Dissertations / Theses on the topic 'DNA hybrid'
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Farrow, Paul J. "Development of hybrid mRNA/DNA vectors for gene therapy." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426407.
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Novoa, Carolina. "RecQ-like helicase SGS1 counteracts DNA : RNA hybrid induced genome instability." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/60964.
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Dividing cells are constantly under threat from both endogenous and exogenous DNA damaging stresses that can lead to mutations and structural variations in DNA. One contributor to genome instability is three-stranded DNA:RNA hybrid structures called R-loops. Though R-loops are known to induce DNA damage and DNA replication stress, it is unclear whether they are recognized and processed by an established DNA repair pathway prior to inducing DNA breaks. Canonically, DNA repair proteins work downstream of R-loop-induced DNA damage to stimulate repair and suppress genome instability. Recently, the possibility that some DNA repair pathways actively destabilize R-loops, thus preventing unscheduled DNA damage has emerged. Here we identify the helicase SGS1 as a suppressor of R-loop stability. Our data reveals that SGS1 depleted cells accumulate R-loops. In addition, we define a role for transcription in genome instability of cells lacking SGS1, which is consistent with an R-loop based mechanism. Hyper-recombination in SGS1 mutants is dependent on transcript length, transcription rate, and active DNA replication. Also, rDNA instability in sgs1Δ can be suppressed by ectopic expression of RNaseH1, a protein that degrades DNA:RNA hybrids. Interestingly, R-loops are known to form at rDNA loci. We favour a model in which SGS1 contributes to the stabilization of stalled replication forks associated with transcription complexes, and unresolved DNA:RNA hybrids. Finally, we showed that knockdown of the human Sgs1 orthologue BLM in HCT116 cells also led to the accumulation of more R-loops than control HCT116 cells. In summary, our data supports the idea that some DNA repair proteins involved in replication fork stabilization might also prevent and process R-loops.Science, Faculty of
Graduate
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Käslin, Edgar. "In vitro hybrid DNA formation by proteins from vegetative Schizosaccharomyces pombe cells /." [S.l : s.n.], 1993. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Patel, Chandan. "Hybrid molecular simulations of oxidative complex lesions." Thesis, Lyon, École normale supérieure, 2013. http://www.theses.fr/2013ENSL0835.
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L'ADN est en permanence exposé à un grand nombre d'événements dommageables déclenchées par des agents endogènes et exogènes. De nombreux travaux expérimentaux ont fourni des informations cruciales sur les propriétés structurelles et la réparation de certains des lésions de l'ADN. Cependant, il manque une vision mécanistique ou énergétique sur leur formation. La biochimie computationnelle a émergé comme un outil puissant pour comprendre les réactions biochimiques et les propriétés électroniques de systèmes complexes.Dans cette thèse, nous étudions la formation de lésions complexes intra-brin et inter-brin. Ces lésions tandem constituent une puissant menace à l'intégrité du génome, en raison de leur haute fréquence mutagenique. Tout d'abord, nous discutons l'attaque d'une liaison covalente entre un radical pyrimidinique. En comparant avec les bases isolees, nos simulations hybrides Car-Parrinello demontrent que la reactivité de la thymine et de la cytosine radicalaires sont inversees dans l'environnement B-helical. De plus, nos resultats montrent egalement une deformation plus importante pour la lesion G[8-5]C.Nous rationalisons également la plus grande réactivité des cytosines par rapport aux purines vers la formation multi-etapes de lésions complexes inter-brins par condensation avec un site C4' abasique. Ces résultats bases sur des simulations avec solvatation explicite et combines a la théorie de la fonctionnelle de la densité sont en accord avec les données expérimentalesDNA is continuously exposed to a vast number of damaging events triggered by endogenous and exogenous agents. Numerous experimental studies have provided key information regarding structural properties of some of the DNA lesions and their repair. However, they lack in mechanistic or energetic information pertaining to their formation. Computational Biochemistry has emerged as a powerful tool to understand biochemical reactions and electronic properties of large systems.In this thesis we study the formation of inter- and intra-strand cross-links. These tandem lesions pose a potent threat to genome integrity, because of their high mutagenic frequency. First, we discuss the formation of complex defects which arise from the attack of a pyrimidine radical onto guanine. In comparison with the reactivity of isolated nucleobases, our hybrid Car-Parrinello Molecular Dynamics simulations reveal that the reactivity of hydrogen-abstracted thymine and cytosine is reversed within a B-helix environment. Further, our data also suggest a more severe distortion of the B-helix for G[8-5]C.Second, we rationalize the higher reactivity of cytosine vs. purines toward the multistep formation of inter-strand crosslinks with a C4' oxidized a basic site, which is in qualitative agreement with experiments on isolated nucleobases, using explicit solvent simulations combined to density functional theory
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Diallo, Amy. "The DNA translocation apparatus involved in Streptococcus Pneumoniae transformation." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066334/document.
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La transformation naturelle bactérienne permet aux micro-organismes d'échanger des informations génétiques pour promouvoir leurs réponses adaptatives pour faire face aux changements environnementaux. De l'ADN extracellulaire est incorporé et recombiné au génome de l'hôte. Ce processus augmente la plasticité des bactéries. Chez S. pneumoniae, un pathogène majeur chez l'Homme engendrant des infections pouvant être mortelles, la transformation bactérienne accentue la transmission de gènes de résistance aux antibiotiques. Chez les bactéries à Gram positif, l'opéron comF encode l'expression de deux protéines. L'une est démontrée comme étant essentielle à la transformation, est décrite pour être membranaire. La seconde n'a pas été étudiée. Cependant ces protéines n'ont pas été étudiées d'un point de vue structural ou fonctionnel. Des mutagenèse et le double hybride bactérien ont permis de mettre en évidence que ses protéines sont indispensables pour l'expression de la compétence et interagissent avec de nombreuses protéines du transformasome. De plus, l'expression des deux protéines de manière hétérologue prouve qu'elles sont solubles et forment des oligomères. L'analyse structurale de ComFA, atteste de la conformation atypique de cette helicase trimerique et hexamerique. En outre, l'activité ATPasique simple brin DNA-dépendant de cette protéine est démontrée. Finalement un complexe protéique a été révélé entre ComFA et ComFC dont l'étude microscopique à hautes résolutions prouve l'apparition d'un anneau via l'assemblage de deux hexamères. Ces résultats suggèrent que ComFA est le moteur tirant l'ADN dans la cellule. Quant à ComFC, elle semble aider à la stabilisation de ComFABacterial natural transformation allows microorganisms to exchange genetic information to promote their adaptive responses to cope with environmental changes. The extracellular DNA is incorporated and recombined with the genome of the host. This phenomenon increases the plasticity of Gram positive and negative bacteria. S. pneumoniae is a major pathogen for humans, which is causing infections that can be deadly. In this specie, bacterial transformation increases the transmission of antibiotic resistance.In Gram-positive bacteria, comF operon encodes the expression of two proteins. One of them, shown to be essential for natural transformation, is expected to be a membrane protein. The second is not described. However, up to now neither protein has been studied from a structural or functional point of view. Mutagenesis technique and double hybrid bacterial assay allowed to show that both proteins are essential for the expression of the competence and interact with many proteins of the transformasome. In addition, heterologous expresion of both proteins have shown their solubility and the formation of oligomers. Structural analysis of ComFA demonstrates the unique conformation of this hexameric and trimeric helicase. Furthermore, the ATPase single stranded DNA-dependent activity of this protein could be detected. Finally, a protein complex is formed between ComFA and ComF, and high-resolution microscopic study proves the occurrence of a ring via a two-hexamers. These results suggest that ComFA is the engine pulling the DNA in the cell. As for ComFC, this protein seems to help stabilizing of ComFA
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Kumar, Deepak. "Analysis and confirmation of the results of a yeast two-hybrid screen carried out to identify proteins that interact with drosophila XRCC2." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/622.
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Repairing DNA damage is brought about by highly specific proteins that partake in a variety of DNA repair. Two of the most common types of damage are double-strand breaks (DSBs) and interstrand crosslinks. A single DSB or crosslink can potentially kill a cell if it is not repaired~ In human and other vertebrate cells, DSBs are repaired by two different mechanisms. The nonhomologous end-joining pathway can bring together the broken ends and join them, usually with the loss of some nucleotide sequence. A second pathway, homologous recombinational repair (HRR), is equally important. This repair process utilizes the information provided by another DNA molecule to restore damaged DNA. This molecule is usually a sister chromatid arising from DNA replication. This process is essentially error-free, unlike the end-joining process. Some HRR activity is required for proliferating cells to remain viable. The central protein player is RAD51, which with the help of other proteins such as XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D, performs the critical initiating steps of homologous pairing and strand transfer. The proteins encoded by the familial breast cancer genes, brcal and brca2, also play an important role in HRR.
My project is concerned with studying proteins that interact with Drosophila melanogaster (XRCC2). Proteins interacting with DmXRCC2 were identified by using a yeast two hybrid system. "Bait fusion protein" (DmXRCC2 linked to GAIA BD) was constructed by Dr. Wrischnik. Tanya Dimetrijevich, a graduate student, used this bait to fish for interacting or "target" proteins. About 50 such proteins were found. I began validating these target proteins with the intention of exploring novel interactions and functions of DmXRCC2. The process of validating proteins interacting with DmXRCC2 yielded two very interesting candidate proteins-CaBPl and FAF. · CaBPl, also called protein disulfide isomerase P5, is an endoplasmic-reticulum calciumbinding protein. FAF belongs to a large family of deubiquitinating enzymes that cleave ubiquitin-protein bonds and play diverse roles in the ubiquitin pathway. One of the implications of such discoveries could be to compare and contrast DmXRCC2 and human XRCC2 in terms of their interactions and functions.
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Kirkpatrick, Robert Daniel. "Interactions of the DNA repair protein Rad23 in the yeast two-hybrid system." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ45071.pdf.
