Investigating the role of the JAK/STAT and MAPK ... - UCL Discovery

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This thesis is dedicated to my parents 4
Acknowledgements First and foremost I would like to thank my parents who throughout my life always told me I could be whatever I wanted to and for their constant love and support. All those years of encouragement really paid off! I would like to thank Prof. David Latchman for giving me the opportunity to work in his lab and letting me have the freedom to pursue my scientific interests. My appreciation goes to Paul Townsend for his support and insight over the last few years and also to Steph and Dicko. Thanks to the Bogue foundation for providing me with a fellowship to travel to Yale and to Richard Flavell for giving me the opportunity to work in his lab there. Special thanks to Hongbo Chi for really looking after me at Yale and for all he taught me there. The journey over the last few years would have been far less fun and interesting without all the wonderful people who have been part of MMBU, both past and present. Jimmy, Mattia, Naushaad, Dan, Charis, Emma, because of you guys I got to a rare thing; spend each day working with friends rather than colleagues. My thanks aswell to our tireless lab manager John, who keeps the whole thing together. Finally I would like to thank Sam for her love and support, you make it all worthwhile. grá mo chroí. Declaration: The work described in this thesis was carried out by the author unless otherwise stated 5 Is tú
Page 1 and 2: Investigating the role of the JAK/S
Page 3: Abstract The signal transducer and
Page 7 and 8: Table of Contents Abstract 3 Dedica
Page 9 and 10: 2.4.3 Hypoxia/Reoxygenation of Neon
Page 11 and 12: Chapter 6: Regulation of the MAPK P
Page 13 and 14: Figure 3.11 - STAT3 mRNA expression
Page 15 and 16: Figure 6.6 - Prolonged MAPK activit
Page 17 and 18: Abbreviations 7-AAD 7-amino-actinom
Page 19 and 20: SOCS Supressor of cytokine signalli
Page 21 and 22: 1.1 Myocardial Infarction Coronary
Page 23 and 24: Reperfusion induced arrhythmias are
Page 25 and 26: eceptors such as Fas or TNFR1; thes
Page 27 and 28: 1.2.3 Bcl-2 Proteins The intrinsic
Page 29 and 30: addition to caspase inhibition. XIA
Page 31 and 32: staurosporine induced cell death. L
Page 33 and 34: stress thus occurs when excess ROS
Page 35 and 36: 1.3 The JAK/STAT Pathway 1.3.1 JAK/
Page 37 and 38: Receptor SOCS3 Cytokines/Growth Fac
Page 39 and 40: Dimerization of STATs appears to be
Page 41 and 42: Fig 1.7. STAT import/export cycle.
Page 43 and 44: Kinase Stimulus Cell Type Reference
Page 45 and 46: domain determines other target sele
Page 47 and 48: 1.3.8 Inhibition of STAT DNA Bindin
Page 49 and 50: (BRM/SWI2-related gene 1) the ATPas
Page 51 and 52: deficient mammary glands (Abell et
Page 53 and 54: and COX-2 induction. Adenoviral med
Page 55 and 56:
compensatory hypertrophy, mediated
Page 57 and 58:
1.5.3 Urocortins and Ischaemia As w
Page 59 and 60:
Ott et al. recently showed that the
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1.6.4 The SAM Complex The outer mem
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etain H2AX activity for longer than
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esulting in an ATM-MDC1-H2AX positi
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1.8.3 TLR Adaptors TLR signalling i
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1.9 The Adaptive Immune System 1.9.
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Fig 1.14. Outline of T-helper cell
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1.10.3 IL-12 IL-12 is a potent pro-
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1.11.2 p38 MAPK p38 is induced by a
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negative JNK overcomes LPS induced
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1.12 Inflammatory Diseases 1.12.1 M
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10 producing Th2 response appears t
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2.1 Reagents The following TLR liga
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2.3.2 Endotoxic shock Toxic shock o
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2.4 Cell Culture 2.4.1 Freezing and
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emove red blood cells. CD4 and CD8
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2.5.2 ELISA ELISA was used as a met
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mM NaCl, 1 mM EDTA, 1 mM EGTA and p
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To measure gene promoter regulation
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uffer (200 mM MES buffer, 2 M NaCl,
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was added (10 mM RbCl, 50 mM , 100
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2.9 Cell Death Measurements 2.9.1 T
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Chapter 3: Investigating the Role o
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3.2 Overexpression of STAT3 Protect
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A B % Cell Death 50 40 30 20 10 0 G
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cell death were examined. Using thi
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A B 7AAD GFP %Cell Death 100 75 50
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3.4 Deletion of STAT3 Sensitises Ce
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Next, wild type and STAT3 knockout
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3.5 STAT3 becomes Phosphorylated an
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While STAT3 is phosphorylated at bo
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Two STAT3 target genes, SOCS3 and c
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A STAT3 B C Fold Change Fold Change
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3.6 Oxidative stress induces STAT3
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3. 7 Activation of STAT1 and STAT3
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Next, the extent of DNA damage and
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A B C pSTAT3 Y705 pSTAT3 S727 Total
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3.9 I/R injury in the brain induces
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3.10 Reperfusion Induced Myocardial
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Tempol infusion before the onset of
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In order to examine the tissue dist
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Fig 3.22. Effect of I/R and drug in
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3.12 Discussion STAT transcription
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may be involved. JAK2 activity has
