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Bacterial physiology and behavior is controlled by complex regulatory networks. Chemosensory systems are sophisticated signal transduction systems that can govern a range of cellular functions beyond that of traditional flagellar-based chemotaxis. The soil bacterium Myxococcus xanthus encodes eight chemosensory systems regulating multiple behaviors, including motility, exopolysaccharide production, and development. This work characterizes the Che7 system and demonstrates a role for Che7 in coupling aggregation and sporulation during multicellular development. The regulation requires an interaction between a single domain response regulator (CheY7) and a HEAT-repeat protein (Cpc7). A fatty acid desaturase, Des7, also impacts development in concert with the Che7 signaling system. Genetic analysis indicates the target of Che7 regulation is in the heme biosynthesis pathway, which is one aspect of iron homeostasis. Finally, characterization of iron and iron-responsive elements during development reveal a novel regulator, Fur2, that controls timing of development as well as che7 transcription. This work provides expands the known network regulating development in M. xanthus.

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Wines are composed of numerous compounds that are complex, making them difficult to analyze. Wine evaluation and discrimination is typically done through chemical and human sensory evaluation. Unfortunately, both of these methods are time consuming and expensive. Therefore a new rapid analysis technique for wine discrimination and analysis is desired. The electronic nose has been suggested as an alternative to current wine discrimination techniques. In this study, a quartz microbalance-based electronic nose system was utilized to analyze the overall volatile components of wine. The electronic nose was optimized for Cabernet Sauvignon and Mouvèdre wine to gain maximum sensor response from the sensors. Response surface methodology was used to determine the optimum sensor response by varying three experimental parameters: sensor temperature, sample temperature and equilibrium time. The maximum sensor response occurred at an equilibrium time of 20 min for each varietal and at a sample temperature of 55ºC and 56ºC for Cabernet Sauvignon and Mouvèdre, respectively. The optimum sensor temperature selected for this study was 40ºC for both varietals. Using the optimum sensor settings, the electronic nose was used to analyze Cabernet Sauvignon wines. Grapes were treated with ethanol spray (5%, and 10%) 13 weeks post-bloom, which has been shown to affect the overall quality of the final wine product. Wine samples were evaluated using chemical analyses, human sensory evaluation and electronic nose. Significant differences between the wines were observed based on pH, percent alcohol, and color intensity only. A consumer sensory panel consisting of 81 panelists was unable to differentiate amongst sample treatments. However, the electronic nose was able to differentiate between the control group and the treated samples 100% of the time. Canonical discriminant analysis of the data placed the 5% ethanol treatment as a sub-set of the 10% ethanol treatment. The results indicate that the electronic nose can be used as a discriminatory tool for assessing wines. / Master of Science

In most animal species, reproductive success depends critically on precopulatory or solicitational behaviors that occur prior to mating. The specific sensory systems and behavioral strategies employed in precopulatory behaviors vary across species; in all cases, however, animals must be able to identify potential mating partners and solicit sexual interest. Female Syrian hamsters (Mesocricetus auratus) engage in multiple forms of precopulatory behaviors that are preferentially expressed to males or their odors, including vaginal scent marking and sexual odor preference. Conspecific odors relevant for precopulatory behaviors are processed by a network of forebrain areas that includes the bed nucleus of the stria terminalis (BNST) and the medial preoptic area (MPOA). The precise functional and neurochemical mechanisms whereby these areas regulate the expression of precopulatory behaviors, however, are unknown. Therefore, the aim of this dissertation is to address the following research questions: (1) Is the neuropeptide oxytocin (OT), acting within BNST or MPOA, necessary for the normal expression of odor-­guided precopulatory behaviors? (2) Is BNST or (3) MPOA required for the preferential expression of vaginal marking or investigation towards male odors?, and (4) Does OT interact with social odor processing to regulate vaginal marking? We found that blockade of OT receptors (OTRs) in MPOA and BNST decreased vaginal marking to male odors. There was no effect of OTR blockade on sexual odor preference. Selective lesions of BNST also disrupted preferential vaginal marking responses to male odors, without affecting sexual odor preference. In contrast, lesions of MPOA disrupted odor preference without affecting vaginal marking responses. Finally, central blockade of OTRs eliminated the normal pattern of increased activation of neurons to male vs. female odors in BNST, but not MPOA. Considered together, these results suggest that OT normally acts within BNST to drive preferential vaginal marking responses to male odors via selective facilitation of neural responses to these odors, and further, that there are separate and distinct neural circuits that regulate different forms of odor-guided precopulatory behaviors in females.

Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. Using the Gal4/UAS system, we have characterized the expression of Gr genes in detail, and find that gene expression falls into two distinct groups. The first group, which make up the majority of the genes analyzed, are co-expressed with Gr66a, and functional ablation of taste neurons expressing these genes demonstrates that these neurons mediate sensitivity to bitter substrates. A second, distinct group of taste neurons, express Gr5a and mediate sensitivity to trehalose. We find that these two sub-populations of taste neurons - attractive-type and avoidance-type - project afferents to discrete areas of the primary taste center in the CNS. These results demonstrate how bitter and sweet taste are coded for in the periphery and indicates that information about different taste modalities is initially segregated in the CNS. We have also used the Gal4/UAS system to thoroughly characterize the expression profile of a cluster of six Grs - Gr28a and Gr28b.a-b.e. We find that these receptors are expressed not only in taste neurons, but other sensory neurons, as well as neurons in the CNS. RNA in situ hybridization confirms this unusual expression pattern. In order to explore the function of these Grs, these genes have been deleted using ends-out homologous recombination, to produce Gr28 mutant flies. Initial behavioral experiments with Gr28 mutant flies suggest that these receptors may play a role in detecting aversive substrates and/or modulate avoidance responses to these substrates. / Dissertation

Many insects rely on olfaction as their primary method of interaction with their environment. One of the best examples of this is the olfactory driven host-seeking behavior displayed by female mosquitoes. Although mosquitoes are capable of extracting blood from a variety of hosts many mosquito species show marked preferences for particular host species. Mosquitoes displaying preference for humans above bovines are more likely to be disease vectors. Therefore understanding the molecular basis of this preference is important for public health. These differences may be the result of genetic variations in olfactory signaling components such as mosquito odorant receptors. This hypothesis is supported by several lines of evidence including the highly divergent and lineage-specific nature of this receptor family. Likely these differences are subtle and will be identified in highly focused studies. Even closely related sibling species of mosquitoes can display large behavioral differences. In our current study I have studied several aspects of both Anopheles and Aedes genus odorant receptors with emphasis on comparing receptors in species that are part of the Anopheles genus. The first goal of this project was to study the insect odorant receptor family for potential sites of heterodimer formation. Numerous studies have shown that insect odorant receptors are involved in detection of odorants. More recent studies have demonstrated that odorant receptors are also involved in protein trafficking and in forming cation channels. Both of these activities involve heterodimer formation between odorant receptors that bind odorants and those that are part of the Or83b subfamily. There is little informaiton on how heterodimers are formed and where within the protein heterodimer sites exist. The C-terminal region has been implicated as sites for such heterodimer formation. A hidden markov model based program, Multiple em for motif elicitation (MEME), was used to uncover three motifs in the C-terminus of the odorant receptor peptides from Anopheles gambiae, D. melanogaster, and Apis mellifera. Previous studies have shown that insect odorant receptors are highly divergent between different insect lineages suggesting conservation of these motifs is functionally important. I propose that these motifs are involved in receptor-receptor protein interactions, contributing to the heterodimer formation between Or83b subfamily members and other odorant receptors.The next goal was to identify odorant receptors in closely related mosquito species and compare and contrast them. This was accomplished by using public sequence data of An. gambiae and BAC library screening to identify orthologous gene clusters in An. stephensi and An. quadriannulatus. Although I have identified many different odorant receptor genes the chapter in this dissertation discusses my work with the Or2 gene cluster. Multi-species comparison of these orthologous regions in An. gambiae, An. quadriannulatus, and An. stephensi revealed highly conserved gene structure among the OR genes and the discovery of the An. stephensi Or10x gene (AsOr10x), which is present only in An. stephensi. AsOr10x showed a different expression pattern than AsOr2 and AsOr10, the other members of this gene subfamily in An. stephensi. Therefore AsOr10x might be adapting or has adapted a new function. Analysis of the phylogeny and physical location of all known members of the Or2/Or10 gene subfamily in Anopheles, Aedes, and Culex mosquitoes suggest that a few events of gene duplication and loss resulted in the current gene distribution. The final focus of this project was to develop a method to study the function of mosquito odorant receptors. There is currently no in vivo system to study mosquito odorant receptors, and experimental systems pioneered in D. melanogaster are not transferable to mosquitoes. I decided to employ a reverse genetics strategy involving the silencing of three Aedes aegypti odorant and gustatory receptors of known or suspected function. These gustatory receptors are members of a small subfamily that encode olfactory and not taste receptors. As a preliminary step the expression profiles of these three genes and an additional gustatory receptor were determined using non-quantitative and quantitative RT-PCR. We found that the putative CO₂-detecting gustatory receptors are expressed in Ae. aegypti larvae, and hence these larvae may respond to CO₂, an observation that has not been reported previously. The purpose of silencing these receptors is to generate a loss-of-function behavior phenotype that will allow for inference of receptor function. Recombinant Sindbis viruses were used to knockdown mRNA levels of these receptors. GFP-expressing recombinant Sindbis viruses were shown to infect chemosensory tissue. Additional viruses containing fragments of receptor genes were found capable of lowering odorant and gustatory receptor mRNA levels. Infected mosquitoes displayed varying levels of gene knockdown with one virus generating supression of mRNA levels to 15.0% of normal. These mRNA levels may not be low enough to generate an unambiguous phenotype. Future experimentation is focused on developing more effective recombinant viruses and identifying characteristics of viruses more effective in receptor gene knockdown. A safe and effective behavior assay setup is needed to test the behavioral responses of these infected mosquitoes. In this study I outline a preliminary behavior assay that is being developed and optimized. When established it will provide a powerful tool in the study of both basic mosquito behavior and phenotype screening of recombinant Sindbis virus-infected mosquitoes. / Ph. D.

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