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Identifier 000426463
Title Biotechnology - based approaches for insecticide discovery
Alternative Title Βιοτεχνολογικές προσεγγίσεις για την ανακάλυψη εντομοκτόνων
Author Λαμπρούση Μάνθα Λ.
Thesis advisor Βόντας. Ιωάννης
Reviewer Δελιδάκης, Χρήστος
Σαρρής, Παναγιώτης
Abstract Insecticides resistance is a constantly increasing problem in recent years, as crops and human health are threatened by agricultural pests and disease vectors, respectively. For this reason, it is imperative to find new insecticide targets and also to investigate the role of several mutations in insecticide resistance through functional validation. The midgut of insects is the most important tissue involved in penetration of insecticides and their entrance to the hemocoel in order to cause their toxic effects in specific tissues where their targets lie. Several Lepidopteran species like Helicoverpa armigera are major agricultural pests and they can easily develop resistance. Thus, the investigation of lepidopteran midgut properties is essential. We tried to established a primary midgut cell line from H. armigera in order to investigate the properties of the different midgut cell types (intestinal stem cells, enterocytes, enteroendocrine cells and Goblet cells), with a major focus on intestinal stem cell properties because of their ability to give rise to all the different midgut cell types and to enterocyte properties due to the role of apical- basolateral polarization and the existence of microvilli in absorption, penetration and diffusion of different compounds,. We also investigated certain properties (transfectability and spheroids formation) of an already established lepidopteran midgut cell line from Helicoverpa zea (RP-HzGUT- AW1), a close relative of H. armigera. Transcriptomics analysis was performed for these cells in order to characterize their expression profile. One major question regarding these cells was if the spheroids formed develop smooth septate junctions (sSJs) as expected in midgut epithelia. This was examined by comparing the levels of expression of various genes involved in sSJs formation, in particular three main regulators: Mesh, tetraspanin- 2A (Tsp2A) and snakeskin (Ssk). While various other markers were identified from transcriptomics for characterization of H. zea midgut cell line, the expression levels of sSJs markers were not significantly altered. Regarding functional validation of mutations possibly associated with target-site insecticide resistance, a mutation first identified in glutamate gated chloride 3 subunit (GluCl) of the spider mite Tetranychus urticae and associated with resistance to abamectin was investigated. This mutation (I310T) was introduced to the Drosophila melanogaster GluCl, with the CRISPR/ Cas9 genome editing tool, in order to functionally validate its contribution to abamectin resistance. Finally, in order to facilitate functional characterization of certain cytochrome P450 momoxygenases, a class of enzymes that among other roles, contribute also, to xenobiotic detoxification in insects, we performed, functional expression of three P450s from Apis mellifera using the baculovirus expression system. For this purpose, we used different baculovirus combinations and co-infected Sf9 cells in order to co- express each honeybee P450 with P450 reductase (CPR) to generate a functional complex. We performed Western analysis to detect both P450 and CPR in membrane extracts of infected cells and also performed with CO spectrum and CPR activity assays that indicated that the membrane extracts contain are functional complexes that can be used for downstream assays.
Language English
Subject Baculovirus
Insect cell lines
Insecticide resistance
Ανθεκτικότητα σε εντομοκτόνα
Issue date 2019-11-29
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work
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