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Theses

CE platform for assaying enzymatic activity and modulation : application to lipases and nucleoside kinases

Abstract : Capillary electrophoresis (CE) has shown promising potential to be used as a complementary or an alternative technique to conventional techniques for several pharmaceutical and therapeutic applications. One of the most significant applications of CE is that of screening for enzyme modulating drug candidates. This thesis was set to demonstrate the analytical and technical power of CE as for monitoring enzymatic reactions in the absence or presence of natural or synthetic modulators. Both offline and online transverse diffusion of laminar flow profiles (TDLFP) reaction modes were adopted for mixing the different reactants. Additionally, several techniques for analyte detection such as UV, C4D and MS, were hyphenated to CE and used for assaying the activities and regulation of model enzymes, mainly lipases and nucleoside kinases. The modulatory molecules ranged in complexity from isolate, molecules purified from natural or synthesized chemically to complex plant extracts. Microscale thermophoresis (MST), a relatively novel biophysical technique used for evaluation of binding affinities between interacting partners (Kd), was used to investigate interactions between lipases and small ligands. The complementarity between the results from ligand binding and those from ligand modulation of enzymatic activity allow the construction of a more complete image of the molecular mechanism in which ligands bind and induce changes in the enzymatic activity. Biomolecular crowding was shown through an offline CE-based hyaluronidase assay to have a significant effect on the ability of purified molecules to inhibit hyaluronidase. Bioactivity assays of several plant extracts, using CE in addition to more conventional techniques revealed the benefit of using these extracts as herbal tea infusions given their anti-hyaluronidase, hypotensive and antioxidant properties. In addition to enzymatic assays, the application of CE, more specifically CE-HRMS, for metabolite detection in complex biological samples demonstrated enhanced repeatability upon using the effective mobility scale instead of the conventional migration time scale. Furthermore, a CE-MS based assay was developed and successfully applied to monitor the activity of nucleoside kinases and screen the activity of novel anti-viral substrates.
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Submitted on : Thursday, December 2, 2021 - 3:41:12 PM
Last modification on : Tuesday, January 4, 2022 - 6:48:00 AM

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  • HAL Id : tel-03463674, version 1

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Ghassan Al Hamoui Dit Banni. CE platform for assaying enzymatic activity and modulation : application to lipases and nucleoside kinases. Analytical chemistry. Université d'Orléans, 2021. English. ⟨NNT : 2021ORLE3122⟩. ⟨tel-03463674⟩

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