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Characterization of the Monocyte to Macrophage Differentiation (MMD) Protein and Its Homologue MMD2

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Characterization of the Monocyte to Macrophage Differentiation (MMD) Protein and Its Homologue MMD2 Characterization of the Monocyte to Macrophage Differentiation (MMD) protein and its homologue MMD2 Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr.rer.nat.) der Naturwissenschaftlichen Fakultät III - Biologie und vorklinische Medizin der Universität Regensburg vorgelegt von Carol El Chartouni aus Beirut - Libanon Juni 2006 The work presented in this thesis was carried out in the Department of Hematology and Oncology at the University Hospital Regensburg from June 2002 to Februar 2006. Die vorliegende Arbeit entstand in der Zeit von Juni 2002 bis Februar 2005 in der Abteilung für Hämatologie und Internistische Onkologie des Klinikums der Universität Regensburg. Promotionsgesuch eingereicht am: 27.06.2006 Tag der mündlichen Prüfung: 8.09.2006 Die Arbeit wurde angeleitet von: PD Dr. Michael Rehli - Prof. Dr. Stephan Schneuwly. Prüfungsausshuß: Vorsitzender: Prof. Dr. Christoph Oberprieler 1. Prüfer (Erstgutachten): Prof: Dr. Stephan Schneuwly 2. Prüfer (Zweitgutachten): PD. Dr. Michael Rehli 3. Prüfer: Prof. Dr. Karl Kunzelmann Table of contents 1. Introduction ..................................................................................................... 1 1.1. Mononuclear phagocytes in the immune system ............................................................1 1.1.1. Monocyte heterogeneity and differentiation..............................................................2 1.1.1.1. Monocyte heterogeneity...................................................................................2 1.1.1.2. Monocyte differentiation..................................................................................2 1.1.2. Macrophage heterogeneity.........................................................................................3 1.1.2.1. Brain macrophages...........................................................................................3 1.1.2.2. Alveolar macrophages......................................................................................4 1.1.2.3. Kupfer cells ......................................................................................................4 1.1.2.4. Macrophages in peripheral lymphoid organs ...................................................4 1.1.2.5. Macrophages of the vascular system................................................................4 1.1.2.6. Placenta macrophages ......................................................................................5 1.1.3. Macrophages in inflammation ...................................................................................5 1.1.3.1. Classically activated macrophages...................................................................6 1.1.3.2. Alternatively activated macrophages ...............................................................6 1.2. MMD, a putative seven transmembrane protein............................................................8 1.3. PAQR family......................................................................................................................9 1.3.1. Adiponectin receptor related proteins......................................................................10 1.3.1.1. Adiponectin receptors, PAQR1 and PAQR2 .................................................10 1.3.1.2. PAQR3, PAQR4.............................................................................................11 1.3.2. Membrane progestin receptor (mPR) related proteins.............................................12 1.3.2.1. PAQR5, 7 and 8 .............................................................................................12 1.3.2.2. PAQR6 ...........................................................................................................13 1.3.2.3. PAQR9 ...........................................................................................................13 2. Research objectives ....................................................................................... 15 3. Material .......................................................................................................... 16 3.1. Equipment........................................................................................................................16 3.2. Material ............................................................................................................................16 3.3. Chemicals .........................................................................................................................17 3.4. DNA oligonucleotides ......................................................................................................17 3.5. Antibodies.........................................................................................................................19 3.6. Enzymes and kits .............................................................................................................19 3.7. Molecular weight standards ...........................................................................................20 3.8. Plasmids............................................................................................................................20 i 3.9. E.coli strains.....................................................................................................................20 3.10. Antibiotics ......................................................................................................................20 3.11. Animal cell lines culture................................................................................................21 3.12. Databases research ........................................................................................................21 4. Methods .......................................................................................................... 22 4.1. General molecular biology..............................................................................................22 4.1.1. Bacterial culture.......................................................................................................22 4.1.1.1. Bacterial growth medium ...............................................................................22 4.1.1.2. Preparation of chemically competent E.coli ..................................................22 4.1.1.3. Transformation of chemically competent E.coli ............................................23 4.1.1.4. Glycerol stock ................................................................................................24 4.1.2. Plasmid isolation from E.coli...................................................................................24 4.1.3. Molecular cloning ....................................................................................................24 4.1.3.1. PCR ................................................................................................................24 4.1.3.2. PCR-based site specific mutagenesis .............................................................25 4.1.3.3. Precipitation of DNA using PEG ...................................................................26 4.1.3.4. Purification of DNA fragment by gel extraction............................................27 4.1.3.5. Agarose gel electrophoresis ...........................................................................27 4.1.3.6. Restriction endonuclease digestion ................................................................28 4.1.3.7. Dephosphorylation of DNA with alkaline phosphatase.................................28 4.1.3.8. Fill in 5’-overhang with Klenow DNA-polymerase ......................................28 4.1.3.9. Generation of blunt ends with T4 DNA-polymerase .....................................28 4.1.3.10. DNA sequencing and sequence analysis......................................................28 4.1.3.11. Generation of different MMD tagged constructs .........................................28 4.1.4. RNA related molecular methods..............................................................................30 4.1.4.1. Isolation of RNA by GTC-Phenol-Chloroform extraction.............................31 4.1.4.2. Isolation of total RNA with Qiagen RNeasy Midi Kit...................................32 4.1.4.3. Formaldehyde agarose gel..............................................................................32 4.1.4.4. Northern blot - RNA transfer .........................................................................33 4.1.4.5. Northern blot hybridization............................................................................33 4.1.4.6. Stripping Northern blots.................................................................................34 4.1.4.7. Generation of specific radioactive DNA probes ............................................34 4.1.4.8. Reverse transcription (RT) and quantitative real time-PCR (qRT-PCR).......34 4.1.5. Gene silencing by short interference RNA ..............................................................36 4.1.6. Whole-mount in situ hybridization ..........................................................................36 4.1.6.1. Isolation of embryos.......................................................................................36 4.1.6.2. Synthesis of sense and antisense RNA probe.................................................38 4.1.6.3. Hybridization procedure on whole-mount .....................................................39 4.1.6.4. Post-hybridization and detection procedures .................................................40 4.2. Protein
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