Aleksandra Konovalova
Glucose is one of the most important metabolites that plays a key role in living organisms, as it is the primary energy source. Glucose metabolism is also an integral part in the pathogenesis of diabetes and cancer. Despite this tremendous impact of glucose, there are no tools for imaging of glucose uptake non-invasively and in a real-time manner working in both living cells and animals without utilization of hazardous radiation and suitable for convenient preclinical use. The first part of this thesis is describing the development of a probe for imaging glucose uptake based on a highly sensitive and versatile bioluminescence technique. We set a goal to design a probe, which should be reliable, robust, easy to use and simply represent the glucose uptake in both living cells and animals. In addition the synthesis of the probe should be straightforward and high-yielding. This part of the thesis is composed of two projects utilizing different approaches to the probe design and probeâs mechanism of action. The first project was based on the Staudinger Ligation for imaging of glucose uptake by modifying the glucose molecule with an azide moiety and using luciferin caged with a reactive phosphine as a counter-partner for the assay. The second project describes the development of a cleavable disulfide glucose-conjugate probe. For the first project we aimed to synthesize a library of glucose pro bioluminescent probes, validate them in a cell-free and cell-based assay and identify the hit compound. Then we proved the GLUT-specificity of the uptake of our hit compound in living cells and animals. The robust and reproducible assay method for measurement of glucose uptake was established. The probe was applied for imaging of antidiabetic drugsâ influence on glucose uptake. We have also demonstrated its superior properties over 2-NBDG that is currently the most used probe in preclinical research. However, the probe utilized in the second project namely cleavable disulfide glucose conjugate probe was not so sensitive as glucose-azide. Nevertheless, we showed a successful application of this probe for imaging of glucose uptake in living cells and animals. The second part of this thesis describes the design and evaluation of the bioluminescent probe for imaging of nicotinamide riboside (NR) uptake. NR is a recently discovered NAD+ precursor that is naturally present in cow's milk. Despite its importance in modern research and a high potential as a new medicine for treatment of various types of diseases, including diabetes, there are no tools to visualize NR uptake in preclinical research. Also, there is a lack of knowledge on where and how NR can be absorbed in mammals. Here we established the Staudinger Ligation-based approach to develop an imaging tool for NR uptake. After the successful synthesis of azido-nicotinamide riboside molecule, we demonstrated its specificity and utility for imaging in living cells and animals. Findings described in this part cover not only the imaging of NR uptake, but also the possibility of existence of specific NR transporter in mammalian cells, which has not been discovered and described yet.
EPFL2016
