Дисертації з теми "Fluorescent analysis"
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1
Mangham, Barry. "Synthesis and analysis of fluorescent dye molecules." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.602528.
Повний текст джерелаАнотація:
The nature of non-covalent bonding interactions was investigated through the deposition and subsequent scanning tunnelling microscopy (STM) imaging of tetra-substituted porphyrins. Porphyrins bearing iodo, bromo, nitro, pyridyl and carboxylic acid groups were synthesised and deposited on either Au(110) or Au(l11). STM imaging and analysis showed a variety of different orientations and packing for the different functional groups. The unusual tip induced growth of honeycomb packing orientations was seen for tetra-pyridyl substituted porphyrin. Tetra-bromo substituted porphyrin was observed to adopt different ordered orientations of the saddle shape conformation on Au(111). The synthesis of novel porphyrin dimers bearing carboxylic acid groups was investigated, with a variety of different pathways being identified and explored. Furthermore, upon cooling unusual spectroscopic behaviour was observed for a hexa-phenyl substituted meso-linked porphyrin dim er. The synthesis of novel BODIPY dimers and trimers was investigated. A number of fluoro and catecholate substituted BODIPY compounds were synthesised, bearing a variety of different linkers. Linkers investigated included phenyl, biphenyl, terphenyl, durene and terphenylene. Electrochemical and spectroscopic investigations demonstrated a variety of differences between meta- and para-substitution positions. The extension of the linker length from phenyl through to terphenyl displayed a reduction in communication of BODIPY moieties. The durene linked dimer added steric bulk to the centre of the BODIPY dimer, resulting in increased fluorescence lifetimes and quantum yields. 1
2
Ladyman, Melissa Kate. "Development of fluorescent assays for biological analysis." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17627.
Повний текст джерелаАнотація:
The work in this thesis is divided into two parts; the first is the synthesis of a ‘switch-on’ fluorophore to measure cell viability, and the second is the development of a fluorescent detection method for protein−peptide affinity assays applied in the identification of protein-protein inhibitors. Tetrazolium salts are often used in cytotoxicity assays as indicators of cell viability as they are reduced to deeply coloured formazans exclusively in healthy cells. However, measuring the absorbance of the formazan is prone to bias from other coloured species in the cell media, requires solubilisation and can be difficult to quantify. A preferable method of detection is direct fluorescence as it is easily quantified, more sensitive and would ideally remove the need to solubilise the insoluble dye. The aim of this project was to synthesise a tetrazolium salt that could be reduced to a soluble fluorescent formazan in healthy cells as an indicator of cell viability. A number of fluorescent formazans were synthesised by incorporation of a fluorophore. The corresponding tetrazolium salts were non-fluorescent and could be reduced to the formazan in vitro. Several formazans were synthesised to attempt to increase the emission wavelength and intensity to overcome cellular autofluorescence. Protein-protein interactions have been implicated in the pathogenesis of many human diseases but until recently were considered undruggable. However, peptides have emerged as ideal compounds for targeting the large and relatively featureless protein interfaces. Work focussed on the discovery of peptide inhibitors for the E3 ubiquitin ligase stationary-phase kinase associated protein (Skp2). Potential peptide inhibitors were identified using CelluSpot synthesis and array technology to screen peptide libraries. Qualitative analysis of the protein affinity assay results by enhanced chemiluminescent detection was found to be misleading, and so a quantifiable and more sensitive fluorescent detection method was developed.
3
Martin, Andrew. "Glycosylated green fluorescent protein for carbohydrate binding protein analysis." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/glycosylated-green-fluorescent-protein-for-carbohydrate-binding-protein-analysis(9ddae46e-b4d7-4c08-8240-94b9b804ac68).html.
Повний текст джерелаАнотація:
The interactions of glycoconjugates with carbohydrate binding proteins are responsible for a wide range of recognition events in vivo; including immune response, cell adhesion and signal transduction. Glycoconjugates have already found many medicinal uses as therapeutic and diagnostic agents, but their full potential is yet to be realised. Access to a variety of homogeneously glycosylated glycoproteins is essential for the study of these important carbohydrate binding events. This requires the chemical synthesis and attachment of biologically relevant glycans to unglycosylated protein scaffolds in a site selective manner. Here we describe the use of a range of glycosyl iodoacetamides to glycosylate proteins selectively via their cysteine residues. We have chosen the green fluorescent protein mutant GFPuv for use as a protein scaffold due its known tolerance of two cysteine mutations (E6C and I229C) and the previous successful derivatisation of these cysteines with iodoacetamides.1 The inherent fluorescence of GFPuv also makes it a useful candidate for fluorescence based binding assays or cell labelling studies.16 active, mutants of GFPuv were created using a mixture of site directed mutagenesis and DNA shuffling (including one mutant containing six reactive cysteine residues). This was achieved by producing random combinations of two synthetic variants of GFPuv, one of which contained 33 surface cysteines. 94 bacterial colonies expressing active GFPuv were then sequenced and the new chimeric genes analysed. Four monosaccharides and one trisaccharide (N-glycan core mimic) suitable for the chemical glycosylation via cysteines were synthesised and successfully used to create a selection of homogeneous neoglycoproteins. These neoglycoproteins were demonstrated to interact differently with different lectins (including ConA, GNL and Jacalin) in a qualitative fluorescence based assay. Interactions were shown to vary with glycan structure, position of glycosylation sites and the number of glycosylation sites.
4
Mohammadi, Mahnaz. "Developing an imaging bi-spectrometer for fluorescent materials /." Online version of thesis, 2009. http://hdl.handle.net/1850/9671.
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Hunter, S. Jayne. "The quantitative analysis of green fluorescent protein in plants." Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340248.
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Manolakis, Alexis. "A comparative analysis of traditional and fluorescent spermatozoa staining techniques." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12502.
Повний текст джерелаАнотація:
Thesis (M.S.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.The purpose of this study was to compare the traditional KPIC method for staining sperm to modern fluorescent techniques employed by SPERM HY-LITERTM. Factors such as staining time, sperm recovery, and the ability to quickly and accurately count sperm were evaluated. Dilutions ranging from 1:2 to 1:10,000,000 of semen, post-coital swabs and sperm/epithelial cell suspensions were stained with SPERM HY-LITERTM, SPERM HY-LITERTM Express and KPIC. Case work samples were also analyzed with KPIC and SPERM HY-LITERTM. The sperm counts for the more concentrated dilutions of semen and sperm/e-cell samples were statistically similar in both KPIC and SPERM HY-LITERTM. However, post-coital samples stained with SPERM HY-LITERTM were statistically different than samples stained with KPIC, resulting in the observation of fewer cells. Similar results were observed with SPERM HY-LITERTM Express as well as with case work samples stained with SPERM HY-LITERTM. Although SPERM HY-LITERTM has the benefits of quicker and easier scanning due to the fluorescent filters, it is considerably more costly to employ and did not result in greater sperm recovery. Overall, KPIC provided the best results in the shortest amount of time and within a reasonable budget.
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Wu, Yongqi. "Multi-parameter Fluorescent Analysis of Magnetically Enriched Circulating Tumor Cells." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1408633549.
