Dissertations / Theses on the topic 'Chemosensory proteins'

La perception des signaux chimiques de l’environnement est un processus nécessaire aux interactions sociales entre les animaux. La Drosophile détecte les molécules odorantes et sapides grâce à ses systèmes gustatif et olfactif impliquant plusieurs familles multigèniques de chimiorécepteurs. Ainsi, ces composés chimiques pénétrant dans l'organe sensoriel (sensille) doivent être solubilisés avant d'être transportés à travers la lymphe sensillaire hydrophile baignant les dendrites des neurones chimiosensoriels. Ces événements périrecepteurs font intervenir plusieurs familles de protéines solubles parmi lesquelles se trouvent les odorant-binding proteins (OBPs). Si les OBPs ont été initialement identifiées dans les sensilles olfactives, certaines sont également exprimées dans les sensilles gustatives. La fonction physiologique des OBPs est encore peu connue mais certaines études révèlent que ces protéines agissent comme transporteurs de molécules lipophiles. Les affinités relativement faibles des OBPs pour les odorants ainsi que leur abondance dans la lymphe sensillaire suggèrent que ces protéines peuvent se lier, solubiliser et transporter des molécules hydrophobes jusqu’aux chimiorécepteurs en traversant la lymphe sensillaire hydrophile. De nouveaux rôles ont été attribués aux OBPs, et en particulier leur capacité à «tamponner» des changements soudains de concentrations d'odorants et leur implication dans la détection de l’humidité. Récemment, l’OBP49a exprimée dans les sensilles gustatives, a été montrée comme étant impliquée dans la détection de certains composés amers. Comme le rôle pérircepteur des OBPs reste encore très peu compris, l'objectif de mon projet de thèse a consisté à clarifier l'implication de certaines OBPs dans l'odorat et le goût chez Drosophila melanogaster. Ma thèse a d’abord consisté à mesurer le rôle des OBPs dans la perception des composés alimentaires chez les adultes D. melanogaster. Les OBPs exprimées dans les appendices gustatifs ont été identifiées par q-PCR et produites en utilisant un système d'expression hétérologue, la levure. Les propriétés de liaison des OBPs recombinantes purifiées ont ensuite été testées pour leur capacité à lier de nombreux ligands potentiels. L’OBP19b est capable de lier certains acides aminés. La cartographie des sensilles et des cellules exprimant l’OBP19b révèle que cette protéine est uniquement exprimée dans certaines cellules accessoires de sensilles précises du labellum. L’OBP19b a été aussi localisée dans le tube digestif et dans certains organes reproducteurs. La comparaison des réponses comportementales et électrophysiologiques sensillaires des mouches témoins et des mouches transgéniques a confirmé que l’OBP19b est impliquée dans la détection de certains acides aminés. De plus, la comparaison des séquences protéique a révélé sa relativement haute conservation au sein des espèces de Drosophilidae et même entre Diptères, ce qui suggère qu’elle joue un rôle crucial vis-à-vis de la recherche de nutriments chez ce groupe d’espèces. J’ai ensuite étudié le rôle de l’OBP28a dans l’olfaction. Cette OBP, l’une des plus abondante dans les antennes de Drosophile, a été montrée importante pour tamponner les variations soudaines de concentrations d'odorants. Des études structurales, génétiques, biochimiques, comportementales et électrophysiologiques ont été réalisées en collaboration avec les membres de l’équipe. L’OBP28a a d'abord été exprimée puis purifiée et sa structure 3D a été résolue. L'étude de ses propriétés de liaison a révélé la capacité de l'OBP28a à se lier à des composés floraux tels que la β-ionone. Les mesures comportementales et électrophysiologiques ont confirmé son rôle physiologique dans la détection de la β-ionone. En conclusion, ma thèse de doctorat met en évidence les rôles nouveaux de deux OBPs dans la chimioréception: l’OBP28a est impliquée dans le détection de molécules florales alors que l’OBP19b est nécessaire pour détecter certains acides aminés
