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Analysis of Sp1 associated transcription regulatory factors bound on TSG101 promoter by DAPA and two dimensional gel electrophoresisLIN, I-Ju 25 August 2008 (has links)
TSG101 is a tumor susceptibility gene exhibits multiple biological functions, including the regulation of vesicular trafficking, transcription, cellular growth and differentiation. The intracellular steady-state level of TSG101 was shown to under stringent control in a narrow range. Either deprivation or overexpression of mouse tsg101 in NIH3T3 cells leads to neoplastic transformation and subsequent tumorigenic potential of the transformed cells. However, the detail mechanism for regulation of TSG101 gene promoter activity is not clear. Our results indicated TSG101 is a housekeeping gene and contains a TATA-less and Sp1 binding site promoter. Here, we demonstrate in vivo binding of Sp1 transcription factor on TSG101 promoter region by chromatin immunoprecipitation(ChIP). In addition, Sp1-associated transcription regulators were purified using DNA affinity precipitation assay (DAPA) method and subjected to two-dimensional gel electrophoresis and the subsequent MALDI-TOF analysis. Our results verify the biding of Sp1 transcription on the DAPA probe containing wildtype but not the mutant Sp1 biding sequence by subsequent western blotting. Our MALDI-TOF analysis of protein spots from two-dimensional gel did not reveal the binding of Sp1 protein, instead the identified a number of cellular proteins, such as U5 small nuclear RNP¡BATP-dependent DNA helicase 2 and actin of unknown significance.
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Detection approaches for the analysis of volume limited biological samples /Gostkowski, Michael Leonard, January 2000 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references. Available also in a digital version from Dissertation Abstracts.
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DNA binding activities in cerebellar granule cell neurons recognizing the promoter for the GABA(A)-alpha6 receptor subunitStock, Rachel E. January 2002 (has links)
Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: NF-1 -- granule cell neuron. Includes bibliographical references (p. 38-39).
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I. Flow injection capillary electrophoresis using on-line enzymatic and dye interaction reactions II. Mini-solid phase extraction of pharmaceuticals and phospholipids in conjunction with nano-electrospray mass spectrometryQi, Lining. January 2003 (has links)
Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2003. / Title from first page of PDF document. Document formatted into pages; contains xvi, 254 p. : ill. Includes bibliographical references.
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Applications of gel electrophoresis in quantum dot conjugates' separation and purificationWang, Luxin. Fan, Xudong. Mustapha, Azlin. January 2009 (has links)
Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Xudong Fan and Dr. Azlin Mustapha. Includes bibliographical references.
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Electrophoretic methodologies for the determinations of minerals and trace elements in milkSze, Kwan-Lok., 施均樂. January 2009 (has links)
published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Study of methodologies for detecting bilirubin by electrochemical, UV,fluorescence and chemiluminescence techniques and their applicationfor CE determination of bilirubin and arsenic anions in biofluidMo, Shanlie., 莫善列. January 2012 (has links)
Capillary-based analytical methodologies were developed to meet the need for metabolite determination in two major areas. The first area is the determination of free bilirubin in sera for the management of jaundiced neonates under critical conditions. Three sensitive detection techniques were investigated, Quantum dots (QD) mediated fluorescence, Chemiluminescence (CL) and Microelectrode detection. Four different types of QDs were synthesized for the direct bilirubin determination. The CAH-capped CdTe QDs were selected as it shows the best performance compared to organic dyes and other QDs. Its optimized preparation conditions are: refluxing solution containing Cd/Te/CAH (1:0.5:2.4 w/w) for 4 hours at 100 °C. From Transmission Electron Microscope characterization, nano-size QDs with an uniform size distribution, high luminescence and good stability were obtained. The optimized detection conditions were: incubation of bilirubin with CAH-capped CdTe QDs (5 10-6 mol/L) in water at pH=5.6 and 20 oC for 8 min prior to spectrofluorometric determination (λex=473 nm and λem=580 nm). A linear working range from 0.043-0.86 μg/mL with 0.9943 correlation coefficient and 2 ng/mL detection limit (LOD, S/N=3) were achieved. Results from nFIA-CL indicate a quick response within seconds though a poorer LOD (S/N=3) of 15 μg/mL for the direct bilirubin determination.
The third technique investigated used an enzyme microelectrode and it was found to be able to couple with capillary electrophoresis (CE) in frontal analysis (FA) for the determination of free bilirubin in serum samples. Making use of the micron size of the carbon-fiber electrode, a new MCNTs (Multi-wall Carbon Nanotubes) modified CFMEs (Carbon fiber microelectrodes) was fabricated within a microchip-CE device with three guided channels to enable electrodes alignment. Method to immobilize bilirubin oxidase (BOD) onto the CFMEs surface by the carbodiimide chemistry achieved the highest detection sensitivity. Under optimized conditions (sample introduced by hydrodynamic injection at △H (20 cm), and a running/detection buffer (10 mM phosphate) at pH 7.4, working potential for amperometric detection at +0.8 V), a linear working range between 1-40 μg/mL and a detection limit (S/N=3) at 0.15 μg/mL for free bilirubin was achieved.
