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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Aptamere als neue molekulare Erkennungselemente in Biosensoren /

Reinemann, Christine. January 2007 (has links)
Zugl.: Leipzig, Universiẗat, Diss., 2007.
2

Die Post-SELEX-Optimierung des Trypanosomen-spezifischen RNA-Aptamers 2-16

Adler, Annette. Unknown Date (has links) (PDF)
Darmstadt, Techn. Universiẗat, Diss., 2007.
3

The Selection of Aptamers to CD20 and Their Application as Inhibitors of Complement Dependent Cytotoxicity

Al-Youssef, Nadia January 2015 (has links)
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
4

Selection of aptamers against prostate specific antigen for diagnostic and therapeutic applications

Svobodová, Markéta 26 June 2012 (has links)
Actualmente, el antígeno prostático específico (PSA) se considera el marcador más sensible para la detección de cáncer de próstata. Hasta la fecha anticuerpos monoclonales y policlonales se han utilizado para detectar el PSA debido a su alta especificidad y sensibilidad. Sin embargo, la producción de anticuerpos es lenta y cara. En el otro lado, los aptámeros con especificidad y afinidad iguales a los de los anticuerpos podría ser una forma alternativa para la detección de PSA. El objetivo de este trabajo es la selección de aptámeros contra el PSA. Aspectos fundamentales como la caracterización de la PSA, la evaluación de las estrategias de inmovilización, la preparación de la biblioteca de oligonucleótidos y de la cadena simple de ADN (ssDNA) han sido evaluados con el fin de alcanzar el objetivo principal de esta tesis. Finalmente, la selección de aptámeros contra el PSA y su uso potencial en aplicaciones de diagnóstica y terapéutica han sido descritos. / Currently, PSA is considered the most sensitive marker available for prostate cancer detection and for monitoring its progression. To date monoclonal/polyclonal antibodies have been used to detect PSA due to their high specificity and sensitivity. However, the production of antibodies is time-consuming and expensive. On the other hand aptamers with specificity and affinity equal to those of antibodies could be an alternative way for the detection of PSA. This work overviews the selection of aptamers against prostate specific antigen (PSA). Fundamental aspects such as the characterization of PSA, evaluation of immobilization strategies, the preparation of oligonucleotide library and single stranded DNA (ssDNA) have been evaluated in order to reach the main objective of this thesis. Finally, selection of DNA and RNA based aptamers against PSA and their potential use in diagnostic and therapeutic applications have been described
5

Characterization of RNA aptamers that bind to HIV-1 gp120

Cohen, Carla J. January 2006 (has links)
RNA aptamers with 2'-fluoro-pyrimidine chemistry were previously selected by in vitro evolution to bind to monomeric HIV-1 gp120 from the R5 strain BaL. A group of 36 novel aptamers were cloned and sequenced from the heterogeneous pool and were tested for their ability to bind to gp120. The diversity of the RNA secondary structure of these, and 27 aptamers isolated previously, was analysed using a bioinformatics approach. This showed that eight aptamers contain a common branched motif, and RNA mutagenesis indicated that this structure is probably required for gp120 binding. Chemically synthesised derivatives of one such aptamer, B40, were designed and tested for binding to gp120. Truncation was found to decrease their binding, but the introduction of point mutations to stabilise the branched conformation and 2'-O-dimethylallyl-modified residues to stabilise helices increased binding to levels greater than that of the parental aptamer. The aptamer epitope on gp120 was mapped by testing aptamer binding to alanine-scanning mutants and deletion mutants of gp120 using a novel plate-based assay. This study showed that the aptamer binding site overlaps with the CCR5 epitope and is confined to four key residues at the base of the V3 loop, one of which is highly conserved. This finding may account for the observation that a number of aptamers were shown previously to neutralise a range of HIV-1 R5 clinical isolates in PBMC cultures. Interestingly however, the aptamer was unable to neutralise HIV-1 pseudovirus in a cell line, which is most likely due to the increased levels of cell-surface CCR5 in cell lines compared to PBMC. Future work should focus on identifying the structure and epitopes of other anti-gp120 aptamers as well as testing neutralisation of HIV-1, HIV-2 and SIV by the B40-derived aptamers. These aptamers can be used as tools to investigate the HIV-1 entry pathway and also have the potential to be developed as anti-HIV-1 microbicides.
6