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Cain, Heather. "Fluorescent derivatization of arginine- and carbonyl-containing analytes for analysis by capillary electrophoresis with laser-induced fluorescence detection." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0007/MQ59786.pdf.
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Hu, Qin. "Synthesis, characterization and analytical separation of fluorescent water-soluble carbon nanoparticles." HKBU Institutional Repository, 2015. https://repository.hkbu.edu.hk/etd_oa/156.
Повний текст джерелаАнотація:
This thesis mainly consists of two parts. The first part is concentrated on synthesizing amine/carboxylic-functionalized carbon nanoparticles (CNP) and investigating its fundamental properties in the aid of capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). In this part, CNP is synthesized from citric acid (CA) and 1,2-ethylenediamine (EDA) by a microwave-assisted pyrolysis method. The resultant CNP is characterized by absorption and photoluminescence (PL) spectroscopy, transmission electron microscopy (TEM) and infrared spectroscopy (IR) to determine its overall optical properties, morphology and composition. Followed by this, the CNP product is separated and analyzed by CZE coupled with UV absorption and laser-induced fluorescence (LIF) detections. Under optimal pH and concentration of run buffer, the effect of reaction time and mole ratio of amine (NH2) to carboxylic acid (COOH) moieties of the precursors on the CNP species present in CNP products are studied. Our results show that the synthesis of CNP could be improved by lengthening the microwave irradiation time and optimizing the initial mole ratio of NH2/COOH in the precursors. Negatively charged CNP are obtained only when the amount of CA exceeds that of EDA, i.e., the mole ratio of NH2/COOH is 0.250.80. By contrast, when the quantity (in mole) of NH2 in EDA is equal to or larger than that of COOH in CA, only positively charged and neutral CNP species are formed, inferring that the CNP species are predominantly covered by the surface-attached ammonium and amido moieties. This work highlights the merit of CZE to identify the composition of an as-prepared CNP product which is pretty much dependent on the mole ratio of NH2/COOH. In addition, we carry out reversed-phase high-performance liquid chromatography coupled with fluorescence detection (RP-HPLC-FD) methodology to study the properties of each individual CNP species. Under optimal mobile phase and elution gradient conditions, the effect of mole ratio of NH2/COOH in the initial reagents on CNP product is studied. At NH2/COOH = 0.67, the strongest fluorescence CNP sample is obtained. The separated CNP fractions are collected and further characterized by UV-visible absorption and PL spectroscopy, CE, TEM, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The absorption and PL emission bands (λem) of the fractions are bathochromatically shifted with the elution order of CNP on RP-HPLC. The TEM images prove that CNP are eluted from the smallest to the largest. The MALDI-TOF MS data show that CNP undergo fragmentations, closely relating to their surface-attached carboxylic acid and amide/amine moieties. This work highlights the merit of RP-HPLC coupled with fluorescence detection, TEM and MS for isolation and characterization of individual CNP species present in a CNP sample. By application of CE and HPLC separation for CNP product, better understandings of the hidden fundamental properties of CNP are achieved. The two separation techniques are well complementary to each. On one hand, CE is able to separate both positively and negatively charged CNP species which cannot be retained and separated by HPLC column, facilitating the investigation of different charge states of CNP species present in a CNP product. On the other hand, the preparative property of HPLC allows for multi-collection of the separated CNP fractions which is difficult in the case of CE analysis owning to its low sample injection volume. By using HPLC separation, the individual CNP fractions can be collected for more precious investigation of their unique photophysical and chemical properties by absorption and PL spectroscopy, TEM and MALDI-TOF MS. The second part is focused on preparing CNP from naturally available bioresources and investigating the effect of doped heteroatoms on nitrogen (N) and sulfur (S)-doped carbon nanoparticles (N,S-CNP) based on our proposed modern RP-HPLC-FD methodology which has been proved to be useful in separating and analyzing CNP in the first part of this work. In this part, ultrasmall N,S-CNP is prepared by microwave-assisted pyrolysis of precursors of rice as carbon source and N-acetyl-L-cysteine (NAC) as N and S dopants. The obtained N,S-CNP are fully characterized by elemental analysis, FTIR, x-ray photoelectron spectroscopy, TEM, UV-vis absorption and PL spectroscopy. Meanwhile, undoped CNP (derived from rice only) is also synthesized and characterized. The chemical compositions, sizes and spectral properties of undoped CNP (derived from rice only) and N,S-CNP are demonstrated to be different from each other. With the assistance of RP-HPLC-FD, the effect of different mass ratios of NAC to rice (NAC/rice) on N,S-CNP is investigated. When the NAC/rice increases from 0.20 to 0.80, the signals of the later eluted peaks increase progressively, indicating that higher NAC/rice benefits the generation of CNP with stronger fluorescence emissions. The HPLC-separated N,S-CNP fractions are collected and further characterized by MALDI-TOF MS, UV-vis absorption and PL spectroscopy, showing that the structural changes induced by doping with heteroatoms N and S plays a key role in regulating the PL properties of the N,S-CNP
10
Chen, Kai. "Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/4886.
Повний текст джерелаАнотація:
Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.
11
Crosby, Kevin C. "Macromolecular Organization and Cell Function: A Multi-System Analysis." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/30259.
Повний текст джерелаАнотація:
The interior of the cell is a densely crowded and complex arena, full of a vast and diverse array of molecules and macromolecules. A fundamental understanding of cellular physiology will depend not only upon a reductionist analysis of the chemistry, structure, and function of individual components and subsystems, but also on a sagacious exegesis of the dynamic and emergent properties that characterize the higher-level system of living cells. Here, we present work on two aspects of the supramolecular organization of the cell: the controlled assembly of the mitotic spindle during cell division and the regulation of cellular metabolism through the formation of multienzyme complexes.
During division, the cell undergoes a profound morphological and molecular reorganization that includes the creation of the mitotic spindle, a process that must be highly controlled in order to ensure that accurate segregation of hereditary material. Chapter 2 describes results that implicate the kinase, Zeste-white3/Shaggy (Zw3/Sgg), as having a role in regulating spindle morphology.
The congregation of metabolic enzymes into macromolecular complexes is a key feature of cellular physiology. Given the apparent pervasiveness of these assemblies, it seems likely that some of the mechanisms involved in their organization and regulation might be conserved across a range of biosynthetic pathways in diverse organisms. The Winkel laboratory makes use of the flavonoid biosynthetic pathway in Arabidopsis as an experimental model for studying the architecture, dynamics, and functional roles of metabolic complexes. Over the past several years, we have accumulated substantive and compelling evidence indicating that a number of these enzymes directly interact, perhaps as part of a dynamic globular complex involving multiple points of contact between proteins. Chapter 3 describes the functional analysis of a predicted flavonol synthase gene family in Arabidopsis. The first evidence for the interaction of flavonoid enzymes in living cells, using fluorescent lifetime imaging microscopy fluorescent resonance energy transfer analysis (FLIM-FRET), is presented in Chapter 4.Ph. D.
12
Gösch, Michael. "Microfluidic analysis and parallel confocal detection of single molecules /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-663-4/.
