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Brahmi, Chloé. "Study of scleractinian coral biomineralization using ⁸⁶Sr-labeling and NanoSIMS ion-microprobe imaging." Paris, Muséum national d'histoire naturelle, 2012. http://www.theses.fr/2012MNHN0042.
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Les coraux Scléractiniaires produisent un exosquelette en carbonate de calcium (aragonite). Leurs processus de biominéralisation ont été étudiés chez trois espèces présentant différents taux de croissance et vivant ou non en symbiose avec les zooxanthelles. Une hétérogénéité de composition du tissu et du squelette a été mise en évidence, à différentes échelles, via des techniques d’observations et de micro-analyses, à haute résolution spatiale. Un thème central a été le développement et l’application d’une méthode de marquage de biocarbonates marins basée sur un enrichissement avec un isotope stable d’un élément trace, naturellement présent dans l’eau de mer (86Sr). Le biocarbonate formé, présentant des enrichissements isotopiques correspondants, est ensuite imagé à la NanoSIMS afin de visualiser, à l’échelle submicrométrique, les zones formées durant le marquage. Cette technique permet ainsi de résoudre les détails ultrastructuraux et accéder à la dynamique de croissance squelettiqueScleractinian corals build an aragonitic calcium carbonate exoskeleton. Their biomineralization processes have been studied in three different scleractinian species with or without zooxanthellae and with different growth rates. Heterogeneity of the tissue and the skeletal compositions was revealed at different length scales, using complementary observations and micro-analytical techniques at high spatial resolution. A central theme was the development and application of a method to label marine biocarbonates through a concentration-enrichment of a minor stable isotope of a trace element that is a natural component of seawater (86Sr), resulting in the formation of biocarbonate with corresponding isotopic enrichments. This biocarbonate was subsequently imaged with a NanoSIMS ion microprobe to visualize the locations of the isotopic marker on submicrometric length scales, permitting resolution of all ultra-structural details and access to the skeletal growth dynamics
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Sardana, Malvika. "Development of New Late-Stage Labeling Methods with Labeled Carbon and Fluorine-18." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASF001.
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Le marquage isotopique est un outil précieux pour la découverte de nouveaux médicaments. Nous présentons dans cette thèse la conception et l’application de méthodes de marquage tardif de molécules bioactives avec du carbone et du fluor. Les réactions de carbonylation utilisant le monoxide de carbone sont connues pour être compatibles avec un marquage isotopique tardif des substances bioactives dans des conditions douces. La première partie de cette thèse décrit une réaction de photocarbonylation à partir d’iodures d’alkyles catalysée par le palladium sous lumière visible. Cette réaction polyvalente utilisant le 9-methylfluorene-9-carbonyl chloride (COgen) et réalisée dans des conditions douces est compatible avec un marquage en carbone-14. La synthèse de COgen radioactif nécessitant plusieurs étapes, nos efforts ont porté sur la production de monoxyde de carbone marqué, par réduction du dioxyde de carbone correspondant avec un disilane. La dernière partie de la thèse aborde la synthèse d’un nouveau traceur marqué au fluor-18 pour la tomographie par émission de positrons, spécifique de la P-glycoprotéine (P-gp), un transporteur de la barrière hématoencéphalique. Nous avons ainsi marqué au fluor-18 le Crizotinib, anticancéreux approuvé pour le traitement du cancer du poumon non à petites cellules et qui voit son accumulation fortement diminuée par le P-gp. Les études de biodistribution et d’imagerie cérébrale chez les rongeurs sont actuellement en coursIsotope labeling is a crucial tool in drug discovery. Therefore, expanding the toolbox of a radiochemist with methods that allow late-stage labeling is highly important. The work presented in this thesis describes the development and utilization of late-stage labeling methods with carbon and fluorine. Carbonylation reactions with carbon monoxide are particularly known as mild and compatible with the late-stage labeling. The first part of the thesis describes the development of visible-light mediated palladium-catalysis using alkyl iodides as the coupling partner for the carbonylation. The mild and versatile radical aminocarbonylation protocol has shown good substrate compatibility. The use of 9-Methylfluorene-9-carbonyl chloride (COgen) allowed easy translation between unlabeled and labeled reaction. In order to bypass the synthesis of COgen which proceeds in two steps plus one step for the liberation of CO, we focused our efforts towards the one step reduction of labeled CO₂ to labeled CO using disilanes catalyzed by fluorides. The last part of this thesis discusses the development of a new positron emission tomography (PET) radiotracer for P-glycoprotein (P-gp), an active transporter at the blood-brain barrier. Crizotinib is an approved treatment for non-small cell lung carcinoma and its brain accumulation is restricted by P-gp. Crizotinib was successfully labeled with ¹⁸F, and rodent studies to map P-gp and improve the delivery of crizotinib to the brain are ongoing
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Campanholi, Diana Ruffato Resende. "Oxidação da galactose utilizando 13C em crianças saudáveis e galactosêmicas." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/17/17144/tde-22042014-101217/.
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A Galactosemia é um erro inato do metabolismo da galactose que ocorre em consequência da deficiência de uma das três principais enzimas envolvidas em seu metabolismo. O tratamento da doença se faz por meio de intervenção dietética. Com a necessidade de melhorar nosso conhecimento acerca do metabolismo da galactose, isótopos estáveis estão sendo usados para mostrar o perfil de oxidação da mesma. O parâmetro bioquímico claramente correlacionado com os resultados clínicos e com o genótipo da GALT é o da oxidação da galactose em dióxido de carbono. A capacidade de determinar a oxidação com administração oral ou endovenosa de isótopos marcados (1-13C-galactose) de galactose fez deste um meio prático de se determinar a oxidação da galactose. Os objetivos do trabalho foram: 1. Avaliar a capacidade de oxidação de galactose em crianças brasileiras com o diagnóstico de galactosemia. 2 Avaliar a capacidade de oxidação da galactose em crianças brasileiras saudáveis. 3 Verificar a possibilidade de definir um ponto de corte para a detecção da galactosemia através do teste respiratório com a construção de uma curva ROC de maior sensibilidade e especificidade. A metodologia empregada foi a seguinte: Amostragem: 21 crianças saudáveis e 7 crianças com galactosemia com idade variando de 1 a 7 anos. Teste Respiratório: O teste respiratório de todas as crianças foi quantitativo para o enriquecimento de 13CO2 em ar expirado antes e depois da administração oral de 7mg/kg de uma solução aquosa de 1-13C-Galactose. As amostras foram colhidas antes da administração da solução e 30,60 e 120min após. Mensuração do 13CO2 no ar: Em cada amostra de ar duplicada a razão molar do 13CO2 e 12CO2 foi quantificado pela razão de massa/carga (m/z) dos isótopos gasosos através de um espectrômetro de massa. Análise estatística: Construção de uma curva ROC para determinar o melhor ponto de corte das diferenças em porcentagem da 1-13C-galactose recuperada em 13CO2 que forneça um teste respiratório positivo para detecção da galactosemia de maior sensibilidade e especificidade. As crianças doentes tiveram uma % acumulativa de 13C no ar expirado proveniente da galactose marcada (CUMPCD) variando em média de 0,03% no tempo de 30 minutos a 1,67% no tempo de 120 minutos. Em contrapartida, os indivíduos saudáveis apresentaram enriquecimentos e uma CUMPCD maiores, com valores de 0,4% no tempo de 30 minutos a 5,58% no tempo 120 minutos. Portanto, nesse estudo ficou evidente que há uma diferença marcante na oxidação da galactose em crianças com e sem galactosemia, logo teste respiratório é útil em discriminar crianças com deficiência na GALT.Galactosemia is an inborn error of galactose metabolism that occurs as an outcome of an enzyme deficiency. The current treatment is based on dietary intervention. Stable isotopes have been used to assess galactoses oxidation profile due to an urgency to improve our knowledge on its metabolism. The biochemical parameter clearly correlated to clinical outcomes and to GALT genotype is the galactose oxidation process into carbon dioxide. The ability to assess galactoses oxidation after galactose labeled isotope administration (1-13C-galactose) has become a practical way of determining the metabolism of galactose. The aims of the study were: 1 Assess the galactose oxidation ability in Brazilian galactosemic children. 2 Assess the galactose oxidation ability in healthy Brazilian children. 3 Set a cut off point for detecting galactosemia through breath test by constructing a ROC curve. The methodology employed was: Sampling: 21 healthy children and 7 children with galactosemia with age ranging from 1 to 7 years. Breath test: the breath test of all the children was quantitative for 13CO2 enrichment in exhaled air before and after oral administration of 7mg/kg of 1-13C-Galactose aqueous solution. Samples were collected at baseline time, 30, 60 and 120 min after solution administration. Measurement of the 13CO2 in the air: molar ratio 13CO2 and 12CO2 were quantified by the mass/charge ratio (m/z) of stable isotopes through a mass spectrometer in every air sample. Statistical analysis: ROC curve construction in order to determine the best cutting point of differences in percentage of 1-13C-galactose recovered in 13CO2 that provides a positive breath test for galactosemia detection. Therefore, as a result, sick children had some percentage of cumulative 13C in the exhaled air from labeled galactose (CUMPCD) ranging from 0.03% in 30 minutes time to 1.67% in 120 minutes. In contrast, healthy subjects showed a CUMPCD much more expressive, with values ranging from 0.4% in 30 minutes to 5.58% in 120 minutes. Therefore, in this study has shown that there is a great difference in galactose oxidation in children with and without galactosemia, hence the breath test is useful in discriminating children with GALT deficiencies.
