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Дисертації з теми "Méthode SELEX"
Wang, Ji. "Développement d'une méthode SELEX pour l'identification de ribozymes pour l'aminoacylation et analyse d’ARN aminoacylés dans le transcriptome d'Escherichia coli." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS269/document.
Повний текст джерелаRibozymes are natural or in vitro selected RNA molecules possessing a catalytic activity. Artificial ribozymes have been extensively investigated by in vitro SELEX experiments, and characterized by kinetic assays. Ribozymes are involved in RNA cleavage, ligation, capping, polymerization, phosphorylation and acyl activation. Because it is required for translation, RNA aminoacylation plays an important role in the evolution from the late RNA world to the modern DNA and protein world, and is central to the genetic code. Several ribozymes catalyzing amino acid transfer from various activating groups have already been selected and characterized in the past two decades, documenting the possibility of tRNA aminoacylation in the absence of aminoacyl tRNA synthetase. With a newly designed SELEX protocol based on periodate oxydation, the aim of our investigation is to uncover small ribozymes of the order of 20 nucleotides that could catalyze both amino acid activation and transesterification. Although molecules catalyzing either reaction have been identified, no existing ribozyme could use free amino acids and activating cofactor(s) as substrates for 3' esterification in a single reactional context. The selection of active molecules in a SELEX procedure requires the presence of constant tracks on both ends of the sequences constituting the initial random pools. These tracks are required for PCR amplification, but they impose significant burden to the identification of ribozymes because they can prevent any activity through structural inhibition. We present an optimized protocol that significantly minimizes the size of these constant tracks. At the same time, our newly design protocol is very specific for the selection of 3'-end aminoacylated RNA. Working with this protocol, we performed 6 to 7 cycles of selection with different pools, and observed an enrichement with specific sequences. Although some experiments performed with entire pools did reveal a possible activity, no activity could be so far confirmed with specific sequences. A similar protocol was also applied in a parallel study to identify aminoacylated RNA from total RNA in Escherichia coli. In this other approach, our goal is to possibly identify new classes of aminoacylated RNA while using the deep sequencing technology. Using tRNA to validate our protocol, we realized that a standard RNAseq procedure could not work due to the presence of modified bases. We established a new method for bank preparation to identify any sequence aminoacylated at the 3' end. Ultimately, this new approach will allow us to study the level of aminoacylation of any sequence present in total RNA
Ennehdi, Atef. "Étude du réseau d'interactions tertiaires au sein du site catalytique du ribozyme delta par méthode de SELEX." Mémoire, Université de Sherbrooke, 2004. http://savoirs.usherbrooke.ca/handle/11143/3349.
Повний текст джерелаGong, Meihua. "Developing a new tool to purify methylated peptides from bacteria in order to study bacterial mechanosensing." Electronic Thesis or Diss., Compiègne, 2023. http://www.theses.fr/2023COMP2749.
