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Wolter, Gwen Annette. "Charakterisierung der Strahlen-induzierten Komplexbildung von DNA-Polymerase [alpha]-Primase [Alpha-Primase] mit dem Tumorsuppressorprotein p53." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=967492394.
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Pan, Hu. "Structural and biochemical studies of DNA primase from Bacillus stearothermophilus." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302122.
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Borazjani, Gholami Farimah. "Role of replicative primase in lesion bypass during DNA replication." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/68762/.
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Maintenance of genome integrity and stability is fundamental for any form of life. This is complicated as DNA is highly reactive and always under attack from a wide range of endogenous and exogenous sources which can lead to different damages in the DNA. To preserve the integrity of DNA replication, cells hav evolved a variety of DNA repair pathways. DNA damage tolerance mechanisms serve as the last line of defence to rescue the stalled replications forks. TLS, error-prone type of DNA damage tolerance, acts to bypass DNA lesions and allows continuation of DNA replication. Surprisingly majority of archaeal species lack canonical TLS polymerases. This poses a question as to how archaea restart stalled replication in the absence of TLS or lesion repair pathways. This thesis establishes that archaeal replicative primase (PriS/L), a member of the archaeo-eukaryotic primase (AEP) superfamily, possessing both primase and polymerase activities, is able to bypass the most common oxidative damages and highly distorting lesions caused by UV radiation. It has been postulated that archaeal replicative polymerases (Pol B and Pol D family Pols) can bind tightly to the deaminated bases uracil and cause replication fork stalling four bases prior to dU. A specific mechanism for resuming replication of uracil containing DNA by PriS/L is suggested in this thesis. In this thesis, we also reported how the enzymatic activities of archaeal PriS/L are regulated. Here, it is demonstrated that in contrast to archaeal replicative polymerases, single-strand binding proteins (RPA) significantly limit the polymerase activity of PriS/L. The remaining results chapter interrogates the possible interactions between PriS/L and RPA. Finally, the attempts to reconstitute an archaeal CMG complex in vitro, with the aim of shedding light on the role of archaeal replicative primase in replication-specific TLS are described.
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Desogus, Gianluigi. "Structural studies of lysyl-tRNA synthetases and DNA primases." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369258.
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Liku, Muluye E. "CDK regulation of replication proteins: Mcm2-7 and DNA polymerase alpha primase." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324598.
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Ligat, Gaëtan. "Cytomégalovirus humain, mutations de résistance et nouvelles cibles thérapeutiques." Thesis, Limoges, 2017. http://www.theses.fr/2017LIMO0046/document.
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Le cytomégalovirus humain (CMVH) est un pathogène opportuniste majeur en cas d’immunodépression et représente la principale cause d’infection congénitale d’origine virale. Bien qu’efficaces, l’utilisation des molécules conventionnelles est limitée par l’émergence de résistance et leur toxicité. Il devient alors nécessaire de développer de nouveaux traitements.L’étude des nouvelles mutations émergeant sous traitement antiviral demeure donc essentielle. L’introduction de ces nouvelles mutations, par mutagénèse « en passant », dans un chromosome bactérien artificiel contenant le génome viral nous permet, après transfection en cellules humaines, de tester la sensibilité de la souche recombinantes aux antiviraux.Différentes mutations de résistances ont ainsi été caractérisées. Afin de mettre en évidence de nouvelles cibles antivirales, des analyses bio-informatiques et la production de virus recombinants ont permis d’identifier de potentiels motifs fonctionnels essentiels à la réplication au sein du complexe terminase et hélicase-primase. Ainsi, nous avons montré quela sous-unité pUL56 du complexe terminase appartient à la famille des LAGLIDADG Homing Endonuclease. En effet, pUL56 contient un motif LATLNDIERFL et un motif de liaison à l’ADN. La technologie Alpha utilisant des protéines purifiées a permis de valider le caractère essentiel du fragment WMVVKYMGFF de pUL56 pour l’interaction avec pUL89. Enfin, nous avons mis en évidence les résidus impliqués dans la fixation de l’ATP au sein de l’hélicase et dans la stabilisation du zinc de la primase. Ainsi, la compréhension de la structure de ces protéines pourrait permettent de mieux appréhender leur fonctionnement au sein du processus de réplication du CMVH et le développement de nouvelles thérapies ciblant ces domainesHuman cytomegalovirus (HCMV) is an important opportunistic pathogen for immunecompromised patients and is the leading cause of congenital viral infection. Although they are effective, using of conventional molecules is limited by the emergence of resistance and their toxicity. Then it becomes necessary to develop new treatments. Study of new mutationsemerging under antiviral treatment is therefore essential. Introduction of these new mutations, by « en passant » mutagenesis, into an artificial bacterial chromosome containing the viral genome allows us, after transfection into human cells, testing antivirals sensitivity of the recombinant. Different mutations of resistances have been characterized. In order tohighlight new antiviral targets, bioinformatics and recombinant viruses production allowed to identify potential functional patterns essential for viral replication within terminase and helicase-primase complex. Thus, we have shown that pUL56 subunit of the terminase complex belongs to the LAGLIDADG Homing Endonuclease family. Indeed, pUL56 contains aLATLNDIERFL motif and a DNA binding motif. Alpha technology using purified proteins allowed to validate the essential character of the WMVVKYMGFF fragment of pUL56 for the interaction with pUL89. Finally, we highlighted the residues involved in ATP binding within the helicase and in the stabilization of zinc within the primase. Thus, understanding of these proteins structure could allow us to better understand their role within the viral replication process and the development of new therapies targeting these domains
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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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Merryweather, Andrew. "The role of DNA primases specified by plasmids RP4 and ColIb-P9." Thesis, University of Leicester, 1986. http://hdl.handle.net/2381/34448.
