Chronic skin injuries greatly increase mortality and morbidity by compromising skin barrier function and altering sensitivity to pain, touch, and temperature. These wounds predominantly affect the elderly and diabetics, leading to delayed healing through unique molecular events. Effective treatment is essential to maintain the skin barrier and prevent further damage or infection. Soluble mediators are vital for successful wound healing. Mesenchymal stromal cells (MSCs) act as reservoirs for bioactive factors, providing therapeutic benefits. The MSC secretome, a complex mix of cytokines and growth factors, is crucial in regenerative medicine. Due to its ease of preparation and safety, the MSC secretome is emerging as a promising cell-free alternative to traditional therapies. The aim is to sustain secretome delivery while enhancing efficacy and preserving biological function. Secretome-laden biomaterials show potential in regenerative medicine and tissue engineering. A redox-responsive waterborne polyurethane (W-PU) was synthesized using oxidized glutathione as a chain extender and processed through electrospinning to create nanofibrillar scaffolds. This method avoids toxic solvents, improving compatibility for direct secretome loading. Characterization studies assessed loading efficiency, bioactivity, and redox-responsive release of soluble factors. Preliminary results indicate sustained release of secretome from nanofibers, showing promise for wound healing applications.

The umbilical cord and the amniotic membrane are a precious source of human mesenchymal stem cells (hMSCs), characterized by a reduced immunogenicity, immunomodulant properties and a vast application in regenerative medicine, especially in the field of wound healing. However, there are some issues related to their use in clinical practice, such as their progressive senescence after multiple cell splittings and the research of biomaterials capable of promoting cell viability and long term tissue regeneration. This project aims to establish and characterize hMSCs primary cultures of umbilical cord and amnion from healthy donors, improving methods to enhance their stemness and proliferation with specific molecules to extend their use. Moreover, it focuses also on the research of a biocompatible and functionalized scaffold suitable for cell growth, able to stimulate regeneration and including curative substances and/or cell derivatives such as extracellular vesicles. Specifically, the final objective is a model for the treatment of diabetic foot ulcers (DFUs), complications of diabetes with strong economic and social impact. Wound healing could be altered in diabetic people, making the treatment of DFUs quite difficult and leading to the amputation of the interested area. The use of specific scaffolds and cytotypes, according to the severity and the features of DFUs, is a promising attempt to heal these lesions causing higher health expenses and disability.

Biodosimetry is crucial for assessing the health effects of exposure to ionizing radiation by measuring the absorbed dose through biological markers. While many studies exist on individuals exposed to high doses, research on the effects of low doses is lacking. Human research is impractical, and using animal models is complex due to ethical concerns and the difficulty of obtaining significant data. This research aims to better understand the consequences of ionizing radiation using radiobiology and biodosimetry methods on plants, specifically focusing on the genotoxic effects of radiation through the Allium cepa (onion) model. The micronucleus test and mitotic index analysis are employed to evaluate genotoxicity. The results indicate that Allium cepa is a valid biodosimeter, showing a significant increase in micronuclei with alpha radiation compared to X-rays, with a 20-fold increase at the same dose. Allium cepa offers a practical and ethical choice for genotoxicity evaluation due to its similarities with human cytogenetic reactions and the feasibility of working with an almost unlimited number of samples. This work demonstrates the potential of Allium cepa as a sensitive indicator of genotoxic stress and as a reliable model for research on the biodosimetry of ionizing radiation. The consistent reactions of Allium cepa to genotoxic stress, similar to human cytogenetic responses, making it a valuable tool for future studies in radiation exposure assessment.

Extracellular membrane vesicles (eMVs) are nanosized structures whose cargo consist of macromolecules (i.e. proteins and enzymes, deossiribonucleic and ribonucleic acids) and molecules (i.e. amino acids, sugars, short chain fatty acids, bioactive compounds) that are mainly involved in cell-to-cell communication and in the regulation of different trans-kingdom molecular mechanisms. Studies on eMVs derived from plants and bacteria are increasing due to their multiple possible roles in different and crucial biological processes, as sources of novel bioactive compounds and macromolecules, and as drug carriers in biotechnological applications. Thus, the aim of my research project is the isolation, characterization of cargo, and elucidation of biological activities of eMVs produced by two different biological systems: i) grapevine (Vitis vinifera), a plant known to be a prolific source of antioxidant molecules whose action is associated with the prevention and/or slowing down of human diseases, like tumors and neurodegenerative diseases; ii) the plant growth promoting bacterial strain Streptomyces violaceoruber, a filamentous Gram-positive bacterial specie belonging to the phylum of Actinobacteria that are known to be prolific sources of bioactive compounds like antibiotics. The preliminary data are focused on isolation protocols of V. vinifera and S. violaceoruber eMVs, their characterization and the elucidation of their biological effects.

