Philosophiae Doctor - PhD / Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication.

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

Environmental enrichment is commonly used to improve the welfare of captive animals by offering options to increase behavioural diversity and normal behaviour patterns. The aim of this study was to assess if environmental enrichment has a positive effect on a group of four zoo-housed reindeer, Rangifer tarandus. Three types of environmental enrichment were used: food enrichment, olfactory enrichment, and tactile enrichment, with a spontaneous rotation to prevent habituation. Following a baseline period of ten days without enrichment, the animals’ behaviour was monitored for four months with enrichment. All behaviours shown by the reindeer during the study were categorized into locomotory behaviour, feeding behaviour, passive behaviour, social interactions, and behaviour towards enrichment. The overall level of activity of the reindeer almost doubled between the baseline period (40.8%) and the enrichment period (75.3%), with a significant increase in feeding behaviour (p < 0.001) and a significant decrease in passive behaviour (p < 0.001). Food enrichment had the strongest impact on the reindeer out of the three types of enrichment presented. Even though the reindeer showed a continuous interest towards tactile enrichment, it was low compared to the interest shown towards food enrichment. They also showed little interest in olfactory enrichment. Neophobia may explain the lack of interest by the reindeer to certain types of enrichment that were novel to them. Overall, the presentation of environmental enrichment made the behaviour of the captive reindeer clearly more similar to the behaviour of wild reindeer.

Dementia is a growing burden for society, and it is of interest to discover it at an early stage. Both subjective cognitive decline (SCD) and subjective olfactory decline (SOD) has been associated with future cognitive decline and dementia. However, subjective measures have often been criticized and are still not fully understood. I aimed to examinate the frequency of SCD and SOD and whether they are likely to measure different things and what their longitudinal effects are. The baseline sample (N=784, 35-90 years, 51% female) were split into reported SCD, SOD, combined subjective olfactory and cognitive decline, and controls. Between-subjects and within-subjects statistical tests were conducted on a subset of participants (N=307, 45 to 90 years, 52% female) comparing SCD and SOD and their olfactory ability, cognitive performance, and demographics. In the baseline sample, a total of 21.1% reported a SOD whereas only 9.9% reported a SCD, only 2.7% reported both. SOD individuals had an emerged olfactory decline at follow up, their olfactory performance was associated with performance in several cognitive tests, this was not the case for the SCD individuals. The SOD and the SCD groups differ from each other, and they appear to be rather independent from each other. They might be complementary in understanding the aging brain.

No description available.

Mammalian olfaction requires the enduring expression of a single olfactory receptor (OR) gene for the life of each sensory neuron. This is due to the fact that OR proteins play multiple roles in the coherent perception of odors, first by sensing molecular cues from the external environment, and by directing the wiring of neuronal projections faithfully from the peripheral sensory neurons to the brain. Both of these processes require singular and stable OR expression in olfactory sensory neurons (OSNs. The transcription factor Atf5 has previously been shown to enforce these modes of expression, through a process that requires the unfolded protein response (UPR). The work presented in this thesis deciphers how Atf5 enables proper OR expression and neuronal function in the olfactory system. We identify the developmental window in which UPR is activated, and provide evidence that Atf5 protein expression coincides with the assembly of a multi-chromosomal enhancer hub that drives singular and robust OR transcription, opposing a model in which precocious polygenic OR transcription initiates UPR. Further, we show that Atf5 directly regulates a collection of genes that facilitate proper OR trafficking, axonogenesis, as well as transcription factors and chromatin modifiers, which we propose to be involved in stable OR expression and neuronal maturation. Finally, we find that Atf5 has a special role in the olfactory system that cannot be replaced by its ubiquitously expressed homologue, Atf4, and that this is due to a requisite interaction between Atf5 and the bZIP transcription factor Cebpγ, and potentially other transcription factors known to be critical for olfactory function.

