Dissertations / Theses on the topic 'Chemosensation'

Plants and the insects that feed on them dominate diversity in terrestrial ecosystems: half of all named species are contained within these two groups. Herbivorous insects (herbivores) are abundant and diverse, yet paradoxically, two thirds of insect orders contain no major lineages of herbivores, implying barriers to the evolution of this trophic interaction. How herbivory evolves and why herbivores are so diverse are questions that are key to understanding the processes that have shaped global biodiversity. Yet, most lineages of herbivores are ancient with sister groups either absent or too divergent for a comparative genomic analysis to yield a mechanistic understanding of both their origin and diversification. Many of the exceptions to this pattern are among the Diptera, where lineages such as the leaf-mining drosophilids in the genus Scaptomyza have emerged within the last 10 million years. Scaptomyza is particularly well-suited for identifying the adaptations associated with the evolution of herbivory because it is embedded within the paraphyletic genus Drosophila, which contains species with 25 sequenced genomes, and is closely related to D. melanogaster , the genetic model, and a taxon with one of the most well-studied nervous systems.

Behavior is thought to be one of the earliest adaptations during the evolution of herbivory and niche shifts in general. Insects undergoing a niche shift likely lose their preferences for their ancestral diet, and also evolve an attraction to novel cues indicative of their new oviposition substrate. Once females lay eggs in a new environment, herbivores must consume the new diet, despite the fact that it may contain aversive chemicals and a different balance of macronutrients compared to the ancestral diet. Using the herbivorous Scaptomyza flava as a model system, the primary aim of my dissertation was to use methods in comparative genomics, chemical ecology, ethology, and neural imaging to characterize the mechanistic basis of behavioral changes associated with the evolution of herbivory in insects.

Using a comparative genomics approach, I found that targeted gain- and loss-of-function mutations were associated with the evolution of herbivory in the genus Scaptomyza. First, four Odorant (Olfactory) Receptor (OR) genes were lost in herbivorous species of Scaptomyza , which are deeply conserved among microbe-feeding drosophilids. The OR genes lost code for receptors that detect yeast-volatiles and are known to stimulate oviposition, feeding and attraction behaviors in Drosophila species. Consistent with these losses was also a loss of detection sensitivity to ligands of these ORs, specifically short-chain aliphatic esters such as ethyl and propyl acetate, major yeast-produced odorants. S. flava female flies were also unresponsive to volatiles produced by active yeast cultures, in contrast to D. melanogaster flies.

In contrast to some other specialized lineages of Drosophila , I found no evidence of increased or mass chemosensory gene loss, with one interesting and novel exception. The majority of the genes encoding the Plus-C subfamily of Odorant Binding-like proteins (OBPs) are deleted or pseudogenized in Scaptomyza. Additional conserved cysteine residues that form disulfide bonds that stabilize the tertiary structure characterize this subfamily. Interestingly the extra disulfide bonds in Plus-C OBPs are known to be vulnerable to attack by toxic breakdown products of glucosinolates, isothiocyanates, chemicals that are characteristic of S. flava's host plants in the mustard family. Other than the loss of OBPs, I found S. flava to have multiple duplications of genes encoding ORs, OBPs, gustatory receptors (GRs) and ionotropic receptors (IRs), some of which showed evidence for positive selection (Or67b, Obp49a, Gr33a, Ir67a and Ir76a). Among receptors expressed in the gustatory system, losses, duplications and genes with selection regime changes were more often orthologs of genes expressed in bitter gustatory neurons in D. melanogaster , especially gustatory sensory neurons with a broad expression of gustatory receptor genes. Changes, such as deletions, duplications and increased amino acid substitution rates, were also found among genes encoding receptors implicated in reproductive behavior including the loss of an anti-aphrodisiac receptor, Gr68a, which could be associated with a switch from males chemically guarding mated females with anti-aphrodisiacs to physical guarding behavior where males remain on the backs of females post-mating. (Abstract shortened by ProQuest.)

