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Wang, Tianjiao. "Function and dynamics of aptamers a case study on the malachite green aptamer /." [Ames, Iowa : Iowa State University], 2008.
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Savonnet, Maud. "Développement d'une méthode de détection innovante appliquée au diagnostic terrain des pathologies cardiaques." Thesis, Université Grenoble Alpes, 2020. http://www.theses.fr/2020GRALY061.
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Diagnostiquer au plus tôt les pathologies cardiaques est aujourd’hui un enjeur majeur dans le monde de la santé. En effet, la rapidité du diagnostic de l’infarctus du myocarde a un impact non seulement sur la santé du patient, mais aussi sur la gestion des services hospitaliers d’urgence. Ainsi, l’utilisation de dispositifs de diagnostic au chevet du patient est une solution pertinente pour répondre efficacement à un tel enjeu. C’est pour cette raison que l’on assiste aujourd’hui à une croissance sur le marché du nombre de systèmes Point-Of-Care dédiés au diagnostic terrain des pathologies cardiaques. Ces dispositifs disposent néanmoins d’un certain nombre d’inconvénients qu’il s’agit de surmonter.C’est dans ce contexte que s’inscrit ce travail de thèse. Aussi, un travail de recherche et de mise au point d’une méthode de détection des biomarqueurs cardiaques innovante a été mené. Cette méthode a pour objectif la détection de tout type d’analyte dans un milieu complexe avec une bonne sensibilité permise par l’amplification biomoléculaire mise en œuvre. Cette méthode générique est basée sur l’amplification LAMP d’une sonde oligonucléotidique. Elle emploie des sondes aptamères, spécifiques de la cible à détecter, et qui ont été validées par imagerie de résonance des plasmons de surface. Cette méthode a été mise en œuvre de manière pertinente sur différents modèles et appliquée à la détection d’un biomarqueur cardiaque d’intérêt, la troponine I. L’intégration de cette méthode dans un dispositif microfluidique portable a finalement été abordée dans la perspective d’une utilisation future pour le diagnostic de terrainToday, early diagnosis of cardiac pathologies is a major issue in healthcare world. Indeed, the speed of myocardial infarction diagnosis has an impact not only on the patient's health, but also on the management of emergency hospital services. The use of diagnostic devices at the patient’s bedside is a relevant solution to overcome effectively such a challenge. Consequently, the number of Point-Of-Care systems dedicated to the diagnosis of cardiac pathologies is growing. However, these devices have some disadvantages that need to be overcome.This thesis work has been conducted in this context. Research and development of an innovative method for the detection of cardiac biomarkers has been carried out. The objective of this method is the detection of any type of analyte in a complex medium with a good sensitivity allowed by the biomolecular amplification used. This generic method is based on the LAMP amplification of an oligonucleotide probe. It uses aptamer probes, specific to the target to be detected, which have been validated by surface plasmon resonance imaging. This method has been implemented in a relevant manner on different models and applied to the detection of a cardiac biomarker of interest, troponin I. The integration of this method in a portable microfluidic device was finally addressed for future use in field diagnostics
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Dibenedetto, Silvia. "Direct activation of endogenous Calcineurin A : biological impact of selective peptide aptamers." Phd thesis, Ecole normale supérieure de lyon - ENS LYON, 2011. http://tel.archives-ouvertes.fr/tel-00757018.
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Therapeutic approaches leading to the stimulation of regeneration, and/or inhibition of degeneration processes in neuromuscular disorders are believed to offer valid therapeutic strategies that would preserve muscle tone and contribute to the quality of life while lengthening patient life span. Activation of CalcineurinA (CnA), a threonine-serine phosphatase, controls gene regulatory programs in skeletal muscle by stimulating slow muscle fiber (type I) gene expression. This phosphatase has been also identified as a key mediator in the hypertrophic response and in skeletal muscle regeneration. Activation of CnA is, therefore, considered as a potentially interesting means of stimulating muscle regeneration in myopathies. We have identified a peptide aptamer that activates CnA in vitro, in cells and in vivo. In a mouse model for denervation-induced muscle atrophy, CnA-activating peptide aptamers show significant positive impact. This is reflected in larger overall muscle cross-sectional surface area due to an increased number of fibers and larger individual fiber surface area. Insight into the biological mechanism is afforded by observation of increased levels of nuclear NFAT transcription factor in these fibers, in agreement with peptide aptamer-mediated activation of CnA. Furthermore, a significant increase in central nuclei, characteristic of the presence of new fibers, is observed in muscles treated with the peptide aptamers specifically activating CnA. Identification of the specific binding site of the peptide aptamer on CnA was achieved using several truncations of the phosphatase, offering insight into the molecular mechanism of action. Together, these studies offer the first proof that direct activation of endogenous CnA has a measureable impact on cellular responses resulting in stimulation of muscle regeneration and enhancement of pathophysiological state in selected animal models.
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Kimura, Mari, and 木村摩利. "Towards intracellular aptamers: delivery of anti-SCV helicase aptamers and development of aptamers againstSATB1." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48079893.
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Aptamers are small RNAs or DNAs that specifically bind to targets through complementary three-dimensional structure with high affinity. Aptamers are screened by an in vitro process called SELEX (systematic evolution of ligands by exponential enrichment) against a variety of targets, including small organic and inorganic molecules, cofactors, peptides, proteins and even whole cells, and aptamers hold great potential as diagnostic tools or as therapeutics. Aptamers are alternatives to antibodies with a variety of potential advantages. However, development of aptamers against intracellular targets is limited by delivery methods. To develop an intracellularly acting aptamer, we aimed to 1) establish an aptamer intracellular delivery system; 2) clone, express and purify the intracellular target SATB1 for aptamer selection; and 3) select an intracellularly acting aptamer against SATB1 by SELEX.
An efficient delivery for the anti-SCV helicase aptamer was achieved using the pDNA transfection reagent Lipofectamine2000, whereby the aptamer was delivered exclusively to the nucleus. We also tested and improved methods to study aptamer-liposome complex formation. Expression of the SCV helicase in the cytoplasm could not alter the aptamer location within cell, suggesting that aptamer modification such as attachment of a redirecting signal or conjugation to a polymer would be required for cytoplasmic targeting. In this thesis we switched to a nuclear target, SATB1, to develop a nuclear intracellularly acting aptamer.
SATB1 is a gene regulator found in the nucleus. Upon activation, SATB1 binds to the nuclear matrix and targets the chromosome via the MAR binding domain to regulate histone modification and nucleosome positioning over a long distance. A recent report demonstrated SATB1’s role in breast cancer metastasis, therefore development of aptamers against this protein has great diagnostic and therapeutic potential. We successfully cloned full length SATB1. The full length protein could not be expressed, however the MAR binding domain was successfully expressed with 6xHis tag and purified using a His trap column. ITC analysis with BUR sequence showed proper folding and selectivity of the MAR binding domain. DNA aptamers were selected by SELEX against the MAR binding domain of SATB1. Selection was successful and a highly conserved family of aptamers was observed. Sequence analysis and alignment revealed the presence of many conserved motifs that involve many A and T in a similar manner to the BUR sequence and dsDNA previously found to have high affinity towards the MAR binding domain.
Altogether, we have taken a step closer towards the development of an intracellularly acting SATB1 aptamer. Future efforts involving affinity determination, application of the established delivery method and in vitro study of inhibitory mechanism will be further steps towards intracellular aptamers for cancer diagnosis or therapeutics.published_or_final_version
Biochemistry
Master
Master of Philosophy
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Brothier, Fabien. "Développement d'outils bioanalytiques miniaturisés : greffage de biomolécules sur monolithes en capillaire couplés à la nanochromatographie pour l'analyse d'échantillons complexes." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066299/document.