Chemoperception is used by animals to detect nutritive food and avoid toxic compounds. It also allows animals to identify suitable ecological niche and mating partners. Like many other insects, Drosophila melanogaster possesses a very sensitive chemosensory ability and can detect and discriminate a wide panel of semiochemicals. Chemosensory detection is mostly mediated by olfactory and gustatory systems involving several multigene chemoreceptor families. Volatile and non-volatile chemical compounds entering the sensory organ (sensillum) must be solubilized before being transported through the hydrophilic sensillum lymph bathing the dendrites of chemosensory neurons. These perireceptor events involve a family of soluble proteins named odorant-binding proteins (OBPs). Despite the fact that OBPs were initially found in olfactory sensilla, some OBPs are also expressed in gustatory sensilla. While their physiological roles in olfaction and gustation remain unclear, many studies suggest that OBPs transport lipophilic chemicals. The relatively low affinity of OBPs for odorants and their high abundance in the sensillum lymph both suggest that OBPs can bind, solubilize and transport hydrophobic stimuli to the chemoreceptors across the aqueous sensilla lymph. In addition to this broadly accepted “transporter role” hypothesis, OBPs have also been proposed to buffer sudden changes in odorant levels and to be involved in hygroreception. The role of OBP49a was recently shown in taste: this OBP, expressed in the gustatory system, is required to detect some bitter compounds. However, the role of OBPs in perireceptor events remains largely unknown. The main goal of my thesis project consisted to investigate the involvement of OBPs in the smell and taste sensory modalities using a multi-faceted approach in Drosophila melanogaster.My first research axis consisted to better understand the role of OBPs in the perception of food compounds by using both in vitro and in vivo approaches of OBPs expressed in the gustatory appendages of D. melanogaster adults. After identifying by q-PCR the OBPs expressed in gustatory appendages, we produced them using a heterologous yeast expression system. Then, the binding properties of the recombinant purified OBP were investigated. Our binding assay screen revealed that the taste-expressed OBP19b is able to bind some amino acids. The expression of OBP19b was mapped in specific accessory cells in a subset of proboscis sensilla. This OBP was also expressed in the digestive tract and in some internal reproductive organs. The comparison of behavioural and single-taste sensilla responses between transgenic variants and control flies supported our finding that OBP19b is indeed involved in the detection of some amino acids. Finally, the comparison between various dipteran insects of the OBP19b-like protein coding sequence indicates the relatively high conservation of this protein suggesting its critical role in food search.The second research axis of my PhD thesis focused on the olfactory role of OBP28a. OBP28a was previously shown to be highly expressed in the Drosophila antennae and proposed to buffer quantitative odour variations. To better understand the physiological role of this OBP, and in collaboration with different members of the team, we used structural, genetic, biochemical, behavioural and electrophysiological methods to better understand the role of this OBP. OBP28a was first heterologously expressed and purified. The folding of OBP28a was then determined and the protein was crystallized. The study of the binding properties of OBP28a revealed that it can bind floral compounds such as β-ionone. Behavioural and electrophysiological recordings supported the physiological role of OBP28a in β-ionone detection. In summary, this PhD thesis reveals novel roles of two OBPs in perireceptor chemoreception: OBP28a in the detection of floral compounds and OBP19b in the detection of some amino acids

