It was previously proposed that expression of secondary sexual characteristics may provide cues to individual's immunocompetence. Body odour could partly serve as one of such characteristics, which provides crucial information about potentional partner even in humans. The main aim of the diploma thesis was to test a relationship between body odour quality and reactivity of immune system. In empirical part of the study we collected body odour samples from 21 men aged between 18-40 years before and two weeks after the vaccination against hepatitis A and B (Twinrix) and meningococcus (Menveo). The participant's blood samples were obtained three times to determine levels of IgG and IgM antibodies (markers of reactivity of immune system), testosterone, cortisol and CRP levels. In the second part of the study, a panel of 88 female raters aged 18-40 assessed body odour samples for their attractiveness, intensity and healthiness. In contrast to our expectations, we found no significant association between levels of antibodies induced by vaccination and perceived body odour attractiveness and health. Simultaneously, there were no significant changes in body odour ratings, neither in levels of testosterone and cortisol ratings, before and after the vaccination. However, we found a negative association...

abstract: The ability to detect and appropriately respond to chemical stimuli is important for many organisms, ranging from bacteria to multicellular animals. Responses to these stimuli can be plastic over multiple time scales. In the short-term, the synaptic strengths of neurons embedded in neural circuits can be modified and result in various forms of learning. In the long-term, the overall developmental trajectory of the olfactory network can be altered and synaptic strengths can be modified on a broad scale as a direct result of long-term (chronic) stimulus experience. Over evolutionary time the olfactory system can impose selection pressures that affect the odorants used in communication networks. On short time scales, I measured the effects of repeated alarm pheromone exposure on the colony-level defense behaviors in a social bee. I found that the responses to the alarm pheromone were plastic. This suggests that there may be mechanisms that affect individual plasticity to pheromones and regulate how these individuals act in groups to coordinate nest defense. On longer time scales, I measured the behavioral and neural affects of bees given a single chronic odor experience versus bees that had a natural, more diverse olfactory experience. The central brains of bees with a deprived odor experience responded more similarly to odorants in imaging studies, and did not develop a fully mature olfactory network. Additionally, these immature networks showed behavioral deficits when recalling odor mixture components. Over evolutionary time, signals need to engage the attention of and be easily recognized by bees. I measured responses of bees to a floral mixture and its constituent monomolecular components. I found that natural floral mixtures engage the orientation of bees’ antennae more strongly than single-component odorants and also provide more consistent central brain responses between stimulations. Together, these studies highlight the importance of olfactory experience on different scales and how the nervous system might impose pressures to select the stimuli used as signals in communication networks. / Dissertation/Thesis / Doctoral Dissertation Biology 2018

abstract: Spatiotemporal processing in the mammalian olfactory bulb (OB), and its analog, the invertebrate antennal lobe (AL), is subject to plasticity driven by biogenic amines. I study plasticity using honey bees, which have been extensively studied with respect to nonassociative and associative based olfactory learning and memory. Octopamine (OA) release in the AL is the functional analog to epinephrine in the OB. Blockade of OA receptors in the AL blocks plasticity induced changes in behavior. I have now begun to test specific hypotheses related to how this biogenic amine might be involved in plasticity in neural circuits within the AL. OA acts via different receptor subtypes, AmOA1, which gates calcium release from intracellular stores, and AmOA-beta, which results in an increase of cAMP. Calcium also enters AL interneurons via nicotinic acetylcholine receptors, which are driven by acetylcholine release from sensory neuron terminals, as well as through voltage-gated calcium channels. I employ 2-photon excitation (2PE) microscopy using fluorescent calcium indicators to investigate potential sources of plasticity as revealed by calcium fluctuations in AL projection neuron (PN) dendrites in vivo. PNs are analogous to mitral cells in the OB and have dendritic processes that show calcium increases in response to odor stimulation. These calcium signals frequently change after association of odor with appetitive reinforcement. However, it is unclear whether the reported plasticity in calcium signals are due to changes intrinsic to the PNs or to changes in other neural components of the network. My studies were aimed toward understanding the role of OA for establishing associative plasticity in the AL network. Accordingly, I developed a treatment that isolates intact, functioning PNs in vivo. A second study revealed that cAMP is a likely component of plasticity in the AL, thus implicating the AmOA-beta; receptors. Finally, I developed a method for loading calcium indicators into neural components of the AL that have yet to be studied in detail. These manipulations are now revealing the molecular mechanisms contributing to associative plasticity in the AL. These studies will allow for a greater understanding of plasticity in several neural components of the honey bee AL and mammalian OB. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2014

