Investigating the localization of an avian hairy homolog (c-Hairy1) protein

Cravo, Janine Anselmo

http://hdl.handle.net/10400.1/3555

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Authors

Cravo, Janine Anselmo

Advisor(s)

Palmeirim, Isabel

Gonçalves, Lisa

Abstract(s)

The embryo segmental pattern is first established with the formation of somites. Somites are embryonic segments of vertebrates, periodically formed in a strict temporal precision, which has been believed to be governed by a biological clock, called the ‘segmentation clock’. In the mid-70s, the “Clock and Wavefront Model” was proposed, predicting the existence of an intracellular clock or oscillator in the presomitic mesoderm (PSM) cells. Several years later, Palmeirim et. al., identified the first molecular evidence of this clock by discovering and characterizing the oscillatory expression of c-hairy1 in the chicken embryo paraxial PSM, which strikingly matched the period of somite formation (90 min). This present work aims to characterize the chicken Hairy1 protein, and to study its expression both in early embryo stages and chicken embryonic fibroblasts (CEFs). Bioinformatic tools have predicted the biochemical properties, primary and secondary structure, post transcriptional modifications and subcellular localization of c-Hairy1 protein. By western blot were established the optimal working conditions of the customized monoclonal antibody, as well as the expression of the protein in both chick embryos and CEFs. The protein distribution and its subcellular localization in CEFs was assessed by immunofluorescence. Results of western blot have demonstrated sensitivity of the antibody, although its specificity for c-Hairy1 protein remains debatable. Even more, both immunofluorescence and bioinformatics analysis showed c-Hariy1 to be localized both in nucleus and cytoplasm. Interestingly, it was also demonstrated that the nucleus:cytoplasm ratio distribution varied between cells. These work’s findings suggest that c-Hairy1 protein holds much investigation potential, and the optimization of the antibody working conditions enables its use for further studies.