Post-graduate theses
Current Record: 16 of 802
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Identifier |
000460185 |
Title |
Epitranscriptomic control of DNA damage induced R-loops |
Alternative Title |
Επιμεταγραφικός έλεγχος των επαγωμένων από βλάβες στο DNA δομών R-loops |
Author
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Πατεράκη, Νικολέτα Ι.
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Thesis advisor
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Ντίνη, Ευγενία
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Reviewer
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Βιδάκη, Μαρίνα
Γαρίνης, Γεώργιος
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Abstract |
The “epitranscriptome” refers to the total pool of biochemical modifications of RNA within
a cell that do not alter the sequence itself. The most abundant RNA modification on RNA
transcripts is N6-methyladenosine (m6A), which plays a regulatory role in many biological
processes, including transcription, splicing and RNA metabolism. Recent studies have
focused on unraveling potential roles of m6A in response to various stress factors, including
those that induce DNA damage. However, the mechanisms underlying the connection
between m6A, and DNA damage response have not been fully elucidated. In this project, I
aim to characterize the potential role of m6A in response to DNA damage and its involvement
in regulating R-loop formation and/or resolution. To resolve this question, my primary focus
is on the nuclear m6A reader YTHDC1, which has been shown to localize to double-strand
DNA breaks. It is worth noting that this m6A reader also regulates responses to heat stress,
suggesting a potential role in DNA damage response as well. In this Master’s thesis,
bioinformatic, molecular and biochemical methods were used to approach this question.
Firstly, reanalysis of published RNA-seq data upon YTHDC1 knockdown in HeLa cells was
performed, in which several genes that take part in the DNA damage response were
identified. Consequently, experiments using DNA damage induced methods (UV radiation)
were performed to evaluate the expression levels as well as the localization of YTHDC1.
Additionally, in order to better dissect the role of YTHDC1 in response to DNA damage, a
specific protein degradation tag (dTAG) system against YTHDC1 was designed and
incorporated in HCT116 colon cancer cells. This tool enables the targeted and controlled
depletion of the protein of interest. Last but not least, the RChIP protocol was established in
order to be able to map R-loops specifically by using dRNase H fused with V5 tag. The
dRNase H has a mutation on its catalytic domain but not on the RNA:DNA binding domain,
therefore, it can recognize the R-loops without cleaving the RNA moiety. These tools will be
used in future experiments to unravel the role of YTHDC1 in stress responses.
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Language |
English |
Subject |
DNA damage |
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Epitranscriptome |
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RNA methylation |
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RNA μεθυλίωση |
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Βλάβη DNA |
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Επιμεταγράφωμα |
Issue date |
2023-11-24 |
Collection
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School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
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Type of Work--Post-graduate theses
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Permanent Link |
https://elocus.lib.uoc.gr//dlib/f/d/7/metadata-dlib-1699261074-626038-24378.tkl
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Views |
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