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Joyce, Donna Marie. "The Development of DNA-Based Bio-Polymer Hybrid Thin Films for Capacitor Applications." University of Dayton / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1389285491.
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Morley, Stewart Anthony. "Interactions Between the Organellar Pol1A, Pol1B, and Twinkle DNA Replication Proteins and Their Role in Plant Organelle DNA Replication." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8128.
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Plants maintain organelle genomes that are descended from ancient microbes. Ages ago, these ancient microbes were engulfed by larger cells, beginning a process of co-evolution we now call the endo-symbiotic theory. Over time, DNA from the engulfed microbe was transferred to the genome of the larger engulfing cell, eventually losing the ability to be free-living, and establishing a permanent residency in the larger cell. Similarly, the larger cell came to rely so much on the microbe it had engulfed, that it too lost its ability to survive without it. Thus, mitochondria and plastids were born. Nearly all multicellular eukaryotes possess mitochondria; however, different evolutionary pressures have created drastically different genomes in plants versus animals. For one, animals have very compact, efficient mitochondrial genomes, with about 97% of the DNA coding for genes. These genomes are very consistent in size across different animal species. Plants, on the other hand, have mitochondrial genomes 10 to more than 100 times as large as animal mitochondrial genomes. Plants also use a variety of mechanisms to replicate and maintain their DNA. Central to these mechanisms are nuclear-encoded, organelle targeted replication proteins. To date, there are two DNA polymerases that have been identified in plant mitochondria and chloroplasts, Pol1A and Pol1B. There is also a DNA helicase-primase that localizes to mitochondria and chloroplasts called Twinkle, which has similarities to the gp4 protein from T7 phage. In this dissertation, we discuss the roles of the polymerases and the effects of mutating the Pol1A and Pol1B genes respectively. We show that organelle genome copy number decreases slightly and over time but with little effect on plant development. We also detail the interactions between Twinkle and Pol1A or Pol1B. Plants possess the same organellar proteins found in animal mitochondria, which are homologs to T7 phage DNA replication proteins. We show that similar to animals and some phage, plants utilize the same proteins in similar interactions to form the basis of a DNA replisome. However, we also show that plants mutated for Twinkle protein show no discernable growth defects, suggesting there are alternative replication mechanisms available to plant mitochondria that are not accessible in animals.
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Islam, Mohammad Kaisarul. "Novel ligands targeting the DNA/RNA hybrid and telomeric quadruplex as potential anticancer agents." Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/novel-ligands-targeting-the-dnarna-hybrid-and-telomeric-quadruplex-as-potential-anticancer-agents(ce8f3d0e-317d-4c2e-b64a-e13e283f7b95).html.
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Telomeres are repetitive sequences of DNA at the ends of chromosomes that become progressively shorter during cell division, acting as a form of “biological clock” causing cell death once they have reached a certain length. Almost 90% of cancer cells overexpress the enzyme telomerase which can lengthen telomeres and confer immortality to the tumour cells. Thus, telomerase has become an important target for drug discovery in the oncology area, and there is also interest from researchers investigating the aging process. During the catalytic cycle of telomerase, a unique DNA/RNA hybrid duplex (DRH) forms that is typically between 6-11 base pairs long and is key to extending the telomere. There is interest in discovering small drug-like molecules that can recognize and bind to this hybrid duplex to inhibit selectively telomerase, either by stabilizing the structure and thereby preventing telomerase dissociation (a key step in the catalytic cycle) or by sufficiently distorting the hybrid duplex to cause the misalignment of key catalytic groups. This project began by using oligonucleotides representing DNA/RNA hybrid duplex (DRH), telomeric G-quadruplex and control duplex DNA sequences to screen against the National Cancer Institute compound libraries (i.e., Diversity Set II, Mechanistic Set and Natural Product Set) using a high throughput Fluorescent Resonance Energy Transfer (FRET)-based DNA thermal denaturation assay to determine binding affinity and specificity. Thirteen novel chemical scaffold families were identified in the assay, compounds which showed a >5 °C selective stabilization of the DNA/RNA hybrid duplex at a 1 μM ligand concentration. Chemical modifications were then made to these scaffolds to generate focused libraries of analogues to improve selectivity, potency and drug-likeness, and to provide Structure-Activity Relationship (SAR) information. A total of 49 novel molecules were synthesized and then screened against an expanded range of four different nucleic acid constructs including telomeric and DNA/RNA hybrid duplex sequences. A number of compounds showed selective DNA/RNA hybrid stabilization potential with some compounds also showing notable telomeric G-quadruplex stabilization without significant affinity for promoter G-quadruplexes (i.e., c-Kit1, c-Kit-2 and c-Myc) and control duplex DNA sequences. The compounds from library-1 provided DNA/RNA hybrid duplex stabilization in the 0.5-7.2 C range and telomeric G-quadruplex stabilization in the 0.2-6.5 C range at a 1 μM ligand concentration. Molecular modelling and molecular dynamics studies confirmed that the methylene spacer between the benzimidazole and phenylene moieties of molecules within library-1 is perfectly shaped to fit within the DRH sequence. In addition, it was confirmed that minor-groove binding and simultaneous intercalation between the nucleobases of a DNA/RNA hybrid duplex requires a linker of specific length (i.e., an eight methylene spacer as in compound 3.3). Selected compounds were then studied further using a variety of biological techniques to confirm selective telomerase inhibition and cell-based assays to utilize their potential as antitumour agents.
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Petzold, Herman E. III. "Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpa." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/89363.
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The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield.Ph. D.
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Komori, Toshiyuki. "Application of DNA marker technology to hybrid breeding and development in rice (Oryza sativa L.)." Kyoto University, 2004. http://hdl.handle.net/2433/145411.
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Kyoto University (京都大学)0048
新制・論文博士
博士(農学)
乙第11497号
論農博第2532号
新制||農||897(附属図書館)
学位論文||H16||N3958(農学部図書室)
22629
UT51-2004-J769
(主査)教授 谷坂 隆俊, 教授 山田 利昭, 教授 遠藤 隆
学位規則第4条第2項該当
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Thomas, Chris W. "Ruthenium-DNA hybrid materials for supramolecular synthesis investigations into osmotic effects ionomeric polymer-metal composites /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2001. http://wwwlib.umi.com/cr/ucsd/fullcit?p3022196.
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Lee-Kirsch, Min-Ae, Kerstin Engel, Ekkehart Paditz, Angela Rösen-Wolff, Young-Ae Lee, and Manfred Gahr. "Assignment of the human homeobox 11-like 2 gene (HOX11L2) to chromosome 5q34→q35 by radiation hybrid mapping." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-137555.
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Bertucci, Alessandro. "Hybrid organic-inorganic interfaces for biomedical applications." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF008/document.
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Le travail de recherche de cette thèse consiste en le développement de nouveaux matériaux hybrides organiques-inorganiques pour des applications en nanotechnologie, nanomédicine et diagnostic. Dans ce contexte, des cristaux poreux de zéolite-L ont été utilisé comme nano-vecteur pour faire de la transfection d’ADN et d’ANP, en combinaison avec le relargage de molécules hôtes placées dans les pores. Des nanoparticules de silice mesoporeuses multifonctionnelles ont été utilisées pour traiter le glioblastome, en combinant la thérapie génique avec l’administration durable d’un principe actif. Des nano-coquilles hybrides biodégradables ont été encore développés pour encapsuler des protéines et les relâcher dans les cellules vivantes. Dans le domaine de la détection d’acides nucléiques, des fibres optiques à cristal photonique, fonctionnalisées avec des sondes d’ANP, ont été exploitées comme plateformes optiques pour faire de la détection ultra-sensible d’oligonucléotides ou d’ADN génomique. Enfin, la squelette de l’ANP a été modifié à créer des sondes fluorescentes pour reconnaître et détecter la présence des séquences cibles spécifiquesThe research work presented throughout this thesis focuses on the development of novel organic-inorganichybrid materials for applications in nanotechnology, nanomedicine and diagnostics. In such a context, porous zeolite-L crystals have been used as nanocarriers to deliver either DNA or PNA in live cells, in combination with the release of guest molecules placed into the pores. Multifunctional mesoporous silica nanoparticles have been designed to treat glioblastoma, combining gene therapy with the sustained delivery of a chemotherapy agent. Biodegradable hybrid nano-shells have been furthermore created to encapsulate proteins and release them in living cells upon degradation of the outer structure in reductive environment. In the field of nucleic acid detection, photonic crystal fibers, functionalized with specific PNA probes, have been exploited as optical sensing devices to perform ultra-sensitive detection of DNA oligonucleotides or genomic DNA. Eventually, the PNA backbone has served as scaffold to synthesize fluorescent switching probes able to recognize and to detect the presence of specific target sequences
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Wang, Peng. "Development of Nanoparticle-based Platforms for Potential Applications in Biosensing and Therapeutics." University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin151186771296011.
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D'ALESSANDRO, GIUSEPPINA. "THE ROLE OF RNA AND DNA:RNA HYBRIDS AT DNA DOUBLE-STRAND BREAKS." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/562552.
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The stability of our genome is constantly challenged by several genotoxic threats. DNA
double-strand breaks (DSBs) are the most dangerous DNA lesions that, if not repaired, can
lead to cancer initiation and progression and/or ageing. These detrimental consequences can
only be avoided if cells promptly recognize the lesions and signal their presence, thus
promoting either efficient repair and transient cell cycle arrest or cell death and cellular
senescence. This is the role of the DNA damage response (DDR) proteins and the newly
identified damage-induced non coding RNAs. We recently discovered that RNA polymerase
II is recruited to DSBs and synthetizes damage-induced non-coding RNAs (dilncRNAs).
DROSHA- and DICER-mediated processing of dilncRNAs generates small RNA species,
named DNA damage response RNA (DDRNAs) (Francia, 2012), that localize to DSBs via
pairing with dilncRNAs and promote DDR signaling (Michelini et al., in press). Similar
small non-coding RNA species discovered in plants are involved in DNA repair by
homologous recombination (HR) (Wei, 2012, Gao, 2014, Wang, 2016). In line with these
results, I report that transcriptional inhibition impairs recruitment of the HR proteins
BRCA1, BRCA2, and RAD51 to DSBs, while partially promoting DNA end resection.
