LIFE09200604007 Tabish - Homi Bhabha National Institute

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Results Fig. 19: Standardisation of radiation dose for foci formation assay for γH2AX. The foci formed at 10 Gy radiation exposure were not discrete while foci formed at 2 Gy and 5 Gy radiation exposure were distinct. 4.2.2 DNA damage and repair following γ-radiation exposure γ-radiation causes DNA double strand breaks. Following DNA damage, appearance and the disappearance of phosphorylated H2AX (γ-H2AX) can be used as a parameter to measure DNA damage and repair respectively. 4.2.2.1 Immunofluorescence foci formation assay for γ-H2AX To compare the extent of DNA damage between MPN and control cell lines, cells were irradiated with 2 Gy and 5 Gy radiation and γ-H2AX foci were measured by immunofluorescence at 30 min time point. The experiment was conducted on randomly selected 4 MPN and 4 control cell lines. The number of γ-H2AX foci were approximately same between the two groups at 30 min indicating that the amount of DNA damage was almost equal (Fig. 20a, Fig. 20b, Fig. 20c, Fig. 20d). To evaluate the extent of DNA repair between the two groups, LCLs were incubated further for 4 h, providing time for the cells to repair the damage. 100
Results At 4 h time point, at both the radiation dose, number of γ-H2AX foci disappeared in LCLs from healthy controls indicating that DNA repair was complete, while in case of the MPN cell lines approximately half of the foci were still present at 4 h indicating that DNA repair was either delayed or impaired (Fig. 20a, Fig. 20b, Fig. 20c, Fig. 20d). 4.2.2.2 Assessment of γ-H2AX by flow cytometry In order to have a quantitative measurement, DNA damage and repair was measured in cell lines by flow cytometry. This experiment was performed on all 20 MPN and 10 control cell lines by analyzing percent positive cells for γ-H2AX at 30 min and 4 h time points. After radiation exposure, percent γ-H2AX positive cells at 30 min were approximately same in both MPN and control cell lines. As shown in Table 6 average percent γ-H2AX positive cells were 43.40% and 36.43% at 2 Gy; and 77.01% and 76.08% at 5 Gy in patient and control groups respectively. This indicated that the extent of DNA damage was almost equal between patient and control cell lines (Fig. 21, Fig. 22). Fig. 21: Percent relative γ-H2AX positive cells. Percent positive cells in MPN cell lines (a) percent positive cells in control cell lines (b). The black peak corresponds to unstained cell, green peak corresponds to percent positive cells after 2 Gy radiation exposure at 30 min, pink peak corresponds to percent positive cells after 5 Gy radiation exposure at 30 min, blue peak corresponds to percent positive cells after 2 Gy radiation exposure at 4 h and orange peak corresponds to percent positive cells after 5 Gy radiation exposure at 4 h. 105
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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
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Review of Literature 2.9.2 Gene inv
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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 110 and 111: Results Objective 2 To compare the
Page 112 and 113: Results Fig. 17: Standardization 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
Page 160 and 161: Summary and Conclusions Statistical
Page 162 and 163: 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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