5-HT 5-hydroxytryptamine, Serotonin
AC Adenylate cyclase
cAMP Cyclic adenosine monophosphate
CD (i.e. CD28) Cluster of differentiation
CFP Cyan fluorescent protein
CV Coefficient of variation
DPBS Dulbecco’s phosphate buffered solution DMEM Dulbecco’s modified Eagle’s medium
Epac Exchange protein directly activated by cAMP
FCS Fetal calf serum
FRET Förster resonance energy transfer GPCR G protein coupled receptor MAD Microscope analog-digital units
SNR Signal to noise ratio
TC Tandem construct
TCSPC Time correlated single photon counting
V Volts
YFP Yellow fluorescent protein
Symbols
fA ratio of FRET complexes over total acceptor, considering intact fluorophores only
fD ratio of FRET complexes over total donor, considering intact fluorophores only fa fraction of acceptor-type molecules participating in complexes, irrespective of
their labeling state
fd fraction of donor-type molecules participating in complexes, irrespective of their labeling state
A concentration of free acceptor fluorophores101 | P a g e
D concentration of free donor fluorophores
DA concentration of complexes carrying both intact donor and acceptor fluorophore a , d , da ‘chemical‘ concentrations of free acceptor , free donor and complexes, irrespective of their labeling state
Aref
concentration of intact acceptor fluorophore in the calibration samples Dref
concentration of intact donor fluorophore in the calibration samples At
total concentration of labeled acceptors with intact fluorophore Dt
total concentration of labeled donor with intact fluorophore
pa,tc, pd,tc labeling probabilities of donors and acceptors within the tandem construct pd' abbreviation for pdpa,tc/ pd,tc (see above)
i apparent relative acceptor concentrations
i apparent relative donor concentrations
Fi measured spectrum (linear combination of FDi,ref andFAi,ref )
, i ref
FA reference fluorescence emission spectra of pure acceptor
, i ref
FD reference fluorescence emission spectra of pure donor
rex,i scaling factor reflecting the excitation ratios of two fluorophores at the given excitation wavelength
E characteristic FRET efficiency
ETC FRET efficiency of the tandem construct
Kd dissociation constant
Ai,
Di extinction coefficients of acceptor and donor QA,QD quantum yields of acceptor and donoreA
,eD
standard emission spectra of the two fluorophores normalized to unit areaIi,ref excitation intensity
i
detection efficiencies of the instrument used102 | P a g e 7.2 Appendix 2 – Full derivation of error propagation equations
Gaussian error propagation equations
Gaussian error propagation was used to define the variance of the luxFRET quantities, the emission ratio, and the ligand concentration. Using the original equation and derived variance the CV2 of the quantities was solved.
Error propagation through equation 1.37: Derivation of equation 1.46 With
we can define the variance of Epd’ from Gaussian error propagation as,
By defining the squared coefficient of variation of each variable,
'
we can substitute the error propagation into CV2 of Epd’ and simplify to obtain the final form.
103 | P a g e Error propagation through equation 1.39: Derivation of equation 1.47
With
we can define the variance of Epa from Gaussian error propagation as,
By defining the squared coefficient of variation of each variable,
2
we can substitute the error propagation into CV2 of Epa and simplify to obtain the final form.
104 | P a g e
Error propagation through equation 1.40: Derivation of equation 1.48.
With
we can define the variance of Epd’ from Gaussian error propagation as,
By defining the squared coefficient of variation of each variable,
2
we can substitute the error propagation into CV2 of Epd’ and simplify to obtain the final form.
105 | P a g e Error propagation through emission ratio analysis
With
2 1
R F
F , (A19)
we can define the variance of R from Gaussian error propagation as,
Error propagation through ligand concentration estimation: Derivation of equation 1.51 (analogous to equation 1.50 for FRET efficiency).
With
we can define the variance of X from Gaussian error propagation as,
106 | P a g e
Acknowledgements
I would like to express my sincerest gratitude to my supervisors Prof. Dr. Erwin Neher and Prof. Dr. Evgeni Ponimaskin, not only for the opportunity to perform this work, but also for their invaluable guidance, discussions, feedback, challenges, criticism, and support throughout its completion.
I would also like to thank Prof. Dr. Dr. D. Schild for his suggestions and help in steering this project from his position on my thesis committee. My many thanks go out to Prof. Dr. Michael Hörner and Ms. Sandra Drube for their help and support over my time in Göttingen. I am also grateful to the Study Committee of the Neurosciences program for the opportunity of performing my graduate studies in Göttingen.
