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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

On-chip Labeling via Surface Initiated Enzymatic Polymerization (SIEP) for Nucleic Acids Hybridization Detection

Tjong, Vinalia January 2013 (has links)
<p>Current techniques for nucleic acid analysis often involve extensive sample preparation that requires skilled personnel and multiple purification steps. In this dissertation, we introduce an on-chip, isothermal, post-hybridization labeling and signal amplification technique that can directly interrogate unmodified DNA and RNA samples on a microarray format, eliminating the need for microarray sample pre-processing. </p><p>We name this technique Surface Initiated Enzymatic Polymerization (SIEP), where we exploit the ability of a template independent DNA polymerase called Terminal Deoxynucleotidyl Transferase (TdT) to catalyze the formation of long single-stranded DNA (ssDNA) chain from the 3'-end of a short DNA primer, which is tethered on the surface, and TdT's ability to incorporate unnatural reporter nucleotides, such as fluorescent nucleotides. We hypothesize that polymerization of a long ssDNA chain while incorporating multiple fluorescent nucleotides on target DNA or RNA hybridized to probe printed on a surface will provide a simple and powerful, isothermal method for on-chip labeling and signal amplification. </p><p>We developed the SIEP methodology by first characterizing TdT biochemical reaction to polymerize long homopolymer ssDNA (> 1000 bases) starting from the 3'-OH of ten bases oligonucleotides. We found that the preferred monomers (deoxynucleotide, dNTP) are dATP and dTTP, and that the length of the ssDNA extension is determined by the ratio of input monomer (dNTP) to initiator (short oligonucleotides). We also investigated TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the initiation efficiency, the degree of fluorophore incorporation, the length and the polydispersity of the polymerized DNA strand. These experiments allowed us to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using SIEP as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a up to ~45 fold increase in signal amplification compared to the incorporation of a single fluorophore.</p><p>SIEP was then employed to detect hybridization of DNA (25 bases), short miRNA (21 bases) and long mRNA (1400 bases) by the post-hybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of hybridized target strands. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection (LOD) and a 2-log linear dynamic range while the detection of short miRNA and fragmented mRNA targets resulted in ~2 pM and ~10 pM LOD, respectively with a 3-log linear dynamic range.</p><p>We further developed SIEP for colorimetric detection by exploiting the presence of negatively charged phosphate backbone on the surface as target DNA or RNA hybridizes on the immobilized probe. The net negative charge on the surface is further increased by TdT catalyzed polymerization of long ssDNA. We then used positively charged gold nanoparticles as reporters, which can be further amplified through electroless metallization, creating DNA spots that are visible by eye. We observed an increase of 100 fold in LOD due to SIEP amplification.</p><p>Overall, we demonstrated the use of SIEP methodology to label unmodified target DNA and RNA on chip, which can be detected through fluorescence signal or colorimetric signal of metallized DNA spots. This methodology is straightforward and versatile, is compatible with current microarray technology, and can be implemented using commercially available reagents.</p> / Dissertation
2

Occurrence of a Terminal Deoxynucleotidyl Transferase-Like Activity in N-2-Fluorenylacetamide-treated Rat Liver

KOJIMA, KIYOHIDE, NAKAMURA, HIROMU, YOSHIDA, SHONEN 01 1900 (has links)
No description available.
3

Molecular mechanisms leading to the emergence of mouse regulatory T lymphocytes specific to non-inherited maternal antigens