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Jameson, Elizabeth Frances Mary. "Development of solid phase-based PET isotope labelling methods." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/17973.
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Positron Emission Tomography (PET) has great value in research and clinical applications from oncology to neurodegenerative disorders. However, there is a barrier in translating biological knowledge into new PET applications due in part to the lack of efficient, widely applicable methods for labelling compounds with PET radioisotopes. Herein, a generic approach to radiolabelling is presented which is direct, broadly applicable and potentially adaptable to either of the two most commonly used PET radioisotopes, 11C and 18F. This approach employs the advantages of solid phase synthesis to achieve selective release of only the desired radiolabelled product from a solid support in a single step, simplifying purification and hence improving synthetic efficiency. Polystyrene resin was functionalised with a 1,2-diol group; this allowed the covalent attachment of compounds bearing boronic acid groups via formation of a boronate ester linkage. A Suzuki-Miyaura reaction with methyl iodide was used to cleave a model compound from the resin in 61% conversion after five minutes. This reaction was adapted to develop a fully automated radiosynthesis with [11C]- methyl iodide which generated a radiolabelled model compound in 2 – 7% non-decay-corrected radiochemical yield. This provided proof of concept for the simultaneous cleavage of compounds from the resin and radiolabelling with 11C. A boronic acid precursor of the known radiotracer [11C]-M-MTEB was attached to the resin and successfully radiolabelled with 11C in 2.4% non-decay-corrected radiochemical yield and 96 – 100% radiochemical purity under the same conditions. Furthermore, the potential adaptability of this solid phase approach to 18F radiolabelling was demonstrated by treatment of the resin-bound small molecules and peptides with potassium bifluoride, which released the compounds rapidly as trifluoroborate salts.
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Khaw, Lake Ee. "Isotopic labelling of dihydrofolate reductase for NMR studies." Thesis, Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/25179.
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Smyth, Patrick. "Studying the Temporal Dynamics of the Gut Microbiota Using Metabolic Stable Isotope Labeling and Metaproteomics." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42344.
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The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15-N hydrolysate from Ralstonia eutropha, we identified 15,297 non-redundant unlabeled peptides of which 10,839 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse gut microbiome.
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Castleberry, Colette M. "Quantitative Identification of Non-coding RNAs by Isotope Labeling and LC-MS/MS." University of Cincinnati / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1258474676.
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Alghamdi, Waleed. "New isotopic labelling methodology and its application in phosphoproteomics." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/new-isotopic-labelling-methodology-and-its-application-in-phosphoproteomics(7d39b2dd-14aa-47f5-a4fa-0228b83b5a3d).html.
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The kinetics of protein phosphorylation and dephosphorylation are tightly controlled by specific kinases and phosphatases; disturbances are often disease-causing. Phosphorylation kinetics are normally monitored using radioactive isotopes of phosphorus, or by using stop-flow techniques. Approaches using mass spectrometry are severely limited by the lack of a stable isotope of phosphorus (other than 31P). The principal aim of this study is to develop a new method to incorporate 18O label into phosphorylation sites of phosphoproteins with a view of applying this method to enhance the detection of phosphorylation by mass spectrometry and to analyze the phosphorylation kinetics of proteins. Aurora-A kinase was selected to explore the possibility of using 18O-labelling to monitor phosphorylation kinetics. The kinase is well characterized, phosphorylated both in human cells and when expressed in recombinant form in E. coli and it contributes to development of some cancers when deregulated. Applying different mass spectrometric approaches resulted in the identification of 19 phosphorylation sites of Aurora-A including five new sites. Using H3P18O4 as a label donor to incorporate 18O into Aurora-A phosphorylation sites showed partial and inconsistent label incorporation. Alternatively, H218O was used to investigate the possibility of label incorporation. Preliminary results, however, showed high complex data which hampered precise identification of phosphopeptides and their labelling state. The labelling experiment was then redesigned in which induction took place in label free medium to allow the light version of the kinase to accumulate, before chasing with 18O label. This design successfully introduced fully labelled P18O3 into Aurora-A phosphorylation sites. LC ESI Q-ToF analysis of 18O labelled Aurora-A sample isolated according to this protocol identified 30 phosphopeptides showing label incorporation, which is double the number of phosphopeptides identified by MASCOT using the same MS analysis. The method was also used to investigate phosphorylation kinetics of Aurora-A. The results suggested differential regulation of phosphorylation sites of Aurora-A as some sites showed early phosphorylation while others were phosphorylated at later stages. Overall, a new approach was developed for enhanced detection of phosphorylation sites and analysis of phosphorylation kinetics.
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Li, Siwei. "High Throughput Automated Comparative Analysis of RNAs Using Isotope Labeling and LC-MS/MS." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1384427990.
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Jagdeo, Julienne. "Identification of novel picornavirus proteinase substrates using terminal amine isotopic labeling of substrates." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/61277.
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Viruses have exploited strategies of proteolysis for the purposes of processing viral proteins and manipulating cellular processes to direct synthesis of new virions and subvert host antiviral responses. Many viruses encode proteases within their genome, of which many have been well studied among the family of positive-sense single-stranded RNA picornaviruses. A subset of host proteins have already been identified as targets of picornaviral proteinases; however, the full repertoire of targets is not known. In this thesis, a novel proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) was used to conduct a global analysis of protease-generated N-terminal peptides by mass spectrometry and identify novel substrates of the 3C (3Cpro) and 2A (2Apro) proteinases from poliovirus and coxsackievirus type B3 (CVB3). TAILS was performed on HeLa cell extracts subjected to purified poliovirus 3Cpro or CVB3 2Apro, and on mouse HL-1 cardiomyocyte extracts subjected to purified CVB3 3Cpro. A list of high confidence candidate substrates for all three proteinases was generated, which included a peptide corresponding to the known poliovirus 3Cpro substrate polypyrimide tract binding protein at a known cleavage site, thus validating this approach. Furthermore, three identical peptides in both the poliovirus and CVB3 3Cpro list of high confidence substrates were identified, suggesting that cleavage of these substrates may contribute to general strategy of picornaviral infection. A total of seven high confidence substrates were validated as novel targets of 3Cpro in vitro and during virus infection. Moreover, mutations in the TAILS-identified cleavage sites for these candidates blocked cleavage in vitro and during infection. Depletion of these proteins by siRNAs modulated virus infection, suggesting that cleavage of these substrates either promotes or inhibits virus infection. In summary, an in vitro TAILS assay can be utilized to identify novel substrates of viral proteinases that are cleave during infection. Moreover, TAILS can identify common substrates of viral proteinases between different viral species, revealing general strategies of infection utilized by related viruses. Finally, the identification of novel host substrates provides new insights the viral-host interactions mediated by viral proteinases that are required for successful infection.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Dean, Matthew, Geoffrey Findlay, Michael Hoopmann, Christine Wu, Michael MacCoss, Willie Swanson, and Michael Nachman. "Identification of ejaculated proteins in the house mouse (Mus domesticus) via isotopic labeling." BioMed Central, 2011. http://hdl.handle.net/10150/610018.
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BACKGROUND:Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins.RESULTS:We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract.CONCLUSION:Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract.
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Serveta, Irena. "Towards the use of Alcohol dehydrogenases as biocatalysts for stereoselective isotope labeling of aromatic alcohols." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-368315.
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The enzyme ADH-A and one of its mutants ADH-A C1B1 from Rhodococcus ruber, have in previous studies been proved to act as proper biocatalysts, fully capable of performing redox reactions. Two redox reactions were studied during this project, were those enzymes act as catalysts. For that matter, ADH-A wild type and ADH-A C1B1 genes were expressed in E. coli and the encoded enzymes were purified and used for kinetic studies with a final goal on studying the kinetic isotope effect that is generated between them and the molecules that contain deuterium. HPLC analysis on these products showed that the reactions were not thermodynamically favored and conclusions on the best reaction conditions for both enzymes as well as for further improvements are discussed.
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Chen, Jianqing. "Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96 potential use for extracorporeal immunoadsorption with enhanced tumor radioactivity retention of iodine, indium and rhenium /." Lund : Lund University, the Jubileum Institute, Dept. of Radiation Physics, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39725797.html.
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Li, Ping. "Expression, purification and isotopic labelling of HAV and HRV14 3C protease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0003/MQ59833.pdf.
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Noban, Catherine. "Development of methods for isotopic labelling of aromatics using ipso-fluorodegermylation." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429416.
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Edwards, Andrew John. "An NMR isotope labelling analysis of calmodulin interactions with high affinity chiral inhibitors." Thesis, University of Hertfordshire, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267964.
