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1
Button, Eric A. "Regulation of T-DNA gene 7." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26177.
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The purpose of this study was two-fold. The first objective was to determine if Saccharomyces cerevisiae is a useful system for investigating the expression of T-DNA (it takes several months to obtain sufficient bacteria-free transformed plant tissue to investigate T-DNA transcription). A short fragment of T-DNA carrying T-DNA gene 7 was cloned into a yeast plasmid in an attempt to investigate the expression of gene 7 in yeast. The second objective was to determine the significance of a heat shock related sequence identified in the 5¹ region of T-DNA gene 7.
Primer extension analysis, SI nuclease mapping, and Northern hybridizations indicate that transcription of T-DNA gene 7 in yeast is different from that of transcription of gene 7 in crown gall tumors. Transcription is different because the distance between the TATA box and the transcription initiation sites must be at least 40 nucleotides in yeast. Therefore, Saccharomyces cerevisiae does not appear to be a useful system for investigating the expression of T-DNA.
Crown gall tumors were subjected to a number of stress agents, including heat shock, to determine the significance of the heat shock related sequence identified in gene 7. Primer extension analyses indicate that only cadmium and mercury have a significant effect on the expression of T-DNA gene 7. Although gene 7 responds to cadmium and mercury, the increase in transcription does not appear to be heat shock or metallothionein related, indicating that another mechanism is involved in the enhanced transcription of T-DNA gene 7 in crown gall tumors.<br>Medicine, Faculty of<br>Medical Genetics, Department of<br>Graduate
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Simanis, V. "T antigen binding sequences in cellular DNA." Thesis, Imperial College London, 1985. http://hdl.handle.net/10044/1/37854.
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Joseph, Ansamma K. "DNA sequence analysis of T cell receptors." Thesis, University of Bath, 1996. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321849.
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Lindencrona, Jan Alvar. "Enhancing T cell mediated immunity in DNA vaccination /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-710-x.
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Sunstrom, Noëlle-Ann. "Specific DNA binding by polyomavirus large T antigen." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=70257.
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To define the DNA binding domain of polyomavirus large T antigen, systematic deletion mutagenesis was carried out on large T antigen. A set of plasmids coding for unidirectional carboxy- or amino-terminal deletion mutations in large T antigen were constructed. Deleted proteins were expressed by plasmid transfection of Cos-1 cells. Analysis of origin-specific DNA binding by mutant proteins revealed that the C-terminal boundary of the DNA-binding domain is at or near Glu 398. Fusion proteins of large T antigen lacking the first 200 N-terminal amino acids bound specifically to polyomavirus origin DNA; however, deletions beyond this site resulted in unstable proteins which could not be tested for DNA binding. Testing of point mutants and internal deletions by others suggest that the N-terminal boundary of the DNA-binding domain lies between amino acids 282 and 286. Taken together, these results locate the DNA-binding domain of polyomavirus large T antigen to the 116- amino acid region between residues 282 and 398.<br>Polyomavirus large T antigen binds to four discrete regions within the regulatory region of the viral genome. The relative arrangement of these binding sites may be important for the distinct regulatory functions performed by the protein. Each of the four large T antigen binding sites was separately cloned into a test plasmid by the use of oligonucleotide-directed mutagenesis. The binding affinity of large T antigen was analyzed for each individual site and for combinations of sites. The results of this analysis showed that there exists an hierarchy of binding strength among individual and combinations of large T antigen binding sites as measured by DNA immunoprecipitation. In addition, the DNA binding of large T antigen involves cooperative interaction of protein molecules between adjacent binding sites, and the integrity of the amino terminus is required for cooperativity.
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Measures, Kathryn M. "Computational approaches to studying protein structures and reactivity." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321374.
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Stoll, Marion. "Aktivierende T-DNA-Mutagenese in Nicotiana-tabacum-Mesophyll-Protoplasten." [S.l. : s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=96491638X.
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Narita, Takeo. "Human replicative DNA polymerase δ can bypass T-T (6-4) ultraviolet photoproducts on template strands". Kyoto University, 2014. http://hdl.handle.net/2433/188688.
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Fontenot, Jason David. "The role of Foxp3 in CD4⁺ T cell development and function /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8336.
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Forsbach, Alexandra. "T-DNA-Integration und Regulation von Transgenexpression in Arabidopsis thaliana." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=96268368X.
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Ho, Siu-ki, and 何肇騏. "DNA methylation patterns in t(8;21) acute myeloid leukemia patients." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47151389.
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Acute myeloid leukemia (AML) is a heterogeneous disease both clinically and
biologically. Approximately 55% of AML harbour karyotypic changes, and one of the most common chromosomal aberrations is the t(8;21)(q22;q22), which leads to the AML1-ETO fusion protein. Previous studies have found that this fusion protein recruits the N-CoR/mSin3A/HDAC complex, thereby acts as a transcriptional repressor. Recently, DNA methylation array studies have shown that DNA methylation patterns can stratify AML cases into different subgroups, and some of these correspond to certain chromosomal abnormalities, such as the t(8;21). These findings suggest a possible link between the fusion transcript AML1-ETO and epigenetic modifications. Additionally, c-kit mutations have emerged as an important disease modifier in the t(8;21) AML and are correlated with poor overall survival and event free survival in patients with t(8;21) AML. We therefore sought to investigate whether there are different DNA methylation patterns in t(8;21) AML with or without c-kit mutations. In our series, 52.2% of the t(8;21) AMLs harbored c-kit mutations, which were correlated with poor event free survival. We next performed pyrosequencing on a selected panel of genes and pinpointed the THBS4 and PAWR genes as hypermethylated in their promoter CpG islands in 86.4% and 59.1% of the t(8;21) AML patients, respectively. These data suggest that THBS4 and PAWR may be important in the pathogenesis of t(8;21) AML.<br>published_or_final_version<br>Pathology<br>Master<br>Master of Philosophy
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Creusot, Remi Jerome. "Particle-mediated DNA immunisation : CD+T cell priming and cooperation." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399172.
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Feltquate, David Marc. "Helper T Cell Differentiation in DNA-Immunized Mice: A Dissertation." eScholarship@UMMS, 1998. http://escholarship.umassmed.edu/gsbs_diss/181.
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DNA immunization, inoculation with an antigen-expressing plasmid DNA, is a new method for generating an antigen-specific immune response. At the time these investigations began, very little was known about the immune response produced by DNA vaccines. Thus, the first aim of our studies was to perform a detailed examination of the antibody response generated by DNA immunization with an influenza hemagglutinin (HA)-expressing DNA in BALB/c mice. Using several different routes and methods of DNA immunization, we observed a number of findings. Although all three forms of DNA immunization elicited strong anti-HA antibody responses, i.m. and i.d. saline DNA immunization required approximately 100 times more DNA than a gene gun DNA immunization to raise an equivalent titer of anti-HA antibody. Indeed, as little as one inoculation and one boost by gene gun of 0.0004 μg of DNA produced a measurable antibody response in 50% of mice. Unexpectedly, we found the isotype of the antibody response differed among groups of mice immunized by different forms of DNA immunization. Intramuscular and i.d. saline DNA immunization produced predominantly an IgG2a anti-HA antibody response, whereas gene gun DNA immunization elicited mostly an IgG1 anti-HA antibody response.
Considering that IgG2a and IgG1 antibody isotypes were known to correlate with Th1 and Th2 immune responses, respectively, we analyzed the type of immune responses produced by i.m., i.d., and gene gun DNA immunization. We found that i.m. and i.d. saline DNA immunization produced a Th1 predominant cellular immune response. In contrast, gene gun DNA immunization produced a Th2 cellular immune response. The differences in the type of immune responses were found to be due to the method of DNA immunization, and not due to the route of DNA inoculation. A gene gun DNA immunization of muscle produced the same IgG1, Th2 immune response as a gene gun DNA immunization of skin, while a saline DNA immunization of muscle and skin produced mostly an IgG2a, Th1 immune response. Each method of DNA immunization created good memory Th cell responses. The type of immune response created by an initial DNA immunization remained fixed even after multiple boosts with the identical method of DNA immunization, following a boost with the alternative method of DNA immunization, or after a viral challenge.
The differentiation of naive Th cells into Th1 or Th2 cells depends on a variety of factors. We performed many experiments to elucidate which factors played a role in the generation of Th1 or Th2 immune responses following saline DNA immunization and gene gun DNA immunization. DNA dose response studies revealed the use of different doses of DNA between groups of saline DNA and gene gun DNA immunized mice did not account for the differentiation of distinct Th cell subsets. Cytokine production inducible by a number of factors inherently associated with either saline DNA or gene gun DNA immunization did not affect Th differentiation. For instance, contamination of plasmid DNA with lipopolysaccharide did not account for differences in the immune response. Immunostimulatory CpG sequences did not affect Th differentiation following DNA immunization, but they did enhance the IgG2a antibody response to coinoculated HA protein. Finally, cotransfection of IFNγ or IL-4 expressing plasmids with an HA-expressing plasmid by gene gun inoculation or as a saline DNA injection did not shift the type of immune response in a Th1 or Th2 direction, respectively. Thus, it appeared that increased cytokine stimulation was not responsible for selective Th subset differentiation.
