Dissertations / Theses on the topic 'Odorant Binding Proteins'

This PhD project aimed to investigate the possibility of using Odorant Binding Proteins (OBPs) as sensing layers of chemical sensors, for the detection of organic compounds in both vapour and liquid phases. OBPs are small soluble proteins present in high concentrations in the olfactory system of vertebrates and insects. OBPs are attractive in the biosensor field since they can bind odorants and pheromones in a reversible way. They are resistant to high temperatures and protease activity and they can be easily expressed in large amounts. OBPs belonging to different species of mammals and insects were utilised for developing biosensors relied on different transduction mechanisms. Recombinant OBPs were grafted on the gold electrode of transducers by using Self-assembled monolayers (SAMs) of alkanethiols. The efficiency of the immobilisation method was proved by using electrochemical techniques. Quartz crystal microbalances (QCMs), screen-printed electrodes (SPEs) and interdigitated electrodes (IDEs) were employed for developing three types of OBP-based biosensors. I. QCMs functionalised with OBPs were tested against pheromones (i.e. bombykol and bombykal) and volatile compounds found in foodstuffs (i.e. pyrazine derivatives and geosmin) in vapour phase. The QCM based biosensors showed a good degree of selectivity and a detection limit of the order of parts per billion, in air. II. In liquid phase, impedimetric biosensors based on SPEs also showed a good selectivity and sensitivity being able to detect analyte concentrations of the order of 10-9 M. III. OBPs immobilised on the gold electrodes of IDEs were instead tested against S-(+) carvone vapour, proving that the binding activity of the proteins was preserved in vapour phase and can be quantified as variation of capacitance. The developed OBP biosensors showed good selectivity, sensitivity and stability over time in both liquid and vapour phase. The responses of the sensors were reversible, allowing to the device to be used several times. Moreover, the biosensors were label-free, hence the interaction between OBPs and ligand was directly detected without using auxiliary probes/species. With these findings, we envisage the use of our biosensors in several applications, including monitoring of the quality of food along the transportation and storage, controlling of pests and useful insects in agriculture, or as analytical devices for studying the dynamics in binding processes.

La perception des signaux chimiques de l’environnement est un processus nécessaire aux interactions sociales entre les animaux. La Drosophile détecte les molécules odorantes et sapides grâce à ses systèmes gustatif et olfactif impliquant plusieurs familles multigèniques de chimiorécepteurs. Ainsi, ces composés chimiques pénétrant dans l'organe sensoriel (sensille) doivent être solubilisés avant d'être transportés à travers la lymphe sensillaire hydrophile baignant les dendrites des neurones chimiosensoriels. Ces événements périrecepteurs font intervenir plusieurs familles de protéines solubles parmi lesquelles se trouvent les odorant-binding proteins (OBPs). Si les OBPs ont été initialement identifiées dans les sensilles olfactives, certaines sont également exprimées dans les sensilles gustatives. La fonction physiologique des OBPs est encore peu connue mais certaines études révèlent que ces protéines agissent comme transporteurs de molécules lipophiles. Les affinités relativement faibles des OBPs pour les odorants ainsi que leur abondance dans la lymphe sensillaire suggèrent que ces protéines peuvent se lier, solubiliser et transporter des molécules hydrophobes jusqu’aux chimiorécepteurs en traversant la lymphe sensillaire hydrophile. De nouveaux rôles ont été attribués aux OBPs, et en particulier leur capacité à «tamponner» des changements soudains de concentrations d'odorants et leur implication dans la détection de l’humidité. Récemment, l’OBP49a exprimée dans les sensilles gustatives, a été montrée comme étant impliquée dans la détection de certains composés amers. Comme le rôle pérircepteur des OBPs reste encore très peu compris, l'objectif de mon projet de thèse a consisté à clarifier l'implication de certaines OBPs dans l'odorat et le goût chez Drosophila melanogaster. Ma thèse a d’abord consisté à mesurer le rôle des OBPs dans la perception des composés alimentaires chez les adultes D. melanogaster. Les OBPs exprimées dans les appendices gustatifs ont été identifiées par q-PCR et produites en utilisant un système d'expression hétérologue, la levure. Les propriétés de liaison des OBPs recombinantes purifiées ont ensuite été testées pour leur capacité à lier de nombreux ligands potentiels. L’OBP19b est capable de lier certains acides aminés. La cartographie des sensilles et des cellules exprimant l’OBP19b révèle que cette protéine est uniquement exprimée dans certaines cellules accessoires de sensilles précises du labellum. L’OBP19b a été aussi localisée dans le tube digestif et dans certains organes reproducteurs. La comparaison des réponses comportementales et électrophysiologiques sensillaires des mouches témoins et des mouches transgéniques a confirmé que l’OBP19b est impliquée dans la détection de certains acides aminés. De plus, la comparaison des séquences protéique a révélé sa relativement haute conservation au sein des espèces de Drosophilidae et même entre Diptères, ce qui suggère qu’elle joue un rôle crucial vis-à-vis de la recherche de nutriments chez ce groupe d’espèces. J’ai ensuite étudié le rôle de l’OBP28a dans l’olfaction. Cette OBP, l’une des plus abondante dans les antennes de Drosophile, a été montrée importante pour tamponner les variations soudaines de concentrations d'odorants. Des études structurales, génétiques, biochimiques, comportementales et électrophysiologiques ont été réalisées en collaboration avec les membres de l’équipe. L’OBP28a a d'abord été exprimée puis purifiée et sa structure 3D a été résolue. L'étude de ses propriétés de liaison a révélé la capacité de l'OBP28a à se lier à des composés floraux tels que la β-ionone. Les mesures comportementales et électrophysiologiques ont confirmé son rôle physiologique dans la détection de la β-ionone. En conclusion, ma thèse de doctorat met en évidence les rôles nouveaux de deux OBPs dans la chimioréception: l’OBP28a est impliquée dans le détection de molécules florales alors que l’OBP19b est nécessaire pour détecter certains acides aminés
Chemoperception is used by animals to detect nutritive food and avoid toxic compounds. It also allows animals to identify suitable ecological niche and mating partners. Like many other insects, Drosophila melanogaster possesses a very sensitive chemosensory ability and can detect and discriminate a wide panel of semiochemicals. Chemosensory detection is mostly mediated by olfactory and gustatory systems involving several multigene chemoreceptor families. Volatile and non-volatile chemical compounds entering the sensory organ (sensillum) must be solubilized before being transported through the hydrophilic sensillum lymph bathing the dendrites of chemosensory neurons. These perireceptor events involve a family of soluble proteins named odorant-binding proteins (OBPs). Despite the fact that OBPs were initially found in olfactory sensilla, some OBPs are also expressed in gustatory sensilla. While their physiological roles in olfaction and gustation remain unclear, many studies suggest that OBPs transport lipophilic chemicals. The relatively low affinity of OBPs for odorants and their high abundance in the sensillum lymph both suggest that OBPs can bind, solubilize and transport hydrophobic stimuli to the chemoreceptors across the aqueous sensilla lymph. In addition to this broadly accepted “transporter role” hypothesis, OBPs have also been proposed to buffer sudden changes in odorant levels and to be involved in hygroreception. The role of OBP49a was recently shown in taste: this OBP, expressed in the gustatory system, is required to detect some bitter compounds. However, the role of OBPs in perireceptor events remains largely unknown. The main goal of my thesis project consisted to investigate the involvement of OBPs in the smell and taste sensory modalities using a multi-faceted approach in Drosophila melanogaster.My first research axis consisted to better understand the role of OBPs in the perception of food compounds by using both in vitro and in vivo approaches of OBPs expressed in the gustatory appendages of D. melanogaster adults. After identifying by q-PCR the OBPs expressed in gustatory appendages, we produced them using a heterologous yeast expression system. Then, the binding properties of the recombinant purified OBP were investigated. Our binding assay screen revealed that the taste-expressed OBP19b is able to bind some amino acids. The expression of OBP19b was mapped in specific accessory cells in a subset of proboscis sensilla. This OBP was also expressed in the digestive tract and in some internal reproductive organs. The comparison of behavioural and single-taste sensilla responses between transgenic variants and control flies supported our finding that OBP19b is indeed involved in the detection of some amino acids. Finally, the comparison between various dipteran insects of the OBP19b-like protein coding sequence indicates the relatively high conservation of this protein suggesting its critical role in food search.The second research axis of my PhD thesis focused on the olfactory role of OBP28a. OBP28a was previously shown to be highly expressed in the Drosophila antennae and proposed to buffer quantitative odour variations. To better understand the physiological role of this OBP, and in collaboration with different members of the team, we used structural, genetic, biochemical, behavioural and electrophysiological methods to better understand the role of this OBP. OBP28a was first heterologously expressed and purified. The folding of OBP28a was then determined and the protein was crystallized. The study of the binding properties of OBP28a revealed that it can bind floral compounds such as β-ionone. Behavioural and electrophysiological recordings supported the physiological role of OBP28a in β-ionone detection. In summary, this PhD thesis reveals novel roles of two OBPs in perireceptor chemoreception: OBP28a in the detection of floral compounds and OBP19b in the detection of some amino acids