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13
Lamb, J. C. "Fluorescent derivatives of tubulin as probes for the analysis of microtubule dynamics." Thesis, University of East Anglia, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372350.
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Yarbrough, Daniel Kenneth. "Structural and mutational analysis of chromophore maturation in long wavelength fluorescent proteins /." view abstract or download file of text, 2004. http://wwwlib.umi.com/cr/uoregon/fullcit?p3120630.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--University of Oregon, 2004.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 142-152). Also available for download via the World Wide Web; free to University of Oregon users.
15
Ahlford, Annika. "Applications of Four-Colour Fluorescent Primer Extension Technology for SNP Analysis and Discovery." Doctoral thesis, Uppsala universitet, Molekylär medicin, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-129221.
Повний текст джерелаАнотація:
Studies on genetic variation can reveal effects on traits and disease, both in humans and in model organisms. Good technology for the analysis of DNA sequence variations is critical. Currently the development towards assays for large-scale and parallel DNA sequencing and genotyping is progressing rapidly. Single base primer extension (SBE) is a robust reaction principle based on four-colour fluorescent terminating nucleotides to interrogate all four DNA nucleotides in a single reaction. In this thesis, SBE methods were applied to the analysis and discovery of single nucleotide polymorphism (SNP) in the model organism Drosophila melanogaster and in humans. The tag-array minisequencing system in a microarray format is convenient for intermediate sized genotyping projects. The system is scalable and flexible to adapt to specialized and novel applications. In Study I of the thesis a tool was established to automate quality control of clustered genotype data. By calculating “Silhouette scores”, the SNP genotype assignment can be evaluated by a single numeric measure. Silhouette scores were then applied in Study I to compare the performance of four DNA polymerases and in Study III to evaluate freeze-dried reagents in the tag-array minisequencing system. The characteristics of the tag-array minisequencing system makes it suitable for inexpensive genome-wide gene mapping in the fruit fly. In Study II a high-resolution SNP map, and 293 genotyping assays, were established across the X, 2nd and 3rd chromosomes to distinguish commonly used Drosophila strains. A database of the SNP markers and a program for automatic allele calling and identification of map positions of mutants was also developed. The utility of the system was demonstrated by rapid mapping of 14 genes that disrupt embryonic muscle patterning. In Study III the tag-array minisequencing system was adapted to a lab-on-a-chip format for diagnostic testing for mutations in the TP53 gene. Freeze-drying was evaluated for storing reagents, including thermo-sensitive enzymes, on the microchip to reduce the complexity of the integrated test. Correct genotyping results were obtained using freeze-dried reagents in each reaction step of the genotyping protocol, both in test tubes and in single polymer test chambers. The results showed the potential of the approach to be implemented in fully integrated systems. The four-colour chemistry of SBE has been developed further to allow massively parallel sequencing (MPS) of short DNA fragments as in the Genome Analyzer system (Solexa/Illumina). In Study IV MPS was used to compare Nimblegen arrays and the SureSelect solution-based system for targeted enrichment of 56 continuous human candidate-gene regions totalling 3.1 Mb in size. Both methods detected known SNPs and discovered novel SNPs in the target regions, demonstrating the feasibility for complexity reduction of sequencing libraries by hybridization methods.
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Toomre, Derek K. "Structural and functional analysis of oligosaccharides with a novel and versatile fluorescent tag /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9719872.
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Brown, Lynda Jane. "Development of novel fluorescent oligonucleotide probes for use in nucleic acid sequence analysis." Thesis, University of Southampton, 2000. https://eprints.soton.ac.uk/386336/.
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Gustafsson, Nils S. "Enabling live-cell super-resolution microscopy by computational analysis and fluorescent probe design." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10025032/.
Повний текст джерелаАнотація:
Super-resolution fluorescence microscopy has become a well-established tool for structural cell biology, achieving lateral resolutions on molecular scales. Limitations to the application of super-resolution in live-cell imaging remain however. Particularly, the requirement for intense phototoxic illumination, prolonged acquisition times, non-physiological media and extensive computational analysis. This thesis aims to address these issues, through both analytical and biochemical approaches. Three analytical methods are presented and evaluated, including a method for nano-meter precision microtubule end tracking applicable to live-cells, and a Hidden Markov model of fluorophore photoswitching, for use in live-cell experimental design. The third, Super-Resolution Radial Fluctuations (SRRF), is a novel image processing algorithm that forms the basis of a new approach to live-cell super-resolution. Low-illumination, live-cell imaging of actin dynamics in T-cell synapses at resolutions better than 100 nm are demonstrated in physiologically relevant media using SRRF. In addition, SRRF enables super-resolution in modern widefield or confocal microscopes using conventional fluorophores with illumination orders of magnitude lower than methods such as single molecule localisation or stimulated emission depletion. The computational framework developed for SRRF, NanoJ, is an open-source java library of high performance algorithms that has enabled the development of a number of GPU accelerated ImageJ/FIJI plugins for super-resolution. While SRRF allows super-resolution in live-cells, achieving resolutions on the order of 10s of nm remains challenging. To this end, Super-Beacons (SBs), novel DNA-based super-resolution probes that photoswitch spontaneously under environments relevant for live-cell imaging, are presented. The design of SBs allows the engineering of photoswitching within the physical environment of the cell, mediated through structural, chemical or thermal control. Single molecule imaging and characterisation of the photoswitching of the probes is performed. The use of SBs is verified in fixed-cell imaging of benchmark structures and live-cell super-resolution of interferon inducible transmembrane proteins is demonstrated resolving the endosomal membrane with 65 nm resolution.
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Davies, G. "Calibration of droplet spectra using fluorescent tracers and their use and quantification in spray experiments." Thesis, Cranfield University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379520.
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Kurishita, Yasutaka. "Development of Molecular Tools for Analysis and Imaging of ATP and Other Biomolecules Based on Coordination Chemistry." 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/188614.
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Wakelin, Stuart. "Fluorescence and transient kinetic analysis of the dictyostelium myosin-II motor domain using green fluorescent protein (GFP) and its variants as probes." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/29706.
Повний текст джерелаАнотація:
The determination of the structure of the myosin motor domain has made it possible to introduce fluorescent probes at defined sites, thereby allowing the resolution of the mechanochemical steps in greater detail. Here, modern genetic cloning techniques were utilised to create and express within Dictyostelium discoideum novel myosin-II motor fusion proteins containing various fluorescent probes, in an attempt to investigate conformational changes within the motor domain during the actin-bound stages of the crossbridge cycle. Stopped-flow analysis showed that the myosin ATPase of the single tryptophan myosin-II motor W501 was unaffected by N- and C-terminal YFP and CFP probes, whereas ATP-induced actomyosin dissociation was disrupted (potentially by the probes' propensity to dimerise in close proximity), thus rendering the system unsuitable for investigation of the actin-bound stages of the actomyosin ATPase. By combining total internal reflection microscopy with flash photolysis of an inert caged-ATP precursor, it was shown that kinetic information for the ATP-induced dissociation of fluorescently-labelled myosin motor domains may be achieved using only nanogram quantities, while simultaneously avoiding bundling artefacts common to solution kinetics. A slight adaptation of this process could yield a highly sensitive assay for the processivity of non-classical myosin types, yielding information on their rotational and lateral movement concurrently. Lever arm movement was assessed via the analysis of fluorescence resonance energy transfer (FRET) changes between the YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein) probes. A FRET efficiency increase was observed upon nucleotide occupancy of the active site, in direct contrast to previous FRET studies. Anisotropy studies showed no change upon nucleotide binding, suggesting the FRET increase was due to a distance change, rather than a variation of relative dipole orientations. However, due to the high anisotropy (i.e. slow rotation in solution) of the protein, it was also shown that results from this type of FRET system are qualitative rather than quantitative.
