O sistema olfatório de mamífero pode discriminar milhares de odores presentes no meio ambiente. Aproximadamente 1000 diferentes receptores olfatórios (ORs) são expressos no epitélio olfatório (OE) do nariz, Os ORs detectam os odores e transmitem os sinais resultantes para o bulbo olfatório (OB) no cérebro. Os ORs pertencem a super família dos receptores acoplados a proteína G (GPCR) e apresentam sete domínios transmembrânicos putativos. Por razões desconhecidas, os ORs são retidos no retículo endoplasmático quando expressos em linhagens de células de mamíferos heterólogas. Provavelmente, proteínas acessórias sejam requeridas para o endereçamento dos Ors para a superficie celular. No presente estudo, utilizamos o OR M93 para estudar os mecanismos de expressão de um ORo A dissertação teve como objetivos específicos: (l) construção de um vetor para expressão do OR M93 em fusão com GFP em levedura e análise de sua localização celular; (2) identificar proteínas expressas no epitélio olfatório de camundongo que interajam com os ORs. A análise por microscopia de fluorescência revelou que a expressão do OR M93 fusionado a GFP demonstrou um padrão de fluorescência que sugere a retenção do OR M93 no retículo endoplasmático. Nós utilizamos o sistema de duplo híbrido em levedura para varrer uma biblioteca de cDNA de epitélio olfatório de camundongo com uma isca correspondente à região N-terminal do OR M93. Quatro proteínas candidatas foram identificadas: HLA-B associado ao transcrito 3 (BAT-3/ Scythe), superfamília transmembrana 4 (membro CD82), superfamília transmembrana 4 (membro OAP-I) e sindecan (membro SDC2) (\"GenBank accession numbers\": BC026647, D14883, BC0430n e BC047144). A análise da hibridação in situ destas proteínas, revelou que a proteína OAP-1 é a melhor candidata a interação com OR M93. Dessa maneira, nós indicamos a proteína OAP-1 como possível proteína candidata a auxiliar o OR a ser expresso de maneira funcional em sistemas heterólogos. / The mammalian olfactory system can discrim inate thousands of odorants present in the environrnent. Approximately 1000 different olfactory receptors (ORs) are expressed in the olfactory epithelium (OE) of the nose. The ORs detect odorants and transmit the resulting signals to the olfactory bulb (OB) of the brain. ORs belong to the G-protein-coupled receptor (GPCR) super family and have seven putative transmembrane domains. For unknown reasons, the ORs are retained in the endoplasmatic reticulum when expressed in heterologous mammalian cell lines. Probably accessory proteins are required for the sorting of the ORs to the cell surface. In the present work, we used the OR M93 to study the mechanisms of OR expression. Our goals were to (1) construct an expression vector for OR M93 in fusion with GFP in yeast and (2) to identify proteins expressed in the mouse OE that interact with ORs. The analysis by fluorescence microscopy suggested that OR M93 in fusion with GFP was retained in the endoplasmic reticulum (ER) of yeast. We used the yeast two-hybrid system to screen a mouse OE cDNA library with a bait corresponding to the N-terminal region ofthe üR M93. Four potential candidates were identified: HLA-B associated transcript 3 (BAT-3/Scythe), transmembrane 4 superfamily (CD82 member), transmembrane 4 superfamily (TSPN-3 member) and syndecan (SDC2). In situ hybridization analysis suggests that OAP-l protein represents the best candidate for interaction with OR M93. We suggest the OAP-l protein could be an accessory protein required for the sorting of the ORs to the cell surface in heterologous cell lines.

