Undergraduate honors paper--Mount Holyoke College, 2005. Dept. of Chemistry. / Includes bibliographical references (leaves 107-111).

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008. / Wartell, Roger, Committee Member ; Bunz, Uwe, Committee Member ; Doyle, Donald, Committee Member ; Fahrni, Christoph, Committee Member ; Schuster, Gary, Committee Chair.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 161-174).

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Η αδειοδότηση εξασφαλίζει τη χωροχρονική ρύθμιση της αντιγραφής του γενετικού υλικού. Στα ευκαρυωτικά κύτταρα, η πρωτεΐνη Cdt1 καθορίζει πότε θα λάβει χώρα η αδειοδότηση και η έκφρασή της είναι αυστηρώς ρυθμισμένη, διαμέσου πολλαπλών μονοπατιών. Διατάραξη της ισορροπίας της ρύθμισης της αντιγραφής οδηγεί σε γονιδιωματική αστάθεια, ανεξέλεγκτο πολλαπλασιασμό ή σε κυτταρικό θάνατο. Γονιδιωματική αστάθεια σε ένα οργανισμό επίσης προκαλείται από βλάβες στο γενετικό υλικό, είτε εξαιτίας περιβαλλοντικών παραγόντων, είτε εξαιτίας τυχαίων αλλαγών που συμβαίνουν κατά τη διάρκεια του μεταβολισμού του. Για την αντιμετώπιση των βλαβών έχουν εξελικτικά προκύψει επιδιορθωτικοί μηχανισμοί εξειδικευμένοι στην αντιμετώπιση κάθε τύπου βλάβης. Ο παράγοντας Cdt1 φαίνεται να διασυνδέει τα μονοπάτια της αδειοδότησης της αντιγραφής με αυτά της απόκρισης σε βλάβη στο DNA, γεγονός που τον καθιστά ενδιαφέροντα για περαιτέρω μελέτη. Στο πρώτο μέρος της μελέτης θα γίνει χαρακτηρισμός μια μεθόδου που προκαλεί ολική ή εντοπισμένη βλάβη στο γενετικό υλικό κυττάρων με χρήση υπεριώδους ακτινοβολίας. Με τη τεχνική αυτή θα μελετηθεί σε καρκινικές σειρές η πρωτεΐνη Cdt1 και η ταχύτητα πρωτεόλυσής της.Σε μικροσκόπιο φθορισμού θα μελετηθεί ο εντοπισμός και σε συνεστιακό μικροσκόπιο η κινητική της πρωτεΐνης αυτής, καθώς και η μεταλλαγμένη μορφή Cdt1 (Cdt1+4A), η οποία δεν αλληλεπιδρά με το πρωτεολυτικό μηχανισμό Cul4-DDB1Cdt2,. Η μελέτη της κινητικής γίνεται σε ζωντανά κύτταρα καρκινικών σειρών με σκοπό την κατανόηση της ιεράρχησης των γεγονότων που συμβαίνουν σε περιοχές στοχευμένης βλάβης από υπεριώδη ακτινοβολία στο γενετικό υλικό. Με την τεχνική επαναφοράς φθορισμού μετά από φωτολεύκανση (Fluoresent Recovery After Photobleaching) θα παρακολουθηθεί η επαναφορά του σήματος με σκοπό την ποσοτικοποίηση της δεσμευμένης πρωτεΐνης. Στο δεύτερο μέρος της μελέτης θα αναπτυχθούν μέθοδοι κανονικοποίησης και ανάλυσης 6 πειραματικών δεδομένων λειτουργικής μικροσκοπίας με εργαλεία το οποία αναπτύχθηκαν ειδικά για την ανάλυση πειραμάτων FRAP (easyFRAP). Επίσης αναλύεται η δημιουργία κατάλληλου λογισμικού και η βελτιστοποίηση τεχνικών παρατήρησης της επαναφοράς του φθορισμού μετά από μεγάλα χρονικά διαστήματα, της τάξεως των ωρών έναντι της κλασικής μεθόδου όπου η παρατήρηση διαρκεί κάποια δευτερόλεπτα. Τέλος ακολουθεί σύγκριση της εφαρμογής easyFRAP με την εφαρμογή FRAPcalc, η οποία χρησιμοποιείται επίσης για την κανονικοποίηση και οπτικοποίηση δεδομένων FRAP. / The process of licensing of DNA replication ensures that the cell cycle proceeds to timely regulated replication. In metazoa, Cdt1 is the factor that ensures the regulation of licensing. Cdt1 is tightly regulated through various biological pathways and it is functionally conserved through evolution. If the regulation of Cdt1 is disturbed, genomic instability, uncontrolled replication and apoptosis may occur. Genomic instability may also occur in an organism after DNA damage, either due to environmental factors, or due to random mutations. Evolution has provided cells with various DNA damage response mechanisms for all kinds of damages. The cell cycle regulatory protein Cdt1 has been postulated to link the cell cycle and the DNA damage responses, therefore Cdt1 is a very interesting protein for further studying. At the first part of our study we introduced a method for total or localized DNA damage in live cells with the use of ultraviolet radiation. With this technique we showed that on different cancer cell lines the protein Cdt1, accumulate fast in the sites of damage. With a fluorescent microscope we studied the localization and with a confocal microscope we investigated the kinetics of wild type Cdt1 linked with a green fluorescent protein tag, along with the study of a Cdt1 mutant (Cdt1+4A) again linked with a green fluorescent protein tag. Cdt1+4A has lost the ability to associate with the proteolytic mechanism of Cul4-Ddb1Cdt2. In live cells, in order to investigate the spatiotemporal regulation of DNA damage response we used Fluorescent Recovery after Photobleaching (FRAP) technique and we studied the protein kinetics and we quantified the percentage of the bound protein on the chromatin. At the second part of our study, we present the normalization method we used for our raw experimental data. For the purpose of this analysis, tools and softwares were developed for accurate normalization. We also aimed to optimize a technique for the observation of fluorescent recovery after photobleaching during long periods of time, in contrast with the typical short FRAP experiments. Finally, we describe a brief comparison between the FRAP analysis tool we developed(easyFRAP) and another software commercially available (FRAPcalc) for that purpose.