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L’analyse de traces dans des matrices complexes (environnementales, alimentaires ou biologiques) requiert très souvent une étape de purification et de préconcentration avant une analyse via des méthodes chromatographiques. Dans cette optique, des supports d’extraction basés sur des mécanismes de reconnaissance moléculaire ont été développés et appliqués à l’extraction de composés cibles rendant ainsi l’analyse plus sensible et plus fiable. Ces supports sélectifs peuvent entre autres résulter de l’immobilisation de biomolécules tels que les anticorps ou les aptamères (i.e. des oligonucléotides présentant une séquence capable de se lier spécifiquement à une molécule). Cette étape de traitement de l’échantillon est particulièrement nécessaire lorsqu’il s’agit de développer des systèmes séparatifs miniaturisés, tels que les microsystèmes séparatifs sur puce, du fait de la diminution de la résolution qui résulte de l’utilisation d’un canal de séparation de faible longueur. Dans ce contexte, ce projet de recherche a consisté à développer des systèmes bioanalytiques miniaturisés pour l’analyse de petites molécules ou protéines dans des échantillons complexes. Pour développer ces systèmes, la synthèse d’un monolithe hybride organique-inorganique in situ dans des capillaires de 100 µm d.i. a, dans un premier temps, été optimisée via une approche sol-gel puis caractérisée en termes de répétabilité. Dans une deuxième partie, deux toxines modèles de faible poids moléculaire ont été choisies : la microcystine-LR (MC-LR) et l’ochratoxine A (OTA). Des anticorps monoclonaux et des aptamères, spécifiques de l’une et l’autre des toxines ont ensuite été greffés sur des monolithes en capillaire. Les immuno- et oligoadsorbants miniaturisés obtenus (respectivement mIS et mOS) ont été couplés en ligne avec la nanoLC. La rétention spécifique des toxines cibles sur les mIS et mOS a été démontrée dans l’eau pure. La répétabilité de la synthèse et du greffage a été évaluée et la capacité de chacun des supports miniaturisés a été déterminée. Enfin, mIS et mOS ont été appliqués avec succès à l’extraction sélective de la MC-LR et de l’OTA à partir d’extraits de cultures de cyanobactéries, d’eaux environnementales ainsi que d’échantillons de bière dopés. Dans un troisième temps, de façon à transposer les outils sélectifs développés à l’analyse de protéines, des microréacteurs enzymatiques (IMER) ont été préparés par greffage de deux enzymes protéolytiques (pepsine et trypsine) sur des monolithes. Ces outils ont ensuite été couplés avec la nanoLC-MS² pour l’analyse d’une protéine modèle, le cytochrome C. Les rendements de digestion sur IMER se sont avérés présenter une bonne répétabilité. Toutefois, l’efficacité de la digestion sur les IMER à base de pepsine reste à ce jour insuffisante et nécessite de réadapter la procédure de greffage et/ou de digestionThe analysis of ultra-traces from complex matrices (environmental, foodstuff or biological) often requires a step of purification and preconcentration before their analysis by chromatographic separation methods. Therefore, extraction sorbents based on a molecular recognition mechanism can be developed and used for the selective extraction of target molecules thus rendering their quantitative analysis in complex samples more reliable and sensitive. These extraction sorbents may result, among others, from the immobilization of biomolecules such as antibodies and aptamers (i.e. oligonucleotides whose sequence is specific for a target molecule). This selective sample pretreatment step is particularly necessary when developing miniaturized devices such as separative microsystems on chip because of the decrease of the resolution that results from the use of a shorter length separation channel. In this context, the aim of our study was to develop miniaturized bioanalytical devices for the analysis of small molecules or proteins in complex samples. For the development of these devices, in-situ synthesis of a porous hybrid organic-inorganic monolith in capillaries (100 µm i.d.) by sol-gel approach was firstly optimized and characterized in terms of repeatability. Secondly, two model toxins of low molecular weight were chosen: microcystin-LR (MC-LR) and ochratoxin A (OTA). Monoclonal antibodies and aptamers specific to one and the other target molecules were then grafted on the monolithic capillaries. The resulting miniaturized immunosorbent (mIS) and oligosorbent (mOS) were then coupled on-line to nanoLC. Specific retention of MC-LR and OTA on the mIS and the mOS, respectively, was demonstrated in pure water. Synthesis repeatability and capacity of the miniaturized sorbents were evaluated. Finally, these miniaturized tools were applied to the selective extraction of MC-LR or OTA from complex samples, i.e. blue-green algae extracts, environmental waters or beer. In a third part, immobilized enzyme reactors (IMERs) were prepared by grafting two proteolytic enzymes (pepsin and trypsin) on monoliths in order to transpose the developed selective tools to the analysis of proteins. These IMERs were then coupled on-line to nanoLC-MS² for the analysis of a model protein, cytochrome C. Digestion yields on IMERs presented a good repeatability. However, digestion efficiency on the pepsin-based IMERs remains so far insufficient and grafting or digestion procedure needs to be readjusted
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Aschl, Timothy. "Biochips based on silicon for detecting the interaction between aptamers and pathogens." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLX103/document.
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La détection rapide et sensible des agents pathogènes est d’une très grande importance pour la biosécurité. Les biopuces sont bien adaptées à cet effet, car elles permettent la détection multiplexe des cibles. Une limitation cruciale des biopuces est leur manque de fiabilité et de sensibilité. L’objectif de cette thèse est de développer une architecture reproductible de biopuces à base de couche mince de silicium amorphe carboné (a-SiC:H) déposée sur un réflecteur en aluminium pour une détection fiable et sensible des pathogènes. Nous avons choisi comme système modèle l’interaction de la toxine alimentaire ochratoxine A (OTA) avec son aptamère AntiOTA de longueur 36mer. Les aptamères (simples brins d’ADN) sont de plus en plus utilisés comme sondes en raison de leur grande spécificité et affinité vis-à-vis d’une large gamme de cibles (i.e. protéines, bactéries…). La stratégie de fabrication consiste en un greffage de monocouches organiques d’acides carboxyliques via des liaisons Si-C robustes, suivi de l’accrochage covalent des aptamères par un couplage peptidique. Les processus de greffage ont été mis au point sur silicium cristallin permettant la quantification des couches greffées par spectroscopie infrarouge en mode ATR (Attenuated total reflexion). La quantification IR des interactions OTA – AntiOTA a été montrée pour la première fois sur des surfaces par IR-ATR. La spécificité de l’aptamère a été démontrée en utilisant une molécule chimiquement similaire (warfarin), pour laquelle l’AntiOTA ne montre aucune affinité. Ces protocoles bien contrôlés ont été transférés sur l’architecture de la biopuce a-SiC:H. Les aptamères immobilisés sont hybridés avec des brins complémentaires marqués avec des fluorophores. En présence de l’OTA une déshybridation des brins complémentaires est attendue, conduisant à une diminution du signal fluorescent. Différentes longueurs de brins complémentaires ont été comparées, montrant jusqu’à 13% de diminution due à l’interaction de l’OTARapid and sensitive detection of pathogenic targets play a crucial role in biosecurity. Biochips are ideal for this, as they allow easy and multiplex detection of targets. A crucial limitation in biochips is that they often suffer from low reliability and sensitivity. The goal of this thesis is to develop a stable and reproducible architecture for biochips based on an amorphous silicon carbon alloy (a-SiC:H) deposited on an aluminium back-reflector for reliable and sensitive detection of pathogens. On these biochips we introduced the interaction of the food and feed toxin ochratoxin A (OTA) with its 36mer aptamer AntiOTA as a model system. Aptamers (single strands of DNA) are ideal as probes for biochips as they display high specificity and affinity towards a wide range of targets (i.e. proteins, bacteria…). The well-controlled multi-step fabrication process consists of the reliable photochemical grafting of acid-terminated organic monolayers on silicon surfaces by robust Si C bonds, which in turn were functionalized with aptamers by stable peptide coupling. Carrying out this process on crystalline silicon allowed monitoring and quantification of every step by infrared spectroscopy (IR-ATR). The interaction OTA – AntiOTA was shown for the first time on surfaces by IR, and an IR in situ calibration allowed the quantification of OTA which was bound by the aptamers on the surface. The specificity of AntiOTA towards OTA was demonstrated by using a chemically similar molecule (warfarin), for which AntiOTA shows no affinity. The well-controlled protocols were transferred to the a-SiC:H biochip. The immobilized aptamers were hybridized with complementary and fluorescent-labeled DNA-strands. In presence of OTA, dehybridization of the complementary strands is expected, resulting in a decrease of fluorescent signal. Different lengths of complementary strands were compared, exhibiting up to 13% signal decrease due to OTA
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Daniel, Camille. "Biopuce à aptamères anti-thrombine : exploration d'une technique alternative de détection." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00954086.
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Du fait de leur haute stabilité et bas coût de production, les aptamères suscitent un intérêt croissant, depuis près de 20 ans, dans le design de biocapteurs en tant qu'élément de reconnaissance idéal. Le but de ce travail de thèse est de démontrer l'intérêt et la pertinence d'un outil tel qu'une biopuce à aptamères, associant les avantages des sondes aptamères à ceux d'une détection par SPRi (Surface Plasmon Resonance imaging), permettant une détection sans marquage et en temps réel d'interactions moléculaires. Dans ce but, deux aptamères anti-thrombine (APT1 = 5′- GGT-TGG-TGT-GGT-TGG -3′ et APT2 = 5′-AGT-CCG-TGG-TAG-GGG-AGG-TTG-GGG-TGA-CT-3′) ont été choisis comme objets d'étude modèles. Ce choix a permis d'orienter différents axes de recherche : utilisés indépendamment comme sondes lors de l'élaboration de notre biopuce, ils ont tout d'abord permis de réaliser une détection cinétique optimisée de la thrombine, avec des performances remarquables pour une détection de ce type, ainsi que le calcul de constantes de dissociation en solution et à la surface des biopuces. Mais au-delà d'un simple biocapteur, la biopuce a également pu être utilisée comme véritable plateforme d'étude de la thrombine et de ses interactions, au sein de structures plus complexes telles que la structure " sandwich " entre les deux aptamères, ou d'autres interactions impliquant la thrombine en tant qu'acteur de la cascade de coagulation (inhibition de la thrombine par l'antithrombine III et le cofacteur II de l'héparine, transformation de la prothrombine au sein du complexe prothrombinase).