Obesity results from the complex interplay of environmental, genetic and epigenetic factors. Among these, DNA methylation has emerged as a key mechanism regulating gene expression, particularly in pathways linked to adipogenesis. This study explores the methylation status of the PTHLH gene proposed as a potential biomarker for obesity-related metabolic dysfunction. Previous studies suggest a dual role for PTHrP, the protein encoded by PTHLH gene, in the processes of browning and whitening of adipose tissue (BAT and WAT), both of which are crucial in the development of obesity. We investigated DNA methylation at specific CpG sites within PTHLH gene promoters in peripheral blood mononuclear cells (PBMCs) before and after dietary iron supplementation. Our results revealed that there is a demethylation for the majority of subjects in PTHLH gene promoters. Furthermore, we established a protocol to isolate adipose-derived mesenchymal stem cells (ADSCs) to study DNA methylation in these cells as an in vitro model, aiming to better understand the epigenetic regulation of adipogenesis. These findings suggest that DNA methylation changes in the PTHLH gene could serve as novel biomarkers for obesity risk and open new perspectives for personalized diet targeting epigenetic modifications in the prevention and treatment of metabolic disorders.

Cross-cell communication is a key feature of cancer progression and metastasis. Increasing interest is focusing on the role of extracellular vesicles (EVs), as key mediators able to transfer bioactive factors both in pathological/physiological conditions. [1] EVs contain bioactive cargo, including proteins, lipids, RNA, DNA, and other factors, such as P-glycoprotein (P-gp), involved in acquired multidrug resistance (MDR) [2]. The horizontal transfer of resistance from MDR to sensitive cells by EVs is widely demonstrated and constitutes a pharmacological target. The aim of my work was determine the effect of two natural drugs, curcumin and heptacosane in the regulation of resistance transfer mediated by EVs in two MDR cancer model: MCF-7R cells (breast cancer) and HL-60R cells (acute myeloid leukemia). WB analysis confirmed the presence of P-gp only in the EVs from MDR lines. The transfer of P-gp through EVs was verified, by immunofluorescence assay, using three co-colture methods. Analysis of P-gp expression showed that EVs-mediated transfer is able to induce phenotypic changes in sensitive cells. Both natural molecules revert acquired resistance and restore the sensitivity of recipient cells. Data observed with natural drugs showed as they are able to counteract EVs process, this take on a relevant importance and could have clinical impact.

Multidrug resistance (MDR) is a major problem for cancer treatment. In fact, overcoming MDR still represents a challenge for oncology research worldwide [1]. The aim of this study was to investigate the inhibitory effect of two natural compounds (phytol and lupeol) on the activity of P-glycoprotein (P-gp), a cell membrane transporter involved in drug resistance in several tumors, including hepatocellular carcinoma (HCC) and breast cancer (BC) [2]. P-gp level in different cell lines of an acquired MDR model (HCC) was evaluated by qPCR, flow cytometry and western blot. Among those analyzed, cell line with the highest P-gp expression is HepG2, the one with the lowest is SNU-475; HuH-7 have an intermediate P-gp expression. The effect of the two natural substances was tested on the 3 selected HCC cell lines and on cell lines of a model of acquired resistance (BC), MCF-7 (low P-gp level) and MCF-7R (high P-gp level) cells. Cell viability assays (MTS) demonstrated the low toxicity of phytol and lupeol administered individually and to show the advantage, in terms of increased cytotoxicity of anticancer drugs substrates of the efflux pump (P-gp), when associated with these molecules. Lupeol appears to be the compound that most enhances the effect of the anticancer drugs doxorubicin and paclitaxel in MCF-7R cells and sorafenib and lenvatinib in HCC cells. The mechanism of action probably depends on P-gp inhibition in the acquired resistance model but not in the innate resistance model.

