LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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1572 I. J. Radiation Oncology d Biology d Physics Volume 72, Number 5, 2008 Fig. 6. Gene expression of NF-kB (p65) and iNOS gene in EL-4 cells that had received medium from irradiated RAW 264.7 cells. EL-4 cells cultured for 2 h in presence of different conditioned media. b-Actin gene expression in each group was used as an internal control. Ratio of intensities of NF-kB (p65) and iNOS band to that of respective b-actin band as quantified from gel pictures are shown above each gel picture. Key: Lane 1, control EL-4 cells; Lane 2, EL-4 cells receiving medium from lipopolysaccharide (LPS)-stimulated (500 ng/mL) and g-irradiated RAW 264.7 cells; Lane 3, EL-4 cells receiving medium from g-irradiated RAW 264.7 cells without LPS stimulation; Lane 4, EL-4 cells receiving medium from LPSstimulated RAW 264.7 cells; Lane 5, EL-4 cells receiving medium from unirradiated RAW 264.7 cells. Data represent means SE of three independent experiments; significantly different from unirradiated controls: *p < 0.05, **p < 0.01. upregulation and increased production of NO, many forms of reactive nitrogen species would be formed leading to covalent modification of proteins and the expression of various other genes (24). Having confirmed the existence of bystander effect between similar cells (EL-4 Vs EL-4), the work was extended to look for the existence of cross-bystander effect between EL-4 and RAW 264.7 cells (i.e., lymphocytes and macrophages). There was a significant upregulation of NF-kB and iNOS genes in EL-4 cells that had received medium from LPS stimulated and irradiated RAW 264.7 cells (Fig. 6, Lane 2) and expectedly the DNA damage was also significantly higher in these cells (Fig. 7, Lane 2). A noteworthy finding of this study is that the unirradiated EL-4 cells that had received medium from LPS-stimulated RAW 264.7 cells also showed upregulation of iNOS gene but not NF-kB gene (Fig. 6, Lane 4). The LPS treatment of macrophage upregulates many genes in the macrophage hence their response to irradiation will be different from unstimulated macrophage. Another noteworthy finding of this study is that the addition of medium from nonirradiated RAW 264.7 cells resulted in significantly decrease in iNOS gene expression in EL-4 cells Fig. 7. DNA damage in EL-4 cells cultured for 2 h in presence of different conditioned media was measured using single-cell gel electrophoresis (‘‘comet assay’’) and expressed as tail length. Key: Lane 1, control EL-4 cells; Lane 2, EL-4 cells receiving medium from LPS-stimulated (500 ng/mL) and g-irradiated RAW 264.7 cells; Lane 3, EL-4 cells receiving medium from g irradiated RAW 264.7 cells without lipopolysaccharide stimulation; Lane 4, EL-4 cells receiving medium from LPS-stimulated RAW 264.7 cells; Lane 5, EL-4 cells receiving medium from unirradiated RAW 264.7 cells. Data represent means SE of three independent; significantly different from unirradiated controls: *p < 0.01. relative to the control (Fig. 6, Lane 5). Nonirradiated macrophages may release some factor that is responsible for down regulation of iNOS in EL-4 cells. Further studies in this direction will reveal the nature of the factor. It is clear from these results that effective cross-bystander effects exists between these two cell lines only when the RAW 264.7 cells are both stimulated and irradiated. Other studies have looked at only the clonogenic survival of dissimilar bystander cells (25). Numerous studies have implicated extracellular secreted signaling proteins in mediating the bystander effect (5), but the strongest contenders have been reactive oxygen and nitrogen species (26). To investigate what factors are involved in radiation induced bystander effect, the cells were treated with either L-NAME (NOS inhibitor) or cPTIO (NO scavenger). The effect of cPTIO and L-NAME in bystander response between similar cell (EL-4 Vs EL-4) was different in that treatment with L-NAME reduces the bystander induction of NF-kB as well as iNOS gene expression in EL-4 cells receiving medium from irradiated EL-4 cells (Fig. 8, Lane 4), but the treatment with cPTIO reduces the bystander induction of only NF-kB gene expression and not iNOS gene expression (Fig. 8, Lane 5). These results demonstrate that the activated NOS in the irradiated cell is essential for gene expression in the bystanding cell. The addition of either L-NAME or cPTIO reduces the bystander induction of NF-kB as well as iNOS gene expression in EL-4 cells receiving medium from LPS stimulated and irradiated RAW 264.7 cells (Fig. 8, Lanes 6, 7), indicating that both activation of iNOS in the irradiated cell and NO production play a role in cross-bystander effect.