Повний текст джерелаFlagella have been described as an important virulence factor for initial attachment to the host or to the surface of hospital equipment. However, how bacteria use their flagella to switch from a floating (planktonic) state to an immediately attached (sessile) state remains an open question. This transition involves flagellar mechanosensing or surface-sensing. It has been illustrated that lysine methylations in S. Typhimurium flagella facilitate bacterial adhesion to the surface or receptor via hydrophobicity. However, the study of the entire methylome, including non-histone methylation, remains a major challenge because of the lack of efficient methyl protein/peptide enrichment techniques. Here, to investigate protein methylation and its mechanisms in S. Typhimurium and its mutants, we applied two complementary strategies for methyl protein enrichment: aptamer-based enrichment technology and high pH SCXtips separation strategy. The specific DNA aptamers were obtained after performing several rounds of positive selection and one round of negative selection from a random oligonucleotide library through the FluMag-SELEX procedure. The selected ssDNA pools were sequenced and 10%, 11%, and 33% of redundant sequences for MML, DML, and TML, respectively have been observed. The highest redundancy was observed with oligonucleotides directed against TML. The interaction study of this aptamer with its target was then performed by the methods like ITC, PCR, and bead-based binding assays followed by mass spectrometry titration. The specificity was confirmed while the affinity was determined to be KD=2.48±0.14 mM. Then, the selected aptamer has been used as an enrichment tool based on the aptamer-bound beads method, in order to isolate methylated proteins or peptides on various cell lines. After the validation of our approach using a positive control (HEK293 cells), we identified 19 lysine methylation sites on 5 proteins from Salmonella Preliminary results from proteomic analysis confirmed that the selected aptamer was indeed able to distinguish and enrich methyllysine-containing proteins from S. Typhimurium strains and human cell lines. In parallel with this, the high pH SCXtips technology was performed as a complementary separation strategy when cultivating the strains in the hM-SILAC medium. Using this method, 23 unique methylation sites in ΔmetE, 51 unique methylation sites in ΔmetEΔmotAB, and 18 unique sites in ΔmetEΔfliB on 19 methylated proteins were identified. By comparing the differences in protein methylation, our results suggest that when bacteria lack motility, lysine methylations in bacterial proteins seem to be upregulated. Besides being methylated by the methylase FliB, a large number of methylation events could be regulated by other methylases
Ungeheuer, Sarah. "Conception d'une technique d'évolution moléculaire couplée à la chimie bioorthogonale pour l'identification de ligands." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL042.
Повний текст джерелаThe thesis project involves designing a new method for the molecular evolution of nucleic acids. Coupling the SELEX method with bioorthogonal chemistry will make it possible to identify ligands with chemical modifications. Increasing the chemical diversity of nucleic acids in this way will provide the opportunity to obtain high-affinity ligands that can be used for diagnosis and therapy.Proof of concept for this technique at the interface between biology and chemistry first required the synthesis of compounds that increase the molecular diversity of oligonucleotides and therefore potentially their affinity for the target. Nucleic acids are then functionalized by these compounds using click chemistry. The enzymatic synthesis and amplification steps using chemically modified oligonucleotides were optimized by high-throughput sequencing analysis. Our method was validated by selecting ligands against Streptavidin, whose interaction require chemical modifications. The affinity of the ligands was measured using different technics (Fluorescence and Bio-Layer Interferometry)
Loussouarn, Claire. "Sélection et caractérisation d’aptamères oligonucléotidiques régulateurs de la protéine STAT5B, impliquée dans les leucémies." Thesis, Compiègne, 2014. http://www.theses.fr/2014COMP2137.
Повний текст джерелаLeukemias are due to abnormal cell proliferation, which is the result of intracellular over-expression or excessive activation of protein due to oncogenic event. Still today, it is necessary to find new therapeutic molecules, which specifically target these proteins. STAT5, via the JAK/STAT signaling pathway, controls fundamental cellular processes, including .cell survival, proliferation and differentiation. To struggle against tumorigenesis, JAK/STAT signaling pathway has to be inhibited. The aim of this project is to target specifically STAT5 factors to restore healthy signal transduction. We generated aptamers by an iterative in vitro selection. Aptamers are short-structured single strand DNAs or RNAs that bind with high affinity and specificity to their target. Once STAT5B recombinant proteins are produced, they are subjected to SELEX process. The number of rounds depends on various parameters. After seven rounds, two sequences are retrieved. The specificity and affinity of these aptamers are assessed by fluorescent immunoassays. Binding affinity and kinetics of interaction are characterized by SPR. Aptamer anti proliferative effects are determined by evaluation of the growth of cells depending on STAT5. Finally, we developed several .assays aiming at understanding the mechanism of an aptamer action on STAT5B such as phosphorylation measurement and EMSA. Aptamers are now emerging therapeutic tools; they exhibit significant advantages relative to protein therapeutics
Rhouati, Amina. "Développement de méthodes bioanalytiques à base d’aptamères pour la détermination de l’ochratoxine A dans les denrees alimentaires." Perpignan, 2013. http://www.theses.fr/2013PERP0003.