Mitral regurgitation (MR) is one of the most common and severe valvulopathies worldwide. One of the leading causes is leaflet prolapse due to the elongation or rupture of the Chordae Tendineae (CT), tendon-like structures preventing backflow in the atrioventricular valves. In my research, I apply a mandrel-less electrodeposition technique to fabricate engineered CT, BioChords, that mimic the structure and mechanics of native CT. BioChords are designed to provide mechanical support to the mitral valve (MV) apparatus and promote progressive tissue regeneration. BioChords were fabricated via mandrel-less electrodeposition and analyzed using Scanning Electron Microscopy (SEM). The SEM images were post-processed to determine the fibers’ orientation index (OI), which quantifies the anisotropy of a network. The mechanical properties of BioChords were also assessed using Uniaxial Tensile Testing (UTT), focusing on the Initial Modulus (IM). The results demonstrated that BioChords fabrication is a versatile technique, allowing the production of cylindrical scaffolds using a plethora of polymers (biodegradable and non-biodegradable), while maintaining the scaffold’s macrostructure and microarchitecture. The OI of 0.82±0.11 indicates a highly aligned fibrous network. UTT showed that BioChords can reach an IM of 88.15±12 MPa, comparable to that of human CT (91.05±6 MPa). These findings support the potential suitability of BioChords for MV repair applications.

DNA methylation is an epigenetic modification that allows chromatin condensation and thereby regulates gene transcription and chromatin structure. Loss of DNA methylation at repetitive sequences, the most abundant elements of the genome, is recurrent in many types of tumors. This widespread epigenetic change could have implications beyond transcriptional regulation, since DNA methylation contributes to maintain repetitive sequences in a heterochromatic compacted state. Thus, heterochromatin disruption might affect chromatin organization in the nucleus jeopardising chromosomal stability and nuclear mechanics as well, both conditions associated with cancer. To determine the role of DNA methylation in the maintenance of nuclear mechanics and proper chromosome dynamics, our study uses a non-tumor immortalized cell line engineered to induce DNA hypomethylation (RPE-1 NGA-DNMT1) by the rapid degradation of the maintenance DNA methyltransferase DNMT1 for several cell cycles. Our results show that hypomethylated cells acquired aneuploidy and increased mitotic defects. By microscopy, we also noticed that hypomethylated nuclei exhibit enlarged nuclear size and increased nuclear blebs. Furthermore, the localization of proteins of the nuclear envelope is impaired. Altogether these findings suggest that DNA hypomethylation could undermine chromosome segregation, nuclear envelope composition, and nuclear deformability, potentially promoting cell transformation.

Investigating the antimicrobial activity of new extracts from soil actinomycetes against ESKAPE pathogens. Antibiotic resistance is a major challenge for healthcare systems. The World Health Organization reports that multi-drug resistant (MDR) strains cause over a million deaths annually. The 'ESKAPE' bacteria group causes most difficult-to-treat hospital infections, leading to high morbidity and mortality rates. This has increased the need for new antimicrobial products. Actinomycetes, Gram-positive bacteria, produce important secondary metabolites, such as antibiotics like erythromycin and streptomycin. My PhD project aims to: - identify novel actinomycetes that produce novel bioactivities using extracts, obtained using different organic solvents, - to perform Whole genome sequencing (WGS) of actinomycetes of interest to identify, through genome mining studies, previously uncharacterized natural product biosynthetic gene clusters – to evaluate the antibacterial properties against ATCC bacterial strains and ESKAPE pathogens. The deep molecular characterization of ~100 actinomycetes is ongoing. A preliminary screening led to find 48 actinomycetes having antibacterial activity. These results will be further confirmed in secondary screening assays.

