Toward a 3D in vitro model based on decellularized thymus to maintain adult thymic ephitelial cells functionality

During my PhD,I have been develop an innovative technique to reproduce in vitro the 3D thymic microenvironment, to be used for growth and differentiation of thymocytes, and possible transplantation replacement in conditions of depressed thymic immune regulation. The work has been developed in the laboratory of Tissue Engineering at the University Hospital in Basel, Switzerland, under the tutorship of Prof.Ivan Martin. Since a number of studies have suggested that the 3D structure of the thymic microenvironment might play a key role in regulating the survival and functional competence of thymocytes, I’ve focused my effort on the isolation and purification of the extracellular matrix of the mouse thymus. Specifically, based on the assumption that TEC can favour the differentiation of pre-T lymphocytes, I’ve developed a specific decellularization protocol to obtain the intact, DNA-free extracellular matrix of the adult mouse thymus. Two different protocols satisfied the main characteristics of a decellularized matrix, according to qualitative and quantitative assays. In particular, the quantity of DNA was less than 10% in absolute value, no positive staining for cells was found and the 3D structure and composition of the ECM were maintained. In addition, I was able to prove that the decellularized matrixes were not cytotoxic for the cells themselves, and were able to increase expression of MHC II antigens compared to control cells grown in standard conditions. I was able to prove that TECs grow and proliferate up to ten days on top the decellularized matrix. After a complete characterization of the culture system, these innovative natural scaffolds could be used to improve the standard culture conditions of TEC, to study in vitro the action of different factors on their differentiation genes, and to test the ability of TECs to induce in vitro maturation of seeded T lymphocytes.

In vitro application of biocontrol agents to improve the safety and quality of grapevine products

Pathogenic fungi are responsible for vine diseases affecting the grapevine yield and the organoleptic quality of the final wine products. Using of biocontrol agents can represent a sustainable alternative to the use of synthetic fungicides whose intense use can have negative effects on the ecosystem and cause increase resistant pathogen population to synthetic agents. The principal aim of my PhD thesis was the isolation and characterization of new yeast strains and Bacillus subtilis SV108 as biocontrol agent and the comprehension of the mechanism of their antimicrobial action. Accordingly, twenty wild yeast and one selected bacterium isolated among 62 samples, isolated from different Italian and Malaysian regions and molecularly identified, were evaluated in a preliminary screening test on agar. Results showed the highest effects on inhibiting mycelial growth by Starmerella bacillaris FE08.05, Metschnikowia pulcherrima GP8 and Hanseniaspora uvarum GM19. On the other side, Bacillus subtilis SV108 showed the ability of inhibit the mycelial growth of selected fungi by producing antimicrobial compounds on Malt Extract Broth medium recovered by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by electrospray ionization (ESI) tandem mass spectrometer Triple TOF 5600. Moreover, in order to analyze the volatile fraction of compounds, the quantitative analysis of the VOCs profiles was performed by GC/MS/SPME. The analysis highlighted the presence of isoamyl and phenylethyl alcohols and an overall higher presence of low-chain fatty acids and volatile ethyl esters. All the data collected suggest that the tested yeasts, found among the epiphytic microbiota associated with grape berries, can be potentially effective for the biological control of pathogenic moulds. On the other hand, the proteomic study conducted on B. subtilis SV108 revealed that there are two cyclic antifungal peptides which can explain the antimicrobial effect of Bacillus subtilis SV108 acting as biocontrol agent against fungal pathogens in grapevine.