Identification and characterization of a Y chromosome-encoded gene overexpressed in acute myeloid leukaemia cells [Elektronische Ressource] / Antonio Gallo

Antonio Negri

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• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 17
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN



Institut für Experimentelle Genetik




Identification and characterization of a Y
chromosome-encoded gene overexpressed in acute
myeloid leukaemia cells


Antonio Gallo




Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines


Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. J. J. Hauner

Prüfer der Dissertation: 1. apl. Prof. Dr. J. Adamski
2. Univ.-Prof. Dr. A. Gierl
3. Univ.-Prof. Dr. H.-J. Kolb
(Ludwig-Maximilians-Universität München)


Die Dissertation wurde am 26.05.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 11.09.2009 angenommen.


La scienza è sempre imperfetta. Ogni volta che risolve
un problema, ne crea almeno dieci nuovi.
George Bernard Shaw








Tra tutte le cose sicure, la più sicura è il dubbio.
Bertolt Brecht






















Alla mia famiglia, ai miei amici e ad Olga.

Table of contents


Table of contents................................................................................................................i
Abstract............................................................................................................................. 1
Zusammenfassung ............................................................................................................ 3
1 Introduction..................................................................................................................5
1.1 Acute myeloid leukaemia...................................................................................... 5
1.1.1 Definition, etiology, and epidemiology ........................................................... 5
1.1.2 Classification of acute myeloid leukaemia ...................................................... 7
1.1.3 Diagnosis of acute myeloid leukaemia............................................................ 9
1.1.4 Treatment of AML patients ........................................................................... 10
1.2 Clinical observation.............................................................................................13
1.3 Aim of the study .................................................................................................. 16
2 Results........................................................................................................................ 18
2.1 Y chromosome gene expression profile in AML and healthy cells .................... 18
+2.1.1 Isolation of leukaemic and healthy CD14 cells............................................ 18
2.1.2 Low density arrays......................................................................................... 19
2.1.3 Candidate genes are not expressed in non-leukaemic cells of the patients ... 22
2.1.4 Candidate genes expression in THP-1 cells................................................... 24
2.2 Investigating TGIF2LY function.........................................................................26
2.2.1 Introduction....................................................................................................26
2.2.2 TGIF2LY protein expression in THP-1 cells ................................................ 27
2.2.3 Subcellular localization of TGIF2LY............................................................ 30
2.2.4 Protein function prediction using bioinformatics .......................................... 33
2.2.5 TGIF2LY-DNA interaction...........................................................................37
2.2.6 Identification of TGIF2LY binding site ........................................................ 41
2.3 Immunological potential of the candidate genes................................................. 42
2.3.1 Introduction....................................................................................................42
2.3.2 Prediction of HLA-A0201-restricted peptides............................................... 42
2.3.3 Peptide-HLA-A0201 binding ........................................................................43
2.3.4 Antigenicity of the peptides 44
3 Discussion..................................................................................................................47
3.1 Study design........................................................................................................47
3.1.1 General remarks.............................................................................................47
3.1.2 Samples’ choice48
3.2 Y chromosome gene expression evaluation ........................................................ 49
3.2.1 Why looking at the Y chromosome ............................................................... 49
3.2.2 Specificity of the TaqMan probes.................................................................. 50
3.2.3 Y chromosome gene expression in AML and healthy cells .......................... 51
i Table of contents

3.2.4 Candidate genes.............................................................................................52
3.2.5 Candidate genes are up-regulated in leukaemic cells.................................... 54
3.2.6 THP-1 as cell line model ............................................................................... 55
3.3 TGIF2LY – molecular analysis...........................................................................56
3.3.1 Introductory remarks .....................................................................................56
3.3.2 TGIF2LY can have transcription factor activity ........................................... 58
3.3.3 Interaction of TGIF2LY with DNA............................................................... 61
3.3.4 Genes potentially regulated by TGIF2LY ..................................................... 66
3.4 Immunological studies on the candidate genes ................................................... 72
3.4.1 Introductory remarks72
3.4.2 Immunological potential of the candidate genes ........................................... 72
3.5 Outlook................................................................................................................77
3.5.1 The “time factor” ........................................................................................... 77
3.5.2 Further steps...................................................................................................78
4 Material and Methods ................................................................................................ 79
4.1 Working with Escherichia coli............................................................................ 79
4.1.1 Culture media.................................................................................................79
4.1.2 Inhibitory and selective media supplements.................................................. 79
4.1.3 Growing of bacteria.......................................................................................80
4.1.4 Short- and long-term storage of bacteria cultures.......................................... 80
4.1.5 Production of chemocompetent E. coli.......................................................... 80
4.1.6 Transformation of chemocompetent E. coli by heat shock ........................... 81
4.2 Working with eukaryotic cell lines...................................................................... 81
4.2.1 Cultivation of cell lines.................................................................................. 81
4.2.2 Maintenance of cell culture: Splitting, Thawing, Freezing and long-term
storage of eukaryotic cells ............................................................................. 82
4.2.3 Transfection of eukaryotic cells..................................................................... 83
4.2.4 Growing of HeLa cells for subcellular localization studies........................... 84
4.3 DNA-based molecular biological methods ......................................................... 85
4.3.1 Isolation and purification procedures ............................................................ 85
4.3.2 Measurement and quality assessment of DNA solutions .............................. 86
4.3.3 Cloning strategies ..........................................................................................88
4.4 RNA-based molecular biological methods.......................................................... 89
4.4.1 Isolation and purification procedures RNA methods .................................... 89
4.4.2 Determination of RNA integrity.................................................................... 90
4.4.3 Reverse transcription of RNA into cDNA..................................................... 90
4