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Engineering microbial rhodopsins to expand the optogenetic toolkit.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	Engineering microbial rhodopsins to expand the optogenetic toolkit./
Author:	Venkatachalam, Veena.
Description:	1 online resource (176 pages)
Notes:	Source: Dissertation Abstracts International, Volume: 76-03(E), Section: B.
Subject:	Biophysics. -
Online resource:	click for full text (PQDT)
ISBN:	9781321335620

Engineering microbial rhodopsins to expand the optogenetic toolkit.
Venkatachalam, Veena.

Engineering microbial rhodopsins to expand the optogenetic toolkit. - 1 online resource (176 pages)

Source: Dissertation Abstracts International, Volume: 76-03(E), Section: B.

Thesis (Ph.D.)--Harvard University, 2014.

Includes bibliographical references

Cellular lipid membranes can---and often do---support a transmembrane electric field, serving as biological capacitors that maintain a voltage difference between their two sides. It isn't hard to see why these voltage gradients matter; the electrical spiking of neurons gives rise to our thoughts and actions, and the voltage dynamics of cardiomyocytes keep our hearts beating. Studies of bioelectricity have historically relied on electrode-based techniques to perturb and measure membrane potential, but these techniques have inherent limitations. I present new optogenetic methods of studying membrane potential that will broaden the scope of electrophysiological investigations by complementing traditional approaches.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2018

Mode of access: World Wide Web

ISBN: 9781321335620Subjects--Topical Terms:

581576
Biophysics.
Index Terms--Genre/Form:

554714
Electronic books.

Engineering microbial rhodopsins to expand the optogenetic toolkit.
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245 1 0 $a Engineering microbial rhodopsins to expand the optogenetic toolkit.
264 0 $c 2014
300 $a 1 online resource (176 pages)
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500 $a Source: Dissertation Abstracts International, Volume: 76-03(E), Section: B.
500 $a Adviser: Adam E. Cohen.
502 $a Thesis (Ph.D.)--Harvard University, 2014.
504 $a Includes bibliographical references
520 $a Cellular lipid membranes can---and often do---support a transmembrane electric field, serving as biological capacitors that maintain a voltage difference between their two sides. It isn't hard to see why these voltage gradients matter; the electrical spiking of neurons gives rise to our thoughts and actions, and the voltage dynamics of cardiomyocytes keep our hearts beating. Studies of bioelectricity have historically relied on electrode-based techniques to perturb and measure membrane potential, but these techniques have inherent limitations. I present new optogenetic methods of studying membrane potential that will broaden the scope of electrophysiological investigations by complementing traditional approaches.
520 $a I introduce the microbial rhodopsin Archaerhodopsin-3 (Arch), a transmembrane protein from Halorubrum sodomense. The fluorescence of Arch is a function of membrane potential, allowing it to serve as an optical voltage reporter. We use time-dependent pump-probe spectroscopy to interrogate the light- and voltage-dependent conformational dynamics of this protein, to elucidate the mechanism of voltage-dependent fluorescence in Arch.
520 $a I then present two new methods for imaging voltage using engineered variants of Arch. Both techniques take advantage of the unique photophysical properties of Arch(D95X) mutants. The first method, Flash Memory, records a photochemical imprint of the activity state---firing or not firing---of a neuron at a user-selected moment in time. The Flash Memory technique decouples the recording of neural activity from its readout, and can potentially allow us to take large-scale snapshots of voltage (e.g. maps of activity in a whole mouse brain). The second method allows for the quantitative optical measurement of membrane potential. This technique overcomes the problems that typically hinder intensity-based measurements by encoding a measurement of voltage in the time domain.
520 $a Finally, I present a method to visualize cellular responses to changes in membrane potential. I engineer mutants of Channelrhodopsin-2 (ChR2), a light-gated cation channel from Chlamydomonas reinhardtii that is used for optical control of neural activity, and use these optogenetic actuators in conjunction with GFP-based sensors to study the activity-dependent behavior of cultured neurons.
533 $a Electronic reproduction. $b Ann Arbor, Mich. : $c ProQuest, $d 2018
538 $a Mode of access: World Wide Web
650 4 $a Biophysics. $3 581576
650 4 $a Neurosciences. $3 593561
655 7 $a Electronic books. $2 local $3 554714
690 $a 0786
690 $a 0317
710 2 $a ProQuest Information and Learning Co. $3 1178819
710 2 $a Harvard University. $b Biophysics. $3 1186714
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3645086 $z click for full text (PQDT)