Li, Shuang 08 June 2021 (has links) (PDF)
[EN]It is well illustrated that the generation of Tregs is the main mechanism responsible for maintaining immune tolerance during developmental exposure to non-inherited maternal antigen (NIMA). Moreover, the presence of NIMA-specific Tregs in the uterus of pregnant mice promote reproductive fitness by enforcing maternal tolerance to overlapping paternal antigens expressed by the fetus during next-generation pregnancies. However, the reason why perinatal T cell lineage is biased towards immune tolerance is poorly understood. Due to the fact that terminal deoxynucleotidyl transferase (TdT) is not expressed in neonatal T cells in the mouse, neonatal T cells have a less diverse TCR repertoire. This is known to limit their specificity and to increase their affinity for MHC/peptide complexes. At the start of the present work, we postulated that expressing high affinity TCR might be the reason that forces the development of antigen-specific Tregs in neonates. We undertook our study with the aim to investigate the mechanisms underlying mouse NIMA-specific Treg development in the perinatal period. Using 2W1S-OVA+ heterozygous mouse model in which 2W1S antigen was transformed into surrogate NIMA for half of the offspring, we observed an increased frequency of 2W1S-specific Tregs in NIMA-2W1S-exposed animals. Moreover, we also observed that periphery-derived NIMA-2W1S Tregs had a less diverse TCR repertoire and were phenotypically distinct from thymus-derived SELF-2W1S-specific Tregs. In order to investigate whether the lack of diversity was responsible for the development of neonatal NIMA-specific Tregs, we generated transgenic mice where TdT expression was enforced in T cells before birth. We found that transgenic TdT added clonal TCR diversity but did not prevent the development of T cell clones with neonatal type TCR repertoire and did not modify the frequency of neonatal NIMA-specific Tregs. On the contrary, TdT expression increased significantly generation of SELF-specific Tregs to levels similar to that of NIMA-specific Tregs. Taken together, our data indicate that the developmental pathways of NIMA- and SELF-specific Treg repertoire are different in terms of inducing and maintaining neonatal tolerance. / [FR]Il est bien illustré que la génération périnatal de Treg est le principal mécanisme responsable du maintien de la tolérance immunitaire fœtale qui se développe suite à l'exposition aux antigènes maternels non-hérités (NIMA). De plus, la présence de Tregs spécifiques des NIMA dans l'utérus des femmes enceintes favorise la capacité de reproduction en renforçant la tolérance maternelle aux mêmes antigènes paternels exprimés par le fœtus pendant les grossesses de prochaine génération. Cependant, la raison pour laquelle la lignée des cellules T fœtales est biaisée en faveur de la tolérance immunitaire est mal comprise. Chez la souris, en raison du manque d'expression de la désoxynucléotidyl transférase terminale (TdT), les cellules T néonatales ont un répertoire de TCR moins diversifié. Ceci est connu pour limiter leur spécificité et augmenter leur affinité pour les complexes CMH / peptide. Au début du présent travail, nous avons émis l'hypothèse que l'expression de TCRs de haute affinité pourrait être la raison qui force le développement de Treg spécifiques chez les nouveau-nés. Nous avons plus particulièrement entrepris notre étude dans le but d'étudier les mécanismes sous-jacents au développement de Tregs spécifiques des NIMA chez la souris pendant la période périnatale. En utilisant le modèle de souris hétérozygotes pour 2W1S-OVA+ dans lequel l'antigène 2W1S a été transformé en NIMA pour la moitié de la progéniture, nous avons observé une fréquence accrue de Tregs spécifiques de 2W1S chez les animaux exposés au NIMA. De plus, nous avons également observé que les Treg NIMA-2W1S dérivés de la périphérie avaient un répertoire de TCRs moins diversifié et étaient phénotypiquement distincts des Tregs spécifiques de SELF-2W1S dérivés du thymus. Afin de déterminer si le manque de diversité était responsable du développement de Tregs néonataux spécifiques de NIMA, nous avons généré des souris transgéniques où l'expression de TdT était appliquée dans les cellules T avant la naissance. Nous avons constaté que le TdT transgénique ajoutait une diversité de TCR clonale, mais n'empêchait pas le développement de clones de cellules T avec un répertoire TCR de type néonatal et ne modifiait pas la fréquence des Treg néonataux spécifiques du NIMA. Au contraire, l'expression de TdT a augmenté de manière significative la génération de Tregs spécifiques de SELF-2W1S à des niveaux similaires à ceux des Treg spécifiques de NIMA-2W1S. Prises ensembles, nos données indiquent que les voies de développement du répertoire des Tregs néonataux spécifiques de NIMA et SELF sont différentes en termes d'induction et de maintien de la tolérance néonatale. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished
4

Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress conditions

Swain, Lija 07 July 2014 (has links)
No description available.
5

SCF cdc4 regulates msn2 and msn4 dependent gene expression to counteract hog1 induced lethality

Vendrell Arasa, Alexandre 16 January 2009 (has links)
L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.

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