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Kittichan, Kanokphandharangkul. "Aptamer biosensors." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/39048.
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Aptamers are single stranded nucleic acids, typically composed of between twenty to eighty nucleotides in length, capable of binding selectively to non-nucleic acid ligands. Aptamers are selected through a combinatorial chemistry process called Systematic Evolution of Ligands by Exponential enrichment (SELEX), which is composed of successive cycles of selection based on target affinity, followed by amplification. This results in the Darwinian evolution of the nucleic acid library resulting in increasing library homogeneity and target affinity over time. Aptamers have been extensively investigated for potential application as sensing molecules, with roles similar to those traditionally occupied by antibodies. Aptamers and monoclonal antibodies have similar sensitivity in the pico to micro molar range. However aptamers have a number of advantages over protein antibodies, such as greater thermal stability, ease of chemical amplification, and amenability to modification, especially at the 5' and 3' prime ends. The work performed in this Thesis is divided into three categories. The first section describes the development of voltammetric Kanamycin and Tetracycline biosensors based on electrode immobilized, redox label bioconjugated nucleotide molecular beacons. These sensors relies on the target-aptamer binding induced spatial displacement of the redox label towards or away from the electrode surface as a means of signal generation. Further study was conducted to test the feasibility of this sensor design under likely field operation environments such as in soil sample analysis for microbial product discovery and in agricultural effluence for regulatory purposes. The biosensor was also enhanced by gel encapsulation for defense against nuclease degradation. Negative control was performed against structurally similar antibiotics of the same family in order to prove the specificity of the biosensor. Lastly, the sensor was moved onto an automated platform in a multichannel format in order to improve the utility of the sensor. The second section describes the development of a voltammetric biosensor based on Enzyme-Linked Oligonucleotide Assay (ELONA) technology. Two sub-types of ELONA-like biosensors were originally envisioned, based respectively on direct and indirect ELONA. Both sub-types depend on the mass of redox label rich Gold Nanoparticles (GNP) at the electrode surface as a means of signal generation. Negative controls was performed against globular proteins Bovine Serum Albumin and Lysozyme, the former since it is the most ubiquitous protein component of serum (the most likely biosensor operational environment), the latter as a worst case scenario for non-specific false positive results due to its positive charge. The last section describes an attempt to develop an automated SELEX device based on mesofluidic flow channels. It was hoped that by using flow channels of a millimeter scale it would be possible to retain both the advantages of the conventional auto sampler based SELEX protocols (large library and sequence variation), while also gaining the primary advantages of microfluidic SELEX (reduced contamination risk, low initial cost and maintenance). Essential components of the SELEX device, such as thermal cycler, liquid handling, electronics infrastructure, and software control were designed, tested and integrated. Lastly an attempt was made to perform automated SELEX against Lysozyme targets using the device, though no nucleic acid with high affinity to target had yet been successfully isolated by the end of this study.
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Bini, Alessandra. "Aptamers for biosensors." Thesis, Cranfield University, 2008. http://dspace.lib.cranfield.ac.uk/handle/1826/4004.
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Aptamers are single-stranded DNA or RNA molecules isolated in vitro by a selection and amplification method. Aptamers bind with high specificity and affinity to a wide range of target molecules, with dissociation constant comparable to antibodies. In this work aptamers were employed as a new kind of bio-recognition element in affinity biosensors for the detection of clinically relevant proteins in heterogeneous assay, using Piezoelectric Quartz Crystal Microbalance and Surface Plasmon Resonance as transducers. The work was focused on two case studies, i.e. the Thrombin-binding aptamer and the aptamer against C-Reactive Protein. From an analytical point of view, the work was devoted to the optimisation of the analytical performance of a piezoelectric and an optical aptasensor for Thrombin and C-Reactive Protein detection, respectively. Efforts towards the application of these aptasensors in complex matrices, such as human plasma and serum, were also undertaken, in order to demonstrate the wide applicability of aptamers, as an alternative to antibodies. In this work, the possibility of introducing a computationally-assisted method to study aptamer-protein interaction and aptamer selection was also evaluated. For this purpose, the Thrombin-binding aptamer was chosen as a model and a retrospective docking study was performed by comparing the affinity of mutated sequences for thrombin with that of the Thrombin-binding aptamer, on the basis of a computationally-derived binding score. Finally, the reliability of computational results was tested by experimental measurements. For this purpose, the Thrombin-binding aptamer and other mutated sequences, selected on the basis of their binding score, were employed for the development of optical biosensors and the resulting analytical performances were compared. Even if further studies should be carried out in order to validate the proposed computational approach to aptamer selection, this work can have a significant impact on future aptamers selection for sensors and diagnostics.
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Dalton, Colette. "Aptamers as biosensors." Thesis, University of Strathclyde, 2010. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=15484.
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Le, Thao Thi. "Aptamers for proteomics." Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/1385.
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Changes in post-translation modifications are very important in the regulation of biological processes. Many modifications occur at very low levels, resulting in a low-abundance of the modified proteins in cells, and therefore assessing those modifications is not an easy task. Modern proteomics needs improved methods for identifying such changes. In this thesis, we focus on generating aptamers that can bind phosphoproteins with high affinities and therefore would be able to detect even low-abundance proteins. Aptamers are short sequences of nucleic acids that can be selected from libraries through a process called SELEX to bind targets of interest with high affinity and specificity. In this work, a phosphotyrosine (pY) peptide in a consensus sequence, commonly found in a class of phosphoproteins recognised by SH2 domains of signalling cascades in cells, was chosen as the target. By choosing this peptide target, we aim to create aptamers that can bind a class of proteins that carry this peptide sequence, mimicking the action of the intracellular SH2 domains. An RNA library with 7×1014 molecules with 30 nucleotides in the random region was employed for the selection and aptamers that bind the pY peptide were selected. Using surface plasmon resonance (SPR), binding affinities of these aptamers with their peptide target were determined (Kd values in high nanomolar (nM) range). In addition, aptamers that bind streptavidin tightly (Kd values in low nM range) were also isolated, as streptavidin was used as the matrix in partitioning step during the selection. Affinities of these aptamers were also determined by SPR. Moreover, fluorescence quenching suggested that the streptavidin binding aptamers bound in or near the biotin binding site. These aptamers can be used as affinity tags for RNA molecules. The secondary structures of both types of the aptamers were predicted based on their random-region sequences using the Mfold program.
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Mejri, Nawel. "Development of biosensors based on DNA aptamers for direct mycotoxins detection." Thesis, Perpignan, 2016. http://www.theses.fr/2016PERP0010.
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Le travail réalisé au cours de cette thèse a porté sur le développement de biocapteurs électrochimiques d’affinité, sensibles et sélectifs, pour la détection de l’ochratoxine A (OTA) et l’aflatoxine M1 (AFM1). Les biocapteurs développés reposent sur l’association de différents nanomatériaux pour une meilleure performance analytique. Pour construire notre transducteur, nous avons associé le polypyrrole à des dendrimères poly(amido-amine) PAMAM, ce qui a permis d’avoir de très bon rendements grâce au propriétés électriques du polypyrrole et à l’augmentation de la surface active due à la structure tridimensionnelle des dendrimères. L’utilisation d’aptamères spécifiques pour la détection des différentes mycotoxines a permis leur détection et quantification à des concentrations de l’ordre des nM, ainsi que l’élargissement des gammes dynamiques. Nous avons pu démontrer grâce à l’utilisation de dendrimères de différentes tailles que la sensibilité des biocapteurs ne provient pas uniquement de l’affinité qui existe entre les biorécepteurs et leurs molécules cibles, mais aussi des propriétés physico-chimiques du biocapteurThis aim of this work is to develop ultrasensitive electrochemical biosensors with high affinity toward ochratoxine (OTA) and aflatoxine M1 (AFM1). In order to obtain the best analytical performances, we associated nano-materials in the transducer construction: conducting polypyrrole polymer and poly(amido-amine) dendrimères. Thanks to this association, we benefited from the conducting material’s electrical properties, and the large active detection surface dendrimers. For the bimolecular sensing part, we used specific DNA aptamers which allowed us to quantify mycotoxines at nM concentrations. In addition, the different aptamer based biosensors present a very large dynamic ranges. We also demonstrated through the use of different sizes of dendrimers, that the sensitivity depend not only in the affinity between bioreceptors and their target molecules, but also in the physico-chemical properties of the biosensor
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Milovskij, Aleksandr Sergeevič. "Oligosaccharid-gerichtete RNA-Aptamere." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=981439365.