Functional DNA Aptamers as Biotherapeutic Molecules

Orava, Erik 14 January 2014 (has links)
Aptamers are single-stranded oligonucleotides, DNA or RNA, which can bind to a myriad of targets such as ions, peptides, proteins, drugs, organic and inorganic molecules with high affinity and specificity. Aptamers are derived using combinatorial libraries comprised of a variable region flanked by two primer regions used for a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The central theme of my thesis was to use this technology to develop aptamers able to bind to validated therapeutic targets, specifically the Tumour Necrosis Factor alpha (TNFα) and Carcinoembryonic Antigen (CEA), and block their biological functions. As well, I investigated the use of CEA and MUC1 binding aptamers as targeting agents to guide and detect the delivery of contrast agent-loaded liposomes in tumour-bearing mice using computed tomography (CT) imaging. Aptamer selections successfully identified a 25-base aptamer (VR11) that can bind with high affinity and specificity to TNFα. VR11 blocked TNFα signaling, prevented apoptosis, reduced nitric oxide (NO) production in cultured cells and was non-immunogenic when injected into C57BL/6 mice. As well, aptamers were derived to the IgV-like N-domain of CEA. Two DNA aptamers were isolated containing a 40-base variable region, N54 and N56, bearing anti- CEA homotypic adhesive properties. These aptamers are not cytotoxic or immunogenic and III are able to prevent CEA-mediated homotypic and heterotypic cell adhesion events. In addition, the pretreatment of murine cancer cells expressing CEA with these aptamers prior to their intraperitoneal injection into C57BL/6 mice resulted in the prevention of tumour foci formation. Finally, the in vivo targeting of nanoparticles such as pegylated liposomes to tumour cells was enhanced by introducing tumour marker-specific DNA aptamers on their surface. The CEA-specific aptamer N54 and a 40-base second generation aptamer MUC1-VR1 that recognizes the tumour-associated mucin MUC1 were incorporated into liposomes containing the CT contrast agent Omnipaque350™ and Cy5 to characterize their binding to CEA and MUC1-expressing cancer cells in vitro. Pharmacokinetic studies also revealed that the incorporation of these aptamers into pegylated liposomes significantly lenghthened their circulation half-lives to values that parrallel that of untargeted pegylated liposomes.
7

Functional DNA Aptamers as Biotherapeutic Molecules

Orava, Erik 14 January 2014 (has links)
Aptamers are single-stranded oligonucleotides, DNA or RNA, which can bind to a myriad of targets such as ions, peptides, proteins, drugs, organic and inorganic molecules with high affinity and specificity. Aptamers are derived using combinatorial libraries comprised of a variable region flanked by two primer regions used for a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The central theme of my thesis was to use this technology to develop aptamers able to bind to validated therapeutic targets, specifically the Tumour Necrosis Factor alpha (TNFα) and Carcinoembryonic Antigen (CEA), and block their biological functions. As well, I investigated the use of CEA and MUC1 binding aptamers as targeting agents to guide and detect the delivery of contrast agent-loaded liposomes in tumour-bearing mice using computed tomography (CT) imaging. Aptamer selections successfully identified a 25-base aptamer (VR11) that can bind with high affinity and specificity to TNFα. VR11 blocked TNFα signaling, prevented apoptosis, reduced nitric oxide (NO) production in cultured cells and was non-immunogenic when injected into C57BL/6 mice. As well, aptamers were derived to the IgV-like N-domain of CEA. Two DNA aptamers were isolated containing a 40-base variable region, N54 and N56, bearing anti- CEA homotypic adhesive properties. These aptamers are not cytotoxic or immunogenic and III are able to prevent CEA-mediated homotypic and heterotypic cell adhesion events. In addition, the pretreatment of murine cancer cells expressing CEA with these aptamers prior to their intraperitoneal injection into C57BL/6 mice resulted in the prevention of tumour foci formation. Finally, the in vivo targeting of nanoparticles such as pegylated liposomes to tumour cells was enhanced by introducing tumour marker-specific DNA aptamers on their surface. The CEA-specific aptamer N54 and a 40-base second generation aptamer MUC1-VR1 that recognizes the tumour-associated mucin MUC1 were incorporated into liposomes containing the CT contrast agent Omnipaque350™ and Cy5 to characterize their binding to CEA and MUC1-expressing cancer cells in vitro. Pharmacokinetic studies also revealed that the incorporation of these aptamers into pegylated liposomes significantly lenghthened their circulation half-lives to values that parrallel that of untargeted pegylated liposomes.
8

Partially Robotic Selection of Aptamers to Red Blood Cell Protein Glycophorin A

Bushnik, Evan 24 September 2018 (has links)
Aptamers are small DNA ligands that have been manually selected to strongly and specifically bind a target of interest. These molecules may prove superior to modern antibodies in a number of ways including price and reproducibility. One of the major advantages of using aptamers as opposed to antibodies is the relative speed of development. This, coupled with the repetitive nature of aptamer selection, means that the entire process is a possible target for automation. In the following experiments, a ssDNA aptamer is developed against the human red blood cell protein glycophorin A, partially through the novel use of a robotized benchtop. The process also utilizes an adapted protocol for emulsion PCR to further increase the efficiency of the selection process. After 11 rounds of selection, the DNA pools were sequenced leading to the generation of 14 potential aptamers. These aptamers were tested with the isolated protein and with human red blood cells resulting in several of the aptamers being deemed potential binders. Further work with these identified sequences could result in aptamers that can be reliably used to tag and delicately separate red blood cells from other cells of interest within blood, such as stem cells. The novel approaches to selection used in this work may also lead to quicker and more efficient generation of future aptamers.
9

Detection of changes related to breast cancer using charge sensors or mass sensors coupled to DNA monolayers / Detecção de alterações relacionadas ao câncer de mama através de sensores de carga ou de massa acoplados a monocamadas de DNA

