by Ho Wai Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 207-243). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.viii / Table of Contents --- p.x / List of Abbreviations --- p.xviii / List of Figures --- p.xxiv / List of Tables --- p.xxix / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Traumatic brain injury --- p.1 / Chapter 1.2 --- Ceils of the nervous system: glia --- p.1 / Chapter 1.2.1 --- Astroglia - / Chapter 1.2.1.1 --- Molecular markers of astroglia --- p.3 / Chapter 1.2.1.2 --- Functions of astroglia --- p.3 / Chapter 1.2.2 --- Oligodendrocyte --- p.5 / Chapter 1.2.2.1 --- Molecular markers of oligodendrocyte --- p.6 / Chapter 1.2.2.2 --- Functions of oligodendrocyte --- p.6 / Chapter 1.2.3 --- Microglia --- p.7 / Chapter 1.2.3.1 --- Molecular markers of microglia --- p.7 / Chapter 1.2.3.2 --- Functions of microglia --- p.8 / Chapter 1.3 --- Cytokine and brain injury --- p.8 / Chapter 1.4 --- Tumor necrosis factor alpha (TNF-α) --- p.9 / Chapter 1.5 --- TNF-α receptor --- p.10 / Chapter 1.6 --- Biological activities of TNF-α --- p.11 / Chapter 1.7 --- Signaling mechanism --- p.13 / Chapter 1.7.1 --- Protein kinase C --- p.13 / Chapter 1.7.2 --- Protein kinase A --- p.14 / Chapter 1.7.3 --- p38 mitogen-activated protein kinase (p38 MAPK) --- p.15 / Chapter 1.7.3.1 --- Biological activities of p38 MAPK --- p.18 / Chapter 1.7.4 --- Inducible nitric oxide synthase (iNOS) --- p.20 / Chapter 1.7.5 --- cAMP responsive element binding protein (CREB) --- p.21 / Chapter 1.7.6 --- Transcription factor c-fos --- p.23 / Chapter 1.7.7 --- Nuclear factor kappa-B (NF-kB) --- p.24 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.26 / Chapter 1.9 --- β-adrenergic receptor (β-AR) --- p.28 / Chapter 1.9.1 --- Functions of β-AR in astrocytes --- p.29 / Chapter 1.10 --- Why do we use C6 glioma cell? --- p.31 / Chapter 1.11 --- Fluorescent differential display (FDD) --- p.34 / Chapter 1.12 --- Aims and Scopes of this project --- p.36 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Material --- p.40 / Chapter 2.1.1 --- Cell line --- p.40 / Chapter 2.1.2 --- Cell culture reagents --- p.40 / Chapter 2.1.2.1 --- Complete Dulbecco's modified Eagle medium (CDMEM) --- p.40 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.41 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.41 / Chapter 2.1.3 --- Recombinant cytokines --- p.41 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.42 / Chapter 2.1.4.1 --- Modulators of p38 mitogen-activated protein kinase (p38 MAPK) --- p.42 / Chapter 2.1.4.2 --- Modulators of protein kinase C (PKC) --- p.42 / Chapter 2.1.4.3 --- Modulators of protein kinase A (PKA) --- p.42 / Chapter 2.1.4.4 --- β-Adrenergic agonist and antagonist --- p.43 / Chapter 2.1.5 --- Antibodies --- p.44 / Chapter 2.1.5.1 --- Anti-p38 mitogen-activated protein kinase (p38 MAPK) antibody --- p.44 / Chapter 2.1.5.2 --- Anti-phosporylation p38 mitogen-activated protein kinase (p-p38 MAPK) antibody --- p.44 / Chapter 2.1.5.3 --- Antibody conjugates --- p.44 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.45 / Chapter 2.1.7 --- Reagents for DNase I treatment --- p.45 / Chapter 2.1.8 --- Reagents for reverse transcription of mRNA and fluorescent PCR amplification --- p.45 / Chapter 2.1.9 --- Reagents for fluorescent differential display --- p.46 / Chapter 2.1.10 --- Materials for excision of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.11 --- Reagents for reamplification of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.12 --- Reagents for subcloning of reamplified cDNA fragments --- p.47 / Chapter 2.1.13 --- Reagents for purification of plasmid DNA from recombinant clones --- p.47 / Chapter 2.1.14 --- Reagents for DNA sequencing of differentially expressed cDNA fragments --- p.47 / Chapter 2.1.15 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.1.16 --- Reagents for electrophoresis --- p.50 / Chapter 2.1.17 --- Reagents and buffers for Western blot --- p.50 / Chapter 2.1.18 --- Other chemicals and reagents --- p.50 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.51 / Chapter 2.3 --- RNA isolation --- p.52 / Chapter 2.3.1 --- Measurement of RNA yield --- p.53 / Chapter 2.4 --- DNase I treatment --- p.53 / Chapter 2.5 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.54 / Chapter 2.6 --- Fluorescent differentia display --- p.55 / Chapter 2.7 --- Excision of differentially expressed cDNA fragments --- p.59 / Chapter 2.8 --- Reamplification of differentially expressed cDNA fragments --- p.59 / Chapter 2.9 --- Subcloning of reamplified cDNA fragments --- p.60 / Chapter 2.10 --- Purification of plasmid DNA from recombinant clones --- p.63 / Chapter 2.11 --- DNA sequencing of differentially expressed cDNA fragments --- p.64 / Chapter 2.12 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.66 / Chapter 2.13 --- Western bolt analysis --- p.67 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- DNase I treatment --- p.71 / Chapter 3.2 --- FDD RT-PCR and band excision --- p.71 / Chapter 3.3 --- Reamplification of excised cDNA fragments --- p.74 / Chapter 3.4 --- Subcloning of reamplified cDNA fragments --- p.77 / Chapter 3.5 --- DNA sequencing of subcloned cDNA fragments --- p.77 / Chapter 3.6 --- Confirmation of the differentially expressed cDNA fragments by RT-PCR and Western blotting --- p.84 / Chapter 3.6.1 --- Effects of TNF-α on p38a mitogen protein kinase (p38 α MAPK) --- p.84 / Chapter 3.6.2 --- Effects of TNF-α on p38 a MAPK and p-p38 α MAPK protein level --- p.86 / Chapter 3.7 --- Effects of TNF-α on p38 MAPK --- p.88 / Chapter 3.7.1 --- "Effects of TNF-α on p38 α, β,γ andδ MAPK" --- p.88 / Chapter 3.7.2 --- Role of TNF-receptor (TNF-R) subtype in the TNF-α-induced p3 8 MAPK expression in C6 cells --- p.89 / Chapter 3.7.3 --- The signaling system mediating TNF-α-induced p38 a MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.1 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.2 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.98 / Chapter 3.7.4 --- The relationship between p38 MAPK and β-adrenergic mechanisms in C6 cells --- p.99 / Chapter 3.7.4.1 --- Effects of isoproterenol and propanol on p38 MAPK mRNA levels in C6 cells --- p.103 / Chapter 3.7.4.2 --- Effects of β1-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.106 / Chapter 3.7.4.3 --- Effects of β2-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.107 / Chapter 3.8 --- The relationship between p3 8 MAPK and inducible nitric oxide synthase (iNOS) expression --- p.113 / Chapter 3.8.1 --- Effects of TNF-α on the iNOS expression in C6 cells --- p.113 / Chapter 3.8.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3 --- The signaling system mediating TNF-α-induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3.1 --- The involvement of p38 MAPK in the TNF-α-induced iNOS expression in C6 cells --- p.117 / Chapter 3.8.3.2 --- The involvement of PKA in the TNF-α-induced iNOS expression in C6 cells --- p.119 / Chapter 3.9 --- The relationship between p38 MAPK and cAMP-responsive element binding protein (CREB) expression --- p.120 / Chapter 3.9.1 --- Effects of TNF-α on the CREB expression in C6 cells --- p.120 / Chapter 3.9.