Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease

(English) Genomic instability, caused by DNA damage, is the main determinant for cancer and aging. To safeguard genomic integrity, cells evolved complex mechanisms to ensure error-free DNA replication and DNA damage repair. However, cells are not always able to repair DNA damage, and have to halt pr...

ver descrição completa

Detalhes bibliográficos
Autor: Knapp, Christian
Formato: tesis doctoral
Fecha de publicación:2024
País:España
Recursos:Universitat Politècnica de Catalunya (UPC)
Repositorio:UPCommons. Portal del coneixement obert de la UPC
Idioma:inglés
OAI Identifier:oai:upcommons.upc.edu:2117/452670
Acesso em linha:https://hdl.handle.net/2117/452670
https://dx.doi.org/10.5821/dissertation-2117-452670
Access Level:acceso abierto
Palavra-chave:621.3 - Enginyeria elèctrica. Electrotècnia. Telecomunicacions
577 - Bioquímica. Biologia molecular. Biofísica
612 - Fisiologia
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació
Àrees temàtiques de la UPC::Ciències de la salut
id ES_17f0f453cccfb15be04a81925c5dbfb0
oai_identifier_str oai:upcommons.upc.edu:2117/452670
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
title Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
spellingShingle Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
Knapp, Christian
621.3 - Enginyeria elèctrica. Electrotècnia. Telecomunicacions
577 - Bioquímica. Biologia molecular. Biofísica
612 - Fisiologia
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació
Àrees temàtiques de la UPC::Ciències de la salut
title_short Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
title_full Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
title_fullStr Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
title_full_unstemmed Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
title_sort Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and disease
dc.creator.none.fl_str_mv Knapp, Christian
author Knapp, Christian
author_facet Knapp, Christian
author_role author
dc.subject.none.fl_str_mv 621.3 - Enginyeria elèctrica. Electrotècnia. Telecomunicacions
577 - Bioquímica. Biologia molecular. Biofísica
612 - Fisiologia
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació
Àrees temàtiques de la UPC::Ciències de la salut
topic 621.3 - Enginyeria elèctrica. Electrotècnia. Telecomunicacions
577 - Bioquímica. Biologia molecular. Biofísica
612 - Fisiologia
Àrees temàtiques de la UPC::Enginyeria de la telecomunicació
Àrees temàtiques de la UPC::Ciències de la salut
description (English) Genomic instability, caused by DNA damage, is the main determinant for cancer and aging. To safeguard genomic integrity, cells evolved complex mechanisms to ensure error-free DNA replication and DNA damage repair. 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. The significance of DNA damage repair is underlined by mutations in genes encoding DNA repair proteins, which lead to premature 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 lamin A. Nonetheless, DNA damage is considered a main driver of this disease. The 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 mechanism how the mutant form of lamin A leads to DNA damage in HGPS is poorly understood. Here, we propose our hypothesis that this mutations disturbs the interactions between the nuclear lamina and peripheral DNA in a manner that mechanically interferes with the local progression of DNA replication sites. Consequently, our hypothesis predicts that DNA damage predominantly arises during DNA replication of peripheral DNA in close proximity to the nuclear lamina. This creates a spatial correlation between the occurrence of DNA damage and the nuclear periphery, as well as a temporal correlation with DNA replication of peripheral DNA which occurs during late S-phase of the cell cycle. Hence, in Chapter 3, we present our approach to characterize the spatiotemporal dynamics of DNA damage throughout the cell cycle. This approach employs simple reporter cell models of DNA damage and DNA replication, along with long-term multi-color fluorescence live cell imaging microcopy, and a quantitative analysis pipeline. This analysis pipeline monitors and follows cells over multiple days and quantifies DNA damage foci formed by fluorescent DNA damage repair proteins, and employs machine learning-based algorithms to classify distribution patterns of the DNA replication protein PCNA to perform post hoc in silico synchronization of cell cycles. In Chapter 4, we describe how we employed this approach to quantify DNA damage foci and to characterize their distributions throughout the cell cycle in cell models of HGPS. We conducted these experiments under different conditions and with different cell lines, however we could not detect differences between HGPS cell models and healthy controls. Finally, we discuss our findings as well as technical and biological aspects of our approach in the context of literature. In Chapter 5, we present an approach which we developed to study the influence of the mutant form of lamin A on the mobility of DNA replication sites, and thus to test the mechanical aspects of our hypothesis. This approach is based on single molecule tracking of the DNA replication protein PCNA. While we could not detect differences between HGPS cell models and healthy controls with this approach, we revealed two slow mobility states of PCNA within DNA replication sites. These two mobility states are consistent the the PCNA meshwork model proposed by Boehm et al. in 2016 and may represent DNA replication condensates. Finally, in Chapter 6, we summarize the main results of this thesis and discuss future and potential applications of our approaches to advance our understanding of the cell cycle-dependent dynamics of genome maintenance, and the structural organization of DNA replication sites.
publishDate 2024
dc.date.none.fl_str_mv 2024
2024-06-03
2026
2026-02-04
dc.type.none.fl_str_mv doctoral thesis
http://purl.org/coar/resource_type/c_db06
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/doctoralThesis
format doctoralThesis
dc.identifier.none.fl_str_mv https://hdl.handle.net/2117/452670
https://dx.doi.org/10.5821/dissertation-2117-452670
url https://hdl.handle.net/2117/452670
https://dx.doi.org/10.5821/dissertation-2117-452670
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2