RIC-8B é uma proteína que apresenta, in vitro, atividade de fator de troca de nucleotídeos guanina (GEF). No entanto, seu papel in vivo não é conhecido. Dados anteriores do nosso laboratório demonstraram que essa proteína interage especificamente com Gαolf, que é uma proteína G exclusiva do sistema olfatório, presente nos cílios dos neurônios olfatórios, onde ocorre a transdução de sinal ativada pelos odorantes. No camundongo adulto verificou-se, por meio de ensaios de hibridização in situ, que RIC-8B está presente somente em regiões de expressão de Gαolf: no epitélio olfatório maduro e no núcleo estriado do sistema nervoso central. Para avaliar a função fisiológica de RIC-8B in vivo, resolvemos gerar uma linhagem de camundongo knockout para Ric-8B. Verificamos que a linhagem é inviável devido à letalidade dos embriões já em fases precoces do desenvolvimento (por volta de E8,5 e E9,0). A coloração de embriões com X-gal mostra que RIC-8B é especificamente expressa em regiões que darão origem ao sistema nervoso, como na região ventral do tubo neural, e em regiões cefálicas. Interessantemente, mostramos que RIC-8B é expressa na placa do assoalho do tubo neural, de uma maneira muito semelhante ao padrão de expressão de Sonic Hedgehog (SHH), que apresenta um papel fundamental para a organização do sistema nervoso, entre outras funções. Nossos resultados indicam, portanto, que RIC-8B desempenha um papel crucial durante a embriogênese, e que este papel pode estar relacionado com o papel exercido por SHH. Além disso, como a via de sinalização de SHH ocorre em cílios primários nas células alvo, nossos dados levantam a interessante possibilidade de que RIC-8B apresenta funções relacionadas a cílios, tanto no camundongo adulto (neste caso nos cílios dos neurônios olfatórios) como no embrião (neste caso nos cílios primários). / RIC-8B is a protein that, in vitro, acts as a guanine nucleotide exchange factor (GEF). However, its role in vivo remains unknown. Previous data from our laboratory demonstrated that this protein is able to interact specifically with Gαolf, a G protein found only in the olfactory system. This G protein is located in the cilia from olfactory neurons, where odorant signaling occurs. In situ hybridization experiments showed that RIC-8B, in adult mice, is expressed only in regions where Gαolf is expressed, such as the olfactory epithelium and the nucleus striatum in the central nervous system. In order to determine the function of RIC-8B in vivo, we decided to generate a knockout mouse strain for Ric-8B. We found that this strain is not viable due to the lethality of embryos in the early stages of development (around days E8.5 and E9.0). X-gal staining of embryos shows that RIC-8B is specifically expressed in regions that originate the nervous system, such as the ventral neural tube and also cephalic regions. Interestingly, we show that RIC-8B is restrictedly expressed in the floor plate of the neural tube, in a pattern that is very similar to the one shown by Sonic Hedgehog (SHH). The SHH protein plays a fundamental role in the organization of the nervous system, among other functions. Therefore, our results indicate that RIC-8B plays an essential role during the embryogenesis, and that this role can be related to the role played by SHH. Furthermore, because the SHH signaling pathway occurs in primary cilia in the target cells, our data raise the interesting possibility that the role played by RIC-8B is related to ciliary functions, both in adult mice (in this case, in olfactory cilia), and in the embryo (in this case, in primary cilia)