One factor related to the method of DNA immunization did seem to correlate with Th1 differentiation. Deposition of plasmid DNA extracellulary by saline DNA injections (as opposed to intracellular DNA delivery by gene gun) may have stimulated Th1 immune responses. Manipulating a gene gun DNA immunization to deliver DNA to the dermis (and thus extracellularly) shifted the immune response from that of a Th2 type to a mixed Th1/Th2 type. Furthermore, evidence was gathered demonstrating that pDNA can interact with cell surface molecules and that specific sequences in pDNA can act as a ligand and bind to molecules. Taken together, our data led us to propose a new model for Th1 differentiation following saline DNA immunization. We believe extracellular pDNA binds to an APC cell surface molecule which activates the cell. The activated APC preferentially stimulates naive Th cells to differentiate into Th1 cells.
Finally, studies using a variety of mice differing in their genetic backgound and MHC genotype demonstrated the generality of our findings regarding i.m. saline DNA inoculations of an HA-expressing pDNA. Saline DNA immunization produced IgG2a, Th1-predominant immune responses independent of the genetic background and MHC genotype of the mice. In contrast, the type of immune response elicited by a gene gun DNA immunization was dependent on the MHC genotype of mice. Thus the type of immune response produced by gene gun DNA immunization probably depends on the specific antigen (and its effect on MHC-peptide/TcR interaction and signaling) and is less likely due to any inherent feature associated with the process of gene gun DNA delivery.
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Fobert, Pierre R. (Pierre Rheal) Carleton University Dissertation Biology. "Characterization of chromosomal sites of T-DNA integration by activation of a promoterless B-glucuronidase (GUS) gene linked to the T-DNA right border repeat." Ottawa, 1992.
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Soares, Daiane da Rocha. "Modulação de Orc1/Cdc6 de Trypanosoma brucei pela ligação e hidrólise de ATP." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-13082014-184135/.
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O Complexo de pré-replicação em T.brucei é composto por Orc1/Cdc6 e as helicases MCMs. Em um trabalho anterior mostramos que TbOrc1/Cdc6 pode ligar e hidrolisar ATP in vitro. Neste sentido, o objetivo deste trabalho é avaliar a importância da hidrólise e ligação de ATP para a formação e estabilidade do complexo pré-replicação de T.brucei. Para tanto, foram geradas proteínas recombinantes Orc1/Cdc6 de T. brucei mutadas nas regiões Walker A (TbOrc1/Cdc6K79T) ou sensor 2 (TbOrc1/Cdc6R251,252E) incapazes de ligar ou hidrolisar ATP, respectivamente. Finalmente, as células expressando TbOrc1/Cdc6K79T ou TbOrc1/Cdc6R251,252E foram avaliadas quanto a (i ) estabilidade da interação Orc1/Cdc6 -DNA, (ii) capacidade de estabilizar MCM no DNA, (iii) capacidade de replicar seu DNA. A mutação na região sensor 2 de T.brucei (TbOrc1/Cdc6R251,252E) reduziu drasticamente a atividade de ATPase em comparação com a proteína selvagem . TbOrc1/Cdc6 mutado no sitio de ligação ao ATP perdeu a capacidade de interagir com o ATP (TbOrc1/Cdc6K79T). A super expressão desses genes inibiu de forma significativa a proliferação celular, causou ineficiência no carregamento de MCM para o DNA e ocasionou falhas na progressão do ciclo celular, atrasando a fase S.<br>The pre-replication complex in T.brucei is composed of at Orc1/Cdc6 and MCMs helicases. In a previous paper we showed that TbOrc1/Cdc6 can bind and hydrolyze ATP in vitro. Based on that, the objective of this study is to evaluate the importance of ATP binding and hydrolysis to the formation and stability of the pre - replication complex in T.brucei. For this purpose, T. brucei Orc1/Cdc6 recombinant proteins were generated mutated at regions on Walker A (TbOrc1/Cdc6K79T) and sensor 2 (TbOrc1/Cdc6R251 , 252E) in order to unable the ATP binding and hydrolyzation respectively . Finally , cells expressing TbOrc1/Cdc6K79T or TbOrc1/Cdc6R251 , 252E were evaluated for (i) stability of Orc1/Cdc6 - DNA interaction , (ii) ability to stabilize MCM in DNA , (iii) ability to replicate its DNA . The mutation in the sensor 2 region of T.brucei (TbOrc1/Cdc6R251 , 252E) drastically reduced the ATPase activity compared to the wild-type protein. TbOrc1/Cdc6 mutated in the ATP binding site has lost the ability to interact with ATP (TbOrc1/Cdc6K79T). The overexpression of these genes significantly inhibited cell proliferation causing inefficient loading of MCM DNA and led to failure in cell cycle progression by delaying the phase S.
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Bourras, Salim Ahmed. "Pathogenomics of the fungal phytopathogen Leptosphaeria maculans : exploitation of a large T-DNA mutagenized collection to elucidate T-DNA integration patterns and identify new pathogenicity determinants." Thesis, Paris 11, 2012. http://www.theses.fr/2012PA112072.
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Leptosphaeria maculans est un Dothideomycète phytopathogène capable d’alterner des modes de vie saprophyte, hemibiotrophe endophyte et nécrotrophe durant son cycle infectieux sur le colza. Afin de comprendre cette plasticité infectieuse, une mutagenèse aléatoire à grande échelle par ATMT a permis de générer une collection de 5000 transformants. L’objectif principal de cette thèse était d’exploiter cette collection en prenant avantage de la disponibilité d’outils de génomique, pour, d’une part, comprendre les mécanismes d’intégration de l’ADN-T dans le génome, et d’autre part, identifier des déterminants du pouvoir pathogène ciblés par l’ADN-T dans des mutants affectés dans leur capacité infectieuse. Une analyse systématique de 318 pieds d’insertion a été réalisée dans le but de d’évaluer les caractéristiques de l’insertion de l’ADN-T. Ce dernier est fréquemment inséré dans les régions actives riches en gènes, et favorise des gènes impliqués dans des processus biologiques indicatifs d’une conidie germante. Un biais marqué des insertions en faveur des régions régulatrices est observé ainsi qu’une corrélation entre la densité des insertions et le skew CG près du site d’initiation de la transcription. Ces observations sont cohérentes avec le modèle de ciblage intranucléaire de l’ADN-T. Enfin, l’existence de micro-homologies entre le site de pré-insertion et la bordure gauche de l’ADN-T indique une intégration par voie de recombinaison homologue (HR) et/ou microhomologue (MMEJ). Une approche multicritère a été utilisée pour caractériser cette collection. Un test d’inoculation a permis d’identifier 166 mutants altérés dans leur pouvoir pathogène. La validation génétique de la co-ségrégation entre le phénotype altéré et la présence de l’ADN-T indique que 44% des mutants montrant un déterminisme monogénique de l’altération sont effectivement étiquetés. Parmi les mutants altérés, 35 ont été analysé pour : (i) le phénotype de croissance en conditions usuelles de culture et en réponse aux stress oxydatif, UV et nutritif, (ii) le lien entre altération de la germination et pouvoir pathogène. Les gènes affectés par l’intégration de l’ADN-T, ont été identifié et analysé dans la souche sauvage à l’aide de données de transcriptomique. Ces cribles ont permis de décomposer le phénotype d’altération in planta en plusieurs phénotypes in vitro. Le plus fréquemment, les mutants ne sont pas altérés dans leur in vitro croissance, mais la plupart sont sensibles aux ROS. Les analyses d’expression au cours de l’infection indiquent que les gènes codant pour des effecteurs et ceux impliqués dans la détoxification des ROS, ont des dynamiques d’expression inverses et bi-phasiques, en lien avec le style de vie hemibiotrophe de L. maculans. Les 42 gènes ciblés par l’ADN-T dans ces mutants ont été identifiés et leur fonction putative disséquée par bioinformatique et transcriptomique. Au final, nous avons identifié six mutants d’intérêt pour une caractérisation fonctionnelle. Deux de ceux-ci deux ont atteint un niveau de caractérisation suffisant pour l’émission d’une hypothèse de travail. Dans le mutant µ1165, l’ADN-T cible un gène codant pour une protéine ribosomale S17 (LmRPS17), dont la régulation dépendrait de la voie de signalisation du complex TORC1. Nos résultats préliminaires suggèrent, d’une part, que TORC1 est une cible potentielle de LmRPS17 et, d’autre part, que la phase biotrophe de l’infection chez L. maculans est probablement régulée par TORC1 via l’enzyme de détoxication des ROS SOD2. Dans le mutant µ444, l’ADN-T cible un gène codant pour un élément rétroviral putatif LmHYP1, largement conservé parmi les ascomycètes. Nos résultats suggèrent que LmHYP1 interviendrait dans la régulation de gènes impliqués dans le pouvoir pathogène, via la production de petits ARN interférents issus hypothétiquement du clivage de son ARN messager par la machinerie de défense anti-rétrovirale. La validation de ces deux hypothèses est en cours au laboratoire<br>The Dothideomycete phytopathogen Leptosphaeria maculans is capable to alternate saprophytic, hemibiotrophic, endophytic and necrotrophic life styles during a single infectious cycle