IV. CONCLUSION AND PERSPECTIVES
36. Mitochondrial calcium measurements
INS-1E cells (7.5 x 104 cells) were plated on polyornithin-treated 15 mm diameter Thermanox coverslips (Nalge Nunc, Rochester, NY, USA, Cat. No. 174969) in 4-well tissue culture plates (Nunc, Cat. No. 176740). The day after plating, cells were transfected with siRNAs as described (see chapter 22). 48 h post-transfection cells were infected for 2 h with adenovirus expressing mitochondrially-targeted aequorin under the control of the rat insulin promoter (Ad-RIP-mitoAequorin). 96 h post-transfection cells were transferred for 2 h to RPMI-1640 medium containing 4 mM glucose, 1% (v/v) FCS and 5 µM coelenterazine (Calbiochem, Cat.
No. 233900). Then cells were perifused at a rate of 1 ml/min with KRBH containing different stimuli and emitted photons were counted with a photomultiplier apparatus (Thorn-EMI Electron Tubes, UK). At the end of the measurement cells were permeabilized with 0.1 mM digitonin and 10 mM CaCl2. The aequorin luminescence signal produced during permeabilization was used to calculate calcium concentrations.
37. [1-14C] pyruvate oxidation measurements
INS-1E cells were plated in 6-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ, USA, Cat. No. 353046) at a density of 0.5 x 106 cells/well. Throughout the experiment the volume of media and buffers used for incubations and washes was 3ml/well. Experiments were performed 48 h after plating the cells. Before the experiment, cells were switched for 2 h to RPMI-1640 medium containing 4 mM glucose and 1% (v/v) FCS and kept at 37°C in a cell culture incubator. Then cells were washed once with KRBH containing 2.5 mM glucose and left for 30 min at 37°C in a waterbath. Then cells were switched to KRBH containing 2.5 or
129 16.7 mM glucose plus 0.1 mM cold pyruvate and 0.02 nCi/µl of [1-14C] pyruvate (American Radiolabeled Chemicals, Cat. No. ARC 0223, 55 mCi/mmol). Specific activity in the resulting solution was 0.208 mCi/mmol. In order to capture 14CO2, Whatman filters (6.5 x 2 cm) were soaked with 2M NaOH (400 µl) and attached using strips of adhesive tape to the inner side of the culture plate lid. The plates were sealed with parafilm and incubated for 1 h at 37°C on the shaking platform (50 rpm). For each experimental condition a separate plate was used to avoid the exchange of CO2 between the different conditions studied. Plate without cells was used as control in each experiment. Following incubations, each filter was collected in a scintillation vial (Wheaton, Millville, NJ, USA, 6 ml HDPE vial, Cat. No.
986644). Medium from two neighboring wells (6 ml) were pooled in 20 ml glass vial (PerkinElmer, Waltham, MA, USA, Econo Glass Vial, Cat. No. 6000097). Then each well was washed with 1.5 ml of PBS w/o Ca2+ and Mg2+ and this buffer was added to the same glass vial (total volume of liquid 9 ml). Each vial received an Eppendorf tube containing Whatman filter (3 x 1.5 cm) soaked with 2M NaOH (250 µl). Vials were closed with rubber caps (Precision Seal rubber septa, Sigma, Cat. No. Z553964). Then 400 µl of 5M HCl was injected into the medium using syringe with a needle. Following HCl injection, the vials were sealed with parafilm and incubated for 2 h at 37°C on the shaking platform (50 rpm). Then filters from each condition were combined in a scintillation vial and 4 ml of scintillation fluid (Packard, USA, Emulsifier Safe, Cat. No. 6013389) added. 14C counting was performed on LS6500 liquid scintillation counter (Beckman Instruments) 24-48 h after adding the scintillation fluid. Earlier measurements gave very large artefactual variations.
130 ACKNOWLEDGEMENTS
First of all, I would like to express my gratitude to Prof. Claes Wollheim for giving me the opportunity to complete my Ph.D. studies in his lab. During this 4 years and 3 months he shared his expertise, gave precious suggestions and support. His energy and enthusiasm were always encouraging and stimulating.
I am very grateful to my direct supervisor Dr. Andreas Wiederkehr who was always ready to help me design experiments, analyze results, prepare talks and write the thesis. He was truly interested in my work and his advices were invaluable. I am very thankful to him for the opportunity to discuss every small experimental detail. This work would not have been possible without his guidance and help.
I appreciated the opportunity of working with Dr. Kyu Sang Park and to learn several techniques from him. I am indebted to the lab technicians Olivier Dupont, Danielle Nappey, Nicole Aebischer, Dale Brighouse, Dominique Duhamel and Daniela Cornut-Harry for teaching me many techniques. I am thankful to Asllan Gjinovci who patiently taught me surgery.
I thank Prof. Pierre Maechler for his interest in my work and for kindly allowing me performing several experiments in his lab. I am also grateful to his group members Clarissa Bartley and Ning Li for the help with radioactive measurements and mitochondria isolation.
I thank Dr. Françoise Assimacopoulos for sharing her expertise in ATP measurements.
I appreciate the help of Prof. Jean-Charles Sanchez and members of his lab in planning 2D gel experiments.
The study on pyruvate dehydrogenase phosphorylation would not have been possible without the precious antibodies gifted by Dr. Henriette Pilegaard from the University of Copenhagen.
I thank my colleagues and friends Deborah Aeberhard, Elodie Husi, Dr. Nicole Feldmann, Dr.
Kai Hu He, Dr. Mathurin Baquie for the friendly and supportive atmosphere in the lab.
I express my appreciation to the members of the thesis jury Prof. Mary Sugden and Prof.
Jean-Claude Martinou who kindly agreed to evaluate my work.
I am extremely grateful to my mother and brothers who have been supporting and encouraging me every day during my Ph.D. studies.
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