Proteins from the lipocalin family play significant roles during the transport of chemical signals. These globular proteins (molecular mass: 17,7 - 21,7 kDa) are able to bind a wide spectrum of volatile ligands. Since they decay continuously, the scent impact is prolonged. Major urinary proteins (MUPs) are lipocalins, typically found in the urine and used during olfactory communication in the house mouse. But the urine is not the only source of chemical signals. Many species of mammals perform selfgrooming - a technique in which saliva is transported into the fur. The goal of the thesis is to identify main proteins that are involved in chemical communication by selected species of the genus Apodemus, and to discover the source of production (i.e. saliva or urine). The Experion capillary electrophoresis system was used to confirm the absence of lipocalins in the urine in two Apodemus species (A. uralensis, A. sylvaticus). Furthermore, a lot of lipocalins is present in the saliva of above-mentioned species. The quantity and types of salivary lipocalins was identified by 2D electrophoresis along with mass spectrometry analyses (MALDI MS/MS) in three species of the genus Apodemus (A. uralensis, A. sylvaticus and A. flavicollis). The conclusion of this inter-specific comparison suggests, that sociality...

La détection de molécules odorantes représente un enjeu important dans divers domaines tels que l’industrie alimentaire, le diagnostic médical et la sécurité du territoire, par exemple. En effet, les odorants, présents par milliers dans notre environnement, véhiculent de nombreuses informations, via leur nature chimique ou leur concentration. Notre système olfactif est capable de discriminer des milliers de molécules différentes via des mécanismes biochimiques impliquant l’association de nombreux partenaires protéiques et un codage combinatoire de l’information. Ces biomolécules, qui englobent notamment les récepteurs olfactifs et les protéines de liaison aux odorants (OBP), constituent une source intéressante d’éléments de détection pour la conception de biocapteurs. Les OBP sont de petites protéines solubles présentent dans le mucus nasal à des concentrations de l’ordre du millimolaire. Leur poche de liaison hydrophobe leur confère la capacité de lier de façon réversible les molécules odorantes. Leur robustesse et leur manipulation aisée en font de bonnes candidates pour l’élaboration de biocapteurs. Au cours de ce travail, nous nous sommes intéressés à la détection de molécules odorantes en associant des OBP comme biorécepteur et l’électrochimie comme méthode de transduction. Par une méthode de mutagenèse dirigée, nous avons montré qu’en modifiant un seul des acides aminés dans la poche de liaison de deux OBP de rat (rOBP2 et rOBP3), il était possible de moduler leurs affinités envers les odorants. En parallèle, nous avons décrit la détection qualitative et quantitative de molécules volatiles à partir d’OBP. Nous avons montré que rOBP3 lie la 2-méthyl-1,4-naphtoquinone (MNQ), une sonde électrochimique. La quantité de MNQ déplacée de la poche de liaison de rOBP3 par la 3-isobutyl-2-méthoxypyrazine (IBMP), un odorant modèle, a été mesurée par voltammétrie cyclique à vagues carrées. Nous avons déterminé les constantes de dissociation des complexes rOBP3 / MNQ et rOBP3 / IBMP. Les valeurs mesurées par électrochimie ont été confirmées par compétition avec une sonde fluorescente et par titration calorimétrique isotherme. En combinant cette nouvelle méthode analytique à des variants de rOBP3 qui présentent des profils de liaison différents et complémentaires, nous avons détecté sélectivement chacun des constituants d’un mélange ternaire d’odorants. Ces travaux, qui allient ingénierie des OBP et électrochimie, offrent des perspectives intéressantes dans le domaine des nez électroniques. / The detection of odorant molecules has become an important challenge in different research area, such as the food industry, medical diagnostics and homeland security. Indeed, the thousands of odorants in our environment provide information on their chemical nature or their concentration. Human olfactory system is capable of discriminating thousands of different molecules thanks to biochemical mechanisms involving multiple protein receptor partners and a combinatorial coding. These biomolecules that include olfactory receptors and odorant-binding proteins (OBP) represent an interesting source of detectors for the design of biosensors. OBPs are small soluble proteins present in nasal mucus at millimolar concentrations. Their hydrophobic binding pocket gives them the ability to reversibly bind odorant molecules. OBPs are robust and easy to produce and are thus good candidates for the design of biosensors. In this work, we focused on the detection of odorant molecules associating OBPs as a bioreceptor and electrochemistry as a transduction method. Using site-directed mutagenesis, we have shown that by substituting a single amino acid in the binding pocket of two rat OBPs (rOBP2 and rOBP3), it is possible to modulate their binding affinities towards odorants. In parallel, we described a qualitative and quantitative method for the detection of volatile molecules using OBPs. We have shown that rOBP3 binds 2-methyl-1,4-naphtoquinone (MNQ), an electrochemical probe. The amount of MNQ displaced from the binding pocket of rOBP3 by the model odorant 3-isobutyl-2-methoxypyrazine (IBMP), was measured using square-wave voltammetry. We determined the dissociation constants of the rOBP3 / MNQ and rOBP3 / IBMP complexes. These values measured by electrochemistry were confirmed by a competitive fluorescent assay and isothermal titration calorimetry. By combining this new analytical method to rOBP3 variants with different and complementary binding profiles, we were able to selectively detect each of the components of a ternary mixture of odorants. This work, that combines the engineering of OBPs and electrochemistry, offers us interesting perspectives in the field of electronic noses.