I would like to thank the past and present members of the Department of Neuro and Sensory Physiology for the particularly positive working environment. I am grateful to Dr. Ute Renner, Dr. Andre Zeug, Gaby Klaen, and Dagmar Crzan for all the help over the past few years. I am especially grateful to Dr. Petrus Salonikidis, Dr. Jakub Wlodarczyk and Fritz Kobe for their close friendship and the frequent coffee breaks.
I am fortunate to have found myself studying, working, and living with among the most interesting and incredible people I have met. I owe a tremendous debt of gratitude to Natalia, Adema, Marija, Regis, Victorija, Achim and Katharina for the ceaseless encouragement and inspiration.
None of this would have been possible without my parents, Craig and Linda Danielson, my grandparents, Rodney and Margaret Danielson and my brother William. I am eternally grateful for their unquestioning love, unending sacrifice and constant support.
107 | P a g e
Curriculum Vitae
Education
2005 – Present PhD Student - International Max Planck Research School - Neurosciences Graduate Program, Georg August University Göttingen, Germany.
Quantitative analysis of Förster resonance energy transfer from spectral fluorescence measurements. Supervisors - Prof. Erwin Neher and Prof.
Evgeni Ponimaskin
1999 – 2004 B.S.E. Bioengineering - Harrington Department of Bioengineering, Arizona State University. Magna Cum Laude, GPA 3.7.
Design and Development of a System for the Controlled Electrical Neural Stimulation of Epileptic Rats. Supervisor - Prof. Leon Iasemidis, ASU Brain Dynamics Lab, Barrow Neurological Institute.
Honors Thesis - Barrett Honors College, Arizona State University
Nonlinear Dynamical Systems and Chaos Theory Application to Human Physiology.
Professional Experience
2004 – 2005 Quality Engineer, General Electric Company, Bio-Sciences, Molecular Diagnostics, Chandler, AZ
2002 – 2003 Product Development Engineering Intern, Alliance Medical Corporation, Phoenix, AZ
2003 – 2004 Academic Tutor - Arizona State University, Learning Resource Center.
Tempe, Arizona
2003 Program Coordinator - Arizona State University, Center for Outreach and Recruitment. Tempe, Arizona.
Awards
2006 – 2008 George-Christoph-Lichtenberg-Stipendium, awarded by the State of Lower Saxony, Germany
2005 – 2006 Graduate Stipend - International Max Planck Research School, Germany 1999 – 2004 Arizona Regents Scholarship and ASU Presidential Scholarships
108 | P a g e Publications
Woehler A, Wlodarczyk J, Neher E. 2010. Signal to Noise Analysis of FRET-Based Sensors. Submitted.
Woehler A, Ponimaskin EG. 2009. G protein – mediated signaling: same receptor, multiple effectors.
Curr Mol Pharmacol 2(3):237-48.
Woeher A, Wlodarczyk J, Ponimaskin EG. 2008. Specific oligomerization of the 5-HT1A receptor in the plasma membrane. Glycoconj J. 26(6):749-56.
Kobe F, Renner U, Woehler A, Wlodarczyk J, Papusheva E, Bao G, Zeug A, Richter DW, Neher E, Ponimaskin E. 2008.Stimulation- and palmitoylation-dependent changes in oligomeric conformation of serotonin 5-HT1A receptors. Biochim Biophys Acta. 1783(8):1503-16.
Wlodarczyk J, Woehler A, Kobe F, Ponimaskin E, Zeug A, Neher E. 2008. Analysis of FRET signals in the presence of free donors and acceptors. Biophys J. 94(3):986-1000.
Conference Presentations
Woehler A, Wlodarczyk J, Kobe F, Renner U, Neher E, Ponimaskin E. (2008) FRET Investigations of 5HT1A receptor Oligomerization using two wavelength excitation. Presentation given at 87th Annual Deutsche Physiologische Gesellschaft in the News and Notable from Young Physiologists Symposium.
Cologne, Germany.
Woehler A, Wlodarczyk J, Zeug A, Neher E , Ponimaskin E. (2007) FRET Investigations of 5HT1A Receptor Oligomerization. Poster Presented at Focus on Microscopy 2007. Valencia, Spain.