The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW₃ proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.

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.

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.

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. In the sensilla shaft the dendrite of OSNs are housed, which are protected by called the sensillum lymph produced by support cells and contains a variety of olfactory proteins, including the odorant binding protein (OBP) and chemosensory proteins (CSP). While on the dendrite of OSNs are expressed olfactory receptors. In my PhD, studies I tried to decipher the sense of smell in tsetse fly. In the second chapter, I demonstrated that G. f. fuscipes is equipped with diverse olfactory sensilla, that various from basiconic, trichoid and coeloconic. I also demonstrated, there is shape, length, number difference between sensilla types and sexual dimorphism. There is a major difference between male and female, while male has the unique basiconic sensilla, club shaped found in the pits, which is absent from female pits. In my third chapter, I investigated the odorant receptors which are expressed on the dendrite of the olfactory sensory neurons (OSNs). G. f. fuscipes has 42 ORs, which were not functionally characterised. I used behaviourally well studied odorants, tsetse repellents, composed of four components blend. I demonstrated that tsetse repellent is also a strong antifeedant for both G. pallidipes and G. f. fuscipes using feeding bioassays as compared to the attractant odour, adding the value of tsetse repellent. However, the attractant odour enhanced the feeding index. Using DREAM (deorphanization of receptors based on expression alterations of mRNA levels). I found that in G. f. fuscipes, following a short in vivo exposure to the individual tsetse repellent component as well as an attractant volatile chemical, OSNs that respond to these compounds altered their mRNA expression in two opposite direction, significant downregulation and upregulation in their number of transcripts corresponding to the OR that they expressed and interacted with odorant. Also, I found that the odorants with opposite valence already segregate distinctly at the cellular and molecular target at the periphery, which is the reception of odorants by OSNs, which is the basis of sophisticated olfactory behaviour. Deorphanization of ORs in none model insect is a challenge, here by combining DREAM with molecular dynamics, as docking score, physiology and homology modelling with Drosophila a well-studied model insects, I was able to predict putative receptors of the tsetse repellent components and an attractant odour. However, many ORs were neutral, showing they were not activated by the odorants, demonstrating the selectivity of the technique as well as the receptors. In my fourth chapter, I investigated the OBPs structures and their interaction with odorants molecules. I demonstrated that OBPs are expressed both in the antenna, as well as in other tissues, such as legs. I also demonstrated that there are variations in the expression of OBPs between tissues as well as sexes. I also demonstrated that odorants induced a fast alteration in OBP mRNA expression, some odorants induced a decrease in the transcription of genes corresponding to the activated OBP and others increased the expression by many fold in OBPs in live insect, others were neutral after 5 hours of exposure. Moreover, with subsequent behavioural data showed that the behavioural response of G. f. fuscipes toward 1-octen-3-ol decreased significantly when 1-octen-3-ol putative OBPs were silenced with feeding of double-stranded RNA (dsRNA). In summary, our finding whereby odorant exposure affects the OBPs mRNA, their physiochemical properties and the silencing of these OBPs affected the behavioural response demonstrate that the OBPs are involved in odour detection that affect the percept of the given odorant. The expression of OBPs in olfactory tissues, antenna and their interaction with odorant and their effect on behavioural response when silenced shows their direct involvement in odour detection and reception. Furthermore, their expression in other tissues such as legs indicates they might also have role in other physiological functions, such as taste.

Cellular activities constantly change to precisely respond to their biological needs. In many cases, proteins carry out these activities because they can exhibit graded and dynamic responses to perform an array of cellular functions. To study these biological activities and to repurpose proteins for novel uses such as cell therapies, we must be able to control protein activity with synthetic inducers, such as chemical ligands. Multiple chemical inducers have been employed to achieve protein control, but there remains a need for inducer ligands that minimally interact with endogenous pathways, display high bioavailability, and are absent or minimally present from dietary sources. In this work, we control protein activities with the Hepatitis C virus cis-protease NS3 and its numerous clinically validated, highly specific inhibitors. First, we use NS3 to create a Ligand Inducible Connection (LInC) to chemically control gene expression, protein localization and cell signaling in mammalian cells. We then extend the use of catalytically inactive NS3 as a high affinity binder in conjunction with genetically encoded approaches to inhibit NS3, including peptides and RNA aptamers. Using catalytically inactive NS3, genetically encoded peptides, and small molecule drugs, we conditionally control peptide docking with antiviral drugs. We apply this concept to control mammalian gene expression, cell signaling, enzyme activity, and develop a new mechanism for allosteric regulation of Cre recombinase. Altogether, we have developed a new toolkit for controlling diverse protein activities with highly orthogonal, antiviral drugs.
2023-05-15T00:00:00Z

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.

Thesis (Ph. D.)--University of Georgia, 2006.
Directed by Lawrence Shimkets. Includes an article published in Molecular microbiology, and articles submitted to Molecular microbiology and Journal of bacteriology. Includes bibliographical references.

