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research day - University of Toronto Department of Obstetrics and ...

research day - University of Toronto Department of Obstetrics and ...

28 ABSTRACT #O3* VEPH1

28 ABSTRACT #O3* VEPH1 IS A NOVEL REGULATOR OF TGF-ß SIGNALING IN OVARIAN CANCER CELLS. Premalatha Shathasivam[G](1,2,3) J Wrana(1,4), and TJ Brown(1,2,3). (1)Samuel Lunenfeld Research Institute, Mount Sinai Hospital; Departments of (2)Physiology, (3)Obstetrics and Gynaecology, and (4)Medical Genetics and Microbiology, University of Toronto, Toronto *Abstract available in hardcopy version only.

29 ABSTRACT #O4 DISSECTING THE MOLECULAR PATHWAY OF NALP5 IN MURINE EMBRYO DEVELOPMENT (Work in Progress) Alagammal Perumalsamy [PD] (1), Roxanne Fernandes (1,3), Andrea Jurisicova (1,2,3) (1)Samuel Lunenfeld Research Institute, (2) Department of Obstetrics & Gynaecology, Mount Sinai Hospital, (3) Department of Physiology, University of Toronto. Objective: To determine molecular mode of action mediated by NALP5 and test whether NALP5 forms a signaling complex involved in NFκB activation in mouse embryos. NALPs mediate molecular responses to a variety of signals such as bacterial peptide signaling, and foreign DNA/RNA responses. Also, due to NALPs interaction with cell death proteins, caspases, they have been implicated in cell death/survival decisions. However, the specific pathways in response to NALPs in germ cells have not been determined. To investigate the biological role of NALP5 as a cell survival molecule, we established whether altered NFκB translocation and response occurs in NALP5 deficient (KO) zygotes. NALP5 can be a modulator of NFκB activity, and thus contributing to efficient activation of embryonic genome. Methods: To determine pattern and level of expression of various components of canonical and non-canonical NFκB signaling in wildtype (WT) and NALP5 KO murine oocytes and zygotes, real time qRT-PCR and immunocytochemistry will be employed. To determine whether NALP5 also act as other NALPs, immunoprecipitation experiments will be performed using HEK293 cells preceded by transient transfection of epitope tagged ASC and NALP5. The effect of NALP5&ASC interaction on NFκB activity will be analyzed by performing NFκB DNA binding reporter assays. We will also investigate if NALP5 facilitates caspase activation, via recruitment of ASC protein, resulting in efficient NFκB nuclear translocation and gene activation. Results: We determined the expression of ASC in NALP5 oocytes and two cell embryos. While transcript levels of ASC were not altered, ASC protein formed clear specks in the nuclei of WT embryos, whereas in the KO embryos, specks were mislocalized, as they were seen in both the cytoplasm and nuclei. This altered distribution between WT and KO embryos may indicate altered signaling platform. Additionally, embryos lacking NALP5 also exhibit decreased spontaneous caspase activity as measured by cell permeable fluorescently labeled caspase substrate. In order to determine if decreased level of caspase activity contribute to abnormal embryo development, we cultured zygotes in presence of pan-caspase inhibitor. These results suggest that proper ASC protein localization and maternal caspases may be required for setting up events leading to proper initiation of embryonic development. NFκB subunit RelA (p65) expression levels are also altered between WT and KO oocytes and zygotes, and cytoplasmic localization, determined by immunocytochemistry analysis with NFκB p65 antibodies. While elevated cytoplasmic NFκB expression was observed in NALP5 KO oocytes, decreased nuclear of NFκB was detected in NALP KO zygotes. Conclusions: Further elucidation of the precise molecular role of NALP5 in embryo development is an important finding and the results can have broad applications to understanding the basic biology of mammalian embryo development but also may have further implications on the understanding of early embryo loss in humans.

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