Like many other organisms, insects use chemical stimuli to regulate behaviours including feeding, egg-laying, and mating. Odorant binding proteins (OBPs) are one of the crucial components of insect chemosensory system, which play an essential role in transporting the hydrophobic volatile odorant molecules to the olfactory receptors. Most of insect OBPs were investigated by using recombinant technology in bacterial cells due to its fast, cost-effective, and high production mechanism. However, one of the major concerns of bacterial expression system is that the protein is frequently expressed in an unfolded state in inclusion bodies (IBs), which requires further in-vitro protein refolding step to make the protein biologically active. While doing this, there are always high chances of protein misfolding which results in soluble or insoluble protein aggregation. Thus, it is highly important to confirm the efficiency of each refolding method, used for OBP refolding, in terms of getting the correctly folded structure of the target protein. Unfortunately, it was neglected in many previous studies, resulting in significant doubts on various functional studies of insect OBPs. In this study, I used three Helicoverpa armigera OBPs, HarmOBP2, HarmOBP5, and HarmGOBP2, as model proteins to compare the different protein refolding strategies in producing correctly folded recombinant OBPs. Along with that, I have developed a novel pH-dependent method of protein refolding which demonstrated as a more efficient and productive approach for selected HarmOBPs’ refolding compared to other used methods. Further, I also developed a novel reverse chemical ecology method to isolate and identify the candidate natural ligands from host plants for HarmOBPs. This study points out a crucial but largely ignored step of insect OBP research, protein refolding and the loopholes associated with it in previous studies, which will improve our understanding of insect chemosensation and help develop more efficient and environmentally friendly insect control strategies.

Biological functions take place within tightly controlled parameters, including pH, which is managed in part through the ventilatory chemoreflex. This reflex is mediated by central respiratory chemoreceptors (CRCs) specialized to detect blood pH/CO2. Two neuronal populations are thought to mediate this response: the serotonergic (5-HT) neurons of the medullary raphé, and the Phox2b expressing neurons of the retrotrapezoid nucleus (RTN). These groups are both responsive to CO2 stimuli in vivo and in vitro. There are also apparent one-way connections from the raphé to the RTN, which is sensitive to 5-HT. Due to its complex innervation, study of RTN neurons while isolated from other cells, especially 5-HT neurons, has been limited. Here, we developed a culture model that simplifies this circuit, limiting cell types to those found in the rostral ventral medulla. This protocol yielded healthy RTN and 5-HT neurons in vitro, as well as other cell types from that area. Upon study with patch-clamp electrophysiology, cultured RTN neurons responded to CO2 and 5-HT in similar ways to what is reported for different RTN neuron preparations. Using this model, RTN neuron chemosensitivity was significantly decreased during application of 5-HT7 antagonists (SB258719, SB269970) and a 5-HT2A antagonist (MDL 11,939). The effect of 5-HT7 antagonists was recapitulated in slice recordings. Therefore, signaling at 5-HT7 and 5-HT2A receptors is necessary for RTN neuron chemosensitivity. Exogenous 5-HT application also increased RTN neuron firing rate without potentiating the response to CO2, most likely indicating that the necessary 5-HT stimulation must come from neurons that can alter their activity during acidosis. We conclude that RTN neuron chemosensitivity is largely driven by chemosensitive 5-HT neurons, and should be considered an integrative or relay center, rather than an independently chemosensitive one.

Les animaux utilisent les signaux de leur environnement pour guider leurs comportements. De nombreux comportements cruciaux, tel que le choix du site de ponte chez les insectes, sont le résultats d’adaptation à divers signaux. Un même signal sensoriel peut être perçu et interprété différemment par deux espèces, mais les mechanismes responsables de l’evolution du comportement sont encore mal-connus. Dans la nature, la majorité des Drosophiles préfèrent pondre dans les fruits en décomposition. A l’inverse, D. suzukii préfère pondre dans les fruits mûrs. Ce comportement spécifique a fait de D. suzukii un ravageur de culture important. Le changement de préférence de ponte de D. suzukii du fruit pourri vers le fruit mûr est une opportunité pour étudier les mechanismes de l’evolution du comportement. Mon projet de thèse vise à identifier les signaux gustatifs et les composants du système sensoriel périphérique (récepteurs, neurones) impliqués dans le comportement de ponte de D. suzukii. Dans les fruits mûrs, les sucres sont présents en abondance, et pourraient être un signal chimique important pour guider la préférence de ponte de D. suzukii.Pour répondre à cette hypothèse, j’ai utilisé une approche comparative entre D. suzukii et D. melanogaster incluant (1) des test comportementaux de ponte variés et (2) l’établissement du profile transcriptomique des organes gustatifs.Ensemble, mes résultats suggèrent que la préférence de ponte de D. suzukii pour les fruits mûrs pourrait être guidée par sa forte préférence pour le fructose et le glucose. Des changements importants dans le pool des GRs pourraient être à l’origine de cette plus forte réponse aux sucres des fruits
Animal’s behavior is the direct result of its perception of the outside world. Numerous crucial behaviors, like the egg-laying site choice in insects, are the product of adaptations to specific sensory cues. Two species can detect and respond differently to the same sensory cue, but not much is known about the mechanisms underlying the evolution of behavior.The majority of Drosophila prefers to lay eggs on rotten fruits in nature. On the contrary, D. suzukii prefers to lay eggs on ripe fruits. Because of this specific behavior, D. suzukii became a major crop pest during the last decade. D. suzukii’s host shift from rotten to ripe fruits is a unique opportunity to study the mechanims of behavior evolution. My thesis project seeks to identify the gustatory cues and components of sensory system (receptors, neurons) involved in the egg-laying preference of D. suzukii for ripe fruits.In the ripe fruits, sugars (fructose, glucose, sucrose) are present in abundance, and could be an important chemical cue that guide D. suzukii egg-laying choice.To test this hypothesis, I used a comparative approach between D. suzukii and D. melanogaster which includes (1) various egg-laying behavior assays, and (2) the transcriptomic profiling of taste organs by mRNA sequencing.Together, my results suggest that D. suzukii oviposition preference for ripe fruits could be the result of its strong preference for fructose and glucose. Important changes in the GRs’ pool could be at the origin of this response to fruit sugars, by enhancing the detection of fructose and glucose notably

