The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects

A Comprehensive Analysis of the Chorion Locus in Silkmoth

Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig, and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as “middle”, and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization, and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins

Construction, Complete Sequence, and Annotation of a BAC Contig Covering the Silkworm Chorion Locus

The silkmoth chorion was studied extensively by F.C. Kafatos’ group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties

The role of independent authorities in the implementation of EU Competition Law

Η παρούσα διπλωματική εργασία επιχειρεί να προσεγγίσει τον ρόλο που επιφυλάσσει το ενωσιακό δίκαιο στις εθνικές ανεξάρτητες αρχές προς το σκοπό της αποτελεσματικής και ομοιόμορφης εφαρμογής των κανόνων ανταγωνισμού της Ε.Ε. Η προσέγγιση αυτή πραγματοποιείται μέσα από μια διμερή οπτική: στο πρώτο μέρος εξετάζεται ο ρόλος των εθνικών ανεξάρτητων αρχών ως θεσμικών εγγυητών της ορθής εφαρμογής του ενωσιακού δικαίου ανταγωνισμού, ενώ στο δεύτερο μέρος, εξετάζονται τα καθ'εαυτό εργαλεία που διαθέτουν στο "οπλοστάσιό" τους οι εθνικές ανεξάρτητες αρχές προκειμένου να επιτελέσουν την αποστολή τους.The present master thesis attemps to approach the role that EU law grants to national independent authorities in order to achieve effective and uniform implementation of EU competition rules. Our approach is achieved through a two-fold analysis: on the first part we examine the role of independent authorities as institutional guarantors for the implementation of EU competition law, whereas on the second part we examine the per se tools that independent authorities are equipped with in order to fullfill their mission

Positive allosteric modulation of insect olfactory receptor function by ORco agonists

Insect olfactory receptors (ORs) are heteromeric ligand-gated cation channels composed of a common olfactory receptor subunit (ORco) and a variable subunit (ORx) of as yet unknown structures and undetermined stoichiometries. In this study, we examined the allosteric modulation exerted on Anopheles gambiae heteromeric ORx/ORco olfactory receptors in vitro by a specific class of ORco agonists (OAs) comprising ORcoRAM2 and VUAA1. High OA concentrations produced stronger functional responses in cells expressing heteromeric receptor channels relative to cells expressing ORco alone. These OA-induced responses of ORx/ORco channels were also notably much stronger than those obtained upon administration of ORx-specific ligands to the same receptors. Most importantly, small concentrations of OAs were found to act as strong potentiators of ORx/ORco function, increasing dramatically both the efficacy and potency of ORx-specific odorants. These results suggest that insect heteromeric ORs are highly dynamic complexes adopting different conformations that change in a concerted fashion as a result of the interplay between the subunits of the oligomeric assemblies, and that allosteric modulation may constitute an important element in the modulation and fining tuning of olfactory reception function

