LIFE09200604007 Tabish - Homi Bhabha National Institute

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Results Objective 2 To compare the response of MPN patients with tobacco habits and appropriate controls in vitro, to exposure to DNA damaging agents such as γ-radiations and Benzo[a]pyrene-diol-epoxide (BPDE, a tobacco specific carcinogen), by assessing DNA damage and repair, cell cycle profiling, apoptosis and global gene expression profiling Elevated levels of cellular damage caused by excessive exposure to genotoxic agents like γ-radiation and BPDE can elicit protective cellular responses like cell cycle regulation, DNA repair and apoptosis. Inter-individual difference leading to inherited deficiency in any of the above cellular mechanisms may increase an individual‟s risk to cancer. Using various in vitro phenotypic assays we have assessed the difference in the DNA repair capacity, cell cycle regulation and apoptotic response, after radiation and BPDE exposure, between MPN patients and controls. In addition whole genome expression profiling was also done to evaluate difference in the gene expression profile after γ-radiation exposure between the two groups. 4.2.1 Standardization of γ-radiation dose and BPDE concentration for phenotypic assays For apoptotic response assay standardization of γ-radiation dose and time point was done by irradiating few randomly selected MPN and control cell lines with γ- radiation dose ranging from 2 Gy-10 Gy and measuring percent cell death using Annexin-V-FLUOS-PI staining by flow cytometry at 48 h time points (Fig. 16). Radiation doses of 5 Gy and 10 Gy were found to be optimum for comparing cell death between MPN and control cell lines. To further standardise the time, percent cell death was measured at 24 h and 48 h time points. The difference in cell death between MPN and control cell lines was more pronounced at 48 h. Hence 5 Gy and 10 Gy radiation dose and 48 h time point were selected to assess percent cell death after γ-radiation exposure (Fig. 16). 96
Results Fig. 16: Standardization of γ-radiation dose to measure percent cell death using Annexin-V- FLUOS-PI staining by flow cytometry. Standardization of radiation exposure using E264 (MPN) and E249 (Control) cell lines. A range of radiation doses was selected to assess cell death response (a). Standardization of radiation exposure using E265 (MPN) and E250 (Control) cell lines. Vertical line on each bar shows standard deviation (b). Comparison of percent cell death after 5 Gy and 10 Gy radiation exposure at 24 h time point between MPN (n=3) and control (n=2) cell lines (c). Comparison of percent cell death after 48 h time point between MPN (n=2) and control (n=3) cell lines. The difference in the percent cell death after 48 h was significant between MPN and control group with *p value=0.04 at 5 Gy and **p value=0.01 at 10 Gy (d). Similarly a range of BPDE concentrations, time of treatment duration and various time points post BPDE exposure were evaluated to standardise the optimum BPDE concentration and time of exposure. Few representative graphs showing BPDE concentration standardisation are shown in Fig. 17. A concentration of 5 μM and 8 μM, and a 6 h time of treatment duration were found to be best suited for comparing percent cell death after BPDE exposure between MPN and control cell lines. 97
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Genotype-Molecular Phenotype Correl
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CONTENTS Page No Synopsis 1 List of
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Synopsis
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Synopsis Genotype-Molecular Phenoty
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Synopsis Materials and Methods: 1.