Batistuti, Marina Ribeiro 26 May 2017 (has links)
The electrochemical biosensor has been extensively used due to its capacity for rapid and accurate detection of a wide variety of target molecules or biomarkers. DNA hybridization sensors are based on the increase of negative charge on the electrode surface after the DNA target hybridize to the immobilized probes. The development of this platform requires first an understanding of the immobilization process and optimization of surface probe density. In this thesis the electron transfer is investigated on a label-free DNA hybridization detection by its intrinsic charge. The investigation using different immobilization buffers shows a strong dependence on their composition and concentration, and also the influence of the probe and spacer co-immobilized to obtain an organized and compact self-assembled monolayer. The probe density is determined using the chronocoulometry method with hexaammineruthenium (III) chloride, where the value is calculated from the number of cationic redox molecules electrostatically associated with the anionic DNA backbone and presented a linear relationship between thiol molar fraction and probe density from 2 to 5 x 1012 molecules/cm2. The effect of hybridization was determined using electrochemical impedance spectroscopy using negatively charged ferri/ferrocyanide redox couple in solution. After probe surface density optimization, the maximum shift of charge transfer resistence (20%) upon 1 M complementary sequence was obtained with around 25% probe fraction immobilized vii viii on surface. This electrochemical platform developed was able to detected 100 pM of target sequence and distinguish mismatched sequences. The limit of detection is higher when compared to the literature, however, this system can be further improved by amplifing the signal. The same platform is reproduced in the quartz crystal microbalance system and with field-effect transistor, comparing the different detections. The same platform is tested using two different HER2 aptamer sequences. Biological aspects are explored for a better understanding of the system / O biossensor eletroquímico foi amplamente utilizado devido à sua capacidade de Detecção rápida e precisa de uma grande variedade de moléculas alvo ou biomarcadores. Os sensores de hibridização do DNA são baseados no aumento da carga negativa na Superfície do eletrodo depois que o alvo do DNA se hibridar com as sondas imobilizadas. o O desenvolvimento desta plataforma exige primeiro uma compreensão da imobilização Processo e otimização da densidade da sonda de superfície. Nesta tese, o elétron A transferência é investigada em uma detecção de hibridação de DNA sem rótulas por sua intrínseca carregar. A investigação usando diferentes buffers de imobilização mostra uma forte Dependência de sua composição e concentração, e também a influência da Sonda e espaçador co-imobilizados para obter uma auto-montagem organizada e compacta Monocamada. A densidade da sonda é determinada usando o método de cronoculometria Com cloreto de hexaamminerutenio (III), onde o valor é calculado a partir do Número de moléculas redox catiónicas associadas eletrostática com o aniónico DNA backbone e apresentou uma relação linear entre a fração molar tiol E densidade de sonda de 2 a 5 x 1012 moléculas / cm2. O efeito da hibridação Foi determinado usando espectroscopia de impedância eletroquímica usando negativamente Casal redox ferri / ferrocianeto carregado em solução. Após a densidade da superfície da sonda Otimização, a mudança máxima da resistência à transferência de carga (20%) após 1 M A sequência complementar foi obtida com cerca de 25% de fração de sonda imobilizada Vii Viii Na superfície. Esta plataforma eletroquímica desenvolvida foi capaz de detectar 100 pM Da sequência alvo e distinguir sequências incompatíveis. O limite de detecção É maior quando comparado à literatura, no entanto, esse sistema pode ser mais Melhorou ampliando o sinal. A mesma plataforma é reproduzida no Sistema de microbalança de cristal de quartzo e com transistor de efeito de campo, comparando o Detecções diferentes. A mesma plataforma é testada usando dois diferentes HERMETO aptamer Sequências. Os aspectos biológicos são explorados para uma melhor compreensão do sistema
10

Characterization of the binding activity of immobilized DNA aptamers for nucleotide and non-nucleotide targets

Dunaway, Adam Blake 07 January 2016 (has links)
Deoxyribonucleic acid (DNA) aptamers are oligonucleotides with high specificity and affinity for non-nucleotide targets ranging from molecular species to cellular proteins. Their high affinity, rapid synthesis, and the ease with which they can be chemically modified to include convenient chemical groups (e.g. amine group on 5’ end) make them excellent adaptable ligands for use in colloidal drug delivery vehicles for both uptake and release of therapeutic agents. This work uses pre-identified aptamers for vascular endothelial growth factor (VEGF) to investigate the design of one such vehicle for controlled uptake and release of target therapeutics and analyzes the ability of particle-immobilized aptamers to bind both nucleotide and non-nucleotide targets. Aptamer sequences are immobilized on colloidal microspheres and binding activity of both the primary DNA and protein targets are directly monitored using flow cytometry. Additionally, the dual nature of aptamer-target binding is further investigated by evaluating the effects of simultaneous and serial incubation of the primary targets. Finally, the ability to recover the functionality of the aptamer is evaluated after displacement of the primary DNA target through DNA mediated interactions. It has been shown that the nature of aptamer-target interactions are complex in nature, requiring optimization for each species incorporated into a delivery vehicle; however, partial recovery of aptamer functionality was achieved after hybridization with the primary DNA target.

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