terminal, and postsynaptic target) at hands in the same preparation, we will using PV-Cre/+:Ai9Tg/Tg and SST-Cre/+:Ai99Tg/Tg mice. To study the localization of mGluIIIRs and GLT-1 in PV-Cre/+:Ai99Tg/Tg and SST-Cre/+:Ai99Tg/Tg mice, we will combine pre- and post-embedding techniques. These studies will define whether both PV and SST terminals co-express mGluIIIRs and GLT-1 (and their proportion); and the spatial relationship between mGluIIIRs and GLT-1 in these subsets of GABAergic terminals.

Importo totale UniUrb: € 62.311 di cui € 24.311 co-finanziati

Periodo: 17/10/2023 – 16/10/2024

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Andrea Minelli

Codice progetto: 2022BZWEKA

CUP: H53D23005460006

Multifaceted Approach to the Study of Neuropathy-causing mutations of Actin (MAS-NeurActin)

Breve descrizione, finalità e risultati attesi: Actin is a globular protein (G-actin, 43 kDa in size) and polymerizes to form actin filaments. Within the cell, actin filaments are organized into higher-order structures, forming bundles or three-dimensional networks, fundamental constituents of the cytoskeleton, the architecture that provides a shape to the cell and organizes its constituents. The assembly and disassembly of actin filaments are regulated by a variety of actin-binding proteins and are the basis of the dynamic remodelling of the cytoskeleton. Actin filaments are particularly abundant below the plasma membrane, where they form a network that provides mechanical support, determines cell shape, and allows movement of the cell surface, thereby enabling cells to migrate (1–3). As a consequence of gene mutation, actin isoforms with different polymerisation properties are incorporated into actin filaments, altering the actin monomer-polymer dynamics, and creating dramatic changes in cellular properties or even organism’s phenotype. A large spectrum of diseases, including hearing loss, isolated ocular colobomas, pleotropic neurodevelopmental disorder and dystonia-deafness syndrome, classified as Non-muscle Actinopathies (NMAs) are associated with mutations in the ACTG1 and ACTB genes which affect non-muscular tissues and primarily neuronal cells. Studies addressing actin isoform expression in the neuronal tissue are conflicting and no data are available regarding actin isoform specific functions in the neurons. Although a lot of progress has already been made in identifying mutations and their effects on actin polymers, several questions remain unanswered, especially regarding the structural changes of the actin-based complexes and the resulting consequences on cellular morphology, functionality, and development. MAS-NeurActin aims at addressing these questions and characterizing structural, mechanical, and functional properties of both wild-type and actin-mutated neuronal progenitor cells and mature neurons, combining different biophysical and physiological advanced techniques. We plan to correlate the mechanobiological properties of actin-mutated neurons determined by optical tweezers mechanics and atomic force indentation to their morphology and functionality, studied by well-assessed approaches like immunofluorescence, confocal microscopy, calcium imaging, and cutting-edge methods such as multi-electrode arrays (MEA) electrophysiological recordings and high-resolved and enhanced Raman spectroscopy (TERS). The relation between the cytoskeletal dynamic remodelling and the neuronal activity in presence of mutations of actin will provide important insights on the mechano-sensing properties of neurons carrying mutations. That is a fundamental step in the path towards understanding human actinopathies and developing therapeutical interventions.

Importo totale UniUrb: € 61.104

Periodo: 27/09/2023 – 26/09/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Rossana Rauti

Codice progetto: 2022XJ29R7

CUP: H53D23000870001

Oxalate metabolism and hyperoxaluria: from genetic predisposition and biochemical analyses to the development of new treatment strategies.