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Bayes, Michelle. "A molecular phylogenetic study of the galagos, strepsirrhine primates and archontan mammals." Thesis, Oxford Brookes University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266454.
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Rudd, Sean G. "Cellular and biochemical characterisation of PrimPol, a novel eukaryotic primase-polymerase involved in DNA damage tolerance." Thesis, University of Sussex, 2013. http://sro.sussex.ac.uk/id/eprint/45543/.
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Genome stability is of upmost importance to life. DNA polymerases are essential for the duplication and maintenance of the genome but they cannot themselves begin synthesis of a DNA chain, and require the activity of specialised RNA polymerases called primases. In eukaryotic cells distinct enzymes catalyse these two essential processes. This thesis contains the characterisation of coiled-coil domain containing protein (CCDC)111, a previously uncharacterised protein conserved in a broad range of unicellular and multicellular eukaryotes including humans. CCDC111 is a member of the archaeaoeukaroytic primase (AEP) superfamily and uniquely for a eukaryotic enzyme possesses both primase and polymerase activities, and was thus renamed PrimPol. The work in this thesis implicates PrimPol in the process of DNA damage tolerance, a universal mechanism by which cells complete genome duplication in spite of potentially lethal DNA damage. The first results chapters detail the essential role of a PrimPol homologue (TbPrimPol2) in the important protozoan pathogen Trypanosoma brucei. A combination of molecular, cell biology, and biochemical analyses indicate a role for TbPrimPol2 in the post-replication tolerance of endogenously occurring DNA damage using its trans-lesion DNA synthesis activity. The remaining results chapters characterise PrimPol in human cultured cells, and demonstrate that this enzyme is present in both the nucleus and mitochondria. In the nucleus PrimPol functions in the cellular tolerance of ultraviolet (UV)- induced DNA damage, and is required to protect xeroderma pigmentosum variant (XP-V) cells, deficient in the UV lesion bypass polymerase Pol !, from the cytotoxic affects of UV radiation. Together, this thesis establishes the involvement of PrimPol in DNA damage tolerance from one of the earliest diverging eukaryotic organisms to man.
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Bianchi, Julie. "Investigating the role of a novel primase-polymerase, PrimPol, in DNA damage tolerance in vertebrate cells." Thesis, University of Sussex, 2013. http://sro.sussex.ac.uk/id/eprint/45544/.
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Genome duplication is an essential task our cells have to achieve prior to cell division, and requires a highly specialized replication machinery to ensure it is performed in an accurate and complete manner. DNA primase and polymerases are essential components of the replisome. Primases initiate DNA replication by synthesising short RNA primers that are then elongated by faithful and processive replicative DNA polymerases. However, both exogenous and endogenous agents can damage DNA and hinder progression of the replicative machinery. Translesion synthesis DNA polymerases assist in bypassing these DNA lesions in a process called DNA damage tolerance that enables chromosomal replication to proceed in in spite of damaged templates. This thesis details the characterisation of a novel eukaryotic DNA primase, coiled-coil domain containing protein (CCDC111), a member of the Archaeo Eukaryotic Primase (AEP) superfamily. Preliminary in vitro characterisation of CCDC111 demonstrated that the recombinant protein is capable of both DNA-dependant priming and polymerase activities, which is unprecedented for a eukaryotic polymerase, and it was therefore renamed Primase-polymerase (PrimPol). The aim of this thesis was to provide one of the first cellular characterisations of PrimPol by generating a knockout of the gene in avian DT40 cells and also depleting the protein in human cells using RNAi. In vivo evidence supports the involvement of this novel polymerase in replication fork progression following replicative stress, such as exposure to UV light, but also during unperturbed DNA replication. Work in this thesis also indicates a role for PrimPol in mitochondrial DNA maintenance. Together, the data presented here establish a role for PrimPol in DNA damage tolerance in avian and human cells.
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Lynch, Magnus David. "The role of primary DNA sequence in templating chromatin states." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543027.
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Reeves, Richard Guy. "Molecular zoogeography and evolution of primary freshwater fishes in Central-America." Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340798.
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Henderson, Dorian Travis. "Activity of the long and short forms of the plasmid-encoded primase, MobA and RepB', in vegetative and conjugal replication of the broad-host-rang plasmid R1162 /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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Bailey, Elisabeth Ann 1967. "Mutagenesis by the primary aflatoxin B₁ DNA adduct in Escherichia coli." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/40572.
