This experimental thesis project, conducted at the Istituto Cardiocentro Ticino (EOC) in Bellinzona, aimed to develop and validate a molecular system for the selective induction of mitophagy in cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs), within a context of premature cellular senescence. The approach is based on the use of intrinsically disordered regions (IDRs), capable of stimulating mitochondrial autophagy through the direct recruitment of the LC3 protein, fused to mitochondrial proteins. Three plasmid constructs (MITOC, MITOF, MITOFD) were designed and subsequently converted into lentiviral vectors containing a cardiomyocyte-specific promoter (cTnT), in order to ensure stable and efficient expression. Senescence was induced through treatments with D-galactose, doxorubicin, and a genetic model of progeria (HGPS). The effectiveness of the system was assessed using confocal microscopy and automated image analysis with the LysoQuant software, revealing significant activation of mitophagy in the D-Gal model and, to a lesser extent, in the Doxo and HGPS models. The results support the hypothesis that mitophagy blockade represents a common mechanism in cellular senescence. The MITOFD system proved effective in restoring mitochondrial degradation, laying the foundation for potential therapeutic strategies aimed at maintaining mitochondrial homeostasis in aged cardiac tissues. As a future development, the use of inducible promoters responsive to senescence markers is proposed to increase the specificity and clinical translatability of the approach.
Il presente progetto di tesi sperimentale, condotto presso l’Istituto Cardiocentro Ticino (EOC) di Bellinzona, ha avuto come obiettivo lo sviluppo e la validazione di un sistema molecolare per l’induzione selettiva della mitofagia in cardiomiociti derivati da cellule staminali pluripotenti indotte (hiPSC), in un contesto di senescenza cellulare precoce. L’approccio si basa sull’impiego di regioni intrinsecamente disordinate (IDR), capaci di stimolare l’autofagia mitocondriale mediante il reclutamento diretto della proteina LC3, fuse a proteine mitocondriali. Tre costrutti plasmidici (MITOC, MITOF, MITOFD) sono stati progettati e successivamente convertiti in vettori lentivirali contenenti un promotore specifico per cardiomiociti (cTnT), al fine di garantire un’espressione stabile ed efficiente. La senescenza è stata indotta attraverso trattamenti con D-galattosio, doxorubicina e un modello genetico di progeria (HGPS). L’efficacia del sistema è stata valutata tramite microscopia confocale e analisi automatizzata delle immagini con il software LysoQuant, evidenziando una significativa attivazione della mitofagia nel modello D-Gal e, in misura minore, nei modelli Doxo e HGPS. I risultati supportano l’ipotesi che il blocco della mitofagia rappresenti un meccanismo comune nella senescenza cellulare. Il sistema MITOFD si è dimostrato efficace nel ripristinare la degradazione mitocondriale, ponendo le basi per potenziali strategie terapeutiche mirate al mantenimento dell’omeostasi mitocondriale nei tessuti cardiaci invecchiati. Come sviluppo futuro, si propone l’utilizzo di promotori inducibili sensibili a marcatori di senescenza per aumentare la specificità e la traslabilità clinica dell’approccio.
Potenziare il Turnover Mitocondriale per Alleviare la Senescenza Indotta dallo Stress nei Cardiomiociti Umani
ZEGLIO, SILVIA
2024/2025
Abstract
This experimental thesis project, conducted at the Istituto Cardiocentro Ticino (EOC) in Bellinzona, aimed to develop and validate a molecular system for the selective induction of mitophagy in cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs), within a context of premature cellular senescence. The approach is based on the use of intrinsically disordered regions (IDRs), capable of stimulating mitochondrial autophagy through the direct recruitment of the LC3 protein, fused to mitochondrial proteins. Three plasmid constructs (MITOC, MITOF, MITOFD) were designed and subsequently converted into lentiviral vectors containing a cardiomyocyte-specific promoter (cTnT), in order to ensure stable and efficient expression. Senescence was induced through treatments with D-galactose, doxorubicin, and a genetic model of progeria (HGPS). The effectiveness of the system was assessed using confocal microscopy and automated image analysis with the LysoQuant software, revealing significant activation of mitophagy in the D-Gal model and, to a lesser extent, in the Doxo and HGPS models. The results support the hypothesis that mitophagy blockade represents a common mechanism in cellular senescence. The MITOFD system proved effective in restoring mitochondrial degradation, laying the foundation for potential therapeutic strategies aimed at maintaining mitochondrial homeostasis in aged cardiac tissues. As a future development, the use of inducible promoters responsive to senescence markers is proposed to increase the specificity and clinical translatability of the approach.| File | Dimensione | Formato | |
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TESI STUDENTESSA SILVIA ZEGLIO PDF-A.pdf
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Descrizione: Sviluppo di un sistema molecolare in grado di stimolare la mitofagia in cardiomiociti senescenti derivati da hiPS.
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https://hdl.handle.net/20.500.14239/30193