Moreover, I show DNA:RNA hybrids accumulation at DSBs in mammalian cells by both
DRIP analyses and imaging techniques. Damage-induced DNA:RNA hybrids form upon the
hybridization of RNA species, likely dilncRNAs, to the resected DSBs DNA ends generated
during the S/G2 cell cycle phase. I also report that purified recombinant BRCA1 binds
DNA:RNA hybrids in vitro; moreover, DNA:RNA hybrids in vivo contribute to BRCA1
recruitment to DSBs. Consistent with the need to tightly regulate DNA:RNA hybrid levels,
I demonstrate that RNase H2, the major RNase H activity in mammalian nuclei, is recruited
to DSBs through direct interaction with RAD51. In summary, I report for the first time that
DNA:RNA hybrids accumulate at DSBs in mammalian cells in a cell-cycle- and DNA end
resection-depended way. At DSBs, BRCA1 directly recognizes DNA:RNA hybrids and
likely controls their turn-over by mediating the recruitment of RNase H2 via RAD51.
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Rutter, Brian Douglas. "Catch of the Day: A yeast One-Hybrid Assay Identifies a Novel DNA-Binding Domain in Phytophthora Sojae." Bowling Green State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1339861904.
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Keilwagen, Jens [Verfasser], Ivo [Akademischer Betreuer] Grosse, and Gunnar [Akademischer Betreuer] Rätsch. "Predicting DNA binding sites using generative, discriminative, and hybrid learning principles / Jens Keilwagen. Betreuer: Ivo Grosse ; Gunnar Rätsch." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2010. http://d-nb.info/1024976939/34.
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Lee-Kirsch, Min-Ae, Kerstin Engel, Ekkehart Paditz, Angela Rösen-Wolff, Young-Ae Lee, and Manfred Gahr. "Assignment of the human homeobox 11-like 2 gene (HOX11L2) to chromosome 5q34→q35 by radiation hybrid mapping." Karger, 2001. https://tud.qucosa.de/id/qucosa%3A27741.
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Miller, Jeffrey Christopher 1974. "A two-color bacterial two-hybrid system for selecting sequence-specific DNA-binding proteins via fluorescence activated cell sorting." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/8388.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002.Vita.
Includes bibliographical references.
Proteins that recognize specific DNA sequences play a major role in many biological processes, and the ability to select or design novel DNA binding proteins could have a major impact on many areas of biotechnology and medicine. This thesis begins with background information on zinc finger DNA-binding proteins and on methods to select these proteins, and how they can be used to regulate endogenous human genes. Next, I describe a structural and biochemical study of a DNA-binding protein which demonstrates some of the complexities of the protein-DNA interface, and which highlights difficulties for designing sequence-specific proteins via a simple code. I then develop an experimental system (starting with a pre-existing bacterial two-hybrid selection system) which allows the selection of proteins based on their preference of one DNA site over another. I use this system to attempt to attenuate the affinity of a zinc finger protein without destroying its specificity. Finally, I describe an experiment in which I select a new domain that adds sequence specificity to a pre-existing protein from a library of completely random peptides.
by Jeffrey Christopher Miller.
Ph.D.
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Lopez, Christina. "Inhibition of amikacin resistance in bacteria by a peptide conjugated 2',4'-bridged nucleic acid-NC-DNA hybrid oligomer." Thesis, California State University, Fullerton, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10117089.
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Multidrug resistant Acinetobacter baumannii, a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors aac(6’)-Ib. This gene codes for an aminoglycoside acetyltransferase that modifies amikacin and other aminoglycosides of clinical relevance. The goal of this work was to interfere with expression of this resistance gene and induce susceptibility to amikacin in resistant pathogens. In vitro translation assays led to the identification of an antisense oligodeoxynucleotide (ODN4) that targets the initiation of translation region of aac(6’)-Ib mRNA. An isosequential nuclease-resistant chimeric oligomer composed of 2’,4’-bridged nucleic acid-NC (BNANC) residues and deoxynucleotides (BNANC-DNA) covalently bound to the cell-penetrating peptide (RXR)4XB (where “X” and “B” stand for 6-aminohexanoic acid and β-alanine, respectively). This compound, called CPPBD4, inhibited translation at similar levels observed with ODN4. Addition of a combination of Amikacin and CPPBD4 to a culture of an Acinetobacter baumannii clinical strain harboring aac(6’)-Ib resulted in growth inhibition indicating that CPPBD4 reached the cytosol and interfere with the expression of the resistance enzyme.
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Sessa, Gaetana. "Role of the Interaction of BRCA2 and DDX5 in the DNA Damage Response BRCA2 promotes DNA-RNA hybrid resolution by DDX5 at DNA double strand breaks to facilitate homologous recombination Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS116.
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Un nombre croissant d’études soutiennent le fait que les protéines majeures du métabolisme des ARN, telles que les hélicases ARN, sont impliquées dans la réponse aux dommages à l’ADN. Cette activité est généralement accomplie par leur interaction avec des facteurs de réparation de l’ADN. BRCA2, une protéine suppressive de tumeurs, joue un rôle crucial dans la réparation des cassures double-brin (CDB) de l'ADN par recombinaison homologue (RH) et donc, est un facteur essentiel pour l’intégrité du génome. Les cellules déficientes pour BRCA2 accumulent des hybrides ADN-ARN ou R-loops, une source de dommage à l'ADN, suggérant ainsi l’importance de cette protéine dans la prévention ou la suppression de ces structures. Toutefois, le rôle spécifique de BRCA2 dans la résolution des hybrides ADN-ARN reste inconnu.Afin de connaître des potentiels partenaires de BRCA2, une analyse par spectrométrie de masse réalisée dans notre laboratoire a révélé un enrichissement en protéines impliquées dans le métabolisme de l'ARN, comme les hélicases ARN. Ces résultats nous ont menés à examiner la coopération entre BRCA2 et les hélicases ARN dans la séparation des structures ADN-ARN. Nous avons d’abord confirmé l'interaction entre l'hélicase ARN DDX5 et BRCA2, qui est améliorée dans les cellules exposées à γ-irradiation. Ensuite, nous avons réduit l’interaction aux premiers 250 aa de BRCA2 (BRCA2T1) et avons constaté que celle-ci est directe en utilisant des protéines purifiées. En collaboration avec le laboratoire du docteur A. Aguilera (Cabimer, SP), nous avons montré que la déplétion de DDX5 conduit à une accumulation des hybrides ADN-ARN dans l’entièreté du génome, particulièrement aux sites de dommages à l’ADN. De plus, nos résultats indiquent que DDX5 localise aussi aux hybrides ARN-ADN qui se forment à proximité de CDB.De manière intéressante, nous avons constaté que BRCA2 est important pour la rétention de DDX5 aux sites de dommage à l’ADN induit par l’irradiation laser. Notamment, des tests de déroulement de brins in vitro en utilisant les protéines purifiées DDX5 et BRCA2 ont révélé que BRCA2 stimule l’activité de déroulement des R-loops de DDX5.Un variant de signification inconnue (VSI) trouvé dans de patients atteints de cancer du sein situé dans la région BRCA2T1 (T207A) réduit l’interaction de BRCA2 avec DDX5 et conduit à l’accumulation des hybrides ADN-ARN. Les cellules exprimant stablement BRCA2-T207A montrent également une diminution de l’association de DDX5 avec les hybrides ARN-ADN, en particulier lors d’une exposition de cellules à l’irradiation. L’analyse de l’efficacité de la réparation des CDB par RH dans les cellules déficientes en DDX5 ou exprimant BRCA2-T207A, montre une cinétique retardée de l’apparition des foyers de réparation RAD51 lors de l’irradiation, ce qui suggère un rôle actif de l’interaction BRCA2-DDX5 pour assurer la réparation par RH efficacement. En accord avec cette hypothèse, la ribonucléase RNAseH1, qui dégrade spécifiquement la fraction d’ARN dans les structures d’ADN-ARN, restaure partiellement le phénotype de cinétique des foyers RAD51 dans les cellules BRCA2 T207A. De plus, les cellules portant le variant BRCA2-T207A ont également montré un nombre réduit de foyers RPA par rapport aux cellules qui expriment BRCA2 sauvage, témoins d’un défaut dans l’étape qui précède le chargement de RAD51 aux CDB.Ensemble, nos résultats suggèrent que les hybrides ADN-ARN représentent un obstacle à la réparation des CDB par RH et révèlent BRCA2 et DDX5 en tant que facteurs actifs dans leur suppressionIncreasing evidence support the idea that proteins involved in RNA metabolism such as RNA binding proteins (RBPs) and RNA helicases are directly implicated in the DNA damage response (DDR). This activity is generally achieved through their interaction with DNA repair factors.BRCA2 is a tumor suppressor protein that plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) as well as protecting stalled replication forks from unscheduled degradation; therefore, it is essential to maintain genome integrity. Interestingly, BRCA2 deficient cells accumulate DNA-RNA hybrids or R-loops, a known source of DNA damage and genome instability, providing evidence for its role in either R-loop prevention or processing. However, the specific role of BRCA2 on these structures remains poorly understood.A mass spectrometry screen to identify partners of BRCA2 performed in our laboratory revealed an enrichment of proteins involved in RNA metabolism such as RNA helicases. These findings led us to investigate whether BRCA2 could cooperate with these candidate interacting RNA helicases in processing DNA-RNA structures. First, we confirmed the interaction of BRCA2 and the DEAD-box RNA helicase DDX5, which we found is enhanced in cells exposed to -irradiation. Then, we narrowed down the interaction to the first 250 aa of BRCA2 (BRCA2T1) and found that it is direct using purified proteins. In collaboration with A. Aguilera lab (Cabimer, SP), we could show that depletion of DDX5 leads to a genome-wide accumulation of DNA-RNA hybrids that is particularly enriched at DNA damage sites. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs. Interestingly, we found that BRCA2 is important for the retention of DDX5 at laser irradiation-induced DNA damage. Notably, in vitro R-loop unwinding assays using purified DDX5 and BRCA2 proteins revealed that BRCA2 stimulates the R-loop helicase activity of DDX5.A breast cancer variant of unknown clinical significance (VUS) located in BRCA2T1 (T207A) reduced the interaction between BRCA2 and DDX5 and led to the accumulation of DNA-RNA hybrids. Cells stably expressing BRCA2-T207A also showed a decreased association of DDX5 with DNA-RNA hybrids, especially upon irradiation. Notably, monitoring RAD51 foci to evaluate HR-mediated DSBs repair efficiency in either DDX5-depleted cells or in BRCA2-T207A cells resulted in a delayed kinetics of appearance of RAD51 foci upon irradiation suggesting an active role of BRCA2-DDX5 interaction in ensuring timely HR repair. In agreement with this, overexpression of the RNAseH1 ribonuclease, that specifically degrades the RNA moiety in DNA-RNA structures, partially restored RAD51 kinetics phenotype of BRCA2-T207A cells. Moreover, cells bearing BRCA2-T207A variant also showed a reduced number of RPA foci compared to BRCA2 WT expressing cells, a step that precedes RAD51 loading at DSBs.Taken together, our results are consistent with DNA-RNA hybrids being an impediment for the repair of DSBs by HR and reveal BRCA2 and DDX5 as active players in their removal