Like many other organisms, insects use chemical stimuli to regulate behaviours including feeding, egg-laying, and mating. Odorant binding proteins (OBPs) are one of the crucial components of insect chemosensory system, which play an essential role in transporting the hydrophobic volatile odorant molecules to the olfactory receptors. Most of insect OBPs were investigated by using recombinant technology in bacterial cells due to its fast, cost-effective, and high production mechanism. However, one of the major concerns of bacterial expression system is that the protein is frequently expressed in an unfolded state in inclusion bodies (IBs), which requires further in-vitro protein refolding step to make the protein biologically active. While doing this, there are always high chances of protein misfolding which results in soluble or insoluble protein aggregation. Thus, it is highly important to confirm the efficiency of each refolding method, used for OBP refolding, in terms of getting the correctly folded structure of the target protein. Unfortunately, it was neglected in many previous studies, resulting in significant doubts on various functional studies of insect OBPs. In this study, I used three Helicoverpa armigera OBPs, HarmOBP2, HarmOBP5, and HarmGOBP2, as model proteins to compare the different protein refolding strategies in producing correctly folded recombinant OBPs. Along with that, I have developed a novel pH-dependent method of protein refolding which demonstrated as a more efficient and productive approach for selected HarmOBPs’ refolding compared to other used methods. Further, I also developed a novel reverse chemical ecology method to isolate and identify the candidate natural ligands from host plants for HarmOBPs. This study points out a crucial but largely ignored step of insect OBP research, protein refolding and the loopholes associated with it in previous studies, which will improve our understanding of insect chemosensation and help develop more efficient and environmentally friendly insect control strategies.

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of G-protein-coupled olfactory receptors (ORs) and olfactory carriers. Two such carrier gene families are found in insects: the odorant binding proteins (OBPs) and the chemosensory proteins (CSPs). In olfactory sensilla, OBPs and CSPs are believed to deliver hydrophobic air-borne molecules to ORs, but their expression in non-olfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. ¶ Bioinformatics and experimental approaches were used to characterise the OBP and CSP gene families in a highly social insect, the western honey bee (Apis mellifera). Comparison with other insects reveals that the honey bee has the smallest set of these genes, consisting of only 21 OBPs and 6 CSPs. These numbers stand in stark contrast to the 66 OBPs and 7 CSPs in the mosquito Anopheles gambiae and the 46 OBPs and 20 CSPs in the beetle Tribolium castaneum. The genes belonging to both families are often organised in clusters, and evolve by lineage specic expansions. Positive selection has been found to play a role in generating a greater sequence diversication in the OBP family in contrast to the CSP gene family that is more conserved, especially in the binding pocket. Expression proling under a wide range of conditions shows that, in the honey, bee only a minority of these genes are antenna-specic. The remaining genes are expressed either ubiquitously, or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs and CSPs are not restricted to olfaction, and are likely to be involved in broader physiological functions. ¶ Finally, the detailed expression study and the functional characterization of a member of the CSP family, uth (unable-to-hatch), is reported. This gene is expressed in a maternal-zygotic fashion, and is restricted to the egg and embryo. Blocking the zygotic expression of uth with double-stranded RNA causes abnormalities in all body parts where this gene is highly expressed. The treated embryos are `unable-to-hatch' and cannot progress to the larval stages. Our ndings reveal a novel, essential role for this gene family and suggest that uth is an ectodermal gene involved in embryonic cuticle formation.

L'olfaction est un des modes sensoriels predominant chez les lepidopteres crepusculaires et nocturnes, et ces animaux s'averent un modele de choix pour l'etude des mecanismes moleculaires de la reception des signaux olfactifs et de leur transduction vers des niveaux superieurs d'integration. Depuis leur decouverte dans le systeme olfactif peripherique des insectes et des vertebres, le degre d'implication des odorant-binding proteins (obp) dans les processus de discrimination des odeurs n'est toujours pas elucide. Dans ce cadre, nous avons entrepris l'etude de telles proteines chez la noctuelle du chou mamestra brassicae l. , ravageur des cultures dont le systeme de communication chimique est bien connu. L'utilisation combinee de differentes techniques biochimiques et immunologiques nous a permis de caracteriser et de purifier plusieurs proteines de type pbp (pheromone-binding protein) et gobp (general odorant-binding protein) dans les antennes des males et des femelles. Cette microdiversite est en faveur d'une participation active de ces proteines dans le tri des molecules odorantes. L'etude fonctionnelle de ces proteines a ete effectuee, en utilisant des proteines purifiees et le compose majoritaire de la pheromone sexuelle femelle tritie. Ce compose est fixe de facon specifique par la proteine pbp qui est la plus abondante dans les extraits antennaires males, et il peut egalement etre fixe par les pbp de la femelle. Ces resultats montrent la specificite de l'interaction proteine-compose pheromonal et confortent l'hypothese selon laquelle seul le complexe specifique entre une proteine et son ligand serait capable d'activer le recepteur olfactif. L'utilisation de la biologie moleculaire a permis de cloner les genes codant differentes pbp et une gobp chez le male, et il a ete possible de correler les proprietes fonctionnelles de ces differentes proteines avec des differences dans leur structure primaire. Notamment, des variations localisees dans le site potentiel de fixation des molecules odorantes pourraient expliquer l'affinite opposee de deux des pbp du male envers le compose pheromonal majoritaire. L'etude de la structure et de l'expression des genes correspondant a ete initiee et l'expression preliminaire d'une proteine de type gobp recombinante a ete effectuee dans un systeme procaryote. L'objectif vise par la production de proteines recombinantes est d'en etudier la structure tridimensionnelle, encore inconnue chez les insectes. L'ensemble de ces resultats apportent une meilleure comprehension des mecanismes moleculaires du codage des odeurs pheromonales chez m. Brassicae. Ils permettent d'attribuer aux pbp un role de filtre vis-a-vis des molecules odorantes, et dans l'hypothese de l'activation du recepteur par le complexe pbp-pheromone, un role protecteur a l'egard du recepteur olfactif.

