LIFE09200604007 Tabish - Homi Bhabha National Institute

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Results Table 6: Demographics of healthy control subjects LCL Type Age Sex Habit E245* Control 52 M Smoking E246* Control 62 M Smoking E247* Control 52 M Smoking E248* Control 58 M Smoking+Tobacco chewing E249* Control 46 M Smoking+Tobacco chewing E250* Control 41 M Tobacco chewing E313 Control 49 M Pan+Smoking+Tobacco chewing+Alcohol E314 Control 61 M Pan+Smoking+Tobacco chewing+Alcohol E334* Control 45 F Masheri (pyriform tobacco) E335* Control 65 F Masheri (pyriform tobacco) Key: * cell lines prepared by Ashwin Kotnis A total of 10 control cell lines were used for the study in which 8 samples were obtained from males, while 2 were from females. The age range for healthy controls was from 41-65 years with a median age of 52 years. In the control group as well, tobacco and alcohol habit varied between different individuals in terms of type and amount (Fig. 9). A comparison of demographics of cases and controls showed that the controls were fairly matched for age and habit. The patients and controls were mainly residents of Indian states of Maharashtra, Assam, Mizoram, Bihar, Uttar Pradesh, West Bengal, Gujarat, Karnataka and the families belonged to upper middle to lower income groups. Fig. 9: Frequency of tobacco and alcohol habit in healthy control samples. Control subjects had mixed type of tobacco habit including smoking, tobacco chewing, pan (betel quid). Two controls had a habit of using Masheri and two had a habit of alcohol along with tobacco. 88
Results 4.1.2 Establishment of LCLs and morphological analysis A total of 34 LCLs from UADT MPN patients (n=24) and healthy controls (n=10) were prepared from PBLs isolated from blood samples. Sixteen UADT MPN patient (n=8) and healthy control cell lines (n=8) were prepared as a part of earlier study in the lab (marked with * in Table 5 and Table 6, prepared by Ashwin Kotnis); eighteen MPN patient (n=16) and control cell lines (n=2) have been prepared in the present study. Blood sample (3 ml) from MPN patients and cancer free control individuals was obtained and subjected to PBL isolation by ficoll-hypaque density gradient centrifugation. Approximately 4x10 6 cells were obtained from 3 ml blood. After lymphocytes isolation, LCLs were generated by infecting isolated lymphocytes with EBV which is known to infect only B cells in a mixed population of B, T and natural killer cells present in PBLs. Presence of complement receptor type 2, commonly known as CR2 (CD21) on B cells creates a route for virus entry into the cell. Considerable cell death of the PBLs was observed 24 h post EBV infection; however virus infection promoted B cells to re-populate the culture. The time taken for each LCL preparation varied, on an average culturing the cells 3-4 weeks post infection was sufficient to produce >1 million cells. Once the cell lines started growing, initial passage cells were cryopreserved for future use. The LCLs in the present study were established as a model to study carcinogen sensitivity in vitro and to understand the aetiology of UADT MPN. Hence it was necessary to ensure that the process of EBV transformation did not affect the basic genotypic and phenotypic properties of the cells and LCLs were comparable to isolated lymphocytes. Therefore, random LCLs were undertaken for partial characterization with respect to cell population doubling time (PD), ploidy status, cell surface marker and expression and activity of ATM gene. 89
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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
Page 51 and 52: Review of Literature Table 1: Steps
Page 53 and 54: Review of Literature future analysi
Page 55 and 56: Review of Literature Inefficient ce
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 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 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
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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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