Academic literature on the topic 'Affinity-Based probe'
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Journal articles on the topic "Affinity-Based probe"
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Lafreniere, Matthew A., Geneviève F. Desrochers, Kedous Mekbib, and John Paul Pezacki. "An affinity-based probe for methyltransferase enzymes based on sinefungin." Canadian Journal of Chemistry 95, no. 10 (2017): 1059–63. http://dx.doi.org/10.1139/cjc-2017-0168.
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Epigenetics control numerous cellular processes such as gene transcription, signal transduction, and protein stabilization. An understanding of epigenetic mechanisms can lead to the development of therapeutic agents for various diseases. Herein, we report the design and synthesis of a sinefungin affinity-probe (BpyneSF) that targets methyltranferase enzymes and proteins involved in recognition of methylation. This probe contains a bioorthogonal alkyne residue for conjugation using the copper-catalyzed azide–alkyne cycloaddition and a photoactivatable crosslinker group for covalent attachment of the probe to its proteomic targets. We investigate the efficiency and selectivity of the probe to inhibit and label methyltransferase enzymes, and we demonstrate, through in-gel fluorescence, on-bead digestion, and tandem mass spectrometry, that BpyneSF can label methyltransferase SETD2 and reader proteins in vitro. These results establish the utility of BpyneSF as a tool for affinity-based protein profiling in complex biological environments.
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Tano, Hanna, Maryam Oroujeni, Anzhelika Vorobyeva, et al. "Comparative Evaluation of Novel 177Lu-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting." Cancers 13, no. 3 (2021): 500. http://dx.doi.org/10.3390/cancers13030500.
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Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe ZHER2:342-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with 177Lu. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [177Lu]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all 177Lu-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe’s size and decreased with an increased number of nucleobases. The shortest PNA probe, [177Lu]Lu-HP16, showed the highest tumor-to-kidney ratio. [177Lu]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.
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Yang, Xue, Thomas J. M. Michiels, Coen de Jong, et al. "An Affinity-Based Probe for the Human Adenosine A2A Receptor." Journal of Medicinal Chemistry 61, no. 17 (2018): 7892–901. http://dx.doi.org/10.1021/acs.jmedchem.8b00860.
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Schiedel, Matthias, Tobias Rumpf, Berin Karaman, et al. "Structure-Based Development of an Affinity Probe for Sirtuin 2." Angewandte Chemie International Edition 55, no. 6 (2016): 2252–56. http://dx.doi.org/10.1002/anie.201509843.
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Qiao, Chunhua, Daniel J. Wilson, Eric M. Bennett, and Courtney C. Aldrich. "A Mechanism-Based Aryl Carrier Protein/Thiolation Domain Affinity Probe." Journal of the American Chemical Society 129, no. 20 (2007): 6350–51. http://dx.doi.org/10.1021/ja069201e.
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Stubbs, Keith A., and David J. Vocadlo. "Affinity-Based Proteomics Probes; Tools for Studying Carbohydrate-Processing Enzymes." Australian Journal of Chemistry 62, no. 6 (2009): 521. http://dx.doi.org/10.1071/ch09140.
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As more information becomes available through the efforts of high-throughput screens, there is increasing pressure on the three main ‘omic’ fields, genomics, proteomics, and metabolomics, to organize this material into useful libraries that enable further understanding of biological systems. Proteomics especially is faced with two highly challenging tasks. The first is assigning the activity of thousands of putative proteins, the existence of which has been suggested by genomics studies. The second is to serve as a link between genomics and metabolomics by demonstrating which enzymes play roles in specific metabolic pathways. Underscoring these challenges in one area are the thousands of putative carbohydrate-processing enzymes that have been bioinformatically identified, mostly in prokaryotes, but that have unknown or unverified activities. Using two brief examples, we illustrate how biochemical pathways within bacteria that involve carbohydrate-processing enzymes present interesting potential antimicrobial targets, offering a clear motivation for gaining a functional understanding of biological proteomes. One method for studying proteomes that has been developed recently is to use synthetic compounds termed activity-based proteomics probes. Activity-based proteomic profiling using such probes facilitates rapid identification of enzyme activities within proteomes and assignment of function to putative enzymes. Here we discuss the general design principles for these probes with particular reference to carbohydrate-processing enzymes and give an example of using such a probe for the profiling of a bacterial proteome.
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Chattopadhaya, Souvik, Elaine W. S. Chan, and Shao Q. Yao. "An affinity-based probe for the proteomic profiling of aspartic proteases." Tetrahedron Letters 46, no. 23 (2005): 4053–56. http://dx.doi.org/10.1016/j.tetlet.2005.04.015.
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Bi, Xihe, Anton Schmitz, Alaa M Hayallah, Jin-Na Song, and Michael Famulok. "Affinity-Based Labeling of Cytohesins with a Bifunctional SecinH3 Photoaffinity Probe." Angewandte Chemie International Edition 47, no. 49 (2008): 9565–68. http://dx.doi.org/10.1002/anie.200803962.
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Chakraborty, Goutam, Alok K. Ray, Prabhat K. Singh, and Haridas Pal. "A styryl based fluorogenic probe with high affinity for a cyclodextrin derivative." Organic & Biomolecular Chemistry 17, no. 28 (2019): 6895–904. http://dx.doi.org/10.1039/c9ob01349k.
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A styryl-based fluorogenic near-IR probe registers a very high association constant with sulfobutylether substituted β-cyclodextrin host, having prospects as biological marker and improved pH and temperature sensor.
