Abstract

Grape pomace, a by-product of the winemaking process, is rich in bioactive compounds like polyphenols, known for their health benefits. Our previous research has highlighted the therapeutic potential of a hydroalcoholic extract (HE) from white grape pomace in cancer cell lines. This study explores a more sustainable approach for unlocking these benefits. We employed subcritical water extraction (SWE), an innovative and eco-friendly method that avoids harmful organic solvents. SWE works by using water at high pressure and temperatures just below boiling point to release bioactive compounds. The characterization revealed the presence of key compounds such as gallic acid, protocatechuic acid, and catechin. We analysed SWE effects on non-tumor Human Bronchial Epithelial (HBE) cells. SWE did not significantly reduce HBE cell viability, while, similarly to HE preparation, SWE (160 °C) displayed remarkable anti-cancer properties. In HBE cells the extract acted as a powerful antioxidant by reducing basal ROS levels, also in the presence of H2O2. ROS reduction was associated with the increase in two main antioxidant enzymes, HO-1 and SOD-2. Looking ahead, we aim to explore the molecular pathways which are related to the SWE antioxidant potential and to its eventual anti-aging application. By embracing SWE, we not only enhance the therapeutic potential of grape pomace but also provide a sustainable, organic solvent-free method that maximizes the value of winemaking by-products.

This study focuses on the development of injectable hydrogels for intratumoral responsive drug release. By blending thiol-functionalized, glutathione-based waterborne polyurethane (WPU-GSH) with hyaluronic acid divinylsulfone(HA-DV), hydrogels responsive to the tumor microenvironment were obtained. WPU-GSSG was produced using a PCL-PEG-PCL, DMPA, isophorone diisocyanate and L-glutathione oxidized as a chain extender. Reductive depolymerization with DTT produced thiol-bearing WPU-GSH, which re-polymerizes under physiological conditions. Various DV/SH crosslinking ratios were established resulting in hydrogels double-crosslinked via Michael-type reaction and oxidative polymerization of WPU-GSH. Rheological characterization assessed the gelation process and mechanical properties, confirming that hydrogels with enhanced mechanical stability were obtained. A thiol-bearing doxorubicin(Dox) derivative was conjugated to WPU-GSH through trans-thiolation before crosslinking with HA-DV. Release studies, conducted in a tumor-simulating environment,demonstrated pH and redox-responsive Dox release. The hybrid hydrogels exhibited strong mechanical properties, hydrophilicity and bio-responsiveness, making them promising for innovative biomaterials. Michael-type and oxidative crosslinking allow for a flexible injectable platform to treat solid tumors,enabling the linking of proteins and bioeffectors.This opens the way to the development of DDS for more tailored treatments, like immunotherapy. 

Oxidative stress and inflammation are commonly present in many chronic diseases. Several evidences suggested that plants as Brassicaceae act as antioxidant and/or anti-inflammatory agents, suggesting a possible use in the treatment of many pathologies. In this study we aimed to evaluate in a model of copper sulfate (CuSO₄)-induced inflammation in zebrafish embryos the toxicity, behavior and antioxidant properties of methanol extract of Brassica macrocarpa leaves. Embryos were exposed to various concentrations of extract for 96 h and the LC50 value was 762 µg/mL, then sub-lethal concentrations were used for further analyses. Brassica macrocarpa extract itself induced antioxidant activity after 96 h, increasing the levels of superoxide dismutase (SOD) and catalase (CAT) activity, and reducing in lipid peroxidation. In the CuSO₄-induced oxidative stress model (20μM/5h), pretreatment with extract for 21h significantly reduced reactive oxygen species and nitric oxide levels, as well as lipid peroxidation, CAT and SOD levels. However, the extract did not reverse DNA damage or protein oxidation. Behavioral tests revealed no significant changes in anxiety-like or exploratory behaviors, although locomotor activity was affected at higher concentration. Our data indicate that Brassica macrocarpa demonstrated promising antioxidant properties, suggesting its potential applications in the management of oxidative stress conditions.