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Proske, Daniela. "Neuropeptid Y- und Prionprotein- spezifische Aptamere." Diss., lmu, 2001. http://nbn-resolving.de/urn:nbn:de:bvb:19-3017.
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Nick, Thomas [Verfasser]. "Stability of split-aptamers / Thomas Nick." Mainz : Universitätsbibliothek Mainz, 2017. http://d-nb.info/1122865783/34.
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Tan, Kei Xian. "Aptameric Formulation for Enhanced Biopharmaceutical Delivery." Thesis, Curtin University, 2018. http://hdl.handle.net/20.500.11937/68408.
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This research work developed a novel polymeric carrier system for targeted drug delivery to specific body sites. It utilized unique molecular probes called aptamers that navigate the complexity of mammalian cell systems and present drug molecules to desired locations. The aptamer molecules were tagged onto polymer drug carriers as binding ligands to direct an efficient path towards targeted delivery, avoiding unwanted effects on nontargeted cells and potentially addressing side effects of chemotherapy.
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Pohl, Andrea. "Beiträge zur chemisch-biologischen Oberflächenmodifikation von Nanodiamanten aus der Detonationssynthese." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-232137.
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Die vorliegende Arbeit behandelt die Oberflächenmodifikation von Nanodiamanten (ND) aus der Detonationssynthese und die anschließende Konjugation von einzel- bzw. doppelsträngiger DNA an die zuvor eingeführten funktionellen Gruppen. Als Ausgangsmaterialien wurden zwei Nanodiamantpulver mit unbekannter Oberflächenbelegung eingesetzt, deren Charakterisierung durch elektronenmikroskopische Methoden erfolgte. Weiterhin wurden kommerziell modifizierte ND mit definierter Oberflächenbelegung (Amino- und Hydroxylgruppen) verwendet. Für potenzielle Anwendungen von ND wird eine monofunktionale Oberfläche angestrebt, die u. a. über Oxidation oder Reduktion der durch den Herstellungsprozess eingeführten primären funktionellen Gruppen realisiert werden kann. Die dadurch erzeugten sekundären Funktionen ermöglichen die kovalente bzw. nichtkovalente Anbindung weiterer Substanzen, z. B. von Biomolekülen, an die Oberflächen der ND-Partikel. Die hier beschriebene Konjugation von DNA, an die mit Carboxyl-, Hydroxyl- oder Aminogruppen modifizierten Partikeloberflächen, erfolgte durch die Generierung von Amid-, Phosphodiester- und Isoharnstoffbindungen. Der Erfolg der Konjugationen wurde mit Hilfe von Infrarotspektroskopie und Fluoreszenzmikroskopie untersucht. Die Fluoreszenz der Konjugate beruhte dabei auf Fluoreszenzfarbstoffen, die an die DNA-Moleküle gebunden waren. Darüber hinaus wird die Herstellung einer kolloidalen ND-Suspension beschrieben, von der die Partikelgrößen und das Zeta-Potenzial bestimmt wurden. Kolloidale Suspensionen ermöglichen aufgrund der geringen Partikelgrößen diverse biologische und medizinische Anwendungen von ND. Mit den hier präsentierten Ergebnissen erweitert sich der Kenntnisstand zur Konjugation von DNA an ND aus der Detonationssynthese. Die angewandte Methodik kann ebenso auf andere Substanzen wie Proteine oder Chemotherapeutika übertragen werden. Derart funktionalisierte Partikel besitzen ein großes Potenzial für die weitere Anwendung in der Biomedizin und NanotechnologieThe present study deals with the surface modification of nanodiamonds (ND) from detonation synthesis and the subsequent conjugation of both single and double stranded DNA to previously introduced functional groups. As starting materials two kinds of nanodiamond powders with unknown surface configuration were used. Both types of ND were characterized by electron-microscopic methods. Furthermore, commercially modified ND with defined surface configuration (amino and hydroxyl groups) were applied. Potential applications of ND require a mono-functional surface, that can be realized e. g. via oxidation or reduction of the primary functional groups introduced during the production process. The thereby generated secondary functions permit the covalent or non-covalent linking of further substances onto the surfaces of ND particles. Conjugation of DNA, as described here, onto the carboxyl-, hydroxyl- or aminomodified particle surfaces was accomplished by generating of amino, phosphodiester and isourea bonds. The success of conjugations has been examined by infrared spectroscopy and fluorescence microscopy. The fluorescence of conjugates based on fluorescent dyes bound to the DNA molecules. Furthermore, the fabrication of a colloidal ND suspension is described, of which the particle sizes and the Zeta potential have been determined. Colloidal suspensions facilitate various biological and medical applications of ND on the basis of low particle sizes. The presented results enlarge the state of knowledge about the conjugation of DNA on ND from detonation synthesis. The applied methodology may also be transferred to other substances like proteins or chemotherapeutics. In this way, functionalized particles have a big potential for further application in biomedicine and nanotechnology
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Reinemann, Christine. "Aptamere als neue molekulare Erkennungselemente in Biosensoren /." Leipzig : Helmholtz-Zentrum für Umweltforschung, 2007. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=016271117&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Elsaadani, Moez. "Détection des Ochratoxines A dans la production alimentaire par l’utilisation d’aptacapteur capacitif." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTG077.
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La contamination par les mycotoxines est une menace pour la santé et la vie des humains et des animaux. L’Ochratoxine A (OTA) est une mycotoxine des plus courantes qui contaminent les aliments. L'OTA a un effet toxique chronique et s'est révélée mutagène, néphrotoxique, tératogène, immunosuppresseur et cancérogène. Il est donc important de pouvoir détecter la présence de cette mycotoxine. Certains aptamères ont une affinité spécifique pour l’OTA et peuvent être utilisés pour créer une technique analytique. Plusieurs méthodes ont été décrites pour la détermination de l'OTA dans les aliments. Cependant, la plupart de ces méthodes ne pouvaient pas être appliquées à un aliment complexe tel que le café vert car les produits fluorescents interférents natifs du grain de café rendaient la quantification très difficile. Dans ce travail, nous avons mélangé deux techniques basées sur la séparation pour identifier et quantifier l'OTA dans le café vert. L'ultrafiltration assistée par aptamère comme technique de séparation basée sur la taille des molécules a été appliquée pour séparer l'OTA libre. La quantification de l'OTA a été réalisée par chromatographie en phase liquide à haute performance (HPLC-FLD) avec une limite de détection (LOD) de 0,05 ng/mL pour l'OTA. La récupération de l’OTA dans le café vert artificiellement contaminé présentait une bonne gamme de récupération jusqu’à 97,7%. L’aptamère sélectionné a ensuite été utilisé pour fonctionnaliser un support flexible d’alumine poreuse afin de former un aptacapteur capacitif, facilement manipulable, et spécifique envers l’OTA. Une preuve de concept a été développé pour la quantification de l’OTA et les premiers résultats ont montrés une limite de détection (LOD) de 10-4 ng/mL. Cette méthode a pu être appliquée à la détermination quantitative de l'OTA dans le café vert à des niveaux inférieurs aux niveaux maximaux proposés par la Commission européenne pour le café vert (0.25 ng/mL). Cette étude confirme également que les aptamères peuvent être utilisés comme élément de bioreconnaissance dans les tests de diagnostic ayant une application commerciale pour l'analyse des mycotoxines. À notre connaissance, il s'agit du premier aptacapteur capacitif électrique sans marquage utilisé pour détecter l'OTAMycotoxin contamination is a threat to the health and life of Humans and animals. One of the most common mycotoxin contaminating feed and foodstuffs is Ochratoxin A (OTA). OTA has a chronic toxic effect and has proved to be mutagenic, nephrotoxic, teratogenic, immunosuppressive, and carcinogenic molecule. Aptamer with their specific affinity for OTA was used in this paper to create an analytical technique. Several methods have been reported for the determination of OTA in foods. However, most of these methods could not be applied to a complex food as green coffee because the interfering native fluorescent molecules made the quantification very difficult. In this work, we mixed two separations based techniques to identify and quantify OTA in green coffee. Aptamer assisted ultrafiltration as separation technique based on the size of molecules was applied to separate the free OTA; the quantification of OTA was established by a high-performance liquid chromatography (HPLC-FD) with LOD of 0.05 ng/mL for OTA. Artificially contaminated green coffee displayed a good range of OTA recoveries up to 97.7%. The selected aptamer was used as a biorecognition element to functionalize a capacitive sensor for OTA detection. A capacitive aptasensor was developed for quantification of OTA based on modified anodized aluminum oxide with a LOD of 10-4 ng/mL. This method can be applied to the quantitative determination of OTA in green coffee at levels below the maximum levels proposed by the European Commission for green coffee (0.25 ng/mL). It also confirm that aptamers can be used as biorecognition element in diagnostic assays with commercial application for mycotoxin analysis. To our knowledge, it is the first label-free and electric capacitive aptasensor used to detect the OTA
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Weinert, Ulrike. "Erzeugung funktionaler Schichten auf Basis von bakteriellen Hüllproteinen." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-119909.
