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Regulation of human protein S gene (PROS1) transcription Wolf, Cornelia de

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(1)Regulation of human protein S gene (PROS1) transcription Wolf, Cornelia de. Citation Wolf, C. de. (2006, May 29). Regulation of human protein S gene (PROS1) transcription. Retrieved from https://hdl.handle.net/1887/4413 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/4413. Note: To cite this publication please use the final published version (if applicable)..

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