on its host plant, Brassica napus. However, little is known about the determinants of such plasticity. As part of a large-scale insertional mutagenesis project aiming at the discovery of pathogenicity factors a collection 5000 transformants has been generated by ATMT. The main objective of my PhD was to take advantage of “omics” to extract biological value from L. maculans mutants collection for a better understanding of T-DNA integration features and further identification of pathogenesis determinants in this fungus. A systematic analysis of 318 T-DNA tags was performed in a large collection of transformants in order to evaluate the features of T-DNA integration in its particular TE-rich compartmentalized genome. The T-DNA integration was mainly targeted to gene-rich, transcriptionally active regions, and favored biological processes that are consistent with the physiological status of a germinating conidia. In addition, T-DNA integration was strongly biased towards regulatory regions, and mainly promoters. Consistently with the T-DNA intranuclear targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination and/or the microhomology-mediated end joining pathways. Based on this data, a multi-criteria approach was used to draw the more possible information from this collection by identifying all 166 mutants reliably affected in pathogenicity, and expanding the genetic analysis. Considering single-gene segregation of the pathogenicity phenotype, our data indicate a 44% ratio of actual tagging. In parallel, for a subset of 35 isolates of the collection, we (i) described growth patterns in regular culture conditions or in response to oxidative, UV or starvation stresses, (ii) evaluated the link between altered germination and pathogenicity, (iii) identified and annotated the genes putatively affected by the T-DNA integration, and (iv) analyzed kinetics of expression of these genes in the WT isolate using available microarrays. Our results showed that pathogenicity alteration phenotype could be broken down into a pattern of phenotypes in vitro including growth, germination defect and susceptibilities to oxidative burst, starvation and UV stresses. Our results showed that most of these mutants were not altered in axenic growth but showed enhanced sensitivity to reactive oxygen species (ROS). Furthermore, our results showed that effectors and ROS scavengers have inverted dynamics during plant infection, consistently with the biphasic hemibiotrophic growth of L. maculans. Also, 42 genes targeted by the T-DNA in these mutants were recovered and dissected. This catalogue allowed us to identify a series of promising mutants for further functional studies. Based on this screening, six mutants were subjected to further analyses but only two reached sufficient advance for hypothesis building. In mutant µ1165, the T-DNA targeted a ribosomal protein S17 (LmRPS17), a downstream component of target of rapamycin complex 1 (TORC1) pathway. Our preliminary results suggested that TORC1 is a potential a target of LmRPS17. Also, biotrophic growth is probably tuned by TORC1 via Super oxide dismutase 2 (SOD2). In mutant µ444, the T-DNA targeted a retroviral-like element LmHyp1 widely conserved among ascomycetes. Our results suggest that LmHyp1 probably acts as a regulatory element during plant infection as its cleavage by the antiretroviral defences is hypothesized to generate siRNAs that silences distant genes. Work on these mutants is in progress
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Amorim, Juliana de. "Geração de células T de memória e linfócitos T reguladores em camundongos BALB/c vacinados com vetor plasmidial contendo o inserto P10 de Paracoccidioides brasiliensis." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-07102010-094556/.
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Paracoccidioides brasiliensis é um fungo dimórfico patogênico agente etiológico da paracoccidioidomicose (PCM), uma micose endêmica no Brasil. A busca por alternativas para reduzir o tempo de tratamento da PCM levou ao desenvolvimento de uma vacina de DNA contendo a sequência do peptídeo P10 de P. brasiliensis. Neste trabalho, avaliamos a geração de células T de memória e células T reguladoras em camundongos imunizados com esta vacina de DNA antes e após o desafio com o fungo, através da análise de seus esplenócitos e linfócitos pulmonares por citometria de fluxo. Os resultados mostram um aumento no percentual de células T reguladoras e de memória no baço e pulmões dos animais imunizados antes e depois de 30, 60 e 120 dias do desafio em comparação com os grupos controle e não imunizado. Outro experimento revelou que o modelo experimental da PCM in vivo é capaz de induzir a expressão de RORγt. Este estudo mostra que nossa vacina de DNA contra a PCM gera células com fenótipo de reguladoras e de memória, caracterizando seu potencial para o tratamento desta micose.<br>Paracoccidioides brasiliensis is a dimorphic fungal pathogen that is the etiological agent of paracoccidioidomycosis (PCM), a mycosis endemic in Brazil. The search for new alternatives to reduce the duration of PCM treatment led to the development of a DNA vaccine encoding the peptide P10 of P. brasiliensis. Presently, we evaluated the generation of memory and regulatory T cells in mice immunized with this DNA vaccine, before and after the challenge with the fungus by analizing their splenocytes and pulmonary lymphocytes by flow cytometry. The results show an increase in the percentage of regulatory and memory T cells on spleens and lungs of immunized mice before and after 30, 60 and 120 days of challenge compared with the control and untreated groups. Another experiment revealed that the PCM in vivo infection model is capable of inducing RORγt expression. This study demonstrates that our DNA vaccine against PCM generates cells with a regulatory and memory phenotype, which shows its potencial in the treatment of this mycosis.
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Rocha, Pedro. "Promoter trapping in Arabidopsis thaliana : characterization of T-DNA tagged lines." Thesis, University of Leicester, 1999. http://hdl.handle.net/2381/29798.
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Two A. thaliana lines transgenic for the promoter-trap vector pgusBin19 were studied. Line AtEN101 lacked a phenotype but reporter gene (gus) expression exhibited temporal and spatial regulation, including in the developing embryo. This pattern was unaltered when AtEN101 was used as a functional marker in two embryo-defective mutant backgrounds tested. In roots of AtEN101 seedlings, gus expression was repressed by ABA and modified by auxins, in a sucrose-modulated manner. The tagged genomic region in the mutant and wildtype, and cDNAs mapping to it were isolated. The cDNAs derived from two overlapping genes (PKT1 and PKT2) almost identical except for their first exons and introns and encoding putative peroxisomal 3-ketoacyl-CoA thiolases. The T-DNA was inserted in the long intron1 (966 bp) of PKT1, and 0.3 kb upstream from exon1 of PKT2. gus copies were found at both T-NDA junctions. Fusion transcripts including exon1 of PKT1 and T-DNA sequences were identified, and a cryptic 3' splice site identified in the left border region. The genes were found to be independently expressed, at low levels, in all organs by Northern blot hybridization and RT-PCR. Both putative promoter regions were capable of driving the expression of gus in transient assays. That of PKT1 was also functional in reverse orientation. The thiolase genes are part of a family of at least four members, PKT1 being unique in lacking the characteristic N-terminal peroxisomal targeting signal, PTS2. A new T-DNA linked semi-dominant mutation causing dwarfism (bashful) was identified in line P26K4, gus-derived sequences were present in its genome but no GUS activity was observed. The mutation affected cell growth and was rescued by epibrassinolide but not gibberellic acid (GA3). Skotomorphogenic development is also affected in the mutant. The utility of pgusBin19 as a promoter-trap and insertional mutagen in Arabidopsis, and the possible functions of PKT1/PKT2 are discussed.
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Iwasaki, Akiko. "Cytotoxic T lymphocyte induction by plasmid DNA immunization, mechanism and applications." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq35195.pdf.
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Pavlenko, Maxim. "Induction of T-cell responses against PSA by plasmid DNA immunization /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-385-X/.
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Fullner, Karla Jean. "The pilus assembly and T-DNA transfer machinery of Agrobacterium tumefaciens /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/11497.
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Bauer, Brigitte J. "A bifunctional selectable marker gene for T-DNA tagging of plant promoters." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0030/NQ63839.pdf.
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Yao, Edmund. "Effects of DNA methyltrasferase-inhibition on FOXP3 expresssion in CD4+ T cells." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110525.