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced CREB expression in C6 cells --- p.124 / Chapter 3.9.3 --- The signaling system mediating TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.1 --- The involvement of p38 MAPK in the TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.2 --- The involvement of PKC in the TNF-α-induced CREB expression in C6 cells --- p.128 / Chapter 3.9.3.3 --- The involvement of PKA in TNF-α-induced CREB expression in C6 cells --- p.129 / Chapter 3.9.4 --- The relationship between CREB and β-adrenergic mechanisms in C6 cells --- p.136 / Chapter 3.9.4.1 --- Effects of isoproterenol and propanol on CREB mRNA levels in C6 cells --- p.136 / Chapter 3.9.4.2 --- Effects of β1-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.139 / Chapter 3.9.4.3 --- Effects of (32-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.142 / Chapter 3.10 --- The relationship between p38 MAPK and transcription factor c-fos expression --- p.146 / Chapter 3.10.1 --- Effects of TNF-α on the c-fos expression in C6 cells --- p.146 / Chapter 3.10.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced c-fos expression in C6 cells --- p.146 / Chapter 3.10.3 --- The signaling system mediating TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.1 --- The involvement of p38 MAPK in the TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.2 --- The involvement of PKC in the TNF-α-induced c-fos expression in C6 cells --- p.151 / Chapter 3.10.3.3 --- The involvement of PKA in TNF-α-induced c-fos expression in C6 cells --- p.154 / Chapter 3.10.4 --- The relationship between c-fos and β-adrenergic mechanisms in C6 cells --- p.157 / Chapter 3.10.4.1 --- Effects of isoproterenol and propanolol on c-fos mRNA levels in C6 cells --- p.157 / Chapter 3.10.4.2 --- Effects of β1-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.160 / Chapter 3.10.4.3 --- Effects of β2-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.164 / Chapter 3.11 --- The relationship between p38 MAPK and transcription factor NF-kB expression --- p.168 / Chapter 3.11.1 --- Effects of TNF-α on the NF-kB expression in C6 cells --- p.168 / Chapter 3.11.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced NF-kB expression in C6 cells --- p.168 / Chapter 3.11.3 --- The signaling system mediating TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.1 --- The involvement of p38 MAPK in the TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.2 --- The involvement of PKC in the TNF-α-induced NF-kB expression in C6 cells --- p.173 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of tumor-necrosis factor-alpha (TNF-α) on C6 cell proliferations --- p.176 / Chapter 4.2 --- The Signaling System Involved in TNF-α-Induced p38 MAPK Expression in C6 cells --- p.178 / Chapter 4.3 --- The Signaling System Involved in TNF-α-Induced iNOS Expression in C6 cells --- p.184 / Chapter 4.4 --- The Signaling System Involved in TNF-α-Induced CREB Expression in C6 cells --- p.186 / Chapter 4.5 --- The Signaling System Involved in TNF-α-Induced c-fos Expressionin in C6 cells --- p.190 / Chapter 4.6 --- The Signaling System Involved in TNF-α-Induced NF-kB Expression in C6 cells --- p.193 / Chapter 4.7 --- Conclusions --- p.195 / Chapter 4.8 --- Possible application / References

by Wong Kin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 348-373). / Abstracts in English and Chinese. / Abstract --- p.ii / 撮要 --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xviii / List of Tables --- p.xxi / List of Figures --- p.xxii / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1. --- Events happened in brain injury --- p.1 / Chapter 1.2. --- An alternate approach based on neuronal regeneration --- p.3 / Chapter 1.3. --- Fate of astrocytes after brain injury --- p.4 / Chapter 1.3.1. --- General information of astrocytes --- p.4 / Chapter 1.3.2. --- Functions of astrocytes --- p.5 / Chapter 1.4. --- Factors relate to astrocytes proliferation --- p.7 / Chapter 1.4.1. --- TNF-α --- p.8 / Chapter 1.4.2. --- β adrenergic mechanism and astrocyte proliferation --- p.11 / Chapter 1.5. --- Cell cycle-related proteins --- p.13 / Chapter 1.5.1. --- Maturation promoting factor (MPF) --- p.15 / Chapter 1.5.2. --- Early G1 phase --- p.16 / Chapter 1.5.3. --- Retinoblastoma protein (pRb) --- p.18 / Chapter 1.5.4. --- Cyclin-dependent kinase (cdk) activating kinase (Cak) --- p.19 / Chapter 1.5.5. --- "Cyclin, cdks, cki" --- p.20 / Chapter 1.5.5.1. --- Cyclins --- p.20 / Chapter 1.5.5.1.1. --- Cyclin D --- p.21 / Chapter 1.5.5.1.2. --- Cyclin E --- p.22 / Chapter 1.5.5.1.3. --- Cyclin A --- p.23 / Chapter 1.5.5.1.4. --- Cyclin B --- p.23 / Chapter 1.5.5.2. --- Cyclin-dependent kinases (cdks) --- p.24 / Chapter 1.5.5.3. --- Cyclin-dependent kinase inhibitor (cki) --- p.24 / Chapter 1.5.5.3.1. --- INK4 proteins (inhibitors of cdk-4 and cdk-6) --- p.25 / Chapter 1.5.5.3.2. --- p21 family proteins --- p.25 / Chapter 1.5.5.3.2.1. --- p21 --- p.25 / Chapter 1.5.5.3.2.2. --- p27 --- p.25 / Chapter 1.6. --- Apoptosis related proteins --- p.26 / Chapter 1.6.1. --- bcl-2 family --- p.26 / Chapter 1.6.1.1. --- bcl-2 --- p.26 / Chapter 1.6.1.2. --- bcl-x --- p.27 / Chapter 1.6.1.3. --- bcl-xα --- p.27 / Chapter 1.6.1.4. --- bcl-w --- p.28 / Chapter 1.6.1.5. --- Myeloid cell leukemia factor 1 (Mcl-1) --- p.28 / Chapter 1.7. --- C6 glioma cell line --- p.28 / Chapter 1.8. --- Aim of this project --- p.30 / Chapter CHAPTER 2. --- MATERIALS & METHODS / Chapter 2.1. --- Materials / Chapter 2.1.1. --- Rat C6 glioma cell line --- p.32 / Chapter 2.1.2. --- Cell culture materials preparation / Chapter 2.1.2.1. --- Complete Dulbecco's Modified Medium (cDMEM) --- p.32 / Chapter 2.1.2.2. --- Serum-free Dulbecco's Modified Medium (sDMEM) --- p.33 / Chapter 2.1.2.3. --- Phosphate buffered saline (PBS) --- p.33 / Chapter 2.1.3. --- Drug preparation / Chapter 2.1.3.1. --- Recombinant cytokines --- p.34 / Chapter 2.1.3.2. --- Antibodies / Chapter 2.1.3.2.1. --- Antibodies used in expression analysis --- p.34 / Chapter 2.1.4. --- Antibodies used in Western blotting --- p.34 / Chapter 2.1.5. --- Reagents for RNA isolation --- p.36 / Chapter 2.1.6. --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.36 / Chapter 2.1.7. --- Reagents for Electrophoresis --- p.38 / Chapter 2.1.8. --- Reagents and buffers for Western blotting --- p.38 / Chapter 2.1.9. --- Other chemicals and reagents --- p.39 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Maintenance of C6 cells --- p.39 / Chapter 2.2.2. --- Preparation of cells for assays --- p.40 / Chapter 2.2.3. --- Drugs preparation --- p.40 / Chapter 2.2.4. --- Determination of RNA expression by RT-PCR analysis / Chapter 2.2.4.1. --- RNA extraction --- p.41 / Chapter 2.2.4.2. --- Spectrophotometric Quantitation of DNA and RNA --- p.43 / Chapter 2.2.4.3. --- RNA gel electrophoresis --- p.43 / Chapter 2.2.4.4. --- Reverse transcription-polymerase chain reaction (RT- PCR) --- p.43 / Chapter 2.2.4.5. --- Separation of PCR products by agarose gel electrophoresis --- p.43 / Chapter 2.2.4.6. --- Quantification of band density --- p.45 / Chapter 2.2.4.7. --- Restriction