Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Author:
Alexopoulou Eirini
Dissertation committee:
Βουλγαρέλης Μιχαήλ, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Βλαχογιαννόπουλος Παναγιώτης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Θάνος Δημήτρης, Ακαδημαϊκός Ερευνητής Α΄, Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών
Μόσιαλος Γεώργιος, Καθηγητής, Αριστοτέλειο Πανεπιστήμιο Θεσσαλονίκης
Κλινάκης Απόστολος, Ερευνητής A,΄ Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών
Ξάνθου-Τσιγκόγλου Γεωργία, Ερευνήτρια Β΄, Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών
Καψογεώργου Ευσταθία, Επ. Καθηγήτρια, Iατρική Σχολή, ΕΚΠΑ
Original Title:
Μοριακοί μηχανισμοί κυτταρικού επαναπρογραμματισμού
Translated title:
Molecular mechanisms of cellular reprogramming
Summary:
The present doctoral dissertation studies the mechanisms regulating the inducible gene expression of eukaryotic cells at whole genome level and more specifically, the identification of molecular switches, which based on the coordinated cells response, they control the function of enhancers in different cell types before and after virus infection. The characterization of enhancers that respond to viral infections in the human genome is required in order to understand the coordination of the immune response and the molecular characteristics that range from the binding of transcription factors, co-activators, modifiers, and chromatin remodellers to transcription activation. Our research was based on a pioneering and holistic approach of the phenomenon of virus infection, the application of which combines all the data derived from a variety of genomic techniques. More than 150 analyses of RNA-seq, ChIP-seq, DNaseI-seq, FAIRE-seq, STARR-seq methods and multi-level bioinformatics analyses were performed, which have as a common method of analysis the Next Generation Sequencing (NGS) technology. In order to compare the difference expression regulatory programs during virus infection, our study was performed on different cell types, at HeLa epithelial cells and Namalwa B-lymphocytes, which were transfected with Sendai virus as an environmental stimulus. Chromatin characteristics, epigenomic profiling, as well as transcription machinery involvement in genomic coordinates, where enhancers are located and a large number of well-characterized anti-viral genes, led us to the construction of a "molecular-digital encyclopedia" of genes and enhancers. The encyclopedia is characterized and can reveal a high potential for anti-viral properties and markers, and is called Human-ATLAS-of-Virus-Infection. The finding of Human-ATLAS were further validated by applying the ChIP-STARR-seq method. This innovative, large-scale method allow us to simultaneously examine the virus inducible activity of the numerous binding sites of IRF3 and p65, the transcriptional regulators of the anti-viral response. Sequence-based analyses have revealed that functional anti-viral enhancers containing IRF motifs are highly conservative throughout evolution, as they are found in the genomes of different species representing vertebrates, invertebrates, plants, and even microbes. These repeat sequences are the first examples of repeating sequences with primary origin for anti-viral response regulation. In order to systematically study the function of the conserved sequences from the above different organisms in human cells, and also in order to determine whether these sequences also show conserved enhancer activity, we cloned them into STARR vectors. HeLa cells were transfected and we monitored their ability to activate the expression of the GFP reported gene in vivo. This is one of the few times that enhancers are conserved in different phyla, and primordial sequences appear to function as the regulators of inducible gene expression during the anti-viral cellular response.Based on the above, the completion of this dissertation leads to a detailed observation of the mechanism used by human cells to respond during viral infections. It also lays the groundwork for further investigation as well as for the discovery of unknown elements that participate in this process. Decoding the regulatory logic behind genome remodeling after viral infection will lead to a deeper understanding of the basic principles of genome organization and the ability to predict patients' response to treatment and prevention with the ultimate goal of being applied on an individual basis.
Main subject category:
Health Sciences
Keywords:
Viral infections, Inducible gene expression, Regulation mechanisms of inducible gene expression, Enhancers, Next generation sequencing, RNA-sequencing, DNaseI-sequencing, FAIRE-sequencing, Chip-sequencing, STARR-sequencing
Number of references:
249
