| Abstract |
Vector-borne diseases are threatening the global public health due to the deaths that they cause annually and result in enormous environmental, economical and social cost. Especially, malaria infectious disease is transmitted via the bites of infected female mosquitoes. Anopheles gambiae mosquitoes are concluded among the most serious vectors of malaria’s parasite P.falciparum (the most serious malarial pathogen) and characterized as the biggest killer worldwide.
More specifically, the goal of the present study was the characterization of An. gambiae CYP4G16/CYP4G17 and the study of the putative role of Membrane Steroid Binding Protein (MSBP) as a binding partner and regulator of Drosophila’s melanogaster CYP4G1. CYP4g genes are insect-specific with orthologs present across the Insecta, key evolutionary genes for the latter’s successful transition to terrestrial environment (Feyereisen 2006). Anopheles gambiae has two CYP4g genes, the CYP4g16 (three transcripts –RA,-RB,-RC, which encode the –PA isoform and the –RD transcript, which encodes the –PD isoform) and the CYP4g17 (one transcript that corresponds to one isoform). About the dmCYP4G1, it has been characterized as oxidative decarbonylase, catalyzing the terminal step in cuticular hydrocarbon biosynthetic pathway (Qiu et al. 2012). Cuticular hydrocarbons (CHs) serve as the first barrier of protection for insects, for instance, against insecticides. As mentioned above, CYP4gs genes may play an important role in cuticular hydrocarbon biosynthetic pathway. This putative function may contribute to reduced penetration resistance mechanism that can be achieved due to the enrichment of cuticle. According the aforementioned, it is obvious that the characterization and functional study of CYP4gs is crucial.
In this thesis, transcript, tissue-specific expression and proteomic analysis in conjuction with immunohistochemistry approaches were used for the characterization of An.gambiae CYP4Gs. An in vivo functional RNAi technique was also employed for the functional characterization of the putative role of the Membrane Steroid Binding Protein (MSBP) as a binding partner and regulator of dmCYP4G1 function.
Firstly, there has been effort to achieve the transcript analysis of CYP4g16 by sequencing cDNA clones which confirm the abundance of –RA and –RB. Also, tissue-specific analysis by RT-PCR identifies that CYP4g16 -RABC and -RD transcripts are mainly on abdomen walls, as well as, that the –RABC ones are expressed 100x more than –RD. The focus was on specific tissues, head and abdomen walls, which have been associated with the expression of the above genes (Balabanidou et al. 2013) (Ingham et al. 2014). MS-analysis of Anopheles gambiae CYP4G16 on dissected head and abdomen wall tissues from adult female mosquitoes confirm the presence of CYP4G16-PA isoform at both tissues. The immunolocalization experiments on 4th instar larvae of An.gambiae indicate the presence of both CYP4Gs together with CPR on larval oenocytes and newly emerged adult oenocytes. Additionally, inmmunolocalization analysis (supplemental to previous work by Balabanidou) on the adult abdomen wall shows the co-localization of CYP4Gs on oenocytes along with the presence of CPR. The proposed localization of the two variants of Anopheles gambiae, CYP4G16 and CYP4G17, on the oenocytes supports the belief that these proteins play a potential role in CH biosynthesis.
Secondly, at Drosophila melanogaster model organism, there has been an effort to study the regulation of CYP4G1 function. Specifically, based on tissue-specific expression data (www.flyatlas.org) and experimental studies (Qiu et al. 2012) (Fujii-taira et al. 2009) (Hughes et al. 2007) there has been an attempt to identify whether the MSBP (CG9066) regulates the function of CYP4G1. Finally, phenotyping results of the effect of in vivo RNAi silencing of CYP4G1, CPR and Membrane Steroid Binding Protein (MSBP, CG9066), confirm that MSBP (CG9066) is not essential for the regulation of CYP4G1 function.
The abovementioned study can produce fundamental data about the characterization (and putative regulation) of CYP4Gs sub-family P450s that could be useful for the orientation to new targets, as far as insect control is concerned.
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