IOC60

O2k-Workshop on High-Resolution Respirometry - Introductory and Advanced

Schröcken, Vorarlberg, Austria 11-16 Dec, 2010.

More information on IOC60: http://www.oroboros.at/index.php?nextcourse

Format

Hot Topics in Mitochondrial Physiology: 10+5 min

Abstracts

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Combined introductory and advanced O2k-Workshop with parallel sessions

A: Introductory lectures and hands-on experiments for basic HRR in small working teams:

A1. General HRR topics

• O2k-assembly,
• Polarographic oxygen sensor (OroboPOS) service,
• POS calibration,
• DatLab software: principles and operation,
• Titration-Injection-microPump - TIP2k.

A2. Protocols in HRR

• Introduction to fundamental elements of mitochondrial respiratory physiology,
• Design and examples of experimental protocols with intact cells, permeabilized cells and tissues, isolated mitochondria.

A3. Hands-on experiments with the O2k

• Measurement of instrumental background oxygen flux, manual and TIP2k-feedback control,
• Intact cells: Respirometric reference assay with yeast, phosphorylation control protocol,
• Preparation of permeabilized muscle fibres,
• HRR with permeabilized muscle fibres.

A4. Presentation and interpretation of oxygen flux

• Normalization of respiration per tissue or per mitochondrial marker,
• Coupling control ratios,
• Substrate control ratios,
• Generalized flux control ratios.

A5. Overview on O2k-MultiSensor applications - Simultaneous measurement of respiration and:

• Mitochondrial membrane potential (TPP+ electrode),
• Acidification (pH electrode),
• Nitric oxide (amperometric),
• Spectrophotometry (cytochrome spectra),
• Spectrofluorimetry (Amplex red, safranin, etc.)

B: Advanced users will join in small working teams in parallel sessions, with specific topics:

B1. O2k-MultiSensor and TIP2k

• Hands-on MultiSensor TPP⁺ applications,
• Oxygen kinetics: experimental examples with TIP2k-feedback control (oxystat) and data analysis,
• ADP kinetics: experimental examples with TIP2-steady state control and data analysis,
• pH-stat feedback control,

B2. Theoretical perspectives

• Advanced experimental protocols,
• Quality control - towards quality assurance;

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Tutors

• Erich Gnaiger (Innsbruck Medical University, AT; OROBOROS INSTRUMENTS, AT)
• Mario Fasching (OROBOROS INSTRUMENTS, AT)
• Dominik Pesta (Innsbruck Medical University, AT)
• Zuzana Sumbalova (OROBOROS INSTRUMENTS, AT)
• Anita Wiethuechter (OROBOROS INSTRUMENTS, AT)

Invited guest tutors

• Goncalves Renata (IBqM/UFRJ, Rio de Janeiro, BR)
• Jacobs Robert (University of Zurich, CH)

• Contact: Philipp Tsolakis, administration; Email [email protected]