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Athanason, Mark Gabriel. "Quantitative Proteomic Investigation of Disease Models of Type 2 Diabetes." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6460.
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PANcreatic DERived factor (PANDER, FAM3B) is a member of a superfamily of FAM3 proteins that are uniquely structured and strongly expressed from the endocrine pancreas and co-secreted with insulin. Unique animal models available to our lab have indicated that PANDER can induce a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased. The complexity of the biological networks involved with this process warranted the logical approach of employing quantitative mass spectrometry based proteomic analysis using stable isotope labeling of amino acids in cell culture (SILAC) to identify the global proteome differences between the PANDER transgenic (TG) overexpressing murine model to matched wild-type mice under three metabolic states (fasted, fed and insulin stimulated). Additionally, this technique was used to compare the hepatic proteome of mice on a high fat diet to elucidate early and late mechanisms of disease progression. The “spike-in” process was employed by equal addition of lysate obtained from livers of heavy L-Lysine (13C6, 97%) fed mice to the mice liver protein lysate (PANTG and WT) for relative quantitative analysis. Upon acquisition of the dataset by use of liquid chromatography tandem mass spectrometry (LC-MS/MS, LTQ Orbitrap), geometric means and Uniprot Protein identification numbers were uploaded to Ingenuity Pathway Analysis (IPA) to reveal the effect of PANDER on hepatic signaling. IPA identified lipid metabolism and fatty acid synthesis as top cellular functions differentially altered in all metabolic states. Several molecules with a role in lipid metabolism were identified and include FASN, ApoA1, ApoA4, SCD1, CD36, CYP7A1 and ACC. Furthermore, central to the differentially expressed proteins was the revealed activation of the liver X receptor (LXR) pathway. In summary, our SILAC proteomic approach has elucidated numerous previously unidentified PANDER induced molecules and pathways resulting in increased hepatic lipogenesis. In addition, we have demonstrated strong utility of this approach in comprehensively phenotyping animal models of hepatic insulin resistance. PANDER may potentially propagate pro-hepatic lipogenic effects by LXR activation in contrast to increased LXRα expression. This can be evaluated through the use of LXR agonists (T0901317) antagonists (GSK 2033). LXR activity can be measured by luciferase assays using an LXRE response plasmid. Our central hypothesis is that PANDER induces activation of LXR and is measured and predicted in our line of experiments. In general, PANDER induced LXR activation will be enhanced by T0901317 and diminish effects of GSK 2033 along with direct correlation of downstream metabolic effects such as increased hepatic lipogenesis and fatty acid metabolism. Taken together, PANDER strongly impacts hepatic lipid metabolism and may induce a SHIR phenotype via the LXR pathway. Additionally, phosphoproteomic analysis uncovered large-scale differences in protein phosphorylation states as PANDER impacts insulin signaling. A notable finding was the increased phosphorylation of glycogen synthase (GSK), possibly responsible for the decreased hepatic glycogen content in the PANTG mouse. In an effort to map out critical molecules involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis, the same proteomic approach was carried out, providing a unique dataset of differentially expressed hepatic proteins due to a high at diet.
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Barnett, Derek W. "PART 1. SYNTHESIS OF STABLE-ISOTOPE LABELED AMINO ACIDS PART 2. SYNTHESIS OF MECHANISTIC PROBES OF RETINOID ACTION." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1038951598.
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Ghirardo, Andrea [Verfasser]. "Studies of plant terpenoid biosynthesis using 13C stable isotope labeling techniques (KIT Scientific Reports ; 7583) / Andrea Ghirardo." Karlsruhe : KIT Scientific Publishing, 2011. http://d-nb.info/1185581405/34.
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Johansson, Anna Karin. "Linking structure and function of the asialoglycoprotein receptor H1-CRD using site-directed mutagenesis and isotope labeling /." [S.l.] : [s.n.], 2007. http://edoc.unibas.ch/diss/DissB_8736.
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Jiang, Lin. "Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling." FIU Digital Commons, 2012. http://digitalcommons.fiu.edu/etd/774.
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Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme.
In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide.
Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins.
For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.
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Rinkel, Jan [Verfasser]. "Establishing a Comprehensive Toolbox for Isotopic Labelling Studies on Terpene Synthases / Jan Rinkel." Bonn : Universitäts- und Landesbibliothek Bonn, 2019. http://d-nb.info/1200019881/34.
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Beller, Nicole C. "Selective Pulse Chase-SILAC Labeling of Three-Dimensional Multicellular Spheroids for Global Proteome Analysis." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586292839111678.
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Chaput, Dale. "Mass Spectrometry-Based Investigation of APP-Dependent Mechanisms in Neurodegeneration." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5921.
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Alzheimer’s disease (AD) is the most prevalent form of dementia affecting the elderly, and as the aging population increases the social and economic burden of AD grows substantially. Pathological hallmarks of AD include the accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs), as well as significant neuron loss. Amyloid plaques consist of aggregated amyloid beta (Aβ) peptide, which is generated from the proteolytic processing of amyloid precursor protein (APP) in addition to several other peptides. While the processing of APP has been characterized, its primary physiological function and its involvement in AD pathology are poorly understood. Developing a greater understanding of the function of APP, and the molecular and cellular functions it is involved in or other proteins it is associated with, could provide insight into its role in AD pathology. To investigate the function of APP695, the neuronal isoform of APP, we used mass spectrometry to compare changes in protein expression and phosphorylation between APP-null B103 and APP695-expressing B103-695 rat neuroblastoma cells.
Mass spectrometry-based proteomics has become a powerful technique for the unbiased identification of proteins from complex mixtures. Quantitative proteomics using labeling techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), allow relative quantitation of multiple samples at once. More recently, with advances in mass spectrometer technology, label-free quantitation has become a reliable quantitative proteomics approach. Additionally, mass spectrometry can be used for the analysis of post-translational modifications, such as phosphorylation, a dynamic modification involved in the regulation of many cellular processes. Phosphoproteomics identifies site-specific phosphorylation and surrounding sequence information, which can be used for consensus motif analysis to provide further information about potential changes in kinase activity. Identifying changes in phosphorylation and kinase activity also provides information about signaling pathways and functions that may be affected by APP695 expression. Comprehensive proteomic and phosphoproteomic datasets can be used to gain insight into the molecular mechanisms that may be regulated by APP695 expression, or involved in AD progression and pathology, leading to the development of novel therapeutic and preventative strategies for AD.
Proteomic and phosphoproteomic analysis of B103 and B103-695 cells identified several significant protein expression and phosphorylation changes that may be mediated by APP695-expression. Global-scale proteomic analysis identified increased expression of Ras and ƴ-synuclein in B103-695 cells, which was further validated in human AD brain tissue. Phosphoproteomic analysis showed increased phosphorylation of Histone H4 at Ser47, and led to the investigation of PCTAIRE-2 (Cdk17), and PCTAIRE-3 (Cdk18) expression, which were all shown to be increased in AD transgenic mouse tissue, culture primary rat neurons treated with Aβ, as well as mild cognitive impairment (MCI) and AD human brain tissue.
Label-free quantitative proteomics was used for the analysis of human brain tissue from the cortex of individuals affected by AD, MCI, Parkinson’s disease (PD), and progressive supranuclear palsy (PSP) compared to cognitively normal, control samples. A number of differentially expressed proteins were identified in AD, MCI, PD, and PSP tissue. Bioinformatic analysis of the comprehensive proteomic datasets from AD, MCI, PD, and PSP human brain tissue identified several proteins consistent with corresponding disease pathology and neurodegeneration, such as inflammatory proteins. While some of the molecular and cellular functions were unique among neurodegenerative diseases, there also appears to be overlap of affected functions, suggesting there may be a more common mechanism of neurodegeneration.
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Chesters, Nicola C. J. E. "Biosynthetic studies on tropic acid and piliformic acid." Thesis, Durham University, 1995. http://etheses.dur.ac.uk/5211/.
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This thesis is divided into two parts and covers biosynthetic studies on two secondary metabolites, tropic acid in Part I and piliformic acid, in Part II.(S)-Tropic acid is the acid moiety of the alkaloids hyoscyamine and scopolamine, which are produced by a number of plants of the Solanacae family. An intriguing rearrangement of the L-phenylalanine side chain gives rise to the isopropanoid (S)-tropic acid skeleton. The detailed nature of the rearrangement has however remained elusive despite continued interest over the years. In chapter two the identification of intermediates between L-phenylalanine and (S)-tropic acid is discussed, which has placed (R)-D-phenyllactic acid as an immediate precursor. The stereochemical features of the rearrangement are described in chapter 3 and finally in chapter 4 a mechanism for the rearrangement is proposed. This is based on information obtained from the incorporation of various isotopically labelled precursors to tropic acid into two of the minor alkaloids, 3a-2'-hydroxyacetoxytropane and 3a- phenylacetoxytropane. This work was carried out in collaboration with Dr Richard Robins at the AFRC Institute of Food Research in Norwich. Piliformic acid is elaborated by the slow growing fungus Poronia piliformis. The incorporation of a number of isotopically labelled substrates into piliformic acid has revealed a mixed biosynthetic origin, comprising C(_8) and C(_3) fragments. These have been shown to be of acetogenic and citric acid cycle origins respectively. The C(_8) fragment has been further demonstrated to be a degradation product of a longer chain fatty acid. The mode of coupling of the two fragments has been investigated and suggests the intermediacy of a novel a-carboxyoctanoate. A pathway for the assembly of piliformic acid, involving a 1,3-hydrogen shift, is proposed, consistent with the above findings. These results are the subject of chapter 6.