The role of odorant binding proteins in the olfaction of mosquitoes, the primary mechanism of human host recognition, has been an important focus of biological research in the field of infectious disease transmission by these insects. This thesis provides an in depth knowledge of these proteins in three mosquito species Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. A large scale analysis on these genomes has been carried out towards the identification of the odorant binding proteins in the mosquito genomes. Identification of many new OBP members, in particular in the Aedes aegypti and Culex quinquefasciatus species, and an extensive phylogenetic analysis presenting a novel classification of the OBP subfamilies of these mosquito species has been proposed. This results further demonstrates the extraordinary multiplicity and diversity of the OBP gene repertoire in these three mosquito genomes and highlights the striking sequence features that are nevertheless highly conserved across all mosquito OBPs. Owing to the availability of homologous structures from mosquitoes or related species, the 3D structure modelling of all the Classic OBPs from the three genomes (representing in total 137 structures) has been performed. This was completed by large scale docking studies on these structures by screening a large set of compounds that are known to be mosquito attractants or repellents. These provide many exciting new insights into the structural and functional aspects towards understanding the efficacy of some repellents and of some attractants from human emanations. Through molecular dynamics simulation, the structural changes observed in an OBP bounded to an odorant when pH conditions are modified were characterized and the probable mechanism of ligand binding and release is presented. This work provides the first insights to many of the long awaited questions on the genomic, structural and functional characterization of mosquito OBPs and can be viewed as a reliable starting point for further experimental research focussed on these aspects.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
This dissertation is divided into three chapters. In the first chapter, we provide a concise literature review that discusses key theoretical concepts, the rationale, and main objectives outlined for this study. The second chapter investigates the molecular evolution of the gene family of odor receptors (ORs) identified in the transcriptomes of two species of fruit flies of great economic importance: Anastrepha fraterculus and A. obliqua. The results showed a high percentage of average identities between ORs from these species, as well as recent gene expansions with signs of positive selection. A comparison of rates of synonymous and nonsynonymous substitutions among Anastrepha species detected evidence of positive selection in the gene Or7c, which is associated to an important potential role in aggregation behavior and host choice for oviposition in D. melanogaster. The third chapter investigates patterns of molecular evolution in pheromone binding proteins (PBPs), also identified in A. fraterculus and A. obliqua, as well as studied pattern of polymorphisms, divergence and populational structure of four chemosensory genes amplified in four species of tephritid flies of fraterculus group: A. fraterculus, A. obliqua, A. sororcula and A. turpiniae. This study contrasted previously identified genes with evidence of positive and purifying selection in order to investigate whether they are contributing to the differentiation among some of the species of this group. We found no evidence of positive selection in PBPs studied in a more global comparison, although we found positive selection signals in some of the genes and studied strains. Population analysis of chemosensory genes in different species of Anastrepha detected high levels of intraspecific nucleotide and haplotype diversity. Divergence tests showed that A. obliqua is the most different species of the ones here investigated, having, in general, high levels of nucleotide substitutions, non-synonymous divergence, as well as fixed species specific differences, whereas we failed to find similar differences amongst the other species here studied. The genes Obp28a, Or7c and Or7d were differentiated in A. obliqua, indicating a potential role in the differentiation of other species in the group, or in this species’ diversification and adaptation.
A presente dissertação encontra-se dividida em três capítulos. O primeiro capítulo apresenta uma concisa revisão bibliográfica que aborda os principais conceitos teóricos, a justificativa e os objetivos delineados para este estudo. O segundo capítulo apresenta um estudo da evolução molecular da família gênica dos receptores de odores (ORs) identificados nos transcriptomas de duas espécies de moscas-das-frutas de grande importância econômica: Anastrepha fraterculus e A.obliqua. Os resultados mostraram uma alta porcentagem de identidade média entre os ORs destas espécies, assim como expansões gênicas recentes com sinal de seleção positiva. Quando comparamos as taxas de substituições sinônimas e não-sinônimas entre as espécies de Anastrepha encontramos evidências de seleção positiva no gene Or7c, que está associado em D. melanogaster a um potencial importante papel nos comportamentos de agregação e escolha de frutos para oviposição. No terceiro capítulo apresentamos um estudo do padrão de evolução molecular dos genes que codificam para proteínas ligantes aos feromônios (PBPs), também identificados em A. fraterculus e A. obliqua, assim como também estudamos o padrão de polimorfismos, divergência e estrutura dos genes quimiossensoriais Obp28a, Obp84a, Or7c e Or7d os quais foram amplificados em quatro espécies de moscas-das-frutas do grupo fraterculus, A. fraterculus, A. obliqua, A. sororcula e A. turpiniae. Este estudo foi realizado contrastando genes identificados com sinais de seleção positiva e seleção purificadora com o intuito de investigar se eles estão contribuindo para a diferenciação entre algumas das espécies desse grupo. Não encontramos evidências de seleção positiva nas PBPs estudadas em uma comparação mais global, embora tenhamos encontrado sinais de seleção positiva em alguns dos genes e linhagens estudadas. A análise populacional de genes quimiossensoriais em diferentes espécies de Anastrepha detectou níveis altos de diversidade nucleotídica e haplotípica dentro das espécies. Os testes de divergência mostraram que a espécie A. obliqua é a espécie mais diferenciada, apresentando, em geral, altos níveis de substituições nucleotídicas, divergência não-sinônima, assim como diferenças fixadas quando comparada com as outras espécies. Os genes Obp28a, Or7c e Or7d mostraram-se diferenciados em A. obliqua, indicando um potencial papel na diferenciação desta espécie com respeito às outras espécies estudadas.

Recombinant bovine odorant binding protein (bOBP) is a very promising platform for building protein-based biosensors. The protein possesses a broad binding specificity for hydrophobic molecules with affinities in the sub-micromolar range. Previous work has shown non-covalent binding of 1-aminoanthracene (1-AMA) in the internal cavities of bOBP that results in a large enhancement of fluorescence intensity. We have shown fluorescence titrations of recombinant bOBP with 1-AMA yielded a single type of binding site with a Kd of 0.16 ± 0.023 μM. Competitive displacement assays between 1-AMA and other ligands such as thymol were established and the results indicated their binding to bOBP. The strategy of competitive binding with 1-AMA was thus employed to quantify thymol concentration for the bOBP biosensors. Ten different solid supports for the bOBP biosensors were examined for their biocompatibility with bOBP function using 1-AMA as a probe. The result was that nitrocellulose was chosen as the best membrane for immobilization, probably due to its 3-D micro-porous matrix (sponge structure) that provides a much larger surface area for protein binding compared with 2-D surfaces. It was found that the optimum operating concentrations of bOBP and 1-AMA and the method for the immobilization was incubation of nitrocellulose with the complex of 100μM bOBP and 100μM 1-AMA solution. The amounts of the total and functional protein binding to nitrocellulose were 7 ± 0.1 and 7 ± 0.4 nmol bOBP per cm2 of membrane, respectively. A fibre-optic biosensor based on bOBP has therefore been constructed. It has been an extrinsic sensor with bOBP immobilized on a nitrocellulose membrane placed at the tip of a probe of a bifurcated fibre-optic bundle that was in turn connected to the LLS-385 LED light source and the HR2000 spectrometer. The light emitted by fluorescent 1-AMA bound bOBP was detected by 2048-element CCD array of the spectrometer. The LODs for thymol in the liquid phase were found to be 14 ± 6 μM (calculated as S/N = 3), which is less than the guideline values considered to be toxic to humans. Moreover, this fibre-optic bOBP biosensor was also capable of sensing thymol vapour, and some potential uses of this sensor will be described.

The development of an inexpensive, portable, stable, sensitive and selective biosensor for detection of odorants is a daunting task. Here, we hypothesized the development of a detector layer composed of the protein groups; the olfactory receptors (ORs) and the odorant binding proteins (OBPs), known to bind odorants in animal sensing. We report the design of 13 OR gene-vector constructs, and their subsequent transformation into Escherichia (E.) coli (BL21 (DE3)-STAR-pLysS) strain. Moreover, we report the expression of several ORs into an in vitro wheat germ extract using three separate detergent mixes for protein solubilization.   In addition, we describe the design of an odorant binding protein from the Aenopheles gambiae mosquito PEST strain (OBP-PEST) gene-vector construct under an IPTG (Isopropyl β-D-1-thiogalactopyranoside) inducible promoter. OBP-PEST was heterologously expressed in E.coli with an 8 amino acid long sequence (WSPQFEK) attached C-terminally, via a thrombin cleavage site and a flexible linker (GSSG). The WSPQFEK sequence, commonly referred to as a Strep-tag, enabled subsequent affinity chromatography purification of the protein, via binding to an engineered Streptavidin equivalent. Surprisingly, the OBP-PEST was found to contain a signal sequence leading to its truncation and secretion when expressed in E.coli.   Biophysical analyses were established using Circular Dichroism (CD) for the analysis of two lipocalins: Beta-lactoglobulin (BLG) and OBP-PEST. We studied the solubility, refoldability and the conformational transitions of BLG, as a result of change in solvent, pH and temperature. The secondary structure of OBP-PEST and its thermal stability was investigated.   In conclusion, this thesis work has enabled biophysical analyses of OBP-PEST and future analogs of interest to the development of a stable protein detector layer. Although further experiments are needed to fully characterize the biophysical properties, and to find odorant substrates of OBP-PEST, it was found to be a suitable alternative to ORs in a biosensor detector layer application. More importantly, an inherent OBP-PEST signal sequence was found to mediate protein secretion when expressed heterologously in E.coli. To the best of our knowledge this is the first lipocalin discovered to be secreted upon heterologous expression in E.coli.   We hypothesize that this signal peptide could be used as a means for targeted secretion and, hence, efficient protein purification.