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Palstrøm, Nicolai Bjødstrup, Lars Melholt Rasmussen, and Hans Christian Beck. "Affinity Capture Enrichment versus Affinity Depletion: A Comparison of Strategies for Increasing Coverage of Low-Abundant Human Plasma Proteins." International Journal of Molecular Sciences 21, no. 16 (2020): 5903. http://dx.doi.org/10.3390/ijms21165903.
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In the present study, we evaluated four small molecule affinity-based probes based on agarose-immobilized benzamidine (ABA), O-Phospho-L-Tyrosine (pTYR), 8-Amino-hexyl-cAMP (cAMP), or 8-Amino-hexyl-ATP (ATP) for their ability to remove high-abundant proteins such as serum albumin from plasma samples thereby enabling the detection of medium-to-low abundant proteins in plasma samples by mass spectrometry-based proteomics. We compared their performance with the most commonly used immunodepletion method, the Multi Affinity Removal System Human 14 (MARS14) targeting the top 14 most abundant plasma proteins and also the ProteoMiner protein equalization method by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MSMS) analysis. The affinity-based probes demonstrated a high reproducibility for low-abundant plasma proteins, down to picomol per mL levels, compared to the Multi Affinity Removal System (MARS) 14 and the Proteominer methods, and also demonstrated superior removal of the majority of the high-abundant plasma proteins. The ABA-based affinity probe and the Proteominer protein equalization method performed better compared to all other methods in terms of the number of analyzed proteins. All the tested methods were highly reproducible for both high-abundant plasma proteins and low-abundant proteins as measured by correlation analyses of six replicate experiments. In conclusion, our results demonstrated that small-molecule based affinity-based probes are excellent alternatives to the commonly used immune-depletion methods for proteomic biomarker discovery studies in plasma. Data are available via ProteomeXchange with identifier PXD020727.
Dissertations / Theses on the topic "Affinity-Based probe"
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Ahmad, Sheraz. "Applications of NMR techniques: Hyphenations (LC-SPENMR), affinity (DOSY) and NOE based (STD and Tr-OESY) to probe the binding interactions of ligands (synthetic and natural) towards protein." Universidade Federal de São Carlos, 2014. https://repositorio.ufscar.br/handle/ufscar/6289.
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Previous issue date: 2014-01-17<br>Universidade Federal de Sao Carlos<br>O foco principal desse trabalho foi a implementação, otimização e aplicações práticas de métodos de ressonância magnética nuclear (RMN) com o propósito de avaliar as interações entre moléculas de diferentes massas molares, sendo que essas técnicas foram implementadas pela primeira vez no laboratório de RMN do DQ-UFSCar. Existem várias abordagens que podem ser utilizadas com esse propósito, e dentre elas, destacamos: STD NMR, Tr-NOESY, WaterLOGSY, SALMON, INPHARMA, DOSY, SAR. Esses métodos são muito úteis para detectar mudanças de comportamento, a nível molecular, quando adicionamos macromoléculas em um meio contendo somente micromoléculas. O entendimento desse comportamento molecular ajuda a desvendar sistemas complexos de interações moleculares existentes no corpo humano, e que, são muito importantes para o descobrimento de novos medicamentos. O primeiro passo para a implementação das técnicas foi a utilização da proteína de soro bovino (do inglês BSA) e proteína de soro humano (do inglês HSA) como fonte de macromoléculas e micromoléculas orgânicas isoladas da fração etanólica do extrato bruto (1 mg) de Rauia resinous e da fração acetato de etila de Strypnodendron polyphyllum, utilizando cromatografia líquida de alta eficiência, extração por fase sólida e a ressonância magnética nuclear (CLAE-EFS/RMN) para a completa elucidação estrutural quando necessário. As técnicas utilizadas foram: saturation transfer difference (STD), transfer nuclear Overhauser spectroscopy (Tr-NOESY) e STD-TOCSY (total correlation spectroscopy). Essa mesma metodologia além de representar um importante mecanismo para avaliar as interações entre moléculas, também pode ser utilizada para outras matrizes variando tanto as macro quanto as micromoléculas.<br>The main focus of this work was the implementation, optimization and practical applications of nuclear magnetic resonance (NMR) methods for the purpose of evaluating the interactions between molecules of different masses, and these techniques were implemented for the first time in the laboratory NMR DQ - UFSCAR. There are a number of ligand-based screening approaches available, such that, STD NMR, Tr-NOESY, WaterLOGSY, SALMON, INPHARMA, DOSY, SAR by NMR etc. These methods are sensitive towards the perturbations as results of the macromolecular addition in a medium containing the small molecules. The molecular understanding of this behavior helps to uncover the complex systems of molecular interactions existing in the human body, which are very important for the discovery of new medicines. In the first step while implementing these techniques, Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) as a source of organic macromolecules were used. While, for the small organic molecules, a 1 mg crude extract from the hydroethanolic fraction of the Rauia resinous and ethyl acetate fraction of Strypnodendron polyphyllum was utilized, however, for the complete structural characterization, solid phase extraction following high-pressure liquid chromatography in an integrated fashion and then nuclear magnetic resonance (LC-SPE/NMR) was employed when necessary. On the other hand, the ligand screening techniques used were the saturation transfer difference (STD), Nuclear Overhauser Transfer Effect SpectroscopY (Tr- NOESY), Diffusion-Order Spectroscopy (DOSY) and STD- TOCSY (TOtal Correlation SpectroscopY). More importantly, this methodology also represents an important mechanism to evaluate the interactions between molecules or the first hand detection of the active constituents/inhibitors, which can also be used for other matrices varying both the macro and the small molecules.
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Masselin, Arnaud. "Synthèse chimio-enzymatique de sondes moléculaire pour la caractérisation de protéines affines des chitinoligosaccharides." Thesis, Université Grenoble Alpes (ComUE), 2018. http://www.theses.fr/2018GREAV054/document.