Participants and areas of interest

• Christiansen Liselotte, Denmark: Veterinary clinical medicine. Mitochondrial function in cats with hypertrophic cardiomyopathy. A model of blood platelets, heart and skeletal muscle fibers.
• Drabarek Beata, Poland: Respiration measurements in endothelial cells under hyperglycemic conditions.
• El-Bacha Tatiana, Brazil: My research interests are studies on mitochondrial physiology of mammalian cells and animal models of virus infections (alphavirus and flavivirus). Currently, we are investigating the relationship of hepatic function and oxidative stress in mice infected with dengue virus and the possible implications for the severe forms of infection. Keywords: virus infection; dengue; mitochondrial bioenergetics; mice; liver dysfunction; HepG2 cells.
• Felser Andrea, Switzerland: Drug mediated effect on mitochondrial function and proteome. Investigation of individual complexes of the respiratory system, membrane potential and apoptosis (isolated mitochondria, intact and permeabilized cells).
• Fischer Silvia, Germany
• Gatterer Hannes, Austria: Sports science.
• Goncalves Renata, Brazil (invited guest tutor): linking the mt metabolism and mosquito immune response, pointing the regulation of mt ROS as an effective immune tool for A. gambiae against Plasmodium infection; mitochondria, flight muscle, vector disease and hematophagous.
• Hanackova Lenka, Czech Republic: Toxic liver injury, liver regeneration and nonalcoholic fatty liver disease. Keywords: Hepatocytes, liver mitochondria, oxidative stress, ROS, respirometry, mitochondrial membrane potential.
• Itkis S. Yulia, Russia (OROBOROS IOC-Scholarship): Respirometry application in Leigh disease diagnostic and treatment. Keywords: mt disease, respirometry.
• Jacobs Robert, Switzerland (invited guest tutor): Mitochondrial function in human muscle biopsies, exercise physiology.
• Krivakova-Stankova Pavla, Czech Republic: Toxic liver injury, liver regeneration and nonalcoholic fatty liver disease. Keywords: Hepatocytes, liver mitochondria, oxidative stress, ROS, respirometry, mitochondrial membrane potential.
• Łukasiak Agnieszka, Poland, Measurement of oxygen consumption by endothelial cells exposed to proinflammatory compounds as well as compounds influencing mitochondrial state and mitochondrial potassium channel openers/inhibitors (isolated mitochondria, intact and permeabilized cells).
• Manzke Lars, Ireland: Mitochondrial oxygen consumption of UCP-knock out mice under various conditions.
• Mark Felix C., Germany: Mitochondrial respiration and membrane potential in polar and temperate fish and cephalopods. Contributions and capacities of the different complexes. Measurements in isolated mitochondria and permeabilised fibres. Keywords: Mitochondria, membrane potential, fish, cephalopods, cold adaptation, hypoxia.
• McKee Edward, USA: Mitochondrial toxicity of antiviral nucleoside analogs. Interested in metabolism of deoxynucleosides in mitochondria of non-replicating tissues and the effect of antiviral nucloeside analogs on this metabolism as a potential mechanism of toxicity. We are interested in using the Oroboros equipment to study respiration and coupling in isolated mitochondria from tissues exposed to long-term antiviral analog treatment. Keywords: Nucleoside analog reverse transcriptase inhibitors (NRTIs), deoxynucleotide pools (dNTP pools), mitochondrial toxicity, mitochondrial DNA replication.
• Rosenfeld Eric, France: Characterization of non-respiratory oxygen consumption pathways in yeast (oxygen and ROS fluxes); study of redox-active molecules targeting eukaryotic or prokaryotic microorganisms (i.e. Pseudomonas aeruginosa strains involved in cystic fibrosis). Keywords: Yeast, bacteria, respiration, non-respiratory pathways, ROS, redox active molecules.
• Rousar Tomas, Czech Republic: I will be using the respirometry during evaluation of oxidative stress in hepatocytes and mitochondria after treatment with a variety of hepatotoxins. Especially, I am interested in mitochondria-mediated ROS production, disappearance of antioxidants and oxidative stress detection. Keywords: Antioxidants, glutathione, oxidative stress.
• Schauer Irene, USA: Effects of fatty acid lowering and/or insulin; sensitization on mitochondrial function and cardiovascular function in type 1 diabetes. Keywords: Fatty acids, insulin resistance, diabetes.
• Staples James, Canada: Suppression of metabolism in hibernation. Keywords: hibernation, torpor, mitochondria.
• Strobel Anneli, Germany: Mitochondrial respiration and membrane potential in polar and temperate fish and cephalopods. Contributions and capacities of the different complexes. Measurements in isolated mitochondria and permeabilised fibres. Keywords: Mitochondria, membrane potential, fish, cephalopods, cold adaptation, hypoxia.
• Wojciechowska Sylwia, Poland: Respiration measurements in different types of cells in insulin resistance state.

Discussion

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