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Pears, Katrina. "Investigating nitrogen transfer between plants in agricultural grassland by using a 15N stable isotope labelling approach." Thesis, University of Bristol, 2018. http://hdl.handle.net/1983/bce1a3d6-218a-43aa-9033-788fc3432c72.
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The world’s population is predicted to reach 9.5 billion by 2050. This will put increasing pressure on already stretched food supplies. Previously, food supply has been increased by the use of synthetic fertilisers, particularly the use of nitrogen (N). However, fertilisers provide an unsustainable source of N, due to high energy demands for production as well as over-application and inadequate matching of fertiliser application to crop demand (synchrony). One solution to this global problem is the use of legumes, such as white clover (Trifolium repens L.), which are capable of fixing atmospheric N2, N can then be supplied to an associated non-legume crop. To date, legume and non-legume cropping systems have seen little application due to a lack of understanding of the unique N-transfer pathway. Three major belowground pathways have been identified: plant exudation, legume decomposition and mycorrhizae associations. A better understanding of the different N-transfer pathways is needed to maximise the benefits of the association and to develop appropriate land-use management strategies, this is addressed by this research. The research has focused on developing and validating a method for introducing a 15N-label to white clover and following the N-transfer through the plant and soil systems into associated perennial ryegrass (Lolium perenne L.). The method developed comprised a split-root labelling technique, enabling CO(15NH2)2 to be injected into a sand-filled labelling compartment. This allowed substantial 15N enrichment to be achieved, facilitating the investigation of the routing and controls on N-transfer within an agricultural soil. Laboratory experiments revealed that under normal conditions N-transfer from clover to ryegrass, as a proportion of non-legume N derived from the transfer of legume root N (NdftR), provided on average 2.67% of N. However, similar amounts of N were transferred in the reverse direction (1.98%), showing evidence for bi-directional flow. Incorporation of clover shoots into ryegrass soil, significantly increased NdftR (9.34%), whilst, clover exudates are likely to represent about one-third of total N-transfer. Perturbing N-transfer through modifications to the soil biota was shown to increase N-transfer (sterilised soil > weevil addition > fungi addition), although not significantly. Application of compound-specific amino acid (AA) techniques enabled the investigation of whether different N-transfer pathways influenced the distribution of 15N-label within the pool of soil AAs, thereby assessing microbial N assimilation and routing of N. Overall, there was a very low percentage incorporation of the applied 15N-label into individual AAs, although the percentage depended on the individual experiment, with total incorporation into the soil protein pool ranging from 0.1 to 2.4%. The majority of experiments revealed preferential routing into glutamic acid due to its central role within AA biosynthesis, which was seen to be similar to those AAs with the closest biochemical proximity. A key achievement from this research was the development of a robust repeatable method which allows easy manipulation and the investigation of a range of different treatments on N-transfer from clover-to-ryegrass. New insights into the effect of plant stress through 15N leaf-labelling or clover shoot removal were observed, resulting in significant reductions in the concentrations of soil hydrolysable AAs, questioning the use of the commonly used leaf-labelling technique and the effects of defoliation on N cycling and ecosystem functioning. The results generated from studying different N-transfer pathways revealed the importance of decomposition in N-transfer, revealing the rapid decomposition and N release of clover shoot material. This finding is extremely useful in developing land-use management strategies, where incorporation of clover shoot residues into soil can provide sustainable amounts of N in the short-term, which can improve the synchrony between clover and ryegrass, potentially increasing productivity and sustainability.
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Alexakis, Efstathios. "Isotopic studies of the hydrogenation and exchange-labelling of unsaturated hydrocarbons with heterogeneous catalysts." Thesis, University of Surrey, 2005. http://epubs.surrey.ac.uk/843816/.
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This thesis describes investigations utilising isotopic hydrogen gas (D2 and DT) and covering several areas of chemistry. The initial studies involved the selective alpha-labelling of pyridine and other N-heterocyclics via new hydrogen-deuterium exchange catalysts, identified via a parallel-chemistiy screening process. The new catalysts and associated methodology are applicable to labelling with tiitium as well as deuterium and are a significant improvement upon existing labelling approaches. The remaining studies involved the application of isotopes to studies of the hydrogenation of unsaturated hydrocarbons. initially the hydrogenation, and isotopic exchange reactions, of simple imbranched C5 alkenes and alkynes with D2 gas were examined. Although many aspects of the hydrogenation chemistry of pentenes and pentynes have been studied there is an interest in obtaining close to 100% selectivity in the reactions of these important industrial feedstocks. The studies were subsequently extended to the hydrogenation of the less-volatile phenyl-C3 unsaturated hydrocarbons, allowing studies with DT gas as well as D2. The work carried out in these two chapters includes a comparison of a novel l%Pd/Al2O3 catalyst developed by Johnson Matthey (JM)/Synetix with a standard catalyst 5% Pd/C catalyst routinely used for the hydrogenation of double bonds. Although these investigations are still in their initial stages, the results obtained suggest that the JM catalyst could prove more selective than the commonly used Pd/C. The above DT studies also enabled an investigation of the application of a new development in 3H-NMR spectroscopy, the 3H cryo-probe. This new technology was shown to provide a significant advance for the analysis of tritiated compounds and mixtures containing low levels of radioactivity. The last investigations concerned the facile exchange of isotope during the hydrogenation of terminal alkenes. This process was shown to be general and could well provide a novel methodology for the tritium labelling of this class of compounds.
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Barra, Lena [Verfasser]. "Studies on the Biosynthesis and Structure Elucidation of Terpene Natural Products by Isotopic Labeling Experiments / Lena Barra." Bonn : Universitäts- und Landesbibliothek Bonn, 2019. http://d-nb.info/1177881667/34.
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Kollmeier, Annette [Verfasser]. "Mass Spectrometry of Drug Derivatives: A Contribution to the Characterization of Fragmentation Reactions by Labelling with Stable Isotopes / Annette Kollmeier." Berlin : Freie Universität Berlin, 2021. http://d-nb.info/1235400328/34.
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Dahlberg, Tobias. "The first order Raman spectrum of isotope labelled nitrogen-doped reduced graphene oxide." Thesis, Umeå universitet, Institutionen för fysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-116699.
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The topic of this thesis is the study of nitrogen functionalities in nitrogen-doped reduced graphene oxide using Raman spectroscopy. Specifically, the project set out to investigate if the Raman active nitrogen-related vibrational modes of graphene can be identified via isotope labelling. Previous studies have used Raman spectroscopy to characterise nitrogen doped graphene, but none has employed the method of isotope labelling to do so. The study was conducted by producing undoped, nitrogen-doped and nitrogen-15-doped reduced graphene oxide and comparing the differences in the first-order Raman spectrum of the samples. Results of this study are inconclusive. However, some indications linking the I band to nitrogen functionalities are found. Also, a hypothetical Raman band denoted I* possibly related to \spt{3} hybridised carbon is introduced in the same spectral area as I. This indication of a separation of the I band into two bands, each dependent on one of these factors could bring clarity to this poorly understood spectral area. As the results of this study are highly speculative, further research is needed to confirm them and the work presented here serves as a preliminary investigation.
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Petersson, Hanna M. "Isotopic labelling of dietary antioxidants: Synthesis of indoles using functionalised titanium benzylidenes reagents for solid-phase." Thesis, University of Glasgow, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484837.
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A route to l3C-Iabelled quercetin-3-0-D-glucoside l3C_7 was developed. Our route gives the flavonol glucoside 13C_7 with an isotopic label at C-2 in 18% yield over five steps from the point of introduction of the isotopic label. Attempts were also made to synthesise the isotopically labelled cyanidin-3-0-D-glucoside 13C_8 using methods described in the literature, but these gave low yield of the anthocyanin. A new diversity-based a route for the solid phase synthesis of N-Bn-2,S-disubstited iodoles 15 has been developed. Aniline derivative 9 was reduced using a low-valent titanium species, Cp2Ti[P(OEt)3)2 10, to give a novel titanium(IV) alkylidene reagent 11 which allq,lidenated resinbound esters 12 and gave resin-bound enol ethers 13. Suzuki cross-couplings were then performed on solid phase to introduce an aryl group at the 5 position. Treatment with mild acid (1% TFADCM) cleaved the resin bound enol ether 14 from the resin. Simultaneously Boc deprotection and cyclisation under acidic condition (10% TFA-DCM) gave N-Bn-2,S-disubstited indoles 15 in good purity and moderate yield. In a similar way using N-prenyl-N-Boc aniline 16 gave N-prenyl-2,S-disubstituted indoles 17 in good purity and moderate yield using the same solid-phase method as above. A synthetic route to the N-TMS-N-Boc aniline 18 has also been developed, which should give unprotected 2,5-disubstitued indoles 19 in high purity.
