On 24 August at 10 Olga Mazina will defend her PhD thesis titled "Development and application of the biosensor assay for measurements of cyclic adenosine monophosphate in studies of G protein-coupled receptor signaling" in Chemistry in the Faculty of Science and Technology in the University of Tartu.
Thesis supervisors: Professor Ago Rinken (PhD), University of Tartu
Research Fellow Sergei Kopantšuk (PhD), Insitute of Chemistry, University of Tartu
Opponent: Professor Alexander Savitsky, (PhD, D.Sc), A.N. Bach Institute of Biochemistry Russian Academy of Science, Moscow, Russia
World’s population is constantly growing and aging. As a result, the number of patients suffering from some sort of illness or disease is rising as well. Most of the illnesses are caused by disturbances in cellular signaling or metabolism of the organism. G protein-coupled receptors (GPCR) form a large family of cell surface proteins responsible for the regulation of signal transduction. Over one third of all todays’ prescription drugs are targeted to GPCRs.
In search for new synthetic compounds targeting these receptors functional testing is of great importance. Obtained experimental data need to be evaluated and compared with data from structurally similar or endogenous ligands. This yields information about the properties of the new ligand and for molecular design of future compounds. In the current thesis an assay system for characterization of different biologically active molecules on living cells was developed based on the use of fluorescent biosensor proteins.
To use a biosensor, it needs to be present inside the cells of interest. To achieve a reliable and uniform cellular level of the biosensor protein, a gene delivery system based on the use of insect viruses was employed. These viruses, harmless to humans, transport the biosensor gene sequence in to the cells, where a new functional protein is synthesized and is ready to be used in functional experiments. The level of protein expression can be regulated by the amount of the applied virus, which results in homogeneous expression and a reliable assay with high reproducibility.
During the studies the applicability of the new biosensor assay was demonstrated by monitoring the activation of very different GPCRs in various cells. Due to high sensitivity of the assay it is also suitable for quantification of endogenous hormones in different probes and preparations. This is of importance for the field of medicine as well as for the development of novel technologies. The result of this work is the developed biological tool that enables us to monitor GPCR activation for measurement of biological activity of compounds with various structural properties.