Bystander effect: role of iNOS d S. GHOSH et al. 1573 Fig. 8. Gene expression of NF-kB (p65) and iNOS gene in EL-4 cells that had received medium from irradiated cells. EL-4 cells cultured for 2 h in presence of different conditioned media. b-Actin gene expression in each group was used as an internal control. Ratio of intensities of NF-kB (p65) and inducible nitric oxide synthase band to that of respective b-actin band as quantified from gel pictures are shown above each gel picture. Key: Lane 1, control EL- 4 cells; Lane 2, g-irradiated EL-4 cells; Lane 3, EL-4 cells receiving medium from g-irradiated EL-4 cells; Lane 4, EL-4 cells receiving medium from L-NAME treated and g-irradiated EL-4 cells; Lane 5, EL-4 cells receiving medium from cPTIO-treated and g-irradiated EL-4 cells; Lane 6, EL-4 cells receiving medium from LPS-stimulated (500 ng/mL), L-NAME treated and g-irradiated RAW 264.7 cells; Lane 7, EL-4 cells receiving medium from lipopolysaccharide-stimulated (500 ng/mL), cPTIO-treated and g-irradiated RAW 264.7 cells. Data represent means SE of three independent experiments; significantly different from unirradiated controls: *p < 0.05, **p < 0.01. The treatment with L-NAME or cPTIO also reduces the bystander induction of DNA damage in EL-4 cells receiving medium from irradiated EL-4 cells or LPS-stimulated and LPS-irradiated RAW 264.7 cells (Fig. 9, Lanes 4–7), demonstrating that NO contributes to the DNA damage in bystander EL-4 cells. NO is generated endogenously from L-arginine by inducible NO synthases (27, 28) that can be stimulated by radiation in mammalian cells (8). It is believed that NO itself does not induce DNA strand breaks, although one of its oxidation product, peroxynitrite, is toxic and can cause DNA Fig. 9. DNA damage in EL-4 cells cultured for 2 h in presence of different conditioned media was measured using single-cell gel electrophoresis (‘‘comet assay’’) and expressed as tail length. Key: Lane 1, control EL-4 cells; Lane 2, g-irradiated EL-4 cells; Lane 3, EL-4 cells receiving medium from g-irradiated EL-4 cells; Lane 4, EL-4 cells receiving medium from L-NAME treated and g-irradiated EL- 4 cells; Lane 5, EL-4 cells receiving medium from cPTIO treated and g-irradiated EL-4 cells; Lane 6, EL-4 cells receiving medium from lipopolysaccharide (LPS)-stimulated (500 ng/mL), L- NAME-treated, and g-irradiated RAW 264.7 cells; Lane 7, EL-4 cells receiving medium from LPS-stimulated (500 ng/mL), cPTIO-treated, and g-irradiated RAW 264.7 cells; Lane 8, EL-4 cells treated with L-NAME; Lane 9, EL-4 cells treated with cPTIO. Data represent means SE of three independent experiments; significantly different from unirradiated controls: *p < 0.05, **p < 0.01. damage by both attacking deoxyribose and direct oxidation of purine and pyrimidine (29). Radiation-induced NO has the possibility of attacking nearby cells within their diffusion distance of less than 0.5 mm (30, 31). However, because of their having very short half-lives, they may not be direct contributors to the cellular damage in the bystander population. This is confirmed by the fact that inclusion of cPTIO in the medium only marginally inhibits the bystander response in similar cells (Fig. 8, Lane 5). Contrarily, it has also been reported that NO is involved in the bystander responses caused by the conditioned medium harvested from irradiated cells (32). Therefore some long-lived bioactive factors downstream of NO are most likely involved in this bystander response. REFERENCES 1. Azzam EI, De Toledo SM, Spitz DR, et al. Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alphaparticle- irradiated normal human fibroblast cultures. Cancer Res 2002;62:5436–5442. 2. Azzam EI, De Toledo SM, Gooding T, et al. Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of a particles. Radiat Res 1998;150:497–504. 3. Mothersill C, Seymour C. Radiation-induced bystander effects, carcinogenesis and models. Oncogene 2003;22:7028–7033. 4. Mothersill C, Seymour C. Radiation-induced bystander effects: Are they good, bad or both? Med Confl Surviv 2005;21: 101–110. 5. Azzam EI, De Toledo SM, Little JB. Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect. Oncogene 2003;22:7050–7057.