Submitted by (ana.araujo@ufrpe.br) on 2016-05-20T13:54:50Z No. of bitstreams: 1 Ana Virginia Pedrosa Figueiredo.pdf: 2045507 bytes, checksum: 8ec16ce7fe3a69992ddd7f91512eb706 (MD5) / Made available in DSpace on 2016-05-20T13:54:50Z (GMT). No. of bitstreams: 1 Ana Virginia Pedrosa Figueiredo.pdf: 2045507 bytes, checksum: 8ec16ce7fe3a69992ddd7f91512eb706 (MD5) Previous issue date: 2007-02-26 / One of the human senses that has several aspects get to be elucidated is the olfactory sense. Therefore, many scientists have been studying this sense in order to better understand how does the information processing happens until the brain recognize it. There were lots of theories regarding olfactory system functioning, in which its authors try to explain how the reception, the analysis and the odor detection occur. Many people still use their own noses as a working tool. In this kind of job, people are trained to inhale and detect different odors. It is considered as an exhausting and risky job for those professional that, for example, could inhale toxics gases. In order to solve this problem, many systems that try to simulate a biological nose were developed. These systems are known as artificial noses. An artificial nose is an equipment composed of sensors and a pattern recognition system. The sensors are responsible for detecting odor signs from the external environment. The pattern recognition system is used to classify the signs sent by sensors and to provide a result from these signs.In the present work, artificial neural network techniques were used for the patternrecognition process, once these techniques are non-parametric and usually nonlinear. The usage of artificial neural networks as an odor recognition system has been quite advantageous. These networks are capable of working with non-linear data and also have an adaptation capability, they are tolerant to errors and noise, and have parallel processing. MLP, RBF e PNN were used in the development of an odor recognizing system based on a biologic system model and its results were compared, using the Wilcoxon test on the respective network models without the adaptation to the biologic model. / Um dos sentidos humanos que apresenta vários aspectos que ainda precisam ser elucidados é o sentido do olfato. Para isto muitos pesquisadores vêm estudando este sentido para melhor entender como ocorre o processamento das informações até a etapa de reconhecimento feita pelo cérebro. Desses estudos muitas foram as teorias propostas sobre o funcionamento do sistema olfativo, onde seus autores procuram esclarecer como ocorre a recepção, análise e detecção do odor. Muitas pessoas ainda têm como ferramenta de trabalho seus próprios narizes. Nesse tipo de trabalho pessoas são treinadas para inalar e detectar odores. Esse trabalho é considerado exaustivo e de risco para o profissional que, por exemplo, pode vir a inalar gases tóxicos. Para solucionar esse problema foram criados sistemas que simulam o nariz biológico. Esses sistemas são chamados de narizes artificiais. O nariz artificial é um equipamento formado por sensores e um sistema de reconhecimento de padrões. Os sensores são responsáveis por captar do meioexterno os sinais de odor. O sistema de reconhecimento de padrões é utilizado para classificar os sinais enviados pelos sensores e apresentar um resultado. No presente trabalho, foram utilizadas as técnicas de redes neurais artificiais para o reconhecimento de padrões. Pois essas técnicas são não-paramétricas e geralmente são não-lineares. A utilização de redes neurais artificiais como sistema de reconhecimento de odor tem sido bastante vantajosa. Elas têm a capacidade de trabalhar com dados não-lineares, possuem capacidade de adaptação, são tolerantes a erros e a ruídos e também possuem processamento paralelo. Foram utilizadas as redes MLP, RBF e PNN para o desenvolvimento de um sistema de reconhecimento de odor baseado em um modelo do sistema olfativo biológico e seus resultados foram comparados, utilizando o teste de Wilcoxon, com os respectivos modelos de redes sem a adaptação ao modelo biológico.

Orientador: Maria Aparecida Azevedo Pereira da Silva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T19:30:51Z (GMT). No. of bitstreams: 1 Natalicio_MariaAngelica_D.pdf: 1137447 bytes, checksum: bf1a6d401d0100649213c09ece37f7f9 (MD5) Previous issue date: 2012 / Resumo: Testes sensoriais para a avaliação da função olfatória de indivíduos têm sido validados e utilizados em diagnósticos da população em geral, e em portadores de desordens cerebrais e pesquisas em neurociência em particular. No Brasil, testes para serem utilizados com segurança e eficiência para a avaliação da capacidade de identificação e discriminação de odores de indivíduos ainda não foram adequadamente desenvolvidos e validados. Assim, os objetivos da presente pesquisa foram: i) desenvolver, testar e validar testes de identificação e discriminação de odores para avaliar a função olfatória de indivíduos brasileiros, ii) avaliar a potencialidade das metodologias desenvolvidas para uso em pré-diagnóstico clínico de indivíduos da terceira idade e pacientes portadores de epilepsia, iii) verificar o desempenho, em portadores de epilepsia, de uma metodologia já validada e utilizada mundialmente para avaliar a capacidade de identificação de odores de indivíduos e, iv) avaliar em portadores de epilepsia, funções que são processadas por substratos neurais comuns à função olfatória, neste caso, a capacidade de reconhecimento de emoção facial e vocal. Para o teste de identificação de odores, foi desenvolvido um instrumento intitulado &quot;Pastilhas de Odor¿ contendo em pastilhas individuais, 36 odores familiares aos brasileiros. Os odores foram caracterizados quanto à intensidade, aceitação, pungência, refrescância e familiaridade, sendo considerados adequados para comporem um teste de