碩士
中臺科技大學
醫學生物科技研究所
96
Forcipomyia taiwana is a blood-sucking insect, which is found in Taichung County by Japanese entomologist Siraki in 1913, and has the serious influence in Taiwan area in recent years. The place bitten by it may cause rash, intense pruritus, swelling, and even allergy. In the previously study, F. taiwana has the chemosensory protein (CSP) in chemosensory organs. CSP has been identified in the several sense organs and could bind the hydrophobic molecular in the environment, such as CO2, fatty acid and pheromone, then affects the olfactory receptors and cause a succession of chemical signal transmission. So, CSP influences insect’s reproduction and the behavior of looking for food, and it acts the quite important role to the insect’s multiplied. In this research, we want to explore the tertiary structure of CSP of F. taiwana and look for suitable ligand to interfere the information transmission and reduction bite on the human. In the experiment, we expressed the gene of CSP of F. taiwana in Kluyveromyces lactis, and obtained the high-purity CSP protein after a series process of ammonium precipitation, cation-exchange and gel filtration chromatography, and demonstrated that CSP had not post-translation modification by LC/MS/MS. Then, we expressed the gene of CSP of F. taiwana in Escherichia coli, and obtained the high-purity CSP protein by the same purified method. By further analyzing its secondary structure and functionality, we found CSP of F. taiwana is mainly composed by α-helix and it has activeness. We combined the CSP of F. taiwana with different ligands, and used the hanging drop vapor diffusion method to sieve out the crystal generated conditions, and then we found star shaped crystals in the 23th condition of the commercialized reagent “Crystallization Basic”. But those crystals are too small to assay by X-ray diffraction method.

碩士
中臺科技大學
醫學生物科技研究所
99
Chemosensory proteins (CSPs) are secreted proteins of 10-12kDa. The conformation of CSPs is globular with a hydrophobic channel within it. The channel is responsible for binding with ligands as long-chain aliphatic on aromatic compounds. The family of CSPs can be divided into several types according to expression profile and function. There is one CSP characterized in Forcipomyia taiwana by its sequence similarity with that of other insects. This protein has a secondary structure rich in α-helice and can associate with hydrophobic compounds. Through Northern and Western blotting, the characteristics of the expression profile of F. taiwana CSP at different stages were identified. The results revealed that level CSP of F. taiwana was low since larvae stage and reached peck in the adult stage. Moreover, no significant difference of expression levels of CSP was identified between genders. According to the identity of the results of Northern and Western blotting, the expression of CSP from F. taiwana is mainly regulation at the transcriptional stage.

碩士
中臺科技大學
生命科學研究所
100
Forcipomyia taiwana belongs Diptera, Ceratopogonidae, Forcipomyia, Lasiohelea, and its body length is about 1.4 mm, and the female addicted to suck human blood. The physique sensitive person will cause pruritus and swelling if bitten by them, so it’s a nuisance pest. In the previous studies, it was found F. taiwana has CSP. The amino acid sequence of its CSP is highly similarity with CSP of other insects. And it was also confirmed by the secondary structure analysis and binding test. Chemosensory proteins (CSPs) are small and soluble proteins, and usually in the lymph of insect sensillar. The molecular weight of CSPs is about 12-13 kD. The amino acid sequence contains four cysteines, and it forms two disulfide bridges. The structure contains six α-helices constituting a hydrophobic channel, and could bind hydrophobic odorant molecules in the environment。By binding hydrophobic molecules of the environment, and active the olfactory neural dendrites in the sensory organs of insects, the insects can identify the external environment, and adjust physiological and behavioral responses. In this research, we use the characterization of CSP that can bind odorant molecules in the insect sensillar, and screen out the high affinity ligands of CSP. After screened the purified CSP , we find the most suitable condition is pH 7 and 30℃. Based on this condition, the experiment result shows CSP has good affinity with components of Antheraea polyphemus pheromone (Z)-11-hexadecenal, and with the fruits compounds of Hexyl benzoate and Butyl benzoate. By using Y-tube to test affinity ligands , and it shows there is 60% attractive effect for F. taiwana. Then we tried different types of traps, the trap of placed (Z)-11-hexadecenal has the best attractive effect for trapping F. taiwana.