No description available.

No description available.

The effect of FdUrd, zeocin, caffeine and their combination on the cell cycle of green algae Chlamydomonas reinhardtii and Scenedesmus quadricauda and response of these model organisms to DNA damage were studied. Both, FdUrd and zeocin, caused DNA damage that led to cell cycle arrest in these algae. In contrast, caffeine partially abolished G2 phase block imposed by zeocin. Protein levels of three crucial cell cycle regulators - CdkA, CdkB and Wee1 kinases were measured to identify mechanisms controlling reaction to DNA damage.

The human thyroid epithelial cell HTori-3 has been transformed with doses of either chronic and acute x-rays or strontium beta particles. Models of the past have relied upon animal cell systems to mimic in vitro carcinogenesis. The HTori-3 system hoped to overcome the limitations associated with these types of models by using a human thyroid cell line immortalised with the SV40 virus. HTori-3 human thyroid epithelial cells were irradiated in vitro, passaged and then transplanted into nude mice. Tumours that grew over a 2-6 month period were excised and re-established in culture. Samples were stored and all tumours were taken for histological examination. Chromosome spreads confirmed the human nature of all tumours. Following exposure to acute x-rays in the range of 0.25-2.0 Gy 13 tumours were observed in 25 recipients. Following 0.25-2.0 Gy of chronic x-rays 10 tumours from 25 recipients were observed. From a single 2 Gy exposure of strontium beta particles 3 primary tumours from 5 recipients were observed. The largest of these was re-transplanted in nude mice resulting in 100% incidence. All tumours were classified as undifferentiated anaplastic carcinomas. A small number of tumours were observed in the control cell lines, these may be the result of a general instability found with the partial transformed parental cell line. All 2Gy tumours and those previously established from this laboratory after alpha or gamma radiation were used to test for the presence of the delayed lethal death phenotype. A number of cell and molecular endpoints were used. These included plating efficiency, cell adherence, micronucleus formation and p53 status. In all incidences, the reproductive viability of irradiated cells was below that of non- irradiated cells at up to 4 weeks post-irradiation. The HTori-3 cell line and the techniques used to study the delayed effects of radiation may be applicable to other cell systems and may be a useful model to study the long-term effects of radiation induced genomic instability.

Genetically identical cells show a heterogeneous response to a multitude of signals such as growth factors and DNA damage. While this heterogeneity has been shown to be a major determinant of treatment success in several diseases including cancer, little is known about how differences in biochemical signalling networks underlie such heterogeneity. State-of-the-art methodologies to study biochemical networks are often invasive and enable to quantify biochemical events only on cell populations or at a single point in time for a single cell, and therefore, cannot adequately quantify the fast, asynchronous and heterogeneous responses. In order to address these limitations, we have developed a unique sensing platform based on fluorescence lifetime imaging microscopy (FLIM) capable to multiplex at least three biosensors by utilizing Förster Resonance Energy Transfer (FRET) efficiently. After an overall introduction in Chapter 1, I describe the rational design and characterization of novel FRET pairs aiming to utilize the visible spectrum efficiently in combination with FLIM in Chapter 2. We combined blue, green and red donor fluorescent proteins that are excited at the same wavelength (840 nm for two-photon excitation) with genetically encoded quenchers, i.e. non-fluorescent chromoproteins as acceptors. This sensing platform enables the simultaneous detection of three biochemical reactions within single living cells providing new opportunities to characterize and understand non-genetic heterogeneity. In Chapter 3, I will demonstrate the first application of this novel platform by studying the activity of three key enzymes in DNA damage-induced cell death, caspase-2, -3, and -9. We confirm the heterogeneous nature of Cisplatin-induced cell death in genetically identical cells but reveal the existence of at least three subpopulations of cells characterized by distinct caspase dynamics. By combining biochemical and morphological information we infer the existence of different biochemical network topologies that are associated with alternative death phenotypes each cell adopts, such as apoptosis and programmed necrosis. Finally, deconvolution of cellular populations and direct measurement of a three-node caspase network - formerly impossible - permitted us to design perturbations of cell fate choices utilizing clinically relevant inhibitors. These perturbations resulted in changes in cell fate in response to Cisplatin, a clinically desirable outcome that suggests new avenues for combinatorial drugging and a new strategy to reveal cancer vulnerabilities that may be otherwise confounded by typical genetic and non-genetic heterogeneity.