No description available.

Dans le système olfactif des moustiques, les protéines liants les molécules odorantes ou odorant binding proteins (OBPs) interviennent dans les toutes premières étapes permettant d'aboutir à la reconnaissance de leurs hôtes et font l'objet d'un intérêt croissant dans les recherches sur la transmission des maladies infectieuses par ces insectes. Le travail présenté a pour objet d'approfondir les connaissances sur ces OBPs dans trois génomes de moustiques, tous vecteurs de maladies infectieuses : Anopheles gambiae, Aedes aegypti et Culex quinquefasciatus. Une analyse à l'échelle de ces génomes a été réalisée et a permis d'identifier un nombre important de nouveaux gènes d'OBPs notamment chez les espèces de moustiques Aedes aegypti et Culex quinquefasciatus. Complétée par une étude phylogénétique du répertoire complet de ces gènes dans les trois génomes étudiés, cette analyse a permis d'établir une nouvelle classification des sous familles des OBPs. Ce résultat démontre l'extraordinaire multiplicité et diversité des gènes impliqués dans l'olfaction chez ces espèces de moustiques tout en mettant en lumière certaines propriétés des séquences des OBPs qui sont hautement conservés chez les moustiques. Grâce à la disponibilité de certaines structures d'OBPs de moustiques ou d'autres insectes apparentées, des modèles structuraux de tous les OBPs de la sous famille dites Classic dans les trois génomes, soit au total 137 structures, ont été construits. Ces structures ont servi de base pour le criblage à grande échelle par docking moléculaire d'une chimiothèque de 126 molécules odorantes connues pour leurs propriétés attractives ou répulsives vis-à-vis des moustiques. Ces résultats fournissent pour la première fois, les bases structurales et fonctionnelles pour la compréhension au niveau moléculaire de l'efficacité de certains agents répulsifs tout comme de l'attractivité de certains agents provenant des émanations humaines. Par simulation de dynamique moléculaire, les changements qui s'opèrent dans une de ces OBPs lorsque celle ci, liée à une molécule odorante, se retrouve dans des conditions de pH modifiée ont été caractérisée et un mécanisme probable par lequel ces OBPs participeraient à la reconnaissance et la libération des molécules odorantes est proposée. Cette thèse fournit des éléments de réponses importants quant à la caractérisation génomique, structurale et fonctionnelle des OBPs de moustiques et peut servir de base de départ pour des recherches expérimentales plus approfondies sur ces aspects. / 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.

No description available.

No / For decades, the development of the Internet was driven by the purpose of providing applications to non-mobile users. No specific Quality of Service (QoS) requirement is necessary, other than ensuring reliability in the end-to-end data transfers. As such, best effort service model was deemed more than appropriate to satisfy the users' needs. Nevertheless, the scenario has changed in the last few years. A new population of nomadic users, who requires access to Internet services regardless of their location and mode of transportation is growing, while new typologies of Internet applications are being continuously developed, in which best effort service level may no longer be adequate. The SUITED project has been devised to address the emerging issues generated by this new and challenging scenario. SUITED aims at contributing towards the design and deployment of the Global Mobile Broadband System (GMBS), a unique satellite/terrestrial infrastructure, which will ensure that nomadic users have access to Internet services with a negotiated QoS. An overview of the main results achieved in the SUITED project is provided in this article. Some of the most innovative solutions developed by the SUITED team for the joint management of global mobility and end-to-end QoS support are presented. The SUITED demonstrator platform, which was developed to validate the system specifications, is also described. Finally, the results of the experimental measurement campaigns carried out with this platform are reported.