enzyme (RE) digestion --- p.45 / Chapter 2.2.5. --- Determination of protein expression by Western blotting / Chapter 2.2.5.1. --- Total protein extraction --- p.46 / Chapter 2.2.5.2. --- Western blotting analysis --- p.46 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1. --- Effects of TNF-α on cell cycle related genes and proteins expression --- p.49 / Chapter 3.1.1. --- Effects of TNF-α on the time courses of cyclin D1 gene and protein expression --- p.49 / Chapter 3.1.2. --- Effect of TNF-α on the time course of cyclin D2 gene expression --- p.50 / Chapter 3.1.3. --- Effects of TNF-α on the time courses of cyclin D3 gene and protein expression --- p.53 / Chapter 3.1.4. --- Effects of TNF-α on the time courses of cdk-4 gene and protein expression --- p.55 / Chapter 3.1.5. --- Effects of TNF-α on the time courses of cyclin E gene and protein expression --- p.55 / Chapter 3.1.6. --- Effects of TNF-α on the time courses of cdk-2 gene and protein expression --- p.58 / Chapter 3.1.7. --- Effects of TNF-α on the time courses of p15 gene and protein expression --- p.61 / Chapter 3.1.8. --- Effects of TNF-α on the time courses of p27 gene and protein expression --- p.61 / Chapter 3.1.9. --- Effects of TNF-α on the time courses of p21 gene and protein expression --- p.64 / Chapter 3.1.10. --- Effects of TNF-α on the time courses of p130 gene and protein expression --- p.66 / Chapter 3.1.11. --- Effects of TNF-α on the time courses of Cak gene and protein expression --- p.66 / Chapter 3.1.12. --- Effects of TNF-α on the time courses of cyclin H gene and protein expression --- p.68 / Chapter 3.1.13. --- Effects of TNF-α on the time courses of cyclin B gene and protein expression- --- p.71 / Chapter 3.1.14. --- Effect of TNF-α on the time course of bcl-2 protein expression --- p.71 / Chapter 3.1.15. --- Effects of TNF-α on the time courses of bcl-XL gene and protein expression --- p.73 / Chapter 3.1.16. --- Effect of TNF-α on the time course of bcl-xα gene expression --- p.73 / Chapter 3.1.17. --- Effects of TNF-α on the time courses of bcl-w gene and protein expression --- p.76 / Chapter 3.1.18. --- Effects of TNF-α on the time courses of Mcl-1 gene expression --- p.76 / Chapter 3.2. --- Effects of TNF-R1 and -R2 on cell cycle related genes and proteins expression --- p.81 / Chapter 3.2.1. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin D1 gene and protein expression --- p.81 / Chapter 3.2.2. --- Effect of blocking TNF-R1/ -R2 on the time course of cyclin D2 gene expression --- p.82 / Chapter 3.2.3. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin D3 gene and protein expression --- p.85 / Chapter 3.2.4. --- Effects of blocking TNF-R1/ -R2 on the time courses of cdk-4 gene and protein expression --- p.90 / Chapter 3.2.5. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin E gene and protein expression --- p.93 / Chapter 3.2.6. --- Effects of blocking TNF-R1/ -R2 on the time courses of cdk-2 gene and protein expression --- p.93 / Chapter 3.2.7. --- Effects of blocking TNF-R1/ -R2 on the time courses of p15 gene and protein expression --- p.96 / Chapter 3.2.8. --- Effects of blocking TNF-R1/ -R2 on the time courses of p27 gene and protein expression --- p.99 / Chapter 3.2.9. --- Effects of blocking TNF-R1/ -R2 on the time courses of p21 gene and protein expression --- p.103 / Chapter 3.2.10. --- Effects of blocking TNF-R1/ -R2 on the time courses of pl30 gene and protein expression --- p.106 / Chapter 3.2.11. --- Effect of blocking TNF-R1/ -R2 on the time course of Cak gene expression --- p.110 / Chapter 3.2.12. --- Effects of blocking TNP-R1/ -R2 on the time courses of cyclin H gene and protein expression --- p.110 / Chapter 3.2.13. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin B gene and protein expression --- p.112 / Chapter 3.2.14. --- Effect of blocking TNF-R1/ -R2 on the time course of bcl-2 protein expression --- p.116 / Chapter 3.2.15. --- Effects of blocking TNF-R1/ -R2 on the time courses of bcl-xL gene and protein expression --- p.119 / Chapter 3.2.16. --- Effect of blocking TNF-R1/ -R2 on the time course of bcl-xα gene expression --- p.122 / Chapter 3.2.17. --- Effects of blocking TNF-R1/ -R2 on the time courses of bcl-w gene and protein expression --- p.124 / Chapter 3.2.18. --- Effect of blocking TNF-R1/ -R2 on the time course of Mcl-1 gene expression --- p.124 / Chapter 3.3. --- "Effects of other cytokines (IL-6, IL-lα, IL-lβ, IFγ) on cell cycle related genes and proteins expression" --- p.129 / Chapter 3.3.1. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on cyclin D1 gene and protein expression" --- p.129 / Chapter 3.3.2. --- "Effects of TNF-a, IL-6, IL-lα, IL-lβ, IFγ on cyclin D2 gene and protein expression" --- p.132 / Chapter 3.3.3. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin D3 gene and protein expression" --- p.136 / Chapter 3.3.4. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cdk-4 gene and protein expression" --- p.140 / Chapter 3.3.5. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin E gene and protein expression" --- p.144 / Chapter 3.3.6. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cdk-2 gene and protein expression" --- p.148 / Chapter 3.3.7. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on pl5 gene and protein expression" --- p.152 / Chapter 3.3.8. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on p27 gene and protein expression" --- p.152 / Chapter 3.3.9. --- "Effects of TNF-α, IL-6, IL-lα, IL-ip, IFγ on p21 gene and protein expression" --- p.159 / Chapter 3.3.10. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on pl30 gene and protein expression" --- p.162 / Chapter 3.3.11. --- "Effects of TNF-α, IL-6, IL-lα, IL-lp, IFγ on Cak gene expression" --- p.166 / Chapter 3.3.12. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFy on cyclin H gene and protein expression -" --- p.170 / Chapter 3.3.13. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin B gene and protein expression" --- p.174 / Chapter 3.3.14. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on bcl-2 gene and protein expression" --- p.178 / Chapter 3.3.15. --- "Effects of TNF-a, IL-6, IL-lα, IL-1β, IFγ on bcl-xL gene and protein expression" --- p.178 / Chapter 3.3.16. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on bcl-xα gene expression" --- p.184 / Chapter 3.3.17. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on bcl-w gene and protein expression" --- p.187 / Chapter 3.3.18. