The Influence of Oxidative Stress, Carcinogens and Cloning on DNA ...

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Index 5.2.4.2 Dialysis <strong>of</strong> the Micrococcal nuclease enzyme (MN) ................................. 124 5.2.4.3 Production <strong>of</strong> the MN/SPD enzyme mixture ............................................. 124 5.2.5 Other preparations .............................................................................................. 125 5.3 Methods .................................................................................................................. 127 5.3.1 Common methodology for the preparation <strong>of</strong> DNA samples (1 µg or 10 µg) .. 127 5.3.1.1 Hydrolysis <strong>of</strong> genomic DNA samples (1 µg or 10 µg) .............................. 127 5.3.1.2 Derivatisation <strong>of</strong> genomic DNA samples (1 µg or 10 µg) ......................... 128 5.3.1.3 Precipitation <strong>of</strong> the excess <strong>of</strong> Bodipy <strong>and</strong> EDC (1 µg or 10 µg) ............... 128 5.3.1.4 CE-LIF measurement ................................................................................. 129 5.3.2 Synthesis <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’- monophosphate .............................. 130 5.3.3 Electrospray ionisation mass spectrometry (ESI-MS) <strong>of</strong> 8-oxo-2’-deoxyguanosine-3’- monophosphate ........................................................................... 130 5.3.4 Preparation <strong>of</strong> the capillary for CE measurement .............................................. 131 5.4 Instrumentation ....................................................................................................... 132 5.4.1 CE-LIF system ................................................................................................... 132 5.4.2 UV-VIS spectrophotometer specifications for DNA quantification .................. 132 5.4.3 General laboratory instruments .......................................................................... 132 5.4.4 LC-UV system specifications for HPLC separation <strong>of</strong> 8-oxo-2’-deoxyguanosine- 3’-monophosphate from its starting material ..................................................... 134 5.4.5 SPE system ......................................................................................................... 135 5.4.6 Mass spectrophotometer: .................................................................................... 135 5.5 Statistical methods .................................................................................................. 135 5.6 S<strong>of</strong>tware ................................................................................................................. 136 6 Appendix ........................................................................................................................ 137 7 List <strong>of</strong> figures ................................................................................................................. 149 8 List <strong>of</strong> tables ................................................................................................................... 152 9 Literature ........................................................................................................................ 154 4
1 Introduction 1.1 Epigenetics 1 Introduction <strong>The</strong> term epigenetics * defines all mitotically <strong>and</strong> meiotically heritable changes in gene expression that are not coded by the DNA sequence (1, 2) . Epigenetics literally means ‘on top <strong>of</strong> ge- netics’. In addition to the DNA sequencing * , epigenetics explains the contribution <strong>of</strong> another system, one that is responsible for the direct translation <strong>of</strong> the genetic information to control the genes influencing our growth <strong>and</strong> development (2) . Additionally, it explains why different cell types <strong>of</strong> an organism share identical DNA sequences but show amazingly diverse functions <strong>and</strong> phenotypes (2-4) . Epigenetics is important for development <strong>and</strong> cell differentiation but could contribute to congenital disorders <strong>and</strong> health problems (3) . Several epigenetic processes have been described in humans. DNA methylation <strong>and</strong> histone modifications, main components <strong>of</strong> epigenetic code, are the mechanisms involved side by side for the regulation <strong>of</strong> epigenetic processes (5) . 1.1.1 Histone modification DNA * is coiled around histones (the main protein components), which means that both to- gether compose the chromatin (6) . DNA is wrapped around nucleosomes (core histones) (7) <strong>and</strong> fixed in place with small proteins called linker histones (Fig. 1). <strong>The</strong> supercoiling <strong>of</strong> this necklace helps the fitting <strong>of</strong> DNA into the tiny nucleus (6) . Additionally, chromatin controls the gene expression, DNA replication <strong>and</strong> cell functions. Histone modifications including methylation, acetylation, phosphorylation <strong>and</strong> others affect gene expression, DNA replication, mitosis <strong>and</strong> DNA repair mechanism (8-10) . * : See Appendix Be less curious about people <strong>and</strong> more curious about ideas Marie Curie
Page 1 and 2: The Influe
Page 3 and 4: The work at h<stro
Page 5 and 6: First, I feel always grateful to Al
Page 7: I would like to extend my thanks to
Page 11 and 12: The following acro
Page 13 and 14: FDA Food and drug
Page 15: RIA Radioimmunoassay ROS Reactive o
Page 18 and 19: Index 3.1 Spectrophotometeric deter
Page 22 and 23: Introduction 1.1.2 DNA methylation
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Page 28 and 29: Introduction Cancer is classified i
Page 30 and 31: Introduction DNA methylation Gene h
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Page 36 and 37: Introduction chemicals, alkylating
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Page 42 and 43: Introduction Finally, flavo- <stron
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Page 58 and 59: Introduction - Radioactive-mediated
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Summary 4 Summary DNA methylation i
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Summary The same s
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Experimental part Sodium tetrapheny
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Experimental part � Sodium tetrab
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Experimental part framework was fas
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Experimental part propanol
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Experimental part 12.5 mU/µL SPD)
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Experimental part Condition Value D
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Experimental part 5.4 Instrumentati
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Experimental part Graduated cylinde
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Experimental part 3-Nitrobenzanthro
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Appendix In-vitro: Denoting a react
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Appendix Laboratory Document Hydrol
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Appendix BODIPY and</strong
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Appendix Description of</st
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Appendix Transmission [%] 120 100 8
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Appendix Figure 62: Mass spectrum E
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List of figures Fi
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List of tables 8 L
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Literature Literature 1. Egger, G.;
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Literature 41. Bestor, T.H.; "<stro
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Literature 78. Venitt, S.; "Mechani
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Literature 113. Sinclair, K.D.; Mce
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Literature 148. Kadan-Lottick, N.S.
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Literature cyte DNA by gas chromato
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Literature 221. Mirand</str
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Literature 257. Lingaraju, G.M.; Sa
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Literature different tissues <stron
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Literature 324. Vishwanath, B.S.; F
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Literature 359. Wei, L.; Li, R.K.;
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Literature 394. Ramsahoye, B.H.; "M
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Literature 431. Wirtz, M.; Schumann
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Literature 467. Cornelius, M.; Wort
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Literature 502. Issa, J.P.J.; Ottav
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Literature 536. Pinheiro, J. <stron
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