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Waldecker, Bernd. "Investigations towards the design, synthesis and application of new sulfur-based transfer reagents." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C138-4.
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Pfeifer, Viktor. "Tritium and Deuterium Labelling of Bioactive Molecules Catalyzed by Metallic Nanoparticles." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS275/document.
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Cette thèse vise à développer de nouvelles méthodes efficaces pour incorporer des isotopes de l’hydrogène dans les molécules organiques complexes, après une introduction portant sur les applications et la synthèse des molécules marquées par le deutérium et tritium. Les méthodes permettant le marquage, par échange isotopique direct, d’hétérocycles azotés par des isotopes de l’hydrogène restent perfectibles, voire inexistantes dans certains cas, malgré la récurrence de ce type de sous-structures dans les molécules d’intérêt pharmacologique. Pour cette raison, la majeure partie de ce travail a consisté au développement de nouvelles méthodes d’incorporation d’atomes de deutérium et de tritium sur des hétérocycles azotés catalysées par des nanoparticules métalliques. Dans un premier chapitre, la mise au point, le champ d’application d’une méthode de marquage mettant en jeu l’utilisation de nanocatalyseurs de ruthénium seront discutés. Dans ce cadre, des calculs théoriques ont permis de rationaliser les regiosélectivités obtenues expérimentalement et d’identifier notamment des intermédiaires clefs inédits. D’un point de vue applicatif, cette méthode a permis de synthétiser des étalons internes deutérés pour la quantification LC-MS mais aussi des molécules complexes tritiées ayant des activités spécifiques élevées en une étape de synthèse. Dans un autre chapitre, la synthèse et la réactivité de nouveaux nanocatalyseurs de nickel permettant de réaliser des échanges isotopiques sélectifs seront discutésThis PhD thesis deals with the development of new efficient methods for the incorporation of hydrogen isotopes into organic molecules, which represents a serious issue especially for drug discovery and drug development processes. After giving an introduction about hydrogen isotopes and their applications in organic molecules, the course will proceed to an overview of different chemical transformations for establishing deuterium or tritium labels on molecular frameworks. The possibilities to label N-heterocycles by hydrogen isotopes through hydrogen isotope exchange (HIE) are still very restricted and even impossible for some representatives despite the strong recurrence of these substructures in numerous biologically active molecules. For this reason, the emphasis of the practical part will lie on the development of new methods for the incorporation of deuterium and tritium on N-heterocycles through metal nanoparticle catalysis. In the first chapter, HIE through ruthenium nanocatalysts will be optimized and the application range will be demonstrated. In this context, DFT-based calculations allowed to explain experimental regioselectivities and to identify new keyintermediates. In terms of application, it was shown that the ruthenium-catalyzed method is useful for the synthesis of deuterium labelled internal standards for LC-MS quantifications and for the tritiation of complex molecules displaying satisfying specific activities. In the next chapter, the synthesis of new nickel nanoparticles and their potential to catalyze selective HIE on N-heterocyclic derivatives will be discussed
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Al-Qahtani, Mohammed H. S. "Development of new and improved labelling procedures for introducing isotopic hydrogen and carbon-11 into organic compounds." Thesis, University of Surrey, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300367.
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Chu, Fong Lam. "Elucidation of selected Maillard reaction pathways in alanine and phenylalanine model systems through isotope labelling and pyrolysis-GC/MS based techniques." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66729.
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Alanine and phenylalanine based model systems were utilized in this thesis to elucidate selected Maillard reaction pathways through isotope labelling, Fourier Transform Infrared Spectroscopy (FTIR) and Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS) based techniques. The formation of glycosylamines from reducing sugars and amino acids is a well-known process in the initial phase of the Maillard reaction. They play a critical role in both the initiation and propagation stages, however, little attention has been paid so far on the ability of these imines to undergo isomerization and thus contribute to the diversity of Maillard reaction products. In this study, imine isomerization through 5-oxazolidinone formation was explored in phenylalanine and alanine sugar models systems. Spectroscopic evidence was provided for its formation by taking advantage of the strong carbonyl absorption band centered at 1784 cm-1 in the phenylalanine/glyceraldehyde and at 1778 cm-1 in phenylalanine/glycolaldehyde model system. The importance of 5-oxazolidinone formation lies in its ability to decarboxylate to azomethine ylide and subsequently form two isomeric imines, each capable of producing distinct Maillard reaction products. Evidence for the formation of such ylides was also provided through their ability to undergo 1,3-dipolar cycloaddition with dipolarophiles. Regarding the role of oxygen in the Maillard reaction, it was found that molecular oxygen can influence carbon-carbon bond cleavage through the formation and degradation of 1,2-dioxetane moieties generated from enol structures abundantly formed in the Maillard reaction from their corresponding ketones and aldehydes such as phenylacetaldehyde the Strecker aldehyde of phenylalanine and subsequently can be oxidized into benzaldehyde. Furthermore, the α-dicarbonyl compounds generated during the Maillard reaction play a significant role as precursors of important flavour-activeCette thèse comporte une étude approfondie des routes réactionnelles de la réaction de Maillard dans des systèmes modèles à base d'alanine et de phénylalanine à l'aide de techniques basées sur les principes d'incorporation d'isotopes lourds avec la pyrolyse couplée à la chromatographie phase gazeuse et la spectrométrie de masse (Py-CG/SM) et ainsi que la spectroscopie infrarouge à transformée de Fourier (IR-TF). La formation des glycosylamines par des sucres réducteurs et des acides aminés est un processus bien connu dans la phase initiale de la réaction de Maillard. Ceux-ci jouent un rôle critique dans les étapes de déclenchement et de propagation. Cependant, peu d'attention est orientée vers la capacité de ces imines à subir l'isomérisation et de contribuer à la diversité des produits de la réaction de Maillard. Dans cette étude, l'isomérisation d'imine par la formation du 5-oxazolidinone fut explorée dans des systèmes modèles de phénylalanine/sucre et alanine/sucre. Les preuves spectroscopiques pour la formation du 5-oxazolidinone furent obtenues par la bande intense d'absorption carbonylique centrée à 1784 cm-1 dans le système modèle phénylalanine/glycéraldéhyde et à 1778 cm-1 dans le phénylalanine/glycolaldéhyde. L'importance de la formation du 5-oxazolidinone résulte dans sa capacité à se décarboxyler formant ainsi un ylide d'azomethine ayant l'habileté de produire deux imines isomériques, chacune capable de fabriquer des produits distincts de Maillard. De plus, la formation de tels ylides fut également démontrée par la réaction de leur groupement 1,3-dipolaire avec des dipolarophiles par cycloaddition. Parallèlement, une étude sur le rôle de l'oxygène dans la réaction Maillard, nous a permis de constater que l'oxygène moléculaire peut influencer la rupture des liens carbone-carbone par la formation et la dégradation du 1,2-dioxetane. Ceci dit, le 1,2-dio
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Daou, Fatma. "Etude expérimentale d'un procédé de dépollution par décharge couronne à barrière diélectrique type pointe(s) - plan : rôle de la simulation numérique et du marquage isotopique." Paris 6, 2002. http://www.theses.fr/2002PA066488.
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Ahmed, Raya. "Measurement of cell proliferation using in vivo stable isotope labelling : application to T-cell homeostasis in young and elderly healthy human subjects." Thesis, St George's, University of London, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.753986.
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Immune homeostasis is achieved through a long-term balance between proliferation and cell death. With ageing of the immune system, immunosenescence, dysfunctional highly differentiated (CD57high CD28|OW) T-cells accumulate. Their presence correlates with functional impairments which may contribute to the increased risk of infectious and age-related diseases in an expanding elderly population. Whether such cells proliferate in vivo is contentious. Answering that question is the focus of this thesis, but methodological aspects of measurement of cell proliferation by either heavy water (D2O) or deuterium-labelled (deuterated) glucose needed to be addressed first as, historically, they appear to give different results. This thesis describes a series of experiments: (i) To identify the source of the disparity between heavy water or deuterated- glucose, using in vitro tissue culture and in vivo murine models, and (ii) To investigate the kinetics of senescent T-cells and T-cell memory precursors (stem cell memory cells, TScm) in healthy young and elderly subjects. In vitro experiments were conducted using Jurkat cells labelled with deuterated-glucose and/or heavy water. In Murine in vivo experiments, mice received liquid feed labelled with either heavy water or deuterated-glucose. Disparities in lymphocyte proliferation rate estimates were the consequence of different normalisation approaches and were resolved when similar normalisation was applied. Eight healthy human subjects (4 young and 4 elderly, all CMV-seropositive) were given heavy water orally for seven weeks. T-cell subsets were sorted by flow cytometry and analysed for DNA deuterium content by gas-chromatography mass-spectrometry to derive modelled proliferation rates. We found that: (i) CD57+ "senescent" cells (CD4 and CD8) retain proliferative capacity, and have relatively short life-spans (high turnover). (ii) Tscm represent a rapidly-dividing subpopulation (iii) Similar patterns were observed in young and elderly These studies demonstrate how isotope-based measurements may be applied to decipher T- cell differentiation and homeostasis.