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Kasprzyk, Arkadiusz. "Investigation of clonality and minimal residual disease in haematological malignancy using fluorescent in situ hybridization." Thesis, University College London (University of London), 1998. http://discovery.ucl.ac.uk/1317904/.
Повний текст джерелаАнотація:
Cytogenetic analysis of the malignant clone is clinically important in haematological malignancy. Analysis by metaphase cytogenetics is restricted to the small proportion of malignant cells which are actively dividing. This thesis explores the dynamics of malignant clones using the technique of fluorescence in situ hybridization (FISH) to visualize chromosomal abnormalities in interphase (non-dividing) cells. Hyperdiploid (>46 chromosomes) clones have been investigated by interphase FISH in acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) using appropriate chromosome-specific probes. A hyperdiploid clone was detected in interphase cells in 9/65 patients with ALL in whom metaphase cytogenetics had failed or was normal. A single hyperdiploid cell was identified as clonal in one patient with MDS but not in six others with AML, MDS or ALL. The involvement of different cell lineages in the malignant clone was investigated by simultaneous FISH and identification of the cell type by morphology or monoclonal antibodies. In ALL, hyperdiploid clones were restricted to the lymphoid blasts in 9/9 cases, while Philadelphia (Ph) positive clones, (identified by probes to the genes m- BCR or M-BCR and ABL which fuse as a result of the translocation) were found either in lymphoid blasts alone (1/3 cases) or in both lymphoid and myeloid cells (2/3 cases). In AML trisomy 8 (using a chromosome 8-specific probe) and an 11q23 abnormality (which split YAC 13HH4) were both found only in the myeloid blasts, in 3/3 and 2/2 cases respectively. A sensitive method for the detection of hyperdiploid \geq 50 clones in ALL was developed for minimal residual disease detection. Simultaneous probing of three chromosomes enabled detection of one hyperdiploid cell in 10,000. Heterogeneity in the speed with which the clone was eliminated in remission was seen in 16 patients and early relapse was detected in one patient.
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Bruggeman, Andrew. "Generation of fluorescent recombinant listeriolysin O toxin for analysis of interactions with host protein." Connect to resource, 2008. http://hdl.handle.net/1811/32222.
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Zheng, Huanquan. "Green fluorescent protein as a marker for genetic analysis of the plant secretory pathway." Thesis, Oxford Brookes University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341680.
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Wilson, Keith Michael. "Single particle fluorescent imaging analysis of cell surface HLA-DR and associated CD74 antigens." Thesis, University of Essex, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361022.
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Benton, Matthew Alan. "Analysis of embryonic development in Tribolium castaneum using a versatile live fluorescent labelling technique." Thesis, University of Cambridge, 2014. https://www.repository.cam.ac.uk/handle/1810/275921.
Повний текст джерелаАнотація:
Studies on new arthropod models are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, most insect embryos exhibit the short or intermediate-germ type and become enveloped by extensive extraembryonic membranes. The genetic basis of these processes has been the focus of active research in several insects, especially Tribolium castaneum. The processes in question are very dynamic, however, and to study them in depth we require advanced tools for fluorescent labelling of live embryos. In my work, I have used a transient method for strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labelling the chromatin, membrane, cytoskeleton or combinations thereof. I have used several of these new live imaging tools to study the process of cellularisation in Tribolium, and I found that it is strikingly different to what is seen in Drosophila. I was also able to define the stage when cellularisation is complete, a key piece of information that has been unknown until now. Lastly, I carried out extensive live imaging of embryo condensation and extraembryonic tissue formation in both wildtype embryos, and embryos in which caudal gene function was disrupted by RNA interference. Using this approach, I was able to describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. As well as uncovering several of the cellular mechanisms underlying condensation, I have proposed testable hypotheses for other aspects of embryo formation. The work presented in this thesis will serve as a foundation for future studies on cellularisation and tissue morphogenesis in Tribolium. Furthermore, the live imaging method, the fluorescent labelling constructs, and the analysis I carried out should be easily adaptable to other non-model arthropod species.
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Watson, Amy Dawn. "Binding studies of near infrared cyanine dyes with human serum albumin anf poly-l-lysine using optical spectroscopy methods." unrestricted, 2006. http://etd.gsu.edu/theses/available/etd-01042008-154159/.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--Georgia State University, 2006.Title from file title page. Gabor Patonay, committee chair; Zhen Huang, Alfons Baumstark, committee members. Electronic text (236 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 28, 2008. Includes bibliographical references.
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O'Neill, Wendi. "Construction of a Fluorescent Reporter Gene for the Analysis of OGDH2 Protein Stability in Hypoxia." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437737682.
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Fu, Yutao. "Analysis of Protistan Grazing on Bioremediative Bacteria Using IN WVO Fluorescent Protein Expression and Flow Cytometry." Fogler Library, University of Maine, 2002. http://www.library.umaine.edu/theses/pdf/FuY2002.pdf.
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Altenbach, Kirsten. "Development and analysis of recombinant fluorescent probes for use in live cell imaging of filamentous fungi." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4739.
Повний текст джерелаАнотація:
The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.
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Mafina, Marc-Krystelle. "Novel fluorescent probes for analysis of protein interactions under truly physiological conditions with real medical devices." Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/2972.
Повний текст джерелаАнотація:
Protein adsorption under physiological conditions was recognised to be the key step in the modulation of biological responses between materials and an osseous environment. Many studies have shown variation of adsorption behaviour on medical materials but most experiments were performed under non-physiological idealised conditions with idealised samples. The aim of this thesis was to develop a method to analyse protein adsorption on real clinically relevant samples under physiologically relevant conditions. The use of fluorescent probes was identified as a methodology which would facilitate analysis under a range of conditions including fully competitive with real samples that required no specialised surface pre-treatment. Fluoresceinthiureidoaminocaproic acid (FTCA) and a sulforhodamine derivative (SR101), were identified as suitable for coupling to proteins. FTCA labelled bovine serum albumin (BSA) was initially used to validate the technique and found to have several advantages over commercially available total protein assays; including greater sensitivity and facilitate its use under competitive conditions. These experiments also confirmed sensitivity to temperature and test media as well as demonstrating that the technique could be used on idealised dense and real porous granular (as used clinically) samples. Conformational changes associated with protein-surface interactions were observed through variation in protein auto-fluorescence and confirmed with CD analysis, therefore, care in selection of appropriate experimental conditions and fluorophore probes was required. Additionally, labelling facilitated the visualisation in differences in the morphological habit of the surface adsorbed protein species. Investigation of differential response in protein exchange with hydroxyapatite (HA) and 0.8wt% silicon substituted hydroxyapatite (SA) with more biologically relevant proteins; such as bone morphogenetic protein-2 (BMP-2), fibronectin (FN) and osteopontin (OPN) individually and/or competitively in phosphate buffered saline (PBS) or minimum Eagles medium (MEM) supplemented with 10 % foetal bovine serum (FBS) demonstrated SA to have a greater capacity to adsorb selected osteogenic proteins under competitive conditions as compared to HA. Particularly interesting was the BMP-2 findings, which highlighted the role of media in promoting BMP-2 adsorption and the conformation sensitivity of traditional ELISA assays giving rise to unreliable results.