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Les oligosaccharides de chitine (COs) jouent des rôles majeurs chez les plantes. Alors que les COs longs (6-8 unités saccharidiques) sont des éliciteurs c’est-à-dire qu’ils activent leurs mécanismes de défenses vis-à-vis de microorganismes pathogènes, les COs courts (4-5 unités saccharidiques) participeraient à l’établissement de symbioses avec des microorganismes bénéfiques permettant une meilleure assimilation des nutriments du sol. Afin de mieux comprendre comment les plantes discriminent ces signaux moléculaires, il est nécessaire de disposer de molécules pures aux structures chimiques parfaitement contrôlées pour caractériser les récepteurs protéiques mis en jeu. Dans le cadre de cette thèse nous nous sommes intéressés à la synthèse de COs de degré de polymérisation contrôlé et leur modification pour obtenir de nouvelles sondes d’affinité. Pour cela un plan d’expérience a été développé afin d’optimiser la production de COs et plus particulièrement d’oligosaccharides longs par hydrolyse enzymatique de chitine avec une enzyme commerciale, le lysozyme du blanc d’œuf. Par la suite, un nouveau type de sonde d’affinité permettant le marquage spécifique de protéines affines des COs a été mis au point. Nous avons en effet montré pour la première fois que des glycosides de triazinyle peuvent être efficacement utilisés pour introduire un groupe fluorescent sur une protéine interagissant avec l’oligosaccharide sans aucune activation chimique ou physique extérieure. Après avoir démontré la preuve de concept avec des lectines, une sonde d’activité permettant de mesurer l’activité de chitinases par fluorescence et de réaliser leur marquage dans le même temps a été synthétisée. Ces nouveaux outils devraient permettre de progresser dans la caractérisation des récepteurs de COs chez les plantes mais également permettre à terme de découvrir de nouvelles protéines lectines ou enzymes qui interagissent avec les sucres<br>Chitinoligosaccharides (COs) play major roles in plants. While long COs (6-8 saccharide units) are elicitors activating plant defense mechanisms against pathogenic microorganisms, short COs (4-5 saccharide units) would participate in the establishment of symbioses with beneficial microorganisms allowing better assimilation of soil nutrients. Identifying the receptors involved in these processes to understand how plants discriminate these signal molecules requires having access to pure molecules with well-defined degrees of polymerization. In this thesis, we focused on the synthesis of well-defined COs and their modification to obtain new affinity probes. For this purpose, a design of experiments was developed in order to optimize the production of COs and more particularly of long ones by enzymatic hydrolysis of chitin with a commercial enzyme, hen egg-white lysozyme. Subsequently, a new type of affinity-based probe allowing the specific labeling of CO-binding proteins has been developed. We have shown for the first time that triazinyl glycosides can be effectively used to introduce a fluorescent group on an oligosaccharide-binding protein without any external chemical or physical activation. After demonstrating the proof of concept with lectins, a fluorescent activity-based probe allowing continuous assay of chitinases and their labeling at the same time was synthesized. These new tools offer exciting perspectives for the characterization of CO receptors in plants as well as for the discovery of new lectins and carbohydrate-active enzymes
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Lenger, Janina [Verfasser]. "Activity-based sulfatase and affinity-based metalloprotease probes: ready for take-off / Janina Lenger. Fakultät für Chemie - Organische Chemie III." Bielefeld : Universitätsbibliothek Bielefeld, Hochschulschriften, 2011. http://d-nb.info/1017073872/34.
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Mahajan, Shikha. "Protein Profiling of Adenine Nucleoside and Nucleotide Analogs Binding Proteins Using N6-Biotinylated-8-azidoadenosine Analogs as Affinity Based Protein Profiling Probes." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4139.
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Identification of differential expressions of proteins in proteomic profiles of biological samples shows great potential as a valuable technique for the early diagnosis of various diseases. An important challenge in modern protein profiling approaches is to reduce the complexity of the samples by limiting the number of proteins that need to be evaluated for distinction in the expression between normal and deceased cells. In this research, an affinity based approach for the enrichment of nucleotide and nucleoside binding proteins from a complex cell proteome has been developed. To achieve this goal, new N6-biotinylated-8-azido-adenosine probes (AdoRs) have been designed and synthesized to photolabel the nucleotide and nucleoside binding proteins. These probes contain a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Further, a mass spectrometric protein profiling approach is employed to quantitatively identify small variations in expression of nucleoside and nucleotide binding proteins in samples of interest.
Mouse neuroblastoma N18TG2 cell proteome has been used as a model system for the development of the LC-MS/MS based proteomic analysis of these affinity enriched protein fractions. Upon enrichment, the photolabeled proteome exhibited an approximately four-fold abundance of nucleoside and nucleotide binding proteins over
nonlabeled proteome. The approach was extended to compare the proteomic profiles of nucleotide and nucleoside binding proteins in cancerous (Hey) and non-cancerous (T-80) human ovarian cell proteome. Certain proteins that were not detected in cell lysate were also identified in labeled proteome, thereby demonstrating the strength of our approach in enriching low abundant proteins.
To substantiate the qualitative analysis, we have employed the Stable Isotope Labeling in Amino Acid Cell Culture (SILAC) for the quantitative study of the protein expression in cancerous and non-cancerous human ovarian cells. A modest panel of proteins with differential expressions in these cell lines was identified, a few of which have been correlated to various forms of cancer. Vimentin, stress induced phosphoprotein-1, and heat shock protein 90 that were identified to have altered expressions in these cell lines are among some of the proteins associated with ovarian cancer.
