PhD defence by Christos Theodorou

Untargeted Metabolomics as a tool for the elucidation of transporter protein substrate specificity

In plants, transporter proteins are involved in multiple cellular processes such as maintaining homeostasis, signalling and cell communication as well as uptake or efflux of specialized metabolites among different compartments. However, our knowledge about the function of the many transporters in the genomes is limited. In the DynaMo Center, Christos Theodorou has been part of the VILLUM investigator project aimed at identification of transporter substrate specificity in a major transportomics endeavor.

Christos has played a key role in development of a novel transportomics methodology that combines the use of uptake assays with plant extracts as complex substrate mixtures, and untargeted metabolomics as an analytical tool, therefore allowing for the simultaneous testing of multiple compounds against the same transporter. In the thesis, Christos describes all steps, from the creation of the substrate mixture to the generation of the analytical pipeline for screening transporter proteins with the ‘transportomics’ pipeline. This includes the experimental set-up and the subsequent data acquisition, preprocessing and statistical analysis techniques comprising the developed pipeline based on a dataset of transporter proteins with unknown functionality.

Christos has conducted a thorough investigation on different extracts that can be used as substrate mixtures in the transportomics pipeline and use an innovative approach to maximize the metabolic diversity contained within that mixture. He performed an in-depth metabolic profiling of the selected plant extract based on a combinatorial technique leading to the identification of more than 200 metabolites.

Through collaboration with other members who are part of this VILLUM investigator grant, the performance of the developed pipeline was validated based on two different study cases that are used as a proof of concept and present the first-of-its-kind export transportomics assay. Several substrates for four different transporter proteins belonging to different protein families were identified.

Supervisors

Professor Barbara Halkier

Facility Manager, Christoph Crocoll

Assessment committee

Assessment Committee – Chair: Associate professor Giorgia La Barbera, Department of Nutrition and Health (Chair)

Opponent 1: Professor Robert David Hall, Wageningen University and Research, the Netherlands (External member)

Opponent 2: Daniel Petras, Department of Biochemistry, University of California, US (External member)

Time and Venue

7 October at 14:00 in H/K 117

You are all welcome to join the Reception after the defence in H/K 117