Risposta genetica e molecolare ai quantum dot in Saccharomyces cerevisiae

Quantum dots are colloidal semiconductor nanocrystals with peculiar optical properties, widely studied for industrial and biomedical applications. Zinc sulphide quantum dots (ZnS QDs) are commonly utilized as shell material in core-shell QD structures, being considered more biocompatible compared to other metal-based QDs. However, limited research has been conducted regarding their effects as ZnS QDs core-only. This Ph.D. thesis aims to utilize the yeast Saccharomyces cerevisiae as a model organism to investigate the genetic and molecular response associated with exposure to this type of QDs. Following preliminary analyses on ZnS QD characterization and their effects on wild type growth, the initial phase of the project focused on screening a YKO (Yeast Knock-Out) collection, comprising haploid mutants deleted in non-essential genes. The identification of sensitive mutants deleted in genes encoding for proteins localized in mitochondria, involved in response to chemicals and with oxidoreductase activity, prompted further investigation into the phenotype of mutants deleted for genes with crucial roles in maintaining redox homeostasis in mitochondria: the medium sensitive mutants sod1? and glr1? and the hypersensitive mutant pos5?. The ability of these mutants to grow on non-fermentable carbon sources supplemented with ZnS QDs has been tested, revealing that respiratory metabolism could be involved in shaping ZnS QD response. Interestingly, when growth in the presence of CdS QDs, a type of cadmium-based QD previously reported to induce higher toxicity in this model organism, they revealed different degrees of sensitivity, suggesting that different detoxification mechanisms could be activated in response to specific types of QDs. Specifically, the hypersensitivity exhibited by the pos5?, deleted for a mitochondrial NADH kinase, was specific for ZnS QDs. To further dissect its phenotype, pos5? was exposed to other positively charged ENMs, cerium oxide nanoparticles (CeO2 NPs) and ferrous ferric NPs (Fe3O4 NPs), or to the presence of ZnSO4, to assess the effect of a potential release of zinc ions. Overall, the analyses revealed that surface charge and a potential release of Zn2+ ions likely do not play significant roles in shaping the ZnS QD-hypersensitivity. Flow cytometry analyses conducted on the wild type strain and the pos5? deletion mutant did not reveal an increase in reactive oxygen species following ZnS QD treatment; however, the analyses highlighted an increase in the percentage of dead cells in pos5?. Lately, the Ph.D. project availed of another omics approach to study ZnS QD response. RNA samples were extracted from wild type strain and pos5? at two different time points after treatment with ZnS QDs and ZnSO4, to compare the early and the late responses. RNA-sequencing has been performed to analyse the transcriptional changes induced in the different tested conditions. Along with some overlap in the annotation terms enriched in the presence of ZnSO4 and ZnS QDs, this latter specifically modulates proteins associated with membrane, metal-binding proteins, and impacts protein translation processes in the wild type strains. The alteration of various biosynthetic processes in the transcriptional landscape of the untreated pos5? deletion mutant highlighted the pleiotropic effect of POS5 deletion, also revealing a significant modulation of mitochondrial proteins and proteins with oxidoreductase activity. Furthermore, the analysis revealed that exposure to ZnS QDs selectively induced modulation of genes encoding proteins involved in mitochondrial processes, metal binding, and intracellular trafficking in the pos5? deletion mutant. The peculiar modulation observed in some metallochaperones, transcription factors, and metal transporters across different strains emphasized a potential influence of ZnS QDs on metal homeostasis. Within the context of an altered regulatory profile induced by pos5? genetic background, this could contribute to the specific hypersensitivity exhibited by pos5?. Overall, this study investigated the cellular response elicited by exposure to ZnS QDs in Saccharomyces cerevisiae, availing of different omics methodologies and exploiting the distinctive sensitivity phenotypes of different deletion mutants. Saccharomyces cerevisiae has proved to be a valuable model organism for studying the intricate dynamics of ENM interaction within biological systems.