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Die hier vorliegende Arbeit beschäftigt sich mit Eignung bakterieller Hüllproteine als Bindungsmatrix für die Kopplung funktionaler Moleküle mit dem Ziel, sensorische Schichten zu erzeugen. Bakterielle Hüllproteine sind biologische SAMs, anderen Oberfläche sich modifizierbare COOH-, NH2- und OH-Gruppen befinden. Die Ausbildung polymerer Strukturen erfolgt dabei in wässrigen Systemen und auf Oberflächen. Im Zuge der boomenden Entwicklung von Biosensoren werden insbesondere Biotemplate gesucht, die zwischen biologischer Komponente und Sensoroberfläche vermitteln. Bakterielle Hüllproteine stellen eine solche Zwischenschicht dar. Als Anwendungsbeispiel wurden die Proteine daher mit einem FRET-Paar und Thrombin und Kanamycin-Aptameren modifiziert. Hierbei wurden das FRET-Paar H488 und H555 an die bakteriellen Hüllproteine der beiden Haldenisolate A12 und B53 mittels EDC mit einer Modifizierungsrate von 0,54 molFarbstoff/molProtein kovalent gebunden. Bei der vorhandenen p4-Symmetrie bedeutet dies, dass ein FRET-Paar pro Einheitszelle vorhanden war. Der Nachweis eines Energietransfers zwischen den beiden am Protein gebundenen Fluoreszenzfarbstoffen H488 und H555 erfolgte mittels statischer und zeitaufgelöster Fluoreszenzmessung.
Die Ergebnisse zeigten, dass ein Energietransfer nur möglich war, wenn die Proteine in polymerer Form vorlagen, unabhängig davon, ob sich die Proteine immobilisiert an einer Oberfläche oder in wässriger Lösung befanden. Mittels Variieren des Donor-Akzeptor-Verhältnisses konnte ein maximaler Energietransfer von 40 % generiert werden, wenn das Verhältnis der Fluoreszenzfarbstoffe von Donor und Akzeptor 4 betrug. Die Fluoreszenzintensität der Fluorophore wurde durch die Bindung an die Proteine nicht verringert oder gelöscht. Dies legt nahe, dass die Farbstoffe in den hydrophoben Poren immobilisiert wurden und die Poren die Fluoreszenzfarbstoffe schützen. Um weitere Aussagen über die Lage der gebundenen Fluoreszenzfarbstoffe zu erhalten, wurden die bakteriellen Hüllproteine der Stämme A12 und B53 enzymatisch verdaut und die Fragmente mittels SEC und SDS-PAGE untersucht. Dabei zeigten sich je nach Enzym und Protein unterschiedliche Bandenmuster bezüglich modifizierter und nativer Hüllproteine. Dies belegt, dass die Fluoreszenzfarbstoffe an NH2-und COOH-Gruppen der Proteine gebunden wurden und so teilweise den enzymatischen Verdau hinderten. Die SEC deutet an, dass die Fluoreszenzfarbstoffe an verschiedenen Stellen am Protein gebunden wurden.
In einem zweiten Beispiel wurde das bakterielle Hüllprotein von A12 mit einem Aptamer modifiziert. Aptamere sind kurze einzelsträngige Oligonukleotide, die u.a. mittels ihrer ausgebildeten 3D-Struktur spezifisch Zielstrukturen reversibel binden können. Die hier verwendeten Aptamere binden spezifisch Thrombin und Kanamycin. Die Aptamere wurden mit Hilfe einer der beiden Vernetzer PMPI oder Sulfo-SMCC an die bakteriellen Hüllproteine kovalent gebunden. Nach dem Modifizieren der Proteine wurden diese auf entsprechenden Sensorchips immobilisiert und die Aktivität des gekoppelten Aptamers mittels Affinitätsmessungen, SPR-Spektroskopie und QCM-D-Messungen analysiert. Die Funktion des gebundenen Thrombinaptamers konnte mittels Affinitätsmessungen und QCM-D nachgewiesen werden und entspricht in beiden Fällen einer Bindung von 2 nmol Thrombin pro Quadratzentimeter. Die Funktionalität des Kanamycinaptamers sollte mittels SPR bestimmt werden, jedoch konnte keine Funktionalität des gekoppelten Kanamycinaptamers nachgewiesen werden. Alle Messungen bestätigten jedoch, dass die Bindungsmatrix aus bakteriellen Hüllproteinen keinerlei oder nur ein sehr geringes Hintergrundsignal liefert.
Werden nun beide Komponenten, FRET-Paar und Aptamere, an das Protein gebunden, ist es möglich, eine sensorische Schicht zu erzeugen. Die Zielstruktur, welche detektiert werden soll, wird an das Aptamer gebunden und so in räumliche Nähe zur Sensorfläche gebracht. Stell die Zielstruktur einen Fluoreszenzlöscher dar, so wird der Energietransfer durch die räumliche Nähe des Fluoreszenzlöscher gestört. Die Detektion des Zielmoleküls erfolgt nun über die Änderung von Fluoreszenzintensitäten. Die hier vorgelegte Arbeit soll einen Grundstein legen für die Entwicklung eines solchen Sensors und insbesondere die Detektion eines Energietransfers optimieren und Schwachstellen in der Detektion nachweisen. Die systematische Untersuchung der Fluoreszenzfarbstoffe auf dem Protein ermöglichen es, in zukünftigen Arbeiten einen FRET zweifelsfrei zu detektieren. Die Modifizierung von bakteriellen Hüllproteinen von A12 mit Aptameren und die Detektion der Funktionalität der Aptamere mittels verschiedener Methoden zeigte auf, dass die bakteriellen Hüllproteine als universelle Bindungsmatrix für sensorische Moleküle dienen können, bei denen Affinitätsmessungen, SPR- oder QCM-D-Messungen genutzt werden. Besonders hervorzuheben ist, dass bakterielle Hüllproteine nahezu kein Hintergrundsignal liefern und aufgrund ihrer dünnen Monolage von etwa 6 - 9 nm die Sensitivität der Messungen nur gering beeinträchtigen.
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Urmann, Katharina [Verfasser]. "Aptamer-based optical biosensors / Katharina Urmann." Hannover : Technische Informationsbibliothek (TIB), 2018. http://d-nb.info/1166271978/34.
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Zhang, Yangyang. "A functionalised aptamer electrochemical biosensor platform." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6438.
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The ability to utilise new knowledge of biomarkers from genomic and proteomic data will have a great impact on molecular diagnosis. Biomarker detection could be achieved by utilising a capture molecule that associates specifically with the target biomarker. The work described in this thesis focuses on a platform comprising a lysozyme binding aptamer and an amperometric electrode (an electrochemical aptasensor). To couple the binding reaction to a change in current, the aptamer is modified with a redox group, ferrocene. Two types of signalling aptamer were constructed, one comprised the aptamer self-assembled on gold and hybridised to a short complementary oligonucleotide carrying a ferrocene group. The second incorporated the binding sequence into a molecular beacon, one end of which self-assembled onto the electrode, the other end carried the ferrocene group. Both of these showed a lysozyme dependent change in current on a gold electrode. Further characterisation of the first aptasensor suggested that the nucleic acid formed a multilayer structure on the electrode surface and that lysozyme binding induced conformational change moved ferrocene close to the surface, increasing the current. In contrast, the second aptamer usually showed a decrease in current in the presence of lysozyme suggesting that the binding resulted in the ferrocene moving away from the surface. In order to evaluate the possible use of these aptasensors for continuous in vivo measurement, needle shaped microelectrodes arrays were produced and the beacon aptamer immobilised on the surface. These electrodes had high impedance which resulted in low sensitivity, however lysozyme binding could still be detected using electrochemical impedance spectroscopy with ferrocyanide in solution. These microspike arrays could also be used for glucose sensing following modification with glucose oxidase.
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Chaou, Thinhinane. "Sélection d’aptamères anti-adénine ADN modifiés en présence de solvant organique. Application au développement de biocapteurs." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0385/document.