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JÃnior, Francisco Holanda. "Comparative study of capture hÃbrida-hpv-dna for domiciliary autocoleta and it collects doctor." Universidade Federal do CearÃ, 2005. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=353.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoCoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
A infecÃÃo pelo Papilomavirus humano à a principal causa do carcinoma de colo uterino. No Cearà o cÃncer cervical à o segundo mais freqÃente entre as mulheres, quando se excetua o cÃncer de pele. à uma doenÃa de fÃcil prevenÃÃo, haja vista que quando se dispÃe de um serviÃo bem estruturado a mortalidade à reduzida em atà 80% dos casos. O problema à que muitas mulheres nÃo tÃm acesso a testes de varredura, cujos motivos estÃo relacionados a diversas barreiras culturais e geogrÃficas, bem como a baixa oferta e/ou inexistÃncia de serviÃos. O Cearà conta com cerca de 10.000 agentes comunitÃrios de saÃde, o que representa uma possibilidade de se levar um teste de autocoleta atà estas mulheres, considerando, todavia, que este teste tivesse uma sensibilidade e especificidade aceitÃvel e, assim, atendendo rapidamente um quantitativo significativo de mulheres que nunca fizeram o exame de prevenÃÃo ou estÃo em intervalo recomendado para repeti-lo. Em face ao exposto, este estudo tem por objetivo comparar a sensibilidade e especificidade da Captura HÃbrida de espÃcime cÃrvico-vaginal para HPV-DNA por autocoleta domiciliar em condiÃÃes reais, com a Captura HÃbrida para HPV-DNA coletado por mÃdico ginecologista em consultÃrio e nas condiÃÃes ideais. Os dados foram coletados no perÃodo de agosto a dezembro de 2002, junto a 878 mulheres de cinco cidades cearenses. Estas foram submetidas, na mesma semana, aos testes de Captura HÃbrida por autocoleta, citologia, Captura HÃbrida coletada pelo mÃdico ginecologista, colposcopia e histopatolÃgico, quando necessÃrio. Das 878 mulheres participantes 815 foram consideradas negativas pelo padrÃo ouro, 54 foram consideradas positivas - baixo grau e 9 foram consideradas alto grau - carcinoma. Nos resultados das amostras para Captura HÃbrida HPV-DNA da autocoleta e da coleta mÃdica, obtiveram-se que em 546 casos, ambos os testes concordaram como negativos, em 216 casos concordaram como positivos, em 35 casos houve discordÃncia com a coleta mÃdica positiva e em 81 casos houve discordÃncia com a autocoleta positiva. Os percentuais de sensibilidade encontrados da citologia, Captura HÃbrida por coleta mÃdica e Captura HÃbrida autocoleta foram, respectivamente, 18,3%, 63,3% e 66,7%. Os percentuais de especificidade verificados da citologia Captura HÃbrida coleta mÃdica e Captura HÃbrida autocoleta foram, respectivamente, 98,0%, 73,0% e 68,7%. Comparando-se a concordÃncia entre ambas as coletas de Captura HÃbrida, obteve-se o coeficiente de Kappa (K=0,693) com um erro padrÃo de 0,026 embora existe diferenÃa significativa da prevalÃncia detectada por ambos os testes, teste de McNemar (p<0,001). Avaliando-se as Ãreas da curva ROC para ambos os testes, mostrou-se coleta mÃdica Ãrea=0,738 com coeficiente de 95% de certeza - o intervalo de confianÃa IC=[0,673;0,802] e autocoleta Ãrea= 0,670 com 95% de certeza - o intervalo de confianÃa IC=[0,597;0,742] para detecÃÃo do Papilomavirus. Concluiu-se haver boa concordÃncia entre os resultados obtidos pela autocoleta de espÃcime para realizaÃÃo da Captura HÃbrida HPV-DNA com a coleta mÃdica.
Human Papillomavirus infection is the mean cause of the most of cervical cancers. In Cearà this type of cancer is the second in frequency among the women, when the skin cancers are excluded. Cervical cancer is one the most preventable. Where well structured programs exist the mortality has declined, and in some cases in about 80% .The mean problem is that cervical cancer screening is not fully utilized among groups of women, especially those without access or because there are no services offered or when services exist there are many other barriers, since cultural aspects to geographic barriers. In Cearà exist well structured Health communitarian agents program, which we estimate in about 10.000 agents that cover fully the necessities of population in their areas and the all Cearà territory. With this program we can carry on one screening program by self-sampling if this test were acceptable and had a good sensitivity and specificity. By these communitarian agents we could insert in screening program all women who never underwent to pap smear or other type of screening test. The mean objective of this work is to determine whether testing of self-collected vaginal specimen for Human Papillomavirus has the same accuracy of sampling collected by physicians . In order to evaluate this one Cross-sectional observational study was done between August and December of 2002, 878 women from five municipalities were enrolled and the tests were done in the same week, the women started by doing self-sampling at home and after that they were undergone in physicianâs clinics to the others examination in following sequence cytology, hybrid capture HPV-DNA, colposcopy and when were necessary biopsy. OF 878 women that participated in this study , 815 were considered negative by the gold standard ,54 were considered positive low grade and 9 were high grade/ carcinoma . Of 878 samples to HPV-DNA , there was negative concordance to both test in 546 samples, there was positive concordance in 216 samples, there was discordance in 35 samples where the physician collect were positive and finally there was discordance in 81 samples where the self-collect were positive. The results of sensitivity to cytology, Hybrid Capture by physicians, Hybrid Capture by self-sampling were, respectively 18%, 63,3% and 66,7%. The prevalence estimated by the gold standard were 7,2% in this sample. The results of specificity to cytology, hybrid capture by physicians, hybrid capture by self-sampling were, respectively 98%, 73% and 68,7%. There was significant difference between the results of HPV-DNA self-collected and collected by the physicians, McNemar test p<0,001. When we compare the concordance through the Kappa index we have obtained k=0,693 with stand error of 0,026. Compared the areas obtained by the ROC curve as follows area=0,738 that represent the achievement of physician collected with IC=[0,673;0,802] with 95% confidence interval to detect HPV in sample, area=0,670 that represent the achievement of self-collected sample to HPV with the 95% of confidence interval IC=[0,597;0,742].We concluded that Hybrid Capture by self-collected vaginal sample is as good as Hybrid Capture collected by the physicians, and there was good concordance between these tests.
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Murphy, Michael Joseph. "Using microsatellite DNA to genetically identify a potential hybrid population of endangered massasauga rattlesnakes (Sistrurus catenatus) in north central Missouri." Connect to resource, 2009. http://hdl.handle.net/1811/37064.
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Mandal, Amitesh. "Modification of a DNA Vaccine for Oral Administration in Fish for Aquaculture by Using Non-Microbial Nanoparticles." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/33576.
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Utilization of DNA vaccines in aquaculture has been gaining interest and recent efforts have been focused on methods of delivering DNA vaccines to fish. In the present study, a methodology was sought that could protect DNA vaccines such that they could be orally administered. The main objective of the study was to determine if a DNA vaccine could be effectively compounded into an orally administrable formulation with chitosan or polylactide-co-glycolide (PLG). The immune response of hybrid striped bass (Morone saxatilis x Morone chrysops) following oral delivery of a DNA vaccine containing Mycobacterium marinum Ag85A plasmid in either chitosan or PLG nanoparticle encapsulation was evaluated. Hybrid striped bass were divided into four experimental groups: IM immunization of the DNA vaccine as a positive control, oral delivery of uncomplexed DNA vaccine, oral delivery of chitosan or PLG alone as a negative control, and oral delivery of complexed chitosan or complexed PLG DNA vaccine. Fish were bled at regular intervals and an ELISA was used to evaluate antibody levels in individual fish. While the chitosan /plasmid DNA complex containing the Mycobacterium marinum Ag85A gene failed to produce a significant antibody response, the PLG/plasmid DNA matrix stimulated humoral immune response in the fish.Master of Science
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NAVA, GIULIA MARIA. "CHARACTERIZATION OF TLS POLYMERASE ETA FUNCTION UNDER REPLICATION STRESS INDUCED BY LOW DNTP POOLS." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/841241.