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ABSTRACTCD4+Foxp3+ regulatory T cells (TREG) are pivotal in the maintenance of immune tolerance to self-antigens and act as regulators of autoimmune reactions. There are two subsets of TREG: naturally occurring (nTREG) which develop in the thymus and inducible (iTREG) which are generated from conventional Foxp3- effector T cells (TCONV) in the periphery or in vitro. While both subsets require Foxp3 expression to function, iTREG cells gradually downregulate Foxp3 in vitro and lose their suppressive capabilities. In contrast, nTREG cells show constitutive Foxp3 expression and demonstrate stable suppressive function in vitro. Recent studies reveal a region of the Foxp3 locus that is differentially methylated between nTREG and iTREG. The methylation-sensitive region, known as the TREG-specific demethylated region (TSDR), is completely unmethylated in nTREG, partially methylated in iTREG, and heavily methylated in TCONV, suggesting that differential methylation patterns of the TSDR may account for unstable Foxp3 expression in iTREG. Methylation is mediated by DNA methyltransferase (DNMTs) enzymes, and several inhibitors are known to block their activity. In this study, we use 5'-Aza-2'-deoxycytidine (Aza), a nucleoside analog inhibitor, and RG108, a non-nucleoside inhibitor to determine whether inhibition of DNMTs and thus methylation, impacts Foxp3 expression and iTREG development in culture. Our results show that DNMT inhibition in the absence of TGF-β was unable to induce Foxp3 expression in TCONV cells. However, in TCONV cells that were pre-exposed to TGF-β, Aza and RG108 induced Foxp3 expression in a significant number of cells. Moreover, a higher proportion of Foxp3-expressing cells with stronger Foxp3 MFI were induced when Aza was used in conjunction with TGF-β suggesting an additive and non-redundant effect of DNMT-inhibition. Although iTREG cells typically downregulate Foxp3 in the absence of TGF-β, treatment with Aza alone prolongs the persistence of Foxp3 expression. Neither Aza nor RG108 was as effective as TGF-β in maintaining Foxp3 expression. Collectively, our data shows that DNMT inhibition without TGF-β is insufficient to induce Foxp3 expression but in conjunction, can increase induction and strengthen Foxp3 expression. DNMT inhibition can also prolong Foxp3 expression in the absence of TGF-β possibly by disrupting epigenetic silencing mechanisms.<br>ABRÉGÉLes lymphocytes T régulateurs CD4+Foxp3+ (TREG) sont des pivots dans le maintien de la tolérance immunitaire aux antigènes autologues et agissent en tant que régulateurs des réactions auto-immunes. Il y a deux sous-ensembles de TREG : les naturelles (nTREG) qui se développent dans le thymus et les inductibles (iTREG) qui sont générés à partir des lymphocytes T effecteurs Foxp3- conventionnels (TCONV) en périphérie ou in vitro. Bien que les deux sous-ensembles requièrent l'expression de Foxp3 pour fonctionner, les lymphocytes iTREG régulent graduellement de manière négative Foxp3 in vitro et perdent leur capacité suppressive. En revanche, les lymphocytes nTREG présentent une expression constitutive de Foxp3 et démontrent une fonction suppressive stable in vitro. Des études récentes révèlent une région du locus Foxp3 qui est différentiellement méthylée entre les nTREG et les iTREG. La région sensible à la méthylation, connue sous le nom de la région déméthylée spécifique à TREG (TREG-specific demethylated region, TSDR), est complètement non méthylée dans les nTREG, partiellement méthylée dans les iTREG, et fortement méthylée dans les TCONV. Cela suggère que les modèles de méthylation différentielle du TSDR pourraient expliquer l'expression instable de Foxp3 dans les iTREG. Les enzymes ADN méthyltransférases (DNA methyltransferases, DNMTs) servent de médiatrices dans la méthylation, et quelques inhibiteurs sont connus pour bloquer leur activité. Dans cette étude, nous utilisons le 5'-Aza-2'-déoxycytidine (Aza), un inhibiteur analogue nucléosidique, et RG108, un inhibiteur non-nucléosidique, pour déterminer si l'inhibition des DNMTs et ainsi la méthylation, aurait un impact sur l'expression de Foxp3 et le développement de iTREG en culture. Nos résultats montrent que l'inhibition de DNMT en l'absence de TGF-β n'a pas pu induire l'expression de Foxp3 dans les lymphocytes TCONV. Toutefois, dans les lymphocytes TCONV qui étaient pré-exposés au TGF-β, Aza et RG108 ont induit l'expression de Foxp3 dans un nombre important de lymphocytes. De plus, une proportion plus grande de lymphocytes exprimant le Foxp3 avec un Foxp3 MFI plus élevé étaient induits lorsque Aza était utilisé conjointement avec TGF-β, suggérant un effet additif et non redondant de l'inhibition de DNMT. Malgré que les lymphocytes iTREG régulent généralement de façon négative Foxp3 en l'absence de TGF-β, le traitement par Aza seul prolonge la persistance de l'expression de Foxp3. Ni Aza, ni RG108 n'est aussi efficace que TGF-β dans le maintien de l'expression de Foxp3. En somme, nos données montrent que l'inhibition de DNMT sans TGF-β est insuffisant pour induire l'expression de Foxp3, mais que conjointement, peut en augmenter l'induction et la renforcer. L'inhibition de DNMT peut également prolonger l'expression de Foxp3 en l'absence de TGF-β possiblement en perturbant les mécanismes d'inhibition épigénétique.
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Dale, Tracy Maree. "A study of T-DNA integration and transgene expression in Pinus radiata." Thesis, University of Canterbury. Biological Sciences, 2004. http://hdl.handle.net/10092/7223.
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Pinus radiata D Don. is the key resource for the New Zealand forest industry. Genetic engineering could potentially accelerate its genetic improvement by the introduction of novel traits or modification of existing traits, with both environmental and economical importance for the industry.
In this study PCR and Southern hybridisation confirmed stable T-DNA integration in P.radiata plants produced by Agrobacterium-mediated gene transfer. Southern analysis showed a predominance of single T-DNA integration events. However, incomplete transfer leading to truncated copies of the T-DNA appeared to be common. TAIL-PCR was used to identify TDNA/ genomic DNA junctions in five transgenic P.radiata lines. Sequence analysis of these junctions showed attachment of residual vector DNA at the right border for all five lines. We suggest this may reflect the recalcitrance of P.radiata to Agrobacterium infection. Rearrangement of the integrated T-DNA was analysed in detail for two transgenic P.radiata lines and revealed features common to both lines including deletion of T-DNA sequence, presence of plant genomic DNA within the T-DNA loci, and T-DNA rearrangement around the CaMV 355 promoter region.
The visual marker genes β-glucuronidase (uidA) and green fluorescent protein (gfp) were evaluated for their potential use in regenerated P.radiata tissue. Detection of uidA by histochemical staining was poor and unpredictable. Factors within the plant cell appeared to either inhibit the B-glucuronidase enzyme or interfere with the chemistry of the histochemical reaction. GFP could be visualised using epi-fluorescence microscopy in all cell types of needles removed from transgenic P.radiata plants, and could be distinguished from background autofluorescence. An image analysis system was used to compare GFP expression between lines. Transgenic lines with high levels of GFP expression were distinguished from transgenic lines with low levesl of GFP expression. Comparison of GFP expression between clonal shoots propagated from single transgenic P.radiata lines showed that the lines analysed were not
chimeric in origin.
Understanding the predominant characteristics of Agrobacterium-mediated T-DNA integration in P.radiata plants in conjunction with an efficient visual marker-gene system to evaluate transgene expression will improve transformation strategies and thus the stable expression of transgenes.
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Chakravarty, Ashok Hans. "Integration, inheritance and expression of the Agrobacterium rhizogenes Ri plasmid T-DNA." Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334788.
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Yoon, Rosa. "Merkel Cell Polyomavirus Small T Antigen Perturbs the Cellular DNA Damage Response." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17463129.
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Merkel cell polyomavirus (MCPyV) small T antigen (ST) is expressed in the majority of Merkel cell carcinomas (MCC), a highly lethal and aggressive cancer of the skin. Since the discovery of MCPyV in 2008, the role of ST in the context of the virus and MCC has been under intense investigation. Much of our knowledge of polyomavirus ST comes from research on other polyomaviruses, including mouse polyomavirus (MPyV) and simian virus 40 (SV40). Both MPyV and SV40 ST contribute to transformation in part by binding to and inhibiting the cellular phosphatase PP2A. Likewise, MCPyV ST interacts with PP2A, although mutants that are reported to abolish this interaction still transform cells, suggesting that MCPyV ST has PP2A-independent functions. Understanding the unique cellular perturbations induced by MCPyV ST will thus be important for understanding the tumorigenesis of MCC.