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RADIATION INDUCED SIGNALING IN MAMM
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DECLARATION I, hereby declare that
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CONTENTS Page No. SYNOPSIS………
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2.11 Measurement of NO production
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PREAMBLE SYNOPSIS Ionising radiatio
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SYNOPSIS Aims and Objectives: To St
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SYNOPSIS 2.6 Immunofluorescence sta
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SYNOPSIS ATM gene expression, which
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SYNOPSIS Percentage Survival 100 80
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SYNOPSIS Ratio of Rad52 to Actin Ra
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Relative Phosphorylation 45 40 35 3
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SYNOPSIS Fig. 3.3A Clonogenic Cell
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SYNOPSIS (A) Av No. of phospho BRCA
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SYNOPSIS Percentage Survival 120 10
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SYNOPSIS Fig.3.4B. Existance of cro
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SYNOPSIS Fig. 3.4EDNA damage in EL-
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SYNOPSIS subsequent cytotoxicity ca
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SYNOPSIS [4] Hall, E. J. Radiobiolo
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CHAPTER 1 INTRODUCTION INTRODUCTION
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CHAPTER 1 INTRODUCTION 15.8% 9.56%
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CHAPTER 1 INTRODUCTION far apart an
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CHAPTER 1 INTRODUCTION and are more
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CHAPTER 1 INTRODUCTION 1.3 DNA dama
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CHAPTER 1 INTRODUCTION associates w
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CHAPTER 1 INTRODUCTION are unable t
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CHAPTER 1 INTRODUCTION Perhaps one
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CHAPTER 1 INTRODUCTION occurs at DS
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CHAPTER 1 INTRODUCTION related prot
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CHAPTER 1 INTRODUCTION damage must
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CHAPTER 1 INTRODUCTION At the prese
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CHAPTER 1 INTRODUCTION predominant
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CHAPTER 1 INTRODUCTION provide a me
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CHAPTER 1 INTRODUCTION hypersensiti
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CHAPTER 1 INTRODUCTION cycle-specif
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CHAPTER 1 INTRODUCTION 1.4 Overview
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CHAPTER 1 INTRODUCTION 1.4.1 Radiat
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CHAPTER 1 INTRODUCTION interaction
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CHAPTER 1 INTRODUCTION p90S6 kinase
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CHAPTER 1 INTRODUCTION CD4 (formerl
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CHAPTER 1 INTRODUCTION unrepaired d
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CHAPTER 1 INTRODUCTION well to the
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CHAPTER 1 INTRODUCTION 1.6 Radiatio
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CHAPTER 1 INTRODUCTION becomes pote
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CHAPTER 2 MATERIALS AND METHODS MAT
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CHAPTER 2 MATERIALS AND METHODS 2.2
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CHAPTER 2 MATERIALS AND METHODS res
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CHAPTER 2 MATERIALS AND METHODS Arr
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CHAPTER 2 MATERIALS AND METHODS PBS
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CHAPTER 2 MATERIALS AND METHODS the
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CHAPTER 2 MATERIALS AND METHODS 2.1
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CHAPTER 3 RESULTS RESULTS 93
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CHAPTER 3 RESULTS fractionated regi
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CHAPTER 3 RESULTS On comparison of
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CHAPTER 3 RESULTS Table 3.1.2A List
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CHAPTER 3 RESULTS 80 NM_002185 IL7R
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CHAPTER 3 RESULTS SAA2 149 AU134977
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CHAPTER 3 RESULTS 221 LOC643167 RNA
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CHAPTER 3 RESULTS 299 BF244402 FBXO
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CHAPTER 3 RESULTS CDK4) 57 AU152107
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CHAPTER 3 RESULTS 134 AL045793 METT
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CHAPTER 3 RESULTS 58 AF237813 ABAT
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CHAPTER 3 RESULTS 127 AI809749 ORMD
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CHAPTER 3 RESULTS 31 BE615699 UNC84
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CHAPTER 3 RESULTS 47 AF026303 SULT1
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CHAPTER 3 RESULTS 117 BF062193 CYor
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CHAPTER 3 RESULTS 187 AL036662 ---
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CHAPTER 3 RESULTS 52 AV686514 EMP2
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CHAPTER 3 RESULTS exposed to 10 Gy
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CHAPTER 3 RESULTS Fig.3.1.4 Gene ex
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CHAPTER 3 RESULTS Fig. 3.1.6 Radiat
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CHAPTER 3 RESULTS Fig. 3.1.7 Transl
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CHAPTER 3 RESULTS 3.1.9 Stabilizati
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CHAPTER 3 RESULTS 23±1.5% (Fig. 3.