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Lenoir-Capello, Rachel. "Specific labeling strategies for new developments in liquid state protein NMR." Thesis, Sorbonne université, 2020. https://accesdistant.sorbonne-universite.fr/login?url=http://theses-intra.upmc.fr/modules/resources/download/theses/2020SORUS056.pdf.
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La résonance magnétique nucléaire (RMN) fournit des informations structurelles et dynamiques précieuses à l'échelle atomique, cependant, la faible sensibilité et résolution des signaux empêchent l’étude d'objets moléculaires plus importants. Nous présentons 3 stratégies de marquage isotopique pour différentes expériences RMN des protéines en solution et démontrons leur potentiel pour l'étude structurale des biomolécules. Parmi les stratégies envisagées, 2 utilisent l'expression in vitro pour obtenir des protéines marquées sélectivement sur un groupe chimique et/ou acide aminé dans un environnement perdeutéré. Avec l’utilisation de séquences d'impulsions TROSY, ces échantillons ont permis des gains spectraux importants lorsque ils étaient spécifiquement marqués sur des groupes amide ou sur le méthylène des glycines tout en maintenant un taux de deutération élevé sur les autres fonctions chimiques des protéines. La troisième stratégie de marquage protéique utilise des protocoles in vivo pour des applications RMN innovantes: l'hyperpolarisation de noyaux en solution qui augmente leur sensibilité de plusieurs ordres de grandeur. La durée de vie de cette hyperpolarisation est régie par le temps de relaxation longitudinale des noyaux, qui est réduit pour les protéines à température ambiante. En isolant les noyaux d'intérêt dans un environnement perdeutéré, les interactions dipolaires créées par les protons voisins sont éliminées et les noyaux hyperpolarisés relaxent beaucoup plus lentement. L'hyperpolarisation d'un petit domaine protéique a été entreprise avec succès mais les conditions de dissolution doivent encore être améliorées pour conserver une phase aqueuse homogèneNuclear Magnetic Resonance (NMR) provides valuable structural and dynamic information at the atomic scale, however, the low sensitivity and resolution of signals rapidly preclude investigations of larger molecular objects. We present three isotopic labeling strategies for different protein-solution NMR experiments and demonstrate their potential for the structural study of biomolecules in solution. Among the strategies considered, two are based on the use of in vitro protein expression to obtain selectively labeled proteins of a certain chemical group and/or amino acid in a perdeuterated environment. Perdeuteration is essential for the optimal use of Transverse Relaxation Optimized Spectroscopy pulse sequences. They allowed significant spectral gains when samples were specifically labeled on amide groups or on the methylene of glycines while maintaining a very high rate of deuteration on the other chemical functions of the proteins. The third protein labeling strategy employed is based on in vivo protocols but used in innovative NMR applications: a technique of hyperpolarization of nuclei in solution which increases their sensitivity by several orders of magnitude. The lifetime of this hyperpolarization is governed by the longitudinal relaxation time of nuclei, which are reduced for proteins at room temperature. By isolating the nuclei of interest in a perdeuterated environment, dipolar interactions created by neighboring protons were eliminated and hyperpolarized nuclei relaxed much more slowly. Hyperpolarization of a small protein domain was successfully undertaken at 1K but the dissolution conditions need to be improved in order to preserve a homogeneous aqueous phase
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Roy, Chowdhury Taniya. "Tracking Carbon Flow during Methane Oxidation into Methanotrophs using 13C-PLFA Labeling in Pulsing Freshwater Wetlands." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1339084813.
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Nikolov, Plamen. "Isotope labelling studies on the reactivity of n-alpha and n-epsilon of lysine in the presence of glucose and its degredation products." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114216.
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Isotope labelling technique in conjunction with pyrolysis Gas Chromatography/Mass Spectrometry (Py-GC/MS) was utilized to conduct an in-depth investigation into the formation of lysine specific products in the Maillard reaction. Following the pyrolysis of the lysine/glucose models including their labelled counterparts for 20s at 250°C and extensive data analysis it was concluded that lysine can generate piperidine, a reactive secondary amine capable of undergoing Maillard type interactions. The formation of piperidine has been postulated to follow two pathways depending on whether lysine pyrolysis is conducted in the presence or absence of sugars. In the presence of glucose, lysine similar to asparagine and phenylalanine can undergo carbonyl-assisted decarboxylative-deamination reaction to generate Nε-pent-4-ene-1-amine, which is the counterpart of acrylamide - a known food toxicant. Nε-pent-4-ene-1-amine has been shown to cyclize into piperidine. Specifically labelled precursors such as [15N-α]lysine.2HCl, [15N-ε]lysine.2HCl, [U-13C6]lysine.2HCl, [13C-6]lysine.2HCl and [U-13C6]glucose were used to confirm the potential adducts of Nε-pent-4-ene-1-amine and piperidine in the model systems which lead to the characterization of two piperidine and one Nε-pent-4-ene-1-amine derivatives. Products simultaneously possessing Nε nitrogen atom and five carbon atoms from lysine (C2' to C6') in addition to either 3 or 6 glucose carbon atoms were targeted for this analysis. The mechanism of formation of the two piperidine derivatives involved the chemical activation of piperidine with formaldehyde followed by aldol addition. The reactivity of piperidine was further demonstrated through detection of various pyridine derivatives postulated to be formed after oxidation reactions. During the course of this study it was also observed that in the presence of lysine, the glucose moiety was converted into 5-hydroxymethyl-furfural (HMF) and 5-methylfurfural (MF). Analyses of HMF/lysine and glucose/lysine models using high resolution TOF-MS/MS and Py-GS/MS have indicated the formation of Schiff base adducts of HMF with Nε-pent-4-ene-1-amine and piperidine, respectively. The latter being a secondary amine was shown to undergo further stabilization through a vinylogous Amadori rearrangement (vAR) process. Reaction models consisting of HMF with primary and secondary amino acids such as glycine and proline, further confirmed the observed trend that primary amines generated Schiff base adducts and secondary amines resulted in the formation of covalent adducts through vAR. In the absence of amino acids, HMF was discovered to form a dimer through a newly proposed mechanism. The subsequent degradation of the HMF dimer was shown to generate MF and 2,5-furandicarboxaldehyde (FDA), important sugar-specific furans. Furthermore, HMF was shown to form glycosidic linkages with glucose and undergo chain elongation reactions. In addition, reaction of lysine with sugars other than glucose was also explored using ribose/lysine models. These models led to the discovery of furfurylamine, a ribose-specific reactive intermediate whose furfuryl-pyrrole derivatives have been detected in a number of different foods as aroma compounds. The furfuryl-pyrroles were also detected in the model systems generating furfurylamine such as ribose/lysine and in various roasted coffee beans. The formation mechanism of furfuryl-pyrroles was postulated to involve a double adduct of furfurylamine with 3-deoxyribose which was characterized using isotope labelling techniques.La formation des produits dérivés de la lysine lors de la réaction de Maillard est analysée par l'entremise d'une technique utilisant des traceurs isotopique en combinaison avec la pyrolyse couplée à la chromatographie en phase gazeuse et spectrométrie de masse (Py-CG/SM). En étudiant la pyrolyse de différents modèles de lysine/glucose ainsi que celle de leur traceurs isotopiques pendant 20s à 250°C, il appert que la lysine peut générer de la pipéridine, un aminé secondaire très réactif pouvant aussi participer à des interactions de type Maillard. Deux mécanismes de formation de la pipéridine ont été démontrés, variant selon la présence ou l'absence de sucres lors de la pyrolyse de la lysine. En présence de glucose, tout comme l'asparagine et la phénylalanine, la lysine peut subir une déamination decarboxylative lieé à un groupement carbonyle, afin de générer le Nε-pent-4-en-1-amine, ce produit étant un homologue de l'acrylamide, un élément toxique alimentaire reconnu. Il a été démontré que le produit Nε-pent-4-en-1-amine peut se «cycliser» afin de former la pipéridine. Des précurseurs isotopiquement marqués tels que [15N-α/ε],[U-13C6],[13C-6]lysine.2HCl et [U-13C6]glucose ont été utilisés afin de confirmer les composés d'addition potentiels de Nε-pent-4-en-1-amine et de la pipéridine dans les systèmes modèles permettant la caractérisation de deux dérivés de la pipéridine et un dérivé du Nε-pent-4-en-1-amine. Les produits ciblés lors des analyses possédaient un atome d'azote de type Nε et cinq atomes de carbone provenant de la lysine (C2' à C6') ainsi que 3 ou 6 atomes de carbone provenant du glucose. En bref, le mécanisme de formation des deux dérivés de la pipéridine implique l'activation chimique de la pipéridine avec le formaldéhyde suivi d'une addition de type aldol. La réactivité de la pipéridine fut démontrée davantage lors de la détection de plusieurs dérivés de la pyridine quiont été formés suite à des réactions d'oxydation. De plus, il fut aussi observé au cours de l'étude que la présence de la lysine favorisait la conversion du glucose en 5-hydroxymethylfurfural (HMF) et 5-methylfurfural (MF). Des analyses comparatives de modèles HMF/lysine et glucose/lysine à l'aide d'un TOF-MS/MS à haute résolution et du Py-CG/SM ont indiqué la formation de composés d'addition de base de Schiff du HMF avec le Nε-pent-4-ene-1-amine et la pipéridine. Étant donné que le deuxième composé d'addition est un aminé secondaire, il peut se stabiliser davantage par l'entremise du processus de réarrangement vinylogue d'Amadori (vAR). De plus, la réaction de systèmes modèles combinant le HMF avec des acides aminés primaires et secondaires comme la glycine et la proline confirment qu'il y a une tendance pour que les aminés primaires générant des composés d'addition de base de Schiff et les aminés secondaires mènent à la formation de composés d'addition covalents par le processus de vAR. En l'absence d'acides aminés, cette étude démontre que le HMF forme un dimère par l'entremise d'un nouveau mécanisme proposé. La dégradation subséquente du dimère produit deux furanes spécifiques aux sucres, soit le MF et le 2,5-furandicarboxaldéhyde. Cette étude démontre aussi que le HMF forme des liens glycosidiques avec le glucose et participe à des réactions d'élongation de la chaine. De plus, la réactions de la lysine avec de ribose ont permis de faire la découverte du furfurylamine, un intermédiaire réactif spécifique au ribose produisant plusieurs dérivés de furfurylpyrrole qui ont été détectés en tant que composés aromatiques dans plusieurs aliments. Dans les systèmes modèles produisant du furfurylamine, ces furfurylpyrroles furent aussi détectés tout comme dans des grains de café rôtis. Le mécanisme de formation des furfurylpyrroles proposé implique un double composé d'addition du furfurylamine avec le 3-deoxyribose qui fut caractérisé à l'aide de marqueurs isotopiques.