In this dissertation, we sought to understand the manipulation of cellular functions by MCPyV ST. We began by characterizing the MCPyV ST protein itself, starting with structural and functional comparisons with other well characterized polyomaviruses and identifying the interaction of MCPyV ST with cellular proteins. We observed that MCPyV ST uniquely interacts with the TIP60 cellular complex, which contains an ATPase and an acetyltransferase and is involved in histone modifications and DNA damage repair. Through predictions of the structure, we identified a surface-exposed region of ST, loop 4, and observed that regions in this loop were important for regulating the binding of ST to the TIP60 complex.
Functionally, we investigated the role of MCPyV ST in the DNA damage response because of its interaction with TIP60 and because DNA damaging agents are used to treat MCCs. In addition, overcoming checkpoint regulation in the p53 pathway is an open question in MCPyV infection. We determined that ST increased sensitivity to DNA damage by γ-irradiation and etoposide and that expression of ST caused persistence of double strand DNA breaks (DSB) after damage, suggesting that DSB repair was delayed in ST expressing cells. Specifically, we observed that ST expression inhibits repair of breaks by nonhomologous end joining (NHEJ) but does not inhibit repair by homologous recombination (HR). These effects on the DNA damage response are explained in part by a less robust phosphorylation of DNA-PKcs at serine residue 2056, which is important for regulating end processing and repair by NHEJ. Taken together, these results indicate that MCPyV ST disrupts the cellular DNA damage response, which has implications on the viral life cycle and the initiation and treatment of MCC.<br>Medical Sciences
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Ito, Kosei. "c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large T antigen." Kyoto University, 1997. http://hdl.handle.net/2433/202211.
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Chapman, Victoria. "Chemical allergen induced perturbations of DNA methylation : insights into in vivo T cell polarisation." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/chemical-allergen-induced-perturbations-of-dna-methylation-insights-into-in-vivo-t-cell-polarisation(02953c78-3ae7-4d59-9430-690863d56d85).html.
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Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. In particular, they have been implicated in the generation of in vitro cytokine-driven T cell polarization and therefore may determine the vigor, quality and/or longevity of such responses in vivo. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, such as 2,4-dinitrochlorobenzene (DNCB) that result in type 1/type 17 responses in mice, and chemicals that cause sensitization of the respiratory tract and occupational asthma, for example trimellitic anhydride (TMA) that induces preferential type 2 responses in mice. To explore the regulation and maintenance of these divergent responses generated by polarised T cell populations in vivo, BALB/c strain mice were exposed topically DNCB and TMA. DNA from draining lymph nodes (LN) was processed for methylated DNA (5mC) immunoprecipitation (MeDIP) followed by hybridization to a whole-genome DNA promoter array. A higher number of DNCB-associated differently methylated regions (DMR) were identified and there was significant crossover between allergen treatments. Promoter-associated DMR, unique to either DNCB or TMA, were generally hypomethylated. Pathway analyses highlighted a number of immune related pathways, including chemokine and cytokine signalling. A number of these DMR were hypothesised to be candidate biomarkers of chemical allergy. To confirm this, novel analysis of hydroxymethylated (5hmC) DNA in the in vivo allergen-activated LN was compared to analysis of 5mC to identify LN specific DMR. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses and the Nwc DMR was characteristic of TMA treatment, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but also provide a possible basis for differentiation between classes of chemical allergens. Finally, a combined population of effector/effector memory T cells (TEff/TEM) was isolated from the CD4+ and CD8+ populations of allergen-activated draining lymph nodes (LN). Levels of 5mC and 5hmC at T cell lineage cytokine prompters was determined and analysed by comparison with concurrently sorted naïve T cells. In CD8+ TEff/TEM from DNCB-stimulated LN, increased expression of Ifng and Gzmb correlated with a reduction 5mC at their respective promoters. There were also reduced levels of 5mC at an Ifng enhancer. In contrast, TMA-simulated CD4+ TEff/TEM were characterised by high levels of Il4 expression which were associated with a decrease in promoter 5mC and an increase in 5hmC, as well as increased 5hmC at an Il4 enhancer region. These data demonstrate that exposure to chemical allergens results in characteristic DNA methylation patterns indicative of epigenetic regulation of divergent T cell populations in vivo. Furthermore, it highlights a particularly important role for DNA hydroxymethylation at the Th2 locus. In conclusion, exposure to chemical allergens results in divergent patterns of 5mC and 5hmC. These provide possible biomarkers for the different classes of chemical allergens and represent an insight into the importance of 5mC and 5hmC in the control of polarised T cell responses in vivo.
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Chung, Chia-Ling. "Study of DNA- SWNT conjugation for nanoelectronic purposes : realisation of transistors and SWNT positioning in DNA T scaffold." Paris 11, 2010. http://www.theses.fr/2010PA112033.
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Dans cette thèse l’ADN a été envisagé comme un matériau de choix pour l’auto-assemblage de circuits à base de SWNTS. Afin de réaliser cette vision ambitieuse, plusieurs étapes sont nécessaires et dans cette thèse nous nous intéressons à trois d’entre elles : l’assemblage de l’ADN sur nanotubes. Nous avons exploré deux approches : l’approche covalente (chapitre 2) et l’approche non covalente ( chapitre 3). Les résultats obtenus nous ont servi à évaluer quelle méthode était la plus pertinente pour la fabrication de transistors. La conclusion de ces études a été que l’approche non covalente basée sur le système biotin-streptavidine est plus appropriée pour la fabrication de SWNT-FET en raison de son rendement plus élevé. (II) La fabrication d’une nanostructure d’ADN en forme de T, l’idée était ici de créer un échafaudage d’ADN imitant la géométrie d’un transistor a grille individuel. Nos résultats démontrent qu’il est possible de définir le design d’une structure d’ADN pour une fonction donnée et de le positionner sélectivement et spécifiquement un nanotube sur cette structure a trois branches. (III) Finalement nous décrivons la fabrication de dispositifs à base de nanotubes et d’ADN (chapitre 5). Nous avons réalisé des transistors par assemblage biorigide en utilisant les étapes suivantes : A) formation d’un complexe ADN-nanotubes, B) Métallisation sélective de l’ADN, C) fabrication des électrodes sur l’ADN métallisé par lithographie électronique et D) mesure des caractéristiques I/V. Les résultats démontrent la faisabilité des dispositifs, les transistors obtenus présentent un comportement de type P classique pour les nanotubes de carbone<br>In this thesis DNA has been envisioned as a scaffold for self assembly of SWNTS circuits on a substrate to realise this ambitious vision, several steps are needed and we address three of them in this thesis : I) DNA SWNT conjugation experiments. We test two approaches: covalent Approach (chapter2) and non –convalent approach (chapter 3). In order to evaluate which approach is suitable to fabricate DNA assembled SWNT-FET. Our results reveal that non-convalent approach by biotin-streptavidin system is the more appropriate for our DNA-based SWNT-FET fabrication, because of its superior linkage yield. (II) The assembly of a T-Shape DNA scaffold. In chapiter 4, we present a three branched DNA scaffold which can be the template of a SWNT field effect transistor. We design an artificial three branched DNA structure that mimics the geometry of an individual gated transistor. Our results demonstrate that it is possible to design sub-micrometric branched DNA structures for a given function, and directly an specifically localize one SWNT onto a three armed functionalized DNA template. (III) Finally, the fabrication of bio-directed SWNT-FET (chapter 5) through the following steps : (A) formation of DNA SWNT complexes using biotin-streptavidin system, (B) selective DNA metallization, (C) fabrication of electrode contacts on the metallized DNA strand by lithography and (D) the conductivity measurement the results reveal, the feasibility of the approach ant that our bio-directed SWNT-FET presents the typical P type SWNT-FET
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Pugh, Jason L., Sarah A. Foster, Alona S. Sukhina, et al. "Acute systemic DNA damage in youth does not impair immune defense with aging." WILEY-BLACKWELL, 2016. http://hdl.handle.net/10150/622599.
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Aging-related decline in immunity is believed to be the main driver behind decreased vaccine efficacy and reduced resistance to infections in older adults. Unrepaired DNA damage is known to precipitate cellular senescence, which was hypothesized to be the underlying cause of certain age-related phenotypes. Consistent with this, some hallmarks of immune aging were more prevalent in individuals exposed to whole-body irradiation (WBI), which leaves no anatomical repository of undamaged hematopoietic cells. To decisively test whether and to what extent WBI in youth will leave a mark on the immune system as it ages, we exposed young male C57BL/ 6 mice to sublethal WBI (0.5-4 Gy), mimicking human survivor exposure during nuclear catastrophe. We followed lymphocyte homeostasis thorough the lifespan, response to vaccination, and ability to resist lethal viral challenge in the old age. None of the irradiated groups showed significant differences compared with mock-irradiated (0 Gy) animals for the parameters measured. Even the mice that received the highest dose of sublethal WBI in youth (4 Gy) exhibited equilibrated lymphocyte homeostasis, robust T-and B-cell responses to live attenuated West Nile virus (WNV) vaccine and full survival following vaccination upon lethal WNV challenge. Therefore, a single dose of nonlethal WBI in youth, resulting in widespread DNA damage and repopulation stress in hematopoietic cells, leaves no significant trace of increased immune aging in a lethal vaccine challenge model.