Breve descrizione, finalità e risultati attesi: Kidney stones, most of which are formed by calcium oxalate, affect up to 10% individuals worldwide. Oxalate is a metabolic end-products excreted by urine. High urinary oxalate concentrations lead to hyperoxaluria, which thus represents a major risk factor for kidney stones. The amount of urinary oxalate depends on both endogenous production and exogenous absorption by dietary intake, both of which could be impaired leading to pathological conditions. Indeed, increased endogenous oxalate formation, due inherited deficits of enzymes involved in the hepatic metabolism of the oxalate precursor glyoxylate, leads to Primary hyperoxaluria (PH). On the other hand, increased intestinal absorption of exogenous oxalate, e.g. due to fat malabsorption, leads to secondary hyperoxaluria (SH). In other cases hyperoxaluria is idiopathic and leads to recurrent kidney stones or contributes to the onset of end-stage kidney disease (ESKD) in other pathologies. Thus, the identification of genetic biomarkers influencing the onset and/or the progression of hyperoxaluria represents an unmet medical need. Our project combines molecular, preclinical, and clinical approaches to (i) investigate the biochemical effects of genetic variations on proteins involved in oxalate metabolism, (ii) unravel their association with the onset and/or evolution of hyperoxaluria, and (iii) pave the way for new therapies in patients at risk. By whole exon sequencing analyses of different cohorts of patients we will investigate if genetic variations of genes involved in hepatic oxalate metabolism (AGXT1, AGXT2, GRHPR, HOGA1, and HAO1), exogenous oxalate transport (SLC26A1, SLC26A3, SLC26A6, SLC4A1) or both could represent risk factors for the onset of SH, modulate severity and progression of PH, and affect the onset of idiopathic hyperoxaluria and ESKD, respectively. We will perform a detailed analysis of the effects of the identified genetic variations at protein level with multiple approaches ranging from analyses in silico and experiments in purified proteins to cellular models mimicking the natural environment of each protein. We will then pave the way for the development of a new treatment approach based on the use of an available engineered form of oxalate decarboxylase (EngOxDC), showing highly improved stability under physiological conditions. We will test the ability of EngOxDC to degrade both exogenous and endogenous oxalate, using an in-vivo model of SH or ex-vivo models of PH, respectively. The complementary background of the participating units and their previous collaboration, along with the expertise in the field and solid preliminary data, guarantee project feasibility. The expected results will have important relapses for a better understanding of oxalate metabolism and inter-individual variability in hyperoxaluria, the identification of patients at risk, and the development of new treatment strategies that could prevent or mitigate the symptoms.

Importo totale UniUrb: € 68.342 di cui 12.464 co-finanziati

Periodo: 04/10/2023 – 03/10/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Luigia Rossi

Codice progetto: 2022J7CKMJ

CUP: H53D23004800006

PAtient Lipidome As biomarker of Cancer immunothErapy (PALACE study)

Breve descrizione, finalità e risultati attesi: In the last decade, immune checkpoint inhibitors (ICIs) have emerged as effective therapies for advanced solid cancers, such as melanoma, non-small cell lung cancer (NSCLC) and others. ICIs revamped the prognosis of a wide percentage of patients (pts) affected by advanced cancers by enhancing both innate and adaptive immune response against tumor cells. The identification of novel reliable biomarkers of ICI efficacy and resistance is needed to personalize the therapeutic approach towards pts. Lipidomic profile of cancer pts (Lipidome), which include the wide spectrum of lipid biomolecules on cells, blood and tissues, seems to be strictly involved in the modulation of immune response and in recent studies overweight, hypercholesterolemia and use of statins showed an impact on treatment response and outcome of pts receiving ICIs. The goal of our project is to elucidate the role of lipidome in advanced NSCLC pts treated with ICIs, by combining pre-clinical, translational and clinical data, and identify, through the tools of artificial intelligence (AI), a patient's lipidomic signature that may predict ICIs response. Through a multidisciplinary team of experts, we aim to investigate the immune-phenotype of pts with a certain lipidomic signature, to further understand the link between lipid metabolism and the immune system. Advanced NSCLC pts receiving ICIs, both alone or in combination with chemotherapy, will be enrolled in a prospective observational study. A biobank of both tumor tissues and blood samples will be established. Tumor samples collected before ICIs start will be used for lipidomic and genomic analysis and for the characterization of tumor microenvironment (TME). Blood samples will be collected at baseline and after 9 +/- 3 weeks and the circulating lipidomic assessment and the immune cell phenotype will be characterized. Moreover, pre-clinical models made of NSCLC primary cells obtained from tumor tissue will be developed in order to deeply understand the biological mechanisms linked to lipid metabolism. The link between pts lipidomic profile and other clinical factors such as pts nutritional status and body mass composition will be investigated. Since a wide amount of data will be generated by pre-clinical, translational and clinical analyses, a machine learning technology approach will be used to extrapolate a patient's lipidomic signature.