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Chou, Chien-Cheng, and 周建成. "Development of a Nano-based Affinity Probe for Purification and Identification of S-nitrosylated Peptide." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/76581504260912419016.
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碩士<br>國立清華大學<br>化學系<br>99<br>Nitric oxide (NO) influenced on cellular signaling in large part by means of S-nitrosylation/denitrosylation, the NO group covalently attach to the thiol side chain of cysteine resulting in the formation of S-nitrosothiols (RSNOs). RSNOs regulated proteins activity and function in a wide range of cellular pathways and physiological processes. However, the condition and mechanism of RSNOs formation remains uncertain, due to the lability of the S-NO bond caused the challenging for research. To date, numerous methods for assay RSNOs directly or indirectly have been developed. Among methods for studying RSNOs, biotin switch technology (BST) was used most commonly which can be used to detect and isolate SNO proteins from cell extracts in series of capping steps. In 2009, Wang and Ming et al. reported a bis-ligation mechanism of RSNOs which can converts unstable primary RSNOs to stable disulfide-iminophosphorane products in high yields under mild conditions. Based on this study, we conjugated the thioester phosphine ligand to the magnetic nanoparticle(MNP) for identification and purification of RSNOs. To verify the feasibility of the idea, the S-nitrosylated PTP1B peptide mixed in tryptic BSA mixture (50 ug) was successfully captured by the magnet probe and identified by MALDI-TOF. We believe this strategy wiil become a potential tool used in investigate S-nitrosylation proteins.
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Wang, Sin-Ge, and 王欣格. "Gold Nanoparticle-based Affinity Probes and Antibacterial Agents." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/55486490804967399681.
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碩士<br>國立交通大學<br>應用化學系碩博士班<br>105<br>Pathogenic bacteria can contaminate food and cause illness. Staphylococcus aureus, which can cause food poisoning, toxic shock syndrome, and erythematous rash, is one of the common pathogenic bacteria. Traditional analytical methods require at least several hours in characterization of target bacteria, whereas the required reagents such as antibodies are usually expensive. In addition, extensive use of antibiotics has led the emergence of antibiotic-resistant bacterial strains. In this dissertation, functional gold nanoparticles (Au NPs) were generated through facile one-pot reactions, and their functional probes were directly used as reducing and capping agents. The generated functional Au NPs were aimed to be used as affinity probes for development of rapid analytical methods and effective antibacterial therapy. In the first part of this dissertation, functional Au NPs were generated and capped by a peptide with the sequence of DVFLGDVFLGDEC (DD), which possesses selective affinity toward S. aureus. The generated AuNPs@DD were used as sensing probes for S. aureus in localized surface plasmon resonance (LSPR) analysis. Ultraviolet-visible absorption spectroscopy was used as the detection tool. On the basis of the LSPR shift in the absorption band derived from AuNPs@DD, a rapid sensing method for S. aureus was explored. It only took ~15 min to conduct one analysis. In the second part of this work, a polygonal AuNP-based photothermal approach for killing pathogenic bacteria such as vancomycin-resistant Enterococcus faecalis (VRE) was also developed. Polygonal Au NPs immobilized with vancomycin (AuNPs@Van) were generated through a one-pot synthesis method. The results demonstrated that vancomycin on AuNPs@Van still retained its antibiotic activity. The generated AuNPs@Van were consisted of ~47% (w/w) of vancomycin. We demonstrated that VRE in a bacterial sample containing AuNPs@Van (9.77 g/mL) can be used to inhibit ~100% of the growth of VRE, while a similar amount of free-form vancomycin can only be used to inhibit ~13% of bacterial growth. Moreover, the polygonal AuNPs@Van possess an absorption band in the NIR region and photothermal capability. Under irradiation of a near infrared (NIR) laser for ~5 min, the temperature of a low volume of sample (0.1 mL) containing AuNPs@Van (44 g/mL) can be rapidly raised for ~15 oC. When combining the antibacterial property of AuNPs@Van with the photothermal approach, the dosage of vancomycin in AuNPs@Van was 9–fold lower than that when free-form vancomycin was used as the antibacterial agent for obtaining similar antibacterial results. In addition, the cell toxicity of the generated AuNPs@Van was quite low. Although we have demonstrated the effectiveness of this approach toward VRE and the effective dosage of vancomycin on AuNPs@Van is much lower than that of free-form vancomycin, in vivo test is required to further clarify the usefulness of this approach.
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Gonçalves, Pedro Santos. "Development of chemical tools for target identification of Torin-based anti-parasitic agents." Master's thesis, 2015. http://hdl.handle.net/10451/24968.
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Tese de mestrado, Química Farmacêutica e Terapêutica, Universidade de Lisboa, Faculdade de Farmácia, 2016<br>American Trypanosomiasis (AT), Human African Trypanosomiasis (HAT) and Leishmaniasis are neglected tropical diseases (NTDs) caused by the vector-borne infection with parasitic protozoa of the order Trypanosomatid. According to the World Health Organization, these diseases affect millions of people and are responsible for thousands of deaths every year. Despite the high prevalence in developing countries, the available drugs are outdated, present several side effects, lack efficacy and/or are hard to administer. Therefore new drugs are urgently needed.
One powerful approach to fight the dearth of drugs for NTDs has been directed at repurposing established knowledge about classes of molecular targets that the pathogen holds in common with humans. We have recently disclosed Torin2, an ATP-competitive inhibitor of the mammalian target of rapamycin (mTOR), as a potent antimalarial with in vivo activity against both liver and blood stages and a distinct mode of action compared with currently used antimalarials. Although no Plasmodium orthologs of mTOR exist, there are some proteins that show a sequence with relatively high similarity to human mTOR, namely a predicted phosphatidylinositol-3-kinase (PI3K) and two predicted phosphatidylinositol-4 kinases (PI4K), which show conservation primarily in the kinase catalytic domain. This inspired us to screen Torin2 against other protozoan parasites and the results showed that the compound is active against T. brucei, T. cruzi and L. infantum.
