Titolo della tesi: TARGETING MITOCHONDRIAL HOMEOSTASIS WITH SMALL MOLECULES: FROM ENVIRONMENTAL CONTAMINANTS TO INHIBITORS OF NUCLEOTIDE RE-CYCLING
Mitochondria are highly dynamic organelles that play an essential role in cellular energy production, supporting key biosynthetic and metabolic processes necessary for cell growth, maintenance, and survival. These organelles possess their own genome (mtDNA) and machinery for replication and transcription, with mitochondrial function relying on the balanced synthesis and degradation of mtDNA and mitochondrial RNA (mtRNA). Disruptions in mitochondrial homeostasis can lead to significant redox imbalances, contributing to the onset of various diseases.
This thesis focuses on two specific factors that could influence cellular redox homeostasis with broader implications for human health: (1) exogenous environmental contaminants such as bisphenol A (BPA), perfluorooctanoic acid (PFOA), imidacloprid (IMI), and phenmedipham (Phen); and (2) the endogenous RNA-binding protein REXO2, an exoribonuclease involved in mitochondrial RNA metabolism.
Humans are continuously exposed to various anthropogenic environmental pollutants, with BPA and PFOA being industrial contaminants and IMI and Phen categorized as agrochemicals. These substances can accumulate in soil and water, bioaccumulating in organisms, and their combined effects remain underexplored. In first part of the study, two cellular models are employed: HepG2, a human liver cancer cell line widely used to study xenobiotic toxicity, and HaCaT, an immortalized keratinocyte cell line relevant for research on the skin's barrier against environmental pollutants. Using sublethal concentrations of each contaminant, this work investigates their effects on mitochondrial homeostasis, with a focus on nitro-oxidative stress. Techniques such as reactive oxygen/nitrogen species (ROS/RNS) detection, nitro-oxidative stress protein expression, mitochondrial membrane potential measurement, and mitochondrial respiration analysis are employed.
REXO2, on the other hand, plays a crucial role in mitochondrial RNA degradation by eliminating small RNA fragments (nanoRNAs), thereby maintaining nucleotide recycling and mitochondrial homeostasis. Dysregulation of REXO2, whether through overexpression or depletion, can disrupt this balance, affecting mitochondrial function. REXO2 is notably overexpressed in prostate cancer, one of the most prevalent cancers worldwide. To study REXO2's role in prostate cancer cells, two cell lines with different metastatic potentials (DU145 and PC3) are used. Transfection techniques are optimized for both REXO2 silencing (via siRNA) and overexpression, allowing the assessment of cell morphology, viability, and mitochondrial respiration. Based on these results, potential REXO2 inhibitors are further evaluated for their effects on mitochondrial membrane potential and overall mitochondrial function.
This research provides novel insights into the mechanisms by which environmental contaminants and REXO2 dysregulation contribute to mitochondrial homeostasis, with implications for human health and disease.