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RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. Although these structures have physiological relevance, their stable accumulation perturbs DNA replication, inducing replication stress and genome instability. Cells possess two enzymes that process these structures restoring the correct DNA:DNA sequence: RNase H1, which just handles stretches of multiple rNMPs, and RNase H2 that processes either single or multiple ribonucleotides hybridized with DNA. Mutations in the human RNase H2 lead to carcinogenesis and a severe auto-inflammatory disease known as Aicardi-Goutières syndrome. S.cerevisiae yeast cells lacking RNases H enzymes (rnh1Δ rnh201Δ) accumulate ribonucleotides in their genome, becoming sensitive to different replication-stress inducing agents such as hydroxyurea (HU), a compound that decreases the dNTP pools, stalling replication forks. Unexpectedly, we found that this HU-sensitivity is completely suppressed by removing the RAD30 gene, coding for the translesion DNA polymerase η.
In this study, I characterized this novel activity for the yeast Pol η under HU-induced replication stress, and the toxicity observed in RNase H depleted-cells, also focusing on possible regulatory mechanisms. Our data indicate that Pol η acts at HU-stressed replication forks, with recruitment that seems to depend on PCNA mono-Ub, and might also be regulated by the Pol η-SUMOylation levels. We proved that the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. These harmful effects arise during the first replication cycle in HU and seem to be due to the incorporation of stretches of ribonucleotides promoted by Pol η. In agreement, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to HU of cells lacking RNases H enzymes.
All these data are compatible with a model in which Pol η promotes DNA replication from stalled replication forks, inducing the formation or stabilization of RNA:DNA hybrids. These hybrids could result from either direct incorporation of rNMPs into DNA or incorrect Okazaki fragments maturation or R-loops stabilization. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become toxic for cells. Finally, performing an SGA screening, I identified some negative interactors of RAD30 that might compensate for the role played by the polymerase at HU-stalled replication forks.
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Mizaku, Alda. "Biomolecular feature selection of colorectal cancer microarray data using GA-SVM hybrid and noise perturbation to address overfitting." Diss., Online access via UMI:, 2009.
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Thesis (M.S.)--State University of New York at Binghamton, Thomas J. Watson School of Engineering and Applied Science, Department of Bioengineering, Biomedical Engineering, 2009.Includes bibliographical references.
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Rodrigues, Sergio Ricardo Pizano. "Formação de complexos entre compostos híbridos pirrolbenzodiazepinas-cumarinas com DNA por estudos de docking molecular." Universidade Federal de São Carlos, 2011. https://repositorio.ufscar.br/handle/ufscar/6972.
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Compounds of the pirrolbenzodiazepine (PBD) family are known for their promising antitumor activity. Among these, the hybrids, those that have a portion PDB a chain spacer and another functional group, such as the coumarins of this work, have been extensively explored. It is also known that these compounds bind to DNA, but there is no structural data showing how it occurs. To overcome this lack of information molecular docking calculations were performed to study the formation of complexes between these PBD-hybrids and DNA. The compounds were modeled and the coordinates of complexes DNA-receptors with different ligands were obtained from the Protein Data Bank. The redocking served to validate the conditions of the experiments and the scores were used as the parameter to evaluate the complexes formed. The analysis of the intermolecular interactions, an essential knowledge for understanding the obtained structures were analyzed using high-resolution molecular imaging. The results of the in silico experiments showed the formation of complexes in the mixed-mode with the PBD ligand moiety intercalating between the DNA bases and the coumarin portion occupying the minor groove, and a preference for intercalation between GG bases. Moreover, it is possible to postulate that the complex becomes an adduct with the formation of a covalent bond between the intercalated portion PBD and a nucleotide base G. Finally, a correlation between the docking results and the biological activities of the studied compounds was established.
Compostos da família das pirrolbenzodiazepinas (PBD) são conhecidos por apresentarem atividade antitumoral promissora. Dentre elas, as chamadas híbridas que possuem uma porção PDB uma cadeia espaçadora e outro grupo funcional, como as cumarinas deste trabalho, têm sido muito exploradas. Sabe-se que estes compostos se ligam ao DNA, mas não há dados estruturais mostrando como a ligação ocorre. Para suprir esta falta de informação foram realizados cálculos de docking molecular para estudar a formação de complexos entre estas PBDs híbridas e o DNA. Os compostos estudados foram modelados e as coordenadas de complexos DNA-receptores com diferentes ligantes foram obtidas do Protein Data Bank. O redocking serviu para validar as condições dos experimentos e os escores foram utilizados como parâmetro de avaliação dos complexos formados. A análise das interações intermoleculares, conhecimento essencial para o entendimento das estruturas obtidas, foi feita utilizando visualização molecular de alta resolução. Os resultados dos experimentos in silico mostraram a formação de complexos no modo de ligação misto, com os ligantes intercalando a porção PBD entre bases do DNA e a porção cumarina ocupando o sulco menor, mostrando ter preferência pela intercalação entre bases GG. Mais ainda, é possível postular que o complexo se torne um aduto com a formação de uma ligação covalente entre a porção PBD intercalada e uma base nucleotídica G. Finalmente foi estabelecida uma correlação entre os resultados do docking e as atividades biológicas dos compostos estudados.
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Bittorf, Blaine E. "Mapping Hybrid Lethal Genes on the X Chromosome of C. Briggsae." Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright152770556182685.
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Mengue, Me Ngou Milama Krystina. "Caractérisation d'une hybridation naturelle entre Schistosoma haematobium et Schistosoma guineensis au Gabon." Thesis, Tours, 2013. http://www.theses.fr/2013TOUR3304/document.
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La plupart des études sur l’hybride naturel entre Schistosoma haematobium (S.) et S. guineensis sont réalisées sur les vers adultes et contrairement aux études expérimentales sur l’hybridation, on ne retrouve pas de vers adultes hybrides après analyse de leur ADN. Avec cette étude, nous souhaitons mettre en évidence la présence d’hybride naturel entre ces deux espèces au Gabon à partir du premier élément suspect : l’œuf. Nous avons suivi l’œuf de son observation morphologique, à sa coloration par la technique de Ziehl-Neelsen jusqu’à l’amplification par PCR de son ADN et on a pu montrer qu’un œuf de morphologie suspecte observé dans les urines est capable d’amplifier à la fois une région spécifique de S. haematobium et de S. guineensisMost studies on the natural hybrid between Schistosoma haematobium (S.) and S.guineensis are performed on adult worms and contrary to experimental studies of hybridization, we do not find an adult hybrid worm after analysis of their DNA. With this study, we wish to highlight the presence of a natural hybrid between these two species in Gabon from the first suspect element: the egg. We followed the egg from its morphological observation to its staining using Ziehl-Neelsen technique until PCR amplification of its DNA and it has been shown that a suspected egg morphology seen in the urine is able to amplify both a specific region of S. haematobium and S. guineensis
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Noyes, Marcus Blaine. "An Omega-Based Bacterial One-Hybrid System for the Determination of Transcription Factor Specificity." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/407.
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From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome.
The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology.
To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome.
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Dougherty, John Kelly. "Identification of a Hybrid Lethal Gene on the X Chromosome of Caenorhabditis briggsae." Wright State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wright1579011194671611.
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Beckedorff, Felipe César Ferrarezi. "Recrutamento do complexo repressivo polycomb 2 pelo RNA não codificador longo antissenso ANRASSF1 modula a expressão do gene RASSF1A e a proliferação celular." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-23042013-083641/.
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O gene supressor tumoral RASSF1A tem sido associado com redução da proliferação celular em diversos tumores. Sua expressão é regulada por eventos epigenéticos que envolvem o complexo repressivo polycomb (PRC2), no entanto os mecanismos moleculares da modulação do recrutamento deste modificador epigenético para este locus ainda são desconhecidos. Neste trabalho identificamos e caracterizamos ANRASSF1, um RNA não codificador longo (lncRNA) intrônico unspliced, que é transcrito na fita oposta do gene RASSF1A, em várias linhagem celulares e tecidos, e se liga a PRC2. ANRASSF1 é transcrito pela RNAPII, possui cap-5´ e cauda poli-A, além de localizar-se no núcleo e possuir uma meia-vida em média quatro vezes menor comparada com outros lncRNAs ligados à PRC2. A super-expressão ectópica de ANRASSF1 reduziu os níveis de RASSF1A e aumentou a taxa de proliferação em células HeLa, enquanto seu silenciamento provocou efeito oposto. Essas mudanças nos níveis de ANRASSF1 não afetaram a abundância da isoforma RASSF1C em nenhuma das condições. A super-expressão de ANRASSF1 provocou um grande aumento tanto da ocupação de PRC2 como da marca de histona repressiva H3K27me3 especificamente na região promotora RASSF1A. Nenhum efeito da super-expressão de ANRASSF1 foi detectado na ocupação de PRC2 e na histona H3K27me3 nas regiões promotoras de RASSF1C e de outros quatro genes vizinhos, incluindo dois genes supressores tumorais bem caracterizados. Além disso, foi demonstrado que ANRASSF1 forma um híbrido de RNA/DNA e recruta SUZ12, um componente do PRC2, para o promotor de RASSF1A. Notavelmente, foi detectado pelo ensaio de RNase-ChIP que a degradação de ANRASSF1 diminui a ocupação de PRC2 neste promotor. Esses resultados demonstram um novo mecanismo de repressão epigenética do supressor tumoral RASSF1A, envolvendo um lncRNA unspliced antissenso, onde ANRASSF1 reprime seletivamente a expressão da isoforma de RASSF1 que sobrepõe o transcrito antissenso de modo local e específico. Considerando uma perspectiva mais ampla, nossos resultados sugerem que outros lncRNAs intrônicos unspliced não caracterizados no genoma humano podem contribuir para uma modulação epigenética local e específica de cada região em que os lncRNAs são transcritos.Tumor-suppressor RASSF1A gene down-regulation has been implicated in increasing cell proliferation in several tumors. Its expression is regulated by epigenetic events involving polycomb repressive complex 2 (PRC2), however the molecular mechanisms modulating recruitment of this epigenetic modifier to the locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand of RASSF1 gene in several cell lines and tissues, and binds to PRC2. ANRASSF1 is transcribed by RNA Polymerase II, 5\'-capped, polyadenylated, displays nuclear localization, and has on average a four-fold shorter half-life compared to other lncRNAs that bind PRC2. ANRASSF1 ectopic overexpression decreases RASSF1A abundance and increases the proliferation rate of HeLa cells, whereas its silencing causes opposite effects. These changes in NRASSF1 levels do not affect RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase both in PRC2 occupancy and in histone H3K27me3 repressive mark specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression is detected on PRC2 occupancy and on histone H3K27me3 at the promoter regions of RASSF1C and of four other neighbor genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrate that ANRASSF1 forms an RNA/DNA hybrid, and recruits SUZ12, a PRC2 component, to the RASSF1A promoter. Notably, depletion of ANRASSF1 disrupts SUZ12 occupancy on RASSF1A promoter as measured by RNAse-ChIP assay. Together, these results show a new mechanism of epigenetic repression of RASSF1A tumor suppressor gene involving an antisense unspliced lncRNA, in which ANRASSF1 selectively represses expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome may contribute to a location-specific epigenetic modulation of genes.