In order to access the applicability of this new chemotype to other human parasitic protozoan diseases, we have synthesized a small library of Torin2 analogues and tested it against Trypanosoma cruzi, Trypanosoma brucei and Leishmania infantum, the agents of AT, HAT and visceral leishmaniasis (VL) respectively. The results were promising with EC50 values for some compounds in the low nanomolar range, surpassing the reference drugs for AT and VL.
Identifying the target of the Torin-based compounds was also a major interest, thus adequate chemical tools started to be developed to allow proteome profiling and live-cell imaging based on photoaffinity-based probes. A task which was partially accomplished in the present work with the synthesis of the photo-crosslinker moiety and coupling with a synthesized Torin-based compound.<br>A Tripanossomíase Americana (TA), a Tripanossomíase Humana Africana (THA) e Leishmanioses são doenças tropicais negligenciadas (DTNs) causadas pela infeção com parasitas protozoários da ordem Trypanosomatida. De acordo com a Organização Mundial de Saúde, estas doenças afetam milhões de pessoas e são responsáveis por milhares de mortes todos os anos. Ainda que a prevalência das doenças em países em desenvolvimento seja alta, os medicamentos disponíveis são antiquados, apresentam diversos efeitos secundários, são pouco eficientes e/ou são difíceis de administrar. Assim, a descoberta de novos medicamentos é uma necessidade.
Uma abordagem poderosa para combater a escassez de medicamentos para a DTNs passa por reutilizar compostos com alvos comuns entre parasitas e humanos. Recentemente o nosso grupo identificou o composto Torin2, um inibidor competitivo do ATP para a cinase mTOR, como um potente antimalárico com atividade in vivo contra a fase hepática e sanguínea, e um modo de ação distinto dos antimaláricos usados atualmente. Ainda que não existam proteínas ortológas da cinase mTOR no Plasmodium, existem proteínas com sequência relativamente semelhante à mTOR humana, nomeadamente uma PI3K e duas PI4Ks, que conservam o domínio catalítico da cinase. Estes factos levaram-nos a testar o Torin2 contra outros parasitas protozoários e os resultados mostraram que este é ativo contra T. brucei, T. cruzi e L. infantum.
Para testar a aplicabilidade desta molécula para outras doenças de protozoários parasitas em humanos, foi sintetizada uma pequena biblioteca de compostos análogos do Torin2 e testados contra Trypanosoma cruzi, Trypanosoma brucei e Leishmania infantum, os agentes da TA, THA e leishmaniose visceral (LV) respetivamente. Os resultados foram promissores ao revelar compostos com EC50 na ordem do nanomolar, superando os medicamentos de referência para a AT e LV.
A identificação do alvo molecular para estes análogos do Torin2 também era interessante, por isso começou-se a desenvolver ferramentas que possibilitariam o estudo proteómico e a visualização em células vivas baseado em sondas de foto-afinidade molecular. Uma tarefa para a qual se sintetizou um photo-crosslinker que se acoplou a uma estrutura análoga do Torin2.
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Huang, Tzu-Chiao, and 黃子樵. "Synthesis and Application of Affinity-Based Probes: Identification of Pregnenolone Binding Proteins and Study of Telomere-Directed DNA Alkylating Agents." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/21823495243268046594.
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碩士<br>國立臺灣大學<br>化學研究所<br>100<br>Affinity-based probes (AfBPs) have been developed recently by several research groups. Both of Cravatt and Yao groups independently reported the first AfBPs for metalloproteases. AfBPs achieves labelling by binding at a specific site of a protein followed by a non-specific covalent bond-forming event. The seminal works of AfBPs research enlighten us to design and synthesize probes for targeting different biomolecules of interest.
Embryonic cell movement is essential for morphogenesis and establishment of body shape. In the previous research reported by our group, showed the steroid hormone, pregnenolone, can preserve the abundance of microtubules, and thus effectively promotes the cell movement. In light of this study, we speculate that there might be pregnenolone binding proteins (PBPs), which can bind pregnenolone and further control the formation of microtubules. To verify our assumption, the photoreactive probes (P5C7b-O-NBPN) composed of a pregnenolone as the PBPs binding group, benzophenone as the photo cross-linker and a biotin as the reporter were designed and synthesized. It is hoped that by using this set of probes, PBPs could be identified and further purified prospectively.
On the other hand, telomeres are specialized DNA ends providing protection against genomic instability and cell senescence during cell division. The homeostasis of telomere is maintained by telomerase, which is selectively expressed in most tumors, and hence has a crucial role in cellular immortalization.38(?) The G-rich sequence of human telomeric DNA has a strong propensity to form the DNA G-quadruplex secondary structure, which can inhibit the activity of telomerase.53(?) Recently, 3,6-bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) was reported as a G-quartet stabilizer and a fluorescence probe. Preserving the key structural features of BMVC, a series of BMVC-mustard conjugates (BMVC-CnM; n = 2, 3, 6) were designed and synthesized in our group in attempts to develop telomere-directed DNA alkylating agents. The recognition between BMVC-CnM and G-quarduplex were analyzed and confirmed by various spectroscopic tools and DNA-PAGE studies previously. In this thesis, FRET analysis and DNA footprinting were applied to investigate the binding modes of BMVC-CnM with G-quarduplex. Furthermore, the TRAP-assay and cell images experimental results indicate BMVC-CnM have great potentials to be developed as anti-tumor agents.