avaliação da função olfatória. A identificação dos odores de &quot;Pastilhas de Odor¿ foi realizada através de um teste de múltipla escolha contendo quatro alternativas, das quais apenas uma é a correta. Para o desenvolvimento do teste de discriminação de odores, 24 voláteis odoríferos puros (P.A.), associados a 6 diferentes categorias de odor - doce, verde, frutal, cítrico, floral e desagradável - foram selecionados e diluídos em propilenoglicol. Com essas amostras, 36 testes de comparação pareada foram construídos, onde em cada categoria de odor, uma amostra alvo foi selecionada para ser comparada com as demais da mesma categoria. O desempenho de cada indivíduo neste teste é analisado utilizando-se a teoria &quot;signaldetection¿, através dos seguintes parâmetros: taxa de acertos (HR), taxa de falsos alarmes (FR), poder discriminativo (d¿L) e vício de resposta (CL). Os dois testes desenvolvidos - &quot;Pastilhas de Odor¿ e teste de discriminação - foram validados com a participação de três grupos de indivíduos: grupo controle, grupo da terceira idade e grupo de indivíduos portadores de epilepsia do lobo temporal (ELT). A capacidade de identificação de odores, de pacientes norte-americanos portadores de ELT foi também avaliada, utilizando-se com esse fim, o teste já validado, denominado &quot;University of Pennsylvania Smell Identification Test¿ (UPSIT), o qual consiste em um teste de múltipla escolha, composto por 40 estímulos odoríferos microencapsulados. Adicionalmente, avaliou-se nesses pacientes, a capacidade de reconhecimento de emoção facial e vocal, utilizando-se uma nova ferramenta intitulada &quot;Comprehensive Affect Testing System¿ (CATS). Com relação à capacidade de identificação de odores dos indivíduos brasileiros, os resultados obtidos através do teste &quot;Pastilhas de Odor¿ revelaram significância estatística tanto para o efeito &quot;sexo¿ (p=0,0003), como para o efeito &quot;idade¿ (p<0,001). O teste também permitiu identificar que os portadores de ELT, sem cirurgia e após ressecção do lobo temporal, apresentavam menor capacidade de identificação de odores comparativamente ao grupo controle (p= 0,05); este mesmo resultado foi observado ao aplicar o UPSIT em portadores de ELT norte-americanos, antes e após os mesmos terem sido submetidos à mencionada cirurgia. O teste &quot;Pastilhas de Odor¿ mostrou ser de fácil manuseio e aplicação em indivíduos adultos, apresentou alto coeficiente de confiabilidade no teste-reteste (r=0,87, p<0,001) e suas pastilhas apresentaram boa estabilidade ao armazenamento durante 3 meses. Por sua vez, os dados obtidos através do teste de discriminação de odores identificaram que tanto o grupo da terceira idade como o dos portadores de ELT apresentaram poder discriminativo e taxa de acertos inferiores (p=0.05) aos indivíduos do grupo controle. Os resultados obtidos pela aplicação do teste CATS em indivíduos norte-americanos, revelaram que pacientes ELT tanto pré- como póscirúrgicos apresentaram menor reconhecimento de emoção facial e vocal quando comparados com o grupo controle, principalmente para as emoções negativas. O fato dos testes &quot;Pastilhas de Odor¿ e de discriminação de odores desenvolvidos na presente pesquisa terem detectado deficiência olfatória nos indivíduos brasileiros da terceira idade e portadores de epilepsia, constrói validade para a utilização dos mesmos em diagnósticos clínicos associados a essas populações / Abstract: Sensory tests to evaluate olfactory function have been validated and used in the diagnosis of subjects, mainly in brain disorders patients, and in the neuroscience research. In Brazil, reliable tests of odor discrimination and identification have not been appropriately developed and validated. The aims of the present study were: i) to develop, test and validate odor discrimination and identification tests to assess olfactory function of Brazilian population; ii) to evaluate the methodologies performance in the diagnosis of elderly subjects and epilepsy patients. For odor identification test, the developed instrument was entitled &quot;Odor Tablets¿, with 36 different odors familiar to Brazilians; to verify the performance in patients with epilepsy, a methodology previously validated and used worldwide to evaluate the ability to identify odors of individuals, and iv) to evaluate in patients with epilepsy, functions that are processed by common neural substrates for olfactory function, in this case, the ability of recognition of voice and facial emotion. Odors were rated as to their intensity, pleasantness, pungency, coolness and familiarity and they were considered suitable for composing a test to assess olfactory function. &quot;Odor Tablets¿ proceeded through multiple-choice test with four alternatives which only one was correct. For the development of odor discrimination test, 24 pure odorants, associated with six different odor categories ¿ sweet, green, fruity, citric, floral and unpleasant - were selected and diluted in propylene glycol. With these samples, 36 paired comparison tests were constructed, where in each odor category, a target sample was