FRET Probes for Analysis of Serpin-mediated Apoptosis in Focal Brain Ischemia
In light of the profound significance of apoptosis research, it is essential to have probes which can discriminate between different apoptotic pathways. The serine protease-dependent pathway is one of the most recently identified apoptotic programs, which still lacks specific tools for its visualization in situ. SerpinB1 is a key member of this pathway and is an effector of apoptosis. In metabolic stress it transforms into an active endonuclease. The newly formed DNA degrading enzyme promptly moves into the nucleus causing cell death. In spite of the high importance of this non-caspase apoptosis mechanism, there are currently no methods to selectively label it in tissue sections and in live cell cultures. In this project we are developing such methods and applying them to study serpin-mediated apoptosis in focal brain ischemia.
In summary, a successful outcome of this study would provide enabling technologies for apoptosis research in general, and for studies in ischemia in particular. Their systematic application to focal brain ischemia will provide information useful for the future clinical and research investigations of stroke, and for the development of effective therapeutic interventions.
Imaging mRNA Turnover in P-bodies and its Application to Brain Ischemia
Injured cells adapt to an insult by removing their mRNAs for storage or destruction. Both these processes occur in the same cytoplasmic foci referred to as P-bodies. The storage and destruction of transcripts cause the translation block and their relative intensity determines the fate of the cell. The ability to separately visualize these processes directly in P-bodies on a cell-by-cell basis is essential for the assessment and future control of cellular response to injury.
The purpose of this study to develop the first histotechnology for in situ labeling of mRNA turnover in P-bodies. This will allow visualization of previously undetectable molecular pathways in a convenient histotechnology format. Its application to brain ischemia will provide useful information about the dynamics of ischemic cell injury.
Express Assay for Specific Fluorescence Imaging of Apoptosis via Phosphatase-assisted Topoligation
The project will introduce a new ultra-fast bioimaging approach employing the unique properties of Mimivirus topoisomerase. It will close the technological gap and will create an advantageous labeling technique with a wide application field. The new quick and specific assay will be useful in biomedicine, particularly in apoptosis research, in express assessment of pathology samples and in studies where large-volume quantitations of programmed cell death cells are essential, such as in cancers, ischemic disorders, and degenerative diseases.