Book chapters on the topic "Affinity-Based probe"
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Shi, Haibin, Mahesh Uttamchandani, and Shao Q. Yao. "A Method for Small Molecule Microarray-Based Screening for the Rapid Discovery of Affinity-Based Probes." In Methods in Molecular Biology. Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-845-4_5.
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Tiwari, Sandip. "Semiconductor surfaces." In Semiconductor Physics. Oxford University Press, 2020. http://dx.doi.org/10.1093/oso/9780198759867.003.0005.
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This chapter discusses the electronic, phononic and atomic behavior at surfaces. Symmetry breaking at the surface causes the emergence of new properties as the state description changes. Starting with an introduction of the semi-classical view, a bulk-based view of workfunction and electron affinity and cautions related to them, toy models are employed to explore the evolution of states at the surface. For electrons, propagating Bloch states persist to the surface but there are also states that are confined. Properties of these confined states and surface states are explored. The implications of stress and of surface reconstruction are discussed to elucidate the atomic rearranging that arises in semiconductors of interest when symmetries change. The state evolution approach is then extended to probe surface phonons. Again, bulk modes persist up to the surface, but there can also be surface-confined modes as well as surface-propagating modes.
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Söylemez, Fatma, and Çağatay Han Türkseven. "Aptamers and Possible Effects on Neurodegeneration." In Neuroprotection - New Approaches and Prospects. IntechOpen, 2020. http://dx.doi.org/10.5772/intechopen.89621.
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Aptamers are a new class of recognizing agents which are defined as short biomolecules like oligonucleotides and peptides that are used in diagnostics and therapeutics. They can bind to specific targets with extremely high affinity based on their structural conformations. It is believed that in the near future, aptamers could replace monoclonal antibody. The biggest advantage of using aptamers is that the process is in vitro in nature and does not require the use of animals and they also have unique properties, such as thermal stability, low cost, and unlimited applications. Aptamers have been studied as a biomaterial in numerous investigations concerning their use as a diagnostic and therapeutic tool and biosensing probe. DNA aptamers were also used for the diagnosis and treatment of neurodegeneration and neurodegenerative diseases. For example, functional nucleic acid aptamers have been developed to detect Aβ fragments in Alzheimer’s brain hippocampus tissue samples. Aptamers are promising materials for diverse areas, not just as alternatives to antibodies but as the core components of medical equipment. Although they are in the preliminary stages of development, results are quite encouraging, and it seems that aptamer research has a very bright future in neuroscience.
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Mascagni, Paolo. "Purification of large peptides using Chemoselective tags." In Fmoc Solid Phase Peptide Synthesis. Oxford University Press, 1999. http://dx.doi.org/10.1093/oso/9780199637256.003.0016.
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In solid phase peptide synthesis (SPPS), deletion sequences are generated at each addition of amino acid due to non-quantitative coupling reactions. Their concentration increases exponentially with the length of the peptide chain, and after many cycles not only do they represent a large proportion of the crude preparation, but they can also exhibit physicochemical characteristics similar to the target sequence. Thus, these deletion-sequence contaminants present major problems for removal, or even detection. In general, purification of synthetic peptides by conventional chromatography is based on hydrophobicity differences (using RP-HPLC) and charge differences (using ion-exchange chromatography). For short sequences, the use of one or both techniques is in general sufficient to obtain a product with high purity. However, on increasing the number of amino acid residues, the peptide secondary and progressively tertiary and quaternary structures begin to play an important role and the conformation of the largest peptides can decisively affect their retention behaviour. Furthermore, very closely related impurities such as deletion sequences lacking one or few residues can be chromatographically indistinguishable from the target sequence. Therefore, purification of large synthetic peptides is a complex and time-consuming task that requires the use of several separation techniques with the inevitable dramatic reduction in yields of the final material. Permanent termination (capping) of unreacted chains using a large excess of an acylating agent after each coupling step prevents the formation of deletion sequences and generates N-truncated peptides. However, even under these more favourable conditions, separation of the target sequence from chromatographically similar N-capped polypeptides requires extensive purification. If the target sequence could be specifically and transiently labelled so that the resulting product were selectively recognized by a specific stationary phase, then separation from impurities should be facilitated. This chapter deals with such an approach and in particular with the purification of large polypeptides, assembled by solid phase strategy, using lipophilic and biotin-based 9-fluorenylmethoxycarbonyl (Fmoc) chromatographic probes. Assuming that the formation of deletion sequences is prevented by capping unreacted chains, a reciprocal strategy can be applied that involves functional protection of all polymer-supported peptide chains that are still growing, with a specially chosen affinity reagent or chromatographic probe.
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Graves, Steven W., and John P. Nolan. "Molecular Assemblies, Probes, and Proteomics in Flow Cytometry." In Flow Cytometry for Biotechnology. Oxford University Press, 2005. http://dx.doi.org/10.1093/oso/9780195183146.003.0013.
