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.

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.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-05-12T13:15:41Z No. of bitstreams: 1 TeseEBC.pdf: 4307606 bytes, checksum: 49ebc853f6c4c152639d651f942f72b8 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-18T20:31:37Z (GMT) No. of bitstreams: 1 TeseEBC.pdf: 4307606 bytes, checksum: 49ebc853f6c4c152639d651f942f72b8 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-18T20:31:43Z (GMT) No. of bitstreams: 1 TeseEBC.pdf: 4307606 bytes, checksum: 49ebc853f6c4c152639d651f942f72b8 (MD5) / Made available in DSpace on 2017-05-25T18:43:38Z (GMT). No. of bitstreams: 1 TeseEBC.pdf: 4307606 bytes, checksum: 49ebc853f6c4c152639d651f942f72b8 (MD5) Previous issue date: 2016-04-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Odorant-binding proteins (OBPs) are of great importance for survival and reproduction since they participate in initial steps of the olfactory signal transduction cascade, solubilizing and transporting chemical signals to the olfactory receptors. A comparative analysis of OBPs between closely related species may help explain how these genes evolve and are maintained under natural selection and how differences in these proteins can affect olfactory responses, and consequently lead to species differentiation. We studied OBP genes in the closely related species Anastrepha fraterculus and Anastrepha obliqua, which, albeit generalists, have different host preferences, using transcriptomes and real time quantitative PCR data. We identified 24 different OBP sequences from Anastrepha fraterculus and 25 from A. obliqua, which correspond to 21 Drosophila melanogaster OBP genes. Phylogenetic analysis separated Anastrepha OBPs sequences in four branches that represent four subfamilies: classic, minus-C, plus-C and dimer. We found evidence of positive selection in three classic subfamily genes OBP56h-1, OBP56h-2 e OBP57c and in the plus-C subfamily gene OBP50a, and at least one duplication event that preceded the speciation of these two species. Four positively selected sites putatively resulted in radical changes in amino acid properties. Inferences on tertiary structures of putative proteins from these genes revealed that at least one positively selected change involves the binding cavity (the odorant binding region) in the plus-C OBP50a, which is important because changes in the binding cavity could change OBPs specificity. Differential gene expression analysis at different reproductive stages showed that all nine OBP genes tested were significantly differentially expressed between A. fraterculus and A. obliqua at several reproductive profiles, but OBP56a, OBP56d, OBP57c and both OBP56h paralogs showed the highest differences in expression levels. The results generated in this study indicated that at least seven OBP genes may be involved in the A. fraterculus e A. obliqua differentiation, and in the fraterculus group differentiation as well. / As proteínas ligantes a odores (OBPs – odorant-binding proteins) são de grande importância para a sobrevivência e reprodução, pois participam do passo inicial da cascata de transdução dos sinais olfatórios, solubilizando e transportando os sinais químicos (odores e feromônios) até os receptores olfativos. A análise comparativa dos genes OBPs entre espécies próximas pode ajudar na compreensão de como o repertório desses genes é mantido sob seleção natural, além de fornecer informações acerca de como as diferenças observadas podem afetar as respostas olfatórias e, consequentemente, levar à diferenciação dessas espécies. Estudamos genes OBP em duas espécies-irmãs Anastrepha fraterculus e Anastrepha obliqua, as quais têm preferência por diferentes frutos hospedeiros, usando dados de transcriptomas e de PCR quantitativa. Identificamos 24 sequências OBP para A. fraterculus e 25 para A. obliqua, que corresponderam a 21 genes OBP de Drosophila melanogaster. Análises filogenéticas separaram as OBPs de Anastrepha em quatro ramos, que representam quatro subfamílias dessa família gênica: classic, minus-C, plus-C e dimer. Evidências de seleção positiva foram observadas nos genes da subfamília classic OBP56h-1, OBP56h-2 e OBP57c, e para o gene da subfamília plus-C OBP50a, e pelo menos um evento de duplicação gênica que precede a especiação dessas duas espécies. Quatro sítios selecionados positivamente resultavam em mudanças radicais nas propriedades dos aminoácidos. Inferências utilizando a estrutura terciária predita para essas OBPs revelaram que pelo menos um desses sítios faz parte da cavidade ligante ao odor de OBP50a, sendo que uma mudança nessa região pode alterar a especificidade de uma OBP. Análises de expressão por PCR quantitativa em diferentes estágios reprodutivos das moscas mostraram que todos os nove genes testados possuíam expressão gênica significativamente diferente entre A. fraterculus e A. obliqua para mais de um perfil reprodutivo, sendo que OBP56a, OBP56d, OBP57c e os dois parálogos OBP56h foram os que mais apresentaram diferenças entre as duas espécies. Todos os resultados gerados pelo presente trabalho indicam que pelo menos sete genes OBP podem estar envolvidos na diferenciação entre A. fraterculus e A. obliqua e, potencialmente, na diferenciação do grupo fraterculus. / FAPESP: 2012/17160-8. / CAPES: 99999.004252/2014-04