Thesis (Ph.D. in Biochemistry, Biomolecular Structure Program) -- University of Colorado Denver, 2007.
Typescript. Includes bibliographical references (leaves 135-146). Online version available via ProQuest Digital Dissertations.

Insect Odorant-Binding Proteins (OBPs) are small, water-soluble molecules that solubilize hydrophobic odorant molecules in the sensillum lymph and transport them to their cognate receptors in the olfactory receptor neurons. With the availability of the genome sequence of the African malaria mosquito, Anopheles gambiae, there has been a profound interest in the characterization and functional analyses of Obp genes in order to understand the molecular basis of mosquito host-seeking behavior. However, no direct evidence has been found for specific functions of any mosquito OBPs. In this study, I describe the comparative genomics and expression analyses on two mosquito Obp genes (Obp1 and Obp7) as well as efforts to determine their functions. Both of these Obp genes were identified in Anopheles stephensi and only Obp7 gene was identified in Anopheles quadriannulatus by screening bacterial artificial chromosome (BAC) libraries of these species. Comparative analyses revealed several interesting features including segments of conserved non-coding sequences (CNSs) that contain potential regulatory elements relevant to olfactory tissue development and blood-feeding. The expression profiles of these genes were examined in detail in the Asian malaria mosquito An. stephensi. Obp1 and Obp7 transcripts were significantly higher in females than male mosquitoes and they were predominantly found in the antenna, which is the primary olfactory organ of mosquitoes. Twenty-four hours after a blood meal, mRNA levels of these two genes were significantly reduced in the maxillary palp and proboscis, referred to as secondary olfactory organs of mosquitoes. These findings collectively indicate that Obp1 and Obp7 genes in An. stephensi likely function in female olfactory response and may be involved in behaviors related to blood-feeding. To investigate the function of these Obp genes more directly, a Sindbis virus based expression system is established to knockdown the two Obp gene orthologs in Aedes aegypti. The effective knockdown of Obp1 and Obp7 genes (8 and 100-fold, respectively) is accomplished in female mosquito olfactory tissues. The potential for a systematic analysis of the molecular players involved in mosquito olfaction using this newly developed technique is discussed. Such analysis will provide the foundation for interfering with mosquito host-seeking behavior for the prevention of disease transmission.
Ph. D.

La détection de molécules odorantes et de composés organiques volatils (COVs) fait l'objet d'une demande croissante dans divers domaines tels que l’industrie alimentaire, la parfumerie, le diagnostic médical, la surveillance environnementale, etc. Bien que précises et fiables, les méthodes les plus couramment employées - la chromatographie en phase gazeuse couplée à la spectrométrie de masse et les panels de nez humain ou encore des nez de chiens dressés- présentent un certain nombre d'inconvénients notamment en termes de coût et de temps. En réponse à ces contraintes, les nez électroniques (NEs) sont alors apparus comme des outils prometteurs pour l’analyse de COVs. Inspirés du nez biologique, ces dispositifs biomimétiques se composent généralement d'un ensemble de capteurs chimiques à réactivité croisée combinés à un système de reconnaissance des formes. Au cours des trois dernières décennies, les nez électroniques ont démontré leur potentiel significatif pour l'analyse des COV dans de nombreux domaines. Toutefois, l'une des principales faiblesses de la plupart des NEs existants est leur sélectivité limitée. Pour répondre à cette problématique, les efforts de recherche se sont multipliés au cours de la dernière décennie pour explorer l'utilisation de matériaux biologiques issu du système olfactif comme matériaux de détection afin d'améliorer les performances des NEs. Dans ce contexte, notre équipe au sein du laboratoire Systèmes Moléculaires et Nanomatériaux pour l'Energie et la Santé (SyMMES, UMR 5819), a conceptualisé un nez bioélectronique utilisant l’imagerie par résonance de plasmons de surface comme technique de transduction et employant de petits peptides comme matériaux de détection. Cette technologie a conduit à la création d'Aryballe, une société qui a réussi à miniaturiser et à commercialiser le dispositif. Ce projet de thèse fait partie du projet ANR OBP-Optinose (ANR-18-CE42-0012) et vise principalement à explorer le potentiel des protéines liant les odorants (OBPs) en tant que nouveaux matériaux de détection pour le développement de nez bioélectroniques.Au cours de la thèse, nous avons utilisé une combinaison d'OBPs de type sauvage et d’OBPs plus sélectives, qui ont été conçues et génétiquement modifiées pour avoir des propriétés de liaison spécifiques pour certains COVs cibles. Notre approche expérimentale consistait à étudier les divers paramètres susceptibles d’impacter les performances des biocapteurs à base d'OBP pour la détection de COVs en phase gazeuse. Ainsi, dans un premier temps, nous avons entrepris une caractérisation complète des couches d'OBP immobilisée en surface. Nous avons commencé par évaluer la stabilité des protéines en phase gazeuse, un élément crucial pour assurer leur activité. Ensuite, nous avons approfondi notre analyse en examinant la densité et l'orientation des OBPs après leur dépôt sur la surface d’or de la puce. Car ces facteurs peuvent impacter la sensibilité de notre système. Nous avons également examiné l’impact du glycérol sur les couches d’OBPs. En outre, nous avons réalisé des recherches approfondies sur le mécanisme d'hydratation des couches d'OBP qui nous ont permis de mieux comprendre comment les conditions d'humidité influencent la réactivité des biocapteurs. Enfin, nous avons démontré les bonnes performances du nez bioélectronique à base d’OBP en phase gazeuse en termes de sélectivité, stabilité et répétabilité
The detection of odorant molecules and volatile organic compounds (VOCs) is the subject of growing demand in various fields such as food industry, perfumery, medical diagnostics, environmental monitoring and so on. Although accurate and reliable, the most commonly used methods - gas chromatography coupled with mass spectrometry and panels of human noses or trained dogs- have a number of drawbacks, particularly in terms of cost and time. In response to these limitations, electronic noses (eNs) have emerged as promising tools for the analysis of VOCs. Inspired by the biological nose, these biomimetic devices generally consist of a set of cross-reactive chemical sensors combined with a pattern recognition system. Over the past three decades, eNs have demonstrated their great potential for VOC analysis in many areas. However, one of the main weaknesses of most existing eNs is their limited selectivity. In response to this problem, research efforts have multiplied over the last decade to explore the use of biological materials from the olfactory system as sensing materials in order to improve the performance of eNs. In this context, our team at the Molecular Systems and Nanomaterials for Energy and Health laboratory (SyMMES, UMR 5819), has conceptualized a bioelectronic nose using surface plasmon resonance imaging as a transduction technique and employing small peptides as sensing materials. This technology led to the creation of Aryballe, a company that has successfully miniaturized and commercialized the device. This thesis project is a part of the ANR project OBP-Optinose (ANR-18-CE42-0012), which aims to explore the potential of odorant binding proteins (OBPs) as novel sensing materials for the development of bioelectronic noses.During the thesis, we used a combination of wild-type and more selective OBPs, which were designed and genetically modified to have specific binding properties for target VOCs. Our experimental approach was to study various parameters that could have an impact on the performance of OBP-based biosensors for the detection of VOCs in the gas phase. First, a complete characterization of the OBP layers after immobilization on surface was carried out. The stability of the proteins in the gas phase was assessed, which is crucial to ensure their activity. The density and orientation of the OBPs were also studied since they may have impact on the sensitivity of the system. In addition, the impact of glycerol and humidity on the OBP layers was investigated. In particular, in-depth research into the hydration mechanism of the OBP layers was carried out, which enabled us to gain a better understanding of how humidity influences the reactivity of the biosensors. Finally, we demonstrated the good performance of OBP-based bioelectronic nose in the gas phase in terms of selectivity, stability, and repeatability