Gerüche repräsentieren unterschiedliche physiologische Informationen, die auf zentraler Ebene integriert und im Anschluss weiterverarbeitet und psychologisch interpretiert werden. Die Interpretation eines Geruchsobjekts und die daraus resultierende Verhaltensantwort basiert auf unzähligen Variablen, die durch vergangene Erfahrungen sowie den gegenwärtigen Kontext geformt werden. Das olfaktorische und trigeminale System (V1 und V2), zwei frühe chemosensorische Verarbeitungsebenen, liegen beiden in der Nasenhöhle und repräsentieren dort den Geruchs- sowie den taktilen Sinn. Um die Reizantworten dieser Systeme besser zu verstehen, wurden eine Literaturrecherche (Review) angefertigt und zwei Studien durchgeführt. Im Review haben wir das Phänomen der Habituation und Adaptation als den darunterliegenden Mechanismus in der menschlichen Geruchswahrnehmung untersucht. Anhand der Literaturrecherche konnte gezeigt werden, dass Habituation bzw. eine verringerte Intensitätswahrnehmung über die Zeit, relativ schnell stattfindet und dass Adaptation auf höherer zerebraler Ebene schneller abläuft als in der Peripherie. Außerdem konnten wir Merkmale der Habituation auf menschliche Geruchswahrnehmung übertragen und anhand bisher unberücksichtigter Merkmale weitere Forschungsfelder und –designs darstellen, die mit neuen Technologien einhergehen. In der ersten Studie wurden Beeinträchtigungen im olfaktorischen System (ca. 15-20 % Betroffene in der Bevölkerung) und ihre Auswirkungen auf in der Geruchsverarbeitung beteiligte neurologische Prozesse untersucht. Elf hyposmische (teilweiser Geruchsverlust) und 12 normosmische Versuchspersonen wurden während einer funktionellen Magnetresonanztomographieuntersuchung (fMRT) mit zwei Gerüchen beduftet, die mit Essen assoziiert waren (Kaffee und Pfirsich). Es zeigten sich ähnliche Aktivierungen in der hyposmischen wie in der gesunden Gruppe (Insula, OFC, limbisches System). In der hyposmischen Gruppe wurde jedoch weniger Aktivierung in der Amygdala, im linken anterioren cinulären Cortex und im rechten OFC gefunden. Außerdem zeigte sich in dieser Gruppe höhere Aktivierung im rechten parahippocampalen sowie im rechten und linken posterioren cingulären Gyrus. Diese Regionen sind für Verarbeitung und Abruf von Erinnerungen wichtig. Die Ergebnisse sprechen für eine ähnliche zentrale olfaktorische Verarbeitung zwischen den Gruppen. Hyposmische Patienten scheinen die vorhandene Beeinträchtigung jedoch durch verstärkte Geruchserinnerung kompensieren zu wollen. In einer weiteren Studie haben wir einzelne Komponenten intranasaler Geruchswahrnehmung mittels fMRT untersucht. Dafür wurden gesunden Versuchspersonen unter- oder überschwellig konzentrierte Gerüche entweder unimodal oder bimodal (mit zusätzlicher trigeminaler Komponente) präsentiert. Während der bimodalen Bedingung (kontrastiert mit der unimodalen Bedingung) waren viele der zu erwartenden geruchsverarbeitenden Areale (Insula, Thalamus, Cerebellum, postzentraler Gyrus und Cingulärer Cortex) aktiviert. Zusätzlich war eine Aktivierung des Thalamus in der frühen bimodalen Verarbeitung zu sehen, was für seine Rolle in der Aufmerksamkeitslenkung bei mehreren Stimuli sprechen könnte. Die Intensitätsverarbeitung während der bimodalen Bedingung zeigte überlappende Aktivierungen in den einfachen trigeminalen (MCC) und den komplexeren olfaktorischen Verarbeitungsebenen (bilaterale Insula, Superiorer temporaler Gyrus, OFC und cerebellum), allerdings nicht in der Amygdala. Insgesamt tragen diese Studien zu unserem Verständnis von orthonosaler Geruchwahrnehmung bei und zeigen neue Wege für zukünftige Forschung auf.

付記する学位プログラム名: 京都大学大学院思修館 / 京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20432号 / 農博第2217号 / 新制||農||1048(附属図書館) / 学位論文||H29||N5053(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 河田 照雄, 准教授 井上 和生 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Domestication is accompanied by marked changes in the phenotype of the domesticated form of a species relative to its wild ancestor. The domestic dog and its wild ancestor, the wolf, are a widely used model system to study the effects of domestication on cognition and behaviour. The aim of this study was therefore to compare the behavioural responses of wolves (Canis lupus) and domestic dogs (Canis lupus familiaris) to different odour stimuli. To this end, wooden logs were impregnated with one of four odours and repeatedly placed into the enclosure of a wolf or dog pack. The dogs (n=10) and wolves (n=9) both performed a significantly higher number of interactions with the wooden logs when these were impregnated with real blood compared to the blood odour component trans-4,5-epoxy-(E)-2-decenal, the fruity odour iso-pentyl acetate and the solvent diethyl phthalate. No significant difference in the frequency of interactions per animal was found between the wolves and dogs with any of the four odour stimuli. Sniffing was the most frequent behaviour in both wolves and dogs, followed by licking, biting, toying, and pawing. The only qualitative difference in behaviour was that the dogs, but not the wolves, displayed guarding behaviour when presented with the odorized wooden logs. The results of the present study suggest that domestication had little, if any, effect on olfactory behaviour in domesticated dogs. The results also indicate that odour-impregnated wooden logs are a suitable method of olfactory enrichment for captive wolves and domestic dogs.

This project aims to find how, why and by which means the sense of smell can create meaningful interactions in the field of escape rooms, by taking a main role in the interactions that occur in said games, by adding an additional layer of complexity and embodiment. The project finished with the finding that it is not only possible to create meaningful interactions and expand on the possibilities of smell interaction, but also by encouraging future exploration and discussion of the sense of smell as an interactive medium and material.