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The many proteins and nucleic acids encoded in the genome predominantly perform their functions as macromolecular assemblies. In fact, modern biomedical research often targets the interactions of individual molecules of these assemblies, usually by disrupting or enhancing specific contacts, to provide treatment for many different diseases. Therefore, efficient pharmaceutical design requires knowledge of how macromolecular assemblies are built and function. To achieve this goal, sensitive and quantitative tools are essential. This chapter will discuss the use of flow cytometry as a general platform for sensitive measurement and quantification of molecular assemblies. First, this chapter will introduce general methods for analysis of molecular interactions along with a comparison of flow cytometry with these methods. Second, an overview of current flow cytometry instrumentation, assay technologies, and applications in molecular assembly analysis will be given. Third, the implementation of the above approaches in molecular assembly will be discussed. Finally, potential future directions of flow cytometry in molecular assembly analysis will be explored. At present, the analysis of macromolecular assemblies is performed by a wide variety of techniques that are chosen for the target molecules under study (proteins, DNA, lipids, etc.), the type of measurement required (kinetic or equilibrium), and whether the assembly of interest needs to be studied in vivo or in vitro. This continuum of techniques can be divided into the heterogeneous assays, which require a separation step to resolve products from reactants, and homogeneous assays, which can measure interactions without a separation step. Heterogeneous assays, in general, use radioisotopes, which are not perturbing; offer excellent sensitivity; and provide accurate quantification. The products are quantified after a separation step such as gel filtration, gel electrophoresis, or centrifugation. Rapid quench methods can provide subsecond kinetic resolution; however, the added separation steps are tedious and make collection of kinetic time courses difficult, as each time point must be separated and measured individually. Furthermore, in the time it takes the separation to occur, the interaction of interest can dissociate, which is a problem specific to low-affinity assemblies. Nonetheless, by using rapid chemical quench techniques, reaction times as short as a few milliseconds can be observed. Homogenous assays can be separated into solution- or surface-based assays. Solutionbased assays measure an optical signal generated by the assembly to quantify an interaction. High component concentrations (micromolar) allow changes in intrinsic molecular properties, such as protein fluorescence or circular dichroism, to be used to study molecular assemblies. For greater sensitivity (nanomole component concentrations), resonance energy transfer or polarization assays using exogenous fluorescent labels can be used. In combination with stopped-flow spectroscopy methodologies, solution-based assays allow reactions to monitored in a continuous fashion with submillisecond dead times.
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Sukina, Liudmila B. "On a Special Edition of Manuscript Synodikoses — Literary Collections of the 18th Century." In Hermeneutics of Old Russian Literature: Issue 20. А.M. Gorky Institute of World Literature of the Russian Academy of Sciences, 2021. http://dx.doi.org/10.22455/horl.1607-6192-2021-20-391-407.
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The type of Synodikos with a literary preface appeared at the end of the 16th century and developed and spread in the 17th century. It is believed that the study of the copies of the 18th century does not add essential information about the repertoire of collections of Synodikos. However, manuscripts can still be found that do not completely fit into the general picture of the ideas available in current science about the composition of literary collections of the late Synodikos. The article examines three handwritten front Synodikos books identified by the author, which differ significantly in the composition of literary prefaces from the general mass of those Synodikoses of the third edition common in the 18th century (I.V. Dergacheva). They are based on miniatures copied from engravings of various editions of the Synodikos by Leonty Bunin, and selected texts that match their meaning. A similar principle of compiling a moralistic collection was used in the Patriarch Adrian Synodikos (O.R. Khromov). The manuscripts in question have different origins and history of existence, but demonstrate thematic and stylistic affinity. They are also united by a curious combination in preface collections of poetic and prose texts. All this gives reason to say that in this case we are dealing with a special edition of the Synodikos — a literary collection that existed in the 18th century.
Conference papers on the topic "Affinity-Based probe"
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Wang, Ziqian, Zhichao Zhang, Ting Song, and Zongwei Guo. "Abstract 2754: Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor." In Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.am2019-2754.
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Wang, Ziqian, Zhichao Zhang, Ting Song, and Zongwei Guo. "Abstract 2754: Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor." In Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.sabcs18-2754.
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Geddes, V. A., G. V. Louie, G. D. Brayer, and R. T. A. MacGillivray. "MOLECULAR BASIS OF HEMOPHILIA B: IDENTIFICATION OF THE DEFECT IN FACTOR IX VANCOUVER." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643872.
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Factor IX Vancouver (fIX-V) is the cause of a moderate form of hemophilia B. An individual presenting with this disorder had 2.6% of normal procoagulant activity in his plasma but had 62% of the normal factor IX antigen level. Specific antibodies showed that fIX-V contains epitopes for both the heavy and light chains of factor IXa. To identify the defect involved, DNA was isolated from the lymphocytes of the male hemophiliac. Southern blot analysis using a full-length factor IX cDNA as a hybridization probe showed no gross differences between the fIX-V gene and the normal factor IX gene. The DNA from the hemophiliac was then partially digested with Sau3A and the resulting fragments (10-20kbp in size) were ligated into the BamHI site of λEMBL3. The DNA was then packaged into phage particles in vitro, and the recombinant phage were screened with the factor IX cDNA as a probe. Eight phage were isolated that contained overlapping DNA covering the complete gene for fIX-V. DNA sequence analysis of the protein-encoding regions, the intron/exon junctions and 5'-and 3'-flanking sequences revealed a single nucleotide change from the normal factor IX gene. The codon for amino acid 397 was changed from ATA (lie) to ACA (Thr). This mutation is in the catalytic domain of factor IXa and is novel amongst those hemophilia B mutations reported to date. Based on the known three dimensional structures of the pancreatic serine proteases, trypsin, elastase and chymotrypsin, models have been constructed for the structures of the catalytic domains of both the normal and Thr-397 mutant of factor IXa. These results suggest that the Thr-397 mutation may alter the conformation of the substrate binding region in the active site of factor IXa Vancouver through the formation of a hydrogen bond between the hydroxyl group of the Thr-397 side chain and the main chain carbonyl group of Trp-385. The postulated conformational change would lead to reduced binding affinity for the factor IXa substrate resulting in a reduction in the catalytic activity of fIXa-Vancouver.Supported in part by grants from the Medical Research Council of Canada (to GDB and RTAM).
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Edgington, T. S., J. H. Morrissey, and H. Fakhrai. "MOLECULAR CLONING OF HUMAN TISSUE FACTOR cDNA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643740.
