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Zafiropoulos, Demetre (2015) Dosimetria biologica delle radiazioni ionizzanti: valutazione della dose con metodologie citogenetiche tramite la costruzione di curve di calibrazione. [Ph.D. thesis]

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Abstract (english)

The assessment of the risk for biological damage of an individual after overexposure to ionizing radiation due to an accident or a terroristic attack passes through the evaluation of the absorbed dose received from the person/s involved. This evaluation, corresponding also to how dangerous the overexposure is, using methodologies typical of the biological dosimetry, is done with the help of reference calibration curves and it presumes that all individuals exposed have the same individual radiosensitivity, which is true when the biological system into account are human lymphocytes of the peripheral blood (PBL). It supposes that a certain dose of radiation produces the same quantity of variation (chromosomal aberrations) in that biological system in all the exposed.
The goal of this Ph.D. thesis is the application of various cytogenetic methodologies to detect in a reliable and possibly fast way chromosomal aberrations non stable (dicentrics and centromeric rings) in the lymphocytes of the human peripheral blood after gamma irradiation and the construction of calibration curves dose effect able to give rapidly a response of the absorbed dose in case of radiation accident. The time needed to give a reliable response on the absorbed dose is crucial and this is especially true in radiation emergency medicine.
The objective was to standardize, develop and apply sophisticate, sensitive and reliable methodologies for the estimation of the absorbed doses after accidental gamma radiation overexposures for radiation protection purposes.
Specifically, in the present study the following were performed:
I. Application of the classic “golden standard” methodology of biological dosimetry which provides stimulation of the lymphocytes and Giemsa staining of the chromosomes at mitosis
II. Development, consolidation and application of original methodologies in biological dosimetry using fluorescence in situ hybridization (FISH) and peptide nucleic acid (PNA) probes for painting specifically chromosome telomers and centromeres
III. Application and consolidation of the method of premature chromosome condensation (PCC) where no stimulation at lymphocytes is needed
IV. Development of a fast, sensitive and reliable methodology, absolutely original in the field of biological dosimetry for dose evaluation combining the PCC method with the FISH using PNA probes for centromers and telomers
V. Construction for all the above methodologies, using in vitro experiments, of calibration curves dose-effect where unstable chromosomal aberrations where plotted against gamma dose given.
The dose interval investigated was between 0.3 Gy and 8 Gy; more than 12.500 slides with metaphases and interphases were scored using Microscopic analysis of chromosome aberrations. Dose effect curves are linear quadratic and for their construction a minimum of 500 cells or 100 dicentrics per dose were analysed. The Poisson distribution of the chromosomal aberrations was tested for every dose and the coefficients alpha and beta of the linear quadratic equation, their errors and p value were calculated.
Finally, all calibration curves obtained with the different methodologies were compared and the advantages of each methodology reported.

Abstract (italian)