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on Mcl-1 gene expression" --- p.191 / Chapter 3.4. --- Effects of P-ARs on cell cycle related genes expression --- p.194 / Chapter 3.4.1. --- Effects of β-AR agonists and antagonists on cyclin D1 gene expression --- p.195 / Chapter 3.4.2. --- Effects of β-AR agonists and antagonists on cyclin D2 gene expression --- p.198 / Chapter 3.4.3. --- Effects of β-AR agonists and antagonists on cyclin D3 gene expression --- p.201 / Chapter 3.4.4. --- Effects of β-AR agonists and antagonists on cdk-4 gene expression --- p.204 / Chapter 3.4.5. --- Effects of β-AR agonists and antagonists on cyclin E gene expression --- p.207 / Chapter 3.4.6. --- Effects of β-AR agonists and antagonists on cdk-2 gene expression - --- p.210 / Chapter 3.4.7. --- Effects of β-AR agonists and antagonists on p15 gene expression --- p.213 / Chapter 3.4.8. --- Effects of β-AR agonists and antagonists on p27 gene expression --- p.216 / Chapter 3.4.9. --- Effects of β-AR agonists and antagonists on p21 gene expression --- p.219 / Chapter 3.4.10. --- Effects of β-AR agonists and antagonists on p130 gene expression --- p.222 / Chapter 3.4.11. --- Effects of β-AR agonists and antagonists on Cak gene expression --- p.225 / Chapter 3.4.12. --- Effects of β-AR agonists and antagonists on cyclin H gene expression --- p.228 / Chapter 3.4.13. --- Effects of β-AR agonists and antagonists on cyclin B gene expression --- p.231 / Chapter 3.4.14. --- Effects of β-AR agonists and antagonists on bcl-XL gene expression --- p.233 / Chapter 3.4.15. --- Effects of β-AR agonists and antagonists on bcl-xα gene expression --- p.236 / Chapter 3.4.16. --- Effects of β-AR agonists and antagonists on bcl-w gene expression --- p.239 / Chapter 3.4.17. --- Effects of β-AR agonists and antagonists on Mcl-1 gene expression --- p.243 / Chapter CHAPTER 4. --- DISCUSSION & CONCLUSION --- p.247 / Chapter 4.1. --- Effects of TNF-α on the induction of cell cycle regulatory genes/proteins expression --- p.248 / Chapter 4.2. --- Effects of TNF-α on bcl-2 family apoptotic inhibitor genes expression --- p.250 / Chapter 4.3. --- The TNF-R subtype(s) responsible for the TNF-a-induced cell cycle regulatory genes and proteins expression --- p.251 / Chapter 4.4. --- Is the TNF-α-induced cell cycle regulatory genes and proteins expression cytokine specific? --- p.253 / Chapter 4.5. --- The relationship between TNF-α and β-adrenergic mechanism in C6 cell proliferation --- p.254 / Chapter 4.6. --- General Discussion --- p.256 / Chapter 4.7. --- Possible treatments for brain injury --- p.258 / APPENDIX --- p.259 / REFERENCES --- p.348

"Magnetic resonance imaging (MRI) has increasingly become the preferred imaging modality in modern day research to study disease. MRI presents an imaging technique that is practically non-invasive and without any ionizing radiation. This dissertation presents the use of contrast agents in MRI studies to differentiate compartments, to study dose dependence of relaxation times, and to characterize tumors using signal amplifying enzymes in the brain. Differentiating compartments in the brain can be useful in diffusion studies to detect stroke at an early stage. Diffusion-weighted NMR techniques have established that the apparent diffusion coefficient (ADC) of cerebral tissue water decreases during ischemia. However, it is unclear whether the ADC change occurs due to changes in the intracellular (IC) space, extracellular (EC) space, or both. To better understand the mechanism of water ADC changes in response to ischemic injury, making IC and EC compartment specific measurements of water diffusion is essential. The first study was done where manganese (Mn2+) was used as an IC contrast agent. Mn2+ uptake by cells causes shortening of the T1 relaxation time of IC water. The relative difference in T1 relaxation times between the IC and EC compartments can be used to discriminate between the MR signals arising from water in the respective compartments. Mn2+ is also widely used in manganese-enhanced MRI (MEMRI) studies to visualize functional neural tracts and anatomy in the brain in vivo. In animal studies, the goal is to use a dose of Mn2+ that will maximize the contrast while minimizing its toxic effects. The goal of dose study was to investigate the MRI dose response of Mn2+ in rat brain following SC administration of Mn2+. The dose dependence and temporal dynamics of Mn2+ after SC injection can prove useful for longitudinal in vivo studies that require brain enhancement to persist for a long period of time to visualize neuroarchitecture like in neurodegenerative disease studies. Contrast agents, in addition to their use in compartmental differentiation and dose studies, can be used for imaging tumors. The last study in this dissertation focuses on imaging EGF receptors in brain tumors. We tested a novel pretargeting imaging approach that includes the administration of humanized monoclonal antibody (anti-EGFR mAb, EMD72000) linked to enzymes with complementing activities that use a low-molecular weight paramagnetic molecule (diTyr-GdDTPA) as a reducing substrate administered following the mAb conjugates. We analyzed the differential MR tumor signal decay in vivo using orthotopic models of human glioma. The patterns of MR signal change following substrate administration revealed differences in elimination patterns that allowed distinguishing between non-specific and specific modes of MR signal decay. "

Made available in DSpace on 2015-04-14T14:51:17Z (GMT). No. of bitstreams: 1 438124.pdf: 1241897 bytes, checksum: 68c450ddba15c588f04067f7bd75dae9 (MD5) Previous issue date: 2012-03-02 / Glioblastoma multiforme (GBM) is the most aggressive tumor of the CNS and most deadly primary tumors. Despite the malignant gliomas are generally treated with radiotherapy, often they exhibit a significant radioresistance that limits the treatment success. It can be postulated that molecular changes commonly observed in these tumors contribute to this resistance. The nucleotide ATP is an important signaling molecule in CNS and it is a selective P2X7 purinergic receptor (P2X7R) ligand at high concentrations. Some studies have reported that P2X7R is responsible for ATP-induced cell death in various cell types, but some human glioma cells were resistant to death induced by ATP. In addition to ATP resistance, patients with GBM develop spontaneous antitumor immune responses. These studies show the requirement for an exogenous induction of the immune system to generate an antitumor response. IL-17 is a pro-inflammatory cytokine and its role in cancer is already unknown. Herein, we first aimed to characterize a radiosensitive human glioma cell line for sensitivity to ATP and to investigate whether activation of the P2X7R could be involved in the death of this cell line. Furthermore, aimed to elucidate the IL-17 receptor susceptibility to irradiation, as well as, elucidate the effect of IL-17 and a possible interaction between this cytokine and P2X7R in human glioma cells. The human glioma cell lines U-138 MG and U-251 MG were resistant to death when treated with either ATP (5 mM) or BzATP (100 μM), a selective P2X7R agonist, whereas in the radiosensitive M059J glioma cell line, the high ATP (5 mM) or BzATP (100 μM), significantly diminished the cell viability (32.4% ? 4.1 and 24.6% ? 4.0, respectively). The M059J lineage expresses significantly higher P2X7R levels when compared to the U-138 MG and U-251 cell lines (0.40 ? 0.00; 0.28 ? 0.01 and 0.31 ? 