A functional sense of smell is mediated by Olfactory Receptor proteins (ORs), which reside in olfactory sensory neurons (OSNs) in the epithelium of our nose. Only a singular OR allele out of roughly 2,400 other OR alleles is expressed in every OSN⁽¹˒ ²⁾. Singular expression of the active OR gene occurs in a unique transcriptional hub⁽³⁻⁵⁾. This hub contains one OR promoter and many interchromosomal enhancers that converge upon the hub from far nuclear distances⁽⁵˒ ⁶⁾. Once in the hub, the enhancers work in tandem with each other and with the transcription factors (TFs) Lhx2, EBF, and their cofactor, Ldb1⁽⁵˒ ⁷˒ ⁸⁾ The Greek islands contain a novel “composite” motif containing an Lhx2 and EBF binding site directly next to each other⁽⁸⁾. My work aims to understand how these proteins interact with each other and the composite motif to contribute to the accumulation of many enhancers around only a single promoter in the hub. Our findings illuminate how transcription factor interactions contribute to the hub's unique DNA architecture. To investigate the biochemical foundation of OR hubs, we used 𝑒. 𝑐𝑜𝑙𝑖 to grow and purify full-length and truncated forms of Lhx2, Ebf1, and Ldb1. We used the recombinant proteins with other biochemical methods to characterize the interactions between Lhx2, Ebf1, Ldb1, and different types of DNA found in the OR hub. We used EMSAs to quantify the binding affinity that Lhx2 and Ebf1 have for promoter versus enhancer DNA. Finally, we mixed the purified full-length proteins and used fluorescence microscopy to visualize their behavior in solution. This research combined with in vivo imaging in the Lomvardas lab provides a better understanding as to how molecular interactions 𝑖𝑛 𝑣𝑖𝑡𝑟𝑜 contribute to the hub’s architecture 𝑖𝑛 𝑣𝑖𝑣𝑜, and ultimately, stable OR expression. Our biochemical studies suggest that Lhx2 and Ebf1 can bind at the same time to a single composite motif yet they seem to bind independently of one another. We have used EMSAs to determine that Lhx2 binds much better to OR enhancer DNA compared to Ebf1 and that it might stabilize enhancer contacts. We have also found that Lhx2 and Ebf1 do not cooperatively bind to enhancers- indicating that affinity alone does not explain the accumulation of TFs to the OR hub. Our 𝑖𝑛 𝑣𝑖𝑡𝑟𝑜 imaging shows that Lhx2, Ebf1, and Ldb1 self-assemble into rigid nucleoprotein condensates driven by the composite motif of enhancer DNA. This imaging work also reveals that Lhx2 and Ldb1 are scaffolding proteins with low mobility which drive rigid condensate formation over enhancers. Ebf1 displays more plasticity and turnover into condensates indicating that it plays a more complex role as a recruited factor to these assemblies. We have coupled this data with 𝑖𝑛 𝑣𝑖𝑣𝑜 imaging of endogenous Lhx2, Ebf1, and Ldb1 to find that these factors display similar binding and dynamics 𝑖𝑛 𝑣𝑖𝑣𝑜. This data helps to provide a biophysical model of how OR hubs support multi-enhancer and protein-rich environments that are succinct from their surrounding environment. Our studies suggest that the OR hub forms a rigid phase separated compartment in the nucleus- driven by Lhx2 and Ldb1. This solid-like phase separation may contribute to how singular OR expression is achieved. Such long-range enhancer contacts must stay assembled long-term for continuous OR transcription. Therefore, traditional TF DNA binding dynamics would not explain the longevity of these contacts in the OR hub. This work challenges the traditional model of liquid phase separated nuclear compartments and may provide a broader understanding to how long range and inter-chromosomal compartments are maintained.

Molécules du goût et odeurs se fixent sur des récepteurs dits gustatifs et olfactifs, présents dans la bouche et le nez. Ils sont donc en contact avec le monde environnant. Toutefois, on les trouve également dans des organes isolés de l’extérieur, comme le pancréas ou le cerveau, où ils ne sont plus impliqués dans la détection du non-soi. Ils y régulent la glycémie ou l’activation du système immunitaire. Dans le cerveau, leurs rôles demeurent mystérieux. Mon travail a consisté à déterminer: 1) si, et où les récepteurs gustatifs et olfactifs sont présents dans le cerveau humain, 2) quand, où et pourquoi les récepteurs olfactifs sont présents dans le cerveau de souris et 3) si une maladie comme Alzheimer peut modifier leur expression. Mes résultats montrent qu’ils sont présents dans l’ensemble du cerveau humain et particulièrement dans le «cerveau émotionnel». De plus, le cerveau de souris «Alzheimer» surexprime des récepteurs olfactifs, notamment dans les neurones. Le cerveau est donc capable de goûter et sentir son monde intérieur. On peut imaginer que ces récepteurs jouent un rôle dans la détection de la maladie et, qui sait, qu’ils participent à la lutte contre ses effets néfastes. / Taste molecules and odours bind to so-called gustatory and olfactory receptors present in the mouth and nose. They are therefore in contact with the surrounding world. However, they are also found in organs isolated from the outside, such as the pancreas or brain, where they are no longer involved in the detection of non-self. They regulate blood sugar levels or the activation of the immune system. In the brain, their roles remain mysterious. My work consisted in determining: 1) if, and where, taste and smell receptors are present in the human brain, 2) when, where and why smell receptors are present in the mouse brain, and 3) whether a disease like Alzheimer's can change their expression. My results show that they are present in the entire human brain and particularly in the "emotional brain". In addition, the brains of "Alzheimer" mice overexpress olfactory receptors, particularly in neurons. The brain is therefore able to taste and feel its inner world. It is conceivable that these receptors play a role in detecting the disease and, who knows, in combating its harmful effects.

