LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis 3.2.1 S phase arrest at 6 h time point: Mean percent S phase arrest in MPN patient cell lines was 18.54% (range 8.84% - 31.06%) at 5 µM; and 19.14% (range 10.49% - 35.52%) at 8 µM and in control cell lines it was 13.85% (range 4.77% - 22.09%) at 5 µM; and 13.58% (range 7.5% - 16.2%) at 8 µM. 3.2.2 S phase arrest at 24 h time point: Mean percent S phase arrest in MPN patient cell lines was 19.53% (range 3.26% - 31.33%) at 5 µM; and 20.09% (range 3.73% - 30.72%) at 8 µM and in control cell lines it was 17.68% (range 8.10% - 27.24%) at 5 µM; and 16.67% (range 6.28% - 28.09%) at 8 µM. 3.2.3 G2/M phase arrest at 6 h time point: Mean percent G2/M arrest in MPN patient cell lines was 4.86% (range 0 - 9.25%) at 5 µM; and 4.87% (range 0 - 9.48%) at 8 µM and in control cell lines it was 9.57% (range 5.30% - 16.82%) at 5 µM; and 9.55% (range 4.03% - 17.05% ) at 8 µM. 3.2.4 G2/M phase arrest at 24 h time point: Mean percent G2/M arrest in MPN patient cell lines was 3.65% (range 0 - 9.9%) at 5 µM; and 3.39% (range 0 - 8.8%) at 8 µM and control cell lines it was 5.19% (range 0.01% - 17.01%) at 5 µM; and 4.83% (range 0.01% - 15.9%) at 8 µM. 4. Apoptotic response in MPN and control samples in vitro: This experiment was done on 20 MPN and 10 control cell lines. 4.1 Percent cell death after γ-radiation exposure: Percent cell death after radiation exposure was higher in control cell lines (19.22%, range 9.05% - 42.82% at 5 Gy; and 22.09%, range 12.04% - 42.4% at 10 Gy) as compared to MPN patient cell lines (9.84%, range 8.3% - 12.7% at 5 Gy; and 10.84%, range 8.3% - 12.7% at 10 Gy). The mean difference between the two groups was 9.38% at 5 Gy and 11.25% at 10 Gy which was statistically significant (p=0.0003 at 5 Gy; p=0.0002 at 10 Gy). 4.2 Percent cell death after BPDE exposure: Similarly percent cell death after BPDE exposure was higher in control cell lines (23.25%, range 10.49% - 41.79% at 5 µM; and 24.98%, range 10.23% - 51.76% at 8 µM) as compared to MPN patient cell lines (13.57%, range 8.02% - 21.77% at 5 µM and 14.02%, range 8.46% - 24.69% at 8 µM). The mean difference between the two groups was 9.68% at 5 µM and 10.98% at 8 µM which was statistically significant (p=0.0009 at 5 µM and 8 µM BPDE concentration). 13
Synopsis 5. Genotyping of genes: Genotyping of 22 candidate single nucleotide polymorphisms (SNPs) in 17 candidate genes involved in DNA repair, apoptosis, cell cycle regulation and carcinogen metabolism was done and G Score was calculated. For all samples (n=30) value of G Score ranged from 10-22. G Score of only DNA repair genes (XRCC1-Arg 194 Trp, XRCC1-Arg 280 His, XRCC1-Arg 399 Trp, XRCC3-Thr 241 Met, XPD- Lys 751 Gln, hOGG1-Ser 326 cys, BRCA2-5‟UTR, BRCA2-Asn 372 His) ranged from 3-9 and MPN risk association signature (SULT1A1-Arg 213 His, hOGG1-Ser 326 cys, BRCA2- Asn 372 His, mEH-Try 113 His, XRCC1-Arg 280 His) ranged from 0-6. G Score for DNA repair genes and MPN risk association signature could significantly differentiate between MPN patient and control group with a p=0.012 and p=0.039 respectively. 6. Global gene expression profiling: Data obtained after microarray experiment was analysed using GeneSpring GX 10.0 software. A differential expression of few genes associated with various cancers or involved in signal transduction or transcriptional regulation was observed. Genes with differential expression of more than 2 fold (ARHGAP25, MAFB, AREG, ZNF429 and TMEFF) were selected and validated using real time PCR. The difference in the expression of genes that was observed in microarray experiment was also observed in the real time validation. 7. Genotype phenotype correlation: Phenotypic responses after radiation and BPDE exposure were either correlated with total G Score or G Score of DNA repair genes or MPN risk association signature only. Either Pearson or Spearman correlation was used to calculate the relationship between phenotype and genotype on the basis of whether the data followed normal distribution respectively. A comparison between G Score and phenotypic response between most groups revealed statistically significant correlation. Discussion: In this study we investigated the combined effect of multiple genetic variations and phenotypic effects in vitro in order to assess UADT MPN risk, by developing a genotype-phenotype correlation. The advantage of undertaking multigenic pathway based approach in studying complex diseases like cancer is well evident from various association studies 16 . Use of patient derived cell lines is of immense practical value for such long term genotype-phenotype correlation studies involving execution of a number of experiments on single sample. In order to have a continuous supply of 14
Page 1: 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: Synopsis range 41.77% - 90.95% at 5
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 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
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Materials and Methods cDNA it can b
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Materials and Methods Cobalt-60 iso
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Materials and Methods specific bind
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Materials and Methods h half of the
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Materials and Methods phenol and th
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Materials and Methods 7. Filter ste
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Materials and Methods viz. EXO-SAP
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Materials and Methods Table 2: List
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Materials and Methods SMAD6 AREG HM
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Materials and Methods 3.9 Effect of
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Materials and Methods fresh eppendo
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Materials and Methods Power SYBR Gr
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Results Objective 1 To generate EBV
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Results Fig. 8: Frequency of second
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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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