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Le développement d’outils de détection in situ (IDT) est indispensable pour rapporter entemps réel la présence d’une signature moléculaire spécifique. L’efficacité d’un IDT estliée à son affinité, à sa spécificité, mais aussi à son potentiel à opérer dans des conditionsimposées par le contexte d’application. Parmi les contraintes imposées, la variation detempérature, le pH et la présence de solvant d’extraction. Le but du projet est dedévelopper des aptamères capables d’opérer en présence de solvant organique ; à ceteffet, nous avons opté pour une sélection en présence de méthanol. La limitation decette stratégie est principalement liée à la nature chimique des acides nucléiques. Parconséquent, nous avons choisi d’utiliser une banque ADN incorporant le (5-(octa1,7-diynyl)-2’-deoxyuridine) dDOTP, au lieu du dTTP. Notre stratégie expérimentale aabouti à la sélection d’un aptamère qui lie spécifiquement l’adénine en présence de 25 %de méthanol. Nous avons montré que les dDOTP sont essentiels à l’interaction del’aptamère avec l’adénine ; l’aptamère sélectionné comporte un motif riche en G partagéavec l’aptamère anti-adénosine, cependant, l’aptamère sélectionné dans le méthanolcomporte un motif structural indispensable à l’interaction avec la cible en présence deméthanol. Par ailleurs, nous avons montré que l’aptamère sélectionné pourraitfonctionner comme un module de reconnaissance spécifique d’un biocapteurDevelopment of in situ detection tools (IDT) is required for specific real time monitoringof chemical species. Efficient IDT is not only related to its affinity and ability todiscriminate between molecular variants, but moreover it must be adapted for operatingunder conditions imposed by the context of application. Among others, hightemperature, pH variation and presence of organic solvents may be mentioned. The aimof our project is to develop an aptamer operating in the presence of organic solvents. Forthis purpose, we opted for selection in presence of methanol. Limitations of this strategyare adaptability of SELEX technology and chemical diversity of nucleic acids. For thispurpose, we used library incorporating (5-(octa1,7-diynyl)-2’-deoxyuridine) dDOTPinstead of conventional thymine nucleotide. Our experimental strategy led to theselection of an aptamer that specifically recognises adenine in the presence of 25 % ofmethanol. dDOTP nucleotides are essential for adenine recognition; the selectedaptamer shares a purine rich motif with a previously described adenosine aptamer, butdisplays a specific structure motif, essential for operating in the presence of methanol.Furthermore, the ability of truncated variants of the selected aptamer to form arecognition module of biosensor was assessed in this study
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Frigotto, Laura. "Studies on novel RNA ligands for CD4." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249252.
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Muharemagic, Darija. "Aptamers as Enhancers of Oncolytic Virus Therapy." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32170.
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Oncolytic viruses promise to significantly improve current cancer treatments through their tumour-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapies is the intravenous delivery of the virus, as it can be cleared by neutralizing antibodies (nAbs) from the bloodstream before it reaches the tumour cells. In our group, we have succeeded in developing aptamers to vesicular stomatitis virus (VSV), as well as to rabbit anti-VSV polyclonal neutralizing antibodies (nAbs). We tested these aptamers’ biological activity with a cell-based plaque forming assay and found that the aptamers prevented in vitro neutralization of VSV by nAbs and increased the virus infection rate of transformed cells up to 77%.
In line with this approach, we enhanced the delivery of oncolytic viruses by selecting aptamers to the CT26 colon carcinoma cell line. The binding of aptamer pools has been tested on flow cytometry and the best pools were subjected to high throughput sequencing. Selected aptamers were linked to anti-VSV aptamers and applied for target delivery of the virus to cancer cells. Development of this aptamer-based technology aims to improve viral anti-cancer therapies, with a potential to be applied as treatment for patients affected with cancer.
Finally, in collaboration with a group from Erlangen University, we performed an aptamer selection using capillary electrophoresis and cell-SELEX. The target, the extracellular domain of human CD83, is a maturation marker for dendritic cells and is involved in the regulation of the immune system. Selected aptamer sequences bound selectively to mature dendritic cells, in comparison to immature dendritic cells, and thus hold promise to be applied for further studies leading to a better understanding of CD83’s mechanism of action.
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Jones, Louisa Alice School of Biotechnology And Biomolecular Sciences UNSW. "Aptamers to the hepatitis C virus polymerase." Awarded by:University of New South Wales. School of Biotechnology And Biomolecular Sciences, 2005. http://handle.unsw.edu.au/1959.4/32734.
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Treatments for the hepatitis C virus (HCV) are currently only partially effective. Research into antivirals directed at HCV viral proteins are commonly based and tested on a single genotype, namely genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach for the isolation of antiviral agents. SELEX allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNAdependent RNA polymerase (RdRp) [non-structural protein B (NS5B)] protein of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 and resultant aptamers cloned from rounds 2, 4, 8 and 10. Sequences of aptamers were aligned to elucidate common motifs and a proportion of the aptamers from rounds 8 and 10 (29/48) were screened for binding ability using the Biacore. The five ???best binding??? aptamers were investigated for inhibition of 3a polymerase activity in an in vitro polymerase assay. Two aptamers, r10/43 and r10/47, were chosen for further studies based on their ability to inhibit polymerase activity. The inhibition constants (Ki) of r10/43 and r10/47 were estimated to be 1.4 + 2.4 nM and 6.0 + 2.3 nM respectively. The affinity (Kd) of these aptamers for the 3a polymerase was estimated to be 1.3 + 0.3 nM (r10/43) and 23.5 + 6.7 nM (r10/47). The estimated inhibition and dissociation constants of these two aptamers are among the best for inhibitory aptamers of the HCV enzymes (polymerase and protease). Inhibition of HCV 3a polymerase appeared to be specific for r10/47, whilst r10/43 also had some inhibitory effect on norovirus and ??6 polymerase activity. This study is the first description of an inhibitor to the HCV subtype 3a polymerase that investigates genotypic specificity of targeted antivirals.
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Al-Youssef, Nadia. "The Selection of Aptamers to CD20 and Their Application as Inhibitors of Complement Dependent Cytotoxicity." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/33182.
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CD20 is an important oncological B-cell marker. Immunotherapy, using anti-CD20 antibodies, has revolutionized the treatment of B-cell cancers. Aptamers are highly specific DNA ligands, raised to identify virtually any target molecule through an iterative process known as SELEX (systematic evolution of ligands by exponential amplification). Aptamers rival antibodies in both binding affinity and specificity. We developed a novel CD20 specific SELEX method, using a lentiviral system to transfect CD20 cDNA into HEK293 cells. Selection using CD20+HEK cells evolved pools of aptamers with stepwise increases in binding affinity for the transfected cell line. Sequenced aptamer clones exhibited an antagonistic effect with anti-CD20 antibody; and in a biological assay possessed a protective capacity, limiting the extent of antibody induced complement dependent cytotoxicity. Overall, genetic transfection is a novel targeted approach of ligand generation, producing aptamers endowed with both physical and biological capabilities
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Ding, Shu Gu Li-Qun. "Aptamer encoded nanopores as single molecule sensors." Diss., Columbia, Mo. : University of Missouri--Columbia, 2008. http://hdl.handle.net/10355/5767.
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The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on September 21, 2009). Thesis advisor: Liqun Gu Includes bibliographical references.
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Nguyen, Thi-Huong [Verfasser]. "Rupture forces of split aptamers / Thi-Huong Nguyen." Mainz : Universitätsbibliothek Mainz, 2013. http://d-nb.info/1034486209/34.
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Simmons, Suzanne Clare. "Development of Aptamers as Diagnostic and Therapeutic Agents." Thesis, Open University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.524784.
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Aptekar, Shraddha Ashok. "Selective targeting to glioma with nucleic acid aptamers." Thesis, University of Central Lancashire, 2015. http://clok.uclan.ac.uk/11801/.
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The term glioma encompasses brain tumours arising from the glial cells. Malignant glioma are characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue, hence diagnosis and treatment is difficult, and patient survival is poor. Aptamers are small molecular ligands composed of short oligonucleotides that bind to a target with high specificity and affinity. They are produced in vitro through a method called systematic evolution of ligands by exponential enrichment (SELEX). The aim of the study was to examine the binding selectivity of DNA aptamers on commercial glial cell lines and primary glioma tissues. RNA aptamers and their DNA homologues (SA44, SA43, SA56) were selected for study which showed strong binding affinity to the target U87MG cells as measured by flow cytometry. SA44 and SA43 showed higher uptake and cytoplasmic localisation in U87MG and 1321N1 glioma cell lines compared to non-cancerous SVGP12 cells and non-glioma MCF-7 and T24 cells as measured by confocal microscopy. The data was confirmed quantitatively by flow cytometry analysis, which showed that the aptamers were able to actively internalise in U87MG and 1321N1 tumorigenic cells compared to the non-cancerous and non-glioma cell types. Histochemistry staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was found to be significantly higher for only SA43 aptamer (p < 0.05) in glioma tissues (grade I, II, III and IV) compared to the non-cancerous and tissues. Aptamer SA43 also showed cell type selectivity within the tissue. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, show promise for histological diagnosis of glioma and targeted delivery. In the future, targeting tumour cells and tissues through the use of SA43 aptamer will help develop molecular imaging, targeted delivery by reduction of the non-specific toxicity of chemotherapy and selectively directing anti-cancer drugs to tumour cells.