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Isaeva, O. A., O. G. Avrunin, and О. Г. Аврунін. "Basic skin pathologies and the possibilities of their diagnosis." Thesis, Дніпро, 2019, 2019. http://openarchive.nure.ua/handle/document/9954.
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Zhao, Xi. "Analysis of a Low Pressure UV reactor under Multiple Upstream Elbow Configurations using UV Sensitive Fluorescent Microspheres." NCSU, 2007. http://www.lib.ncsu.edu/theses/available/etd-08092007-131823/.
Повний текст джерелаАнотація:
Upstream piping configuration has been known to impact the UV reactor validation using biodosimetry tests. However, the influence of upstream configuration on the UV dose distribution has not been experimentally investigated. This research was performed to evaluate the UV reactor dose distribution under multiple upstream configurations using UV sensitive fluorescent microspheres. The upstream hydraulics configurations included two kinds of 90- degree bends and one straight pipe configuration. Experimental tests were performed at 51 gpm flow rate, 91% UV transmittance (UVT) on a single lamp low-pressure high-output (LPHO) UV reactor. The UV irradiation kinetics of the photo-chemically active fluorescent microspheres was performed with bench-scale collimated beam experiments. The correlation with microspheres fluorescence intensity distribution to UV fluence distribution was achieved by a statistical process involving Bayesian and Markov chain Monte Carlo integration technique. The results of this study showed that the straight pipe configuration produced a shift in UV fluence distribution to a higher UV fluence range compared to the two elbow configurations. No significant difference was formed between the two elbow configurations. The fluorescent microspheres Bayesian method can serve as an additional test to the traditional biodosimetry for UV reactor validation by providing sensitivity in detecting design parameter change and added confidence in the results by providing unbiased UV dose behavior.
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Leppälä, Daniel. "Analysis of surface coverage in regards to surface functionalization : A microscopic approach." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-140994.
Повний текст джерелаАнотація:
The understanding of how white blood cells react when coming into contact with various surfaces is of major importance for a wide range of biomaterials and biosensor applications. In this study it is investigated if it is possible to determine how neutrophils react to a certain type of sensor chip called cell clinic being developed. This study investigates the cell surface coverage on the sensor chip and how it correlates to the signal response of the sensor at hand. Neutrophils, as other white blood cells, are cells that quickly adhere to surfaces and during the adhesion process they activate at different levels depending on i.e. type of surface or surface functionalization, this activation can be visualized by the change in morphology. While measuring the change of capacitance with the cell clinic sensor during cell adhesion, the cell surface coverage is of main importance. The main focus of this diploma work has been to develop an image analysis script capable of conducting automated analysis on a large body of images estimating the surface coverage. Input data for this modeling is taken from fluorescent microscopy images. The experiments conducted during this project have indicated that white blood cells adhered to the sensor surface shows signs of being activated also without external activation. This clearly shows that knowledge of how neutrophils react to surface modifications is of great importance as well as the awareness that any surface may trigger a response from the immune system i.e. neutrophil activation, so also in the cell clinic. It is a fact that it might be difficult to evaluate the effect of a foreign substance on the neutrophils while a significant amount is activated from being in contact with the surface. Regarding different surfaces the white blood cells does not display any preference of adhering to any specific surface. The surfaces used in this project was silicon oxide wafers, silicon oxide wafers with a nitride surface functionalization and the intended sensor chip; however the addition of PMA clearly shows an effect on how many cells that adheres to the surface as well as the average area of each cell.
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Brind'Amour, Julie. "Flow cytometry analysis and sorting of chromosomes following hybridization with fluorescent probes that target specific DNA repeat sequences." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/35973.
Повний текст джерелаАнотація:
Traditional cytogenetic approaches allow analysis of the chromosomal composition (karyotype) of mitotic cells fixed on slides cells by microscopy. The combination of karyotyping and Fluorescence In Situ Hybridization (FISH) enables the detection of specific target sequences on individual chromosomes. Disadvantages are that traditional cytogenetic approaches are very labor and time consuming and that chromosome specific information from only a few dozen cells has poor statistical power. An alternative is flow karyotyping, a method to analyze chromosomes in suspension by flow cytometry. For flow karyotyping, the DNA composition of specific chromosomes in suspension is measured based on the DNA-specific dyes Hoechst 33258 (HO) and Chromomycin A3 (CA3). My thesis work has focused on the development of a new method to analyze and sort chromosomes using FISH with labeled peptide nucleic acid (PNA) probes on chromosomes in suspension. I found that, following FISH, flow karyotyping can be used to detect and quantify repetitive DNA sequences within individual chromosomes.
Using chromosome flow FISH (CFF), chromosomes isolated from cells of various species were hybridized to PNA probes and analyzed by flow cytometry. CFF was used to detect a variety of repeats; interstitial telomeric sequences in Chinese Hamster chromosomes, major satellite in mouse chromosomes and D18Z1 alpha satellite repeats in human chromosomes. Quantitative measurements of repeat length by CFF were validated by comparison with measurements obtained using Q-FISH. We found that parental homologs of human chromosome 18 with different D18Z1 satellite repeat array size could be purified using CFF and Fluorescence Activated Cell Sorting (FACS). Illumina short read sequencing of libraries built from these purified chromosomes enabled us to determine, with a high resolution, the allelic phasing of each homolog over the entire chromosome 18. Finally, CFF was modified to study sister chromatids separately. Using a cell model with inducible separation of sister chromatids, flow karyograms were generated. Using chromosome orientation FISH (CO-FISH) in suspension, we could identify sister chromatids according to the presence of DNA template strands. We anticipate that this approach will allow the purification of sister chromatids to study epigenetic differences between sister chromatids defined on the basis of DNA template strands.
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Park, Kyoung-Joo Jenny. "Multi-Parameter Fluorescent Analysis and Quantitative Magnetophoresis Study as Two Different Technologies to Detect and Characterize Cells and Its Various Applications as Biomarkers." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512018964871473.
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De, Carli Francesco. "Towards genome-wide, single-molecule analysis of eukaryotic DNA replication." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066600/document.
