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Fluctuation analysis reveals protein...

Dalal, Rooshin Bhadrik.

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Fluctuation analysis reveals protein concentration, mobility, and aggregation states in cells.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Fluctuation analysis reveals protein concentration, mobility, and aggregation states in cells./
作者:	Dalal, Rooshin Bhadrik.
面頁冊數:	221 p.
附註:	Adviser: Rick Horwitz.
Contained By:	Dissertation Abstracts International68-11B.
標題:	Biophysics, Medical. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3288360
ISBN:	9780549314257

Fluctuation analysis reveals protein concentration, mobility, and aggregation states in cells.
Dalal, Rooshin Bhadrik.

Fluctuation analysis reveals protein concentration, mobility, and aggregation states in cells. - 221 p.

Adviser: Rick Horwitz.

Thesis (Ph.D.)--University of Virginia, 2008.

Determination of the concentration, aggregation state, and mobility of molecules in living cells is a matter of increasing importance and interest. Cell migration in particular, depends heavily on the ability of adhesion proteins to form aggregates and move to specific locations in the cell. Quantitative measurements of adhesion protein transport and distribution can thus lead to insights about their function or mechanism of action. Unlike intensity-based methods, methods based on analysis of fluorescence fluctuations can determine the aggregation state of molecules. In this study, I have developed and adapted fluctuation-based techniques to determine the concentration, mobility, and aggregation state of fluorescently labeled moelcules.

ISBN: 9780549314257Subjects--Topical Terms:

1017681
Biophysics, Medical.

Fluctuation analysis reveals protein concentration, mobility, and aggregation states in cells.
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100 1 $a Dalal, Rooshin Bhadrik. $3 1280995
245 1 0 $a Fluctuation analysis reveals protein concentration, mobility, and aggregation states in cells.
300 $a 221 p.
500 $a Adviser: Rick Horwitz.
500 $a Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7470.
502 $a Thesis (Ph.D.)--University of Virginia, 2008.
520 $a Determination of the concentration, aggregation state, and mobility of molecules in living cells is a matter of increasing importance and interest. Cell migration in particular, depends heavily on the ability of adhesion proteins to form aggregates and move to specific locations in the cell. Quantitative measurements of adhesion protein transport and distribution can thus lead to insights about their function or mechanism of action. Unlike intensity-based methods, methods based on analysis of fluorescence fluctuations can determine the aggregation state of molecules. In this study, I have developed and adapted fluctuation-based techniques to determine the concentration, mobility, and aggregation state of fluorescently labeled moelcules.
520 $a Fluorescence correlation spectroscopy (FCS) was used over 30 years ago to characterize the fast diffusion of molecules in solution. Image correlation spectroscopy (ICS) was later developed to examine spatial intensity fluctuations of slowly moving molecules. Raster-scan image correlation spectroscopy (RICS) was recently developed to analyze the dynamics of molecules over the time scales accessible by both FCS and ICS. In this work, the parameter limits for RICS were explored. Accurate concentration measurements were made with a standard calibration curve, and the errors associated with performing measurements on image stacks of variable sizes and numbers of frames were determined. Additionally, moment analysis was developed as a way to overcome some limitations of correlation-based approaches. This method obtains the average brightness and number of molecules in a small volume by calculating the average intensity and variance of the intensity for each pixel of an image stack. The requirements for this technique are that the variance of the detector be equal to the average intensity, and that the intensity in any given pixel fluctuates due to the diffusion of molecules in that pixel. The method worked well for systems in which individual photons are recorded, but for systems in which light is accumulated for a certain amount of time (analog detection), the underlying photon count distribution needed to be determined from the digital level distribution. The performance of analog detectors was shown to be acceptable under common experimental conditions. Specifically, the brightness of mEGFP was determined in solution and in cells. Additional methods were developed to correct for photobleaching and to identify the location of immobile components. Lastly, brightness and number count information was recovered from cells expressing alpha5-integrin and paxillin. Since most commercial laser scanning microscopes operate in the analog mode, this analysis can theoretically be applied to data obtained with any of these instruments.
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