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

Made available in DSpace on 2016-08-03T16:34:21Z (GMT). No. of bitstreams: 1 SONIA MARIA MACHADO DE OLIVEIRA NUKUI.pdf: 609257 bytes, checksum: aab8a9dc798a45b0d330d42701e802fa (MD5) Previous issue date: 2012-03-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This research was conducted with two women under situations of domestic violence and was based on theoretical, technical and methodological aspects of psychological intervention called OBP-Operational Brief Psychotherapy. The aim was to analyze the process of OBP of women in situations of domestic violence. The method was a clinical study of the psychoanalytic approach that is based on the study of descriptive case study. Participants performed five diagnostic interviews and were also classified with severe ineffective adaptation featuring most limiting neurotic symptoms, inhibitions and restrictive rigidity of character traits. They went through OBP and after the end of the process the Follow up interviews were reassessed by EDAO. The data that was obtained in follow-up interviews showed that there was a change in adaptive efficiency. Participants were also classified with Moderate Inefficient Adaptation. They had some neurotic symptoms, moderate inhibition, some character traits. Our conclusion is that in the studied cases, though the analysis by sectors, the affective relational sector has established itself as a source of conflict and dissatisfaction bringing very few appropriate solutions, decisively influencing other sectors of adaptation. In addition, we observed the phenomenon of reproduction models of the past in the established relations in the present. In both studied cases there was a situation of physical and sexual abuse, which was configured as an inheritance coming from a relational dynamic of the family of origin and perpetuated freely in their nuclear families. Finally, it should be noticed that the patients participated actively during the therapeutic process. From the theorized interpretations, they were able to reconsider their attitudes towards problematic situations experienced and they were able to take some concrete steps to deal with them more adequately during and after the psychotherapeutic process. It was found that the process of brief psychotherapy and the use of operationalized theorized interpretations seem to have contributed to the change in the quality of adaptive efficacy of two women who took part in this study. / A presente pesquisa foi realizada com duas mulheres em situação de violência doméstica e fundamentou-se nos pressupostos teóricos, técnicos e metodológicos da intervenção psicológica intitulada PBO- Psicoterapia Breve Operacionalizada. O objetivo foi analisar o processo da PBO de mulheres em situação de violência doméstica. Para a realização do diagnóstico adaptativo operacionalizado utilizou-se como instrumento a EDAO - Escala Diagnóstica Adaptativa Operacionalizada. O método utilizado foi o estudo clínico de abordagem psicanalítica que tem como base o estudo descritivo do tipo estudo de caso. As participantes realizaram cinco entrevistas diagnósticas e foram classificadas igualmente com adaptação ineficaz severa apresentando sintomas neuróticos mais limitadores, inibições restritivas e rigidez de traços caracterológicos. Elas foram encaminhadas para um atendimento em PBO. Após o final do processo realizou-se as entrevistas de Follow up que foram novamente avaliadas por meio da EDAO. Os dados obtidos nas entrevistas de follow up mostraram que houve mudança na eficácia adaptativa. As participantes foram classificadas igualmente com Adaptação Ineficaz Moderada. Elas apresentavam alguns sintomas neuróticos, inibição moderada, alguns traços caracterológicos. Conclui-se que nos casos estudados, na análise por setores, pôde-se perceber que o setor afetivo relacional constituiu-se como uma fonte de conflitos e insatisfações gerando soluções em sua maioria pouquíssimas adequadas, obviamente, influenciando decisivamente os demais setores da adaptação. Também, observou-se o fenômeno da reprodução de modelos do passado nas relações estabelecidas no presente. Nos dois casos estudados houve uma situação de violência física e de abuso sexual, que se configurava como uma herança provinda de uma dinâmica relacional da família de origem e se perpetuava livremente em suas famílias nucleares. Por último, cabe assinalar que as pacientes participaram de forma ativa durante o processo terapêutico. A partir das interpretações teorizadas foram capazes de reconsiderarem suas atitudes frente às situações-problema vividas e puderam, durante e após o processo psicoterapêutico, adotar algumas medidas concretas para enfrentá-las de forma mais adequada. Conclui-se que o processo de psicoterapia breve operacionalizado e a utilização das interpretações teorizadas parecem ter contribuído para a mudança na qualidade da eficácia adaptativa das duas mulheres que fizeram parte deste estudo.