Copy number variations of the mitochondrial DNA as potential cause of mitochondrial diseases [Elektronische Ressource] / vorgelegt von Miriam Baron

Miriam Baron

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2010
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Copy number variations
of the mitochondrial DNA as potential cause of
mitochondrial diseases











Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn

vorgelegt von
Miriam Baron
aus
Bonn-Bad Godesberg

Bonn, 2010
































Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn




























1. Gutachter Prof. Dr. Wolfram S. Kunz
2. Gutachter Prof. Dr. Thomas Magin
Tag der Promotion: 09.07.2010
Erscheinungsjahr: 2010 Table of contents I
Table of contents

1. Abstract......................................................................................................................................... 1

2. Introduction ................................................................................................................................. 2
2.1. Roles of Mitochondria ............................................................................................................ 2
2.2. Energy generation via mitochondria....................................................................................... 2
2.3. Mitochondrial DNA (mtDNA) and the interaction of mitochondria with the nucleus............... 4
2.4. Replication of mtDNA molecules............................................................................................ 6
2.5. Mitochondrial distribution ....................................................................................................... 7
2.6. Variations in the mtDNA......................................................................................................... 8
2.7. Diseases associated with mitochondrial defects.................................................................. 11
2.8. Polymerase γ (POLG) and POLG mutations ....................................................................... 15

3. Goals of this study..................................................................................................................... 17

4. Materials and Methods .............................................................................................................. 19
4.1. Materials............................................................................................................................... 19
4.1.1. Synthetic oligodeoxynucleotides .............................................................................. 19
4.1.2. Enzymes, chemicals and solutions .......................................................................... 20
4.1.3. Kits............................................................................................................................ 22
4.1.4. Equipment ................................................................................................................ 23
4.2. Patients and human material ............................................................................................... 24
4.2.1. Patient ascertainment............................................................................................... 24
4.2.2. Human samples........................................................................................................ 24
4.2.3. Cell lines ................................................................................................................... 25
4.3. Cell culture ........................................................................................................................... 25
4.3.1. Thawing cells............................................................................................................ 25
4.3.2. Passaging cells......................................................................................................... 25
4.3.3. Cell counting............................................................................................................. 25
4.3.4. Depletion treatment .................................................................................................. 26
4.3.5. Freezing cells ........................................................................................................... 26
4.4. DNA analysis........................................................................................................................ 26
4.4.1. DNA isolation from blood by salting out ................................................................... 26
4.4.2. DNA isolation from fibroblasts by salting out............................................................ 27
4.4.3. DNA isolation with the QIAamp DNA Mini Kit .......................................................... 27
4.4.4. DNA isolation from liver slices.................................................................................. 27
4.4.5. Photometric quantitation of the nucleic acid concentration...................................... 27
4.4.6. DNA mutation analysis ............................................................................................. 28
4.4.7. Quantitative PCR (qPCR)......................................................................................... 28 Table of contents II
4.5. Enzymatic assays................................................................................................................. 32
4.5.1. Protein content determination .................................................................................. 32
4.5.2. Citrate synthase (CS) assay..................................................................................... 32
4.5.3. Determination of the oxygen consumption............................................................... 34
4.5.4. Immunohistochemistry.............................................................................................. 34
4.6. Statistical analyses............................................................................................................... 35

5. Results ........................................................................................................................................ 36
5.1. Validation of the quantitative PCR (qPCR) method ............................................................. 36
5.2. Tissue-specifity of the mtDNA content................................................................................. 40
5.3. Correlation between citrate synthase (CS) activity and mtDNA copy number in
several tissues...................................................................................................................... 41
5.4. mtDNA depletion in blood specimen of patients with a mild phenotype of PEO
with epilepsy/ataxia .............................................................................................................. 43
5.5. mtDNA depletion in patients with Alpers-Huttenlocher syndrome ....................................... 46
5.6. Reduction of the mtDNA copy number in specific brain regions from Ammon’s horn
sclerosis (AHS) patients....................................................................................................... 51
5.7. Influence of the mtDNA content on the mitochondrial respiration activity............................ 53

6. Discussion .................................................................................................................................. 70
6.1. Effect of mtDNA depletion on the bioenergetic status of the cell......................................... 70
6.2. Importance of the mtDNA content on neurodegeneration ................................................... 76
6.3. Influence of the nuclear gene POLG on the mtDNA copy number...................................... 79
6.4. Tissue-specifity of the mtDNA content................................................................................. 82

7. Summary..................................................................................................................................... 87

8. Appendices................................................................................................................................. 89
8.1. List of references.................................................................................................................. 89
8.2. List of abbreviations ........................................................................................................... 105
8.3. List of figures...................................................................................................................... 108
8.4. List of tables. ...................................................................................................................... 109

List of publications.......................................................