LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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S. <strong>Ghosh</strong>, M. Krishna / Mutation Research 729 (2012) 61–72 63 Table 1 The sequences of gene specific primers and their annealing temperature (T m) used in RT-PCR experiments. Gene Primer sequence T m ( ◦ C) -Actin F 5 ′ -TGGAATCCTGTGGCATCCATGAAA-3 ′ 55 R 5 ′ -TAAAACGCAGCTCAGTAACAGTCCG-3 ′ ATM F 5 ′ -GGACAGTGGAGGCACAAAAT-3 ′ 57 R 5 ′ -GTGTCGAAGACAGCTGGTGA-3 ′ DNA-PK F 5 ′ -TGCCAATCCAGCAGTCATTA-3 ′ 64 R 5 ′ -GGTCCATCAGGCACTTCACT-3 ′ Rad52 F 5 ′ -AGTTTTGGGAATGCACTTGG-3 ′ 50 R 5 ′ -TCGGCAGCTGTTGTATCTTG-3 ′ MLH1 F 5 ′ -GAGGTGAATTGGGACGAAGA-3 ′ 52 R 5 ′ -TCCAGGAGTTTGGAATGGAG-3 ′ CDKN1A (p21) F 5 ′ -CAGCAGAGGAAGACCATGTG-3 ′ 59 R 5 ′ -GGCGTTTGGAGTGGTAGAAA-3 ′ GADD45 F 5 ′ -TGCGAGAACGACATCAACAT-3 ′ 58 R 5 ′ -TCCCGGCAAAAACAAATAAG-3 ′ TLR3 F 5 ′ -GCCTCTTCGTAACTTGACCA-3 ′ 57 R 5 ′ -AAGGATGTGGAGGTGAGACA-3 ′ FDXR F 5 ′ -AGAGAACGGACATCACGAAG-3 ′ 57 R 5 ′ -GTCCTGGAGACCCAAGAAAT-3 ′ F, forward and R, reverse. Percentage Survival 100 80 60 40 20 0 0Gy 2Gy 5X2Gy 10Gy Radiation Dose Fig. 1. Clonogenic cell survival of A549 cells irradiated with 2 Gy, 5 fractions of 2 Gy or 10 Gy -radiation. Data represent means ± SE of three independent experiments; *P < 0.05, **P < 0.01 compared with 10 Gy acute dose. ** 2.8. Semiquantitative reverse transcriptional polymerase chain reaction (RT-PCR) Total RNA from 1 × 10 6 cells was extracted 4 h after irradiation and RT-PCR was carried out as described earlier [44]. Primer sequence and annealing temperature of specific primers are given in Table 1. 2.9. Transfection with ShRNA MLH1, Rad52 and Control shRNA plasmids were procured from Santa cruz biotechnology (Cat No. sc-35943-SH, sc-37399-SH and sc-108060 respectively). In a six well tissue culture plate, A549 cells were grown to 50–70% confluency in antibiotic-free normal growth medium supplemented with FBS and transfection was carried out as per supplier’s instruction. 48 h post-transfection, medium was aspirated and replaced with fresh medium containing puromycin (2 g/ml) for selection of stably transfected cells. Semi-quantitative RT-PCR was performed to monitor MLH1 and Rad52 gene expression knockdown using gene specific primers (Table 1). 2.10. Statistical analysis The data were imported to excel work sheets, and graphs were made using Origin version 5.0. One-way ANOVA with Tukey–Kramer Multiple Comparisons as post-test for P < 0.05 used to study the significant level. Data were insignificant at P > 0.05. Each point represented as mean ± SE (standard error of the mean). 3. Results 3.1. Clonogenic cell survival To determine the sensitivity of cells to different doses of radiation, their ability to form colonies after irradiation was observed. There was 60 ± 2.5% survival of A549 cells that had been exposed to 2 Gy acute dose where as there was 48 ± 2.8% of cells survived that had been exposed to 5 fractions of 2 Gy radiations and 4 ± 0.5% of cells survived that had been exposed to 10 Gy acute dose (Fig. 1). 3.2. Human genome U-133 Plus 2.0 microarray data analysis Global expression analysis of 54,675 probe set using Human Genome U-133 Plus 2.0 microarray was performed in A549 cells. On comparison of control vs 5X2 Gy (henceforth called fractionated dose), 461 genes were identified as differentially expressed; 312 genes up-regulated (defined as a 2.0-fold or greater increase, Supplementary data) and 149 down-regulated (defined as a 2.0- fold or greater decrease, Supplementary data). Up-regulated genes were associated with p53 signaling pathway, cytokine–cytokine receptor interaction, hematopoietic cell lineage, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, cell-cycle check point, B cell receptor signaling pathway. Down-regulated genes were associated with TGF-beta signaling pathway and tyrosine metabolism. In the second group (2 Gy vs 5X2 Gy), 234 genes were identified as differentially expressed; 172 genes as up-regulated (Supplementary data) and 62 as down-regulated (Supplementary