IOC65

O2k-Workshop on High-Resolution Respirometry: O2k-Basic and TPP-Basic

Schröcken, Vorarlberg, Austria 10 - 15 December, 2011.

Registration form:http://www.oroboros.at/index.php?id=ioc-registration

For some pictures, see Workshop photos

Introductory Workshop in Parallel Sessions: O2k Basic and TPP Basic

The workshop is run in parallel sessions: O2k Basic and TPP Basic. Specific emphasis is placed on hands-on practical sessions with the instrument and the software DatLab 4.3, to introduce the participants into the technical and operational details of the method. The workshop programme is complemented by combined lectures (general topics; substrate-uncoupler-inhibitor titration SUIT protocols) and experiments run in "Special interest groups". Short presentations of 'Hot topics in Mitochondrial Physiology' provide participants with the opportunity to present and discuss their work, including projects in progress, methodological details and conceptual aspects. Please register for O2k Basic or TPP Basic according to your specific needs and interest.

O2k Basic includes a basic introduction to the OROBOROS Oxygraph-2k, overview presentations on high-resolution respirometry (HRR), and applications in mitochondrial physiology.

TPP Basic is customized particularly for users interested in MultiSensor applications, particularly ion selective electrodes for measurement of mitochondrial membrane potential (TPP+, TPMP+). It covers an introduction to basic concepts on mitochondrial membrane potential: proton pumps, ion gradients, electrochemical potential (proton motive force) and the measurement of mt-membrane potential in biological samples (homogenate, permeabilized fibres). Furthermore, data analysis (from TPP+ to mt-membrane potential) will constitue a major part. According to interest of the participant other MultiSensor applications, like measurement of Ca2+, calibration and calculation of free Ca2+ in buffered solutions and acidification rate (pH) can be discussed/demonstrated. Please indicate your topics of interest (Ca2+, pH etc.) in advance.

Please register for O2k Basic or TPP Basic according to your specific needs and interest.

Preliminary Programme

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. 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.

A3. 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.

B: Introductory lectures and hands-on experiments for basic TPP⁺ (mitochondrial membrane potential) in small working teams

Hands-on MultiSensor TPP⁺ applications, see TPP special interest group during an International Oxygraph Course
From TPP⁺ to mitochondrial membrane potential: unspecific binding, partitioning, chemical side effects.

C: General topics

Overview on the O2k and DatLab: High-resolution respirometry (HRR)
Overview on O2k-MultiSensor applications

Mitochondrial membrane potential (TPP+ electrode)
Acidification (pH electrode), pH-stat feedback control
Nitric oxide (amperometric)

Introduction to fundamental elements of mitochondrial respiratory physiology
Protocols in HRR: Design and examples of experimental protocols with intact cells, permeabilized cells and tissues, isolated mitochondria.
Quality control - towards quality assurance

D: Special interest groups

Oxygen kinetics: experimental examples with TIP2k-feedback control (oxystat) and data analysis,
ADP kinetics: experimental examples with TIP2-steady state control and data analysis,
Advanced experimental protocols

Participants and areas of interest

Oliveira MF, BR: Studies on the mitochondrial metabolism in the blood flow Schistosoma mansoni.
Pinho Joao Paulo, BR: Studies on the dynamic changes of mitochondrial function following a blood meal in the kissing bug Rhodnius prolixus.
Goncalves Renata de Lima Sales, BR: Mitochondrial membrane potential, metabolic states.
Lau Gigi, CA: Physiological adaptations of mitochondria to hypoxia in fishes.
Mc Donald Allison, CA: Functional and regulatory characterization of alternative oxidases in various kingdoms of life.
Keipert S, DE: Mitochondrial uncoupling in skeletal muscle: Transgenic mice with ectopic expression of UCP1 in skeletal muscle with beneficial effects on energy metabolism, diet-induced obesity and aging.
Ost Mario , DE: Mitochondrial uncoupling in skeletal muscle: Transgenic mice with ectopic expression of UCP1 in skeletal muscle with beneficial effects on energy metabolism, diet-induced obesity and aging.
Gonzalez Franquesa Alba, ES: Mitochondrial dysfunction in insulin-dependent tissues such as liver, skeletal muscle and adipose tissue.
Bechara Hirvonen Nada, FI: Mitochondrial dysfunction in experimental heart failure.
Cannon Megan, NL: Metabolic remodeling in cardiac hypertrophy and heart failure.
Hoffman David L., US: Mitochondrial ROS production / O2; open flow respirometry mitochondrial therapeutics.
Bosch Marta, ES
Strand Harald, NO: Diagnosis of mitochondrial diseases.
Plenge Ulla, DK: Effects of rhEPO in human mitochondria in muscle cells and blood cells.
Cesnekova Jana, CZ: Oxidative phosphorylation system.
Spacilova Jana, CZ: Oxidative metabolism in human skin fibroblast from patients with mitochondrial disorders.
Nielsen Morten Hostrup, DK: Effects of beta2-agonists on human skeletal muscle mitochondrial enzyme activity.

Abstract Submission

Abstracts are welcome for short presentations (hot topics in mitochondrial physiology), providing participants for an opportunity to present and discuss their work, including projects in progress, methodological details and conceptual aspects. Abstracts should be submitted by November 21, 2011 (1 page, single spaced, including title, authors, affiliations, text and 3 references including titles).

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)