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.

TRPV channels play a role in both mammalian insulin signaling, with TRPV1 expression in pancreatic beta-cells, and in C. elegans insulin-like signaling through expression of OSM-9, OCR-1, and OCR-2 in stress response pathways. In response to a glucose-supplemented diet, C. elegans are know to have sensitivity to anoxic stress, exhibit chemotaxis attraction, and display reduced egg-laying rate. Transcriptome analysis reveals that glucose stimulates nervous system activity with increased transcript levels of genes regulating neurotransmitters. Ciliated sensory neurons are needed for a reduced egg-laying phenotype on a glucose-supplemented diet. Egg-laying rate is not affected when worms graze on glucose-supplemented Delta-PTS OP50 E. coli, which is defective in glucose uptake. This suggests a possible sensory neuron obstruction by exopolysaccharides produced by standard OP50 E. coli on glucose, eliciting a starvation response from the worm and causing reduced egg-laying rate. Glucose chemotaxis is affected in specific TRPV subunit allele mutants: ocr-2(vs29) and osm-9(yz6), serotonin receptor mutants: ser-1(ok345) and mod-1(ok103), and G-alpha protein mutant: gpa-10(pk362). TRPV deletion mutants had no effect on glucose chemotaxis, alluding to the modality role pf TRPV alleles in specific sensory neurons. The role of serotonin in a reduced egg-laying rate with glucose remains unclear.