0.01, respectively) and irradiation upregulated P2X7R expression in all lineages. Additionally, the selective P2X7R antagonist A740003 (10 ?M) significantly decreased the cell death caused by irradiation. We provided novel evidence indicating that M059J human glioma cell line is ATP-P2X7R sensitive, pointing out the relevance of the purinergic P2X7R in glioma radiosensitivity. The IL-17 receptor was significantly more expressed after irradiation (2 Gy), showing a possible participation of the IL-17/R on the irradiation susceptibility. The treatment with IL-17 (10 ng/ml) diminished approximately 62% the human glioma cell lines viability. Other preliminary result showed that the P2X7R antagonist was able to partially reverse the toxicity caused by IL-17 in these cell lines. Our data show an antitumor role of this cytokine and corroborate to the idea of a possible interaction between IL-17/P2X7R. This work prospects for studies to be conducted to better understanding the action of IL-17 in gliomas and interaction IL-17/P2X7R / O glioblastoma multiforma (GBM) ? considerado o mais agressivo tumor do sistema nervoso central (SNC), e o mais letal entre os tumores prim?rios. Apesar dos gliomas malignos serem geralmente tratados com radioterapia, muitas vezes estes exibem uma significativa radioresist?ncia que limita o sucesso do tratamento. Pode-se postular que mudan?as moleculares comuns observadas nestes tumores contribuem para essa resist?ncia. O nucleot?deo ATP ? uma importante mol?cula de sinaliza??o no SNC e um ligante seletivo do receptor purin?rgico P2X7 (P2X7R) em altas concentra??es. Estudos mostram que o P2X7R ? respons?vel pela morte induzida pelo ATP em v?rios tipos de c?lulas, por?m, algumas c?lulas de glioma humano mostram-se resistentes ? morte induzida pelo ATP. Al?m da resist?ncia ao ATP, pacientes com GBM espontaneamente desenvolvem resist?ncia a respostas imunes antitumorais. Estudos levantam a necessidade de uma indu??o ex?gena do sistema imunol?gico a fim de gerar uma resposta antitumoral. A IL-17 ? um citocina pr?-inflamat?ria e seu papel no c?ncer ainda ? desconhecido. O presente trabalho primeiramente objetivou caracterizar uma linhagem de glioma humano radiossens?vel quanto ? sensibilidade ao ATP e investigar se a ativa??o do P2X7R poderia estar envolvida na morte destas c?lulas. Al?m disso, visou elucidar a susceptibilidade do receptor da IL-17 ? radioterapia, bem como, o efeito da IL-17 e uma poss?vel intera??o entre esta citocina e o P2X7R em c?lulas de glioma humano. As linhagens celulares de glioma humano U-138 MG e U-251 MG mostraram-se resistentes ? morte, quando tratadas com ATP (5 mM) ou BzATP (100 ?M), agonista seletivo do P2X7R. Na linhagem de glioma radiossens?vel M059J, o tratamento com alta concentra??o de ATP (5 mM) ou com BzATP (100 ?M), diminuiram significativamente a viabilidade celular (32,4% ? 4,1 e 24,6 ? 4,0%, respectivamente). A linhagem M059J expressou n?veis significativamente maiores do P2X7R quando comparada com as linhagens celulares U-138 MG e U-251 MG (0.40 ? 0.00; 0.28 ? 0.01 e 0.31 ? 0.01, respectivamente) e a irradia??o aumentou a express?o do P2X7R em todas linhagens. Al?m disso, o antagonista seletivo do P2X7R, A740003 (10 μM), diminuiu significativamente a morte celular causada pela irradia??o. Nesta primeira parte do trabalho, fornecemos novas evid?ncias indicando que a linhagem de glioma humano M059J ? sens?vel ao ATP-P2X7R, apontando a relev?ncia do P2X7R na radiossensibilidade do glioma. Na segunda parte deste trabalho, observou-se que o receptor da IL-17 foi significativamente mais expresso ap?s a irradia??o (2 Gy) mostrando uma poss?vel participa??o da via IL-17/R na susceptibilidade ? irradia??o. O tratamento com a IL-17 (10 ng/ml) diminuiu em aproximadamente 62% a viablidade de linhagens celulares U-138 MG e M059J de glioma humano. Outros resultados preliminares mostraram que o antagonista do P2X7R foi capaz de reverter parcialmente a toxicidade causada pela IL-17 nestas linhagens. Nossos dados demonstram um papel antitumoral desta citocina e corroboram com a id?ia de uma poss?vel intera??o entre a IL-17/P2X7R. Este trabalho deixa perspectivas de estudos a serem realizados para melhor compreender a a??o da IL-17 nos gliomas e a intera??o IL-17/P2X7R

Glioblastoma multiforme é o tumor maligno mais comum do sistema nervoso central em adultos e a sobrevida média é de apenas 12 a 15 meses após o diagnóstico. Por isso, é extremamente importante desenvolver tratamentos mais eficazes e específicos contra essa neoplasia. A presença do sistema imune, incluindo macrófagos associados ao tumor, promove a proliferação tumoral e está associada com um pior prognóstico em pacientes com essa doença maligna. A sinalização purinérgica e o receptor purinérgico P2X7, um canal iônico, têm sido implicados na progressão de diferentes tipos de tumores tanto in vitro como in vivo. A ciclooxigenase 2 (COX-2) desempenha um papel importante na regulação da proliferação celular, diferenciação e na tumorigênese. O ácido ursólico é um triterpeno pentacíclico encontrado em uma variedade de plantas e exibe diversas atividades biológicas e farmacológicas. O objetivo dessa tese é verificar a participação do sistema purinérgico, sistema imune e COX-2 no desenvolvimento e progressão do glioblastoma multiforme, e também investigar os efeitos citotóxicos do ácido ursólico. Primeiramente, verificamos que macrófagos expostos ao meio condicionado de glioma (GL-CM) foram modulados para um fenótipo do tipo M2 e houve um aumento da liberação de IL-10, IL-6 e MCP-1. Esses efeitos foram diminuídos na presença de antagonistas dos receptores P2X7 e A2A. Portanto, os resultados apresentados contribuem para o melhor entendimento da interação entre inflamação e câncer e demonstram que os receptores purinérgicos são importantes para a progressão do glioma. Após, analisamos o papel do receptor P2X7 na proliferação de células de glioma. Surpreendentemente, in vitro, não se observou nenhuma diferença no crescimento das células quando houve a transfecção com o P2X7. Entretanto, in vivo, essas células geraram tumores maiores quando comparado com o controle. Os nossos resultados demonstram que, como em outros tipos de cânceres, o P2X7 tem um papel importante no desenvolvimento e progressão tumoral. Uma vez verificado o importante papel do receptor P2X7 nos macrófagos associados ao tumor e nas células de glioma, investigamos se esse receptor poderia interagir com a enzima COX-2 em células de glioma. Porém, não houve diferença na expressão do P2X7 ou da COX-2 tanto in vitro como in vivo. E também não houve nenhum efeito adicional entre o antagonista de P2X7 e o inibidor seletivo de COX-2. Esse trabalho fornece evidências de que não há relação entre o P2X7 e COX-2 em células de glioma. Em conclusão, todos esses resultados reforçam a hipótese do envolvimento da sinalização purinérgica na progressão do glioblastoma multiforme e tornam o P2X7 como um interessante alvo terapêutico. Finalmente, também investigamos a possível atividade anticâncer do ácido ursólico contra as células de glioma. Essa molécula foi capaz de diminuir o número de células e induziu parada no ciclo celular. In vivo, o ácido ursólico reduziu ligeiramente o tamanho do tumor, mas não alterou as características malignas. Em conclusão, o ácido ursólico pode ser um potencial candidato como adjuvante para o tratamento do glioblastoma multiforme. Em conjunto, todos os resultados apresentados