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CHAPTER 3 RESULTS whereas only the
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CHAPTER 3 RESULTS with equal doses
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CHAPTER 3 RESULTS Fig.3.3.1.2 Forma
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CHAPTER 3 RESULTS Fig.3.3.1.3 Phosp
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CHAPTER 3 RESULTS (7.5±0.64) (Fig.
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CHAPTER 3 RESULTS Fig.3.3.1.7b Phos
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CHAPTER 3 RESULTS 3.3.2 Oxygen beam
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CHAPTER 3 RESULTS in all the cells.
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CHAPTER 3 RESULTS 3.3.2.3 Radiation
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CHAPTER 3 RESULTS Fig.3.3.2.7 Apopt
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CHAPTER 3 RESULTS PMA stimulated U9
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CHAPTER 3 RESULTS Fig.3.4.2. Exista
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CHAPTER 3 RESULTS up-regulation of
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CHAPTER 3 RESULTS Fig.3.4.3.2 NO pr
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CHAPTER 3 RESULTS 3.4.3.4 Apoptotic
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CHAPTER 3 RESULTS 3.4.4 Bystander e
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CHAPTER 3 RESULTS Fig.3.4.4b DNA da
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CHAPTER 3 RESULTS Fig.3.4.5a Gene e
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CHAPTER 4 DISCUSSION DISCUSSION 185
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CHAPTER 4 DISCUSSION directly expos
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CHAPTER 4 DISCUSSION dose is delive
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CHAPTER 4 DISCUSSION A clear cause
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CHAPTER 4 DISCUSSION 4.2 Proton bea
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CHAPTER 4 DISCUSSION Although the e
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CHAPTER 4 DISCUSSION Similar number
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CHAPTER 4 DISCUSSION the mechanism
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CHAPTER 4 DISCUSSION Thus unlike th
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CHAPTER 4 DISCUSSION different from
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CHAPTER 4 DISCUSSION upregulates ma
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CHAPTER 4 DISCUSSION Numerous studi
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CHAPTER 4 DISCUSSION inhibition of
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CHAPTER 4 DISCUSSION The survival o
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CHAPTER 5 REFERENCES [1] Khan, F. T
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CHAPTER 5 REFERENCES [19] Woo, R. A
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CHAPTER 5 REFERENCES [35] Nghiem, P
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CHAPTER 5 REFERENCES [52] McKay, B.
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CHAPTER 5 REFERENCES [69] Cary, R.
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CHAPTER 5 REFERENCES [84] Uziel, T.
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CHAPTER 5 REFERENCES [98] Liu, Y.;
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CHAPTER 5 REFERENCES [111] Fei, P.;
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CHAPTER 5 REFERENCES [127] Frank, K
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CHAPTER 5 REFERENCES [141] Pierce,
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CHAPTER 5 REFERENCES [156] Roots, R
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CHAPTER 5 REFERENCES [171] Xia, Z.;
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CHAPTER 5 REFERENCES (MAP) kinase c
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CHAPTER 5 REFERENCES phosphorylatio
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CHAPTER 5 REFERENCES [211] Anderson
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CHAPTER 5 REFERENCES [229] Konca, K
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CHAPTER 5 REFERENCES [245] Kastan,
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CHAPTER 5 REFERENCES [261] Miralbel
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