LIFE09200604007 Tabish - Homi Bhabha National Institute

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Review of Literature Comet assay or single cell gel electrophoresis assay is a sensitive technique for the detection of DNA damage at the level of the individual cell. The length of comet tail relative to head is the measure of number of DNA breaks 91, 92 . Comet assay has been a method of choice for undertaking various DNA damage and repair studies deciphering cancer risk association 89, 93 . In Host-Cell Reactivation Assay the host cell is transfected with a damaged plasmid containing reporter gene usually luciferase which has been deactivated due to the damage. The ability of the cell to repair the damage in the plasmid after it is introduced to the cell allows the reporter gene to be reactivated leading it to produce its reporting product and indirectly measuring the ability of the cell to repair DNA damage. This assay has also been frequently reported in assessing altered DNA repair capacity and cancer risk association 94, 95 . Another sensitive assay to measure DNA double strand breaks (DSB) and repair is measurement of activated γH2AX. An early cellular response to DSBs is the rapid phosphorylation of H2AX (histone H2A variant) at Ser-139 to produce γH2AX. Immunofluorescence based assays that allows visualization of discrete nuclear foci formed as a result of H2AX phosphorylation is a very sensitive and reliable methods of detecting DSBs 96 . Activation of H2AX plays an important role in signalling and initiating the DNA repair by facilitating downstream repair proteins to reach the site of damage 96, 97 . Hence after DNA damage, appearance and disappearance of γ-H2AX can be used as a parameter to measure DNA damage and repair. Foci of γ-H2AX can be quantified by immunofluorescence microscopy or flow cytometry. Measurement of association of cancer risk with DNA damage and repair is often done by measuring γ- H2AX 98-100 . In present study we have selected γ-H2AX foci formation assay as a measure of DNA damage and repair. 2.8.2 Cell cycle regulation and cancer After DNA damage cell cycle check points provide time for the cell to repair possible defects upon exposure to DNA damaging agents before entering into the next phase of cell cycle 101, 102 . There are two important check point for cell cycle regulation G1/S and G2/M. The G1/S checkpoint prevents the cell from replicating damaged DNA and G2/M checkpoint is activated by DNA damage and by incompletely replicated DNA. Considerable experimental evidence support the view that alteration in these cell cycle check points can lead to genomic instability and inappropriate survival of genetically damaged cells and contribute to the evolution of cells to malignancy 103 . 42
Review of Literature Inefficient cell cycle regulation with loss of cell cycle arrest after genotoxic exposure is associated with various cancers mainly lung cancer, oral cancer 93, 103-106 . Therefore assessment of cell cycle regulation is important intermediated phenotype to be measured to completely understand cancer predisposition. This is frequently done by staining cells with DNA binding dyes like, Propidium Iodide, Ethidium Bromide and DAPI. 2.8.3 Apoptotic response and cancer Under normal circumstances when a cell is exposed to a genotoxic agent it elicits highly preserved and well-regulated responses including cell cycle check point which allows cell to repair the damaged DNA. However in cases where the damage is severe and cannot be repaired, the cells will go into apoptosis or programmed cell death which is a safe way to inhibit risk acquiring neoplastic autonomy 107 . Studies in transgenic and knockout mice provide direct evidence that disruption of apoptosis can promote tumour development 108 A failure in proper apoptotic response after extreme genotoxic exposure may also contribute to cancer development. There are few reports available where disruption of apoptosis is associated with cancer 53, 104, 105 . In a report by Zheng et al, γ-radiation induced apoptosis has been observed as a biomarker of genetic susceptibility to salivary and thyroid carcinoma along with defects in cell cycle regulation 104 . Similarly defective apoptotic response has also been associated with Lung cancer risk and is suggested to be used a susceptibility marker 53, 105 . Therefore apoptotic response after genotoxic exposure is also an important phenotype to be assessed when developing an association of cancer risk. 2.8.4 Altered gene expression and cancer Differences in cellular responses after genotoxic exposure can be attributed differential gene expression. The tool of microarray analysis is an efficient means to study the differential expression of many genes simultaneously 109, 110 . Differential expression of genes in important carcinogenesis pathways is frequently reported which can be considered as driving malignant changes 111, 112 . Differential expression of genes in DNA repair pathways like nucleotide excision repair genes are found to be associated with in lung cancer 111 . A more than 2 fold increased of risk of head and neck cancer has been observed in individuals expressing lower levels of nucleotide 43
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Genotype-Molecular Phenotype Correl
Page 7 and 8: CONTENTS Page No Synopsis 1 List of
Page 9 and 10: Synopsis
Page 11 and 12: Synopsis Genotype-Molecular Phenoty
Page 13 and 14: Synopsis Materials and Methods: 1.
Page 15 and 16: Synopsis loading control. PCR produ
Page 17 and 18: Synopsis 7.1 Percent cell death aft
Page 19 and 20: Synopsis slides were then dried and
Page 21 and 22: Synopsis range 41.77% - 90.95% at 5
Page 23 and 24: Synopsis 5. Genotyping of genes: Ge
Page 25 and 26: Synopsis G2/M arrest and delaying e
Page 27 and 28: Synopsis may have an important bear
Page 29 and 30: Synopsis References: 1. Bobba, R.;
Page 31 and 32: Abbreviations MOPS : 3-(N-morpholin
Page 33 and 34: List of Figures Fig. 28: Percent re
Page 35 and 36: Chapter 1 Introduction
Page 37 and 38: Introduction In previous studies fr
Page 39 and 40: Introduction important carcinogenes
Page 41 and 42: Review of Literature The yet undeci
Page 43 and 44: Review of Literature Fig. 2: Incide
Page 45 and 46: Review of Literature sustained angi
Page 47 and 48: Review of Literature 2.6 Genotype p
Page 49 and 50: Review of Literature 2.7.2 Biologic
Page 51 and 52: Review of Literature Table 1: Steps
Page 53: Review of Literature future analysi
Page 57 and 58: 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
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Results Fig. 10: Cell population do
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Results Fig. 12: Cell surface marke
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Results 4.1.5 Expression of ATM gen
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Results Objective 2 To compare the
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Results Fig. 17: Standardization of
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Results Fig. 19: Standardisation of
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Results The cells were incubated fu
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Results 4.2.3.1 Percent G2 delay af
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Results 4.2.3.2 Effect of BPDE expo
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Results Fig. 29: Comparison of perc
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Results Fig. 31: Percent cell death
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Results normalization with baseline
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Results 4.2.5.2 Real time PCR valid
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Results homozygous wild-type, heter
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Results Fig. 36b: Electrophoresis o
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Results Fig. 37a: Representative el
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Results Table 13: Consolidated geno
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Results LCL Genes NAT2(1) NAT2(2) N
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Results Fig. 38: Distribution of Ge
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Results Fig. 39: Various combinatio
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Results Table 14: Genotype-phenotyp
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Discussion Introduction Squamous ce
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Discussion stimulated lymphocytes w
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Discussion H2AX is currently the mo
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Discussion or G2/M phase arrest. As
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Discussion differential expression
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Discussion DNA repair genes and MPN
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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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