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General Introduction 10 opposed to wild type flies that avoid high alcohol levels. Further research with LUSH mutants showed that this OBP is involved in activity of the pheromone-sensitive neurons. The activity of the male-specific lipid, 11-cis vaccenyl acetate (cVA) that causes aggregation in wild type flies is lost in mutants. The activity of this pheromone is restored with transgenic expression of the LUSH protein suggesting that the loss of function in mutants is specifically due to absence of LUSH in the pheromone sensitive sensilla (Zhou et al., 2004; Xu et al., 2005). Further to this, Laughlin et al. (2008) showed through solving the structures of various bound and unbound states of LUSH, as well as of several LUSH proteins with point mutations that the cVA bound LUSH acts as an activator of pheromone sensitive neurons (Laughlin et al., 2008). When cVA is bound to LUSH, a conformational change occurs that disrupts a salt bridge in LUSH and causes shifts in its surface loop. This altered state of LUSH then activates the pheromone neuron, as depicted in Figure 1.2. The salt bridge is maintained in the unbound state and when an alcohol such as butanol is bound to LUSH. This indicates that the OBP/ligand complex interacts with the PR (Laughlin et al., 2008). Figure 1.2: Odorant receptor activation as depicted by Laughlin et al. (2008). The formation of the cVA/LUSH complex results in a conformational change of LUSH making it an active ligand that interacts directly with the pheromone receptor. From Stowers and Logan (2008). PR
General Introduction 11 A model has been proposed for ligand deactivation by modification of the PBP in the pheromone complex to a scavenger form (Kaissling, 2009). In B. mori, when the PBP/bombykol complex interacts with the receptor molecule on the dendrite of the ORN, the low pH at the dendrite leads to a conformational change and formation of a C- terminal α-helix in the PBP, thereby releasing the bombykol (Leal et al., 2005). Several mechanisms by which the odorant is conferred to the ORs by the OBP/odorant complex have been proposed. The stable OBP/odorant complex interacts directly with the OR; or the OBP/odorant complex is unstable and at the dendritic membrane, the odorant binds to the OR selectively. It could also be that a dendritic membrane bound docking protein, for example, sensory neuron membrane protein 1, binds the OBP/odorant complex and this in turn releases the odorant from the complex and confers it to the OR (Vogt et al., 1985; Prestwich et al., 1995; Steinbrecht, 1996; Kaissling, 1998). A recent structural based study of A. polyphemus PBP proposes a pH induced release of odorants from the complex (Zubkov et al., 2005). Binding of odorant to PBP occurs at neutral lymph pH and release of the odorant from the complex is achieved by the opening of the ligand binding cavity at the lower pH near the dendritic membrane. Other classes of OBPs include chemosensory proteins (CSPs) and antennal binding proteins (ABPs). The ABPs are expressed specifically in the antennae (Steinbrecht et al., 1995; Zhang et al., 2001). The CSPs bind odorants and pheromones and also have implications in taste reception. The first CSP was identified by subtractive hybridisation experiments in Drosophila melanogaster antennae (McKenna et al., 1994; Pikielny et al., 1994). Refer to Vogt et al. (2003) for a review. Another class of soluble proteins present in the sensillum lymph are the odorant degrading enzymes (ODEs) (Vogt and Riddiford, 1981). Once the signal has been relayed to the neuron, the odorant needs to be cleared from the lymph so that sensitivity of the olfactory system is maintained and new signals can be detected as continuous firing of the neurons with the same odorant can cause a loss of sensitivity to that odorant. Also, not all odorants that enter the sensillum are detected by the olfactory system and need to be cleared from the lymph. This may include odorants toxic to the cells so degradation of these odorants is important in the lymph. This role in insects is suggested to be played by ODEs such as carboxylesterases, glutathione-
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Page 3 and 4: Abstract Abstract Olfaction or the
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The roles of Epiphyas postvittana G
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4.1 Introduction 4 Identification o
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Identification of putative odorant
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Microarray OR 454 Transcriptome OR
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5.1 Introduction 5 Concluding Discu
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Concluding Discussion 129 redundant
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Concluding Discussion 131 against t
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Concluding Discussion 133 sequencin
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Appendix B 135 Buffered TB (Sambroo
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Appendix C 137 C. Appendix C - Clus
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Appendix D 139 D. Appendix D - Solu
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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