Повний текст джерелаАнотація:
Chez les eucaryotes, la réplication de l'ADN démarre au niveau de multiples origines activées suivant un programme précis, qui peut être analysé à l'échelle du génome sur des populations cellulaires. Cependant, l'étude de la variabilité intercellulaire, la détection d'évènements rares et la mesure de la vitesse des fourches de réplication nécessitent des analyses en molécule unique. Avec les techniques actuelles, l'ADN néosynthétisé est marqué avec des analogues de la thymidine et révélé par des anticorps fluorescents. Les molécules d'intérêt sont identifiées par hybridation fluorescente in situ. Ces étapes sont complexes et le débit est faible. Cette thèse développe de nouvelles méthodes de détection et d'identification des molécules d'ADN réplicatives sans anticorps et à haut débit. L'ADN est répliqué en présence d'un dUTP fluorescent, purifié puis marqué en code-barre spécifique permettant l'alignement sur le génome de référence par coupure avec une endonucléase simple brin et incorporation d'un autre dUTP fluorescent. L'ADN est ensuite coloré avec un intercalant fluorescent, le YOYO-1. Les molécules d'ADN, leurs segments néorépliqués et leurs code-barres sont observés en trois couleurs différentes par épifluorescence directe. Les segments répliqués ont une fluorescence YOYO-1 plus intense, ce qui permet de détecter les bulles de réplication sans marquage métabolique. Ces outils ont été couplés à un dispositif nanofluidique dans lequel l'ADN est conduit dans des milliers de nanocanaux et imagé automatiquement, ce qui augmente massivement le débit. L'ensemble de ces résultats ouvre la voie à la cartographie pangénomique de la réplication de l'ADN en molécule uniqueIn eukaryotes, DNA replication starts at multiple origins that are activated following a specific program. Population methods allow genome-wide analysis of DNA replication. However, single-molecule methods are required to monitor cell-to-cell variability, detect rare events and measure individual replication fork speeds. With the existing techniques, newly-synthesized DNA is labelled with thymidine analogs and revealed with fluorescent antibodies. Fibres containing a locus of interest can be identified by fluorescent in situ hybridization. These steps are complex and the throughput is low. This work proposes novel, antibody-free tools to detect replication tracts and identify the locus of origin of all DNA molecules at much higher throughput. DNA replicated in the presence of a fluorescent dUTP was purified and specifically barcoded by using a nicking endonuclease, followed by limited nick-translation with another fluorescent dUTP. This allowed alignment to a reference genome map. DNA was then stained with the fluorescent DNA intercalator YOYO-1. Direct epifluorescence revealed the DNA molecules, their replication tracts and their barcodes in three distinct colours. Replicated segments showed a stronger YOYO-1 fluorescence, demonstrating that replication bubbles can be directly detected without metabolic labelling. Finally, these tools were coupled to a nanofluidic device: DNA was driven into 13,000 parallel nanochannels and automatically imaged, massively increasing the throughput. Altogether, these results provide a starting point for genome-wide, single-molecule mapping of DNA replication in eukaryotic organisms
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Takaba, Kiyofumi. "Charge-density Features of Protein Molecules Revealed with Ultra-high Resolution X-ray Crystallography." Kyoto University, 2018. http://hdl.handle.net/2433/232273.
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Bonar, Michal Mateusz. "Rapid Enumeration, Sorting and Maturation Analysis of Single Viral Particles in HIV-1 Swarms by High-Resolution Flow Virometry." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case149944467787067.
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Korchane, Sonia. "Développement d'une méthode de séparation électrocinétique de biomarqueurs de la polyneuropathie amyloïde familiale à transthyrétine : vers une miniaturisation de l'analyse." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112112/document.
Повний текст джерелаАнотація:
Ces travaux de recherche s’intéressent à la conception de nouvelles méthodologies analytiques destinées à mesurer le bénéfice d’une transplantation hépatique ou bien encore à l’évaluation de nouvelles voies thérapeutiques à l’essai pour des patients atteints de la polyneuropathie amyloïde familiale à transthyrétine (TTR). Cette maladie rare se caractérise par une déstabilisation structurale du tétramère de TTR qui aboutit à l’agrégation de fibrille dans les tissus du système nerveux autonome, au niveau des nerfs périphériques et autour de certains organes dont le cœur. Dans le cadre d’une collaboration entre hospitalo-universitaires, chimistes analystes, électrochimistes, physico-chimistes et technologues, nous nous sommes attachés à développer des séparations en électrophorèse capillaire couplée avec une détection optique de peptides natif et muté qui sont directement associés à une variente de cette maladie rare. La difficulté première de cette recherche concerne le choix même de ces biomarqueurs qui s’est finalement révélé pertinent grâce de la réalisation de cartes peptidiques à partir du sérum. Ensuite deux voies ont été explorées : une séparation électrocinétique avec une détection spectrométrique d’absorbance dans l’ultra-violet et l’autre nécessitant le marquage préalable de peptides par des molécules fluorophores ou fluorogènes pour ensuite faire une séparation en électrophorèse couplée LIF (laser induced fluorescence). Dans les deux cas le critère principal de séparation, la résolution, autorise une quantification et surtout les validations analytiques associées montrent une réelle robustesse des méthodologies développées. L’autre signe encourageant pour la transposition des ces méthodes à l’analyse de prélèvements issus de patients, concerne la limite de quantification qui est inférieure à celle couramment mesuré dans le sérum. La spectrométrie de masse, moyen d’investigation physico-chimique puissant à permis de suivre et de comprendre d’un point vue plus fondamental le produit des réactions de chimie organique de dérivation des peptides par trois marqueurs fluorescents : le TAMRA-SE, le NDA et le FQ. La possibilité de proposer un outil d’analyse miniaturisé et simple d’utilisation pour le monde hospitalier a également été étudiée. Un poste d’analyse sur puce microfluidique permettant l’analyse quantitative et qualitative a été installé pour permettre la réalisation de premiers essais expérimentaux de séparations électrocinétiques sur puce microfluidique. Ces travaux jettent les bases d’une nouvelle voie analytique pour séparer et quantifier les différents biomarqueurs caractéristiques de la polyneuropathie amyloïde familliale à TTRThe purpose of our work was the development of new analytical methodologies to measure the benefit of liver transplantation and also the evaluation of new therapeutic approaches under testing on patients with Transtyretin (TTR) familial amyloid polyneuropathy. This rare disease is characterized by a structural destabilization of TTR tetramer leading to it’s aggregation into amyloïd fibrils that accumulate in the tissues of the autonomous nervous system, peripheral nerves and around certain organs, including the heart. As part of a collaboration between university, hospital, analytical chemists, electrochemist, physical chemists and technologists, we are committed to develop separations in capillary electrophoresis coupled with optical detection of native and mutated peptides that are directly associated with a variant of this rare disease. The first challenge of this research is the choice of these biomarkers that ultimately proved relevant with the realization of peptide maps from the serum. Then two approaches have been explored: electrokinetic separation with absorbance spectrometric detection in the ultraviolet and the other requiring the prior labeling peptides with fluorescent molecules and then to a separation on electrophoresis coupled with LIF (Laser induced fluorescence). In both cases the main criterion of separation, resolution, allows quantification and especially analytical validations show actual strength associated methodologies developed. Another encouraging sign for the transposition of these methods to the analysis of samples from patients regarding the quantification limit is lower than commonly measured in serum. Mass spectrometry, using physico-chemical investigation powerful allowed to follow and understand a more fundamental viewpoint the product of organic chemistry reactions bypass peptides by three fluorescent dyes: TAMRA-SE, the NDA and FQ. The ability to provide a miniaturized analysis and easy to use tool for the hospital environment was also studied. A post analysis on microfluidic chip for quantitative and qualitative analysis was installed to allow the realization of the first experimental tests of electrokinetic separations on microfluidic chip. These studies lay the foundation for a new analytical way to separate and quantify the different characteristics biomarkers family TTR amyloid polyneuropathy
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Buenrostro-Nava, Marco T. "Characterization of GFP Gene Expression Using an Automated Image Collection System and Image Analysis." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1032794680.