Sometimes, internal or external factors can interfere with the wound-healing process, leading to the formation of a chronic wound, commonly known as an ulcer. Chronic wounds are a serious health issue, and current medical treatments often fail to effectively heal them. This project aims to develop a regenerative medicine strategy to improve and accelerate healing using a Poly-L-Lactic-Acid (PLLA) scaffold, combined with species-specific dermal fibroblasts and microvascular fragments. Dermal fibroblasts isolated from the rat skin were seeded onto PLLA scaffolds coated with type I collagen, and their proliferation was assessed through viability assays, confocal microscopy, and scanning electron microscopy (SEM). Simultaneously, microvascular fragments (ad-MVFs) from adipose tissue were cultured in 2D (wells) and 3D (collagen gel and scaffolds) systems. The next step involves co-culturing fibroblasts and microvascular fragments on the scaffolds. In this context, the fibroblasts will boost the healing process by releasing growth factors and extracellular matrix molecules, while the microvascular fragments, by connecting with the host’s blood vessels, will address the hypoxic conditions of the ulcer, ensuring proper nutrient and oxygen supply. This engineered construct will be tested in animal models for treating both skin ulcers and diabetic ulcers and may represent a significant advance in the treatment of chronic wounds.

This work describes the role of two plant growth-promoting bacteria (PGPB) in modulating metabolite production of oregano, a Mediterranean aromatic plant. Plants were sequentially grown in plateaux, pots and, after inoculation with the two PGPB, in open field and monitored until flowering to be, then, harvested. Flowers were used for GC-MS volatilome analyses and then, plants were air-dried to obtain essential oils (EOs) and hydrolates. The results revealed that EOs from consortium-inoculated plants had an increased yield. As a result of the analysis performed on the volatilome, the consortium inoculation increases significantly the amount of p-cymene in oregano flowers. Bacteria isolations from soil were performed from oregano plant pots with the purpose of isolating and characterizing novel PGPB. Thirteen isolates were selected, phylogenetically identified on the basis of 16S rRNA encoding gene and investigated for PGP traits, including indol-3-acetic acid production; organic and inorganic phosphates solubilization. The strain B18, possessing all the investigated PGP traits, was assayed on oregano seeds. In particular, they were inoculated with S. violaceoruber, K. rhizophila, B18 cultures or mixtures thereof, and germinated until seedlings formation under laboratory-controlled conditions revealing significant differences about germination and biomass weight. Thus, PGPB treatments can improve plant growth and affect the production yield of specific plant metabolites.

The tumoral extracellular matrix (TEM), primarily composed of collagens, is crucial in modulating cancer cell behavior and response to chemotherapy. We established an in vitro model using primary cells, isolated from rat mammary tumors, to generate three-dimensional (3D) spheroids with an endogenous ECM. Breast carcinoma in rats was chemically induced in female Wistar rats. The tumor mass in the abdominal region was treated to isolate primary cells, which were used to generate 3D spheroids. Confocal microscopy revealed that the spheroids produce endogenous collagen type I matrix, mainly arranged around the 3D structure. Spheroids were treated with either ultrapure recombinant collagenases, degrading collagen within the spheroid structure. To investigate the impact of ECM degradation on the accessibility of Doxorubicin, uptake and cytotoxicity assays were performed on spheroids pre-treated with collagenases. Collagen digestion significantly improves drug penetration and efficacy within 3D tumor models. In conclusion, this study highlighted the importance of overcoming drug resistance in breast cancer by targeting the ECM and proposing a novel strategy for improving therapeutic outcomes in solid tumors. Finally, stimuli-responsive NPs, composed of bovine serum albumin (BSA) and ultrapure recombinant collagenases were synthesized and characterized, to mask the bacterial enzyme from the immune system and potentially increase doxorubicin uptake.

MMACHC is a crucial protein in the metabolism of vitamin B12 (cobalamin, Cbl), transforming it into its bioactive forms (AdoCbl and MeCbl), which are cofactors in important cellular reactions. Mutations in the gene coding for MMACHC protein are responsible for the metabolic disorder methylmalonic aciduria and homocystinuria cblC type, which affects children causing neurocognitive and cardiovascular dysfunctions. Although the crystal structure of the wild type protein is available, many molecular features of MMACHC physiopathology remain to be understood and a systematic study on the effect of each specific mutation on the resulting protein is still lacking. Hence, by using biophysical methods including spectroscopy, microcalorimetry and molecular dynamics we investigated the differences in stability, binding and functionality between MMACHC wild type and the pathological R161Q mutant. Moreover, we evaluated whether non-specific stabilizers (osmolytes) could restore the functionality of the mutant, and performed a virtual screening of chemical libraries to identify pharmacological chaperones to be tested in vitro. Altogether, our findings demonstrated how the combined use of computational and experimental biophysical approaches deepen the knowledge of the molecular mechanisms underlying the function of MMACHC and provide new insights for potential therapeutic interventions.