博士
國立陽明大學
生化暨分子生物研究所
104
Transmembrane guanylate cyclases (GCs), with activity regulated by peptide ligands and/or calcium-binding proteins, are essential for various physiological and sensory processes. However, the regulation of its activity and protein expression in the mammalian olfactory system remains unclear. In my study, we used the GC subtype GC-G specific antibody, which is the last member of the receptor GC family to validate that the GC-G protein is expressed in Grueneberg ganglion (GG) neurons, a newly recognized olfactory subsystem co-expressing other cGMP signaling components such as the cGMP-regulated PDE2A (phosphodiesterase 2A) and the cGMP-gated ion channel CNGA3 (cyclic nucleotide-gated cation channel α-3). This dissertation aims to clarify the physiological role of GC-G in GG neurons. In chapter 2, molecular and biochemical analyses showed that heterologously expressed GC-G protein, specifically the C-terminal cyclase domain, was directly stimulated by bicarbonate in both in vivo cellular cGMP accumulation assays in human embryonic kidney-293T cells and in vitro GC assays with a purified recombinant protein containing the cyclase domain. In addition, over expression of GC-G in NG108 neuronal cells resulted in a CO2-dependent increase in cellular cGMP level that could be blocked by treatment with acetazolamide, an inhibitor of carbonic anhydrases, which implies that the stimulatory effect of CO2 requires its conversion to bicarbonate. Together, our data suggests that GC-G may be involved in a wide variety of CO2/bicarbonate-regulated biological processes such as the chemosensory function in GG neurons. In chapter 3, we found that its enzymatic activity is directly stimulated by cool temperatures. In this context, it was observed that dimerization/oligomerization of GC-G, a process generally considered as critical for enzymatic activity of GCs, is　strongly enhanced by coolness. Moreover, heterologous expression of GC-G in cultured cells rendered these cells responsive to coolness; thus, the protein might be a sensor for cool temperatures. This concept is supported by the observation of substantially reduced coolness-induced response of GG neurons and coolness-evoked ultrasonic vocalization in GC-G-deficient mouse pups. Hence, GC-G may be a novel thermosensory protein in GG and GC-G activation by coolness is critical for the generation of ultrasound calls by isolated pups to elicit maternal care. In chapter 4, we showed that mouse alarm pheromone (AP) 2-sec-butyl-4,5- dihydrothiazole (SBT) and the predator odor 2,4,5-trimethylthiazoline (TMT), which is structurally-related to SBT can biochemically activate GC-G enzymatic activity, whereas deletion of its extracelluar ligand-binding domain abolishes this stimulatory effect. A direct interaction with notable affinity between these two chemicals and GC-G extracellular domain was confirmed by time-resolved surface plasmon resonance. HEK-293T cells co-expressing GC-G and the cGMP-activated ion channel CNGA3 respond to SBT and other SBT-like kairomones via a rapid influx of calcium. In line with these findings, SBT and TMT-induced calcium transients in the GG as well as TMT-evoked innate fear behaviors and an increase of serum corticosterone (a stress hormone) were markedly attenuated in the GC-G-KO mice compared to wild-type littermates. These results unravel the molecular interaction involved in the inter- and intra-specific olfactory message communication between predators and preys via the GC-G mediated cGMP signaling. This dissertation describes that GC-G is a novel chemosensory and thermosensory protein which could response to danger cues present in the environment, such as cool temperatures, CO2 concentration, APs, and kairomones released by predators. Thus, GC-G is a crucial protein for mice species survival.