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder. The genetic bases for all its different phenotypic variants are mutation in the gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1, which transports very long chain fatty acids from the cytosol into the peroxisome for its degradation. The default manifestation of mutation in ABCD1 is adreno myeloneuropathy (AMN), a slow, progressive dying-back axonopathy affecting both ascending and descending spinal cord tracts as well as in some cases, peripheral neuropathy. In the present study, we use the invertebrate model organism Caenorhabditis elegans to generate a new nematode model of X-ALD. We reveal that the pmp-4(ok396) deletion mutant reproduces the main features of X-ALD such as lipid accumulation, increased mitochondrial oxidative stress, axonopathy and altered locomotion. Given the evidence that oxidative stress plays an important role in VLCFA-induced pathogenesis of ALD, therapeutic efforts aimed at the removal of free-radicals, prevention of their formation, or restoration of ETC function seem promising. Indeed, here we describe the mitochondria-specific pharmacological effects of MitoQ in protecting against VLCFA-induced toxicity and oxidative stress and its ability to rescue the observed X-ALD phenotypes on pmp-4(ok396) mutant worms.
La adrenoleucodistrofia ligada al cromosoma X (ALD-X) es un trastorno neurodegenerativo hereditario, causado por mutaciones en ABCD1. Este gen codifica para un transportador peroxisomal que importa ácidos grasos de cadena muy larga (AGCML) del citosol hacia el peroxisoma para su posterior degradación. La adrenomieloneuropatía (AMN) es la variante fenotípica en adultos y se manifiesta como un axonopatia de progresión lenta en la médula espinal. En este estudio utilizamos el gusano Caenorhabditis elegans para generar un nuevo modelo de ALD-X. Observamos que el mutante pmp-4(ok396) reproduce las principales características de la ALD-X (acumulación de lípidos, incremento del estrés oxidativo mitocondrial, axonopatía y locomoción alterada). Basándonos en evidencias que el estrés oxidativo inducido por acumulación de AGCML juega un papel importante en la patogénesis, estrategias enfocadas en la eliminación de radicales libres, prevención de su formación o normalización de la función de la cadena de transporte de electrones mitocondrial, poseen un prometedor potencial terapéutico. Aquí demostramos que el compuesto MitoQ actua a nivel mitocondrial protegiendo en contra del estrés oxidativo y rescata los fenotipos ALD-X en los gusanos pmp-4(ok396).
La adrenoleucodistròfia lligada al cromosoma X (ALD-X) és un trastorn neurodegeneratiu hereditari, causat per mutacions en ABCD1. Aquest gen codifica per un transportador peroxisomal que importa àcids grassos de cadena molt llarga (AGCML) del citosol cap al peroxisoma per a la seva posterior degradació. La adrenomieloneuropatía (AMN) és la variant fenotípica en adults i es manifesta com una axonopatia de progressió lenta en la medul•la espinal. En aquest estudi utilitzem el cuc Caenorhabditis elegans per generar un nou model d'ALD-X. Observem que el mutant pmp-4 (ok396) reprodueix les principals característiques de l'ALD-X (acumulació de lípids, increment d'estrès oxidatiu mitocondrial, axonopatia i locomoció alterada). Basant-nos en les evidències que l'estrès oxidatiu induït per acumulació d’AGCML juga un paper important en la patogènesi, estratègies enfocades en eliminar radicals lliures, prevenirla seva formació o normalitzar la funció de la cadena de transport d'electrons mitocondrial, posseeixen un prometedor potencial terapèutic. Aquí, demostrem que el compost MitoQ actua a nivell mitocondrial protegint en contra de l'estrès oxidatiu i rescatant els fenotips ALD-X en els cucs pmp-4 (ok396).

Chemoreception is a primitive sense universally employed by organisms for finding and selecting food, rejecting toxic chemicals, detecting mates and offspring, choosing sites for egg-laying, recognizing territories and avoiding predators. Chemosensory responses are frequently modulated based on the internal environment of the organism. An organism’s internal environment undergoes regular changes in anticipation and in response to daily changes in its external environment, e.g., light-dark cycle. A resettable timekeeping mechanism called the circadian clock internally drives these cyclical changes with a ~24 hour period. Using electrophysiological, behavioral and molecular analyses, I tested where and how these two conserved processes, viz., the circadian timekeeping mechanism and the chemosensory pathway, intersect each other at organismal and cellular levels. The presence of autonomous peripheral oscillators in the chemosensory organs of Drosophila, prompted us to test whether chemosensory responses are under control of the circadian clock. I found that local oscillators in afferent (primary) chemosensory neurons drive rhythms in physiological and behavioral responses to attractive and aversive chemical signals. During the middle of the night, high level of G proteincoupled receptor kinase 2 (GPRK2), a clock controlled signaling molecule present in chemosensory neurons, suppresses tastant-evoked responses and promotes olfactory responses. G-protein mediated signaling was shown to be involved in generating optimal response to odorants. Multifunctional chemosensory clocks exert control on feeding and metabolism. I propose that temporal plasticity in innate behaviors should offer adaptive advantages to flies.

In this work, we built an optical setup and defined experimental protocols to assess responses of C. elegans neurons expressing calcium indicators while exposing them to timely controlled stimuli. We then applied this setup to assess the accuracy of C. elegans in discriminating between a group of women affected by breast cancer and a group of healthy donors. To do this we first ran a series of preliminary tests that defined the experimental parameters and protocols to run high-throughput experiments and then tested the response of the AWC neurons to the removal of urine samples collected from both groups. The results obtained prove the impressive accuracy of the AWC neuron to respond with activation upon removal of samples collected from patients affected by breast cancer of both lobular and ductal forms, at different stages. This ability can be quantified by a variable that measures the activation rate of the neuron in response to the stimulus, the neuronal activation index, or NAI. The accuracy associated with the NAI is of 97.22%, which stays the same also when considered in conjunction with the chemotaxis index, CI, through the linear combination defined by the first component of a PCA applied to the plane(NAI, CI).