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Yang, Diya. "Genome-wide Analysis of F1 Hybrids to Determine the Initiation of Epigenetic Silencing in Maize." Miami University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1610098527086245.
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Anderson, Jennifer R. "DOUBLE HELIX: Mapping the generic ethical codes of creative nonfiction writing and medical writing. "THIS WILL PROBABLY HURT": Stories from my student nurse training." Thesis, Queensland University of Technology, 2017. https://eprints.qut.edu.au/104116/1/Jennifer_Anderson_Thesis.pdf.
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This project is a practice-led inquiry into ethical questions that arise when medical writing and creative nonfiction writing – which have different generic and ethical conventions – are combined in a nursing memoir. The thesis presents personal stories from years as a student nurse, alongside an exegetical discussion of how creative goals are informed by the ethics of representation and identity. In so doing, it offers critical and creative insights into creative nonfiction ethics and their application in medical creative writing.
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Essalhi, Kadija. "Interaction entre yOgg1, une ADN glycosylase de la voie BER, et l’ADN polymérase réplicative Polε chez Saccharomyces cerevisiae." Thesis, Orléans, 2013. http://www.theses.fr/2013ORLE2077/document.
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Les dommages oxydatifs de l’ADN sont impliqués dans les processus pathologiques que sont le cancer, les maladies neurodégénératives ou le vieillissement. Ces dommages résultent en partie de l’action des espèces réactives de l’oxygène (ERO), qui proviennent du métabolisme cellulaire ou d’agents exogènes (physiques ou chimiques), et qui conduisent à différents types de lésions parmi lesquelles l’oxydation des bases de l’ADN (8-oxoguanine, 8-oxoG) ou la formation de sites abasiques AP (apurique/apyrimidique). Ces lésions, qui si elles ne sont pas éliminées conduisent à des processus de mutagenèse ou de mort cellulaire, sont prises en charge spécifiquement par le système de réparation de l’ADN par excision de base ou BER. Le BER est initié par l’action d’une ADN glycosylase, telles que la 8-oxoG-ADN glycosylase (Ogg1) chargée d’éliminer la 8-oxoG, une lésion très abondante. Une étude par « double-hybride » initiatrice de ce projet a révélé l’existence d’une interaction in vivo chez S. cerevisiae entre la protéine yOgg1 et la sous-unité catalytique de l’ADN polymérase réplicative Polε (yPol2), également impliquée dans la voie BER chez la levure. Nos travaux démontrent que yOgg1 et yPol2 interagissent bien physiquement entre elles et de façon spécifique. Une étude par troncations et mutagenèse dirigée nous a permis d’identifier le domaine 3’→5’ exonucléase de yPol2 comme faisant partie de la forme tronquée minimale de yPol2 capable d’interagir avec yOgg1. La poche du site actif de yOgg1 et/ou son voisinage immédiat pourrait contenir pour partie le site d’interaction pour yPol2. Nous observons d’ailleurs une corrélation nette entre l’activité de yOgg1 et sa capacité à interagir avec yPol2 dans la levure. De même, l’activité 3’→5’ exonucléase de yPol2 pourrait être liée à son interaction avec yOgg1. D’un point de vue fonctionnel, yPol2 stimulerait l’activité AP lyase de yOgg1 et le couplage entre l’activité ADN glycosylase et AP lyase de l’enzyme, permettant ainsi une meilleure coordination de l’étape d’excision du nucléoside endommagé et l’étape de resynthèse de l’ADN dans la voie BEROxidative DNA damages are involved in pathological processes such as cancer, neurodegenerative diseases and aging. Part of these damages results from the action of reactive oxygen species (ROS), which are produced by cellular metabolism or (physical or chemical) exogenous agents. They lead to different types of DNA lesions including DNA base oxidation (8-oxoguanine, 8-oxoG) and abasic site formation (AP, apuric/apyrimidic). If not removed, these lesions lead to mutagenesis or cell death. Most of base lesions are dealt specifically by the base excision repair (BER) pathway. BER is initiated by a DNA glycosylase, such as 8-oxoG-DNA glycosylase (Ogg1) which is responsible for the removal of 8-oxoG. In previous unpublished work, a yeast two-hybrid study revealed the existence in S. cerevisiae of an interaction between yOgg1 and the catalytic subunit of the replicative DNA polymerase Polε (yPol2), also involved in the BER pathway in eukaryotes. Our work shows that yOgg1 and yPol2 physically and specifically interact with each other. Truncation and site-directed mutagenesis studies allowed us to identify the 3 ' → 5' exonuclease activity domain of yPol2 as part of the minimal form of yPol2 still able to interact with yOgg1. The active site of yOgg1 and/or its immediate vicinity may contain part of its interaction domain with yPol2. Besides, we observe a clear correlation between yOgg1 catalytic activity and its ability to interact with yPol2 in vivo. Similarly, the 3'→5' exonuclease activity of yPol2 could be useful to its interaction with yOgg1. From a functional point of view, yPol2 stimulates in vitro the AP lyase activity of yOgg1 and the coupling of both DNA glycosylase and AP lyase enzyme activity. The interaction yOgg1/yPol2 could allow a better coordination of damaged nucleoside excision and DNA re-synthesis steps in BER
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Veras, TÃnia Maria Cruz Werton. "Estudo da citologia oncÃtica convencional e da detecÃÃo do DNA-HPV pela captura de hÃbridos II no rastreamento primÃrio de lesÃes prÃ-neoplasicas e neoplÃsicas cervicais." Universidade Federal do CearÃ, 2005. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=352.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorObjetivos: avaliar o desempenho da citologia oncÃtica convencional e da captura de hÃbridos II na detecÃÃo de lesÃes cervicais neoplÃsicas e prÃ-neoplÃsicas. Sujeitos e MÃtodos: foram recrutadas aleatoriamente 1685 mulheres, da demanda espontÃnea de postos de saÃde da rede pÃblica, em cinco municÃpios do estado do CearÃ. As pacientes, apÃs assinarem termo de consentimento, responderam a um questionÃrio prÃ-elaborado e, a seguir, foram submetidas à coleta de material para CO, CH II e à realizaÃÃo de colposcopia, que, sendo positiva, levou à imediata biÃpsia dirigida das Ãreas anormais. Os dados foram digitados no programa Microsoft Excel 2000 e analisados no SPSS-for Windows, versÃo 10.0. O desempenho da CO e CH II foram calculados atravÃs da sensibilidade, especificidade, dos valores preditivos positivo e negativo e dos respectivos intervalos de confianÃa de 95%. Considerou-se, para anÃlise, como padrÃo ouro negativo, o resultado da colposcopia negativo ou resultado negativo no exame histopatolÃgico e, como padrÃo ouro positivo, o resultado positivo do histopatolÃgico. Avaliaram-se dois pontos de corte distintos: qualquer achado prÃ-neoplÃsico e neoplÃsico do colo uterino e achados de lesÃes intra-epiteliais de alto grau ou cÃncer. Resultados: 56 mulheres (3,4%) apresentaram atipias celulares na CO, sendo a CH II positiva em 315 (19%). Embora 337(20,32%) mulheres tenham sido positivas em um dos testes, somente 19(1,1%) foram positivas nos dois. Entre as 150 que tiveram colposcopia positiva somente em 53 foram encontradas lesÃes no exame histopatolÃgico, sendo a prevalÃncia estimada de 3,2% para qualquer lesÃo e de 0,4% para lesÃes de alto grau/cÃncer. Considerando o ponto de corte o achado de qualquer lesÃo prÃ-neoplÃsica ou neoplÃsicas, a sensibilidade encontrada para a CO e a CH II foi de 30,2% e de 71,7%, respectivamente. A especificidade dos testes mencionados foi de 97,5% e de 82,7%. O VPP e VPN da CO foram de 28,6% e de 97,7%, respectivamente. Jà o VPP e VPN da CH foram 12,1% e 98,9%. Considerando o ponto de corte lesÃes de alto grau ou cÃncer, temos: sensibilidade e especificidade da CO de 28,6% e de 99,9%, enquanto os VPP e VPN foram de 54,8% e de 99,7%, respectivamente. A CH II alcanÃou 100% de sensibilidade e 81,3% de especificidade. Os VPP e VPN ficaram em 2,2% e 100%. ConclusÃo: o teste de detecÃÃo do DNA-HPV pela CH II foi mais sensÃvel, porÃm menos especÃfico que a CO. Quando associado à CO, melhora significativamente a detecÃÃo das lesÃes cervicais, principalmente as de alto grau e cÃncer. Para este grupo de lesÃes, a CH II isolada apresentou melhor especificidade sem perda da sensibilidade, mostrando-se um bom teste para o rastreamento primÃrio.