DNA methylation is important for heterochromatinization and thus, gene regulation. Also, DNA methylation characterises the repetitive sequences of the centromere and its role in supporting chromosome segregation fidelity is yet to be clarified. So far, studies revealed that loss of DNA methylation in the centromere region correlates with the loss of chromatid cohesion and aneuploidy. To disclose a new putative role of DNA methylation in chromosome segregation, I used engineered cells to allow an inducible DNA hypomethylation. My results showed that DNA hypomethylation in both DLD-1 and RPE-1 cells increases the recruitment of CenpA at the centromere. CenpA is a histone protein that gives identity to the centromere, and thus, these results suggest an impact of DNA methylation on centromere function. Also, I observed an increase of the plus end tracking protein p150Glued in metaphase plates of DLD-1 cells. Finally, Aurora B kinase, important for the correction of aberrant microtubule-kinetochore attachments, was also increased in both cell lines. The studies I conducted so far, reveal that methylation of repetitive DNA at centromere is important to preserve chromosome segregation, probably by maintaining centromere stability and function and by regulating microtubule-kinetochore attachments.

The epithelial-to-mesenchymal transition (EMT) is a key feature of cancer, defined by the shift from epithelial to mesenchymal traits. In breast cancer (BC), EMT is crucial for promoting cell survival and driving cancer progression. To investigate the biological impact of EMT in BC, western blot analysis was performed on a cohort of 95 BC tissue extracts, focusing on the expression of Vimentin (mesenchymal marker) and E-cadherin (epithelial marker). Results revealed high variability in the expression of both proteins, highlighting a positive correlation between Vimentin and E-cadherin levels. To determine if BC patient sera can affect cell migration in an in vitro system, a wound healing assay was conducted using MDA-MB-231 cells, used as a prototype of a highly aggressive tumor model. Cells treated with 1% BC patient sera showed a greater migratory ability compared to untreated (NT) or Fetal Bovine Serum 1% (FBS) treated cells. Moreover, after the wound healing assay, western blot analysis was performed for Vimentin and E- Cadherin, demonstrating again a positive correlation between the two markers.  However, the increased migratory ability observed in serum-treated cells did not directly correlate with Vimentin expression. These results suggest that the EMT process in BC could not be characterized by a complete phenotypic shift, indicating a more complex relationship than the one previously assumed.

The development of new antibacterial strategies is critical to effectively combat infections, particularly given the evolving complexity of global health challenges and the limitations of existing treatments. Here we present an experimental study aimed at evaluating the effect of photocatalytic agents for antibacterial photodynamic therapy (aPDT) on lipid membranes which represent key molecular targets in all bacterial cells. Specifically, we developed a novel approach utilizing blue visible light to activate the photocatalytic activity of titanium dioxide (TiO₂) nanostructures with enhanced activity. TiO₂ bandgap was modified using N-doping procedure to make possible visible light activation, These N-doped structures were combined with gold nanorods (AuNRs) to stabilize charge carriers, thereby amplifying the photocatalytic response. Fluorescence spectroscopy was employed as a powerful tool, offering a highly sensitive method for detecting molecular changes in small unilamellar Vesicles (SUVs) used as model membranes. Fluorescence changes in steady state spectra of  C-11 BODIPY inserted in the membrane bilayer was used to monitor in real time lipid induced by blue light in the presence of N-TiO₂ and AuNRs. Membrane fluidity and changes were assessed using generalized polarization of Laurdan dye, which indicates lipid packing and hydration alterations. Our results demonstrated significant lipid oxidation and membrane damage in the presence of N-TiO₂, which were further amplified by the addition of AuNRs. Notably, lipid oxidation led to substantial membrane stiffening and dehydration.

In recent years, Artificial Intelligence (AI) has emerged as a highly popular field of research, particularly within the biomedical domain. More recently, AI tools have evolved into products and software designed to support diagnosis and treatment planning. Many medical centers are now adopting AI solutions to streamline their clinical workflows. While AI model evaluation is a well-established concept, there are no standardized guidelines for Quality Assurance (QA) protocols specifically tailored to medical scenarios, where a lack of accountability could lead to fatal outcomes. Therefore, the clinical implementation of any AI tool in a medical setting necessitates the development of a robust QA protocol to address potential changes, such as the release of new models, and to ensure reliability to procure patient safety. The aim of this work was to develop a QA protocol for the AI-based software TheraPanacea ART-Plan, designed for the auto-contouring of organs at risk (OARs). This software is currently part of the clinical workflow at the hadron radiotherapy center MedAustron in Lower Austria. Geometrical and dosimetric evaluations were established as part of the protocol. A GUI was designed for intuitive interaction and to avoid the risk of error. As a result, the QA pipeline automatically generates a report that evaluates the tool performance.