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Tissue factor (TF), the cell-surface receptor and allo-steric activator for factor Vll/VIIa, is important in hemostasis andinflammation. The TF apoprotein was purifiedfrom human brain using factor Vll-affinitychromatography and SDS gel electrophoresis,and was found to consist of a 47 kDa heavy chain plus a 12.5 kDa light chain. Approximately one-third of the heavy chain amino acid sequence was determined for four regions by microsequencing the intact protein and peptides derived from V8-protease digestion. A λgtll cDNA library, made from mRNA derived from the human fibroblastic cell line WI38,was screened with (a) affinity-purified rabbit antibodies to human tissue factor, and (b) a 45-mer oligonucleotide probe based on TF heavy chain amino acid sequence. Five overlapping cDNA clones were identifiedand sequenced which confirmed all four partial TF amino acid sequences. Together these clones span the entire heavy chain coding sequence as well as 5" and 3" nontranslated regions. The N-terminusof the TF heavy chain is preceded by an unusually long signal peptide which appears to be cleaved at alternative sites two amino acids apart. This results in two variants of TF heavy chains which differ slightly in length and amino-terminal sequence.The deduced protein sequence shows no major homology to known protein sequences. However,a relatively uncommon tripeptide sequence, Trp-Lys-Ser (WKS), appears three times in the TF heavy chain. This tripeptidesequence also occurs in HMW kininogen, factor VIII,von Willebrand"s factor andant ithrombin-III. Limited sequence similarity is observed in flanking sequences,andthis may indicate a possible functional domain for the recognition of members ofthe vitamin K-dependent serine protease famil.Supported by NIH grants HL-16411 andCA-41085.
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Schulz, Martin, Fritz Klocke, Jan Riepe, Nils Klingbeil, and Kristian Arntz. "Process Optimization of Wire Based Laser Metal Deposition of Titanium." In ASME Turbo Expo 2018: Turbomachinery Technical Conference and Exposition. American Society of Mechanical Engineers, 2018. http://dx.doi.org/10.1115/gt2018-76924.
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Titanium alloys are used instead of steel and nickel-based alloys to lower the weight of turbines whenever it is applicable. Due to the high manufacturing costs of titanium, near-net-shape processes like laser metal deposition (LMD) processes are an approach to improve the production of new turbomachinery components. Additionally, these processes are also suitable for repair. LMD uses wire or powder as additional material. When highly reactive materials like titanium grade 5 (Ti6Al4V) are processed, wire-based laser metal deposition (LMD-W) processes are superior to powder-based processes due to the smaller reactive surface. Nowadays, three main challenges exist when titanium grade 5 (Ti6Al4V) is processed by additive manufacturing (AM): First of all the high affinity to oxygen combined with the increased brittleness of the material in case of a contamination with already low amounts of oxygen has to be faced. Secondly, the material is prone to distortion induced by thermal stress during the manufacturing process. Finally, the material has a complex bimodal microstructure, which has to be adjusted properly to generate optimal strength. The following publication will present how these technical challenges are faced. A local shielding gas concept demonstrates how flooding of the process chamber was avoided. The distortion was lowered by minimizing the heat input. Therefore, the laser spot was adapted. Its size was reduced to physical minimum nearly matching the size of the wire. To avoid process aborts, the proper feeding of the wire was improved. With this optimized process, it was possible to generate several specimens for metallurgical analysis. Finally, treatments to modify the alpha-martensitic-structure into a bimodal structure were performed. Summarizing the results show that the LMD-W process was improved to overcome the main challenges. Thereby the process has become suitable for manufacturing turbomachinery components made from titanium grade 5.
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Herrmann, Marion, Wolfgang Lippmann, and Antonio Hurtado. "The Release of Radionuclides in the Laser Decontamination Process." In 17th International Conference on Nuclear Engineering. ASMEDC, 2009. http://dx.doi.org/10.1115/icone17-75958.
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The decommissioning of nuclear installations requires the decontamination of radioactively contaminated concrete surfaces in order to minimize the amount of radioactive waste to be disposed of as well as the exposure time of the staff during this works. The rapid progress in the development of laser technology has yielded high-performance diode lasers whose radiation can be guided over a long distance by means of glass-fibre optical units. This opens up the possibility of implementing unconventional laser-based decontamination processes. The aim of the method presented here is to combine melting and contactless ablation of a radioactively contaminated concrete surface by means of a laser beam with waste product conditioning. It is intended to design the process in such a way that a maximum of the radioactivity present at the surface is incorporated in the glass melt (= conditioning of waste products). The glassy granulate obtained is very well suited for direct final storage due to its physical and chemical properties. The portion of radioactive isotopes that are released in the process, but not incorporated during the ablation process is selectively deposited in a cooled electro-filter. To prove the effectiveness of the method, research was focused on decontamination experiments conducted on concrete samples contaminated with 137Cs, 60Co and 85Sr. Furthermore, the chemical composition of the concrete samples was varied (quartzitic, quartzitic-calcitic) to take account of the different release conditions in real concrete structures. The experiments showed that 85Sr and 60Co are highly soluble in the glass melt. Their release rate is very low as they have a relatively high boiling point. 137Cs also exhibits a great affinity to the glass melt, but is more easily released again in the high temperature range due to its low boiling point of approx. 700 °C. The released portion of 137Cs is then deposited in the upstream electro-filter. The overall assessment is that the intended decontamination process with simultaneous conditioning of waste products is basically feasible using today’s laser technology. The special advantage can be seen in the great versatility and easy control of the laser unit that is equipped with a fibre-optical system. Furthermore, laser ablation can be set up as a low-dust process, which minimizes problematic secondary contamination.