The mucosa of the small intestine is clearly able to discriminate specific chemical components of ingested meals to stimulate gastrointestinal feedback pathways and reduce further food intake. Luminal carbohydrates delay gastric emptying and initiate satiation, which are mediated by reflexes via the vagus nerve upon activation of vagal afferent endings in the mucosa. Nutrients activate these nerve fibres through intermediary epithelial cells, which release neuromediators upon transduction of luminal signals through the apical membrane. 5-hydroxytryptamine (5-HT) and glucagon-like peptide-1 (GLP-1) are released from enteroendocrine cells in response to luminal carbohydrates and both slow gastric emptying and inhibit food intake via vagal afferent pathways. The molecular mechanisms for carbohydrate detection and transduction leading to 5-HT and GLP-1 release are unknown. However molecules key to transduction of taste by receptor cells in the lingual epithelium are expressed in the gastrointestinal mucosa. The studies in this thesis aimed to investigate 1) the possibility that taste molecules expressed in the intestine form part of the carbohydrate sensing pathway that leads to 5-HT and GLP-1 release, which in turn activate mucosal vagal afferents and 2) to gauge any alterations in taste molecule expression that may relate to adaptation of carbohydrate-induced gastric motility reflexes that occurs in dietary and disease states. Firstly these studies show key taste molecules, including sweet taste receptors T1R2 and T1R3, the Gprotein gustducin (alpha-subunit Gαgust), and the taste transduction channel TRPM5, are expressed in the mouse gastrointestinal mucosa shown by RT-PCR and were further localised to individual epithelial ‘taste’ cells using immunohistochemistry. Quantification of transcript levels by real time RT-PCR revealed the proximal small intestine as the preferential site of sweet taste receptor expression along the gastrointestinal tract. This finding was also confirmed in humans using gastric and intestinal mucosal biopsies obtained at enteroscopy with significantly higher transcript expression levels in the small intestine compared to stomach. In the mouse, double label immunohistochemistry with Gα[subscript]gust antibody, as a marker of intestinal taste cells, was performed using lectin UEA-1, a marker of intestinal brush cells, and 5-HT or GLP-1 to link intestinal taste transduction to 5-HT and GLP-1 release. Results show Gα[subscript]gust is expressed within a subset of all three cell types in the small intestine but predominantly within UEA-1-expressing cells. Although Gα[subscript]gust, 5-HT and GLP-1 are largely expressed in mutually exclusive cells, within the jejunum a portion Gαgust positive cells coexpressed 5-HT or GLP-1. This Indicates a subpopulation of intestinal taste cells may be dedicated to carbohydrate-evoked gastrointestinal reflexes through 5-HT and GLP-1 mediated pathways, however, taste transduction within the small intestine appears to predominantly link to alternate mediators. After nutrient detection at the luminal surface, activation of mucosal afferents by 5-HT released from enterochromaffin cells is well documented, however although vagal afferents express GLP-1 receptors direct activation has not been demonstrated. For this purpose the effects of GLP-1 on gastrointestinal vagal afferents were investigated through single fibre recordings in in vitro tissue preparations. GLP-1 had no effect on the activity of mouse gastroesophageal vagal afferents but a rat duodenal preparation proved too problematic to be able to test GLP-1 specifically on duodenal vagal afferents. Altered gastric motility in response to carbohydrate meals due to prior dietary patterns and diabetes mellitus suggest adaptation in feedback mechanisms. Towards the second aim of this thesis taste molecule expression was quantified in fed and fasted mice by real time RT-PCR and revealed taste gene transcription is altered with the changing luminal environment, specifically transcription of taste genes was significantly decreased after feeding compared to the fasted state. Studies comparing expression in the duodenum of type 2 diabetics and non-diabetic controls show no significant difference in taste transcript levels between the two groups. However taste molecule expression was correlated to blood glucose levels in diabetics suggesting transcription of these signal molecules is adapted to both luminal and systemic carbohydrate levels. Findings in both the mouse and human gastrointestinal tract in terms of intestinal chemosensing are discussed.
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Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2009

As obesity has become one of the most prevalent metabolic diseases, and diabetes mellitus has become the seventh leading causes of death in the United States, alternative food/nutrition-based approaches to tackle obesity that are both efficacious and cost effective are in high demand. Since starch and its derived products are the principal dietary supply of glucose, strategies of using slowly digestible starch to achieve moderated glycemic response and prolonged glucose delivery, as well as to locationally digest starch into the ileum, have shown successful results such as moderation of insulinemia and reducing food intake in obese animals. An important regulator of appetite suppression is the neuroendocrine system of the gut-brain axis. Glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM), and peptide YY (PYY) are the main anorexigenic peptide products of the intestinal enterendocrine L-cells that regulate postprandial insulin levels as well as satiety signals. The stimulation of the enteroendocrine L-cells throughout the gastrointestinal tract through glucose, fatty acids and proteins has been extensively studied and confirmed. However, the stimulatory effect of complex dietary carbohydrates on L-cells is not described. In this dissertation, we investigated the in vitro intestinal cell chemosensation of L-cells to α-amylase starch digestion products, named maltooligosaccharides (MOS), and in the possible application of using slowly digestible starch delivery of MOS in vivo.