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Leonard, Marissa. "Overcoming Breast Cancer Metastasis with Novel RNA Aptamers." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1572879601351414.
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Marquardt, Janice Dionne. "Force interaction characterization between thrombin and DNA aptamers." [Ames, Iowa : Iowa State University], 2008.
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Dassie, Justin Patrick. "Selective targeting of cancer cells with RNA aptamers." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/1310.
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Two of the most commonly diagnosed malignancies in men and women are cancers of the prostate and breast, respectively. Though many advances have been made in reducing the overall morbidity and mortality associated with these diseases, the high number of deaths that still occur emphasizes the need for safer and more effective therapeutic options. To this end, our lab was the first to describe the use of RNA aptamers to specifically deliver cytotoxic siRNAs to PSMA positive prostate cancer cells. This reagent, termed an aptamer-siRNA chimera, was shown to be an effective targeted cancer therapeutic upon intratumoral injection in a pre-clinical, xenograft, mouse model of prostate cancer. However, further work was needed to realize the full clinical potential of RNA aptamer-siRNA chimeras as a targeted therapeutic modality.
The thesis laid out herein, describes work performed to optimize aptamer-siRNA technology in order to enable clinical translation and to increase the scope of this technology (i.e. increase the cancer types for which this technology can be used). We describe several improvements to our first generation PSMA aptamer-siRNA chimera which, include: decreasing the overall nucleotide content to aid in chemical synthesis, altering the siRNA structure to improve RNAi processing and addition of a 20kDa PEG moiety to increase pharmacokinetics/pharmacodynamics. All of these modifications lead to a more effective reagent at lower doses. Importantly, we demonstrate that our optimized reagent is now effective upon systemic administration in an in vivo mouse model of prostate cancer. In addition, we have also identified new aptamers to the receptor tyrosine kinase (RTK) EphA2. Given the broad expression of this RTK on various cancers, this work seeks to extend the scope of targeted aptamer therapeutics beyond that of prostate cancer. Finally, we demonstrate a novel aptamer selection methodology termed cell-internalization SELEX. This approach allowed us to select for aptamers that specifically targeted and internalize into HER2 expressing cells. This allowed us to readily translate all identified aptamers into aptamer-siRNA chimeras. We show that all chimeras tested were able to sensitize HER2+ breast cancer cells to low- dose cisplatin treatment.
Taken together, the work described in this thesis significantly advances the field of targeted cancer therapeutics. Importantly, by demonstrating cancer cell-specific delivery of siRNA, our technology overcomes one of the most significant hurdles to the therapeutic use of siRNAs, delivery.
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Brackett, David Michael. "Ligand binding and catalysis in an RNA aptamer /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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Fordham, Daniel George. "Development of a biosensor using aptamer/antigen interactions." Thesis, University of Portsmouth, 2011. https://researchportal.port.ac.uk/portal/en/theses/development-of-a-biosensor-using-aptamerantigen-interactions(66fc5f4e-0248-41d1-8124-d68b6b040a64).html.
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The use of aptamers acting within novel biosensors as molecular recognition elements is well documented with a wide variety of techniques being adapted to take advantage of the benefits of oligonucleotide detection. A continuing barrier to the commercial use of ssDNA or RNA aptamers remains the lack of a high-throughput system that confers reliable selection and description of suitable species. Here we describe simple methodologies that utilise fusion proteins, modified affinity chromatography, HPLC, and, nanopore assays, along with other techniques to isolate novel aptamers to well-characterised proteins. These methods have yielded novel aptamers to the HsdR, and HsdS subunits of type I restrictionmodification system EcoR124I and to the human rhinovirus 3C protease, along with an enriched libraries for nitrotyrosine. In addition to the isolation of novel aptamer species, methods for the characterisation of the binding capabilities of candidate aptamers are presented. The techniques of SPR, EMSA, and dot blotting are evaluated and utilised in the appraisal of the novel aptamer sequences.
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Laurenson, Sophie. "The development and application of peptide aptamer microarrays." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613244.
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Woodman, Robbie. "The development and application of peptide aptamer technology." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614795.
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Bartley, Amanda Nicole. "Aptamer-Based Assay For Detection Of Ochratoxin A." FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3894.
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Ochratoxin A (OTA) is a potent mycotoxin found in a wide range of agricultural products that has been linked to mitochondrial damage and renal disease. The standard methods for OTA analysis currently rely on the use of high-performance liquid chromatography (HPLC) coupled to fluorescence detection or mass spectrometry. Toward a highthroughput analysis of OTA, a single-stranded DNA aptamer, modified with a fluorophore, coupled to a complementary sequence, modified with a FRET-based quencher that dissociates in the presence of the target toxin, is proposed. In order to integrate “target trapping,” aptamer immobilization methods were explored to mediate interference issues. Assays were evaluated using wine and blood serum matrices. A solution-based assay in a 96-well plate format provided a limit-of-detection of 2.7 ng/mL which would be suitable for many of the proposed applications. Immobilized aptamer formats, however, were not reliable, and a range of limitations to applications of the assay were identified.
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Meini, Nadir. "Conception et réalisation de biocapteurs impédimétriques." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10074/document.
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L'objectif du travail de recherche concerne la conception et la réalisation de biocapteurs à base de mesures impédimétriques, pour lesquels la demande est forte dans différents domaines d'intérêt sociétal, en particulier l'environnement, la sécurité alimentaire et le biomédical. Les biocapteurs sont des moyens d'analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d'outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d'un pouvoir de reconnaissance pour un analyte ou un groupe d'analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans la première partie de ce travail, un aptasensor a été développé pour la détection de la thrombine. Deux aptamères different ciblant la thrombine étaient directement immobilisés sur l'électrode en or. L'aptasensor élaboré présente une grande sensibilité, spécificité et stabilité pour la thrombine. Dans la seconde partie, en utilisant la spectroscopie d'impédance électrochimique (EIS), nous avons surveillé l'immobilisation de protéines et sans marquage sur une surface d'or, au moyen d'une stratégie d'électro-adressage, compatible avec la production de biopuces pour multi-détection.Cette fonctionnalisation est réalisée par la cycloaddition alcyne / azoture, mieux connu comme la réaction «clic». Enfin, un biocapteur utilisant des protéines de phage à été développé pour la détection de E.coliThe objective of the research concerns the design and realization of biosensors based impedimetric measures, for which there is strong demand in various societal benefit areas, particularly the environment, food security and biomedical.Biosensors are rapid, selective and inexpensive devices that combine a biological recognition element, the so-called bioreceptor (e.g. enzymes, antibodies, DNA or microorganisms) to a physical transducer (e.g. electrochemical, optical, thermal or piezoelectrical). They can be used to detect one specific analyte or one family of analytes for a wide range of applications (e.g. environment, food, health). In the first part of this work, an aptasensor was developed for thrombin detection. Two different aptamers targeting thrombin were directly immobilized on the gold electrode. The aptasensor exhibits high sensitivity, specificity and stability in the detection of thrombin. In the second part, using electrochemical impedance spectroscopy (EIS), we have, monitored label-free protein immobilization on a gold surface, through a strategy of electroaddressing, compatible with the production of microarrays for multi-detection. This functionalization is achieved via the alkyne/azide cycloaddition, better known as the "click" reaction.Finally, a biosensor using phage proteins was developed for detecting E. coli
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Haarberg, Hans Eirik. "Theophylline IMAGEtags (intracellular multi aptamer genetic tags) the development and evaluation of an RNA reporter system based on the theophylline aptamer /." [Ames, Iowa : Iowa State University], 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1464205.
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Bayrac, Abdullah Tahir. "In Vitro Selection Of Dna Aptamers To Glioblastoma Multiforme." Phd thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12613769/index.pdf.
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Aptamer probes for specific recognition of glioblastoma multiforme were generated using a repetitive and broad cell-SELEX-based procedure without negative selection. The 454 sequencing technology was used to monitor SELEX, and
bioinformatics tools were used to identify aptamers from high throughput data. A group of aptamers were generated that can bind to target cells specifically with dissociation constants (K d ) in the nanomolar range. Selected aptamers showed high affinity to different types of glioblastoma cell lines, while showing little or no affinity to other cancer cell lines. The aptamers generated in this study have
potential use in different applications, such as probes for diagnosis and devices for targeted drug delivery, as well as tools for molecular marker discovery for glioblastomas.
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Lennarz, Sabine [Verfasser]. "RNA aptamers as selective protein kinase inhibitors / Sabine Lennarz." Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/1077289588/34.
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Meyer, Michael [Verfasser]. "Applications of aptamers in flow cytometry assays / Michael Meyer." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2014. http://d-nb.info/1059512718/34.
