LIFE09200604007 Tabish - Homi Bhabha National Institute

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Results Objective 1 To generate EBV LCLs from peripheral blood lymphocytes (PBLs) isolated from MPN patients and healthy controls - an established, in vitro model for genetic studies Long term genotype-phenotype correlation studies necessitate the availability of patient DNA sample and a continuous source of patient derived cells to avoid repeated blood sampling for multiple phenotypic assays. A major drawback for such studies is the lack of representative human cell lines providing a continuous source of basic biomolecules and a system to carry out various experimental investigations. This can be overcome to some extent by establishing LCLs by infecting peripheral blood lymphocytes with EBV which is known to immortalize human resting B cells in vitro giving rise to actively proliferating B-lymphoblastoid cell lines. Therefore blood samples were collected from patients with multiple primary cancers (MPN) of upper aero digestive tract (UADT) and healthy controls, to develop lymphoblastoid cell lines and isolate genomic DNA. 4.1.1 Accrual and demographic characterization of study samples UADT MPN patients were accrued from the Cancer Genetics Clinic in Tata Memorial Hospital (TMH) where patients with familial history of cancers, rare genetic disorders and multiple primary cancers frequently visit for counselling. For this study blood sample from patients with at least one cancer in UADT were collected following Hong‟s criteria described in material and methods section-3.1. A total of 24 patients were taken for the study and preliminary analysis showed that majority of them had tobacco habits (n=20). The incidence of UADT MPN was almost equal in both the sexes with a male to female ratio of 6:4 in the study subjects with median age 53 years (age range 32-75 years). Tobacco habit varied between individuals in terms of type and amount consumed. Habit included smoking, tobacco chewing, tobacco with pan (betel quid) and areca nut chewing. Four out of 20 patients had a habit of alcohol consumption along with tobacco habit (Table 5, Fig. 6). 84
Results Fig. 6: Frequency of tobacco and alcohol habit in MPN patient samples. The subjects had mixed type of tobacco habit including smoking, tobacco chewing, pan (betel quid). One subject had a habit of areca nut chewing and four subjects had a habit of alcohol along with tobacco. The patients accrued were highly heterogeneous with respect to tumour sites. Major sites of first and second primary cancer included various regions of buccal mucosa (lip, upper and lower alveolus, floor of mouth, retromolar trigone (RMT), gingivobuccal sulcus (GBS) and cheeks), hard palate, tongue, tonsil, larynx, oesophagus, parapharyngeal carcinoma and nasal columella (Fig. 7, Fig. 8). Fig. 7: Frequency of first primary cancers in study subjects (n=24). Values in parenthesis denote the actual number out of 24 subjects in each category. 85
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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
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 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 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 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
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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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