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Kheir, Beik Louay. "Dynamics of soil organic matter amino acids : a carbon isotope approach." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0098.
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Cette thèse aborde un point clé du couplage entre ces cycles: la dynamique des molécules azotées (AAs) des matières organiques du sol (MOS). Par des expériences d'incubation, nous avons estimé que les flux de biosynthèse des AAs par les micro-organismes du sol lors du processus de décomposition sont de l'ordre de 25% de la biomasse nouvellement formée. Le profil des AAs individuels biosynthétisés de novo est plus dépendant du type de sol que de la nature du substrat. Dans chaque sol, il est très similaire à celui des AAs des MOS. La biodégradation de matériaux végétaux marqués en 13C a révélé la transformation rapide des protéines végétales en matériaux microbiens. Ces résultats montrent que les AAs des MOS sont d'origine microbienne. Nous avons mesuré le renouvellement du C des AAs à long terme dans les horizons de surface de neuf sites présentant des végétations, climats et types de sol variés, en utilisant la technique de traçage par les abondances naturelles en 13C. L'âge moyen du carbone des AAs varie de 50 à 200 ans. Un modèle simple permet de discuter les hypothèses du recyclage des AAs des MOS par les micro-organismes. Les rapports isotopiques stables des AAs individuels ont été mesurés par chromatographie en phase gazeuse couplée à la spectrométrie de masse isotopique. À cette fin, nous avons développé une méthode d'étalonnage générique pour la détermination du rapport isotopique des composés spécifiques, par analyse de cultures microbiennes uniformément marquées. Au-delà des résultats présentés, l'étude apporte un large ensemble de données des AAs et examine les variations de l'abondance naturelle en 13C entre les AAs individuelsWe analyzed the coupled dynamics of C and N in Soil Organic Matter (SOM) through the dynamics of N-containing soil organic compounds (amino acids (AAs)) by tracing their carbon atoms. Stable isotope ratios of individual amino acids were measured by gas chromatography coupled with isotope ratio mass spectrometry. For this purpose, we developed a generic calibration method for compound-specific stable isotope ratio analysis, based on the analysis of uniformly labelled microbial cultures. We quantified the biosynthesis of AAs associated with the biodegradation process in four contrasted topsoils through short-term incubation experiments of 13C-labelled substrates. Amino acids-C accounts for ca. 25% of the newly-formed microbial biomass-C. The composition of the de novo biosynthesized individual amino acids was dependent on the soil type, and in each soil was similar to that of SOM amino acids. Biodegradation of 13C-labelled plant materials revealed the rapid conversion of plant proteins into microbial materials. These results together demonstrate that SOM amino acids are of microbial origin. We measured the dynamics of amino acids-C on the long term (decades to centuries) in nine sites using the natural 13C-labelling technique. On average, the age of AAs was equal or slightly inferior to that of bulk soil organic carbon, with mean ages ranging from 50 to 200 years. We built a conceptual model of AAs dynamics to discuss various hypotheses of AAs stabilization. Beyond these perspectives on C and N coupling in soil processes, the overall study brings a broad dataset of amino acids, as well as discuses variations of 13C natural abundance (δ13C) in-between individual amino acids
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Flodell, Sara. "Structure and Dynamics of the Hepatitis B Virus Encapsidation Signal Revealed by NMR Spectroscopy." Doctoral thesis, Umeå : Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-316.
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Zieger, Sarah Lorain Janice [Verfasser], Stefan [Akademischer Betreuer] [Gutachter] Scheu, and Mark [Gutachter] Maraun. "Trophic structure of soil animal food webs of deciduous forests as analyzed by stable isotope labeling / Sarah Lorain Janice Zieger ; Gutachter: Stefan Scheu, Mark Maraun ; Betreuer: Stefan Scheu." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://d-nb.info/1117219585/34.
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Campos, Melo Raúl Iván. "Specific isotope labeling of transthyretin for nuclear magnetic resonance and mass spectrometry studies — Marcaje isotópico específico de transtiretina para estudios en resonancia magnética nuclear y espectrometría de masas." Tesis, Universidad de Chile, 2010. http://repositorio.uchile.cl/handle/2250/105337.
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Memoria para optar al título de BioquímicoLas proteínas son moléculas versátiles que juegan una variedad de roles en la mantención del cuerpo humano, tal como el transporte de nutrientes. La transtiretina (TTR) es una proteína homotetramérica de 55 kDa que se encuentra en el plasma humano y en el cerebro, la cual es responsable del transporte de retinol (vitamina A) y T4 (tiroxina). Sin embargo, probablemente no es necesaria para la vida, puesto que ratones knock out tienen un desarrollo fetal y longevidad normales. La transtiretina, al igual que otras 25 proteínas humanas, ha sido asociada a la deposición de agregados amiloides. Algunas investigaciones anteriores han mostrado que las mutaciones incrementan considerablemente la tendencia de la proteína a formar agregados. A pesar de esto, la proteína wild type (wt-TTR) también muestra la capacidad para agregarse. Esto genera la enfermedad llamada amiloidosis sistémica senil, la cual afecta a 20% de las personas sobre los 80 años de edad. Es sabido que la asociación entre subunidades monoméricas gatilla la enfermedad a través de la disociación del tetrámero, puesto que la estabilización de la estructura cuaternaria suprime la formación de agregados. Recientemente nuestro grupo descubrió que la wt-TTR purificada a 4°C es tan tóxica como los agregados de bajo peso molecular más tóxicos para células de neuroblastoma humanas, sin embargo no lo es cuando se purifica a temperatura ambiente (22°C; RT). Para nuestro asombro, esta actividad citotóxica era inducida por la proteína en su forma tetramérica. Estudios biofísicos revelaron un ligero reordenamiento de la estructura terciaria de la proteína. Es de especial interés si este pequeño cambio estructural puede conducir al descubrimiento de nuevos epítopes citotóxicos. En este trabajo exploramos la estructura proteica en más detalle mediante métodos biofísicos para confirmar la existencia de este cambio conformacional e intentar encontrar dónde específicamente ocurre en la estructura de la proteína. Expresamos wt-TTR recombinante en Escherichia coli y la purificamos mediante cromatografía de intercambio iónico y cromatografía por exclusión de tamaño a 4°C y a RT. Estudiamos la fluorescencia intrínseca de triptófano de la proteína tetramérica para confirmar que wt-TTR purificada y almacenada en frío tiene una menor barrera de activación para la disociación a monómeros comparado con la proteína purificada y almacenada a RT. Esto fue llevado a cabo mediante el desplegamiento de la estructura usando una alta concentración de urea (6,0 M) como agente caotrópico. También realizamos un experimento de intercambio isotópico de hidrógeno y deuterio con espectrometría de masas (HXMS) con el objetivo de estudiar el intercambio local de hidrógenos en los grupos amida del esqueleto polipeptídico, los cuales permiten medir el aumento/disminución de la protección de diferentes segmentos de la proteína en cuanto al intercambio, dependiendo de la temperatura. Por último, un experimento de resonancia magnética nuclear (NMR) llamado correlación heteronuclear de cuanto sencillo (HSQC), poniendo a prueba wt-TTR marcada con el isótopo 15N cultivada en un medio M9 con agua deuterada al ~99%, nos permitió seguir el espectro de acoplamiento-J bidimensional entre 15N-1H de la proteína. El análisis de los espectros bidimensionales obtenidos a ambas temperaturas reveló que algunos residuos experimentan un claro cambio en su desplazamiento químico, indicando que efectivamente ocurren cambios en la estructura terciaria de la proteína. En conclusión, determinamos que wt-TTR sufre un cambio conformacional cuando es purificada a 4°C, a pesar de que la localización específica de éste en la estructura proteica continúa siendo desconocida
