LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis Immunophenotyping revealed that cells from representative randomly selected LCLs showed expression of typical B cell surface marker (CD19) while markers for T cell (CD3) and NK cells (CD56) were absent. DNA ploidy status of the LCLs was assessed immediately after cell line preparation at low population doubling (100 PD). All the cell lines had a DI ratio ranging between 0.9-1.3 and were considered to be diploid. Also there was apparently no change at the expression level of ATM gene between the LCL and respective RNA isolated from the subject. The activity of ATM as assessed by immunofluorescence foci formation assay was comparable between LCLs and PBLs. Out of 34 cell lines 4 MPN patient cell lines could not be grown properly in the continuous culture hence were not undertaken for any phenotypic assay. 2. DNA damage and repair after γ-radiation exposure: DNA damage and repair after γ-radiation exposure was measured by Immunofluorescence foci formation assay for γ- H2AX and measurement of γ-H2AX positive cells by flow cytometry. 2.1 Immunofluorescence foci formation assay for γ-H2AX: After DNA damage appearance and the disappearance of phosphorylated H2AX (γ-H2AX) can be used as a parameter to measure DNA damage and repair. In MPN patient and control cell lines number of γ-H2AX foci at 30 min time point were approximately same indicating that the extent of DNA damage was almost equal. But at 4 h time point most of the γ-H2AX foci disappeared in LCLs from healthy controls (n=4) indicating that DNA repair is active while in case of the MPN patient cell lines (n=4) approximately half of the foci were still present at 4 h indicating that DNA repair is either delayed or impaired in these cells. 2.2 Assessment of γ-H2AX by flow cytometry: This experiment was done on 20 MPN and 10 control cell lines using flow cytometry by analyzing percent positive cells for γ- H2AX at 30 min and 4 h time points. 2.2.1 Measurement of DNA damage and repair at 30 min time point: Percent γ-H2AX positive cells at 30 min time point were approximately same in both MPN patient (42.14%, range 25.08% - 69.38% at 2 Gy; and 76.31%, range 52.06% - 97.69% at 5 Gy) and control cell lines (36.44%, range 14.33% - 55.25% at 2 Gy; and 76.08%, 11
Synopsis range 41.77% - 90.95% at 5 Gy) indicating that the extent of DNA damage was almost equal. 2.2.2 Measurement of DNA damage and repair at 4 h time point: At 4 h time point the number of percent positive cells were more in MPN patient cell lines (29.98%, range 2.80% - 80.2% at 2 Gy; and 43.69%, range 5.82% - 96.89% at 5 Gy) as compared to control cell lines (12.17%, range 4.05% - 21.91% at 2 Gy; and 20.56%, range 0.37% - 43.09% at 5 Gy). The mean difference in percent positive cells at 4 h time point between MPN patient and control cell lines was 17.28% at 2 Gy and 23.13% at 5 Gy which was statistically significant (p=0.0083 at 2 Gy and p=0.0109 at 5 Gy), indicating that DNA repair is either delayed or impaired in MPN patient cell lines which was in concert with immunofluorescence data. 3. Effect of γ-radiation and BPDE exposure on cell cycle profile: After genotoxic exposure cell cycle arrest provides time for the cell to repair damaged DNA before entering into the next phase of the cycle. This experiment was done on 20 MPN and 10 control cell lines using flow cytometry by analysing percent G2 delay after radiation exposure and S and G2/M phase arrest after BPDE exposure. 3.1 Percent G2 delay after γ-radiation exposure: A considerable G2 phase delay was observed in control cell lines (19.18%, range -1.95% - 29.65% at 5 Gy; and 15.83%, range -2.38% - 23.10% at 10 Gy) while the extent of G2 phase arrest was lower in MPN patient cell lines (5.83%, range -1.53% - 14.41% at 5 Gy; and 3.86%, range - 1.59% - 16.98% at 10 Gy) indicating that MPN cell lines bypass the cell cycle arrest. The mean difference of percent G2 delay between the two groups was 13.35% at 5 Gy and 11.97% at 10 Gy which was statistically significant (p=0.0001 at 5 Gy and p=0.0017 at 10 Gy). 3.2 Cell cycle arrest after BPDE exposure: When the cells were treated with BPDE there was no considerable difference in the mean percent cell arrested in S and G2M phase at both 6 h and 24 h time point between MPN patient and control cell lines. 12
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: Synopsis slides were then dried and
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 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
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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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