selected to be compared with the others in the same category. The participant¿s performance in the odor discrimination test was analyzed using the &quot;signal-detection" theory through the following parameters: hit rate (HR), false-alarm rate (FR), discrimination measurement (d 'L) and bias response (CL). Both tests, &quot;Odor Tablets¿ and odor discrimination test, were validated with three groups of subjects: control group, elderly group and temporal lobe epilepsy patients group (TLE). The ability to identify odors from North American TLE patients was also measured, using for this purpose, the validated test "University of Pennsylvania Smell Identification Test" (UPSIT), a multiple choice test consisting of 40 microencapsulated odor stimuli. In addition, the ability of recognition of voice and facial emotion of TLE patients were evaluated using a new tool entitled "Comprehensive Affect Testing System" (CATS). For the subject¿s ability to identify odors, there were significant differences for gender (p=0.0003) and age (p<0.001). Also, for this test, the results showed that pre- and postsurgery epilepsy patients presented lower performance than the control group (p= 0.05); This same result was observed when applying the UPSIT in American TLE patients before and after surgery. &quot;Odor Tablets¿ proved to be easy to administer in adult subjects, showed a high coefficient of the test-retest reliability (r = 0.87, p<0.001), and the tablets presented a storage stability for 3 months. The discrimination test results showed that elderly and epilepsy patient groups presented lower performance in the discrimination measurement and hit rate parameters than the control group. The results obtained using the CATS test in American TLE patients revealed that both pre-and post-surgery showed deficits in the facial and vocal emotions when compared with the control group, especially for negative emotions. The fact that the &quot;Odor Tablets¿ and the discrimination odor test developed in the present study had detected olfactory dysfunction in elderly subjects and epilepsy patients, provides their validity for use in the diagnoses of these population / Doutorado / Consumo e Qualidade de Alimentos / Doutor em Alimentos e Nutrição

Malgré leur domestication il y a plus de 7000 ans, gérer la réponse défensive des abeilles, en particulier contre l'Homme, reste un défi. Cet état de fait est dû en partie à la complexité de cette réponse, qui commence par la détection du danger par quelques individus spécialisés et culmine dans une attaque collective, déclenchée par une phéromone d'alarme. Le comportement agressif des abeilles a fait l'objet de nombreuses études à la fois en laboratoire et sur le terrain, qui ont permis d'identifier les éléments déclencheurs et régulateurs de ce comportement. Cependant les mécanismes neuronaux et moléculaires qui sous-tendent cette réponse agressive sont toujours inconnus. Durant ma thèse, j'ai étudié le rôle des signaux olfactifs et des amines biogènes dans la régulation de l'agressivité des abeilles, en intégrant des approches comportementales, physiologiques et moléculaires. En utilisant un nouveau test pour mesurer la réponse agressive d'abeilles individuelles en conditions contrôlées, j'ai pu déterminer si certaines odeurs de plantes modulent l'agressivité des abeilles, en particulier en interagissant avec la phéromone d'alarme. J'ai identifié deux composés floraux, le linalol et le 2-phenylethanol, qui bloquent la réponse agressive déclenchée par la phéromone d'alarme. Ces odeurs n'empêchent pas les abeilles de sentir la phéromone d'alarme, mais ont une valeur appétitive importante pour les abeilles. Ces résultats suggèrent qu'une intégration sensorielle complexe a lieu lorsque les abeilles décident de participer ou non à la défense de la colonie. De plus, un test de terrain a montré que le linalol peut aussi être utilisé pour diminuer l'agressivité d'une colonie entière, ouvrant la voie pour des applications pratiques. Afin de mieux comprendre les mécanismes neuronaux responsables de cette modulation par des odeurs florales, j'ai ensuite regardé comment ces odeurs affectent la représentation de la phéromone d'alarme dans le centre olfactif primaire du cerveau de l'abeille, le lobe antennaire. J'ai ainsi utilisé l'imagerie calcique in vivo pour visualiser l'activité des neurones de cette région. Les lobes antennaires sont structurés en unités fonctionnelles appelées glomérules, et l'identité d'une odeur est codée par le patron d'activation des glomérules. Notre hypothèse était que la représentation d'un mélange entre une odeur de plante appétitive et la phéromone d'alarme ne peut pas être obtenu linéairement à partir de la représentation de chaque composé, révélant ainsi les mécanismes neuronaux à l'origine de l'effet de ces odeurs florales. Cependant l'analyse des données n'a pas mis en évidence ce phénomène, ce qui suggère que l'intégration de la valeur appétitive des odeurs a lieu dans des centres supérieurs. Finalement, j'ai examiné le rôle des amines biogènes dans le comportement agressif de l'abeille. Les amines biogènes