Повний текст джерелаOchratoxin A (OTA), an isoucoumarinic mycotoxin produced by the genra Aspergillus and Penicillium, is a common contaminant of several foodstuffs. Even in trace amounts, OTA has adverse affects on human and animal health: nephrotoxic, teratogenic, neurotoxic, immunosuppressive and it was considered by the international Agency of Research on Cancer (IARC) as a potential carcinogenic agent (group 2B). In order to meet food safety concerns and official legislated regulations, analytical methods, mainly based on chromatographic techniques, have been reported for OTA detection. Despite their effectiveness, these techniques remain expensive and require qualified skilled personnel, alternatives strategies are emerging and novel technologies based on biochemical methods. These techniques are based on the interaction target-recognition element. We use in this work, a new class of biomolecules called aptamers. They are single or double stranded DNA or RNA, selected by their ability to recognise a target, by a combinatorial method of selection in vitro called SELEX (Systematic Evolution of Ligands by EXponantial enrichment). The aim of this work is the development of new methods based on aptamers for OTA determination in foodstuffs. We will develop two methods, one method for extraction and another one for detection. The first one consists in an aptamer-based affinity column or oligosorbent. This device of extraction was evaluated in term of retention, specificity, selectivity and regeneration. While the second is based on an aptasensor developed in a fully automated system. All the optimizations and manipulations were performed using automated sequences. After optimizations, these two methods are applied for OTA determination in real beer samples. The obtained limits of detection are much lower than the maximum tolerated levels of OTA in set by the European commission. Also, the high recovery yields obtained show the good applicability of the developed oligosorbent and aptasensor in real sample without matrices effect
Daufouy, Guillaume. "Sélection d'aptamères pour la détection de spores de bactéries d'altération alimentaire." Electronic Thesis or Diss., Perpignan, 2023. http://www.theses.fr/2023PERP0004.
Повний текст джерелаDue to their ability to form extremely heat-resistant spores, some non-pathogenic thermophilic bacteria are responsible for many spoilages of canned food products, resulting in high economic losses and food waste. Two species, Geobacillus stearothermophilus and Moorella thermoacetica, are mainly responsible for these contaminations and represent more than 65% of the cases of non-stability detected. The existing cultural and molecular methods have drawbacks in terms of analysis time and applicability in the field, which limits their use for the detection of the spores of these bacteria.This thesis work forms part of the Spores-Quantum project, which aims developing real-time analysis systems for the direct detection and quantification of G. stearothermophilus and M. thermoacetica spores in complex microbial environments. Supported by the Carnot Qualiment programme, it is the result of a collaboration between the Centre Technique de la Conservation des Produits Agricoles (CTCPA), an industrial technical reference center for French canning professionals, and the Biosensors, Analyses, Environment laboratory (BAE-LBBM, UAR 3579) of the University of Perpignan Via Domitia. More precisely, this thesis focused on the development of aptamers as specific recognition elements of the two target species. A Spore-SELEX protocol associated with emulsion PCR and high-throughput sequencing was successfully performed only for the selection of G. stearothermophilus spore specific aptamers. Then, 18 successive SELEX cycles, including 4 counter-selections cycles against 12 bacterial species commonly found along the production lines. Candidate sequences from cycle 18 revealed 70 over-represented sequences with copy numbers exceeding 0.15% of the total sequences obtained. In this group, the G001 aptamer showed a very high enrichment with a relative abundance higher than 18 %. The affinity and specificity for G. stearothermophilus spores of the 13 most abundant candidates at cycle 18 were confirmed by a PCR assay based on aptamer-spore complex formation and a filtration step. Other methods for determining aptamer/spore affinity were also explored and are presented in this manuscript. The obtained aptamers will be used in the near future for developing biosensors dedicated to the detection of G. stearothermophilus spores