Les OBP sont des petites protéines solubles qui se lient avec des molécules odorantes et phéromonales. Le rôle des OBPs n’est pas complètement compris. Une hypothèse suggère que l’OBP solubilise et transporte les ligands aux récepteurs olfactifs et la liaison entre les molécules odorantes et l’OBP est non spécifique. Une autre hypothèse suggère que le complexe formé est une liaison spécifique entre une molécule odorante donnée et une OBP spécifique. Ce travail de thèse montre que les OBPs sont impliquées dans la première étape de la discrimination des odeurs. Dans un premier temps, nous avons montré l’implication de la Phe35 et la Tyr 82 dans la sortie du ligand par l’OBP. Dans un second temps, nous avons mis en évidence la présence de différentes isoformes d’OBP et de VEG qui diffèrent par les modifications post-traductionnelles (phosphorylation et GlcNAcylation) a la fois sur les protéines natives extraites de la muqueuse respiratoire et sur les protéines recombinantes produites par P.pastoris et CHO. Ces isoformes sont capables de discriminer des molécules odorantes et phéromonales. Les OBPs ne sont pas des transporteurs passifs car elles assurent un fin codage des molécules odorantes ou phéromonales avant l’interaction de ce complexe avec un récepteur spécifique
OBPs are small soluble proteins that bind with odorant molecules and pheromones. The role of OBP is not completely understood. A hypothesis suggests that OBP solubilize and transport the ligands to olfactory receptors and the binding between odorant molecule and OBP is unspecific. An other hypothesis suggest that the complex formed is the specific binding between a given odorant molecule and a specific OBP. This work of thesis show that OBP are involved in the first step of odorant discrimination. Initially, we have showed the involvement of the Phe35 and Tyr 82 in the uptake of ligands by OBP. Second, we have given rise to the presence of various isoform of OBP and VEG that differ by post-translational modifications (phosphorylation and GlcNAcylation) both on natives proteins extract of respiratory mucosa and on recombinants proteins produce by P. pastoris and CHO. These isoforms are able to discriminate of odorant molecules and pheromones. OBPs are not passives carriers because they ensure a fine coding of odorant molecules and pheromones before interaction of this complex with specific receptor

Rhipicephalus micropulus, conhecido como carrapato dos bovinos, é um dos parasitas mais importantes para a pecuária, pois causa enormes prejuízos ao produtor. Esses prejuízos se estendem desde a ação espoliativa que o carrapato exerce nos seus hospedeiros até a transmissão de uma diversidade de patógenos. Os carrapatos utilizam vários estímulos térmicos, sonoros, visuais, gustativos, táteis e olfativos na fase de busca pelo hospedeiro. Essas substâncias são conhecidas como semioquímicos e podem atuar em indivíduos de uma mesma espécie como os ferormônios ou entre indivíduos de espécies diferentes como os alomônios e cairomônios. Nos bovinos, já se sabe que uma de suas lipocalinas pode atrair insetos, porém não é sabido se essa atração ocorre para R. microplus. Já é sabido que o carrapato consegue distinguir odores liberados entre bovinos das raças Nelore e Holandês Preto e Branco (HPB), sendo mais atraído para bovinos da raça Holandês. A lipocalina por ser uma proteína globular e estar associada com o transporte de pequenas moléculas hidrofóbicas, tais como odorantes e esteroides, e pode favorecer a atração do R. microplus, uma vez que essa proteína bovina pode ligar a odorantes e desempenhar um papel fundamental na liberação de odores para o ambiente, que por sua vez pode atrair, ou não, o R. microplus. Existem diferentes níveis de infestação de carrapatos entre bovinos das raças Nelore e Holandês, bem como entre os sexos da mesma raça. Além disso, o período do ciclo de vida do bovino também pode influenciar na susceptibilidade a infestação, por exemplo, as vacas no período de lactação são consideradas mais susceptíveis ao parasito. Assim, o objetivo do presente estudo foi avaliar o papel da lipocalina bovina na resistência e suscetibilidade ao carrapato, através da quantificação dessa proteína em líquidos corporais dos bovinos, tais como soro, saliva, urina, suor, secreção nasal e biopsia de pele obtidos de bovinos que apresentam fenótipos contrastantes de infestações, a saber: hospedeiros macho (touros) e fêmea (vacas em lactação) das raças resistentes (Bos indicus) e suscetíveis (Bos taurus). Os resultados deste trabalho tem demonstrado que a lipocalina bovina está presente em todos os fluídos investigados, exceto a urina, como observado por Western blot. Com os resultados deste trabalho podemos concluir que a bcOBP apresenta 9 diferença significativa nos fluidos de saliva e secreção nasal de touros e vacas em lactação da raça HPB quando comparados com touros e vacas em lactação da raça Nelore. Em amostras de biópsia de pele também ocorreu uma maior marcação da bcOBP na raça susceptível (HPB), demonstrando que a bcOBP possivelmente esta auxiliando na susceptibilidade destes bovinos ao carrapato, através do transporte de um maior número de odorantes que estariam atraindo um maior número de carrapatos. Ao analisar os fluidos entre bovinos da mesma raça, porém de sexo diferente, observou-se uma maior quantidade de bcOBP em vacas no período de lactação HPB nos fluidos de saliva e secreção nasal, visto que as vacas estão no período de lactação e são mais susceptíveis a infestações. Consequentemente, a bcOBP poderia estar colaborando para a sua maior susceptibilidade quando comparadas com touros HPB, e o mesmo ocorreu para biopsia de pele. Porém no fluído soro o aumento foi significativo para touro HPB quando comparado às vacas em lactação da raça HPB. Acredita-se que esse fenômeno ocorra devido ao período de lactação nas fêmeas uma vez que a produção da lipocalina bovina esta intimamente relacionada com a produção do leite. Os resultados deste trabalho demonstram que possivelmente a bcOBP na raça HPB está carreando odorantes para o ambiente os quais estariam atraindo mais carrapatos, e auxiliando na susceptibilidade destes bovinos.
Rhipicephalus micropulus, known as bovine tick, is one of the most important parasites for livestock, as they cause enormous damage to the producer. These demages extend from the spoliation action that the tick exerts on the skin of its hosts until the transmission of a diversity of pathogens. Ticks use various thermal, sound, visual, gustatory, tactile and olfactory stimuli in the search phase to the host. These substances are known as semiochemicals and can act within individuals of the same species as the pheromones or among individuals of different species such as alomones and cairomones. In the cattle, it is known that the lipocalin protein may attract insects, but this attraction is not known to influence the R. microplus. It is known that the tick can distinguish released odors between Nelore and Holstein cattle, being more attracted to cattle of the Holstein breed. Lipocalin, being a globular protein and associated with the transport of small hydrophobic molecules, such as odorants and steroids, can favor the attraction of R. microplus, since this bovine protein can bind to odorants and play a key role in the release of odors into the environment, which in turn may or may not attract R. microplus. There are different levels of tick infestation among cattle of the Nelore and Holstein breeds, as well as between the sexes of the same breed. In addition, the life cycle period of the bovine can also influence the susceptibility to infestation. For example, cows in the lactation period are considered more susceptible to the parasite. Thus, the objective of the present study was to evaluate the role of bovine lipocalin in the resistance and susceptibility to tick by quantifying that protein in bovine body fluids such as serum, saliva and urine, sweat, nasal secretions and skin biopsies obtained from resistant (Bos indicus) and susceptible (Bos taurus) cattle breeds. The results of this work have demonstrated that bovine lipocalin is present in all investigated fluids except urine, as observed by Western Blot. With the results of this work we can conclude that bcOBP presents a significant difference in fluids saliva and nasal secretion of bulls and cows in lactation breed HPB when compared to bulls and lactating cows of the Nelore breed. In skin biopsy specimens, there was also a greater labeling of bcOBP in the susceptible strain (HPB), which could then aid in the susceptibility of these cattle to the 11 tick, by transporting a larger number of odorants that would be attracting a greater number of ticks. When analyzing the fluids between bovines of the same breed, but of different sex, a greater amount of bcOBP was observed in cows during the period of HPB lactation in the fluids saliva and nasal secretion, since the cows are in the lactation period and are more susceptible to infestations. Consequently, bcOBP could be contributing to its greater susceptibility when compared to HPB bulls, and the same was done for skin biopsy. However, in the fluid serum, the increase was significant for the bull when compared to the lactating cows of the HPB race. It is believed that this phenomenon occurs due to the lactation period in females since the production of bovine lipocalin is closely related to milk production. The results of this work demonstrate that possibly Lipocalin in Holstein cattle is carrying odorants to the environment which would be attracting more ticks, and enhancing in the susceptibility of these cattle.