In Chapter II of this dissertation, we reported a significantly higher stimulatory effect of MOS on GLP-1 and OXM secretion compared to glucose in mouse and human L-cells, respectively. Additionally, maltotriose enhanced the relative expression of the gastrointestinal peptide, cholecystokinin. Moreover, MOS exhibited protective effects on barrier function and monolayer integrity of intestinal epithelial cells.

In Chapters III and IV, we performed a multiomics approach where transcriptomic analysis and global protein profiling of mouse L-cells treated with different types of MOS showed that the carbohydrates exhibit their effects through the induction of exocytosis of GLP-1- or OXM-containing vesicles and not through a positive regulation of the proglucagon gene expression. It is suggested that MOS induce higher secretion, but not higher synthesis, of the proglucagon gene products. In addition, maltotriose treatment downregulated the relative expression of the glucotoxicity marker, thioredoxin-interacting protein, and upregulated the relative expression of tight junction proteins supporting a role of MOS in barrier function integrity.

Translating the in vitro findings into an in vivo application that is beneficial for human health required the use of controllable tool for the delivery of MOS throughout the small intestine for sensing by a higher number of L-cells. Slowly digestible starch (SDS), compared to rapidly digestible starch, provided such a tool. For this purpose, we used alginate-entrapped SDS microspheres that digest distally into the ileum to examine the role of SDS in the intervention and prevention of obesity in C57BL/6J diet-induced obese (DIO) and lean mice models.

Results showed that 20% SDS in low-fat diets significantly improved weight loss and food intake reduction in DIO mice converted to low-fat diet for 12 weeks. Similarly, 15% SDS in high-fat diets showed significant reduction in body fat percent and significant increase in lean body mass as well as considerable reduction in weight gain rate and food intake in lean mice fed on 45% of calories high-fat diet. Immunohistochemistry of small intestine of mice in both the intervention and prevention studies revealed an even and thorough distribution of GLP-1 positive L-cells.

Overall, this dissertation proposes several insights into L-cell sensation of dietary starch-degraded MOS delivered by the consumption of slowly digestible starch. MOS exhibit unique influences on L-cell sensitivity and gut hormone productivity. Future research investigating the mechanisms of intestinal sensing of MOS, as well as the development of bioactive carbohydrate structures that could preserve body weight and modulate glucose tolerance in vivo is needed to translate these findings into nutritional recommendations and food products beneficial for human health. The intricate role of dietary carbohydrates on gut physiological response, related to satiety and food intake could be a new approach for design of foods for health applications.

Gustation, also known as taste perception, is critical for the survival of most animal species. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of sugars, bitter or toxic compounds, and pheromones. However, with a few notable exceptions, the functions of most GRs involved in feeding are unknown. Our research has focused on a cluster of highly-related Drosophila Grs, known as the Gr64 family, that have been shown to be critical for the perception of multiple sugars. Furthermore, we have demonstrated that another gene related to the Gr64 genes, Gr61a, is a sugar receptor that is narrowly tuned to a subset of pyranose sugars and may (along with the Gr64 genes) be indispensable for early fly development.

As a complementary approach to our behavioral analysis, we have examined the expression pattern of the Drosophila sugar receptors using knock-in driver alleles created by homologous recombination. As expected, most of these drivers have shown strong expression in various taste tissues. Intriguingly, some of these knock-in alleles also show expression in the maxillary palp and antenna, tissues previously thought to be involved only in olfaction. These expression patterns raise interesting questions about the true range of function of these chemosensory receptors and whether or not they might be involved in olfaction as well as gustation.