Per valutare il rischio di danno biologico di un individuo dopo una sovraesposizione alle radiazioni ionizzanti, un incidente o un’azione terroristica che coinvolge le radiazioni è indispensabile valutare la dose assorbita dalla persona esposta. La valutazione della dose assorbita e quindi della pericolosità di una sovraesposizione utilizzando metodologie di tipo biodosimetrico tramite curve di riferimento, presuppone che gli individui della popolazione presentino la stessa sensibilità alle radiazioni ionizzanti. Presuppone cioè che una determinata dose di radiazioni della stessa qualità produca lo stesso numero di aberrazioni cromosomiche a tutti i membri della popolazione. E’ anche noto che tra i membri della popolazione esiste una variabilità nella radiosensibilità e che esistono persone particolarmente radiosensibili.
Argomento di questa tesi di dottorato è l’applicazione di varie metodiche citogenetiche per individuare aberrazioni cromosomiche non stabili, cromosomi dicentrici ed anelli con centromero nei linfociti T del sangue periferico (PBL) umano, formatisi in seguito ad esposizione a radiazioni ionizzanti da sorgente gamma emittente, nonché la costruzione di curve di calibrazione dose-effetto che possono, in caso di sovraesposizione, dare una risposta rapida ed affidabile sulla dose assorbita. La valutazione di tale dose, in caso di incidente, è di fondamentale importanza per la radioprotezione dei lavoratori che operano con radiazioni ionizzanti e, in caso di esposizione, per la popolazione. Di grande rilevanza è anche l’affidabilità e il tempo in cui tale valutazione è ottenuta soprattutto per gli interventi medici di urgenza.
Si è proceduto quindi allo sviluppo, consolidamento e applicazione nel campo della dosimetria biologica di metodologie originali, sensibili e affidabili di analisi citogenetica delle aberrazioni cromosomiche (AC) per la valutazione della dose ricevuta da un individuo dopo una sovraesposizione alle radiazioni ionizzanti.
Oltre all’applicazione classica della colorazione delle AC con la soluzione in Giemsa, quanto sopra riportato ha riguardato:
• sviluppo, consolidamento e applicazione di metodiche originali di dosimetria biologica utilizzando la tecnica dell’ibridizzazione fluorescente in situ (FISH), con sonde peptidiche di acido nucleico (PNA) che colorano i telomeri e il centromero dei cromosomi;
• sviluppo e applicazione della metodica della condensazione prematura dei cromosomi (PCC);
• sviluppo di una metodica veloce, sensibile ed affidabile, assolutamente originale nel campo della dosimetria biologica, per valutare in poche ore la dose ricevuta da un individuo, ottenuta combinando la metodica della PCC e la tecnica FISH che include sonde peptidiche di acido nucleico (PNA).
Con l’applicazione delle varie metodiche si è proceduto alla costruzione di apposite curve di calibrazione ove le aberrazioni cromosomiche riscontrate per cellula (esperimenti in vitro) sono riportate in funzione della dose gamma. Sono stati utilizzati i linfociti di tre donatori sani di età tra i 30 e i 50 anni. L’irraggiamento in vitro è stato effettuato presso i Laboratori Nazionali di Legnaro dell’INFN utilizzando una sorgente gamma (gamma beam) di 60Co con rateo di dose di 0,5 Gy/min. L’intervallo di dose, per creare le curve di calibrazione, è stato tra i 0,3 Gy e gli 8 Gy.
Sono state analizzate in totale circa 12.500 cellule in metafase e interfase, con l’utilizzo di un microscopio ottico munito di fluorescenza; l’irraggiamento con raggi gamma produce nei cromosomi (dicentrici ed anelli) una distribuzione del danno di tipo Poissoniano (Edwards, 1979). Ogni curva lineare quadratica di dose-effetto è stata costruita analizzando un minimo di 500 metafasi o riscontrando 100 aberrazioni per dose (IAEA, Cytogenetic Dosimetry, 2011). Si è proceduto quindi a controllare, attraverso la verifica del test statistico u-test, che la distribuzione delle aberrazioni nelle metafasi soddisfacesse tale distribuzione per ogni dose utilizzata per costruire le curve di calibrazione; inoltre è stata verificata la corrispondenza del fit con il modello lineare quadratico f = y0+aD+bD2 utilizzando il software SigmaPlot versione 13.0 e sono stati calcolati i coefficienti del modello con i rispettivi errori standard e i valori di p value (livello di significatività assegnato, ossia una misura di evidenza contro l’ipotesi nulla) ottenendo delle stime sulla dose. Quindi, sono state costruite per ogni metodica le curve di calibrazione con limite di confidenza al 95% per stimare l’incertezza sulla dose ricevuta una volta noto il numero di aberrazioni per cellula. Le curve di calibrazione ottenute con le varie metodiche sono confrontate tra loro e discussi i vantaggi delle metodiche utilizzate.
Sono state applicate diverse metodologie di dosimetria biologica e sono state costruite curve di calibrazione per ogni metodica. L’equazione per ogni curva è in grado di fornire una valutazione della dose assorbita dall’individuo in caso di esposizione accidentale alle radiazioni ionizzanti. L’applicazione, in dosimetria biologica, della tecnica della Condensazione Prematura dei Cromosomi (PCC) in combinazione con la tecnica dell’ibridizzazione fluorescente in situ (FISH) e le sonde Peptidiche di Acido Nucleico (PNA) è assolutamente originale e pochi Laboratori al mondo la applicano in questo momento. La metodica offre grandi vantaggi di rapidità, accuratezza e sensibilità quando confrontata con quelle convenzionali.

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EPrint type:Ph.D. thesis
Tutor:Bartolucci, Giovanni Battista
Ph.D. course:Ciclo 27 > scuole 27 > TERRITORIO, AMBIENTE, RISORSE E SALUTE
Data di deposito della tesi:27 January 2015
Anno di Pubblicazione:27 January 2015
Key Words:Dosimetria biologica/Biological dosimetry, Condensazione prematura dei cromosomi/Premature chromosome condensation, valutazione dose gamma/gamma dose evaluation, curve di calibrazione/calibration curves
Settori scientifico-disciplinari MIUR:Area 02 - Scienze fisiche > FIS/07 Fisica applicata (a beni culturali, ambientali, biologia e medicina)
Struttura di riferimento:Dipartimenti > Dipartimento Territorio e Sistemi Agro-Forestali
Codice ID:7602
Depositato il:10 Nov 2015 12:54
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