Importo totale UniUrb: € 86.793 di cui 40.062 co-finanziati

Periodo: 04/10/2023 – 03/10/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Annamaria Ruzzo

Codice progetto: 2022J343TP

CUP: H53D23004790006

Structural design of a SARS-COV-2 subunit VAccine by REverse VACCinology 2.0 (COVAREVAX)

Importo totale UniUrb: € 124.433

Periodo: 27/09/2023 – 28/09/2024

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof. Giuseppe Stefanetti

Codice progetto: 2022NC4WHS

CUP: H53D23000720001

Attenuating the stress of the endoplasmic reticulum as a strategy to cure the multi-minicore disease

Breve descrizione, finalità e risultati attesi: Multi-minicore Disease (MmD) is a rare, recessively inherited neuromuscular disorder characterized by histopathological core lesions and clinical features of a congenital myopathy. No FDA-approved drugs are currently available for MmD and current therapies are mainly supportive, so defining their pathogenic mechanisms is an important step towards the development of a pharmacological treatment. The clinical variability of the disease reflects the genetic heterogeneity, due to mutations mainly in two genes Selenoprotein N1, SEPN1, and Ryanodine receptor1, RYR1. Mechanistically, SEPN1 encodes for a calcium sensor of the endo/sarcoplasmic reticulum (ER/SR) which promotes calcium uptake by regulating the sarcoplasmic reticulum calcium pump, SERCA, while RYR1 encodes for a calcium channel, which promotes calcium efflux from the sarcoplasmic reticulum and thus, both, given their role in ER calcium homeostasis, are involved in the mechanism of excitation-contraction coupling in skeletal muscles. Our recent results provide evidence that activation of ER stress and its maladaptive response might be the pathogenic cause of the disease as shown by results in SEPN1 loss-of-function murine model and in a transgenic murine model of RYR1 mutation. CHOP and its downstream target ERO1 are mediators of the maladaptive branch of the ER stress response given their capacity to generate reactive oxygen species (ROS). Thus, to clarify whether CHOP/ERO1 branch of the ER stress response represents a novel pathogenic mechanism underlying MmD, we propose to develop an innovative, multi-faceted approach that combines genetic manipulations of CHOP and ERO1 together with the use of ER stress inhibitors in preclinical models of SEPN1- and RYR1-related myopathy and sensitive calcium and muscle force measurements. The project will be developed through the leading expertise of three Units to leverage on our recently established tools and putative therapeutic targets for SEPN1-related myopathy and RYR1-related myopathy, to address the clinical need of the cohort of MmD patients. The findings will be instrumental for the understanding of the role of the ER stress response mediators CHOP and ERO1 in the muscle dysfunction of preclinical models of MmD. Importantly, achieving these objectives will throw light on novel biomarkers and druggable targets for MmD and may provide a rationale for human clinical trials aimed at evaluating the effects of ER stress inhibitors in MmD.

Importo totale UniUrb: € 104.310 di cui 20.605 co-finanziati

Periodo: 15/10/2023 – 14/10/2025

Struttura UniUrb di riferimento: Dipartimento Scienze Biomolecolari (DISB)

Referente UniUrb: Prof.ssa Ester Zito

Codice progetto: 2022NW44H5

CUP: H53D23006210006