Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...

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Functional characterisation of Epiphyas postvittana odorant receptor 1 44 2.2.7 Data analysis The data collected from the calcium imaging was analysed using MetaFluor Analyst v5.1 (Universal Imaging Corporation). Circles were drawn with the region of interest (ROI) tool around the perimeter of the cells in the field of view and also three background regions free of cells. The fluorescence data for the selected ROIs over the image acquisition period was obtained with the „calculate plot‟ function of the software. The data from the three background ROIs was averaged and the ROI data from the cells was subtracted from this background, to minimise differences in cell image backgrounds. The change in fluorescence (∆F) of the cells that elicit a response upon odorant stimulation was calculated as follows: Where, FBlank is the average fluorescence intensity of the cell, from the first image to the twelfth image before addition of the ligand; FSubstrate is the highest fluorescence intensity of the cell after the addition of the ligand, from the thirteenth to the eighteenth image, and FMax is the maximum fluorescence of the cell achieved after the addition of the ionophore, from image nineteen to twenty-four. The average ∆F of all the cells responding to a particular ligand concentration was take as the mean ∆F for that concentration (with a minimum threshold of at least 3 responding cells) when plotting the dose response curve using OriginPro v7.5 (OriginLab) software. Cells that respond to stimulation with assay buffer only were discarded from the analysis. EC50 (concentration at which 50% of the maximal effect or activation of the ligand is observed) and Hill‟s slope (slope of the dose response curve that gives the level of cooperativity of the ligands) (Motulsky and Christopoulos, 2003) were calculated using Graphpad Prism software. A compound was considered unable to activate EpOR1 if no response was observed in cells upon stimulation with the odorant in triplicates at concentration of 10 -5 M.
Functional characterisation of Epiphyas postvittana odorant receptor 1 45 2.3 Results To determine if the transfection of pIB-EpOR1 into Sf9 cells had been successful, reverse transcription polymerase chain reaction (RT-PCR) was performed on mRNA extracted from the transfected cells using primers specific for the full length coding region of the EpOR1 gene (Figure 2.1A). As a control, cells transfected with the pIB/V5 empty vector were also tested (Figure 2.1B). The empty vector transfected cells did not show the presence of EpOR1 transcripts, while the EpOR1-transfected cells showed the presence of PCR products of the expected size, 1245bp. Figure 2.1: Reverse transcription PCR analysis of transfected pIB-EpOR1 Sf9 cells. A. RT-PCR with myc-EpOR1 specific primers of pIB-EpOR1 transfected Sf9 cells 48 hours post transfection. B. RT-PCR of Sf9 cells transfected with the pIB/V5, an empty vector control, with myc-EpOR1 specific primers. To confirm the expression of myc-EpOR1 protein in transfected Sf9 cells, a western blot of the membrane fraction from these cells was probed with an anti-myc antibody. Three bands of approximately 25 kDa, 50 kDa and 60 kDa were observed on the western blot (Figure 2.2). The band „b‟ observed at approximately 50 kDa corresponds to the theoretical molecular weight of myc-tagged EpOR1. The higher band „a‟ could be aggregation of the protein that did not separate out on the gel or a
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
Page 9 and 10: Contents 4.2.5 Degenerate PCR .....
Page 11 and 12: List of Figures List of Figures Fig
Page 13 and 14: List of Tables List of Tables Table
Page 15 and 16: List of Abbreviations gDNA Genomic
Page 17 and 18: 1.1 General overview 1 General Intr
Page 19 and 20: General Introduction 3 moth in loca
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Page 27 and 28: General Introduction 11 A model has
Page 31 and 32: General Introduction 15 Tanaka et a
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Page 37 and 38: General Introduction 21 et al., 200
Page 39 and 40: General Introduction 23 the attract
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Page 45 and 46: General Introduction 29 Expression
Page 47 and 48: General Introduction 31 BmOr2 HvOr2
Page 49 and 50: General Introduction 33 for lepidop
Page 51 and 52: Functional characterisation of Epip
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Page 73 and 74: 3.1 Introduction 3 The roles of Epi
Page 75 and 76: The roles of Epiphyas postvittana G
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Page 97 and 98: Identification of putative odorant
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Identification of putative odorant
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5.1 Introduction 5 Concluding Discu
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Concluding Discussion 127 VOBA sett
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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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