data). Genes involved in cytokine–cytokine receptor interaction, toll-like receptor signaling pathway, hematopoietic cell lineage, Jak-STAT signaling pathway, MAPK signaling pathway were upregulated. Gene involved in folate biosynthesis, glycine, serine and threonine metabolisms were down-regulated. In the 10 Gy vs fractionated group, 289 genes were identified as differentially expressed; 211 genes as up-regulated (Supplementary data) and 78 as down-regulated (Supplementary data). Genes involved in cytokine–cytokine receptor interaction, toll-like receptor signaling pathway, cell cycle, DNA replication, mismatch repair, homologous recombination were up-regulated. Genes involved in regulation of autophagy, base excision repair, folate biosynthesis were down-regulated. Cluster analysis of the expression of these genes showed that the fractionated group differed most in gene expression compared to the other three groups (control, 2 Gy or 10 Gy) (Supplementary data, TCS2 means 2 Gy treatment group, TCS3 means fractionated group, TCS4 means 10 Gy treatment group). 3.3. Validation of microarray data In order to verify the microarray data, semi-quantitative RT-PCR was performed for four genes from control and fractionated group of A549 cells. These results corresponded very well with those for the microarray data for all four genes, strongly supporting the reliability and rationale of our strategy (Fig. 2A and B). 3.4. Activation of repair related genes When compared to the controls there was a significant upregulation of ATM, DNA-PK, Rad52 and MLH1 genes in A549 cells that had been exposed to fractionated irradiation (Fig. 3A–D, Lane 3). However, in A549 cells that had been exposed to 10 Gy acute dose there was marginal up-regulation of ATM, DNA-PK and Rad52 genes although it was significantly lower than the fractionated group (Fig. 3A–D, Lane 4). In A549 cells which had been exposed to 2 Gy acute dose there was marginal up-regulation of Rad52 and
64 S. <strong>Ghosh</strong>, M. Krishna / Mutation Research 729 (2012) 61–72 (A) rray) Fold Change (Microar (B) Fold Change (RT-PC CR) 3.6 3.4 Control 3.2 5X2Gy 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 CDK1A(p21) GADD45A TLR3 FDXR Genes 3.6 3.4 3.2 Control 3.0 5X2Gy 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 CDK1A(p21) GADD45A TLR3 FDXR Genes Fig. 2. Validation of microarray data by semi-quantitative RT-PCR. (A) Fold changes of genes as determined by microarray analysis. (B) Fold changes of genes as determined by RT-PCR. Four genes from control and 5X2 Gy treatment group of A549 cells were selected and performed three sets of semi-quantitative RT-PCR as described in Section 2. Four genes were CDKN1A (p21), GADD45, Toll-like receptor 3 (TLR3) and Ferredoxin reductase (FDXR). MLH1 genes but these were also significantly lower than the fractionated group (Fig. 3A–D, Lane 2). 3.5. Efficient repair of DNA Repair kinetics was determined by counting the -H2AX foci at 15 min and 4 h after irradiation. As expected maximum numbers of foci were seen at 15 min in A549 cells exposed to 10 Gy followed by cells exposed to fractionated irradiation and very few foci were seen in cells exposed to 2 Gy acute dose. By 4 h the number of foci in cells exposed to fractionated irradiation had been reduced to almost control but in the cells exposed to 10 Gy the number of foci was still very high, indicating that there was efficient repair of DNA in A549 cells that had been exposed to fractionated irradiation (Fig. 4). 3.6. Radiation induced foci of DNA damage response proteins ATM and BRCA1 ATM, an important DNA damage response protein to ionizing radiation and a part of irradiation induced foci (IRIF), is mainly involved in cell cycle arrest and repair. 