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Rojas, Noguera Ingrid Cecilia. "Studies of a matrix attachment region (MAR) adjacent to the mouse CD8a gene, and the MAR-binding proteins, SATB1 and CDP /." Digital version accessible at:, 2000. http://wwwlib.umi.com/cr/utexas/main.
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Alvarez, Martinez Jesus Antonio. "Role of [gamma][delta]-T cells in mycobacterial infection and inflammation /." free to MU campus, to others for purchase, 2000. http://wwwlib.umi.com/cr/mo/fullcitt?p9999268.
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Bishop, David C. "Clinical Translation of CD19-Specific Chimeric Antigen Receptor T cells Generated with the piggyBac Transposon System for the Treatment of B Cell Malignancies." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/22305.
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CD19-specific chimeric antigen receptor (CAR19) T cells are effective against relapsed/refractory B cell malignancy; but, most are individualised autologous products that have been generated using viral vectors. The complexity and cost of this approach is a barrier to widespread use. The plasmid-based piggyBac transposon system is a simple and economical alternative to viral vectors for transgene delivery. However, CAR19 T cells previously generated with piggyBac lacked in vivo efficacy, likely due to deleterious interactions of the CAR with other immune cells. This thesis describes the clinical translation of efficacious piggyBac CAR19 T cells. Pre-clinical evaluation of piggyBac CAR19 T cells expressing re-engineered CARs demonstrated potent efficacy and persistence in vivo, providing the rationale for clinical translation. The influence of CAR design on CAR19 T cell phenotype and function was also demonstrated in vitro. A first-in-human phase I clinical trial of escalating doses of HLA-matched sibling donor-derived piggyBac CAR19 T cells for the treatment of relapsed/refractory B cell malignancies post allogeneic haematopoietic stem cell transplant was initiated. Early results show that donor-derived piggyBac CAR19 T cells effectively induce disease remission, and suggest that expansion, persistence, acute toxicity and relapse profiles may be similar to autologous CAR19 T cells produced with viral vectors. But, development of a CAR19 T cell malignancy in a single patient is a serious safety issue under investigation. CAR19 T cell production with piggyBac was refined by replacing plasmid components with doggybones. These are minimal DNA vectors that can be inexpensively produced enzymatically, and lack bacterial DNA sequences and antibiotic resistance genes that are problematic for clinical use. If serious safety issues can be resolved, simple and affordable CAR T cell production with piggyBac shows promise for accelerating research and improving clinical access.
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McInnes, Elizabeth. "Genetic analysis of the T-DNA genes from the Agrobacterium rhizogenes Ri plasmid." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.276202.
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Ji, Yingjie, Xindi Dang, Lam Ngoc Thao Nguyen, et al. "Topological DNA Damage, Telomere Attrition and T Cell Senescence During Chronic Viral Infections." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etsu-works/6522.
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Background: T cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions.
Results: We demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival.
Conclusion: These findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.
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Carloni, Roberta. "Functional analysis of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) in Trypanosoma brucei brucei." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/18015.
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In order to evaluate the suitability of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) as a potential drug target for an anti-parasite therapy, we are studying its role in the bloodstream form of Trypanosoma brucei brucei, the eukaryotic parasite that causes African Sleeping Sickness. Eukaryotic TDP1 removes covalently trapped topoisomerase IB and other adducts from the 3’ end of the DNA at DNA strand breaks. Covalent topoisomerase IB stalling is caused by endogenous DNA damage and by anti-cancer drugs such as camptothecin (CPT). A potential approach could be to use TDP1 inhibitors synergistically with CPT in a combined anti-parasite therapy. T. brucei TDP1 knock out cells are hypersensitive to CPT and accumulate in the late S phase of the cell cycle upon treatment with the drug. The CPT hypersensitivity of the TDP1-/- cells can be fully rescued through ectopic expression of wild type TDP1. The catalytic activity of TDP1 is required for complementation of the CPT sensitivity since overexpression of a catalytically inactive mutant form of TDP1 further sensitises TDP1-/- cells to CPT. In this context, expression of the mutant H358N, which shows reduced activity, also increases sensitivity of TDP1-/- cells to the drug. Surprisingly, expressing TDP1 carrying an analogous mutation to the one that causes SCAN1, a human neurodegenerative disease, does not sensitise TDP1-/- cells further. With this unique set of mutant TDP1 proteins in a TDP1-/- background we hope to answer questions concerning TDP1 function that have so far been elusive.
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Holt, Sarah Hudson. "Genetic studies of phenotypic variants in the woodland strawberry, (Fragaria vesca)." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/77236.
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The diploid woodland strawberry (Fragaria vesca) is a rapidly developing translational model for members of the family Rosaceae and other plants. This thesis represents some of the first forward genetics studies evaluating putative T-DNA insertional mutants in F. vesca. The observed phenotypes include alterations to floral development, anthocyanin pigmentation and leaf structure.
The floral development mutant named green petal (gp) was not associated with the T-DNA insertions present. Based on similar phenotypes induced by mutation of transcription factors involved in floral development of Arabidopsis thaliana, we used a BLAST search of the F. vesca genome hybrid gene models to identify 30 candidate genes that may have caused the gp phenotype. Expression analysis of these genes revealed that it was due to a 37 bp deletion in a SEPALLATA3-like E-Class MADS box transcription factor. This mutation altered organ structure in the three inner whorls of the flower, affecting fertility and fruit development. The deletion was demonstrated to segregate with the mutant phenotype in a segregating population of 92 individuals, 22 of which had green petals.
The anthocyanin biosynthesis mutant named white runner (wr) lacked red pigmentation in the stems and runners. The T-DNA insertion in this line was located in a highly repetitive LTR retrotransposon region, which complicated analysis. Segregation analysis of the wr lines revealed that the phenotype was unassociated with the T-DNA insertion as well. We used a targeted expression analysis of three critical structural genes in the flavonoid biosynthesis pathway that revealed a 20 bp deletion in the gene encoding flavanone 3-hydroxylase, an enzyme necessary for the production of flavonols, anthocyanins and proanthocyanidins. In an F2 segregating population, this deletion co-segregated with the phenotype.
The third mutant line presented here displayed a curly leaf (cl) phenotype and was found to harbor a T-DNA insertion in a gene encoding a putative erythroblast macrophage attacher protein (EMP). Sequence and protein domain analysis indicated that FvEMP was related to the mammalian EMP protein that functions in cytoskeletal dynamics and red blood cell enucleation. Complementation analysis confirmed that introduction of the wild type FvEMP gene into the cl mutant plants restored wild type leaf phenotype. Further morphological analysis revealed additional pleiotropic effects of the mutation, including abnormalities in seed set and germination, pollen tube growth, adhesion of the abaxial epidermal layer to the mesophyll layer and reduced petiolule length. These phenotypes are consistent with actin binding and microtubule associated protein mutants in other plant species.
Insertional mutagenesis is a critical molecular tool for model crop development. These studies highlight the precautions that must be taken when evaluating insertional mutants. These mutants are excellent tools for studying their respective disrupted gene function. The in depth molecular analysis of the mutants presented in this work was only possible because of the availability of the Fragaria vesca genome which was used extensively to identify T-DNA insertion sites and recover candidate gene sequences for expression analysis.<br>Ph. D.
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Ruiz-Rojas, Juan Jairo. "Characterization of T-DNA integration sites within a population of insertional mutants of the diploid strawberry Fragaria vesca L." Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/77264.