A significant body of research has focused on tracking behaviors of predators responding to prey odor plumes, yet little is known about the specific mechanisms by which predators make decisions during tracking that lead them to a source. This dissertation advances the current knowledge of plume tracking behavior by examining blue crab tracking behavior over a large range of bed-roughnesses (thereby manipulating ambient levels of turbulence), and interpreting these results with respect to chemical signal structure derived from separate examinations of plume characteristics as determined by planar laser induced fluorescence (PLIF). Foraging success and the speed of blue crabs attempting to locate the odorant source both decline consistently with increasing bed roughness. In contrast, steering (path linearity) appears unaffected by bed roughness induced turbulence. The spatial arrangement of blue crab chemosensors combined with the three-dimensional structure of odorant plumes accounts for the differential effects of turbulence on the speed and success of crab tracking behavior. Separate examinations of tracking behavior and plume properties cannot directly examine hypotheses concerning the utility of specific chemical signal properties. In order to make a direct link between cue and behavior, three-dimensional laser induced fluorescence (3DLIF) was used to analyze three-dimensional plume structure and concentration of odor filaments that reach blue crab sensory structures. The corresponding tracking behavior was simultaneously recorded and then analyzed with a motion analysis system. These data provide the most comprehensive examination of odor signal input-behavioral output functions for animals in turbulent plumes. Crabs do not react differentially in response to the absolute concentration of antennule spikes above threshold at their antennules but do show a state-dependent acceleration response to antennule spikes. Signals arriving at the leg sensors of blue crabs help mediate upstream motion and signal change across a single set of leg sensors is sufficient to induce turning during upstream motion. Blue crabs decrease the height of their antennules in correspondence with changing plume properties as they approach the source and the timing of signals arriving at the antennules appears to affect upstream motion.

Dans une société allant de l'avant et voulant couper avec son côté animal, la perception olfactive a été jugée inutile et limitée à sa plus simple expression. Il n'est donc pas étonnant que la perception olfactive soit une des dimensions de notre univers sensoriel les plus difficiles à appréhender. Cette thèse de doctorat étudie la perception des odeurs chez l'Homme. La recherche s'est centrée sur deux aspects importants de l'odorat : la valence hédonique de l'odeur (son caractère agréable ou désagréable) et la qualité de l'odeur (par exemple, son caractère floral ou fruité). L'olfaction a également pour caractéristique d'être très variable d'un individu à l'autre. En effet, bien que certaines odeurs soient perçues de la même manière par la majorité de la population, de nombreuses autres sont perçues de manière très différente entre individus. Ainsi, le principal objectif de la thèse a été d'apporter des éléments d'explication des facteurs déterminant la variabilité interindividuelle dans la perception qualitative et hédonique des odeurs. Sans aller jusqu'à une explication totalement déterministe selon laquelle la perception olfactive serait « inscrite dans la molécule », les résultats de cette thèse renforcent ainsi l'idée que la perception hédonique et qualitative de l'odeur est construite à travers une hiérarchie de traitements débutant par l'association d'une structure moléculaire bien précise (ou d'un attribut particulier) et d'un ensemble de récepteurs olfactifs. L'expression différentielle de ces récepteurs olfactifs selon les individus et les populations, ainsi que l'apprentissage par simple exposition ou par acquisition d'une expertise, coloreront alors de manière personnelle la valence et la qualité perçue des odeurs, conduisant ainsi à une hétérogénéité interindividuelle en matière de perception olfactive / In a society moving forward and wanting to cut with his animal side, olfactory perception has been judged unnecessary and limited to its simplest expression. Unsurprisingly, olfactory perception was found to be one of the most difficult dimensions to understand in our sensory universe. This dissertation examines the perception of odors in humans. The research effort has been focused on two important aspects of smell: the hedonic valence of odor (ie the pleasantness or unpleasantness of the sample) and odor quality (eg, its floral or fruity note). Olfaction is also known to be highly variable from one individual to another. Indeed, although some odors are perceived in the same way by the majority of the population, many others are perceived very differently between individuals. Thus, the main objective of the research was to provide some explanation on important factors of inter-individual variability in the qualitative and hedonic perception of odors. Without making the complete deterministic claim that olfactory perception is "written in the molecule", the results in this dissertation reinforce the idea that the hedonic and qualitative perceptions of smell are built through a hierarchy of treatments starting with the combination of molecular structure (or of a particular structural attribute) and a very specific set of olfactory receptors. The difference in expression of these olfactory receptors as individuals and populations, as well as learning by mere exposure or acquisition of expertise will then color in a personal way the valence and perceived quality of odors, leading to inter-individual heterogeneity in olfactory perception