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LI, BAIKUN. "REDOX POTENTIAL (ORP) REGULATION OF NUTRIENT REMOVAL IN WASTEWATER TREATMENT PROCESSES AND THE STRUCTURE - FUNCTION ANALYSIS OF ACTIVATED SLUDGE FLOC." University of Cincinnati / OhioLINK, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1020695786.
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Hakuna, Lovemore. "Selective Indicators for Optical Determination of Disease Biomarkers." PDXScholar, 2014. http://pdxscholar.library.pdx.edu/open_access_etds/2053.
Повний текст джерелаАнотація:
The most abundant biological thiols, homocysteine (Hcy), cysteine (Cys) and glutathione (GSH) have been the subject of intense research due to their association with a wide range of diseases. They play a key role in maintaining the redox status of biological systems. Selective detection methods for these thiols are challenging due to their similar structures and properties. Current commercially available detection methods use separations, fragile and expensive enzymatic or immunogenic materials and complex instrumentation. This has led to a global effort towards developing simple and inexpensive optical probes and indicators selective for specific biological thiols.
Highly selective chemical probes and simple methods for detection and potential quantification of Hcy and GSH in their natural biological media have been developed. These indicators and methods are relatively simple and inexpensive for potential application at point of care. The selective detection of Hcy using novel asymmetric viologen chemical probes at room temperature is described as well as the use of commercially available materials under photochemical conditions. These probes respond linearly proportional to increasing Hcy concentrations, potentially enabling the monitoring of Hcy levels in human plasma. Additionally, new methods for the selective determination of GSH in human plasma, as well as its quantification in whole blood deposited on filter paper (dried blood spots), is also presented herein.
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Dubrowski, Piotr. "An automated multicolour fluorescence in situ hybridization workstation for the identification of clonally related cells." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/733.
Повний текст джерелаАнотація:
The methods presented in this study are aimed at the identification of subpopulations (clones) of genetically similar cells within tissue samples through measurement of loci-specific Fluorescence in-situ hybridization (FISH) spot signals for
each nucleus and analyzing cell spatial distributions by way of Voronoi tessellation and Delaunay triangulation to robustly define cell neighbourhoods.
The motivation for the system is to examine lung cancer patient for
subpopulations of Non-Small Cell Lung Cancer (NSCLC) cells with biologically meaningful gene copy-number profiles: patterns of genetic alterations statistically
associated with resistance to cis-platinum/vinorelbine doublet chemotherapy treatment.
Current technologies for gene-copy number profiling rely on large amount of cellular
material, which is not always available and suffers from limited sensitivity to only the
most dominant clone in often heterogeneous samples. Thus, through the use of FISH, the
detection of gene copy-numbers is possible in unprocessed tissues, allowing identification of specific tumour clones with biologically relevant patterns of genetic aberrations.
The tissue-wide characterization of multiplexed loci-specific FISH signals,
described herein, is achieved through a fully automated, multicolour fluorescence imaging microscope and object segmentation algorithms to identify cell nuclei and FISH spots within. Related tumour clones are identified through analysis of robustly defined cell neighbourhoods and cell-to-cell connections for regions of cells with homogenous
and highly interconnected FISH spot signal characteristics.
This study presents experiments which demonstrate the system’s ability to
accurately quantify FISH spot signals in various tumour tissues and in up to 5 colours
simultaneously or more through multiple rounds of FISH staining. Furthermore, the
system’s FISH-based cell classification performance is evaluated at a sensitivity of 84% and specificity 81% and clonal identification algorithm results are determined to be comparable to clone delineation by a human-observer. Additionally, guidelines and procedures to perform anticipated, routine analysis experiments are established.
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Patabadige, Damith Randika E. W. "Developing multilayer microfluidic platforms and advancing laser induced fluorescent detection and electrochemical detection to analyze intracellular protein kinases, reactive nitrogen and oxygen species in single cells." Diss., Kansas State University, 2017. http://hdl.handle.net/2097/35299.
Повний текст джерелаАнотація:
Doctor of PhilosophyDepartment of Chemistry
Christopher T. Culbertson
Recent approaches in analytical separations are being advanced towards the “lab-on-a-chip” concept in which multiple lab functions are integrated into micro/nano fluidic platforms. Among the variety of separation techniques that can be implemented on microfluidic devices, capillary electrophoresis is the most popular as it provides high efficiency, simple, fast and low cost separations. In addition, integrating miniaturized fluid manipulation tools into microfluidic devices with separations is essential for a variety of biological applications. Chapter 1 discusses the fundamentals of capillary electrophoresis and miniaturized fluid manipulation tools and provides an over view of single cell analysis in microfluidics. In chapter 2, the integration of miniaturized peristaltic pumps into multilayer microfluidic platforms is discussed. In addition, device characterization, precise fluid control and high throughput single cell analysis are discussed. As a proof of principle, T-lymphocytes were loaded with two fluorescent probes Carboxyfluorescein diacetate (CFDA) and Oregon green (OG). Thousands of single cells were automatically transported, lysed on these devices and analytes from the lysate were electrophoretically separated. 1120 cells were analyzed over the course of 80 min (14 cells/min) and separation characteristics of analytes released from individual cells were investigated. In the third chapter, the development of microfluidic platforms for the electrochemical detection of nitric oxide (NO) and other reactive nitrogen species (RNS) at the single cell level is discussed. A microfluidic system was developed to perform rapid cell lysis followed by electrochemical detection. Miniaturized microband electrodes were designed and integrated with a microfluidic separation channel. Three alignment techniques (in-channel, end-channel and off-channel configurations) were used to detect the electrochemical response of the analyte of interest. Furthermore, a model analyte (CFDA) was used to demonstrate the potential of performing the simultaneous dual detection with electrochemical and laser induced fluorescence detection. In addition, the same microfluidic platform was adapted to detect intracellular superoxide using laser induced fluorescence. In the fourth chapter, the off-chip integration of optical fiber bridges with multilayer microfluidic chips is discussed. A multimode optical fiber (~10cm long) was integrated between the single cell lysing spot and a spot downstream of the separation channel in order to detect both intact cells and the analyte in the lysate. This technique was used to create two detection spots on the microfluidic platform with the use of a single excitation source and single detector. Fluorescently labeled T-lymphocytes were automatically transported and lysed in a manner similar to that described in chapter 2. Hundreds of single cells were analyzed and the absolute migration time was determined for the analytes in the lysate. In addition, the separation characteristics of fluorescently labeled protein kinase B peptide substrates were investigated. Furthermore, this technique was used to measure cell size and the velocity of intact cells (discussed in 5th chapter) by making use of a light tunneling concept available in multimode optical fibers. All the experiments presented in this dissertation exploit the use of multilayer microfluidic platforms to investigate intracellular components in single cells in a high throughput manner that has several advantages over current conventional techniques.
