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Study of the role of Mirk/Dyrk1B kinase in the development of Tau pathology (Bachelor thesis)

Δεδούσης, Αδαμάντιος/ Dedousis, Adamantios

Mirk/Dyrk1B kinase, formally designated as: «Μini brain related kinase/Dual-specificity tyrosine-phosphorylation-regulated kinase 1B», is a member of the DYRK family of protein kinases that have been associated with cell growth and differentiation. This kinase has a particularly notable role in maintaining cellular homeostasis. Dyrk1B cross-talks with several critical signaling pathways that affect both cell cycle progression and apoptosis. Furthermore, Dyrk1B has been investigated with considerable attention in the field of oncology. Its involvement in carcinogenesis and its ability to confer resistance to chemotherapy in various types of cancer has been observed. Specifically, Dyrk1B acts by phosphorylating serine-threonine and tyrosine residues on protein targets regulating transcription and cell cycle progression. Ιn this thesis, the aim was to study the expression of the Dyrk1B kinase in the brain of THY-Tau22 mice, which are an animal model for tauopathies and Alzheimer's disease. Specifically, the ultimate goal was to elucidate the role of the Dyrk1B kinase in adult neurogenesis. The purpose of this research was to investigate Dyrk1B expression in the brain of THY-Tau22 mice in various ages. Before starting the study, it was necessary to generate a specific polyclonal antibody against the Dyrk1B kinase. First, part of the coding nucleotide sequence of the protein kinase Dyrk1B was cloned into the plasmid pGEX-4T-1, in order to be expressed in E. coli BL21 bacteria. In the expression system, the recombinant protein carried the GST tag of plasmid pGEX-4T-1. Upon completion of its production, its expression was confirmed by Western blot using antibodies against both Dyrk1B and GST. The GST tag also assisted in purification of the recombinant protein using specific glutathione-sepharose 4B beads. The recombinant protein Dyrk1B-GST produced in E. coli bacteria will be used as an immunogenic antigen in laboratory rabbit. The antibody resulting from the rabbit's immune response will be used for immunohistochemical studies in the brains of THY-Tau22 mice. In parallel with the antibody production procedures, immuno-histochemical investigations against other proteins that have been associated with adult neurogenesis were performed.