SARS-CoV-2 hijacks cellular host factors to complete its life cycle, including RNA-Binding Proteins. Among these, proteomic studies indicate La-related proteins (LaRPs) and members of the PABPs as involved in CoV-2 infection through interactions with the viral RNA or the main components of its Replication Transcription Complex (RTC): nsp12-8-7. Specifically, LaRP4B and LaRP7 interact with nsp12 and nsp8, respectively, while LaRP1, LaRP4A, LaRP4B and the PABPC1 are enriched in viral RNA. Building on this evidence and to deepen the understanding of the molecular mechanisms in viral replication, we aim at characterizing the protein-protein interactions among the CoV-2 RTC core unit nsp12-8-7 and LaRP4 family members. Thus, we set up the production and characterization of the recombinant proteins and assessed their reciprocal affinities using Bio-Layer Interferometry, Isothermal Titration Calorimetry, and cross-linking assays. In parallel, the impact of LaRPs during CoV-2 infection was assessed in BEAS-2B cells depleted of LaRP4A and LaRP4B. Moreover, since the PABPC1 is involved in viral replication and is known as one of the physiological binding partners of LaRPs4, we are also investigating its role in LaRPs/SARS-CoV-2 binding. These data will be exploited to further investigate around the possible formation of a larger complex LaRP4/SARS-CoV-2/PABPC1 where the PABPC1 and/or the viral RNA act as the factors mediating the interaction.

Duchenne's muscular dystrophy (DMD) is a severe inherited neuromuscular disease caused by mutation of the DMD gene, which results in absence or truncated dystrophin protein. About 10-15% of the cases of DMD result from nonsense mutation. Translational readthrough-inducing drugs (TRIDs) offer a promising therapeutic strategy for suppressing premature termination codons (PTCs). In pursuit of this aim, our research group identified novel chemical scaffolds as potential suppressors of PTCs using a ligand-based pharmacophore approach combined with virtual screening protocols. The compounds were synthesized by following a known procedure.  The products were purified by column chromatography and characterized by HPLC/MS, IR, and 1HNMR spectra analysis. Most of the tested compounds demonstrated good efficacy in inducing PTC readthrough in the FLuc cell-based assay. Similarly, the effect of novel NV molecules on cell viability and proliferation in HCT116 cells was measured using a Trypan Blue Dye Exclusion Assay. The results revealed that the compounds have had no effect on cell viability; the cells grew exponentially after 24-, 48-, and 72-hour treatments. Likewise, the NV848 compound showed a promising result in the recovery of muscle strength in forelimb grip strength test in in vivo mdx mice model. Further studies are ongoing to explore its potential in restoration of dystrophin protein expression and functionality in both skeletal and smooth muscle.

This study focuses on developing innovative drug delivery systems (DDSs) for quercetin, cholecalciferol, and phycocyanin, active pharmaceutical ingredients (APIs) with antioxidant and anti-inflammatory properties, to enhance their therapeutic potential for treating inflammatory bowel diseases (IBD). These APIs are sensitive to pH and temperature, limiting their stability during oral intake. To address this, we employed a spray-drying technique to encapsulate the APIs in microparticle systems using Generally Recognized As Safe (GRAS) excipients, specifically soybean proteins and whey proteins, chosen for their biocompatibility, stability, and pH-dependent solubility. The spray-dried microparticles are designed to protect the APIs from gastric degradation while enabling controlled release in the colon, enhancing the bioavailability of the active compounds, aided by the formation of an amorphous solid dispersion during the formulation phase. These systems were characterized by their release and permeation profiles, solid-state characterization, and evaluation of their antioxidant activity. The therapeutic efficacy of the loaded microparticles was assessed using an in vivo model of 2,4-dinitrofluorobenzene sulfonic acid (DNBS)-induced colitis in rats. The results showed improved bioavailability for the tested systems and a significant reduction in inflammatory markers, positioning these microparticle systems as promising candidates for future clinical trials in the treatment of IBD.