nessa tese indicam possíveis novas abordagens terapêuticas no tratamento e novos conhecimentos em relação a esse maligno câncer cerebral. / Glioblastoma multiforme is the most common malignant tumor of central nervous system in adults and the median survival is only 12 to 15 months after diagnosis. Therefore, it is extremely important to develop more effective and specific treatments. The presence of an inflammatory environment, including tumor-associated macrophages, promotes tumor proliferation and is associated with a poor prognosis in patients with this malignancy. Disruption of purinergic signaling has also been implicated in cancer progression. P2X7R is an ion channel receptor, whose participation in tumor progression has been demonstrated in in vitro and in vivo studies. Cyclooxygenase 2 (COX-2) plays an important role in regulating cell proliferation, differentiation, and tumorigenesis. Ursolic acid is a pentacyclic triterpenoid found in a variety of plants that exhibits several biological and pharmacological activities. The aim of this study is to verify the participation of the purinergic system, immune system and COX-2 in the glioblastoma multiforme development and progression, and also to investigate the anti-proliferative effects of ursolic acid. We first verified that macrophages exposed to glioma conditioned medium (GL-CM) were modulated to an M2-like phenotype and there was an increased IL-10, IL-6 and MCP-1 secretion and these effects were diminished by P2X7 and A2A receptors antagonists. Therefore, the results presented contribute to advancing in the field of cancer-related inflammation and point specific purinergic receptors as targets for glioma progression. After that, we analyzed the role of P2X7 receptor in glioma cell proliferation. Surprisingly, in vitro, no difference in cell growth was observed when the cells were transfected with P2X7R but in vivo these cells generated larger tumors when compared to the control. Our data demonstrate that, as in other type of cancers, P2X7R has an important role in sustaining the development of glioma. Once verified the important role of P2X7 receptor in tumorassociated macrophages and glioma cells, we verified whether this receptor could interact with the COX-2 enzyme in glioma cells. No differences in mRNA expression of P2X7R or COX-2 were verified both in vitro and in vivo experiments. And any additional effect with selective P2X7R antagonist and COX-2 inhibitor were observed in in vitro and in vivo experiments. This work provides evidence that there is no relationship between the P2X7R and COX-2 in glioma. In conclusion, all these results reinforce the hypothesis of purinergic signaling involvement in glioma progression and point to P2X7R as an interesting target for glioma treatment. Finally, we also investigated the potential anticancer activity of ursolic acid against to glioma cells. Ursolic acid decreased the cell number and induced an arrest in the cell cycle in glioma cells. In vivo, ursolic acid slightly reduced the glioma tumor size but did not alter the malignant features. In conclusion, the ursolic acid may be a potential candidate as adjuvant for glioblastoma therapy. Taken together, the results presented herein indicate new adjuvant treatment approaches and new knowledge regarding to this deadliest brain tumor.

Made available in DSpace on 2015-04-14T13:35:24Z (GMT). No. of bitstreams: 1 436876.pdf: 1707187 bytes, checksum: a5856afc9a54d87e34f88e782140d450 (MD5) Previous issue date: 2012-01-20 / Gliomas are the most common and devastating tumors of the central nervous system (CNS). Among the gliomas group, the GBM is the most prevalent, aggressive and deadly malignant. Many pieces of evidence point out the relevance of natural compounds for cancer therapy and prevention, including chalcones. Chalcones are group of natural precursors of flavonoid and display a wide variety of biological and pharmacological proprieties that include anti-proliferative and anti-cancer activities. This study aimed at evaluating the in vitro anti-proliferative activity and cell viability inhibition of nine quinoxaline derived chalcones, structurally based on the selective PI3Ky inhibitor AS605240. These synthetic compounds were tested at different time-periods of incubation (24, 48 e 72 h) and concentrations (0,1; 0,1; 1; 5 e 10 μg/mL) in glioma cell lines from human and rat origin (U-138 MG and C6, respectively). The results showed by MTT assay and cell couting revealed that four chalcones (compounds N2, N9, N10 and N12), displayed higher efficacies and potencies, being able to inhibit either cell proliferation or viability, in a time- and concentration dependent manner. These four compounds which present methoxy groups at A-ring and their efficacy was greater than that seen for the positive control compound AS605240. Flow cytometry analysis demonstrated that incubation of C6 cells with chalcone N9 led to G1 phase arrest, likely indicating an interference with apoptosis. Furthermore, chalcone N9 was able to visibly inhibit AKT activation, allied to the stimulation of ERK 1/2 MAP-kinase. The chalcones tested herein, especially those displaying a methoxy substituent at A-ring, might well represent promising molecules for the treatment of gliomas. / Os gliomas s?o os tumores do SNC mais comuns e devastadores. Dentre os gliomas, o GBM ? o mais prevalente, agressivo, maligno e apresenta um mau progn?stico. A relev?ncia dos compostos naturais, incluindo as chalconas, no tratamento e na preven??o do c?ncer est? sendo muito evidenciada. As chalconas formam um grupo de compostos naturais derivados dos flavon?ides que apresentam diferentes propriedades biol?gicas e farmacol?gicas, incluindo as atividades anti-proliferativas e anti-tumorais. O objetivo deste estudo foi avaliar a a??o anti-proliferativa e a capacidade de inibi??o da viabilidade celular in vitro de nove chalconas derivadas da quinoxalina, baseadas estruturalmente no inibidor seletivo de PI3Ky, o AS605240. Estes compostos sint?ticos foram testados em diferentes tempos de incuba??o (24, 48 e 72 h) e concentra??es (0,1; 0,1; 1; 5 e 10 μg/mL) em linhagens de glioma humano e de rato (U-138 MG e C6, respectivamente). Os resultados observados nos experimentos de MTT e na contagem celular revelaram que quatros chalconas (compostos N2, N9, N10 e N12), apresentaram grande efic?cia e pot?ncia, sendo capazes de inibir a prolifera??o e a viabilidade celular, de maneira tempo e concentra??o dependente. Estes quatro compostos que possuem radicais met?xi no anel A de sua estrutura demonstraram uma efic?cia superior ?quela do composto AS605240, usado como controle positivo. Os resultados da citometria de fluxo demonstraram que a incuba??o das c?lulas C6 com a chalcona N9 levaram a um bloqueio celular na fase G1, possivelmente indicando interfer?ncia com a apoptose. Al?m disto, a chalcona N9 foi capaz de inibir visivelmente a ativa??o da AKT, aliada ? estimula??o de ERK 1/2 MAP-quinase. As chalconas estudadas neste projeto, especialmente as que apresentam o radical metoxi no anel A, representam promissoras mol?culas para o tratamento dos gliomas.