Μελέτη της βιολογικής λειτουργίας επιλεγμένων πρωτεϊνών του ιού της ηπατίτιδας C

Hepatitis C is a major public health problem worldwide, and a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is a single stranded, positive-sense RNA virus that belongs to the family Flaviviridae. HCV has not been definitively visualized and its structure remains to be elucidated. However, it is known that HCV circulates in various forms in the infected host. Although HCV is an enveloped virus, previous observations have suggested that nucleocapsids without a lipid envelope circulate in the serum of HCV-infected patients. However little is known about the nature, the origin and the properties of these naked particles. In addition, defective HCV genomes lacking envelope-coding sequences have recently been identified. Aside from its role in the nucleocapsid formation, HCV core has been shown to be a multifunctional protein and to affect many host cell functions, such as lipid metabolism, apoptosis, gene transcription and signaling pathways. Furthermore, core has been associated with the induction of steatosis and hepatocellular carcinoma, and may also have an immunoregulatory role. Importantly, until recently it was not possible to culture HCV efficiently in vitro. The aim of the present study was the generation of reagents for the study of the biological function of the HCV core protein, the production and purification of recombinant HCV capsids, and the elucidation of the biological significance (functional properties) of the naked capsids. Initially, we focused on the construction , preparation and characterization of reagents, to be used for the expression and study of the biological function of core. These reagents include a specific polyclonal antibody, expression plasmids, a stable inducible cell line, as well as viral vectors, based on the herpes virus and the baculovirus. Use of these reagents has permitted the efficient expression and detection of the HCV core protein in bacteria, insect and mammalian cells (both cell lines and primary culture). We next investigated, by biochemical analysis and electron microscopy, whether these expression systems (mainly the viral vectors) can support the auto-assembly of core protein, in the absence of other viral proteins. Furthermore, using the recombinant herpes virus, the effect of core in the ERK signa transduction pathway was studied. Using the baculovirus expression system, we showed that the HCV core protein is very efficiently expressed in insect cells, and that it can sufficiently direct the formation of capsid-like particles, in the absence of other HCV proteins and of the 5’ untranslated genome. These particles could be purified from cell extracts, and were characterized. Finally, we examined whether recombinant capsid-like particles, produced in insect cells, could bind and enter into cells. This is the first report showing that recombinant non-enveloped capsids are competent for binding and entry into cells of hepatic origin. Moreover, it was shown that the capsid-like particles and the antibody may enter the cells as an immune complex. Binding of naked core particles was also shown to modulate the Ras/ERK pathway.Η ηπατίτιδα C αποτελεί παγκόσµιο πρόβληµα δηµόσιας υγείας. Η λοίµωξη από τον ιό της ηπατίτιδας C (HCV) αποτελεί κύρια αιτία χρόνιας ηπατίτιδας, κίρρωσης του ήπατος και ηπατοκυτταρικού καρκινώµατος. Ο HCV ανήκει στην οικογένεια Flaviviridae. Το γονιδίωµα του ιού είναι ένα µονόκλωνο µόριο RNA θετικής πολικότητας. Το ιικό σωµατίδιο δεν έχει ακόµη παρατηρηθεί οριστικά στο ηλεκτρονικό µικροσκόπιο, και η δοµή του δεν έχει διαλευκανθεί πλήρως. Εντούτοις, είναι γνωστό ότι ο ιός κυκλοφορεί σε διάφορες µορφές στον ξενιστή. Αν και πρόκειται για ιό που διαθέτει έλυτρο, έχει αναφερθεί και η ύπαρξη καψιδίων που δεν διαθέτουν φάκελο. Παρ’ όλα αυτά, πολύ λίγα είναι γνωστά για την φύση, την προέλευση και τις ιδιότητες των γυµνών αυτών καψιδίων. Πρόσφατα αναγνωρίστηκε και η ύπαρξη ελλειπτικών γονιδιωµάτων του ιού, µε ελλείψεις στην περιοχή των γλυκοπρωτεϊνών του φακέλου. Υπεύθυνη για τον σχηµατισµό του νουκλεοκαψιδίου του ιού είναι η καψιδιακή πρωτεΐνη core. Φαίνεται όµως ότι πρόκειται για µία πολυλειτουργική πρωτεΐνη, που µπορεί να επιδρά σε πολλές λειτουργίες του κυττάρου - ξενιστή, όπως τον µεταβολισµό των λιπιδίων, την απόπτωση, την µεταγραφή γονιδίων και την µεταγωγή σήµατος. Επιπλέον, η πρωτεΐνη έχει συσχετισθεί µε την επαγωγή στεάτωσης και ηπατοκυτταρικού καρκίνου, και µπορεί να έχει και ανοσορρυθµιστικό ρόλο. Μέχρι πρόσφατα, δεν υπήρχε διαθέσιµο σύστηµα κυτταροκαλλιέργειας για τον αποτελεσµατικό πολλαπλασιασµό του ιού in vitro. Στόχος της παρούσης µελέτης υπήρξε η ανάπτυξη αντιδραστηρίων για τη µελέτη της βιολογικής λειτουργίας της καψιδιακής πρωτεΐνης του HCV, η παραγωγή και η αποµόνωση ανασυνδυασµένων καψιδίων του ιού, καθώς και η διερεύνηση της βιολογικής σηµασίας των γυµνών καψιδίων. Αρχικά το ενδιαφέρον µας επικεντρώθηκε στην κατασκευή, την ανάπτυξη και τον χαρακτηρισµό αντιδραστηρίων για την έκφραση και τη µελέτη της βιολογικής λειτουργίας της πρωτεΐνης core. Τα αντιδραστήρια αυτά περιλαµβάνουν ειδικό πολυκλωνικό αντίσωµα, πλασµιδιακούς φορείς έκφρασης, σταθερή επαγόµενη κυτταρική σειρά, που βασίζεται στο σύστηµα της εκδυσόνης, καθώς και ιικούς φορείς, που βασίζονται στον ιό του απλού έρπητα και τον βακουλοϊό. Τα αντιδραστήρια αυτά επέτρεψαν την αποτελεσµατική έκφραση και ανίχνευση της καψιδιακής πρωτεΐνης του HCV σε κύτταρα βακτηρίων, εντόµων και κυττάρων θηλαστικών (κυτταρικών σειρών, καθώς και κυττάρων πρωτογενούς καλλιέργειας). Ακολούθησε η διερεύνηση, µε βιοχηµικές µεθόδους και ηλεκτρονική µικροσκοπία, της δυνατότητας των συστηµάτων αυτών (κυρίως των ιικών φορέων) να υποστηρίξουν την αυτοσυγκρότηση της καψιδιακής πρωτεΐνης, απουσία των άλλων πρωτεϊνών του ιού. Επιπλέον, µε την χρήση του ανασυνδυασµένου ερπητοϊού, µελετήθηκε η επίδραση της core στο µονοπάτι µεταγωγής σήµατος των ERK κινασών. Ειδικά για το σύστηµα των ανασυνδυασµένων βακουλοϊών, δείχθηκε ότι η έκφραση της πρωτεΐνης είναι πολύ αποτελεσµατική στα κύτταρα εντόµων, και ότι επαρκεί, απουσία των υπόλοιπων ιικών πρωτεϊνών και της 5΄ µη µεταφραζόµενης περιοχής, για τον σχηµατισµό ανασυνδυασµένων καψιδίων, που αποµονώθηκαν από τα κυτταρικά εκχυλίσµατα και χαρακτηρίσθηκαν. Τέλος, διερευνήθηκε εάν τα ανασυνδασµένα καψίδια, που παρήχθησαν σε κύτταρα εντόµων µε τον ανασυνδυασµένο βακουλοϊό, µπορούν να προσδεθούν και να προσληφθούν από ηπατικά κύτταρα. Η παρούσα µελέτη αποτελεί την πρώτη αναφορά σχετικά µε την ιδιότητα των ανσυνδυασµένων γυµνών HCV καψιδίων να προσδένονται και να εισέρχονται σε κύτταρα ηπατικής προέλευσης. ∆είχθηκε επίσης ότι τα ανασυνδυασμένα καψίδια μπορούν να μπαίνουν στα κύτταρα ως ανοσοσύμπλεγμα με το ειδικό πολυκλωνικό αντίσωμα. Επιπροσθέτως, δείχθηκε ότι η πρόσδεση των γυμνών καψιδίων στα κύτταρα μπορεί να επιδράσει στο μονοπάτι Ras/ERK