The second area for metabolite determination was developing a new analytical method for the management of APL (acute promyelocytic leukemia) patients under arsenic treatment, a drug required continued monitoring. The analytical requirements include a high detection sensitivity and the capability to provide timely results for multiple drug residues. Using a 20 mM phosphate as the running buffer and 0.05mM CTAH (Cetyl-trimethyl-ammonium hydroxide) as an additive for EOF reversal, co-EOF (co-electroosmotic flow) stacking was established to enhance up to 200 times of the detection limit for arsenite. Satisfactory baseline separation for arsenite, arsenate, MMA (Methylarsonic acid) and DMA (Dimethylarsinic acid) was achieved with linear working ranges (correlation coefficients > 0.999) from 1-50 μg/mL for arsenate and DMA, 0.5-50 μg/mL for MMA as well as 0.1-50 μg/mL for arsenite. Detection limits (S/N=3, n=3) achievable for arsenate, arsenite, MMA and DMA were found to be 0.41 μg/mL, 0.01 μg/mL, 0.04 μg/mL and 0.32 μg/mL respectively at levels meeting the requirement for APL patient urine monitoring. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Tracking neuronal content using capillary electrophoresis with multiphoton excitation of fluorescenceWise, Dana Diane 28 August 2008 (has links)
Not available / text
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Applications of multiphoton-excited photochemistry to microsecond capillary electrophoresis, photolithography, and the development of smart materialsRitschdorff, Eric Thomas 20 October 2011 (has links)
Laser-based techniques have become essential tools for probing biological molecules in systems that demand high spatial and temporal control. This dissertation
presents the development of micro-analytical techniques based on multiphoton excitation (MPE) to promote highly localized, three-dimensional (3D) photochemistry of biologically relevant molecules on submicron dimensions. Strategies based on capillary electrophoresis (CE) have been developed for the rapid separation and spectroscopic
analysis of short-lived photochemical reaction products. High-speed separation and
analysis are achieved through a combination of very high electric fields and a laser-based optical system that uses MPE for both the generation and detection of hydroxyindole photoproducts on the time scale of microseconds. MPE was also used for the development of photolithographic techniques for the creation of microstructured protein-based materials with highly defined three-dimensional (3D) topographies. Specifically, a multiphoton lithographic (MPL) technique was developed that used a low-cost microchip laser for the rapid prototyping of 3D microarchitectures when combined with dynamic optical masking. Furthermore, MPL was used to create novel “smart” biomaterials that
reproducibly respond with tunable actuation to changes in the local chemical and thermal environment. The utility of these materials for creating biocompatible cellular
microenvironments was demonstrated and presents a novel approach for studying small populations of microorganisms. Finally, through the development of a multifocal
approach that used multiple laser beams to promote the photocrosslinking of biological
molecules, the speed and versatility of MPL was extended to allow both the parallel
fabrication of 3D microstructures and the rapid creation of large-scale biomaterials with
highly defined spatial features. / text
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I. Hadamard Transform Capillary Electrophoresis for the Analysis of Biologically Active Species II. Characterization and Application of Two-Photon Activatable Proton and Radical GeneratorsBraun, Kevin L January 2005 (has links)
PART I. A modified Hadamard transform has been developed and applied to the analysis of biologically active species using capillary electrophoresis. Hadamard transformations, a matrix based multiplexing technique, when coupled with a capillary electrophoresis instrument capable of rapid sample injection, provides a means to semi-continuously inject samples. The multiple injections separate, interpenetrate, and are detected as the summation of the multiple injections. Deconvolution of the multiplexed signal by multiplication with the inverse of the injection matrix yields a single injection electropherogram that exhibits improved S/N. In modified Hadamard transform capillary electrophoresis (mHTCE), an injection sequence of half the length as conventional HTCE (cHTCE) is utilized. Modifying the manner in which the raw data is manipulated before deconvolution facilitates the reduced injection sequence. When coupled with software, mHTCE can reduce the collection time for a Hadamard sequence by up to 48%. The substantial time reduction afforded by mHTCE is utilized to demonstrate the first time-resolved application of Hadamard transformations for the analysis of neurotransmitters. Additionally, mHTCE has been demonstrated as a means to improve the sensitivity for analysis of amino acids and proteins including gamma-aminobutyric acid, dopamine, and enhanced green fluorescent protein (EGFP) with picomolar detection limits.Part II. Two-photon excitation provides a means to activate chemical and physical processes with high spatial resolution and improved depth penetration compared to one-photon excitation. When combined with three-dimensional lithographic microfabrication (3DLM), these advantages provide a means to fabricate complex structures through radical and cationic two-photon induced polymerization (TPIP). A strategy for realizing high-fidelity microstructures is reported that considers the inherent structural limitations of acrylate monomers. Utilizing this strategy, a series of high-fidelity microstructures is reported for application in microfluidic devices, microelectromechanical systems (MEMS), and microoptical devices such as photonic bandgap (PBG) crystals. Improved periodicity is reported here for f.c.c. PBG crystals compared to earlier examples through addition of micromechanical supports that provide increased strength to the high-aspect ratio crystals. To extend TPIP to cationic polymerization, a series of two-photon activatable photoacid generators has been developed. The new PAGs exhibit one to two orders of magnitude lower polymerization threshold intensities than conventional ultraviolet-sensitive initiators.
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