sont d'importants neuromodulateurs qui ont été impliqués dans le contrôle de l'agressivité de nombreuses espèces, mais leur rôle chez l'abeille n'avait pas été démontré. Les abeilles de colonies agressives ont plus de sérotonine dans leur cerveau central que celles provenant de colonies dociles. / Although honeybees were domesticated over 7000 years ago, finding ways to manage their defensive responses against intruders, including humans, is still a current challenge. This is in part due to the complexity of this behaviour, which starts with the detection of the threat by a few specialized individuals and culminates into a generalized, collective attack triggered by the release of an alarm pheromone. Numerous studies have investigated honeybee aggression and stinging behaviour both in the laboratory and field, including the sensory triggers and the potential regulatory mechanisms. However the specific neural and molecular mechanisms regulating this behaviour are still unknown. In my PhD thesis, I investigated the role of olfactory signals and brain biogenic amines in modulating aggression in honeybees, integrating behavioural, physiological, and pharmacological experiments. Using a novel assay to measure the stinging behaviour of individual bees under controlled conditions, I first explored whether a range of plant odours could modulate aggression, in particular by interacting with the alarm pheromone released by aroused bees. I identified two floral compounds, linalool and 2-phenylethanol, that block the recruitment elicited by the alarm pheromone. These odours do not prevent the bees from perceiving the alarm pheromone. Instead, this blocking effect appears to correlate with the appetitive value of these odours. This suggests that a complex sensory integration takes place when honeybees are faced with the decision of engaging or not into defensive tasks. Furthermore, a field test demonstrated that linalool could also be used to manage aggressive colonies, highlighting the practical application of these findings. To gain a better understanding of the neuronal mechanism underlying this effect of floral odours on honeybee aggression, I next investigated how these floral compounds affect the representation of the alarm pheromone in the primary olfactory center of the honeybee brain, the antennal lobe, using in vivo calcium-imaging to monitor the activity of neurons in this area. The antennal lobes are structured into functional units called glomeruli, and an odour identity and concentration is encoded within the pattern and intensity of activated glomeruli. We expected that the representation of the mixture of an appetitive floral odour with the alarm pheromone may not be linearly obtained from the representation of each compound, thus revealing the neuronal mechanisms at play during our previous aggression assays. However, analysis of the data suggests no such mechanism, which could be a clue that this processing is happening in higher brain centers. Finally, I investigated the role of brain biogenic amines in honeybee aggression. Biogenic amines are important neuromodulators and have been implicated in the regulation of the aggressive behavior of a number of species.

Laurel wilt disease is a vascular wilt affecting the xylem and water conductivity in trees belonging to the family Lauraceae. The disease was introduced by an invasive species of ambrosia beetle, Xyleborus glabratus. The beetle, together with its newly described fungal symbiont Raffaelea lauricola (pathogenic to host trees), has lead to the devastation and destruction of over 300 million wild redbay trees in southeastern forests. Ambrosia beetles make up a very unique clade of beetle and share a co-evolved obligatory mutualistic relationship with their partner fungi. Rather than consuming host tree material, the beetles excavate galleries or canals within them. These galleries serve two purposes: reproduction and fungal gardening. The beetles house fungal spores within specialized sacs, mycangia, and essentially inoculate host trees with the pathogenic agent. They actively grow and cultivate gardens of the fungus in galleries to serve as their sole food source. Once the fungus reaches the xylem vessels of the host tree, it thrives and leads to the blockage of water flow, both because of fungal accumulation and to the host response of secreting gels, gums and tyloses to occlude vessels in an attempt to quarantine the fungus. This disease spreads rapidly, and as a result, once symptoms become visible to the naked eye, it is already too late to save the tree, and it has likely already spread to adjacent ones. The present study presents the first documented study involving the early detection of disease from deep within a tree through the use of scent-discriminating canines. In addition, the present study has lead to the development of a novel sample collection device enabling the non-destructive sampling of beetle galleries. Finally, a metabolomics approach revealed key biochemical pathway modifications in the disease state, as well as potential clues to disease development.

