The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. http://http://dspace.unitus.it:80 2013-05-25T19:56:23Z 2013-05-25T19:56:23Z Belloni, Paola Pepe, Gaetano Palitti, Fabrizio http://hdl.handle.net/2067/1674 2011-06-29T15:33:59Z 2009-12-31T23:00:00Z

Title: Effect of storage conditions of blood on radiation-induced chromosomal aberrations and apoptosis in human lymphocytes Authors: Belloni, Paola; Pepe, Gaetano; Palitti, Fabrizio Abstract: To evaluate the effect of storage conditions of blood on the direct relationship between radiation-induced chromosome aberrations and apoptosis in human peripheral blood lymphocytes,whole bloodwas irradi- atedwith 3Gy X-rays. Directly after irradiation, a sample of bloodwas analyzed for chromosome damage and proliferation index, after phytohaemagglutinin stimulation and incubation at 37 ◦ C for 56 h. Blood samples were stored for 48 h at 4 and 20 ◦ C with or without phytohaemagglutinin and analyzed for cell viability and apoptosis at 0, 24 and 48 h storage time. After 48 h of storage, unstimulated cultures were stimulated to proliferate. These samples and cultures stimulated immediately before storage were incubated at 37 ◦ C for 56 h and analyzed for chromosome damage and proliferation index. Metaphases were examined for the presence of dicentrics, excess acentrics, and rings. Storage at 20 ◦ Cwithout phyto- haemagglutinin for 48 h increases significantly the yield of apoptosis and decreases significantly the yield of dicentrics. During 48 h of storage time the presence of phytohaemagglutinin and the temperature of 4 ◦ C protected the irradiated lymphocytes from apoptosis allowing accurate estimation of the real yield of radiation-induced chromosome damage. Therefore these blood-storage conditions enable analysis in metaphase and may offer some advantages for biodosimetry of absorbed radiation dose. Description: L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com

2009-12-31T23:00:00Z Belloni, Paola http://hdl.handle.net/2067/1934 2011-04-22T00:30:47Z 2008-04-10T22:00:00Z