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Cultivated strawberry (Fragaria × ananassa) is an octoploid (2n=8x=56) species that belongs to the Rosaceae family and the high ploidy level makes genetic and molecular studies difficult. However, its commercial success because of its unique flavor and nutritious qualities has increased interest in the development of genomic resources. Fragaria vesca L. is a diploid (2n=2x=14) species with a small genome size (206 Mbp), short reproductive cycle, and facile vegetative and seed propagation that make it an attractive model for genomic studies. The availability of an efficient transformation methodology for Fragaria vesca has facilitated the use of a T-DNA mutagenesis system to develop a collection of several hundred insertional T-DNA mutants at Virginia Tech, using either of two commercially available vectors, pCAMBIA 1302 and 1304. In this study, we have used expression of the green fluorescent protein (GFP) as a tool to identify homozygous mutant lines. Three different approaches were conducted, first we identified 11 homozygous lines by PCR, then another 55 homozygous lines by absence of segregation of GFP expression in T2 seedlings, and finally we attempted to distinguish homozygous from hemizygous lines by relative GFP expression measured using a commercially available GFP meter. The latter methodology was unsuccessful due to uncontrolled variability in the readings. Continuing the characterization of our mutant population, we used thermal asymmetric interlaced PCR (TAIL-PCR) to obtain the nucleotide sequence of the genomic DNA regions that flank the T-DNA insertion sites in independent transgenic strawberry lines. Primers were designed that would amplify the derived strawberry flanking sequences in the two parents of an interspecific mapping population between the two diploid species, F. vesca x F. bucharica. The amplified products were sequenced and examined for the occurrence of SNPs (single nucleotide polymorphisms). The same primers were then used on the F2 mapping population. Segregation of SNP markers with previously mapped genetic markers allowed us to position 74 SNP markers, and hence their corresponding insertional mutants, on a well-populated genetic linkage map for the diploid strawberry. Finally, we analyzed the insertion site from more than 190 mutants looking at both the right and left borders of the T-DNA where microsimilarities of a few base pairs between ends of T-DNA and genomic DNA were observed, indicating that T-DNA integration had not occurred randomly in strawberry. We have also characterized the insertion sites through gene annotation found in the strawberry genome database.<br>Ph. D.
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Babic, Vivijan. "T-DNA tagging in Brassica carinata with a promoterless gus, nptII gene fusion vector." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0008/NQ37928.pdf.
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Hyrcza, Martin Dominic. "Heat shock-induced global transcriptional changes in T-lymphocytes analyzed using DNA array technology." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ63238.pdf.
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Baumann, Katrin [Verfasser]. "DNA-Methylierungsmuster im foxp3 Gen in humanen CD4+ FOXP3-exprimierenden T-Zellen / Katrin Baumann." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2009. http://d-nb.info/1023494183/34.
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Klaue, Daniel. "DNA Unwinding by Helicases Investigated on the Single Molecule Level." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-97596.
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Each organism has to maintain the integrity of its genetic code, which is stored in its DNA. This is achieved by strongly controlled and regulated cellular processes such as DNA replication, -repair and -recombination. An essential element of these processes is the unwinding of the duplex strands of the DNA helix. This biochemical reaction is catalyzed by helicases that use the energy of nucleoside triphophate (NTP) hydrolysis. Although all helicases comprise highly conserved domains in their amino acid sequence, they exhibit large variations regarding for example their structure, their function and their target nucleic acid structures. The main objective of this thesis is to obtain insight into the DNA unwinding mechanisms of three helicases from two different organisms. These helicase vary in their structures and are involved in different pathways of DNA metabolism. In particular the replicative, hexameric helicase Large Tumor-Antigen (T-Antigen) from Simian virus 40 and the DNA repair helicases RecQ2 and RecQ3 from Arabidopsis thaliana are studied. To observe DNA unwinding by these helicases in real-time on the single molecule level, a biophysical technique, called magnetic tweezers, was applied. This technique allows to stretch single DNA molecules attached to magnetic particles. Simultaneously one can measure the DNA end-to-end distance. Special DNA hairpin templates allowed to characterize different parameters of the DNA unwinding reaction such as the unwinding velocity, the length of unwound DNA (processivity) or the influence of forces. From this mechanistic models about the functions of the helicases could be obtained. T-Antigen is found to be one of the slowest and most processive helicases known so far. In contrast to prokaryotic helicases, the unwinding velocity of T-Antigen shows a weak dependence on the applied force. Since current physical models for the unwinding velocity fail to describe the data an alternative model is developed. The investigated RecQ helicases are found to unwind and close short stretches of DNA in a repetitive fashion. This activity is shown for the first time under external forces. The experiments revealed that the repetitive DNA unwinding is based on the ability of both enzymes to switch from one single DNA strand to the other. Although RecQ2 and RecQ3 perform repetitive DNA unwinding, both enzymes differ largely in the measured DNA unwinding properties. Most importantly, while RecQ2 is a classical helicase that unwinds DNA, RecQ3 mostly rewinds DNA duplexes. These different properties may reflect different specific tasks of the helicases during DNA repair processes. To obtain high spatial resolution in DNA unwinding experiments, the experimental methods were optimized. An improved and more stable magnetic tweezers setup with sub-nanometer resolution was built. Additionally, different methods to prepare various DNA templates for helicase experiments were developed. Furthermore, the torsional stability of magnetic particles within an external field was investigated. The results led to selection rules for DNA-microsphere constructs that allow high resolution measurements<br>Jeder Organismus ist bestrebt, die genetischen Informationen intakt zu halten, die in seiner DNA gespeichert sind. Dies wird durch präzise gesteuerte zelluläre Prozesse wie DNA-Replikation, -Reparatur und -Rekombination verwirklicht. Ein wesentlicher Schritt ist dabei das Entwinden von DNA-Doppelsträngen zu Einzelsträngen. Diese chemische Reaktion wird von Helikasen durch die Hydrolyse von Nukleosidtriphosphaten katalysiert. Obwohl bei allen Helikasen bestimmte Aminosäuresequenzen hoch konserviert sind, können sie sich in Eigenschaften wie Struktur, Funktion oder DNA Substratspezifität stark unterscheiden. Gegenstand der vorliegenden Arbeit ist es, die Entwindungsmechanismen von drei verschieden Helikasen aus zwei unterschiedlichen Organismen zu untersuchen, die sich in ihrer Struktur sowie ihrer Funktion unterscheiden. Es handelt sich dabei um die replikative, hexamerische Helikase Large Tumor-Antigen (T-Antigen) vom Simian-Virus 40 und die DNA-Reparatur-Helikasen RecQ2 und RecQ3 der Pflanze Arabidopsis thaliana. Um DNA-Entwindung in Echtzeit zu untersuchen, wird eine biophysikalische Einzelmolekültechnik, die \"Magnetische Pinzette\", verwendet. Mit dieser Technik kann man ein DNA-Molekül, das an ein magnetisches Partikel gebunden ist, strecken und gleichzeitig dessen Gesamtlänge messen. Mit speziellen DNA-Konstrukten kann man so bestimmte Eigenschaften der Helikasen bei der DNA-Entwindung, wie z.B. Geschwindigkeit, Länge der entwundenen DNA (Prozessivität) oder den Einfluß von Kraft, ermitteln. Es wird gezeigt, dass T-Antigen eine der langsamsten und prozessivsten Helikasen ist. Im Gegensatz zu prokaryotischen Helikasen ist die Entwindungsgeschwindigkeit von T-Antigen kaum kraftabhängig. Aktuelle Modelle sagen dieses Verhalten nicht vorraus, weshalb ein alternatives Modell entwickelt wird. Die untersuchten RecQ-Helikasen zeigen ein Entwindungsverhalten bei dem permanent kurze Abschnitte von DNA entwunden und wieder zusammengeführt werden. Dieses Verhalten wird hier zum ersten Mal unter dem Einfluß externer Kräfte gemessen. Es wird gezeigt, dass die permanente Entwindung auf die Fähigkeit beider Helikasen, von einem einzelen DNA-Strang auf den anderen zu wechseln, zurückzuführen ist. Obwohl RecQ2 und RecQ3 beide das Verhalten des permanenten Entwindens aufzeigen, unterscheiden sie sich stark in anderen Eigenschaften. Der gravierendste Unterschied ist, dass RecQ2 wie eine klassische Helikase die DNA entwindet, während RecQ3 eher bestrebt ist, die DNA-Einzelstränge wieder zusammenzuführen. Die unterschiedlichen Eigenschaften könnten die verschieden Aufgaben beider Helikasen während DNA-Reparaturprozessen widerspiegeln. Weiterhin werden die experimentellen Methoden optimiert, um möglichst hohe Auflösungen der Daten zu erreichen. Dazu zählen der Aufbau einer verbesserten und stabileren \"Magnetischen Pinzette\" mit sub-nanometer Auflösung und die Entwicklung neuer Methoden, um DNA Konstrukte herzustellen. Außerdem wird die Torsions\\-steifigkeit von magnetischen Partikeln in externen magnetischen Feldern untersucht. Dabei finden sich Auswahlkriterien für DNA-gebundene magnetische Partikel, durch die eine hohe Auflösung erreicht wird
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Briju, Betsy J. "Progress of Work towards Cloning Gravity Persistence Signal (gps) Mutants by PCR-Based Methods and Positional Mapping." Ohio University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1320862556.
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Potter, Alan J. "The cell cycle phase specificity of DNA damage induced by radiation, peroxide and chemotherapeutic drugs targeting topoisomerase II, and CD4 and CD8 receptor expression on apoptotic human lymphocytes /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/6338.