Les enzymes du métabolisme des xénobiotiques (EMX) jouent un rôle central dans la biotransformation et l’élimination de composés étrangers à l’organisme. Organisées en réseau de biotransformation, ces enzymes catalysent des réactions variées (oxydations, conjugaisons …) afin de rendre ces composés plus hydrophiles. Les EMX sont fortement exprimées dans l’épithélium olfactif de mammifères et des études in vitro ont montré qu’elles possèdent des activités élevées. Outre un rôle dans le métabolisme de molécules toxiques, ces enzymes pourraient être impliquées dans la modulation périphérique de l'olfaction. Les EMX pourraient contribuer à maintenir la sensibilité du système olfactif en éliminant ou en inactivant les molécules odorantes présentes dans l'espace périrécepteur. Cependant, les preuves scientifiques démontrant l’implication des EMX dans la perception olfactive restent limitées à ce jour. La première étape de ce travail a consisté à montrer que l’expression des EMX olfactives de rat peut être modulée par des traitements chimiques connus pour leur capacité d'induction au niveau hépatique (dexaméthasone, Aroclor, …). Par rapport au foie, les effets observés au niveau de la muqueuse olfactive sont plus modérés. La dexaméthasone, un glucocorticoïde de synthèse, induit significativement l’expression de plusieurs enzymes et transporteurs au niveau du tissu olfactif. Cette étude suggère que l’expression des EMX olfactives soit sous le contrôle de mécanismes spécifiques. Dans une deuxième partie, le métabolisme in vitro de quelques molécules odorantes a été étudié. Nous avons observé que la muqueuse olfactive de rat possède une forte capacité métabolique et catalyse notamment la formation de métabolites hydroxylés. Enfin, les conséquences de ce métabolisme ont été évaluées en enregistrant des électro-olfactogrammes (EOG) au niveau de la muqueuse olfactive de rat. Nous avons montré qu’à concentrations équivalentes, l’amplitude des signaux EOG générés par des métabolites est beaucoup plus faible que l’amplitude des réponses élicitées par les molécules odorantes non biotransformées. De plus, la perfusion de la muqueuse par des inhibiteurs spécifiques de cytochromes P450 ou de carboxylestérases provoque une modification significative des signaux EOG. Ces études démontrent que la biotransformation des molécules odorantes a un impact sur le signal olfactif. L’ensemble de ces études conforte l’hypothèse d’un rôle des EMX dans la première étape de la perception olfactive. Ces enzymes pourraient catalyser la biotransformation des molécules odorantes dans le but d’éviter une saturation du système olfactif. / Xenobiotic metabolizing enzymes (XMEs) play a central role in the biotransformation and the elimination of foreign compounds reaching the body. Organized as a biotransformation network, these enzymes catalyze various reactions (oxidations, conjugations …) in order to increase the water solubility of compounds. XMEs are highly expressed in the mammalian olfactory epithelium and in vitro studies showed that they have important activities. Besides a role in the metabolism of toxic compounds, a role in the peripheral regulation of the olfactory process has been hypothesized. XMEs could contribute to maintain the sensibility of the olfactory system, by eliminating or inactivating odorant molecules present in the perireceptor space. However, scientific proofs of such a role are still limited. In a first study, we demonstrated that olfactory XMEs can be modulated by chemical treatments identified to induce the enzyme expression in the liver (dexamethasone, Aroclor …). The intensities of the effects were lower in the OM than in the liver. Dexamethasone, a synthetic glucocorticoid, was found to increase significantly the expression of several enzymes and transporters in the olfactory mucosa. This study suggests that the expression of olfactory XME is controlled by tissue-specific mechanisms. In the second part, the metabolism of some odorant molecules was studied in vitro. We observed that the olfactory mucosa of rats possesses a high metabolic capacity and catalyses the formation of hydroxylated metabolites. Finally, the consequences of this metabolism were evaluated by recording electro-olfactograms (EOG) from the olfactory mucosa of rats. We showed that, at same concentrations, amplitudes of EOG signals generated by the metabolites were lower than those elicited by the parent molecules. Moreover, the perfusion of the mucosa with specific inhibitors of cytochromes P450 or carboxylesterases induced a significant modification of EOG signals. These studies demonstrate that the biotransformation of odorant molecules have an impact on the olfactory signal. Taken together, these studies support the hypothesis that XME are involved in the first stage of the olfactory perception. These enzymes could catalyze the biotransformation of odorant molecules in order to avoid the saturation of the olfactory system.

