LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis 5.2 Measurement of γ-H2AX by flow cytometry after γ-radiation exposure: 0.5x10 6 cells were irradiated with 2 Gy and 5 Gy γ-radiation in two sets. Unirradiated cell treated in the same way served as control. Following 30 min incubation from one set and 4 h incubation from the other set, cells were collected and washed with PBS and fixed with paraformaldehyde. Permeabilization was performed by addition of 0.3% TritonX-100 in FACS buffer (2% FBS and 0.02% sodium azide in 1X PBS) followed by blocking in 3% BSA in FACS buffer. Cells were incubated with γ-H2AX primary antibody at 1:250 dilution overnight at 4 o C and next day the cells were incubated with Alexa-488 secondary antibody at 1:300 dilution. Cells were further diluted with FACS buffer and acquired by flow cytometry. Data analysis was done using CellQuest Pro software. 6. Effect of genotoxic exposure on cell cycle profile: In order to assess the effect of genotoxic exposure on cell cycle profile the following experiments were done. 6.1 Effect of γ-radiation on cell cycle profile: 1x10 6 cells were irradiated with 5 Gy and 10 Gy radiation dose and allowed to grow for 10 h. Cells were collected, fixed and stained as described in 4.2. Cell cycle phase distribution was obtained after analysing data using ModFit LT V 2.0 software. The differences in G2 cell percentages between treated cells and untreated cells were recorded as the G2 cell cycle delay percentage. 6.2 Effect of BPDE exposure on cell cycle profile: 1x10 6 cells were treated with 5 µM and 8 µM BPDE dissolved in Tetrahydrofuran (THF) and incubated for 6 h. Untreated cells and cells treated with THF alone were taken as controls. After 6 h half of the cells were collected and fixed with 70% alcohol. Rest half were further allowed to grow for 18 h, after removing BPDE containing medium and adding fresh complete medium, so as to complete a 24 h cell cycle. After the 24 h rest half of the cells were collected and fixed with 70% alcohol. The cells for both BPDE concentration and both time points were stained as described in 4.2. Cell cycle phase distribution was obtained after analysing data using ModFit LT V 2.0 software. 7. Measurement of percent cell death after genotoxic exposure: LCLs were treated with γ-radiation and BPDE to assess difference in percent cell death between MPN patient and control groups. 7
Synopsis 7.1 Percent cell death after γ-radiation exposure: 0.5x10 6 cells irradiated with 5 Gy and 10 Gy γ-radiation and were allowed to grow for 48 h. Unirradiated cells treated in the same way were taken as a control. To quantitate percent cell death cells were stained with Annexin-V-FLUOS antibody and Propidium iodide (PI) where Annexin- V-FLUOS stains apoptotic cells and PI stains the necrotic cells. Cells were incubated with Annexin-V-FLUOS antibody at 1:100 dilution and 1 µg PI made in incubation buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl 2 ) and analyze by flow cytometry. Data analysis was done using CellQuest Pro software. 7.2 Percent cell death after BPDE exposure: 0.5x10 6 cells were treated with 5 µM and 8 µM BPDE dissolved in tetrahydrofuran (THF) and incubated for 6 h. Untreated cells and cells treated with THF alone were taken as controls. After 6 h cells were collected and washed with PBS. To quantitate percent cell death Annexin-V- PI staining was performed as described in 7.1. 8. Genotyping of genes: Genotyping of genes was done using standard techniques as described below. 8.1 DNA isolation: RBCs were lysed using sucrose lysis buffer (0.32 M sucrose, 10 mM Tris pH 7.5, 5 mM MgCl 2, 1% Triton X-100). Lysis buffer was added to blood in the ratio of 10:1 and centrifuged to get the nucleated blood cell pellet. Genomic DNA from the pellet was extracted by the lysis of cell membrane with STE buffer (0.1 M NaCl, 0.05 M Tris pH 7.5, 1 mM EDTA pH 8.0, 1% SDS) and proteinase K digestion over night at 37 °C. DNA was isolated by following standard phenol-chloroform extraction method and an aliquot was run on 0.8% agarose gel to check DNA integrity, quality and yield. The samples were stored at 0 °C till further use. 8.2 Polymerase chain reaction: PCR reactions were carried using gene specific primers in sterile thin walled microfuge tubes. The reactions were carried out in 25 μl volumes containing 100 ng of DNA template where reaction mixture without DNA served as negative control and the product was analysed by agarose gel electrophoresis. The remaining product was used for either restriction fragment length polymorphism or for SNaPshot reaction. 8
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: Synopsis loading control. PCR produ
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 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
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Materials and Methods Method: EBV-t
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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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