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. Phospho ATM foci and intensity were looked at 4 h after irradiation. All the cells either exposed to 10 Gy (23 ± 3 foci per cell) or fractionated irradiation (25 ± 2.8 foci per cell) showed ATM foci but only 10% of cells exposed to 2 Gy showed ATM foci and the number of foci per cell was 2 ± 0.08 (Fig. 5A). The intensity of phosphorylation of ATM was quantified by ImageJ software, was significantly higher in the fractionated group as compared to any of the treatment groups (control, 2 Gy or 10 Gy) (Supplementary data, Fig. 5C). BRCA1, which is phosphorylated by ATM, is a part of IRIF and shares many downstream substrates of ATM. 4 h after irradiation, A549 cells that had been exposed to 2 Gy acute dose did not show any BRCA1 foci where as 55% of cells that had been exposed to fractionated irradiation showed BRCA1 foci and the number of foci per cell was 24 ± 4. 22% of cells that had been exposed to 10 Gy acute showed BRCA1 foci and the number of foci per cell was 15 ± 2.1 (Fig. 5B). The intensity of phosphorylation of BRCA1 as determined by ImageJ software was significantly higher in the fractionated group as compared to any of the treatment groups (control, 2 Gy or 10 Gy) (Supplementary data, Fig. 5D). 3.7. Translocation of phospho-p53 18 h after irradiation, phospho-p53 was found to be translocated into the nucleus of A549 cells exposed to fractionated irradiation (Fig. 6A). The intensity of phosphorylation was significantly higher in the cells exposed to fractionated irradiation (Supplementary data, Fig. 6C). Phosphorylation of p53 was further confirmed by Western Blot (Fig. 6B, Lane 3). 3.8. Response of A549 and MCF-7 cells exposed to fractionated irradiation There was 48 ± 2.8% survival of A549 cells exposed to fractionated irradiation where as only 10 ± 1.2% of MCF-7 cells survived exposed to fractionated irradiation (Fig. 7A). 4 h after irradiation, there was a significant up-regulation of ATM, DNA-PK, Rad52 and MLH1 genes in A549 cells but not in MCF-7 cells (Fig. 7B, Lane 3). ATM and BRCA1 phosphorylation was found to be significantly higher 4 h after irradiation in A549 cells but not in MCF-7 cells (Fig. 7C). 18 h after irradiation, phospho-p53 was found to be translocated into the nucleus of A549 cells but not in MCF-7 cells (Fig. 7C). There was efficient repair of DNA by 4 h in A549 cells as determined by counting -H2AX but not in MCF-7 cells (Fig. 7D). 3.9. Role of Rad52 and MLH1 Since there was an intense activation of Rad52 and MLH1 in A549 cells exposed to fractionated irradiation, it was of interest to look for their role in survival of A549 cells. MLH1 and Rad52 gene expression knockdown was carried out in A549 cells using MLH1 and Rad52 shRNA plasmids. Stably transfected cells were selected and the transfection efficiency was found to be 85% for MLH1 gene and 80% for Rad52 gene (Fig. 8A). There was 48 ± 2.8% survival of A549 cells exposed to fractionated irradiation. The survival of A549 cells that had been transfected with MLH1 shRNA plasmid and exposed to fractionated irradiation was found to be 40 ± 1.8% where as the survival of A549 cells that had been transfected with Rad52 shRNA plasmid and exposed to fractionated irradiation was found to be 23 ± 1.5% (Fig. 8B and C). The survival of cells transfected with control shRNA plasmid was the same as unirradiated control cells (data not shown). 4. Discussion Chronic exposure of cells to IR has been reported to induce an adaptive response that results in an enhanced tolerance to the cytotoxicity of subsequent doses of IR [1–4]. There could be many possible mechanisms for the induction of the adaptive response which would eventually determine the cell fate. In the present
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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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Page 222 and 223: CHAPTER 5 REFERENCES [1] Khan, F. T
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Page 252 and 253: CHAPTER 5 REFERENCES [229] Konca, K
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Page 256 and 257: CHAPTER 5 REFERENCES [261] Miralbel
Page 258 and 259: CHAPTER 5 REFERENCES [278] Weizman,
Page 260 and 261: CHAPTER 5 REFERENCES [294] Zhou, H.
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