Os odores são detectados por uma grande família de receptores olfatórios (ORs) que são expressos nos neurônios olfatórios localizados no nariz. Os ORs ativados por um determinado odor acoplam-se à proteína Galfaolf que irá promover a ativação da adenilil ciclase III, resultando na produção de AMPc. O aumento da concentração de AMPc irá ativar canais iônicos dependentes de AMPc, tendo como consequência a despolarização do neurônio olfatório. A informação desencadeada pela ativação de determinados ORs é então transmitida para regiões específicas do cérebro promovendo a percepção do odor. A determinação da especificidade dos ORs para diferentes odores irá contribuir para o entendimento de como os odores são discriminados pelo sistema olfatório, entretanto, poucos ORs tiveram seus ligantes definidos devido a dificuldade de expressão funcional de ORs em sistema heterólogo. Em nosso trabalho, utilizamos o sistema de duplo-híbrido em levedura a fim de determinar potenciais novos reguladores para Galfaolf. Deste experimento, identificamos que Ric-8B (Ric, abreviatura de Resistant to Inhibitors of Cholinesterase), um provável GEF (GTP Exchange Factor), é capaz de interagir com Gaolf. Assim como Gaolf, Ric-8B é predominantemente expresso nos neurônios olfatórios maduros e em regiões específicas do cérebro. A restrita co-localização de Gaolf e Ric-8B fortemente indica que Ric-8B é uma proteína que participa da via de transdução de sinal de Galfaolf. Através de nossos ensaios, utilizando células HEK-293, foi possível mostrar que Ric-8B é capaz de potencializar a atividade de Galfaolf, tendo como consequência o aumento da produção de AMPc em sistema heterólogo. Por fim, nós mostramos que Ric-8B é capaz de promover a expressão funcional de ORs em sistema heterólogo. Nossos resultados demonstram que a expressão de Ric-8B é capaz de aumentar o acúmulo de Galfaolf na periferia de células HEK-293T, indicando que Ric-8B promove a expressão funcional de ORs provavelmente através da melhora da eficiência do acoplamento dos ORs com Galfaolf. Nossos resultados demonstram que o uso de Ric-8B em um sistema em larga escala irá permitir a expressão funcional de diversos ORs, permitindo a identificação de seus respectivos ligantes. Tal análise irá contribuir para o melhor entendimento do mecanismo de percepção dos odores. / Odorants are detected by a large family of odorant receptors (ORs) expressed in the olfactory neurons in the nose. The activated receptors couple to an olfactory-specific G-protein (Galphaolf), which activates adenylyl cyclase III to produce cAMP. Increased cAMP levels activate cyclic nucleotide-gated channels, causing cell membrane depolarization. The information provided by the odorant receptors is transmitted to specific regions of the brain leading to odorant perception. The determination of the odorant specificities of the different ORs will contribute to the understanding of how odorants are discriminated by the olfactory system. However, only a few ORs have been linked to odorants they recognized to date because ORs are not efficiently expressed in heterologous cells since they are poorly expressed on the cell surface. Here we used yeast two-hybrid to search for potential regulators for Galphaolf. We found that Ric-8B (for Resistant to Inhibitors of Cholinesterase), a putative GTP exchange factor, is able to interact with Gaolf. Like Gaolf, Ric-8B is predominantly expressed in the mature olfactory sensory neurons and also in a few regions in the brain. The highly restricted and colocalized expression patterns of Ric-8B and Galphaolf strongly indicate that Ric-8B is a functional partner for Galphaolf. We show that Ric-8B is able to potentiate Galphaolf-dependent cAMP accumulation in human embryonic kidney 293 cells and therefore may be an important component for odorant signal transduction. Finally, we show that Ric-8B promotes efficient heterologous expression of ORs. Our results show that Ric-8B enhances accumulation of Galphaolf at the cell cortex, indicating that it promotes functional OR expression probably by improving the efficiency of OR coupling to Galphaolf. Our results demonstrate that the employment of Ric-8B in a high-throughput system will allow the functional screening of the OR family members and thereby provide further insight into the mechanisms of odor perception.