LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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194 S. <strong>Ghosh</strong> et al. / Mutation Research 723 (2011) 190–198 Fig. 4. Phosphorylation of ATR at 4 h after exposure to 2 Gy and 6 Gy gamma or 2 Gy oxygen irradiation in A549 cells. (A) Representative image showing p-ATR foci 4 h after irradiation. Each phospho-ATR antibody was indirectly labeled with Molecular Probe 488 secondary antibody (green) and cells were mounted with ProLong Gold antifede with DAPI (blue). All images were captured using Carl Zeiss confocal microscope with the same exposure time. (B) Graph represents average numbers of foci per cell, percentage of cells showing the foci is marked above the bars. (C) Graph represents relative intensity of p-ATR as determined by ImageJ software. At least 100 cells per experiment were analyzed from three independent experiments. Data represents means ± SD of three independent experiments; significantly different from unirradiated controls: *P < 0.05, **P < 0.01. Significantly different from 6 Gy gamma: ## P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) lowed only at later time point of 4 h. Moreover, by this time low LET induced damage is mostly repaired and the activation of these factors is reduced to control levels [28] thereby sealing the fate of the cell. Comparing the activation of these molecules at this time could therefore give important insight into differences in damage response signaling with respect to radiation quality. Phosphorylation of H2AX even at 4 h indicates the presence of large number of double strand breaks at that time indicating very slow repair in A549 cells exposed to oxygen beam. 3.3. Radiation induced foci of DNA damage response proteins ATM and ATR ATM is an important DNA damage response protein to ionizing radiation and a part of irradiation induced foci (IRIF). It gets activated after DNA DSB by phosphorylation at ser 1981 and activates many key cellular proteins that include H2AX, BRCA1, Chk1/2 and p53 [14,29]. Phospho ATM foci and intensity were looked at 4 h after gamma or oxygen irradiation. At 4 h after 2 Gy gamma irradiation, most of the cells (93%) cells showed ATM foci (5.9 ± 3.7) as against 25% unirradiated cells (3.4 ± 1.6) (Fig. 3). Like -H2AX foci, lesser number of ATM foci 4 h after 2 Gy gamma irradiation indicates that by 4 h most of the repair due to damage by low LET irradiation would have taken place. 4 h after 6 Gy gamma irradiation, 100% of the cells showed the foci and average ATM foci per cell were 14.5 ± 6.05. However, at 4 h after oxygen ion irradiation, 100% of the cells still showed the foci and average ATM foci per cell (18.6 ± 8.7) were higher than gamma irradiation. Total intensity levels of phospho ATM matched with the number of foci observed (Fig. 3). ATM is major mediator of DSB response and is involved in cell cycle arrest, repair and apoptosis. Persistence of significant pATM foci till 4 h after oxygen ion irradiation indicates the presence of unrepaired DNA, which is still keeping the damage sensor protein ATM in activated form. The slower disappearance of pATM
S. <strong>Ghosh</strong> et al. / Mutation Research 723 (2011) 190–198 195 Fig. 5. Phosphorylation of TP53 at 4 h after exposure to 2 Gy and 6 Gy gamma or 2 Gy oxygen irradiation in A549 cells. (A) Representative image showing p-TP53 4 h after irradiation. Each phospho-TP53 antibody was indirectly labeled with Molecular Probe 488 secondary antibody (green) and cells were mounted with ProLong Gold antifede with DAPI (blue). All images were captured using Carl Zeiss confocal microscope with the same exposure time. (B) Graph represents relative intensity of p-TP53 as determined by ImageJ software. At least 100 cells per experiment were analyzed from three independent experiments. Data represents means ± SD of three independent experiments; significantly different from unirradiated controls: *P < 0.05, **P < 0.01. Significantly different from 6 Gy gamma: # P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) foci from cells, unlike the -H2AX foci (that was seen in 60% of oxygen irradiated cells at 4 h), could reflect their different roles in the repair process with -H2AX foci playing a primary role in activating downstream pathways and initiating DNA repair while ATM foci remaining till broken DNA ends are present. Since the cell survival after 2 Gy oxygen irradiation was only 11%, ATM might be activating downstream apoptotic responses. ATR, another PI3 kinase family member, which is known to respond late to the IR induced damage, was also looked for its activation at 4 h. After gamma irradiation, average foci per cell were very less (2.7 ± 0.95 for 2 Gy and 4 ± 1 for 6 Gy) and that too were visible in only half of the irradiated cells (Fig. 4). On the other hand, although only 36% of oxygen ion irradiated cells showed ATR foci, the average foci per cell were (24 ± 14) at least six times more than that of gamma irradiated cells. Total intensity levels of phospho ATR matched with the number of foci observed (Fig. 4). The massive activation of ATR after oxygen irradiation when compared with gamma (Fig. 4) in 36% of the
