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Index of Figures and Tables
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Index of Figures and Tables
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Figure 1 Sphere-formation of CT1258 cells in serum-free suspension culture medium.
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CT1258 sphere-forming cells were imaged with indicated day of culture. Day 1: After trypsinisation, a defined number of 10000 CT1258 cells/ml was transferred in suspension medium. Day 5: Few floating cells started to construct spheroids and the majority of cells were dead. Day 10: The dead cells reduced compared to day 5 and the number of spheres increased. Day 15: The volume of the formed spheres increased, while a low number of dead cells could be observed.
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Figure 2 Relative expression level of selected markers in CT1258 spheroid s10d and s15d cells compared to adherent CT1258 cells. !
qPCRanalyses of the expression level of 12 selected stem cell markers in spheroid CT1258 cells compared with adherent CT1258 cells. Each colour corresponds to the stem cell marker indicated in the legend. CT1258:
adherent CT1258 cells were used as the calibrator for the spheroid cells. s10d: Spheroid cells cultivated for 10 days. s15d: Spheroid cells cultivated for 15 days!
! Expression of stem cell markers relative to ß-actin in adherent CT1258 cells and generated spheroids T
Index of Figures and Tables
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Figure 3 Flow cytometric analyses of adherent and sphere cells from CT1258 cells.!
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CD44 and CD133 monoclonal antibodies against mouse/dog labelled with FITC and PE fluorophore substances were used. The histograms show the analysed cell lines stained with CD44 and CD133 antibodies compared to the corresponding isotype controls (red). The geometric fluorescence intensities (gMFI) are shown. CD44: X-axes represent for FL1-H the FITC fluorescence intensity (494/24) nm and y-X-axes represent the percentage of counts of viable gated cells. CD133: X-axes represent for FL2-H the PE fluorescence intensity (585/42) nm and y-axes represent the percentage of counts of viable gated.
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Index of Figures and Tables
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Table 1 Primer pairs used in quantitative real-time PCR
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Table 2 Antibodies used for flow cytometric analyses
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Table 3 Normalised geometric mean fluorescence intensity (gMFI) data of the flow cytometric measurements
The normalised gMFI of the specific CD44/ CD133 staining was divided by the respective isotype control staining to define the specific staining ratio
Gene Forward Primer (5’-3’) Reverse Primer (5’-3’) Amplicon (bp)
CD34 ACCAGAGCTATTCCCGCAAG TTTCTCCTGTAGGGCTCCAA 120
CD133 CTTTCTCATGGTCGGAGTTGG TGGAATAGTTTCCTGTTCTGGTAAG 135
C-KIT AGAAACGTGAAGCGCGAGTA ACACAACTGGTACAGCTCGATGG 129
CD44 AATGCTTCAGCTCCACCTG CGGTTAACGATGGTTATGGTAATT 92
OCT4 CGAGGAGTCCCAAGACATCA AACACCTTCCCAAAGAGAACC 138
NANOG CTATAGAGGAGAGCACAGTGAAG GTTCGGATCTACTTTAGAGTGAGG 141
KLF4 CCACATTAATGAGGCAGCCA CTCCCGCCAGCGGTTATT 146
SOX2 GGAAACTTTTGTCGGAGACG CGGGGCCGGTATTTATAATC 103
C-MYC TCGGACTCTCTGCTCTCCTC TTCTTCCTCCGAGTCGCT 108
MELK CCAAGGGTAACAAGGACTAC CTCCAAACATCTGCCTCTGA 112
DDX5 AACTTCCCTGCAAATGTAATGGA AGTCTGTGCTACTCCAACCAT 123
Beta-actin
(ß-act) TCGCTGACAGGATGCAGAAG GTGGACAGTGAGGCCAGGAT 127
Monoclonal
Antibody Specificity Clone Marker identified
Monoclonal Isotype Anti-Canine CD44 FITC
eBioscience Dog YKIX337.8 CD44 Rat IgG2aκ FITC BD Bioscience
Index of Figures and Tables
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9.
Index of Figures and Tables
9.1 Figures
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Manuscript II
Figure 1 Sphere-formation of CT1258 cells in serum-free suspension
culture medium 20
Figure 2 Relative expression level of selected markers in CT1258 spheroid s10d and s15d cells compared to adherent CT1258 cells 22
Figure 3 Flow cytometric analyses of adherent and sphere cells from
CT1258 cells 23
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9.2. Tables
Manuscript II:
Table 1 Primer pairs used in quantitative real-time PCR 31
Table 2 Antibodies used for flow cytometry analyses 32
Table 3 Normalised geometric mean fluorescence intensity (gMFI)
data of the flow cytometry measurements 24!
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Acknowledgments Acknowledgments
In the name of ALLAH the Merciful, Praise to ALLAH and peace and blessings be upon the Messenger of ALLAH.
my ALLAH this is from you and for you.
my ALLAH, Praise for you until you are satisfied and Praise to you If you are satisfied and praise to you after satisfaction.
I have studied in the Clininc of Small Animal, University of Veterinary Medicine Hannover with funding from the DAAD, Deutscher Akademischer Austausch Dienst/ German Academic Exchange. This dissertation would not have been possible without the guidance and the help of Mrs Anke Bahrani and several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study.
First and foremost, I offer my sincerest gratitude to my supervisor, Prof. Dr. Ingo Nolte for his encouragement, guidance, patience, and support enabled me to complete this thesis and to finish my study. Prof. Dr. Steinberg and Prof. Dr. Hassan Naim have been also helpful in academic life.
I owe my deepest gratitude to PD. Dr. Hugo Murua Escobar for his kind support, crucial advices and encouragement during the whole period of my staying in Germany. It is an honor for me to complete this dissertation under his supervision.
Great appreciation and sincere thanks to Mrs Maritta Ledwoch, Mrs Johanna Kroll and Mrs Edith Schneider for their enforcement and appreciated support during the whole period of my stay in Germany.!
It! is! a! pleasure! to! thank!Dr.$ Saskia$ Willenbrock! for! their! help! and! guidance! also! for! the!
friendship.!!
Acknowledgments
I!would!also!like!to!thank!all!staff$ members,$ colleagues$and!my!team!of!work!specially!Wen$
Liu!and!Florenza$Ripoli!for!their!very!nice!friendship!and!their!support.
Finally, I am very grateful to my mother Zohra and my father Al-aalmi, my wife Leyla, parents of my wife Fatma and Hadj, my brothers and sisters and all my family, my second father and my uncle Abd-elkader, uncles Hebri, Omar et Nacer for their endless and unconditional love, support and encouragement which enabled me to conquer all the difficulties in the long run of finishing my Ph.D. thesis. At last, thousand excuses for everybody that I have to thank them and I did not mention their names.
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