Made available in DSpace on 2015-04-14T13:35:33Z (GMT). No. of bitstreams: 1 439149.pdf: 1490829 bytes, checksum: bd616615ee5265db0a960d6f77c8581d (MD5) Previous issue date: 2012-05-29 / Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumours. Current standard treatment for GBM patients involves maximum surgical resection of the tumour, followed by radiotherapy and chemotherapy, usually containing the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. This work consisted in investigating the cytotoxic effects of Akt-inhibition by MK-2206 with irradiation (RT) and TMZ on in vitro human malignant glioma. Seven malignant glioma cell lines were cultured and tested for clonogenic survival, invasion inhibition, tumour spheroid growth and proliferation. The Akt-inhibitor MK-2206 and TMZ were added at different time treatments and in varying doses. Cultures were irradiated with single dose and with fractionated γ-irradiation. Cellular modulation of Akt and p-Akt were assessed by Western blot analysis. MK-2206 reduced the levels of phospho- Akt key protein in the PI3Kinase-Akt pathway, decreased cell survival, and inhibited invasion, proliferation and cell growth. The combination of MK-2206 and RT lead to enhanced inhibition of cell proliferation and invasion, which is not observed with RT alone. The radioenhancing effect of MK-2206 was most striking in inhibition of spheroid volume growth by fractionated RT; the radiosensitizing effect of MK-2206 was stronger than that of TMZ. MK-2206 enhanced the in vitro effects of RT and TMZ in terms of decreased cell survival, invasion, proliferation and growth in malignant glioma. Effects could be ascribed to inhibition of PI3K-Akt pathway / O Glioblastoma multiforme (GBM) ? o tipo mais maligno e mais comum de todos tumores astroc?ticos. O tratamento atual para pacientes de GBM envolve m?xima remo??o cir?rgica, seguida de radio e quimioterapia, normalmente com o agente alquilante Temozolamida (TMZ). Apesar da agressividade da terapia combinada, o tempo de sobreviv?ncia dos pacientes ainda ? baixo. Este trabalho procurou investigar os efeitos citot?xicos do inibidor de Akt MK-2206 em combina??o com irradia??o (RT) e TMZ em um painel de c?lulas de gliomas humanos. Sete linhagens de glioma foram cultivadas e testadas em ensaio de sobreviv?ncia clonog?nica, inibi??o de invas?o, e modelos de prolifera??o e crescimento de volume em esfer?ides. O inibidor MK-2206 e TMZ foram adicionados em diferentes tempos de tratamento e diferentes doses. As culturas foram irradiadas com doses ?nicas ou em terapias fracionadas com irradia??o γ. A modula??o celular de Akt e fosfo-Akt foi checada via Western Blot. O composto MK-2206 reduziu a fosforila??o da prote?na chave Akt na via PI3K, diminuindo a sobreviv?ncia celular e inibindo invas?o, prolifera??o e crescimento celular. A combina??o de MK-2206 com RT levou a uma maior inibi??o de invas?o e prolifera??o, o que n?o ? observado somente com a RT. O efeito radiosens?vel de MK-2206 foi ainda maior na inibi??o do volume dos esfer?ides em terapia combinada com RT fracionada, sendo ainda maior do que o efeito combinado com TMZ. MK-2206 aumentou os efeitos in vitro de RT e TMZ em termos de redu??o de sobreviv?ncia celular, invas?o, prolifera??o e crescimento celular em gliomas malignos. Os efeitos podem ser atribu?dos a inibi??o da via PI3KAkt

O glioblastoma (GBM) é o mais comum dos tumores primários malignos que afetam o Sistema Nervoso Central (SNC), sendo um dos cânceres mais letais. A ressecção cirúrgica desse tumor é o tratamento de intervenção inicial mais utilizado nos pacientes. Embora a radioterapia e a quimioterapia aumente a sobrevida, a maioria dos pacientes chega ao óbito em até um ano após o diagnóstico. Além disso, os GBM estão entre os tumores mais resistentes à radiação e à quimioterapia. Dessa forma, torna-se imprescindível a busca de novas estratégias terapêuticas que visem à melhora da qualidade de vida dos pacientes e ao aumento do tempo de sobrevida. O glutamato (L-Glu) é o aminoácido encontrado em maior concentração no SNC e ele exerce seus papéis fisiológicos e patológicos através da ativação de receptores de membrana metabotrópicos e ionotrópicos. Diversos estudos in vitro e in vivo têm demonstrado que células de GBM liberam altos níveis de L-Glu para o meio extracelular, o que promove a sua proliferação e migração, contribuindo para a malignidade deste tipo de tumor. De fato, é provável que a ligação desse aminoácido aos receptores metabotrópicos de glutamato (mGluR) relacione-se com a agressividade dos GBM, visto que eles são amplamente expressos nestas células. Dessa forma, o objetivo desta tese foi investigar o papel dos mGluR sobre a agressividade de GBM, buscando uma assinatura gênica que tenha valor como potencial biomarcador prognóstico e preditivo de tratamento complementar adjuvante. Através da uma meta-análise de duas coortes de amostras de GBM humanos foi possível identificar uma assinatura gênica de mGluR com valor prognóstico, na qual biópsias com alta expressão gênica de mGluR3 e baixa de mGluR4 e mGluR6 predizem um desfecho antecipado para os pacientes. O potencial desses receptores sobre a malignidade desses tumores foi avaliado por experimentos in vitro utilizando o tratamento de linhagens de GBM com ligantes de mGluR. O bloqueio farmacológico de mGluR do grupo II pelo antagonista LY341495 e a ativação farmacológica de mGluR III por L-AP4 diminuíram a porcentagem de células de linhagem de GBM em 25-28 %. A combinação desses tratamentos não apresentou efeito sinérgico. O potencial da assinatura gênica também foi avaliado in vivo utilizando ratos implantados ortotopicamente com células da linhagem C6 e tratados intracisternalmente com os ligantes de mGluR e intraperitonealmente com quimioterápico padrão, a temozolamida. Os resultados obtidos nos experimentos in vivo, apesar de preliminares, em relação ao tratamento com os ligantes de mGluR, são muito promissores, permitindo várias perspectivas em relação a adaptações de protocolo e a realização de experimentos complementares. Este estudo demonstrou que a avaliação da expressão gênica de mGluR3, 4 e 6 em biópsias de GBM humanos possui um grande potencial prognóstico. A diminuição do número de células da linhagem C6 após o tratamento in vitro com ligantes de mGluR (LY341495 e L-AP4) está de acordo com o comportamento de agressividade previsto nos estudos in silico. Embora mais experimentos in vitro e in vivo sejam necessários para melhor avaliação do potencial dessa assinatura gênica de mGluR, os resultados obtidos indicam que a avaliação da expressão dos oito subtipos de mGluR em biópsias de GBM pode ser considerada em âmbito clínico para guiar futuras intervenções quimioterápicas. / Glioblastoma (GBM) is the most common malignant primary tumor of Central Nervous System (CNS) and one of the most lethal cancers. Surgical resection of this tumor is the most commonly initial intervention treatment used in patients. Although radiotherapy and chemotherapy increase survival, it is expected that the majority of patients will die within a year after diagnosis. In addition, GBM are among the most radiation- and chemotherapy-resistant tumors. Thus, it is imperative to search for new therapeutic strategies that aim to improve patients' quality of life and to increase