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RADIATION INDUCED SIGNALING IN MAMM
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DECLARATION I, hereby declare that
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CONTENTS Page No. SYNOPSIS………
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2.11 Measurement of NO production
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PREAMBLE SYNOPSIS Ionising radiatio
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SYNOPSIS Aims and Objectives: To St
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SYNOPSIS 2.6 Immunofluorescence sta
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SYNOPSIS ATM gene expression, which
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SYNOPSIS Percentage Survival 100 80
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SYNOPSIS Ratio of Rad52 to Actin Ra
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Relative Phosphorylation 45 40 35 3
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SYNOPSIS Fig. 3.3A Clonogenic Cell
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SYNOPSIS (A) Av No. of phospho BRCA
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SYNOPSIS Percentage Survival 120 10
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SYNOPSIS Fig.3.4B. Existance of cro
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SYNOPSIS Fig. 3.4EDNA damage in EL-
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SYNOPSIS subsequent cytotoxicity ca
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SYNOPSIS [4] Hall, E. J. Radiobiolo
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CHAPTER 1 INTRODUCTION INTRODUCTION
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CHAPTER 1 INTRODUCTION 15.8% 9.56%
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CHAPTER 1 INTRODUCTION far apart an
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CHAPTER 1 INTRODUCTION and are more
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CHAPTER 1 INTRODUCTION 1.3 DNA dama
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CHAPTER 1 INTRODUCTION associates w
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CHAPTER 1 INTRODUCTION are unable t
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CHAPTER 1 INTRODUCTION Perhaps one
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CHAPTER 1 INTRODUCTION occurs at DS
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CHAPTER 1 INTRODUCTION related prot
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CHAPTER 1 INTRODUCTION damage must
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CHAPTER 1 INTRODUCTION At the prese
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CHAPTER 1 INTRODUCTION predominant
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CHAPTER 1 INTRODUCTION provide a me
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CHAPTER 1 INTRODUCTION hypersensiti
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CHAPTER 1 INTRODUCTION cycle-specif
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CHAPTER 1 INTRODUCTION 1.4 Overview
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CHAPTER 1 INTRODUCTION 1.4.1 Radiat
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CHAPTER 1 INTRODUCTION interaction
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CHAPTER 1 INTRODUCTION p90S6 kinase
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CHAPTER 1 INTRODUCTION CD4 (formerl
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CHAPTER 1 INTRODUCTION unrepaired d
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CHAPTER 1 INTRODUCTION well to the
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CHAPTER 1 INTRODUCTION 1.6 Radiatio
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CHAPTER 1 INTRODUCTION becomes pote
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CHAPTER 2 MATERIALS AND METHODS MAT
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CHAPTER 2 MATERIALS AND METHODS 2.2
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CHAPTER 2 MATERIALS AND METHODS res
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CHAPTER 2 MATERIALS AND METHODS Arr
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CHAPTER 2 MATERIALS AND METHODS PBS
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CHAPTER 2 MATERIALS AND METHODS the
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CHAPTER 2 MATERIALS AND METHODS 2.1
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CHAPTER 3 RESULTS RESULTS 93
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CHAPTER 3 RESULTS fractionated regi
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CHAPTER 3 RESULTS On comparison of
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CHAPTER 3 RESULTS Table 3.1.2A List
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CHAPTER 3 RESULTS 80 NM_002185 IL7R
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CHAPTER 3 RESULTS SAA2 149 AU134977
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CHAPTER 3 RESULTS 221 LOC643167 RNA