碩士
中臺科技大學
生命科學研究所
100
Forcipomyia taiwana is a diurnal insects, about 1.4 mm body length. The female insects are addicted to suck human blood. Because of minimum size, can''t easily be detected. In the region of high insect density, they often bite in group skin of human. The ones susceptible to F.taiwana often resulted in through biting.Interaction between the insects and environment is usually regulated by chemical communication, either through olfaction or taste. Previous studies found chemosensory proteins (CSPs) in F.taiwana, suggested that CSPs are involved in the olfactory response. The CSP gene has been cloned in the expression vector Escherichia coli. In this study, CSP proteins were expressed and purified. The purified CSP was used to study it characteristics, and screen its binding ligands for interfering prevention of F.taiwana. CSP of F. taiwana expressed and purified from cloned E. coli. The expressed CSP was used to screen ligands by binding assay through fluorescence spectrometry. The repellent response of high-affinity ligand was conducted using Y-tube olfactometer, then for repellents test. Three kinds of aldehyde compounds ( 2-methoxycinnamaldehyde, α-amylcinnamaldehyde, α-hexylcinnamaldehyde ) revealed high affinity for CSP through competitive binding. α-hexylcinnamaldehyde showed the most significant repellent trend for F. taiwana by Y-tube olfactometer. Repellents test showed that the repellent effect 2-Methoxycinnamaldehyde and α-hexylcinnamaldehyde were similar to DEET (N, N-Diethyl-meta-toluamide).

The peripheral sensory system of insects is the first to detect chemical stimuli; it is composed of specialized sensory neurons located within hollow, hair-like sensilla. Chemosensory proteins (CSPs) and odorant binding proteins (OBPs) are small, soluble proteins that transport hydrophobic stimuli across the hydrophilic lymph that separates the sensory receptors from the external environment. Incidental results from various studies indicate that most CSPs, and some OBPs, are expressed broadly in many different tissues, raising the question 'what is their non-sensory function?' In this thesis I explored the nonsensory function of CSPs using three different scopes of investigation: 1) an in silico analysis of all known CSP sequences, 2) a characterization of the expression pattern of four CSP genes from a representative lepidopteran species, and 3) a functional characterization of an individual CSP. I identified 15 new CSP sequences; four from cDNA clones described herein and 11 from sequence databases. Several protein similarity classes, representing CSPs from six insect orders, were identified, and each was characterized by highly conserved sequence motifs, including (A) N-terminal YTTKYDN(V/I)(WD)(L/V)DEIL, (B) central DGKELKXX(I/L)PDAL, and, (C) C-terminal KYDP. Three similarity classes were identified that diverged from these conserved motifs, presumably because they are under new functional and selective pressures. A detailed analysis of the expression pattern of four CSP genes from the Eastern spruce budworm, Choristoneura fumiferana, revealed that one (characterized by the retention of the conserved motifs) was expressed in the adult stage, while two that diverged from the conserved motifs were expressed in the immature stages (larvae and pupae). Furthermore, two of the divergent CSP genes were up-regulated during a natural molt, or during an ecdysteroid agonist induced molt. The ligand binding specificity of CfumAY624538, a divergent CSP, was characterized using the fluorescent reporter 1-NPN. Some CSPs bind to medium chain-length fatty acids; this was not the case for CfumAY624538, rather, a short chain-length alcohol was the only ligand tested that displaced 1-NPN in competition. Collectively, my results indicate that divergent CSPs from the Eastern spruce budworm function in development, including larval molting.
Land and Food Systems, Faculty of
Graduate