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The Visceral Leishmaniasis (VL) is the second most important vector-borne disease in the world, transmitted in the Americas by Lutzomyia longipalpis, vector control is essential for the prevention of the disease. But since it is not possible to identify the oviposition sites, the fight is directed to adult insects, using traps impregnated with chemical attractants. Whereas the Odorant Binding Proteins (OBPs) act in the first level of odor selection, this work used in silico methodology to identify putative vector olfactory chemical modulators based on the structure of OBPs and known ligands. For this, tridimensional (3D) structure of L. longipalpis OBPs were predicted by three comparative modeling methods. The best model, predicted by I-Tasser, was refined by Molecular Dynamics on Gromacs. Then, in a hierarchical virtual screening approach, natural compounds of ZINC12 closer to the typical OBP ligands in global chemical space, provided by ChemGPS-NP, were evaluated and staggered concerning affinity with the orthosteric site from the OBP, by molecular docking on DOCK6. The compounds were scored by GRIDSCORE, then the 100 best classified were submitted to AMBERSCORE, which took into account the flexibility from both OBP and the docked ligands. The lowest energy conformations interacted with a hydrophobic pocket through residues Met6, Gly10, Glu11, Ala9 Arg14, Leu74, Met53, Phe118, Phe119, Pro120, amino groups and formed ionic interaction with carboxyl of Glu11, Furthermore, Phe119, Asn29 and Gln69 formed hydrogen bonds, this last formed donor and acceptor H-bonds.
A Leishmaniose Visceral (LV) ? a segunda doen?a vetorial mais importante do mundo, transmitida nas Am?ricas por Lutzomyia longipalpis, o controle do vetor ? indispens?vel ? preven??o da doen?a. Mas como n?o ? poss?vel identificar onde ocorre a oviposi??o, o combate ? direcionado aos insetos adultos, utilizando armadilhas impregnadas com atrativos qu?micos. Considerando que as Prote?nas Ligadoras de Odor (OBPs) atuam no primeiro n?vel de sele??o dos odores, este trabalho utilizou uma metodologia in silico para identificar potenciais moduladores qu?micos olfativos do vetor baseando-se na estrutura das OBPs e de ligantes conhecidos. Para isso, foram preditas as estruturas tridimensionais (3D) de OBPs de L. longipalpis por tr?s m?todos de modelagem comparativa. O melhor modelo, predito pelo I-Tasser, foi refinado por Din?mica Molecular no Gromacs. Ent?o, numa abordagem hier?rquica da triagem virtual, os compostos naturais do ZINC12 mais pr?ximos dos t?picos ligantes de OBPs no espa?o qu?mico global, fornecido pelo ChemGPS-NP, foram avaliados e escalonados quanto ? afinidade com o s?tio ortost?rico da OBP, pelo acoplamento molecular no DOCK6. Os compostos foram pontuados pelo Gridscore, em seguida, os cem melhores classificados foram submetidos ? pontua??o pelo Amberscore, que levou em conta a flexibilidade tanto da OBP como dos ligantes acoplados. As conforma??es de menor energia interagiram com um bols?o hidrof?bico atrav?s dos res?duos Met6, Ala9, Gly10, Glu11, Arg14, Met53, Leu74, Phe118, Phe119, Pro120; grupamentos amino formaram pontes salinas com a carboxila do Glu11. Al?m disso, os res?duos Phe119, Asn29 e Gln69 formaram liga??es hidrog?nio, sendo que, este ?ltimo res?duo formou liga??es-H aceptoras e doadoras.

Les Olfactory-binding protein (OBP), sont des protéines largement retrouvées dans le mucus olfactif d’un grand nombre de vertébrés mais elles n’ont cependant jamais été mises en évidence chez le poisson. Assez peu de choses sont connues sur leur fonction exacte au sein du système olfactif mais un rôle dans l’adaptation de ce système au milieu aérien est fortement suspecté. Le xénope possède un double système olfactif composé de deux cavités l’une dédiée à l’olfaction aquatique (CM) et l’autre dédiée à l’olfaction aérienne (CP). Cette particularité nous a permis de tester notre hypothèse. Nous avons pour la première fois caractérisée des OBP chez X. Laevis et X. Tropicalis. Grâce aux techniques de transcription inverse et de RACE 3’, deux produits ont été clonés et séquencés. Ces clones ont été utilisé afin d’étudier les patrons d’expression de ces protéines au niveau du système olfactif de ces deux espèces par HIS et immunocytochimie. Les transcrits et les protéines ont été localisés uniquement au niveau de la cavité aérienne de ces deux espèces soutenant donc l’idée que ces protéines seraient impliquées dans une adaptation du système olfactif au milieu aérien. De plus, suite à une étude réalisée sur une banque de gènes étiquetés, nous avons mis en évidence l’existence d’un second gène d’OBP présent chez X. Laevis uniquement et pas chez X. Tropicalis
Olfactory Binding Proteins (OBP), commonly associated with aerial olfaction, are currently found in mammals olfactory mucus, but have never been identified in fish. It is not clear yet if OBP is an adaptation of the olfactory system to an aerial environment. Adult olfactory system Xenopus is organized into two olfactory chambers which are thought to be devoted respectively to aquatic (MC) and aerial olfaction (PC). This specificity provides us the opportunity to test this alternative hypothesis. We have identified for the first time Olfactory Binding Protein in Xenopus laevis and tropicalis. By a reverse transcription and 3’ RACE strategy two products were cloned and sequenced. These cloned sequences were used to analyse the expression pattern of the gene by HIS and immunocytochemistry in the olfactory system of two Xenopus species: X. Laevis and X. Tropicalis. The transcripts and the proteins are only present in the aerial chamber supporting the idea that OBPs are an adaptation to aerial olfaction. Moreover, from an EST (expressed sequence tag) library we also demonstrated that X. Laevis has 2 different OBP genes while X. Tropicalis has only one gene

The scientific and social increasing demand for quantitative/qualitative determination of natural and artificial chemical species in different sector of human life (health, living environment, food, etc.) highlights the need for new strategies and new measurement methods. In particular, the agro-food industries need quick and sensitive tools to identify molecules in its final products or to monitor them during production processes. This need is due to the increase of food frauds. Indeed, in last decades, adulteration and falsification of food become a very serious problems also as regard human health. Biosensors are cutting-edge and the cheapest tools that can satisfy the monitoring of food quality because they represent an intelligent combination of biological components, such as enzymes or bacteria, and technological components that detect physical and chemical changes and transmit them in the form of data. Among agro-food chain products, wine plays an important role in world trade. Wine is one of the most appreciated beverages and it is a complex mixture of sugars, acid and odours that concourse to give it characteristic flavour. Concerning flavours, specific volatile organic compounds (VOCs) in wine are able to give it fruity o floral aroma. These VOCs belong to terpenes class (monoterpenes and sesquiterpenes) and they all together contribute to wine aroma. Analysis of wine aroma is at the base of quality assessment and physiochemical parameters are important to be monitored in order to limit frauds or adulterations. In an automated food production system, devices as biosensors are important to monitor and maintain product quality and uniformity based on aroma characteristics. The nature is the principal source to discover new molecules that could be used to realize biosensors. In this context, insects represent the main source from which to be inspired because they constitute the largest animal phyla. Insects have an important part in all ecosystems considering their role in pollination, biological control and bioconversion. They are a resource of genes, molecules and processes that can inspire several technological innovations. Considerably sophisticated in insect is the “chemoreception” that is a process by which organisms respond to chemical stimuli in their environments that depends primarily on the senses of taste and smell. Chemoreception plays an essential role throughout the life cycle of insects that respond to different arrays of chemical, biological and environmental signals to locate and select food, mates, oviposition sites and avoid predators. To interpret these signals, insects use a range of molecular components that are located in specialized structures called chemosensilla. The last ones are characterized by a variety of forms and by one or more pores on their surface, that allow the access of molecules to the internal aqueous phase, the sensillar lymph, which surrounds the dendrites of sensory neurons. In the sensillar lymph, a large amount of small soluble proteins, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) are present. Some aphids OBPs are able to bind the terpene (E)-β-Farnesene that it is used as alarm pheromone. In Acyrthosiphon pisum, OBP3 and OBP7 are able to bind (E)-β-Farnesene and they are responsible of alarm response. Recently, Acyrthosiphon pisum OBP9 was found to be involved in perception of this compound. Even Megoura viciae use (E)-β-Farnesene as alarm pheromone and its perception is mediate by OBP3. Acyrthosiphon pisum OBP3 are able to bind also farnesol, structurally related to (E)-β-Farnesene and Megoura viciae OBP3 is able to bind limonene. Farnesol and limonene are listed as wine terpenes. Starting from this consideration, aphids OBPs were produced through recombinant procedures and used to develop a biosensor able to bind terpenes in wine. Beside farnesol and limonene, already tested with aphids recombinant proteins, others wine terpenes were tested with Acyrthosiphon pisum OBP3 and Megoura viciae OBP3. A preliminary in silico analysis of aphid OBPs, including also Acyrthosiphon pisum OBP9 and OBP7, was conducted to select other terpenes useful for a rational design of the biosensor. Terpenes selected were geraniol, nerol and citronellol. Concerning biosensor assembly, the choice was to produce optical silicon biosensors. Optical sensors are more versatile than others because they can be made from different materials (silicon, glass, polymers, etc.) and among materials, silicon has gained attention and popularity in recent years because of its ease fabrication, special optical properties and its versatile surface chemistry. Moreover, silicon is readily available, low cost and biocompatible. Acyrthosiphon pisum OBP3 was the first protein used to optimize the optical biosensor assembly protocol because of its previous functional characterization. For biosensor development, Porous Silicon (PSi) surfaces were used and the flow sensing was used as a strategy to verify the behavior of sensing in real time. OBP was adsorbed onto oxidized surface simply flowing the protein on surface and farnesol was tested in different concentration (range: 10 μM – 400 μM). Even if results are not conclusive, they are encouraging considering that terpenes are very small molecules that give a small change in the refractive index.