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Synopsis loading control. PCR produ
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Synopsis 7.1 Percent cell death aft
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Synopsis slides were then dried and
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Synopsis range 41.77% - 90.95% at 5
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Synopsis 5. Genotyping of genes: Ge
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Synopsis G2/M arrest and delaying e
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Synopsis may have an important bear
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Synopsis References: 1. Bobba, R.;
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Abbreviations MOPS : 3-(N-morpholin
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List of Figures Fig. 28: Percent re
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Chapter 1 Introduction
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Introduction In previous studies fr
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Introduction important carcinogenes
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Review of Literature The yet undeci
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Review of Literature Fig. 2: Incide
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Review of Literature sustained angi
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Review of Literature 2.6 Genotype p
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Review of Literature 2.7.2 Biologic
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Review of Literature Table 1: Steps
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Review of Literature future analysi
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Review of Literature Inefficient ce
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Review of Literature XRCC1 (X-ray r
Page 59 and 60: Review of Literature 2.9.2 Gene inv
Page 61 and 62: Review of Literature MPO gene polym
Page 63 and 64: Review of Literature important role
Page 65 and 66: Materials and Methods Source of che
Page 67 and 68: Materials and Methods Method: EBV-t
Page 69 and 70: Materials and Methods Immunophenoty
Page 71 and 72: Materials and Methods with 500 μl
Page 73 and 74: Materials and Methods cDNA it can b
Page 75 and 76: Materials and Methods Cobalt-60 iso
Page 77 and 78: Materials and Methods specific bind
Page 79 and 80: Materials and Methods h half of the
Page 81 and 82: Materials and Methods phenol and th
Page 83 and 84: Materials and Methods 7. Filter ste
Page 85 and 86: Materials and Methods viz. EXO-SAP
Page 88 and 89: Materials and Methods Table 2: List
Page 90 and 91: Materials and Methods SMAD6 AREG HM
Page 92 and 93: Materials and Methods 3.9 Effect of
Page 94 and 95: Materials and Methods fresh eppendo
Page 96 and 97: Materials and Methods Power SYBR Gr
Page 98 and 99: Results Objective 1 To generate EBV
Page 100 and 101: Results Fig. 8: Frequency of second
Page 102 and 103: Results Table 6: Demographics of he
Page 104 and 105: Results Fig. 10: Cell population do
Page 106 and 107: Results Fig. 12: Cell surface marke
Page 108 and 109: Results 4.1.5 Expression of ATM gen
Page 112 and 113: Results Fig. 17: Standardization of
Page 114 and 115: Results Fig. 19: Standardisation of
Page 116 and 117: Results The cells were incubated fu
Page 118 and 119: Results 4.2.3.1 Percent G2 delay af
Page 120 and 121: Results 4.2.3.2 Effect of BPDE expo
Page 122 and 123: Results Fig. 29: Comparison of perc
Page 124 and 125: Results Fig. 31: Percent cell death
Page 126 and 127: Results normalization with baseline
Page 128 and 129: Results 4.2.5.2 Real time PCR valid
Page 130 and 131: Results homozygous wild-type, heter
Page 132 and 133: Results Fig. 36b: Electrophoresis o
Page 134 and 135: Results Fig. 37a: Representative el
Page 136 and 137: Results Table 13: Consolidated geno
Page 138 and 139: Results LCL Genes NAT2(1) NAT2(2) N
Page 140 and 141: Results Fig. 38: Distribution of Ge
Page 142 and 143: Results Fig. 39: Various combinatio
Page 144 and 145: Results Table 14: Genotype-phenotyp
Page 146 and 147: Discussion Introduction Squamous ce
Page 148 and 149: Discussion stimulated lymphocytes w
Page 150 and 151: Discussion H2AX is currently the mo
Page 152 and 153: Discussion or G2/M phase arrest. As
Page 154 and 155: Discussion differential expression
Page 156 and 157: Discussion DNA repair genes and MPN
Page 158 and 159: Chapter 6 Summary and Conclusions
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Summary and Conclusions Statistical
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Bibliography 1. Dikshit, R.; Gupta,
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Bibliography 43. Lei, D.; Sturgis,
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Bibliography 76. Fry, R. C.; Svenss
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Bibliography 115. Kote-Jarai, Z.; M
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Bibliography 149. Hashibe, M.; Bren
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Bibliography 183. Bergamaschi, D.;
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Bibliography 216. Bondy, M. L.; Wan
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Indian J Med Res 135, June 2012, pp
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822 INDIAN J MED RES, JUNE 2012 mar
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824 INDIAN J MED RES, JUNE 2012 (h)
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826 INDIAN J MED RES, JUNE 2012 Tab
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828 INDIAN J MED RES, JUNE 2012 lik
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CASE REPORT Eben L. Rosenthal, MD,
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FIGURE 2. Chromosomal breakage anal
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FA along with ataxia telangiectasia
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LIFE09200604007 Tabish - Homi Bhabha National Institute

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