Animal chemosensation involves several families of G protein-coupled receptors (GPCRs) and, though some of these families are well characterized in vertebrates and nematode worms, receptors have not been identified for most metazoan lineages. In this dissertation, I use a combination of bioinformatics approaches to identify candidate chemosensory receptors in three invertebrates that occupy key positions in the metazoan phylogeny. In the sea urchin Strongylocentrotus purpuratus, I uncovered 192 candidate chemosensory receptors many of which are expressed in sensory structures including pedicellariae and tube feet. In the cephalochordate Branchiostoma floridae, my survey uncovered 50 full-length and 11 partial odorant receptors (OR). No ORs were identified in the urochordate Ciona intestinalis. By exposing conserved amino acid motifs and testing the ability of those motifs to discriminate between ORs and non-OR GPCRs, I identified three OR-specific amino acid motifs that are common in cephalochordate, fish and mammalian ORs and are found in less than 1% of non-ORs from the rhodopsin-like GPCR family. To further investigate the antiquity of vertebrate ORs, I used the OR-specific motifs as probes to search for orthologs among the protein predictions from 12 invertebrates. My search uncovered a novel group of genes in the cnidarian Nematostella vectensis. Phylogenetic analysis that included representatives from the major subgroups of rhodopsin-like GPCRs showed that the cnidarian genes, the cephalochordate and vertebrate ORs, and a subset of genes S. purpuratus from my initial survey, form a monophyletic clade. The taxonomic distribution of these genes indicates that the formation of this clade began at least 700 million years ago, prior to the divergence of cnidarians and bilaterians. Furthermore, my phylogenetic analyses show that three of the four major subgroups of rhodopsin-like GPCRs existed in the ancestor of cnidarians and bilaterians. The utility of the new genes I describe here is that they can be used to identify candidate olfactory cells and organs in cnidarians, echinoderms and cephalochordates that can be tested for function. These genes also provide the raw material for surveys of other metazoans as their genomes become available. My sequence level comparison between chordates, echinoderms and cnidarians exposed several conserved amino acid positions that may be useful for understanding receptor mediated signal transduction. ORs and other rhodopsin-like GPCRs have roles in cell migration, axon guidance and neurite growth; therefore duplication and divergence in the rhodopsin-like gene family may have played a key role in the evolution of cell type diversity (including the emergence of complex nervous systems) and in the evolution of metazoan body plan diversity.
Graduate

During the early life of Matrix-assisted laser desorption/ionization (MALDI), it took the (bio)scientific community a while to realize the great potential of the technique over a wide range of analytical problems, from the discovery or identification of (mostly) organic (macro)molecules through to structure analysis and function. Since then, the practical use of MALDI-MS has grown almost exponentially and it is today an indispensable laboratory tool, particularly in the life sciences. Matrix-assisted laser desorption/ionization (MALDI) is a “soft” ionization technique, which allows for the sensitive detection of large, non-volatile and labile molecules by mass spectrometry. It is fast, allowing the analysis of hundreds of samples per day. It is expensive, as almost all mass spectrometry techniques, yet it is costeffective. Currently the tandem MS and ion different fragmentation capabilities of most MALDI systems add an extra value to this technique as it allows for structural characterization of (bio)molecules. In addition to molecular identification and characterization, MALDI is an invaluable tool to profile and classify complex samples. In spite of its many advantages, MALDI has one achilles tendon. If the sample is not conveniently prepared, the ionization is hampered and then the analysis is not possible. This is the reason why sample treatment for MALDI analysis is a never-ending hot topic in analytical chemistry. Thus, the methods used to identify proteins do not work with polymers and vice versa. In other words, for each new molecule a sample treatment optimization is needed so an adequate, well-defined, method of analysis is obtained. For this reason this thesis focused in the development of new sample treatments for MALDIbased applications. To this end and when possible, ultrasonic energy is used as a tool to simplify the sample handling by matching the analytical minimalism concept as defined by Halls1. The molecules for which the new sample treatments were developed were proteins, polymers and small inorganic molecules. MALDI is the technique of preference for the analysis of polymers as with one single analysis the polymer´s typical values of number-average molecular weight, Mn, (...)