Chez les petits ruminants (mouton et chèvre) la reproduction est saisonnée, c’est-à-dire qu’il y a alternance de période de repos et d’activité sexuelle. Ces deux périodes se caractérises chez les femelles par deux états physiologiques différents : en période d’activité sexuelle, le cycle ovarien des femelles est actif (oestrus) et la reproduction est possible, alors qu’en période de repos sexuel elles sont en anoestrus profond. Dans le but d’induire l’oestrus chez des femelles en repos sexuelle, les éleveurs ont le plus souvent recours à des hormones exogènes. Ces hormones sont de moins en moins acceptable pour le bien-être de l’animal et du consommateur. Cependant la réception d’odeurs émises par un mâle sexuellement actif peut réactiver l’axe gonadotrope des femelles en anoestrus en fin de période de repos sexuel, aboutissant dans la majorité des cas à l’ovulation. Ce phénomène naturel est appelé effet mâle. Les odeurs des béliers et des boucs agissent alors comme des phéromones modificatrices. La plupart des études menée sur l’effet mâle se sont concentrées sur les modifications neuroendocrines induites par la réception des phéromones du mâle, mais l’importance de la plasticité du sécrétome olfactif a longtemps été sous-estimée. De précédentes recherche menée par notre équipe ont montré que chez le porc, le sécrétome olfactif (ensemble des protéines sécrétées dans le mucus nasal) est principalement composé d’isoformes d’OBP (Odorant-Binding Proteins) générées par des modifications post-traductionnelles de type phosphorylations et glycosylations. La composition du sécrétome olfactif est sous contrôle hormonal et dépend du stade physiologique des animaux. Le but de cette thèse est de déterminer sur le sécrétome olfactif de brebis et de chèvre est lui aussi modifié par des facteurs endogènes (hormones) et exogènes (odeurs du mâle), montrant ainsi une adaptation des outils sensoriel des femelles. Nous avons collecté de manière non invasive le mucus nasal des mêmes brebis et de chèvre en période de repos et d’activité sexuelle. Le sécrétome olfactif de 3 femelles de chaque espèce a été analysé par électrophorèses 2D puis par spectrométrie de masse haute résolution. Nos résultats suggèrent que le profil et la composition du sécrétome olfactifs est un marqueur du stade physiologique des femelles, et constitue un phénotype des capacités olfactives. De plus, la réception d’odeurs émise par un mâle induit des changements dans le sécrétome olfactif, démontrant l’effet de facteurs exogène sur celui-ci
Small ungulates (sheep and goat) display a seasonal breeding characterised by successive periods of sexual activity and sexual rest. During these two periods females are in two different physiological status: in sexual activity, the female ovarian cycle is active (oestrus) and ready for reproduction, whereas females are in deep anoestrus during the sexual rest period. In order to induce oestrus in female during the sexual rest periods, breeders are mostly using exogenous hormones. These hormones are less and less acceptable regardless to animal and consumer’s welfare. However, the perception of odours emitted by a sexually active male can reactivate the gonadotropic axis of anoestrus female in the late sexual rest period, leading in most cases to ovulation. This natural process is called “the male effect”. Ram and goat odours act as primer pheromones.Most of studies on male effect focused on the neuroendocrine modifications induced by the reception of male pheromones, but the plasticity of the olfactory system at the peripherical level was underestimated. Previous work of my research team has shown that in pig, the olfactory secretome (secreted proteome) is mainly composed of Odorant-Binding Proteins (OBP) isoforms generated by post-translational modifications, phosphorylation and glycosylation. The composition of this secretome is under hormonal control and depends on the physiological status of animals. The aim of my work is to determine whether the olfactory secretome of ewe and goat is also modified by endogenous factors (hormones) and exogenous factors (male odours), showing an adaptation of the sensory equipment of female. In a first step, we followed the same flocks of ewe and goat during several sexual cycles, and collected their nasal mucus by a non-invasive sampling method. The olfactory secretome of 3 females of each species was analysed by 2D-electrophoresis followed by high-resolution mass spectrometry. Our results suggest that the olfactory secretome profile and its composition is a marker of the physiological status, and constitutes a phenotype of the female receptivity. Furthermore, the males’ odours reception induces changes in the olfactory secretome, demonstrating that exogenous factors can modifies the olfactory equipment of females

Odorant Binding Proteins (OBPs) are small soluble proteins responsible for the binding, solubilization and transportation of Volatile Organic Compounds (VOCs) to olfactory receptors in vertebrates and insects. These proteins have been gaining attraction in artificial olfaction fields due to their natural selectivity toward VOCs. Furthermore, OBPs are reported to be structurally stable, resistant to thermal and proteolytic degradation, and easily produced in E. coli. Therefore, it is possible to implement OBPs in biosensors in order to develop a sensor array with improved selectivity by mimicking the biological olfactory system. The main objective of this work is the expression, purification and characterization of insect OBPs, for further application in an electronic nose biosensor. The design of a vector construct with OBP genes under an IPTG inducible promoter allowed the proteins’ recombinant expression in E. coli. A first expression test revealed the presence of OBPs as inclusions bodies (IBs). Further expression strategies were tested to improve protein solubility during expression, however proved to be ineffective. Thus, CPI (Contact Pathway Inhibitor) from Phlebotomus duboscqi and OBP56a from Phormia regina were selected, expressed as IBs, solubilized and purified. Different purification protocols were assessed. Immobilized Metal Affinity Chromatography (IMAC) in denaturing conditions demonstrated to be the most successful approach, with purities of 86% (CPI) and 92% (OBP56a), and recoveries of 71% (CPI) and 50% (OBP56a). Western blot confirmed the presence of monomeric and dimeric forms of the purified OBPs. The secondary structure of the alpha helix rich insect OBPs was assessed by Circular Dichroism. We consider that further optimizations are still required, mainly in dialysis and a second purification step. Lastly, preliminary results were obtained in gas sensing experiments, in which OBP56a functionalized sensor has shown to be selective toward acetic acid. Overall, this work explored different techniques regarding expression and purification of insect OBPs in inclusion body form. With further optimization, these proteins can be employed in VOC detecting biomaterials, for applications such as the early detection of viral and bacterial infections, cancers and inflammatory diseases.
As proteínas de ligação a compostos odorantes (OBPs) são proteínas solúveis de tamanho pequeno responsáveis pela ligação, solubilização e transporte de Compostos Orgânicos Voláteis (VOCs) para recetores olfativos em vertebrados e insetos. Estas proteínas têm vindo a ganhar atenção em sistemas olfativos artificiais devido à sua seletividade natural para com os VOCs. Além disso, as OBPs são estruturalmente estáveis, resistentes à degradação térmica e proteolítica, e facilmente produzidas em E. coli. Portanto, é possível implementar OBPs em biosensores de modo a desenvolver um conjunto de sensores com uma seletividade melhorada, havendo assim um paralelismo com o sistema olfativo biológico. O principal objetivo deste trabalho é a expressão, purificação e caracterização de OBPs de insetos, para posterior aplicação num biossensor de nariz eletrónico. As proteínas selecionadas foram expressas em E. coli após a clonagem de um vetor com genes que codificam as mesmas OBPs sob um promotor indutível por IPTG. Um primeiro teste de expressão revelou a presença de OBPs como corpos de inclusão(IBs). Estratégias de expressão adicionais foram testadas para melhorar a solubilidade das proteínas durante a expressão, no entanto mostraram-se ineficazes. Assim, a CPI (Contact Pathway Inhibitor) de Phlebotomus duboscqi e OBP56a de Phormia regina foram selecionadas, expressas em IBs, solubilizadas e purificadas. Foram avaliados diferentes protocolos de purificação. A cromatografia de afinidade por iões metálicos imobilizados (IMAC) em condições desnaturantes demonstrou ser a abordagem mais bem sucedida, com purezas de 86% (CPI) e 92% (OBP56a) e recuperações de 71% (CPI) e 50% (OBP56a). Resultados de western blot confirmaram a presença de formas monoméricas e diméricas das OBPs purificadas. A estrutura secundária das OBPs de insetos, composta por hélices alfa, foi avaliada por Dicroísmo Circular. Consideramos que ainda são necessárias otimizações adicionais, principalmente no protocolo de diálise e na aplicação de um segundo passo de purificação. Por último, resultados preliminares foram obtidos em experiências de deteção de VOCs, nas quais o sensor funcionalizado com OBP56a mostrou ser seletivo para o ácido acético. No geral, este trabalho explorou diferentes técnicas de expressão e purificação de OBPs de insetos na forma de corpos de inclusão. Com otimização adicional, estas proteínas podem ser utilizadas em biossensores para a deteção de VOCs, para aplicações como a deteção precoce de infeções virais e bacterianas, cancros e doenças inflamatórias.