Modulation of IL-2 expression after uptake of hepatitis C virus non-enveloped capsid-like particles: the role of p38 kinase

Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response

List of oligonucleotides used in PCR. Restriction sites are underlined; initiation and termination codons are in bold and italics, respectively.

<p>List of oligonucleotides used in PCR. Restriction sites are underlined; initiation and termination codons are in bold and italics, respectively.</p

Expression of <i>A. gambiae</i> OR1, OR2 and OR7 in insect cells.

<p>(<b>A</b>) Schematic representation of the basic backbone vector (pEIA) used for the heterologous expression of various forms (tagged and untagged) of ORs in lepidopteran cells. <i>hr3</i> enhancer, baculoviral (BmNPV) homologous region 3 enhancer sequence; pActin, <i>Bombyx mori</i> A3 cytoplasmic actin promoter; MCS, multiple cloning site; actin pA, 3′untranslated region of <i>B. mori</i> actin gene containing polyadenylation signals; IE1 cassette, baculoviral (BmNPV) DNA fragment containing the <i>ie-1</i> transactivator gene under the control of its native viral promoter; OR; <i>A. gambiae</i> odorant receptor ORF; Myc, Flag and MycHis, epitope tags. (<b>B</b>) Detection of heterologous expression of C-terminally MycHis-tagged ORs in transfected Hi5 cells using Myc monoclonal antibody. (<b>C</b>) Detailed western blot analysis of OR2. Hi5 cells were transfected with plasmids expressing different versions of OR2, and lysates were analyzed using a specific polyclonal antibody against OR2 (left panel, lanes 1–5) or monoclonal antibodies against the Myc (middle panel, lanes 6–9) or the Flag epitope (right panel, lanes 10–11). Arrowheads and arrows indicate major bands corresponding to monomers and putative dimers, respectively. (<b>D</b>) Detailed western blot analysis of OR1. Hi5 cells were transfected with plasmids expressing different versions of OR1, and lysates were analyzed using monoclonal antibodies against the Myc (middle panel, lanes 1–4) or the Flag epitope (right panel, lanes 5–6). In the left panel immunoreactivity of the specific polyclonal antibody against OR1 is shown, with lysates from cells expressing mycOR1 after treatment with the proteasome inhibitor MG132. Molecular weight markers are shown on the left. (<b>E</b>) and (<b>F</b>) Effect of coexpression of OR7 on the expression levels of OR1 and OR2. Hi5 cells were transfected with constant amounts (45% of total DNA) of Myc-tagged OR1 or OR2, along with equal amounts of Flag-tagged OR7 or empty vector (pEIA), and pEIA-GFP (10% of total DNA, for evaluation of the efficiency of transfection). Whole cell lysates (<b>E</b>) and membrane fractions (<b>F</b>) were analysed by SDS-PAGE and western blot. Detection of OR1, OR2 and OR7 was done using the anti-Myc and anti-Flag antibodies either consecutively (in <b>E</b>) or simultaneously (in <b>F</b>). To control for loading, the whole lysate fractions were also probed with an anti-tubulin antibody.</p