Third party funded individual grant
Start date : 01.01.2014
End date : 31.12.2014
Scientific goals of this project:
- to perform confocal live cell imaging in P. falciparum-infected erythrocytes, erythrocyte ghosts, isolated parasites, isolated digestive vacuoles and non-infected erythrocytes.
- to record Ca2+ flux kinetics in the different compartments in response to external pCa-steps and pH step combinations
- to record pH changes in the different compartments in response to external pH steps and pCa2+ step combinations
- to evaluate steady-state fluorescence data in the compartments
- to implement the kinetics and steady state data in a multi-compartment model to predict ion fluxes in live parasites from buffer power curves for each compartment
In the remaining third project year applied through this extension application, we will finalize the efforts of the joint research initiatives set out in the recent 2-year DAAD funding period. The Ca2+ recordings have been mostly covered and are currently written up for publication. There is still need to perform more experiments on Fluo-4 fluorescence uptake in isolated food vacuoles from P.falciparum strains either sensitive or resistant to chloroquine. After isolation, vacuoles can be kept for extended times at -80°C and will be provided by the Canadian partner. As for fluorescence recordings, intact vacuoles will be incubated in appropriate buffers and the dye Fluo4 added. At given time points, confocal images of fluorescence intensities will be obtained for a large number of single vacuoles and uptake rates assessed in the absence and presence of antimalarials. From the kinetics and steady-state data, buffering power curves 'in situ' for the dye will be derived and ion fluxes then modeled using a multicompartment systems approach combining all the data we have from the whole infected and uninfected erythrocytes (already available), isolated parasites (available) and vacuoles (to be done still). As an outlook for a connecting project to be applied for through the Bavarian Research Alliance - Quebec network, we will start with first pH fluorescence recordings in our biological system.