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of G-protein-coupled olfactory receptors (ORs) and olfactory carriers. Two such carrier gene families are found in insects: the odorant binding proteins (OBPs) and the chemosensory proteins (CSPs). In olfactory sensilla, OBPs and CSPs are believed to deliver hydrophobic air-borne molecules to ORs, but their expression in non-olfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. ¶ Bioinformatics and experimental approaches were used to characterise the OBP and CSP gene families in a highly social insect, the western honey bee (Apis mellifera). Comparison with other insects reveals that the honey bee has the smallest set of these genes, consisting of only 21 OBPs and 6 CSPs. These numbers stand in stark contrast to the 66 OBPs and 7 CSPs in the mosquito Anopheles gambiae and the 46 OBPs and 20 CSPs in the beetle Tribolium castaneum. The genes belonging to both families are often organised in clusters, and evolve by lineage specic expansions. Positive selection has been found to play a role in generating a greater sequence diversication in the OBP family in contrast to the CSP gene family that is more conserved, especially in the binding pocket. Expression proling under a wide range of conditions shows that, in the honey, bee only a minority of these genes are antenna-specic. The remaining genes are expressed either ubiquitously, or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs and CSPs are not restricted to olfaction, and are likely to be involved in broader physiological functions. ¶ Finally, the detailed expression study and the functional characterization of a member of the CSP family, uth (unable-to-hatch), is reported. This gene is expressed in a maternal-zygotic fashion, and is restricted to the egg and embryo. Blocking the zygotic expression of uth with double-stranded RNA causes abnormalities in all body parts where this gene is highly expressed. The treated embryos are `unable-to-hatch' and cannot progress to the larval stages. Our ndings reveal a novel, essential role for this gene family and suggest that uth is an ectodermal gene involved in embryonic cuticle formation.

Odorant-binding proteins (OBPs) are small soluble proteins expressed at high levels in the proximity of olfactory receptors. OBPs act as solubilizers and carriers of the lipophilic odorants in the aqueous mucus of mammals and other vertebrates. OBPs have now been studied nearly thirty years, but in comparison to the wealth of data available on their structural chemistry and molecular biology, our knowledge about gene expression and function of these proteins is still insufficient. This work provides new insights into the tissue specificity of OBP and presents several new sequences of genes governing these proteins in selected species of mice.

Tese de Doutoramento em Engenharia Química e Biológica
A atividade diária e o exercício físico são responsáveis pela produção de odores corporais desagradáveis que podem causar ansiedade e embaraço social. A procura de novas soluções que previnam o desenvolvimento desses odores é atualmente objeto de interesse para as indústrias da cosmética e têxtil. Nos mamíferos, as proteínas de ligação a odores (OBPs) são responsáveis pelo transporte de moléculas odoríferas do muco nasal aos recetores olfativos. As OBPs são proteínas extracelulares com uma estrutura robusta e estável em barril β, com grande capacidade para ligar a diferentes ligandos. Estas características têm sido foco de diferentes trabalhos de modo a compreender os mecanismos inerentes à sua função na natureza e a desenvolver aplicações biotecnológicas avançadas. Os resultados levaram-nos a estudar as OBPs como uma solução elegante para prevenir e/ou remover odores desagradáveis dos têxteis, através da captura de odores e libertação controlada de fragrâncias. Inicialmente, a OBP de porco (pOBP) foi fundida com três péptidos de penetração celular (CPPs). Estas proteínas (OBP::CPPs), em conjunto com lipossomas, foram usadas como transportadores e reservatórios num sistema avançado de captura de moléculas odoríferas. A pOBP foi também fundida com o péptido SP-DS3, com e sem o espaçador GQ20 para a ancoragem na membrana lipídica de lipossomas. A transdução/captura de 1-aminoanthraceno (1- AMA, ligando modelo fluorescente) para o interior dos lipossomas revelou ser dependente da proximidade da proteína à membrana lipídica. Estes trabalhos permitiram o desenvolvimento de dispositivos para a encapsulação de fragrâncias e captura de odores pelos lipossomas. Outras proteínas, a OBP truncada com as mutações F44A e F66A, e a OBP::GQ20::SP-DS3, apresentaram uma afinidade ao 1-AMA diferenciada e dependente da temperatura Recentemente, foi desenvolvido um “têxtil inteligente” pela funcionalização do tecido com OBP::GQ20::CBM (OBP fundida com o espaçador GQ20 e um módulo de ligação a carbohidratos). O têxtil funcionalizado revelou capacidade de libertação controlada de fragrâncias em resposta à transpiração (suor).
The daily activity and physical exercise are responsible for the generation of unpleasant body odors that may cause social unrest and embarrassment. The search for new solutions to prevent the development of these odors is nowadays a subject with great interest for cosmetic and textiles industries. In mammals, odorant-binding proteins (OBPs) are responsible to transport odorant molecules across the aqueous nasal mucus until the olfactory receptors (ORs). OBPs are small extracellular proteins with a robust and stable three-dimensional structure in β-barrel with great ability to bind differentiated ligand molecules which has driven the research to understand the mechanisms underlying the OBP function in nature and the development of advanced biotechnological applications. These features inspired us to study OBPs as an elegant solution to prevent and/or remove unpleasant odors from textiles, by the entrapment of odors and the controlled release of fragrances. Firstly, porcine OBP (pOBP) was fused with three cell penetrating peptides (CPPs). A new methodology using liposomes as reservoirs and OBP::CPPs as carriers was developed as an advanced system to capture odorant molecules. pOBP was also fused with an anchor peptide (SP-DS3), without and with a spacer GQ20, and the liposomes were produced anchoring these new fusion proteins in the lipid membrane. The transduction of 1-aminoanthracene (1-AMA, a fluorescent ligand model) into the liposomes revealed to be driven by the proximity of the protein to the liposomal membrane. Both works showed the development of an efficient device for the encapsulation of fragrances or capture of unpleasant odors inside of the liposomes. Other two proteins, truncated OBP with mutation F44A and F66A, and OBP::GQ20::SP-DS3 presented differentiated 1-AMA binding behavior depending on the temperature. Further a smart fabric was developed by functionalization with OBP::GQ20::CBM (OBP fused with a spacer GQ20 and a carbohydrate-binding module). The functionalized fabric exhibited controlled release of fragrances triggered by perspiration (sweat).
Fundação para a Ciência e a Tecnologia (FCT)