Stent-graft is an artificial flexible blood conduit, designed to act as a false lumen at the site of the abdominal aortic aneurysm. It consists of a biocompatible polymeric graft  and a metal frame (stent). Polyethylene terephthalate (Dacron) and expanded polytetrafluoroethylene (ePTFE) are the main materials used for the graft, while nitinol and stainless steel for the stent. Usually, aortic stent-graft  are self-expanding device, standardized in size and shape. The effects of the implantation of the device regard the extra-stiffness of the infrarenal segment  and the geometrical alteration of the AAA flow lumen. The first effects is related to the graft materials which are stiffer than the native vessel and perturbate the Windkessel effects of the aorta, leading to long-term cardiovascular morbidity. As regard the design, the available devices are not able to accommodate the anatomical features of the patient’s aorta. The mismatch between the device and the aorta can cause leakage, endoleak and fluid dynamics alterations. To overcome these operative limitations, in collaboration with AMED, this research project aims to develop an personalized innovative cardiovascular device with a biomimetic graft able to match the biomechanical behavior of aorta. In details, I investigated the application of an electrospun synthetic materials for the graft and I introduced a preliminary study of the geometrical design of a stainless steel structure by means computational tools.

Alzheimer's disease (AD) is a neurodegenerative disorder marked by memory loss and cognitive decline, significantly impacting patients' quality of life and society. Symptoms emerge once the pathology is established, making early diagnosis and better therapies essential. This study aims to address these needs with two objectives: i) profiling cerebrospinal fluid (CSF) of AD patients using proteomics to identify predictive biomarkers, and ii) assessing if preconditioned mesenchymal stem/stromal cell (MSC) secretome can reduce neuroinflammation in an ex vivo AD model. AD is heterogeneous, with subtypes including a typical form with amyloid plaques and tau tangles, and an amyloid-only form without tau pathology. These suggest distinct mechanisms, requiring tailored diagnostic strategies. Machine learning on proteomic data identified 11 proteins capable of distinguishing AD subtypes from controls, with potential as biomarkers. Neuroinflammation is present in both subtypes. MSC secretome, enhanced by preconditioning with hypoxia, IFN-γ, or TNF-α, showed significant shifts in proteins involved in immune regulation and neuroprotection, highlighting therapeutic potential. These findings provide insights into AD subtypes and offer new avenues for biomarker discovery and tailored treatments.

Papillary Thyroid Cancer is the most common form of thyroid cancer. Gene fusion events lead to the formation of chimeric RET products, such as CCDC6-RET, NCOA4-RET, and KIF5B-RET, which result in a gain of oncogenic function of RET. In order to inhibit the enzymatic activity of chimeric RET, several drugs have been developed, with Selpercatinib being the latest generation. However, cases of chemoresistance have already been documented. To understand the resistance mechanisms mediated by Selpercatinib, cell cultures of the Papillary Thyroid Cancer cell line, TPC-1, were established. The drug suppresses cell viability and the expression of Survivin; however, it unexpectedly leads to an increase in the gene expression of the chimeric construct CCDC6-RET. Subsequently, TPC-1 cells were treated with Selpercatinib to induce resistance: the gene expression of CCDC6-RET appears to be increased again compared to the non-resistant control, and the expression of Survivin is almost comparable to the non-resistant control. As Berberine acts as a negative modulator of wild-type RET gene expression, a series of in silico analyses were conducted, demonstrating that the nutraceutical’s DNA binding sites can be mapped to the promoter sequence of CCDC6-RET. However, the nutraceutical did not show effects in vitro on the cell viability of resistant cells. Further in silico analyses were therefore focused on understanding the key transcription factors interacting with the CCDC6-RET promoter.

Melanin pigment, a high-molecular-weight biopolymer, is known for its unique physicochemical properties, such as UV radiation protection, antimicrobial activity, and its role in material synthesis. Recent advancements in melanin-based materials have demonstrated significant potential for bio-applications, particularly in the healthcare sector. Moreover, incorporating metal ions, like copper ions (Cu²⁺), through non-covalent interactions allows for fine-tuning melanin-based material properties, amplifying their application spectrum. Thus, this study uses cell-free spent medium from the Kitasatospora sp. SeTe27 strain, which is known for producing melanin-like metabolites. Also, copper sulfate was used to dope melanin-like compounds, generating copper-melanin formulations (CM-Fs). UV-Visible analysis was performed to indicate melanin's presence within the synthesized material. The CM-F antimicrobial efficacy was evaluated against the Staphylococcus aureus strain, revealing that CM-F promoted oxidative damage by inducing reactive oxygen species production and depleting the intracellular pool of reduced thiols. These findings suggest that copper-doped melanin-based materials significantly enhance their antimicrobial potential, making them promising candidates for biomedical applications.