| Abstract |
Μalaria is one of the major health problems in the world, leading to about 400,000,000 cases per year, of which 2,000,000 result to death. Anopheline mosquitoes transmit the Plasmodium parasites, which cause the disease. Because only little is known about the interactions between the insect vector and the parasite, the aim of this study was to isolate potential receptors that are involved in the invasion of the mosquito midgut wall by the parasite. A PART : Isolation, characterization and functional analysis of the An.gambiae integrin β-subunit Initially the gene that codes for the β integrin subunit of the malaria vector An.gambiae (ΙΝΤΒ) was cloned. The mosquito and the Drosophila (βPS) orthologues share extensive structural similarities through-out the entire molecule. The most recognizable ones are the I/A domains which are responsible for the interaction of the receptor with the ligand, and the EGF-like repeats, known for their involvement in protein-protein interactions. Expression profile determination suggested that INTB is active during all developmental stages and in distinct body parts, including the head, the thorax, the midgut and the abdomen. The modulation of β integrin expression in different time points after blood meal ingestion, was also studied. No dramatic changes were observed, except at 48 hours after bloodfeeding when a threefold increase in transcript concentration was noted. Using the β-integrin antibody we produced, the protein was detected at the basolateral membranes of the midgut epithelial cells. The study of subcellular localization of the protein in mosquito midgut 24 hours after bloodfeeding showed the redistribution of integrin, possibly due to endocytosis processes. Also, after extensive analysis of the protein localization in guts dissected from mosquitoes killed at different time points after feeding with blood infected with P.berghei, it was shown that it covers the surface of both ookinetes and young oocysts, and it was also found at high concentrations in the vicinity of the oocysts later in development. Due to these observations we proceeded to a detailed analysis of the interactions, on the molecular level, of β integrin with Plasmodium surface proteins which are expressed in different developmental stages of the parasite. Using the in vitro Yeast Two-Hybrid System, no such interactions were detected. B PART : Functional analysis of the An.gambiae extracellular matrix protein Laminin Several recent reports have implicated the involvement of the basal lamina surrounding the midgut in the binding of the Plasmodium ookinete. Whether this extracellular structure acts as a mere physical barrier that blocks parasite migration, also masking the parasite from the insects immune system, or whether specific developmental cues are provided through the intimate contact, which eventually lead to the induction of the transformation into an oocyst, is unknown. Initially, the subcellular localization of the mosquito Laminin protein, the main component of basal lamina was studied. In immunofluorescence microscopy experiments using infected mosquito midgets, laminin was detected covering the ookinete surface. Moreover it coated the mature oocysts protruding towards the haemolymph. Extensive analysis using Yeast Two-Hybrid System was undertaken in order to elucidate the molecular interactions of mosquito laminin with parasite surface proteins. It was shown that a subdomain (V) of γ1 laminin subunit strongly interacts with the ookinete surface protein CTRP (Circumsporozoite- and Thrombospondin-related adhesive protein [TRAP]-related protein).
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Απομόνωση, χαρακτηρισμός και λειουργική ανάλυση γονιδίων του κουνουπιού An.gambiae ως υποψήφιων υποδοχέων για τη μετάδοση του παρασίτου P.berghei