Objective: to compare the usual Pap smear (Papanicolaou) and the Hybrid Capture II tests in detecting cervical intraepithelial neoplasia in women of Ceara State. Subjects and Methods: 1685 women were enrolled from routine practice in five municipalities of the main Cearà State Health Regions. The whole study was explained to the volunteers, who accepted to participate by signing an informed consent form. The study procedures included filling a questionaire and a cervical sample collection, done by a physician, for cytology and HPV-DNA Hybrid Capture, followed by a complete colposcopic evaluation with directed biopsy if necessary. Data were analyzed in Statistical Package for Social Sciences - SPSS - for Windows 10.0. The accuracy of both tests â Pap smear and Hybrid Capture II - was evaluated by using the sensitivity, specificity, positive predictive value, negative predictive value and the respective 95% confidence intervals. The negative colposcopic examination or negative histological result were considered gold standard for negative results. Positive histological results were considered gold standard for positive results. Results: 56 women (3,4%) had abnormal pap smear. Hybrid Capture tests were positive in 315 women (19%). Despite 337 (20,32%) tests had positive results for one of the two tests, only 19 (1,1%) were positive in both tests. Lesions were detected in 53 women among those 150 considered positive in colposcopic examination. The prevalence for any lesion was estimated in 3,2% and for high grade lesions and cancer in 0,4%. Using the cut-off point as the finding of any cervical lesion, the sensitivity of pap smear and HC II was 30,2% and 71,7%, respectively. The specificity for pap smear and HC II was 97,5% and 82,7%, respectively. The positive and negative predictive value for pap smear was 28,6% and 97,7%, respectively. The positive and negative predictive value for HC II was 12,1% and 98,9%, respectively. By using the cut-off value as high grade cervical lesions and cancer, the sensitivity and specificity for pap smear were 28,6% and 99,9%, respectively, and the positive predictive value and negative predictive value for the same test were 54,8% and 99,7%. The sensitivity and specificity for HC II were 100% and 81,3%, respectively, as well as 2,2% and 100% for positive and negative predictive value. Conclusions: hybrid Capture II test was more sensitive than pap smear, however Hybrid Capture II test was less specific than pap smear. When both tests were used together for detecting cervical lesions the results improved significantly, mainly high grade lesion and cancer. For this group of lesions, HC II alone, presented better specificity, without loss of the sensitivity, apparently itâs a good test for primary sceening.
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Pont, Geneviève. "Adn circulaires extrachromosomiques dans les embryons de drosophila melanogaster : caracterisation d'une classe moleculaire homologue aux genes histones." Clermont-Ferrand 2, 1987. http://www.theses.fr/1987CLF21068.
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Roncaglia, Maria Teresa. "Valor da captura híbrida para o papilomavírus humano (HPV) no seguimento de pacientes submetidos à conização do colo uterino devido a lesão intraepitelial de alto grau por cirurgia de alta frequência (CAF)." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5139/tde-13082012-093829/.
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INTRODUÇÃO: A lesão intraepitelial cervical de alto grau causada pelo HPV, precursora do câncer cervical, é facilmente diagnosticada e seu tratamento pode ser realizado de maneira ambulatorial, sem muitas complicações. Mesmo assim, o seguimento das pacientes tratadas deve ser feito de maneira criteriosa e sistemática para que a recorrência ou persistência da doença não passe despercebida. Conseguir identificar o grupo de pacientes com maior probabilidade de recorrência ou persistência da doença facilitaria sobremaneira esse seguimento, diminuindo o ônus econômico e psicológico que o seguimento generalizado produz. O objetivo deste trabalho é identificar marcadores que possam indicar o grupo de pacientes com maior possibilidade de recorrência da lesão intraepitelial cervical de alto grau. MÉTODOS: Neste estudo 114 mulheres com diagnóstico de lesão intraepitelial cervical de alto grau foram submetidas à conização cervical por cirurgia de alta frequência (CAF) no Setor de Patologia do Trato Genital Inferior (PTGI) da Divisão da Clínica Ginecológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) no período entre março de 2006 e maio de 2009. O seguimento foi realizado a cada seis meses durante o período de 24 meses. No seguimento foi coletada a citologia cervical, captura híbrida para HPV e realizada a colposcopia. Foram avaliados os testes de captura híbrida para HPV coletadas durante o seguimento, imuno-histoquímicos para oncoproteína viral E6 e proteína p16 na peça cirúrgica como possíveis marcadores de recorrência. RESULTADOS: A avaliação anatomopatológica da peça cirúrgica diagnosticou 85 (74,6%) casos de lesão intraepitelial cervical de alto grau e 29 (25,4%) casos de lesão intraepitelial de baixo grau. Nessas peças, 45 (39,5%) apresentaram expressão positiva para oncoproteína E6 e 69 (60,5%) apresentaram expressão negativa para E6; 74 (64,9%) apresentaram expressão positiva para p16 e 40 (35,1%) apresentaram expressão negativa para p16. A oncoproteína E6 não se associou com a apresentação mais grave da doença. Já a proteína p16 esteve positiva em 68 (80%) casos diagnosticados como lesão intraepitelial de alto grau e negativa em 23 (79,3%) casos com diagnóstico anatomopatológico de lesão intraepitelial de baixo grau ou cervicite crônica. A CH coletada no primeiro retorno apresentou sensibilidade de 83,3%, especificidade de 87,8%, VPP de 50% e VPN de 97,3%. Comparando a CH coletada no primeiro retorno com a citologia coletada após os 24 meses de seguimento, a CH apresentou sensibilidade de 75%, especificidade de 83,1%, VPP de 20% e VPN de 98,3%. CONCLUSÕES: A expressão das oncoproteína E6 e proteína p16 na peça cirúrgica não demonstraram ter valor para predizer recorrência no seguimento de pacientes tratadas por lesão intraepitelial cervical de alto grau, mesmo a p16 estando associada à presença de lesões mais graves. A CH pode ser usada como um teste preditivo de recorrência durante o seguimento de pacientes tratadas por lesão intraepitelial de alto grau. O VPN do teste é bastante alto e seu resultado negativo, seis meses após o tratamento indica uma taxa baixíssima de recorrência da doençaINTRODUCTION: The high grade cervical intraepithelial lesion caused by HPV, a pre-malignant condition, is easily diagnosed and its treatment can be done in outpatients without many complications. Nevertheless the patients follow-up must be done in a very systematic way to avoid any recurrence or persistence of the disease. To be able to identify the group of patients with higher rate of recurrence or persistence of the disease would make this follow-up much easier and decrease the economic and psychological burden of stressed outcome. The goal of our study is to identify markers that could indicate the group of patients more likely to recur. METHODS: In this study, 114 women diagnosed with high grade cervical intraepithelial lesion were treated with LLETZ at the Discipline of Gynecology, Faculty of Medicine, São Paulo University from March 2006 and May 2009. The follow-up visits after the treatment included Pap smear, HPV DNA test and colposcopy and occurred every 6 months for 24 months. The markers evaluated were the HPV DNA test collected during the follow-up and immunohistochemical tests performed on the surgical specimen: E6 oncoprotein and protein p16. RESULTS: We found 85 cases (74,6%) of HSIL and 29 cases (25,4%) of LSIL in the surgical specimen obtained with the LLETZ. The E6 oncoprotein was expressed in 45 (39,5%) and not expressed in 69 (60,5%) of the specimen; 74 (64,9%) expressed p16 and 40 (35,1%) didnt express p16. The E6 oncoprotein was not associated with severe presentation of the disease. The protein p16 was positive in 68 (80%) cases of diagnosed HSIL and negative in 23 (79,3%) cases with diagnosed LSIL or chronic cervicitis. The HPV DNA test collected at the first follow-up consult at 6 months presented a sensitivity of 83,3% specificity of 87,8%, positive predictive value (PPV) of 50% and negative predictive value (NPV) of 97,3%. Comparing the HPV DNA test collected at the first follow-up visit and the cervical cytology collected at the fourth and last follow-up visit at 24 months, the HPV DNA test presented a sensitivity of 75%, specificity of 83,1%, PPV of 20% and NPV of 98,3%. CONCLUSIONS: The E6 oncoprotein and protein p16 expression on the surgical specimen were not able to predict recurrence of the disease during the follow-up of the patients. The HPV DNA test can be used as a marker of the recurrence on the follow-up of patients treated for HSIL with LLETZ. The HPV DNA test negative result at the 6 month follow-up visit represents an extremely low recurrence rate
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Diniz, Ginetom S. "Electronic and Transport Properties of Carbon Nanotubes: Spin-orbit Effects and External Fields." Ohio University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1343143890.
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Liew, Steven Christopher. "Development of novel vaccines for the concurrent immunisation against multiple dengue virus serotypes." Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16199/3/Steven_Liew_Thesis.pdf.
Full textAbstract:
A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated.
In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype.
A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4.
However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes.
In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes.
Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins.
In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies.
In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.
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Liew, Steven Christopher. "Development of novel vaccines for the concurrent immunisation against multiple dengue virus serotypes." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16199/.
Full textAbstract:
A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated. In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype. A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4. However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes. In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes. Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins. In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies. In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.
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Stevens, Amy L. "Energy transfer processes in supramolecular light-harvesting systems." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:43833f3a-96b0-432a-9608-8f08a9096be7.
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This dissertation attempts to understand how energy transfer in a molecular wire and a spherical organic assembly are affected by molecular structure. The molecular wire is a DNA-hybrid structure composed of a strand of thymine bases appended by a cyanine dye. Hydrogen bonded to each base is a naphthalene-derivative molecule. Using time-integrated photoluminescence and time-correlated single photon counting measurements, energy transfer from the naphthalene donors to the cyanine acceptors was confirmed, and its dependence on temperature and DNA-template length investigated. Donor-thymine bonding was disrupted at temperatures above about 25 degrees Celcius resulting in poor donor template decoration and low rates of energy transfer. Increasing numbers of donors attach to the scaffold, forming an orderly array, as the template length increases due to the stabilising effects of the donor-donor pi-stacking interactions. Conversely, modelled energy transfer rates fall as the scaffold length increases because of the longer donor-acceptor distances involved. Therefore, the energy transfer rate was greatest for a template built from 30 thymines. The spherical organic assemblies (nanoparticles) are formed by fast injection of a small volume of molecularly dissolved fluorene-derivative amphiphilic molecules into a polar solvent. The amphiphilic molecules contained either a naphthalene (donor) or a benzothiadiazole (acceptor) core. The donor-acceptor mixed nanoparticles resemble an amorphous polymer film and were modelled as such using the Foerster resonance energy transfer theory. The Foerster radii extracted from the measurements depends intricately on the donor-acceptor spectral overlap and distance. The latter effect was controlled by the stacking interactions between the molecules. Altering the morphology of the structural units is the key to optimising energy transfer in molecular structures. To achieve efficient organic molecule-based devices, the importance of this property needs to be fully appreciated and effectively exploited.
