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Cancer Invest Downloaded from informahealthcare.com by University of Chicago Library on 06/10/11 For personal use only. 27. Wang, H.; Zeng, Z.C.; Bui, T.A.; DiBiase, S.J.; Qin, W.; Xia, F.; Powell, S.N.; Iliakis, G. Nonhomologous end-joining of ionizing radiation-induced DNA double-stranded breaks in human tumor cells deficient in BRCA1 or BRCA2. Cancer Res 2001, 61, 270–277 28. Rogakou, E.P.; Pilch, D.R.; Orr, A.H.; Ivanova, V.S.; Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998, 273, 5858– 5868. 29. Olive, P.L.; Banath, J.P. Phosphorylation of histone H2AX as a measure of radiosensitivity. Int J Radiat Oncol Biol Phys 2004, 58, 331–335. 30. Burma, S.; Kurimasa, A.; Xie, G.; Taya, Y.; Araki, R.; Abe, M.; Crissman, H.A.; Ouyang, H.; Li, G.C.; Chen, D.J. DNA-dependent protein kinase-independent activation of p53 in response to DNA damage. J Biol Chem 1999, 274, 17139–17143. 31. Wang, S.; Guo, M.; Ouyang, H.; Li, X.; Cordon-Cardo, C.; Kurimasa, A.; Chen, D.J.; Fuks, Z.; Ling, C.C.; Li, G.C. The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest. Proc Natl Acad Sci USA 2000, 97, 1584–1588. 32. Fei, P.; Bernhard, E.J.; El-Deiry, W.S. Tissue-specific induction of p53 targets in vivo. Cancer Res 2002, 62, 7316–7327. 33. Yonish-Rouach, E.; Resnitzky, D.; Lotem, J.; Sachs, L.; Kimchi, A.; Oren, M. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 1991, 352, 345–347. 34. Sabbatini, P.; Lin, J.; Levine, A.J.; White, E. Essential role for p53-mediated transcription in E1A-induced apoptosis. Genes Dev 1995, 9, 2184–2192. 35. Caelles, C.; Helmberg, A.; Karin, M. p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature 1994, 370, 220–223. 36. Polyak, K.; Xia, Y.; Zweier, J.L.; Kinzler, K.W.; Vogelstein, B. A model for p53-induced apoptosis. Nature 1997, 389, 300–305. 37. Mihara, M.; Erster, S.; Zaika, A.; Petrenko, O.; Chittenden, T.; Pancoska, P.; Moll, U.M. p53 has a direct apoptogenic role at the mitochondria. Mol Cell 2003, 11, 577–590. 38. Fei, P.; El-Deiry, W.S. p53 and radiation responses. Oncogene 2003, 22, 5774–5783. 39. Mitra, A.K.; Bhat, N.; Sarma, A.; Krishna, M. Alteration in the expression of signaling parameters following carbon ion irradiation. Mol Cell Biochem 2005, 276(1–2), 169–173. 622 S. Ghosh et al.
doi:10.1016/j.ijrobp.2008.08.006 Int. J. Radiation Oncology Biol. Phys., Vol. 72, No. 5, pp. 1567–1574, 2008 Copyright Ó 2008 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/08/$–see front matter BIOLOGY CONTRIBUTION ROLE OF iNOS IN BYSTANDER SIGNALING BETWEEN MACROPHAGES AND LYMPHOMA CELLS SOMNATH GHOSH, M.SC.,* DHARMENDRA KUMAR MAURYA, PH.D.,* AND MALINI KRISHNA, PH.D.* *Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai, India Purpose: The present report describes the bystander effects of radiation between similar and dissimilar cells and the role of iNOS in such communication. Materials and Methods: EL-4 and RAW 264.7 cells were exposed to 5 Gy g-irradiation. The medium from irradiated cells was transferred to unirradiated cells. Results: Irradiated EL-4 cells as well as those cultured in the presence of medium from g-irradiated EL-4 cells showed an upregulation of NF-kB, iNOS, p53, and p21/waf1 genes. The directly irradiated and the bystander EL-4 cells showed an increase in DNA damage, apoptosis, and NO production. Bystander signaling was also found to exist between RAW 264.7 (macrophage) and EL-4 (lymphoma) cells. Unstimulated or irradiated RAW 264.7 cells did not induce bystander effect in unirradiated EL-4 cells, but LPS stimulated and irradiated RAW 264.7 cells induced an upregulation of NF-kB and iNOS genes and increased the DNA damage in bystander EL-4 cells. Treatment of EL-4 or RAW 264.7 cells with L-NAME significantly reduced the induction of gene expression and DNA damage in the bystander EL-4 cells, whereas treatment with cPTIO only partially reduced the induction of gene expression and DNA damage in the bystander EL-4 cells. Conclusions: It was concluded that active iNOS in the irradiated cells was essential for bystander response. Ó 2008 Elsevier Inc. Radiation, Bystander signaling, Nuclear factor-kB, iNOS, p53 and p21. INTRODUCTION It is now apparent that the target for biologic effects of ionizing radiation is not solely the irradiated cell, but also the surrounding cells and tissues. Preliminary evidence indicates that similar signal transduction pathways are activated in directly irradiated cells and the bystander cells and contribute to the induction of bystander DNA damage (1). Many genes have been shown to be activated in bystander cells (2), but evidence for genes that are involved in the signaling pathways is lacking. Several factors involving soluble growth factors (3, 4), oxidative metabolites (5), and those that pass through gap junction (5–7) have been implicated. Among the various compounds that could transmit the signal from irradiated cells to bystander cells, nitric oxide (NO), by virtue of its diffusible nature and long life in vivo is a promising candidate (8–10). The accumulation of inducible nitric oxide synthase (iNOS) and the increased activity of constitutive NOS have been known to be an early signaling event induced by radiation. Gamma irradiation–induced DNA damage is known to enhance NO production in lipopolysaccharide (LPS)-stimulated macrophages (11). It is likely that stimulated and unstimulated macrophage behave differently where bystander response is concerned. Although abscopal effects have been deemed responsible for retarding the growth of a similar tumors located at a distant site in the mice (12), their contribution in transferring signals between dissimilar cells have not been extensively examined. To validate the presence of abscopal effects in vitro, we have used two dissimilar cell lines viz. EL-4 cells, a lymphoma cell line and RAW 264.7 cells, a macrophage cell line. Because there is extensive intercellular communications between dissimilar cells during immune responses, the leukocytes and lymphocytes were deemed suitable candidates to study bystander effects. The present report looks at the mechanism of bystander response in similar cells (EL-4 Vs EL-4 cells) and whether the state of activation of RAW 264.7 cells affects the bystander response in dissimilar cells (EL-4 Vs RAW 264.7). METHODS AND MATERIALS Cell culture, reagents, and irradiation schedule Mouse lymphoma cell line, EL-4 and mouse macrophage RAW 264.7 cells were grown in RPMI 1640 (Sigma, St. Louis, Mo), Reprint requests to: Dr. Malini Krishna, Ph.D., Bhabha Atomic Research Centre, Radiation Biology and Health Science Division, Trombay, Mumbai, Mumbai, Maharashtra 400085, India. Tel: (+91) 22-25593868; Fax: (+91) 22-2550 5151; E-mail: malini@ barc.gov.in 1567 Conflict of interest: none Received April 25, 2008, and in revised form July 30, 2008. Accepted for publication Aug 2, 2008.
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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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Page 262 and 263: PUBLICATIONS AND PRESENTATIONS Publ
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