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Sinha, Raunak [Verfasser], Jürgen [Akademischer Betreuer] Klingauf, Erwin [Akademischer Betreuer] Neher, and Walter [Akademischer Betreuer] Stühmer. "Optical analysis of synaptic vesicle protein molecules during exo- and endocytosis using pH-switchable fluorescent probes / Raunak Sinha. Gutachter: Jürgen Klingauf ; Erwin Neher ; Walter Stühmer. Betreuer: Jürgen Klingauf." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2011. http://d-nb.info/1042305188/34.
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CHEN, HORN NAN, and 陳宏男. "Analysis on Dimming Operation of Fluorescent Lamps." Thesis, 1996. http://ndltd.ncl.edu.tw/handle/37821887196533049534.
Повний текст джерелаАнотація:
碩士國立中山大學
電機工程研究所
84
An electric circuit model is developed for characterizing the electrical behavior of fluorescent lamps with dimming operation at high frequencies. The proposed model can be simply derived from three sets of measured data. This model can be used for predicting circuit behavior of dimmable electronic ballasts, and then to determine the circuit parameters and the adequate operating ranges. An electronic ballast with the series resonant inverter is built to realize two dimming operation methods, voltage control and frequency control. The design considerations including operating frequency, starting voltage, filament current, and control sensitivity factor are discussed in detail. Experimental tests are found to be quite in agreement with the simulation results based on the proposed model.
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Ko, Hung-Yu, and 柯虹毓. "Analysis of fluorescent derivatives of phenylenediamines by micellar electrokinetic chromatography with laser-induced fluorescence detector." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/71893606564708657035.
Повний текст джерелаАнотація:
碩士高雄醫學大學
香粧品學系碩士班
104
Phenylenediamines were ingredients that used as permanent hair dyes. These compounds were reported to cause allergic dermatitis and have genotoxicity and carcinogenicity. Therefore, o-phenylenediamine (OPD) and m-phenylenediamine (MPD) were prohibited to use and the limitation concentrations of p-phenylenediamine (PPD) and toluene-2,5-diamine (PTD) were 2% and 4% in permanent hair dye products in Taiwan. To improve sensitivity, the fluorescent derivatization strategy and micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF) were established to analyze OPD, MPD, PPD and PTD in hair dye products, hair samples and percutaneous absorption experiment. 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) was used as a fluorescent reagent and derived at amino groups of phenylenediamines and underwent nucleophilic substitution reaction. Derivatization solution contained phenylenediamines, DTAF, and internal standard (1-Aminoadamantane hydrochloride, 1-AD) in alkaline conditions. The derivatization condition reacted at 90℃ for 10 minutes. The derivatives were analyzed by MEKC equipped with LIF detection (excitation wavelength: 488 nm; emission wavelength: 520 nm). The optimized separation conditions were as following: separation buffer was 20 mM borate (pH 8.0) containing 10 mM Brij 35 and 35% (v/v) MeOH. Sample was injected at 0.5 psi for 15 sec. The separation voltage was 20 kV. The limit of detections (S/N = 3) for MPD, PTD, PPD and OPD were 25, 25, 50 and 100 nM, respectively. Comparing to previous studies, the sensitivity enhancements were from 30-fold to 81-fold. The correlation coefficients of intra-day and inter-day analysis were all above 0.997. This method showed good linearity from 0.125 to 5.000 μM. Moreover, the relative standard deviation and relative error were less than 4.56% and 4.97%, respectively, with high precision and accuracy. The high sensitive MEKC-LIF method was successfully established and applied to determine the content of phenylenediamines in commercial hair dye products, hair and percutaneous absorption samples.
49
ching, Lee yi, and 李怡青. "Preliminary Study of Fluorescent Particles for Biochemical Analysis." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/95530185166217717512.
Повний текст джерелаАнотація:
碩士國立暨南國際大學
應用化學系
96
The goal of this study is to synthesize functional magnetic particles and combine with immunosorbent assay for biochemical analysis. The sizes of magnetic nanoparticles were 40nm, 80 nm and 150nm, and SiO2 coated Fe3O4 were 80nm, 150 nm and 200nm. Streptavidin or IgG was bound onto Fe3O4@SiO2 particles. Biotin or protein A was covalently bound to fluorescent marker, and applied to magnetic separation in microplate. We studied experimental parameters of magnetic particles size、and particles number for immunoassay. Preliminary study of immunoassay between particles was studied to demonstrate the potential application of this method for biochemical analysis. The interactions of streptavidin-biotin and IgG-protein A that coating on particles was studied. Experimental parameters were studied under different conditions of the particle size and the number of particles. The streptavidin-conjugated magnetic beads and IgG-conjugated magnetic beads can capture fluorescent silica particles labeled with biotin and protein A in the 96 well plate under magnetic filed. The selectivity is high with controlled experiments.
50
Chen, Yu-Rong, and 陳昱榮. "Dynamics Analysis of Fluorescent Lamp and Electronic Ballast System." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/98959989873012526772.
Повний текст джерелаАнотація:
碩士國立成功大學
工程科學系碩博士班
94
The stability of the fluorescent lamp and ballast system is
investigated in this thesis. Fluorescent lamps are gas discharge
lamps. Based on its v-i characteristic curve derived by experimental
results, it reveals that the dynamics of the lamp is a negative
incremental impedance at high frequencies. The small-signal model of
the lamp is obtained by using the modified Francis first-order
differential equation described the behavior of the lamp.
Interestingly, the mathematical model is non-minimum phase with a
right-half plane zero. As a result, the lamp is regarded as a
current controlled device. A current source is thereby applied to
the lamp to stabilize the overall system. The electronic ballast is composed of power transistors and
resonant circuit. Three resonant circuits under investigation are LC
series resonant circuit, LC parallel resonant circuit, and LCC
series and parallel resonant circuit. The phasor transformation is
applied to derive the small-signal model of the resonant circuits.
First, the circuit is transformed from the time domain into the
phasor domain. Then according to the equivalent phasor-domain
circuit, the transfer function is derived by the exact method and
approximate method presented in this thesis. Notably, the transfer
function of the equivalent phasor-domain circuit of the resonant
circuit can be simplified to be first-order lowpass filter on the
basis of the approximate method. According to the small-signal model of the lamp and the first-
order lowpass filter of the resonant circuit, the stability of
system can be analyzed by Routh-Hurwitz criterion. Three systems for
stability investigation are resistive ballast for dc operation,
traditional inductive ballast for utility power of 60 Hz, and
electronic ballast for a square wave power source of 50 kHz
generated from the inverter. The theoretical analysis is validated
by experimental results. For a practical engineer, the theoretical analysis and
experimental results in this thesis are helpful to gain insights of
the electronic ballast design.