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Singh, Arunoday [Verfasser]. "Synthesis and Self Association of Branched DNA Hybrids / Arunoday Singh." München : Verlag Dr. Hut, 2012. http://d-nb.info/1024242803/34.
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Papanicolaou, Irene. "Liposome-polymer nanoparticle hybrids as vectors in DNA vaccine delivery." Thesis, University College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422201.
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Wollman, Adam J. M. "DNA motor-protein hybrids for molecular transport and self-organisation." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:907144ad-2eec-4c01-8f20-217a1b7c122c.
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Kinesin is a molecular motor which walks on microtubule tracks in the eukaryotic cytoskeleton. It transports cargo but is also involved in cytoskeletal organisation. This thesis demonstrates fusing kinesin and DNA to construct a molecular transport system using self-organised tracks and to study the mechanics of the minimal motor unit of kinesin. The programmability of DNA allows for the formation of nanostructures with controllable interactions. Kinesin is conjugated to various DNA nanostructures to accomplish different tasks. Instructions encoded into DNA sequences are used to direct the assembly of a polar array of microtubules, to control the loading, active concentration and unloading of cargo on this track network and to trigger the disassembly of the network. Fluorescence microscopy was used to observe these microtubule arrays and the movement of cargo. It was found that the DNA signals used to control the unloading of cargo and the disassembly of the network had to be actively transported, rather than relying on diffusion, for effective delivery of the signal. This work lead to a first author publication, Wollman et al. (2013). DNA was also used to study kinesin by linking defined numbers of minimal functional motor units, single kinesin heads, into teams of 4-12 heads and observing their movement along microtubules via fluorescent labelling. A minimum of 5 heads were required for sustained movement, in agreement with the predictions of Hancock and Howard (1998). The velocity of teams increased with more heads, up to 8, and then a decrease was observed in teams with more heads.
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Ly, Danith. "Mechanism of electron transfer in double-stranded DNA and PNA-DNA hybrids, and the development of a fluorescence probe for DNA and RNA detection." Diss., Georgia Institute of Technology, 1998. http://hdl.handle.net/1853/30485.
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Jusas, Mantas. "Hibrido Populus tremuloides L. x Populus tremula L. x Betula pendula Roth mikrodauginimo in vitro sąlygų ištyrimas ir augalų regenerantų išauginimas." Master's thesis, Lithuanian Academic Libraries Network (LABT), 2009. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2008~D_20090114_155051-99442.
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Darbo objektas – naujai sukryžminti hibridinės drebulės (Populus tremuloides x Populus tremula) ir karpotojo beržo ( Betula pendula) hibridai. Darbo tikslas – Atlikti tolimąją hibridizaciją ir išauginti augalus-regenerantus nesubrendusių gemalų kultūroje bei įvertinti genetinę įvairovę. Darbo rezultatai. Sukryžminus gauta skirtingi hibridai. Jų įvairovė įvertinta APPD metodu. Išmatavus augimo tempus nustatyta, kad hibridai 16.2 ir 16.4 auga greičiau nei hibridinės drebulės klonai. Ištyrus adaptacijos nesterilioje aplinkoje sąlygas, nustatyta šaknijimosi tempai. Tyrimo metu pastebėta, kad ūgliukų sodinimas į durpių substratą Jiffi tabletėse su šaknimis ir be jų ilgesniam nei 3 mėnesių laikotarpiui, neturi augimo skirtumų.Aim of the work: new crossbred hybrid aspen (Populus tremuloides x Populus tremula) and birch (Betula pendula) hybrids Objekt of the work: make long hybridization and grow up new regeneration plants in unformed embrio culture and rate genetical variation Results: After crossing get new hybrids. His variation rated by RAPD metod. After measure growing speed, set that hybrids 16.2 and 16.4 growing faster than hybriding aspen clons. In adaptation study set root growing speed. In study notice that plants with root and without in Jiffi tablet peat substratum after 3 month get same height .
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Cohen, Sarah. "Le rôle de senataxine dans la résolution des hybrides ARN : ADN aux cassures double brins de l'ADN." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30125.
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Les gènes transcriptionellement actifs peuvent être la source de l'instabilité du génome via de nombreux mécanismes. Ces gènes sont caractérisés par la formation de structures secondaires telles que les hybrides ADN : ARN. Ils se forment lorsque l'ARN sortant l'ARN polymérase II s'hybride au simple brin d'ADN. De nombreuses études ont montrées que l'accumulation de ces hybrides peut mener à la création de dommages à l'ADN. Parmi ces dommages, les Cassures Double Brins (CDB) sont les plus dangereuses pour la cellule puisqu'elles peuvent produire des mutations et des réarrangements chromosomiques. Il existe deux mécanismes de réparation majeurs dans la cellule : la Jonction Non-Homologue des Extrémités (NHEJ) et la Recombinaison Homologue (HR). Mon équipe a récemment montré que les CDB localisées dans les gènes transcrits sont préférentiellement réparés par HR. De plus, de nombreuses études ont montrées une interaction entre transcription et réparation des CDB. Au vue de ces résultats, nous avons donc émis l'hypothèse que les gènes transcriptionellement actifs pourraient être réparés par un mécanisme spécifique nécessitant l'activité de protéines associées à la transcription : "Réparation couplée à la transcription". Durant ma thèse, je me suis intéressée au rôle de deux protéines dans la réparation des régions transcrites en utilisant la lignée cellulaire DIvA (DSB Induction via AsiSI) qui permet l'induction de cassures annotées sur tout le génome. Premièrement, nous avons montré que la réparation des CDB dans des loci transcrits nécessitent une hélicase ADN : ARN connue : sénataxine (SETX). Après induction d'une cassure dans un gène, SETX est recrutée ce qui permet la résolution d'hybride ADN : ARN (cartographié par DRIP-seq). Nous avons aussi montré que SETX permet le recrutement de RAD51 et limite les jonctions illégitimes des CDB et par conséquent promeut la survie des cellules après induction des cassures. Cette étude montre que les CDB dans les loci transcrits requièrent la résolution spécifique des hybrides ADN : ARN par SETX pour permettre une réparation précise et est absolument indispensable pour la survie cellulaire. Deuxièmement, nous avons montré une interaction entre SETX et Bloom (BLM) une G4 DNA hélicase dans la réparation des CDB dans les régions transcrites. Nous avons montré que BLM est aussi recrutée au CDB dans les loci transcrits où elle est nécessaire à la résection et à la fidélité de réparation. De façon importante, nous avons montré que la déplétion de BLM restaure le défaut de survie cellulaire observé dans les cellules déplétées pour SETX après induction des CDB. La déplétion d'autres hélicases G4 (RTEL1, FANCJ) promeut aussi la survie des cellules déplétées pour SETX après dommages. Ces résultats suggèrent une interaction entre les hélicases G4 et la résolution des hybrides ADN : ARN dans la réparation des gènes actifs. En conclusion, ces études permettent une meilleure compréhension de la spécificité de la réparation des régions transcrites du génome, et notamment l'identification de protéines impliquées dans la "Réparation couplée à la Transcription"Actively transcribed genes can be the source of genome instability through numerous mechanisms. Those genes are characterized by the formation of secondary structures such as RNA-DNA hybrids. They are formed when nascent RNA exiting RNA polymerase II hybridizes single stranded DNA. Numerous studies have shown that RNA-DNA hybrids accumulation can lead to DNA damages. Among those damages, DNA double strand breaks (DSB) are the most deleterious for cells since they can generate mutations and chromosomal rearrangements. Two major repair mechanisms exist in the cell: Non-Homologous End-Joining (NHEJ) and Homologous recombination (HR). My lab showed recently that DSB occurring in transcribed genes are preferentially repaired by HR. Moreover, multiple studies have shown a cross talk between transcription and DSB repair. Those results led us to propose that actively transcribed genes could be repaired by a specific mechanism implicating proteins associated with transcription: "Transcription-coupled DSB repair". During my PhD, using the DIvA (DSB Induction via AsiSI) cell line allowing the induction of annotated DSB through the genome, I worked on 2 projects focusing on DSB repair in transcribed genes. First, we showed that DSB repair in transcribed loci requires a known RNA: DNA helicase: senataxin (SETX). After DSB induction in an active gene, SETX is recruited which allows RNA-DNA hybrid resolution (mapped by DRIP-seq). We also showed that SETX activity allows RAD51 loading and limits DSB illegitimate rejoining and consequently promotes cell survival after DSB induction. This study shows that DSB in transcribed loci require specific RNA-DNA hybrids removal by SETX for accurate repair. Second, we showed an interplay between SETX and Bloom (BLM) a G4 DNA helicase in DSB repair induced in transcribed loci. We showed that BLM is also recruited at DSB in transcribed loci where it promotes resection and repair fidelity. Strikingly, we showed that BLM depletion rescued the survival defects observed in SETX depleted cells following DSB induction. Knock down of other G4-helicases (RTEL1, FANCJ) also promoted cell survival in SETX depleted cells upon damage. Those data suggest an interplay between G4 helicases and RNA: DNA resolution for DSB repair in active genes. Altogether, these studies promote a better understanding of the specificity of DSB repair in transcriptionally active genes, and notably identification of proteins involved in "Transcription-coupled DSB repair"