Title: Role of Apoptosis in the final outcome of DNA damage Authors: Belloni, Paola Abstract: Damage to DNA is intrinsic to life, as the cell continuosly suffers from numerous exogenous agents, including radiation and chemicals, and from endogenous sources, such as free radicals generated during essential metabolic processes. The broad spectrum of DNA lesions induced by these agents includes damage to nucleotide bases, DNA protein crosslinks and DNA single- and double-strand breaks (DBSs). Because of their high cytotoxicity (unrepaired or misrepaired DNA DSBs can kill a cell) and their ability to induce chromosomal aberrations (that may ultimately lead to carcinogenesis) cell survival and mantenaince of genome integrity are critically dependent on efficient repair of DNA DSBs. When the burden of genomic insult is too large to be effectively repaired, cells are able to initiate apoptosis (programmed cell death). On the basis of these considerations, the aim of this thesis has been to investigate the role of apoptosis in the final outcome of DNA damage. Since G0 lymphocytes are the most common tissue used in biodosimetry studies, and the amount of chromosomal damage detected depends on the time between exposure and sampling, it was of interest to investigate the relationship between the frequencies of radiation induced chromosomal aberrations and the extent of apoptosis in G0 human lymphocytes (Paper I). In human lymphocytes irradiated in G0 without subsequent stimulation to proliferate, a p53-dependent apoptotic pathway preferentially eliminates cells bearing unstable aberrations. Studies in Paper II demonstrated that in human lymphocytes the type of chromosome damage influences the induction of programmed cell death and provided direct evidence that cells bearing dicentric chromosomes are eliminated by apoptosis. The lymphocytes undergo apoptosis during storage, and this loss of viability is accelerated by increasing both temperature and storage time, was demonstrated in Paper III. Studies on the effects of storage conditions of whole blood on the viability and proliferation of lymphocytes revealed optimal the storage at 4°C for 96 h in the presence of phytohaemagglutinin. The influence of genetic background on the induction of apoptosis in cells of different origin, treated with a potential anticancer drug “STDS2323”, was studied in Paper IV. In conclusion, this thesis suggests that apoptosis has an important role in the final outcome of DNA damage. The thesis further suggests that these results may have relevance for biodosimetrical and molecular epidemiological studies and in evaluations of the efficacy of radio- and chemotherapy.; Il DNA è sottoposto continuamente ad alterazioni strutturali da parte di agenti sia esogeni che endogeni. Le radiazioni ionizzanti (IR) sono tra i più importanti agenti fisici che hanno sulle cellule bersaglio un effetto citotossico, mutageno e cancerogeno. Il danno indotto al DNA consiste principalmente in rotture a singola e doppia elica, legami crociati DNA-proteine e modificazioni delle basi. Le rotture a doppia elica (DSBs) sono le lesioni più dannose per la cellula. Tutti gli organismi hanno evoluto diversi meccanismi per riparare le rotture al DNA. L’effetto delle DSBs può essere osservato principalmente con la produzione d’aberrazioni cromosomiche in cellule in divisione, la cui formazione è data soprattutto da un’errata (misrepair) o mancata (unrepair) riparazione; oppure con l’apoptosi (morte cellulare programmata) la cui induzione è data da un danno troppo complesso perché sia riparato. In conformità a queste osservazioni, lo scopo di questa tesi è stato quello di comprendere il ruolo dell’apoptosi nella manifestazione finale del danno al DNA. Poiché i linfociti quiescenti sono il tessuto più comunemente usato in biodosimetria e il danno cromosomico dipende dal tempo che trascorre dopo l’esposizione “in vivo” alla radiazione, è stato interessante investigare la relazione tra l’induzione di aberrazioni di tipo instabile e l’induzione dell’apoptosi, nei linfociti umani in G0 (Paper I). Nei linfociti umani irradiati in G0 e tenuti per vario tempo in questa fase, un “pathway” apoptotico p53 dipendente elimina preferenzialmente le cellule portatrici di aberrazioni di tipo instabile. Gli studi condotti in “Paper II” hanno dimostrato che, nei linfociti umani in G0 ,il tipo di danno cromosomico influenza l’induzione del processo apoptotico e che esiste un ruolo diretto dell’apoptosi nell’eliminare le cellule portatrici di cromosomi dicentrici. In “Paper III” è stato dimostrato che i linfociti muoiono durante la conservazione (storage) e questa perdita di vitalità è accelerata dall’aumento sia della temperatura che il tempo di conservazione. Studi condotti sull’effetto delle condizioni di conservazione del sangue intero, sulla vitalità e la proliferazione dei linfociti, hanno rilevato che la condizione ottimale di conservazione è quella a 4°C per 96 h ed alla presenza del mitogeno “phytohaemagglutinin”. L’influenza del “background” genetico cellulare nell’induzione dell’apoptosi è stata studiata (Paper IV) in cellule di diversa origine trattate con un potenziale farmaco antitumorale “STDS2323”. In conclusione, questa tesi suggerisce un ruolo importante dell’apoptosi nella manifestazione finale del danno al DNA. Inoltre la tesi suggerisce una rilevanza dei risultati ottenuti in studi di dosimetria biologica e di epidemiologia molecolare e nella valutazione dell’efficacia di una radio- o chemioterapia antitumorale. Description: Dottorato di ricerca in Evoluzione biologica e biochimica

2008-04-10T22:00:00Z Belloni, Paola Latini, Paolo Palitti, Fabrizio http://hdl.handle.net/2067/1694 2011-06-30T15:40:05Z 2009-12-31T23:00:00Z

Title: Relationship between spontaneous or radiation-induced apoptosis and telomere shortening in G(0) human lymphocytes Authors: Belloni, Paola; Latini, Paolo; Palitti, Fabrizio Abstract: To examine the correlation between spontaneous or radiation-induced apoptosis and telomere shortening, G0 human peripheral blood lymphocytes were irradiated with X-rays and analyzed for viability, apoptosis, and telomere length. Part of the lymphocytes was kept under liquid-holding conditions for 48 h, and then loaded onto Ficoll-Paque medium to separate apoptotic (high-density) from normal (normal-density) cells. Then all samples were examined for the same three end-points. To determine whether expression of p53 influences the telomere shortening associated with a spontaneous or radiation-induced apoptotic process, the lymphocytes were also analyzed for expression of p53 at 0 and 48 h recovery times (non-irradiated and irradiated samples) and after 2 weeks in liquid-holding conditions (non-irradiated sample). After 48 h in liquid-holding, the p53-dependent apoptotic lymphocytes in the irradiated cultures presented shortened telomeres. After a 2-week recovery time, non-irradiated cells showed a p53-dependent spontaneous apoptosis, but no telomere shortening. These results demonstrate that radiation-induced apoptosis correlates with shortened telomeres in G0 human lymphocytes. Spontaneous and radiation-induced apoptosis are dependent on expression of p53. In contrast, p53 may not play an effective role in telomere shortening, because spontaneous apoptosis did not correlate with telomere shortening. As most tumours are compromised with respect to p53 function, our findings on the role of p53 in telomere shortening may prove critical for applying therapeutic modalities in the clinic, and may facilitate the design of agents that selectively disrupt telomere integrity in tumour cells. Description: L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com

2009-12-31T23:00:00Z