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studies have begun to address the r
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4.1 Aims In the previous chapter, t
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all 15 arrays to be included in dow
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C D A Log intensity Raw Intensity B
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A B C I/R Ucn1 Probe Sets Annotated
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Table 4.2. The 20 genes with the hi
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A B 209 245 150 111 133 30 44 41 30
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A B 161
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E Activation Inhibition Translocati
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Number of Genes 20 15 10 5 0 No Ide
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4.6 Differential Expression mediate
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Number of Genes Number of Genes 20
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Ucn1 Ucn2 Fig 4.10 Network analysis
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A B D qPCR Expression (log2) qPCR E
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Table 4.5. Genes differentially reg
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A C Fold Change Fold Change 17.5 15
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A C Fold Change Fold Change 30 20 1
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order to examine if neonatal cardia
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4.10 Differential Regulation of MAP
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4.11 Uracil Metabolism is Altered b
Page 187 and 188:
4.12 Reduced Expression of Mitochon
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Gene Gene Title FC p value Nqo2 NAD
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ATP thus allowing protons to be pum
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A Fold Change 1.00 0.75 0.50 0.25 0
Page 195 and 196:
Fig 4.19. Tempol and Ucn1 upregulat
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A B Fold Change [MDA] µmol/g prote
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exogenously added IL-17 could induc
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The subunits that comprise the mito
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Taken together, these observations
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mediator of Ucn1 and Ucn2 cardiopro
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5.1 Aims In chapter 3 it was shown
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To show that reduced DNA repair in
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A pATM S1981 GAPDH C H2AX GAPDH STA
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5.4. STAT3 Facilitates DNA Damage M
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The STAT3 dependent regulation of M
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5.5 Discussion Efficient repair of
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(Cimprich and Cortez, 2008). Chk1 c
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Chapter 6: Regulation of the MAPK P
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700 bp 450 bp +/+ -/- +/- Fig 6.1 M
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The pathological consequences of en
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6.3 MKP-1 Negatively Regulates p38
Page 229 and 230:
A B AP-1 AP-1 Fold Change 1500 1000
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A B C MKP-1 Actin D MKP-1 Actin Act
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A B MKP-1 Actin Fig 6.9. MKP-1 upre
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A B IL-10 (pg/ml) 700 600 500 400 3
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B A Fold Change 1000 750 500 250 0
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6.6 Dynamic Regulation of TNF- is M
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levels seen by 5 hr LPS challenge i
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Previous studies have shown that IL
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6.7 MKP-1 Activity Promotes IL-12 E
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B A Fold Change 6000 4000 2000 0 +/
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A B IL-12p70 pg/ml IL-12p70 500 +/+
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% Weight 105 100 95 90 DSS 0 5 10 1
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6.10 Loss of MKP-1 does not Effect
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Taken together, these results demon
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Since loss of MKP-1 does not appear
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mice leads increased NO levels (Zha
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Fig 6.26. Model of MKP-1 mediated t
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of DSS induced colitis. Other possi
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wild type mice but failed to do so
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STAT3 was found to be active during
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Both Ucn1 and Ucn2 were found to lo
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cytokine production (IL-2, IL-4, IF
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(A) Table of Antibodies Appendix 1
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IL-6_F ACTGCCTTCCCTACTTCACA IL-6_R
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Alexander WS, Starr R, Fenner JE, S
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Bevan MJ: Helping the CD8(+) T-cell
Page 281 and 282:
Chen P, Li J, Barnes J, Kokkonen GC
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Deveraux QL, Takahashi R, Salvesen
Page 285 and 286:
Garcia R, Bowman TL, Niu G, Yu H, M
Page 287 and 288:
Hirota H, Izumi M, Hamaguchi T, Sug
Page 289 and 290:
Kassel O, Sancono A, Kratzschmar J,
Page 291 and 292:
Kuwata H, Watanabe Y, Miyoshi H, Ya
Page 293 and 294:
Lufei C, Ma J, Huang G, Zhang T, No
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Minners J, McLeod CJ, Sack MN: Mito
Page 297 and 298:
KL, JA, TL, R, TE: Activation of ST
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F, Y, D, C, SB, PT, RJ, Monte F. Pr
Page 301 and 302:
Roucou X, Montessuit S, Antonsson B
Page 303 and 304:
Shen Y, Schlessinger K, Zhu X, Meff
Page 305 and 306:
Szabo SJ, Sullivan BM, Peng SL, Gli
Page 307 and 308:
Valledor AF, Xaus J, Comalada M, So
Page 309 and 310:
Yamamoto M, Sato S, Hemmi H, Hoshin
Page 311 and 312:
Zhang J, Yang J, Roy SK, Tininini S
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