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Zhang, Naru, and 张娜茹. "Study on influenza virus-like particles and ssDNA aptamers." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/200167.
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Since there is an urgent need for development of vaccines and antiviral agents to combat influenza pandemics, this study aimed to develop influenza virus-like particles (VLPs) and aptamers targeting the virus particles as vaccine and antiviral agent candidates.
Influenza VLPs containing three structural proteins of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) derived from influenza A/Hong Kong/01/2009 (H1N1) virus (HK/01) were constructed using a Bac-to-Bac baculovirus expression system. The expressed VLPs were purified by sucrose density gradient ultracentrifugation and characterized by Western blotting analysis and transmission electron microscopy. The immune responses and protective efficacy induced by VLPs were compared with those elicited by the clinically used Panenza vaccine in BALB/c mouse model. The results showed that two-dose vaccination with both VLP and the Panenza vaccine could confer complete protection. Single-dose vaccination with VLP could also provide 100% protection against lethal virus challenge, whereas single dose of an equal amount (based on HA content) of the Panenza vaccination just provided incomplete protection (67% survival rate) against the lethal virus challenge. Compared to the Panenza vaccination, the VLP vaccination could induce higher and broader antibody responses and higher viral specific T help (Th) cell and cytotoxic T lymphocyte (CTL) responses. Notably, a novel finding in this study is that the VLP vaccination could induce antibodies to inhibit virus release from infected MDCK cells, although the underlined mechanism needed to be further studied. These results indicated that influenza VLP might be a more effective and safe vaccine candidate which could be developed into an alternative vaccine for the control of epidemic and pandemic influenza in the future.
To develop aptamers as antiviral agents against influenza, I sought to use influenza VLPs as target for ssDNA aptamer selection. After 11 rounds of selection using the systemic evolution of ligandsby exponential enrichment (SELEX),the recovered DNA molecules were PCR-amplified, gel purified and cloned into pCR-Blunt II TOPO vector for sequencing. The sequencing results showed that one aptamer Va-1 was markedly enriched, which was accounted for 59% (13/22) of the selected aptamers. Compared to the other non-enriched aptamers, the enriched aptamer Va-1 showed the highest binding affinity to the UV inactivated influenza HK/01 virus. It was also shown that the aptamer Va-1 specifically bound to the HK/01 stain while it could not bind other respiratory viruses even the PR8 strain within the H1N1 subtype. It was further demonstrated that the aptamer Va-1 could only bind to NA protein in a dose-dependent manner but not bind to HA and M1 proteins. Unfortunately, the selected aptamer did not show any antiviral effects. However, it may be potentially developed into a diagnostic and analytic agent because its binding activity was comparable with that of the commercial anti-NA antibody.
In conclusion, the influenza VLPs may be a promising vaccine candidate for the control of influenza virus infection and the selected aptamer may be potentially developed into an alternative tool for influenza virus detection.published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy
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Ellingham, Mark David. "Selection and characterisation of RNA aptamers to FMDV 3Dpol." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436396.
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Ding, Yindi. "Peptide aptamers : tools for the analysis of RAS signaling." Lyon, École normale supérieure (sciences), 2009. http://www.theses.fr/2009ENSL0535.
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Cette thèse décrit l'utilisation d'aptamères peptidiques (PA) comme outil d'étude de la signalisation de Ras dans des modèles de lignées cellulaires. Elle décrit ensuite les analyses initiales de l'effet biologique de petites molécules sélectionnées pour leur capacité à inhiber l'interaction entre PAs et H-RasV12. Finalement elle présente la caractérisation de précurseurs de neurones de DRG et que nous espérons pouvoir utiliser pour de futures études. Afin d'analyser les interactions entre Ras et différentes protéines et les perturbations de la voie de signalisation, PAs dirigés contre la protéine Ras sauvage et Ras oncogène avaient été sélectionnés. PA 105R présente un phénotype d'interaction en double hybride de levure (Y2H) avec H-, K-, et N-Ras. PA HR3C présente un phénotype d'interaction avec la protéine Ras sauvage et H-RasV12. Dans les cellules PC12, HR3C bloque aussi bien la signalisation de Ras sauvage que celle de H-Ras oncogène. Par contre dans les cellules AsPC1, la protéine oncogène K-RasD12 endogène est inhibée par 105R, alors que HR3C est bien moins efficace. Des mutations dirigées contre les acides aminés localisés à la surface de HrasV12, suivi d'un test d'interaction en Y2H, ont permis de cartographier les sites de liaison des PAs biologiquement actifs sur H-RasV12. L'activité biologique d'un certain nombre de petites molécules sélectionnées pour leur capacité à dissocier l'un des PAs spécifique de Ras. Ces études ont abouti à l'identification d’une molécules qui pourrait être biologiquement pertinente. Des études supplémentaires seront requises afin d'obtenir, à partir de ces molécules, de possibles candidats aux études pré-cliniques. Finalement, nous avons mis en place la technologie des PAs pour une utilisation dans des cellules primaires dérivées rat DRG, et développé une lignée précurseur de cellules neuronales de DRG que nous avons maintenue en culture 2 ans. Nous espérons ainsi utiliser ces précurseurs pour des études futures dans le PNSThis thesis describes the use of peptide aptamers (PA) for the study of Ras signaling in model cell lines. It further describes the initial analysis of biological effects of small molecules identified for their capacity to displace a biologicallyactive PA from H-RasV12. Finally, it offers characterization of DRGn precursors that we hope to be able to use for future studies. To analyze Ras interactions with other proteins and perturbation of signaling pathways, PAs directed towards wt Ras and oncogenic Ras had been selected. PA 105R elicited an interaction phenotype in yeast two–hybrid (Y2H) with H-, K-, and N-Ras. PA HR3C gave an interaction phenotype with H-Ras wt and HrasV12. In PC12 cells, it blocked both wt and oncogenic H-Ras signaling, whereas 105R had little effect on NGF-induced signaling, but inhibited that stimulated by oncogenic Ras. In contrast, in AsPC-1, endogenous oncogenic K-RasD12 is inhibited by 105R, while HR3C is much less effective. Targeted mutation of amino acids located on the surface of H-RasV12, followed by Y2H interaction mating assays, offered the possibility to map the binding sites of the biologically-active Pas. We evaluated biological activity of a number of small molecules selected for their capacity to displace one of the Ras-specific PAs from the target. These studies resulted in the identification of one molecule that may be biologically pertinent. Further studies will be required to develop possible pre-clinical candidates based on these molecules. Finally, we were to implement the PA technology in primary cells derived from rat DRG, and we developed a DRG precursor line that we have maintained in culture for over two years. It is hoped that we use thes precursors for future studies in the PNS
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Gibert, Benjamin. "La protéine de stress Hsp27 / HspB1, une cible de choix en thérapie anti-cancéreuse." Phd thesis, Université Claude Bernard - Lyon I, 2010. http://tel.archives-ouvertes.fr/tel-00733751.
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Hsp27 appartient à la famille des protéines dites de survie comme Bcl2 ou la survivine. C'est une protéine anti-apoptotique qui subit une dérégulation de son expression dans de nombreux types tumoraux. Elle est caractérisée comme étant une cible thérapeutique majeure. Au cours de ma thèse, j'ai isolé des peptides stabilisés, dit aptamères, capables d'inhiber fonctionnellement les activités anti-apoptotiques et tumorigènes d'Hsp27. Ces aptamères perturbent la biochimie structurale de Hsp27 et induisent le blocage du cycle cellulaire in vivo. Parallèlement à cette étude, j'ai caractérisé les effets de la déplétion de Hsp27 sur la formation de métastases et de tumeurs osseuses. J'ai aussi montré que la modification du taux de Hsp27 induisait la dégradation de différentes protéines, dites clientes, comme la caspase3, HDAC6 et STAT2.
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Catlin, Diane M. "DNA Aptamer Confirmation and Utilization for the Cyanotoxin, Cylindrospermopsin." FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/2552.
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Cyanotoxins are posing an increasing threat to the health of humans and wildlife. Cylindrospermopsin is a cyanotoxin that occurs in warm climates and is harmful when ingested. The toxic effects of CYN can affect multiple organ systems. The effects, coupled with the evidence of a mass contamination of a water supply in Australia, prove that CYN needs to be investigated further.
Aptamers have become a desirable method for detection of CYN as a result of an aptamer’s high specificity and the ability to scale up experiments. Aptamers have been designed to bind with a variety of targets, including cyanotoxins. An aptamer for CYN was identified by Elshafey et al.
This study aims to confirm the binding of the aptamer to CYN and the selectivity of the aptamer using fluorescent biosensing and circular dichroism. Aptamer affinity capture was used to investigate the possibility of a real world application of the aptamer.
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Mehanovic, Samir. "Development of an RNA aptamer to PD173955N through SELEX." [Ames, Iowa : Iowa State University], 2008.
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