http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2

http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universitat Politècnica de Catalunya
publisher.none.fl_str_mv Universitat Politècnica de Catalunya
dc.source.none.fl_str_mv reponame:UPCommons. Portal del coneixement obert de la UPC
instname:Universitat Politècnica de Catalunya (UPC)
instname_str Universitat Politècnica de Catalunya (UPC)
reponame_str UPCommons. Portal del coneixement obert de la UPC
collection UPCommons. Portal del coneixement obert de la UPC
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869403952693379072
spelling Quantitative fluorescence imaging of spatiotemporal dynamics of DNA damage and DNA replication in health and diseaseKnapp, Christian621.3 - Enginyeria elèctrica. Electrotècnia. Telecomunicacions577 - Bioquímica. Biologia molecular. Biofísica612 - FisiologiaÀrees temàtiques de la UPC::Enginyeria de la telecomunicacióÀrees temàtiques de la UPC::Ciències de la salut(English) Genomic instability, caused by DNA damage, is the main determinant for cancer and aging. To safeguard genomic integrity, cells evolved complex mechanisms to ensure error-free DNA replication and DNA damage repair. 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. The significance of DNA damage repair is underlined by mutations in genes encoding DNA repair proteins, which lead to premature 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 lamin A. Nonetheless, DNA damage is considered a main driver of this disease. The 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 mechanism how the mutant form of lamin A leads to DNA damage in HGPS is poorly understood. Here, we propose our hypothesis that this mutations disturbs the interactions between the nuclear lamina and peripheral DNA in a manner that mechanically interferes with the local progression of DNA replication sites. Consequently, our hypothesis predicts that DNA damage predominantly arises during DNA replication of peripheral DNA in close proximity to the nuclear lamina. This creates a spatial correlation between the occurrence of DNA damage and the nuclear periphery, as well as a temporal correlation with DNA replication of peripheral DNA which occurs during late S-phase of the cell cycle. Hence, in Chapter 3, we present our approach to characterize the spatiotemporal dynamics of DNA damage throughout the cell cycle. This approach employs simple reporter cell models of DNA damage and DNA replication, along with long-term multi-color fluorescence live cell imaging microcopy, and a quantitative analysis pipeline. This analysis pipeline monitors and follows cells over multiple days and quantifies DNA damage foci formed by fluorescent DNA damage repair proteins, and employs machine learning-based algorithms to classify distribution patterns of the DNA replication protein PCNA to perform post hoc in silico synchronization of cell cycles. In Chapter 4, we describe how we employed this approach to quantify DNA damage foci and to characterize their distributions throughout the cell cycle in cell models of HGPS. We conducted these experiments under different conditions and with different cell lines, however we could not detect differences between HGPS cell models and healthy controls. Finally, we discuss our findings as well as technical and biological aspects of our approach in the context of literature. In Chapter 5, we present an approach which we developed to study the influence of the mutant form of lamin A on the mobility of DNA replication sites, and thus to test the mechanical aspects of our hypothesis. This approach is based on single molecule tracking of the DNA replication protein PCNA. While we could not detect differences between HGPS cell models and healthy controls with this approach, we revealed two slow mobility states of PCNA within DNA replication sites. These two mobility states are consistent the the PCNA meshwork model proposed by Boehm et al. in 2016 and may represent DNA replication condensates. Finally, in Chapter 6, we summarize the main results of this thesis and discuss future and potential applications of our approaches to advance our understanding of the cell cycle-dependent dynamics of genome maintenance, and the structural organization of DNA replication sites.