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Identifier

000443041

Title

Protein biotechnology-based approaches for the functional and structural characterization of insect enzymes

Alternative Title

Μέθοδοι πρωτεϊνικής βιοτεχνολογίας και στον λειτουργικό και δομικό χαρακτηρισμό πρωτεϊνών εντόμων

Author

Σαράφογλου, Χαρά Δ.

Thesis advisor

Βόντας, Ιωάννης
Μπαλαμπανίδου, Βασιλεία

Reviewer

Κοκκινίδης, Μιχαήλ
Παυλίδης, Ιωάννης

Abstract

Since the practice of agriculture first begun, the loss of yield due to several insect pests was apparent. Even in recent years, with advances in agricultural sciences, losses due to pests and diseases range from 10-90%. Not limited to agricultural damage, insects are also responsible for disease transmission having a profound impact on human and livestock health. To address economy and health issues caused by these insects, various field techniques are used for insect control that can be grouped into three categories: mechanical, biological, and chemical. Chemical methods are related to the use of insecticides targeting harmful insect species with the intention of eliminating them. Since the first applications of insecticides, insects, pressured by natural selection, started developing resistance mechanisms against them. This created the need of new methods of action in the form of new chemicals or new targets, forming this never-ending cycle. Protein biotechnology-based approaches is a powerful tool used to win the race against insecticide resistance. In vitro techniques are used to isolate and characterize existing or novel insecticide targets. Furthermore, resistance mechanisms can be studied in order to identify patterns and eventually resolve problems by slightly altering existing commercially available insecticides or even by designing new chemicals that by-pass insect defenses. For the purpose of this work, two such examples are being examined. As a new promising target, type III geranylgeranyl diphosphate synthase from Helicoverpa armigera was isolated and examined. After protein purification, an assay was adapted to measure activity and determine kinetic values. Through homology modeling, substrate binding patterns were evaluated, and new chemicals were designed using in silico techniques. These chemicals were later tested in the assay to determine potency by highthroughput screening. Finally, Cytochrome P450 from Bemicia tabaci, an enzyme with detoxification function, was studied as it is implicated in insecticide resistance. In this case, the main objective was to establish a reproducible protocol of bacterial expression and purification of the enzyme while keeping it functional in lab conditions. Due to this enzyme’s properties the above proved to be challenging and involved protein engineering strategies to improve efficiency.

Language

English

Subject

Geranylgeranyl diphosphate synthase

Insecticide targets

Pest control

Resistance mechanism

Αντιμετώπιση εντόμων

Μηχανισμοί ανθεκτικότητας

Μοριακή εντομολογία

Στόχοι εντομοκτόμων

Issue date