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CHAPTER 3 RESULTS 299 BF244402 FBXO
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CHAPTER 3 RESULTS CDK4) 57 AU152107
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CHAPTER 3 RESULTS 134 AL045793 METT
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CHAPTER 3 RESULTS 58 AF237813 ABAT
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CHAPTER 3 RESULTS 127 AI809749 ORMD
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CHAPTER 3 RESULTS 31 BE615699 UNC84
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CHAPTER 3 RESULTS 47 AF026303 SULT1
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CHAPTER 3 RESULTS 117 BF062193 CYor
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CHAPTER 3 RESULTS 187 AL036662 ---
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CHAPTER 3 RESULTS 52 AV686514 EMP2
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CHAPTER 3 RESULTS exposed to 10 Gy
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CHAPTER 3 RESULTS Fig.3.1.4 Gene ex
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CHAPTER 3 RESULTS Fig. 3.1.6 Radiat
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CHAPTER 3 RESULTS Fig. 3.1.7 Transl
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CHAPTER 3 RESULTS 3.1.9 Stabilizati
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CHAPTER 3 RESULTS 23±1.5% (Fig. 3.
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CHAPTER 3 RESULTS whereas only the
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CHAPTER 3 RESULTS with equal doses
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CHAPTER 3 RESULTS Fig.3.3.1.2 Forma
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CHAPTER 3 RESULTS Fig.3.3.1.3 Phosp
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CHAPTER 3 RESULTS (7.5±0.64) (Fig.
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CHAPTER 3 RESULTS Fig.3.3.1.7b Phos
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CHAPTER 3 RESULTS 3.3.2 Oxygen beam
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CHAPTER 3 RESULTS in all the cells.
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CHAPTER 3 RESULTS 3.3.2.3 Radiation
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CHAPTER 3 RESULTS Fig.3.3.2.7 Apopt
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CHAPTER 3 RESULTS PMA stimulated U9
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CHAPTER 3 RESULTS Fig.3.4.2. Exista
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CHAPTER 3 RESULTS up-regulation of
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CHAPTER 3 RESULTS Fig.3.4.3.2 NO pr
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CHAPTER 3 RESULTS 3.4.3.4 Apoptotic
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CHAPTER 3 RESULTS 3.4.4 Bystander e
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CHAPTER 3 RESULTS Fig.3.4.4b DNA da
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CHAPTER 3 RESULTS Fig.3.4.5a Gene e
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CHAPTER 4 DISCUSSION DISCUSSION 185
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CHAPTER 4 DISCUSSION directly expos
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CHAPTER 4 DISCUSSION dose is delive
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CHAPTER 4 DISCUSSION A clear cause
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CHAPTER 4 DISCUSSION 4.2 Proton bea
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CHAPTER 4 DISCUSSION Although the e
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CHAPTER 4 DISCUSSION Similar number
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CHAPTER 4 DISCUSSION the mechanism
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CHAPTER 4 DISCUSSION Thus unlike th
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CHAPTER 4 DISCUSSION different from
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CHAPTER 4 DISCUSSION upregulates ma
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CHAPTER 4 DISCUSSION Numerous studi
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CHAPTER 4 DISCUSSION inhibition of
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CHAPTER 4 DISCUSSION The survival o
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CHAPTER 5 REFERENCES [1] Khan, F. T
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CHAPTER 5 REFERENCES [19] Woo, R. A
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CHAPTER 5 REFERENCES [35] Nghiem, P
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CHAPTER 5 REFERENCES [52] McKay, B.
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CHAPTER 5 REFERENCES [69] Cary, R.
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CHAPTER 5 REFERENCES [84] Uziel, T.
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CHAPTER 5 REFERENCES [98] Liu, Y.;
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CHAPTER 5 REFERENCES [111] Fei, P.;
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CHAPTER 5 REFERENCES [127] Frank, K
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