Dissertation

Sweet and bitter are two basic taste sensations perceivable thanks to taste receptor cells (TRCs) in the oral cavity. Interestingly, T1R3 sweet receptor and members of the bitter T2Rs family are also localized in different extra-oral tissues (e.g. airways system). In the context of this diffuse extra-oral chemosensory system, airways ciliated cells represent an interesting cellular population. Preliminary studies reported that their cilia exhibit bitter and sweet receptors and some elements of the chemoreception transduction pathway. To the best of our knowledge, no physiological data regarding ciliated cells sweet response in the airways have been reported in the literature. Therefore, to investigate the functional role of sweet receptors within the airways, we chose to explore the physiological behavior of murine tracheal ciliated cells stimulating them with sweet compounds by means of calcium-imaging technique. This method allows to follow the changes in intracellular calcium concentration thus showing possible calcium-mediated sweet responses. We planned to work with acute slices of mice tracheas, instead of using cells cultures, to better preserve the native conditions, avoiding excessive cells handling and artefacts introduction. We decided also to stimulate the ciliated cells with a bitter compound, yet investigated in cultured cells, to see the cellular behavior towards the two different stimulation qualities (sweet and bitter). In addition, we adopted immunohistochemical analysis to verify that the ciliated cells, used for the physiology experiments, retained the phenotypic expression of molecules of the taste transduction pathway normally expressed in native ciliated cells. Our experiments showed that some ciliated cells respond to sweet compounds. In particular, combining two different stimuli, glucose (sweet) first and then bitter (denatonium), 36% of the ciliated cells responded to both stimulations while 37% were responsive only to bitter, 2% only to sweet and 25% of the cells were unresponsive to both stimulations. Another important finding is that, when stimulating the cells with two artificial sweeteners (acesulfame K and sucralose), we observed that 31% of the cells responded to both the sweeteners, 24% only to acesulfame K and 5% only to sucralose. 40% of the cells were irresponsive to both the artificial sweeteners. Combining the artificial stimulus acesulfame K to the bitter one denatonium, 65% of the cells responded to both stimuli, 7% only to acesulfame K while 5% only to denatonium. Moreover, the immunohistochemical results confirmed the presence of α-gustducin and PLCβ2, two markers of taste signaling pathway, in the ciliated cells. The above described data are interesting and clearly demonstrate that the ciliated cells of murine trachea are able to perceive and respond to sweet compounds, natural and artificial, with possible implications in the glucose sensing mechanisms in the airways, especially in relationship with respiratory infections. Moreover, it is clear that the ciliated cells exhibit a different pattern of response suggesting their heterogeneity, according with the previous literature. These data might be important for considering this cellular population a new cellular model of extra-oral chemoreception investigation. Future research could unravel the roles of airways ciliated cells in health and pathological conditions with a possible therapeutic aim.Sweet and bitter are two basic taste sensations perceivable thanks to taste receptor cells (TRCs) in the oral cavity. Interestingly, T1R3 sweet receptor and members of the bitter T2Rs family are also localized in different extra-oral tissues (e.g. airways system). In the context of this diffuse extra-oral chemosensory system, airways ciliated cells represent an interesting cellular population. Preliminary studies reported that their cilia exhibit bitter and sweet receptors and some elements of the chemoreception transduction pathway. To the best of our knowledge, no physiological data regarding ciliated cells sweet response in the airways have been reported in the literature. Therefore, to investigate the functional role of sweet receptors within the airways, we chose to explore the physiological behavior of murine tracheal ciliated cells stimulating them with sweet compounds by means of calcium-imaging technique. This method allows to follow the changes in intracellular calcium concentration thus showing possible calcium-mediated sweet responses. We planned to work with acute slices of mice tracheas, instead of using cells cultures, to better preserve the native conditions, avoiding excessive cells handling and artefacts introduction. We decided also to stimulate the ciliated cells with a bitter compound, yet investigated in cultured cells, to see the cellular behavior towards the two different stimulation qualities (sweet and bitter). In addition, we adopted immunohistochemical analysis to verify that the ciliated cells, used for the physiology experiments, retained the phenotypic expression of molecules of the taste transduction pathway normally expressed in native ciliated cells. Our experiments showed that some ciliated cells respond to sweet compounds. In particular, combining two different stimuli, glucose (sweet) first and then bitter (denatonium), 36% of the ciliated cells responded to both stimulations while 37% were responsive only to bitter, 2% only to sweet and 25% of the cells were unresponsive to both stimulations. Another important finding is that, when stimulating the cells with two artificial sweeteners (acesulfame K and sucralose), we observed that 31% of the cells responded to both the sweeteners, 24% only to acesulfame K and 5% only to sucralose. 40% of the cells were irresponsive to both the artificial sweeteners. Combining the artificial stimulus acesulfame K to the bitter one denatonium, 65% of the cells responded to both stimuli, 7% only to acesulfame K while 5% only to denatonium. Moreover, the immunohistochemical results confirmed the presence of α-gustducin and PLCβ2, two markers of taste signaling pathway, in the ciliated cells. The above described data are interesting and clearly demonstrate that the ciliated cells of murine trachea are able to perceive and respond to sweet compounds, natural and artificial, with possible implications in the glucose sensing mechanisms in the airways, especially in relationship with respiratory infections. Moreover, it is clear that the ciliated cells exhibit a different pattern of response suggesting their heterogeneity, according with the previous literature. These data might be important for considering this cellular population a new cellular model of extra-oral chemoreception investigation. Future research could unravel the roles of airways ciliated cells in health and pathological conditions with a possible therapeutic aim.