Premature-termination codons (PTCs) within CFTR mRNA generate truncated, non-functional CFTR proteins accounting for approximately 10% of patients with cystic fibrosis, for whom no therapies exist. In this regard, we are exploring RNA-editing approaches to correct PTCs in the CFTR mRNA of Human Bronchial Epithelial cells. These systems exploit ADAR enzymes to convert the adenosine (A) within the PTC into inosine (I) that will be read as guanosine (G) by the ribosome, allowing the full-length protein synthesis. With the minixABE base editor (mxABE) system ADAR2 deaminase domain (ADAR2DD), which is fused with a truncated dCAS13x.1, is recruited to the adenosine within the PTC by means of specifically designed guide RNAs. Instead, with the RESTORE system linear and circular specific antisense RNA oligonucleotides (ASOs) targeting the CFTR mRNA region surrounding the PTC promote ADAR recruitment. Our results show the rescue of CFTR protein on the plasma membrane by immunofluorescence, and the increase of CFTR full transcript by RT-qPCR in the mutated cell lines when both systems were applied. Moreover, ADAR mediated RNA editing tools induced the functional rescue of CFTR in these cultures measured by electrophysiological method, indicating their therapeutic potential in the cure of cystic fibrosis.

Dupuytren's disease (DD) is a condition affecting the connective tissue of the hand. It consists of collagen deposition in the palmar fascia, primarily triggered by the formation and proliferation of myofibroblasts. It can cause deformity and finger contracture limiting daily activities in case of high morbidity. Current studies focus on therapies targeting the disease at the molecular level, to prevent disease progression and recurrence after surgery. In DD, there is a complex interaction between myofibroblast-mediated contracture and extracellular matrix remodeling (ECM). MMP-14 (or MT1-MMP), a type-I transmembrane MMP, degrades ECM molecules and is involved in the activation of proMMP-2 by binding the MMP2 physiological inhibitor TIMP-2. MMP14 plays a crucial role in the abnormal contractile response of fibroblasts in DD tissues. Moreover, SNPs in MMP14 gene interferes with the activity of collagen degradation, being implicated in the DD progression. On that note, the current project focused on phenotype characterization of this SNP by investigation of the effects of the anti-MMP14 antibodies on primary fibroblast cells derived from patients with DD, stratified according to the different SNP genotype (GG, GA, AA). Preliminary results show that the activity of proMMP2 in fibroblasts homozygous for recessive homozygous genotypes (AA) is increased, while the response to anti-MMP14 antibodies is different compared to other genotypes, opening new ways for therapeutic research.

Precision medicine represents a new insight in genetic medicine to treat the mutation profile of a patient. Nonsense mutations represent a severe genetic defect in 11% the rare genetic diseases. Currently, suppression therapy is focused on addressing nonsense mutation diseases, for which no treatments that stimulate readthrough are approved. This approach uses translational readthrough-inducing drugs (TRIDs) against premature termination codons (PTCs) occurring in mRNA. Our studies are centered on understanding the mechanism of action (MoA) of three TRID (NV848, NV914, and NV930) molecules. For this purpose, we determined NV TRIDs' effect on readthrough using the reconstituted in vitro model system PURE-LITE, which allows for the separate determination of TRIDs' impact on both readthrough and termination activities. We find that all three NVs induce readthrough in the PURE-LITE system and that their MoA differs from Ataluren, which is, actually, the only approved TRID. Our results exclude eRF1 and eRF3 as targets, but further efforts will be necessary to establish the precise MoA.

Anderson-Fabry disease (AFD) is a Lysosomal disorder caused by mutations on GLA gene, located at Xq22 on the X chromosome. Mutations result in a deficiency or absence of α-galactosidase enzyme, resulting in the accumulation of Globotriaosylceramide (Gb3). In female patients, X-chromosome random inactivation can lead to residual α-galactosidase activity, with variable phenotypic presentation. Beyond the X linked inheritance pattern and α-Gal A mutation, other factors such as genetic, environmental and epigenetics factors may impact disease spectrum and DNA methylation of X-linked CpG islands for this reason is particularly interesting to study the contribution of methylation in AFD. According to Burand, A.J et al. one of the limitations in Anderson Fabry disease research is the necessity to have a cellular model. This lack leads to a slowdown in obtaining the results. Various inherited disorders are caused by mutations in genes for which counterparts with similar function but distinct expression pattern exist. Different transcriptional activators have been linked to catalytically inactive Cas9 (dCas9) proteins, such as dCas9 fused to transcriptional activators (dCas9-VPR) and dCas9 linked with TET1, they can be used to modulate gene expression. Both are innovative approaches for genetic disease management since, to compensate for the missing function of a defective gene, could be possible to transcriptionally activate its functionally equivalent counterpart.