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Håkman, Jonna. "Vitamin C as a modifier of mammalian epigenetics: implications for adaptive immunity." Thesis, Linköpings universitet, Institutionen för klinisk och experimentell medicin, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-105389.
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Ascorbic acid (AA), in popular speech vitamin C, is a commonly known nutrient. It is involved in several biological processes and deficiency can lead to scurvy. Recent publications have shown the impact of AA on epigenetic regulation in mice. Addition of AA, via enzymatic activity, enhances the generation of 5-hydroxymethylcytosine (5hmC), which is an intermediate in active demethylation of DNA. The role of AA on epigenetic changes in humans has to our knowledge never been studied. In this study, naïve CD4+ T cells from blood donors were used as a model system to investigate AAs possible role in methylation changes in the immune system. By using dot-blot assay, hydroxymethylated DNA immunoprecipitation (hmeDIP) and qPCR, changes in methylation executed by AA could be detected. A confirmation of AAs impact on epigenetic changes in mice was observed. AA enhanced the levels of 5hmC compared to untreated cells. The Jurkat cell line, a human T lymphocyte cell line, showed an opposite result. Treatment with AA decreased the levels of 5hmC compared to untreated cells. When comparing this result with the results obtained in human naïve T cells, the same observation was made. The difference between mouse and human in the ability of producing and metabolize AA could be a reason for this opposite result. Since AA had the ability to modify epigenetic changes in primary human CD4+ T cells, the results suggest that AA may have a function in the human immune system.
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Peng, Yu-Cai. "Interactions between polyomavirus large T antigen and the viral replication origin DNA, how and why." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0028/NQ50235.pdf.
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Yang, Rui [Verfasser], and T. [Akademischer Betreuer] Lamparter. "Phytochrome studies via locked chromophores, DNA interference and temperature effects / Rui Yang. Betreuer: T. Lamparter." Karlsruhe : KIT-Bibliothek, 2012. http://d-nb.info/1022626736/34.
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Peng, Yu-Cai 1965. "Interactions between polyomavirus large T antigen and the viral replication origin DNA : how and why." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35927.
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Polyomavirus large T antigen is the major regulatory protein in the polyomavirus life cycle. It binds to multiple G(A/G)GGC pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. The nature of interactions between large T antigen and the viral origin of DNA replication is not fully understood. We set out to produce large T antigen protein in the methylotropic yeast Pichia pastoris by placing the large T antigen gene downstream of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immuno-affinity chromatography by using a monoclonal antibody.<br>While optimizing the conditions for binding of large T antigen to viral origin DNA, we discovered that binding was substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. We showed that increased binding at low pH is due to increased stability of protein-DNA complexes, and that large T antigen molecules self-associated at low pH, forming massive complexes. ATP increased binding of large T antigen to origin DNA by about 2-fold at pH 7.8, but had no detectable effect at pH 7 or below.<br>Enhanced, stable DNA binding by large T antigen to viral origin DNA at pH 6 enabled us to develop a novel gel mobility shift assay using unfixed protein-DNA complexes. We demonstrated that this assay is very sensitive and highly specific. This method can be used both for detection of large T antigen in crude cell lysates and for quantitation of binding of purified large T antigen to target DNAs under various conditions.<br>Using a series of point and deletion mutants in the viral origin of DNA replication, we demonstrated that binding of large T antigen to sites 1/2, A, B, and C is cooperative. Binding of large T antigen to one site stimulated binding to other sites 20 to 100 bp distant, and binding to inherently weak sites was strengthened if two or more such sites were present on the same DNA molecule. These findings suggest that large T antigen molecules bound to DNA interact with each other to mutually stabilise their binding.<br>ATP was shown to stabilise large T antigen-DNA complexes against dissociation only if the DNA contained site 1/2. ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, where hexamers are believed to form and begin unwinding DNA. We propose a model in which large T antigen molecules bound to sites 1/2, A, B, and C on origin DNA form a compact protein-DNA complex via mutual interactions; large T antigen molecules bound to sites A, B, and C are mobilised and "handed over" to site 1/2, where ATP stimulates their assembly into hexamers.
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Akbari, Omid. "Analysis of the basis for induction and maintenance of T cell responses in DNA vaccination." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312898.
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Paes, Fernanda Araujo. "AnÃlise de comunidades microbianas de solo de manguezal por T-RFLP e microarranjos de DNA." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2712.
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FundaÃÃo de Amparo à Pesquisa do Estado do CearÃ<br>The mangroves are essistemas important and productive, found along the coastline of tropical and subtropical regions. Important processes such as nutrient cycling, are directly connected to the activity of soil microbial communities. Given its strategic position the mangroves are very impacted the world. In Brazil, important areas such as the Bay of All Saints (BA) are severely affected by the presence of oil. Understanding the adaptation of the microbiota and its local dynamics, before the environmental change is essential to be able to maintain or restore these ecosystems. This thesis investigated the structure and function of microbial communities in a mangrove area of the Bay of All Saints polluted by the oil industry. Two sites were sampled - one located in the refinement of the industry and another, distant location and used as the control. Besides the measured abiotic data, two independent methods of cultivation were used: T-RFLP and DNA microarranjos. The results of both sites in the experiments of T-RFLP showed that the two bacterial communities differ in their structure. For the site sampled in the refinery, a greater number of Otus suggests an environmental stimulus, the bacterial community, a fact associÃvel the high organic matter content of the site. For microarranjos DNA, similar categories of genes were detected in both places, but the specificity of their sequences were distinct. The genes that encode for the remediation organic, were more representative, more than 40% in total in the two sites. Even detecting similar categories, only 4% of the gene sequences were common to the sites. These data show that different bodies are responsible for similar functions at each point. The difference between the sites observed in the experiments can be related with the fact that Site 1 is located in a region heavily affected by oil. Using the ecological data rates of T-RFLP indicated the site 1 as the most diverse, while the results with the DNA microarranjos show the opposite. In conclusion, the structure and function of the local microbiota are affected by the presence of the oil industry. Additional studies may help understand the dynamics of microbial communities, facing the future exposure to the oil and / or derivatives.<br>Os manguezais sÃo essistemas importantes e produtivos, encontrados ao longo da linha da costa de regiÃes tropicias e subtropicais. Processos importantes como a ciclagem de nutrientes, estÃo diretamente conectados à atividade das comunidades microbianas desses solos. Dada sua posiÃÃo estratÃgica os manguezais sÃo bastante impactados no mundo todo. No Brasil, Ãreas importantes como a da BaÃa de todos os Santos (BA) sÃo severamente afetadas pela presenÃa do petrÃleo. Compreender as adaptaÃÃes da microbiota local e sua dinÃmica, frente Ãs alteraÃÃes ambientais, à essencial para que se consiga manter ou restaurar esses ecossistemas. A presente dissertaÃÃo investigou a estrutura e a funÃÃo de comunidades microbianas em uma Ãrea de manguezal da baÃa de Todos os Santos poluÃda pela indÃstria do petrÃleo. Dois locais foram amostrados - um, situado na Ãrea de refinamento dessa indÃstria e o outro, distante desse local e usado como controle. AlÃm dos dados abiÃticos medidos, dois mÃtodos independentes de cultivo foram utilizados: T-RFLP e microarranjos de DNA. Os resultados de ambos os sÃtios nos experimentos de T-RFLP mostraram que as duas comunidades bacterianas diferem quanto à sua estrutura. Para o sÃtio amostrado na refinaria, um nÃmero maior de OTUs sugere um estÃmulo ambiental, na comunidade bacteriana, fato associÃvel ao alto teor de matÃria orgÃnica desse local. Para os microarranjos de DNA, categorias similares de genes foram detectados, em ambos os locais, mas a especificidade de suas sequÃncias foi distinta. Os genes que codificam para a remediaÃÃo orgÃnica, foram os mais representativos, mais de 40% no total nos dois sÃtios. Mesmo detectando categorias semelhantes, apenas 4% das sequÃncias genÃticas foram comuns aos sÃtios. Estes dados mostram que organismos diferentes sÃo responsÃveis por funÃÃes similares em cada ponto. A diferenÃa entre os locais observadas nos experimentos pode ser relacionada, com o fato do sÃtio 1 situar-se numa regiÃo fortemente afetada por hidrocarbonetos. Utilizando-se Ãndices ecolÃgicos os dados de T-RFLP indicaram o sÃtio 1 como o mais diverso, enquantoo os resultados com os microarranjos de DNA mostram o oposto. Concluindo, a estrutura e funÃÃo da microbiota local sÃo afetadas pela presenÃa da indÃstria do petrÃleo. Estudos adicionais poderÃo ajudar a compreender a dinÃmica dessas comunidades microbianas, frente Ãs futuras exposiÃÃes ao Ãleo e/ou derivados.