Herve Tricoire

Cell-specific inositol 1,4,5 trisphosphate 3-kinase mediates epithelial cell apoptosis in response to oxidative stress in Drosophila.

Organismal stress responses to oxidative stress are relevant to ageing and disease and involve key cell-/tissue-specific signal transduction mechanisms. Using Drosophila, an established in vivo model for stress studies, we show that cell-specific inositol phosphate signalling specifically via inositol 1,4,5 trisphosphate 3-kinase (InsP(3) 3-K, IP(3)K), negatively regulates organismal responses to oxidative stress. We demonstrate that the Drosophila Malpighian tubule (equivalent to vertebrate kidney and liver) is a key epithelial sensor for organismal oxidative stress responses: precise targeting of either gain-of-function constructs of Drosophila IP(3)Ks (IP(3)K-1 and IP(3)K-2), or loss-of-function (RNAi) constructs to only one cell type in tubule reversibly modulates survival of stress-challenged adult flies. In vivo, targeted IP(3)K-1 directly increases H(2)O(2) production, pro-apoptotic caspase-9 activity and mitochondrial membrane potential. The mitochondrial calcium load in tubule principal cells-assessed by luminescent and fluorescent genetically-encoded mitochondrial calcium reporters-is significantly increased by IP(3)K-1 under oxidative stress conditions, leading to apoptosis. The Drosophila orthologues of human apoptotic bcl-2 genes include debcl and buffy. Oxidative stress challenge does not modulate gene expression of either debcl or buffy in tubules; and altered debcl expression does not influence survival rates under oxidative stress challenge. Finally, targeted over-expression of either debcl or buffy to tubule principal cells does not impact on tubule caspase-9 activity. Thus, IP(3)K-1 modulates epithelial cell apoptosis without involvement of bcl-2-type proteins.

A self triggered intensified CCD (STIC)

We are developing a new device based on the results reported previously of the successful coincidence detection of β− particles with a high spatial resolution. The novelty of the device consists in triggering and intensified CCD, i.e. a CCD coupled to an image intensifier (II), by an electrical signal collected from the II itself. This is a suitable procedure for detecting low light and rare events with high efficiency and high resolution. The trigger pulse is obtained from the secondary electrons produced by multiplication in a double microchannel plate (MCP) and collected on the aluminized layer protecting the phosphor screen in the II. Triggering efficiencies up to 80% have already been achieved.

SOFI: A scintillating optical fiber imager

We present characteristics of a charged particle detector which combines optical fibers with a multianode photomultiplier. Our first prototype allows detection of 32P emitters on a 26 × 26 cm2 surface with 1.5 mm and 5 mm resolutions, respectively for X and Y directions, and a 0.3% efficiency. Improvements, presently under study, in both fiber qualities and photocathode efficiency should increase the device efficiency to 40% and its resolution up to the optical fiber diameter (500 μm for the time being). Such characteristics would allow SOFI to replace autoradiographic films in biology and make it a convenient position sensitive detector in nuclear and particle physics.

HRRI: A high-resolution β− imager for biological applications

We have developed a high-resolution β− position detector which combines a thin scintillator sheet and an intensified CCD. This device has been designed for molecular biology purposes (“in situ” hybridization). Therefore, the absolute detection efficiency, S/N ratio and spatial resolution have been studied in particular. With the first version of our detector, we achieved an intrinsic resolution of 10 μm with a relative efficiency for 35S of 99% (i.e. 18% absolute efficiency). We now present results in which another scintillator and a new triggering system have been adopted. The main result is that the absolute efficiency has been improved (up to ≈ 60% for 35S) and the background influence has been significantly reduced. These improvements can be observed on the biological image displayed in this paper.

A high resolution β− detector

Abstract We present a detector which associates a charge coupled device to a light amplifier. The image sensor must detect weak β − activity, with a 10 μ resolution and should replace the autoradiographic films used for molecular hybridization. The best results are obtained with the 35 S emitter, for which the resolution and the efficiency are respectively 20 μm and 100% (relative to the measured standard source).

Scintillating optical fiber detectors for DNA sequencing

Abstract We have developed a two-dimensional detector (SOFI) for 32 P emitting molecules used in molecular biology by combining scintillating optical fibers (SOFs) and a multianode photomultiplier (MAPM). A good efficiency (15%) was obtained by suppressing the internal cross talk of the MAPM with a new electronic device. Using this improvement we are developing two new detectors using SOFs for DNA sequencing. We shall present the basic principle of these detectors and the results in efficiency and position accuracy obtained with the first prototypes. The advantage of these detectors over currently available DNA sequencers will be discussed.