BEGIN:VCALENDAR VERSION:2.0 PRODID:Icfo X-PUBLISHED-TTL:P1W BEGIN:VEVENT UID:69d4c5db33c0a DTSTART:20240603T090000Z SEQUENCE:0 TRANSP:OPAQUE LOCATION:ICFO Auditorium and Online (Teams) SUMMARY:ICFO | CHRISTIAN KNAPP CLASS:PUBLIC DESCRIPTION:Genomic instability\, caused by DNA damage\, is the main determ inant for cancer and aging. To safeguard genomic integrity\, cells evolved complex mechanisms to ensure error-free DNA replication and DNA damage re pair. However\, cells are not always able to repair DNA damage\, and have to halt proliferation in a state of senescence\, or perform the programmed cell death\, apoptosis\, to prevent giving rise to tumors and to protect the organism. Yet\, this loss of proliferative potential ultimately leads to aging of the organism.\nThe significance of DNA damage repair is underl ined by mutations in genes encoding DNA repair proteins\, which lead to pr emature aging diseases associated with a wide spectrum of early-onset age- related diseases. Notably\, Hutchinson-Gildford progeria syndrome (HGPS)\, the most severe premature aging disease\, is not caused by mutations in a DNA repair protein\, but in the nuclear intermediate filament protein lam in A. Nonetheless\, DNA damage is considered a main driver of this disease .\nThe affected protein lamin A is a main component of the nuclear lamina\ , which is an intermediate filament meshwork and one of the layers of the nuclear envelope which surrounds the nucleus. To date\, the pathological m echanism how the mutant form of lamin A leads to DNA damage in HGPS is poo rly understood.\nHere\, we propose our hypothesis that this mutations dist urbs the interactions between the nuclear lamina and peripheral DNA in a m anner that mechanically interferes with the local progression of DNA repli cation sites. Consequently\, our hypothesis predicts that DNA damage predo minantly arises during DNA replication of peripheral DNA in close proximit y to the nuclear lamina. This creates a spatial correlation between the oc currence of DNA damage and the nuclear periphery\, as well as a temporal c orrelation with DNA replication of peripheral DNA which occurs during late S-phase of the cell cycle.\nHence\, in Chapter 3\, we present our approac h to characterize the spatiotemporal dynamics of DNA damage throughout the cell cycle. This approach employs simple reporter cell models of DNA dama ge and DNA replication\, along with long-term multi-color fluorescence liv e cell imaging microcopy\, and a quantitative analysis pipeline. This anal ysis pipeline monitors and follows cells over multiple days and quantifies DNA damage foci formed by fluorescent DNA damage repair\nproteins\, and e mploys machine learning-based algorithms to classify distribution patterns of the DNA replication protein PCNA to perform post hoc in silico synchro nization of cell cycles.\nIn Chapter 4\, we describe how we employed this approach to quantify DNA damage foci and to characterize their distributio ns throughout the cell cycle in cell models of HGPS. We conducted these ex periments under different conditions and with different cell lines\, howev er we could not detect differences between HGPS cell models and healthy co ntrols. Finally\, we discuss our findings as well as technical and biologi cal aspects of our approach in the context of literature.\nIn Chapter 5\, we present an approach which we developed to study the influence of the mu tant form of lamin A on the mobility of DNA replication sites\, and thus t o test the mechanical aspects of our hypothesis. This approach is based on single molecule tracking of the DNA replication protein PCNA. While we co uld not detect differences between HGPS cell models and healthy controls w ith this approach\, we revealed two slow mobility states of PCNA within DN A replication sites. These two mobility states are consistent the the PCNA meshwork model proposed by Boehm et al. in 2016 and may represent DNA rep lication condensates.\nFinally\, in Chapter 6\, we summarize the main resu lts of this thesis and discuss future and potential applications of our ap proaches to advance our understanding of the cell cycle-dependent dynamics of genome maintenance\, and the structural organization of DNA replicatio n sites.\n \;\nThursday June 03\, 11:00 h. ICFO Auditorium and Online (Teams)\nThesis Director: Prof Dr. Marí\;a Garcia-Parajo and Felix C ampelo Aubarell DTSTAMP:20260407T085243Z END:VEVENT END:VCALENDAR