survival time. Glutamate (L-Glu) is the amino acid found in higher concentration in CNS and it exerts its physiological and pathological roles through the activation of metabotropic and ionotropic membrane receptors. Several studies have demonstrated in vitro and in vivo that GBM cells release high levels of L-Glu into extracellular medium. This event was shown to promote GBM proliferation and migration, contributing to the malignancy of this type of tumor. Indeed, it is likely that the binding of that amino acid to metabotropic glutamate receptors (mGluR) relates to GBM aggressiveness, since they are widely expressed in these cells. Thus, the objective of this work was to investigate the role of mGluR on GBM aggressiveness, searching for a gene signature that has potential value as biomarker to predict the prognosis and adjuvant complementary treatment. Through the meta-analysis of two GBM human samples cohorts, it was possible to identify an mGluR gene signature with prognostic value, in which biopsies with high mGluR3 and low mGluR4 and mGluR6 gene expression predict an early outcome for patients. The potential of these receptors on the malignancy of these tumors was assessed by in vitro experiments through the treatment of GBM lineages with mGluR ligands. Pharmacological blockade of group II mGluR by LY341495 and pharmacological activation of group III mGluR by L-AP4 decreased the amount of C6 cells in 25-28 %. The combination of these treatments had no synergistic effect. The potential of the gene signature was also assessed in vitro using GBM-implanted rats treated intracisternally with mGluR ligands and intraperitoneally with standard chemotherapy, temozolomide. The results obtained in in vivo experiments, although very preliminary in relation to treatment with the mGluR ligands, are very promising, allowing several perspectives regarding protocol adaptations and the accomplishment of complementary experiments. This study demonstrated that evaluation of mRNA levels of mGluR3, 4, and 6 in human GBM biopsies has a great prognostic potential. The decrease in number of C6 cells after in vitro treatment with mGluR ligands (LY341495 and L-AP4) is in accordance with the predicted aggressiveness behavior proposed in silico. Although further in vitro and in vivo experiments are required for better evaluation of mGluR gene signature potential, these results indicate that evaluation of the eight mGluR subtypes in GBM biopsies may be considered in clinical scope to guide future chemotherapeutic interventions.

Dong Shumin. / "February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 187-220). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2012-09-21T18:18:29Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AnalisesMolecularesRegiao.pdf: 1463887 bytes, checksum: ca32f1226f6858f28c34cb8da6ba8fc2 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-09-28T15:14:14Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AnalisesMolecularesRegiao.pdf: 1463887 bytes, checksum: ca32f1226f6858f28c34cb8da6ba8fc2 (MD5) / Made available in DSpace on 2012-09-28T15:14:14Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AnalisesMolecularesRegiao.pdf: 1463887 bytes, checksum: ca32f1226f6858f28c34cb8da6ba8fc2 (MD5) Previous issue date: 2012 / O câncer do sistema nervoso central representa 2% de todas as neoplasias malignas na população mundial e 23% dos casos de câncer infantil. No Brasil, estimam-se 4.820 casos deste câncer em homens e 4.450 em mulheres para o ano de 2012. Os gliomas são tumores do sistema nervoso central formados a partir de células da glia e somam mais de 70% do tumores cerebrais. A propriedade mais importante dos gliomas é sua capacidade de evasão imunológica. Idade, etnia, gênero e ocupação podem ser considerados fatores de risco para o surgimento de gliomas, e são duas vezes mais frequentes em afro-americanos. O astrocitoma é o tumor glial mais frequente, constituindo cerca de 75% dos casos de gliomas. Estes tumores são classificados em quatro graus, de acordo com a Organização Mundial de Saúde. O DNA mitocondrial está relacionado com o desenvolvimento e a progressão de vários tipos de tumores. A mitocôndria é responsável pelo balanço energético celular e está envolvida no disparo da apoptose em resposta ao estresse oxidativo. Mutações na D-LOOP podem alterar a taxa de replicação do DNA e aumentar o risco do desenvolvimento do câncer. Neste estudo foram analisadas 29 amostras de astrocitoma classificados de acordo com a OMS. Nossos dados sugerem que os astrocitomas de baixo grau podem estar relacionados à herança genética, tornando portadores de alguns polimorfismos ou mutações específicas, mais suscetíveis ao risco de desenvolver a doença, e os de alto grau podem estar relacionados à exposição prolongada aos agentes carginógenos. Foram identificados polimorfismos e mutações onde alguns apresentaram relação com o risco do desenvolvimento de astrocitomas e com a progressão da doença. A inserção de dois ou mais nucleotídeos nas regiões de microssatélites pode causar sua instabilidade e contribuir com o surgimento do câncer. A deleção no sítio 16132 pode ser um marcador para astrocitoma de alto grau, assim como a inserção de duas ou mais citosinas no sítio 16190 pode ser um marcador específico para astrocitomas. As mutações heteroplásmicas podem ser determinantes para o surgimento e/ou progressão de astrocitomas de alto grau. / The central nervous system cancer represents 2% of all malignancies in the world population and 23% of cases of childhood cancer. In Brazil, an estimated 4,820 cases of cancer in men and women in 4450 to the year 2012. Gliomas are tumors of the central nervous system formed from glial cells, making up over 70% of brain tumors. The most important property of gliomas is the ability of immune evasion. Age, ethnicity, gender and occupation may be considered risk factors for the development of gliomas, and are twice as common in African-Americans. The astrocytoma is the most common glial tumor, constituting about 75% of cases of gliomas. These tumors are classified into four levels according to the World Health Organization. Mitochondrial DNA is related to the development and progression of various types of tumors. Mitochondrion is responsible for cellular energy balance and is involved in triggering apoptosis responding to oxidative stress. Mutations in DLOOP can change DNA replication rates and increase the developing cancer risk. We analyzed 29 samples astrocytoma classified according to the WHO. Our data suggest that low-grade astrocytomas may be related to genetic inheritance, making some patients with specific mutations or polymorphisms more susceptible to the risk of developing the disease, and high grade may be related to prolonged exposure to carcinogenics. Polymorphisms and mutations have been identified which correlate with some risk of developing astrocytomas and disease progression. The insertion of two or more nucleotides at microsatellite regions may cause instability and contribute to the cancer onset. Deletion at the site 16132 may be a high-grade astrocytoma marker, as well as insertion of two or more cytosines to the site 16190 can be an astrocytoma specific marker. Heteroplasmy may be decisive for the emergence and / or progression of high-grade astrocytomas.