(Català) Per mantenir la integritat del seu genoma, les cèl·lules han desenvolupat mecanismes complexos per assegurar una replicació del DNA sense errors i també per a reparar el dany al DNA. No obstant, les cèl·lules no sempre poden reparar el dany al DNA, i han de detenir la proliferació en un estat de senescència o induir la mort cel·lular programada, l'apoptosi, per prevenir la formació de tumors i protegir l'organisme. Aquesta pèrdua de potencial proliferatiu acaba duent a l'envelliment de l'organisme. Les malalties d'envelliment prematur estan normalment associades a mutacions en els gens que codifiquen proteïnes involucrades en la reparació del DNA. No obstant, el síndrome de progèria de Hutchinson-Gilford (HGPS, per les seves sigles en anglès), una de les malalties d'envelliment prematur més severes, està causat per una mutació en la proteïna de filament intermedi nuclear lamina A. La proteïna afectada, la lamina A, és un component principal de la làmina nuclear, una xarxa de filaments intermedis que envolta el nucli per dins. El mecanisme patològic pel qual la forma mutada de la lamina A provoca dany al DNA en HGPS encara es desconeix. En aquesta tesi proposem la nostra hipòtesi, que aquestes mutacions alteren les interaccions entre la làmina nuclear i el DNA perifèric d'una manera que interfereix mecànicament amb la progressió local dels llocs de replicació del DNA. Per tant, la nostra hipòtesi prediu que el dany al DNA arriba predominantment durant la replicació del DNA perifèric, pròxim a la làmina nuclear. Això crea una correlació espacial i temporal entre l'aparició del dany al DNA i la perifèria nuclear, així com la replicació del DNA perifèric que ocorre durant la fase S tardana del cicle cel·lular. En el Capítol 3, presentem el nostre enfocament metodològic per caracteritzar la dinàmica espai-temporal del dany al DNA al llarg del cicle cel·lular. Per això, utilitzem models cel·lulars amb marcadors de dany al DNA i replicació del DNA, i els visualitzem mitjançant tècniques de microscòpia de fluorescència multicolor en cèl·lules vives, conjuntament amb una anàlisi quantitativa. Aquesta anàlisi monitoritza i segueix les cèl·lules durant diversos dies i quantifica els focus de dany al DNA formats per proteïnes de reparació del dany al DNA marcades fluorescentment, i utilitza algoritmes d’intel·ligència artificial per classificar els patrons de distribució de la proteïna de replicació del DNA PCNA i així poder realitzar una sincronització post-hoc del cicle cel·lular. Al Capítol 4, descrivim com vam emprar aquesta metodologia per quantificar els focus de dany al DNA i caracteritzar la seva distribució al llarg del cicle cel·lular en models cel·lulars de HGPS. Tot i això, no vam poder detectar diferències entre aquests models de HGPS i els controls sans. En acabat, discutim els nostres resultats, així com els aspectes tècnics i biològics del nostre enfocament en el context de la literatura científica. En el Capítol 5, presentem un plantejament que hem desenvolupat per provar els aspectes mecànics de la nostra hipòtesi. Aquest plantejament es basa en el seguiment de molècules individuals de la proteïna de replicació del DNA PCNA. Tot i que no vam detectar diferències entre els models de HGPS i els controls sans, vam revelar dos estats de mobilitat lenta de PCNA dins dels llocs de replicació del DNA. Aquests dos estats de mobilitat són consistents amb el model de xarxa de PCNA proposat per Boehm et al. el 2016 i poden representar condensats de replicació del DNA. Finalment, en el Capítol 6, resumim els principals resultats d'aquesta tesi i discutim futures aplicacions dels nostres enfocaments i metodologies per avançar en la nostra comprensió de la dinàmica del manteniment del genoma i de l'organització estructural dels llocs de replicació del DNA en relació al cicle cel·lular.Universitat Politècnica de Catalunya20242024-06-0320262026-02-04doctoral thesishttp://purl.org/coar/resource_type/c_db06VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/doctoralThesisapplication/pdfhttps://hdl.handle.net/2117/452670https://dx.doi.org/10.5821/dissertation-2117-452670reponame:UPCommons. Portal del coneixement obert de la UPCinstname:Universitat Politècnica de Catalunya (UPC)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2http://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:upcommons.upc.edu:2117/4526702026-05-27T15:37:01Z
score 15,812429

Registos relacionados