Proteins are versatile molecules that play a variety of roles in maintaining the human body, e.g. transport of nutrients. Transthyretin (TTR) is a 55 kDa homotetrameric protein found in human plasma and in the brain, responsible for the transport of retinol (vitamin A) and T4 (thyroxine). This protein is probably not essential for life, since TTR knockout mice have normal fetal development and lifespan. TTR, like 25 other human proteins, has been associated to the deposition of amyloid aggregates. Previous research has shown that mutations considerably increase the propensity of the protein to form aggregates. However, the wild type protein (wt-TTR) also exhibits this ability to aggregate, giving rise to the senile systemic amyloidosis disease that affects 20% people over 80 years of age. It is well accepted at the moment that self association of monomeric subunits triggers the disease through tetramer dissociation, since stabilization of the quaternary structure suppresses aggregate formation. Recently, our group discovered that wt-TTR purified at 4°C is just as toxic to human neuroblastoma cells as the most toxic small molecular weight aggregates, while when purified at room temperature (22°C; RT) it is not. Strikingly, this cytotoxicity was exhibited by the protein as a tetramer. Biophysical studies revealed a slight rearrangement of the tertiary structure of the protein. It is of high interest whether this minor structural change can lead to the discovery of new cytotoxic epitopes. Herein, we explored the protein structure in more detail by biophysical methods to confirm the existence of this conformational change and attempt to resolve where it specifically takes place in the protein structure. Recombinant wt-TTR was expressed in Escherichia coli and purified by ion exchange chromatography and size exclusion chromatography at 4°C and RT. We studied the tetramer’s intrinsic tryptophan fluorescence to confirm that cold purified and stored wt-TTR has a lower activation barrier for dissociation into monomers compared to the protein purified and stored at RT. This was performed by submitting the protein to unfolding using a high concentration of urea (6 M) as the chaotropic agent. We also carried out a hydrogen/deuterium isotope exchange mass spectrometry (HXMS) experiment in order to study the local exchange of backbone amide hydrogens, which serve as probes for increased/decreased protection of different segments of the protein towards exchange, depending on the temperature. At last, nuclear magnetic resonance spectroscopy using the heteronuclear single quantum coherence (HSQC) experiment probing a 15N isotope labeled wt-TTR grown in ~99% deuterated water M9 medium, allowed us to track the two dimensional 15N-1H J-coupling spectrum of the protein. Analysis of 2D spectra run at both temperatures revealed that some residues experience a clear change in chemical shift, indicating that changes in tertiary structure occurred. In conclusion, we determined that wt-TTR undergoes a conformational rearrangement when purified and stored at 4°C, although the exact location of this conformational change in the protein structure remains unclear
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Zhou, Wenxuan. "Stoichiometry and Crystal Structure of Poly (Lactic Acid) (PLA) Stereocomplex (SC) in Cold-crystallization and Solution-grown Crystals as Studied by Solid-state NMR and 13C Isotope Labeling." University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron1522239647112751.
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Bayoumi, Soad Abdel Latief Hassan. "Molecular genetic analysis of secondary metabolite biosynthesis in cassava as an economic and nutritious plant." Thesis, University of Bath, 2008. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512260.
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Cassava (Manihot esculenta Crantz Family Euphorbiaceae) is an important tropical food crop. However, harvested cassava roots have a shelf-life of only days due to post-harvest physiological deterioration (PPD). Within 1-3 days of harvesting, the roots show blue-black vascular streaking and are unpalatable. PPD includes altered gene expression and the accumulation of hydroxycoumarin secondary metabolites, e.g. scopoletin and esculetin, and their respective glucosides scopolin and esculin. In this research several important aspects of the biosynthesis of these phytochemically important hydroxycoumarins were resolved. Stable isotopically labelled intermediates on the postulated biosynthetic pathways of scopoletin were fed to cassava cubes and PPD was allowed to occur. Ethanolic extracts of these deteriorated roots were separated (HPLC) and analysed (HRESI-MS). Incorporation (in both scopoletin and scopolin) of only 3 deuterons from E-cinnamic-2,3,2',3',4',5',6'-d7 and E-cinnamic-3,2',3',4',5',6'-d6 is strong support that the E-Zisomerisation step is enzymatic and not photochemical. There are three hypothetical pathways for the biosynthesis of scopoletin via: 2',4'-dihydroxycinnamate, caffeate, or ferulate. High incorporation of label from p-coumaric-2-13C, caffeic-2-13C and ferulic-2-13C acids was observed into labelled scopoletin and scopolin while there was only a small incorporation from 18O-umbelliferone and 18O-esculetin. We conclude that the major biosynthetic pathway to scopoletin and scopolin is via ferulic acid. C18O2-enrichment of E-cinnamic and ferulic acids and feeding gave scopoletin containing only one 18O-labelled oxygen atom. Therefore the lactonisation step is through o-hydroxylation and not via a postulated spirolactone-dienone intermediate. These results were confirmed by feeding experiments in an atmosphere of 18O2-air which showed that the major isotopic peak was 18O3-enriched scopoletin. Three glucosyltransferases were isolated and identified from a cassava PPDrelated cDNA library. These genes are expressed in the cassava storage root during PPD and they are also expressed in the fresh root. While one of these glucosyltransferases was novel, two had previously been isolated from cassava cotyledons.
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COSTA, RENATA F. "Desenvolvimento de métodos de purificação do sup(67)Ge e sup(68)Ge para a marcação de biomoléculas." reponame:Repositório Institucional do IPEN, 2012. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10097.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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MASSICANO, ADRIANA V. F. "Desenvolvimento farmacotécnico de um radioimunoconjugado para terapia de linfoma não-Hodgkin." reponame:Repositório Institucional do IPEN, 2016. http://repositorio.ipen.br:8080/xmlui/handle/123456789/26398.
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Tese (Doutorado em Tecnologia Nuclear)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Fredriksson, Anna. "Tracer development and PET studies : labeled proinsulin C-peptide and an EGFR-TK inhibitor /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-191-8.
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Haynes, Christopher Allen. "Development of an assay for fatty acyl-CoAs using liquid chromatography-electrospray ionization-tandem mass spectrometry and its application to the stable isotope labeling and quantitation of sphingolipid metabolism." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/37171.
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Fatty acyl-Coenzyme As are metabolites of lipid anabolism and catabolism. A method was developed for their quantitation in extracts of cultured mammalian cells using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Palmitoyl-CoA (C16:0-CoA) is utilized for de novo sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. After reduction to form sphinganine (Sa), dihydroceramide synthase (CerS) can N-acylate the Sa using a second fatty acyl-CoA molecule, forming dihydroceramide (DHCer). The CerS enzyme family utilizes different acyl chain lengths of fatty acyl-CoAs in an isoform-specific manner, resulting in DHCer with N-acyl chains ranging from C16 to C26 [and even longer] in mammalian tissues. DHCer is trans-4,5-desaturated to yield ceramide, which is further metabolized by the addition of moieties at the 1-O-position, forming sphingomyelin (SM) and ceramide monohexose (CMH).
The rates of fatty acyl-CoA and sphingolipid biosynthesis were determined using stable isotope-labeling and LC-ESI-MS/MS analysis of the analyte isotopologues and isotopomers. Isotopic labeling of palmitoyl-CoA with [U-13C]-palmitate in HEK293 and RAW264.7 cells was robust and rapid (~ 60% labeling of the metabolite pool in 3 hr). Isotopic labeling of sphingolipids indicated utilization of [M + 16]-palmitoyl-CoA by SPT and CerS isoforms in both cell types. Metabolic flux modeling was applied to the data for [U-13C]-palmitate activation to [M + 16]-palmitoyl-CoA and its subsequent utilization in de novo sphingolipid biosynthesis, and this analysis indicated rapid turn-over rates for palmitoyl-CoA and ceramide in both cell types.
Palmitate treatment of cultured cells alters their metabolic status and gene expression, therefore labeling of palmitoyl-CoA by treatment with [1-13C]-acetate was employed. A distribution of mass-shifted palmitoyl-CoA species (isotopologues) is observed based on the number of incorporations of [1-13C]-acetate during de novo biosynthesis, requiring computational analysis to derive two parameters: the isotopic enrichment of the precursor pool, and the fraction of palmitoyl-CoA that was biosynthesized during the experiment. Previous reports by others describe mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) for this purpose, and both calculation approaches indicated concurrent results.
In summary, the quantitation of fatty acyl-CoAs and their isotopic enrichment during stable isotope-labeling studies of lipid metabolism can provide data that significantly change the interpretation of analyte quantitation in these experiments, as demonstrated here for investigations of de novo sphingolipid biosynthesis.