Signal transduction pathways mediated by G protein-coupled receptors (GPCRs) and their intracellular coupling partners, the heterotrimeric G proteins, are crucial for several physiological functions in eukaryotes, including humans. This thesis describes a broad genomic survey and extensive comparative phylogenetic analysis of GPCR and G protein families from a wide selection of eukaryotes. A robust mining of GPCR families in fungal genomes (Paper I) provides the first evidence that homologs of the mammalian families of GPCRs, including Rhodopsin, Adhesion, Glutamate and Frizzled are present in Fungi. These findings further support the hypothesis that all main GPCR families share a common origin. Moreover, we clarified the evolutionary hierarchy by showing for the first time that Rhodopsin family members are found outside metazoan lineages. We also characterized the GPCR superfamily in two important model organisms (Amphimedon queenslandica and Saccoglossus kowalevskii) that belong to different metazoan phyla and which differ greatly in morphological characteristics. Curation of the GPCR superfamily (Paper II) in Amphimedon queenslandica (an important model to understand evolution of animal multicellularity) reveals the presence of four of the five GRAFS families and several other GPCR gene families. However, we find that the sponge GPCR subset is divergent from GPCRs in other studied bilaterian and eumetazoan lineages. Mapping of the GPCR superfamily (Paper III) in a hemichordate Saccoglossus kowalevskii (an essential model to understand the evolution of the chordate body plan) revealed the presence of all major GPCR GRAFS families. We find that S. kowalevskii encodes local expansions of peptide and somatostatin- like GPCRs. Furthermore, we delineate the overall evolutionary hierarchy of vertebrate-like G protein families (Paper IV) and provide a comparative perspective with GPCR repertoires. The study also maps the individual gene gain/loss events of G proteins across holozoans with more expanded invertebrate taxon sampling than earlier reports. In addition, Paper V describes a broad survey of nematode chemosensory GPCR families and provides insights into the evolutionary events that shaped the GPCR mediated chemosensory system in protostomes. Overall, our findings further illustrate the evolutionary hierarchy and the diversity of the major components of the G protein-coupled receptor signaling system in eukaryotes.

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.

The left-right axis is established during early development in four steps. First the asymmetry is broken through the cilia movement within the left-right organizer (LRO) generating a leftward fluid flow. Second, the flow, or what it transports, is sensed by some organizer cells that produce an asymmetric signal. Such signal triggers a genetic cascade that transmits the asymmetric information from the organizer to the lateral plate mesoderm. Ultimately, leading to an organ-specific morphogenesis with visceral organs being placed in the correct side of the body plan. Two hypotheses try to explain how the fluid flow is sensed. The Chemosensation model proposes that a morphogen accumulates on the left side of the LRO where it is perceived by ciliated cells. To test this model we studied several taste sensing-related genes. We focused on gnaia, a gene encoding a G protein alpha subunit, highly expressed in the zebrafish LRO. However, we could not draw definitive conclusions, as gnaia knockdown did not produce major left-right defects. The second hypothesis, the ‘two cilia model’ is based on mechanosensation and predicts that two cilia populations have different functions in the LRO: the motile cilia generate the directional flow and the immotile cilia sense it through the Pkd1l1-Pkd2 complex. To test this hypothesis we looked for the expression patterns of possible downstream targets of Pkd2, screening for left-right asymmetries. However, we were not able to find new asymmetric genes, confirming that, so far, dand5 is still the only asymmetrically expressed gene in the LRO. The two-cilia model also raised the question of what makes these two cilia populations different. In order to try to understand if the difference between motile and immotile cilia was structural, we looked for the localization of Dnal1, a crucial dynein component of outer dynein arms. Results showed mCherry-Dnal1 is expressed in both cilia types, suggesting that LRO cilia may be structural identical.