Pyrazoloanthrone and its analogues form the central core of the thesis and the work is focused on the evaluation of chemical and biological applications of pyrazoloanthrones. Selective and sensitive detection of biologically, environmentally and industrially important molecular species such as fluoride, cyanide and picric acid by using pyrazoloanthrones as sensors form the first part while the second part deals with selective and specific kinase inhibition by pyrazoloanthrones to moderate inflammation associated disorders like septic shock. All the investigations are based on extensive crystallographic studies of the participating molecules. Chapter 1 provides a brief review on the history and biological importance of 1,9-pyrazoloanthrones. The potential of these molecules as probes in sensor chemistry and protein kinase inhibition is envisaged. A short account of the techniques employed for the investigations along with a preamble is presented. Chapter 2 is divided into two parts. Part A deals with the design of a colorimetric and “turn-on” fluorescent chemosensor based on 1,9-pyrazoloanthrone specifically for cyanide and fluoride ion detection. A remarkable solid state reaction indicated by the development of intense red color occurs when crystals of tetrabutylammonium cyanide/fluoride are brought in physical contact with 1,9¬pyrazoloanthrone resulting in corresponding molecular complexes (Figure 1). X-ray crystal structures of these complexes and also of 1,9-pyrazoloanthrone have been determined and the ion sensing activity has been substantiated on the basis of spectroscopic (absorption, fluorescence and NMR) and structural analyses. The crystal structure of the parent compound exhibits a disorder as a consequence of tautomerism and the disorder gets carried on to the complexes as well with even the cyanide and the fluoride ions showing partial occupancy sites. The presence of the –NH group and associated intramolecular charge transfer upon complex formation is attributed to the extreme sensitivity of 1,9-pyrazoloanthrone for cyanide and fluoride (detection limits of 0.2 ppb and 2 ppb) ions respectively. Figure 1. Development of intense red color during the solid state reaction (shown on left) and the turn on fluorescence behavior (shown to the right) Part B demonstrates the utilization of electron rich N-alkyl substituted pyrazoloanthrones to design sensors for detecting explosive and electron deficient nitro aromatics such as picric acid (PA). The N-alkyl derivative of 1,9-pyrazoloanthrone has been synthesized, characterized by single crystal X-ray diffraction studies and evaluated as a potent sensor for picric acid. NMR and fluorescence lifetime measurements validate that the fluorescence quenching of sensor compound by PA (Figure 2) as due to the formation of excited state charge-transfer complex resulting in dynamic quenching. Figure 2. Fluorescence quenching measurements demonstrating the dynamic quenching in the charge transfer complex. Chapter 3 deals with the biological evaluation of 1,9-pyrazoloanthrone and its alkyl derivatives towards the inhibition of a decisive protein kinase called c-Jun N-terminal Kinase (JNK), an important member of MAP kinase family. JNK controls crucial cellular processes like apoptosis and cell proliferation and is implicated in disorders associated with inflammation such as septic shock, arthritis, inflammatory bowel disease, etc. Therapeutic inhibition of JNK activity by small molecules has proven to be advantageous in the treatment of diseases coupled with derailed inflammation. In this context, it is already established that 1,9-pyrazoloanthrone (SP600125) effectively and selectively inhibits JNK at concentrations beyond 10 M. A series of alkyl isomers of pyrazoloanthrone derivatives have been synthesized to evaluate the structural implications of inhibition and to elevate both selectivity and sensitivity at lower concentrations. The crystal structures of these isomers have been characterized and their utility as inhibitors has been tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). The minimum inhibitory concentrations required by these molecules to inhibit JNK was found to be lesser as compared to 1,9-pyrazoloanthrone (<5 µM; Figure 3). Critically, it turns out that among the various inhibitors synthesized, the lead candidates SPP1 and SPB1 display specific inhibition of JNK among other LPS activated MAP kinases like ERK1/2 and p38. These results suggest that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders. Figure 3. Inhibition of JNK in macrophages by the SPP1 and SPB1 compared to the known SP600125. Inspired by the results reported in the previous chapter, Chapter 4 is devoted to the generation of a library of compounds based on SPP1 and SPB1 with a purpose to design inhibitors of JNK which perform at the lowest possible concentrations and the consequent evaluation of their potential on endotoxin induced septic shock. Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. Figure 4. Two selected molecules for specific inhibition studies of JNK at lower concentrations. It is demonstrated that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Based on the results from both in vitro with macrophages and in vivo with the mouse model of septicemia, the potential role of two selected molecules D1 and D2 (Figure 4) in regulating endotoxin induced inflammation is firmly established. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the in vitro as well as the in vivo data clearly potentiates the selective inhibitory capacity of small molecule inhibitors like D1 and D2 which can facilitate the treatment of current inflammatory disorders when used in combination with the available drugs having varied efficacies. The results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.