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Keen, Benjamin A. "Molecular dissection of PrimPol, a novel primase-polymerase involved in damage tolerance during DNA replication in eukaryotic cells." Thesis, University of Sussex, 2015. http://sro.sussex.ac.uk/id/eprint/54095/.
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PrimPol is a recently identified member of the archaeo-eukaryotic primase (AEP) family of proteins. It possesses both primase and polymerase activities and is involved in the replication of both nuclear and mitochondrial DNA. PrimPol is predicted to possess an AEP polymerase and a UL52-like zinc finger domain. This thesis establishes the roles of these domains in the context of PrimPol's catalytic activities. Although apparently dispensable for polymerase activity, the zinc finger is essential for maintaining primase activity and also appears to play an important role in regulating the processivity and fidelity of PrimPol's extension activities. A recently study identified a PrimPol mutation (Y89D) that is potentially associated with the development of high myopia in humans. Here, the biochemical defects associated with this mutant are analysed and described. This protein variant has a significant reduction in polymerase activity. Mutational analysis suggests that the hydrophobic ring of tyrosine is important for retaining wildtype DNA extension activity. Biophysical analysis of the secondary structure and stability of this PrimPol variant suggests that this PrimPol variant has reduced α-helical content and is less stable than the wild-type protein. Finally, the interaction of PrimPol with single-stranded DNA binding protein replication protein A (RPA) is investigated. Previous studies have identified an interaction of PrimPol with RPA. Here, it is demonstrated that PrimPol has two separate RPA interaction motifs and a crystal structure is presented of one such motif in PrimPol bound to RPA that reveals the molecular basis for this interaction. Together, these studies provide molecular insights into the catalytic mechanism of PrimPol as well as some of the key intramolecular and intermolecular mechanisms of that regulate the activities of PrimPol.
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Lu, Yang. "Functional studies of new protein-protein interactions potentially involved in homologous recombination in hyperthermophilic archaea : study of interactions between PCNA and Mre11-Rad50 complex & Primase and RadA." Thesis, Brest, 2018. http://www.theses.fr/2018BRES0077/document.
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Les archées hyperthermophiles ont une température optimale de croissance supérieure à 80°C.Les cellules exposées à un stress thermique subissent une augmentation de la sensibilité aux agents induisant des cassures double brin de l’ADN. Les études sur les eucaryotes et bactéries ont démontré que la recombinaison homologue joue un rôle essentiel non seulement dans la réparation de l’ADN, mais aussi dans le redémarrage des arrêts de la fourche de réplication. Les enzymes associées aux étapes initiales de la recombinaison homologue chez les archées sont homologues à celles des eucaryotes, et différentes des analogues bactériens. De plus, plusieurs études ont démontré que les protéines impliquées dans la recombinaison homologue sont essentielles chez les archées hyperthermophiles, soulignant l’importance biologique de cette voie de réparation chez ces organismes particuliers. Le rôle de la recombinaison homologue pour la stabilité génomique a été bien étudié chez les eucaryotes et les bactéries, cependant, peu de ses propriétés fonctionnelles ont été étudiées chez les archées hyperthermophiles. Pour mieux comprendre le mécanisme de recombinaison homologue impliquée au niveau de la maintenance génomique chez les archées, un réseau d’interactions protéine-protéines a été révélé précédemment au laboratoire à partir des protéines de Pyrococcus abyssi. Ces travaux ont démontré de nouvelles interactions où interviennent les protéines de la réplication et les protéines de la recombinaison de l’ADN. L’objet de cette étude de thèse est de présenter deux interactions : PCNA/Mre11-rad50 (MR) complexe et Primase/RadA. Pour la première fois chez P. furiosus, une interaction physique et fonctionnelle a été démontrée entre le PCNA et le complexe MR (l’initiateur de HR). Un motif, situé en position Cterminale de Mre11, permet l’interaction avec PCNA.PCNA stimule l’activité endonucléase du complexe MR à distance proche de l’extrémité 5’ d’une cassure double brin. Cette propriété est en accord avec l’intervention ultérieure des enzymes assurant la suite du mécanisme de réparation par recombinaison homologue. Par ailleurs, les protéines RadA, Primase et P41 ont été produites et purifiées. Leurs fonctions enzymatiques ont été confirmées. Cependant, nous n’avons pas pu caractériser la fonction de l’association de RadA/PrimaseHyperthermophilic archaea (HA) are found in high-temperature environments and grow optimally above 80°C. Usually, cells exposed to heat stress display an increased sensitivity to agents inducing double-stranded DNA breaks (DSBs). Studies in Eukaryotes and Bacteria have revealed that homologous recombination (HR) plays a crucial role not only in DNA DSBs repair, but also in the collapsed/stalled DNA replication fork restart.Recombinase and various HR-associated enzymes in archaea specifically resemble the eukaryotic homologues, rather than bacterial homologues.Furthermore, several studies have demonstrated the necessity of HR proteins in HA, suggesting that, HR is an important mechanism in HA. HR influencing genome stability has been well studied in Eukaryotes andBacteria, however, few of its functional properties have been studied in HA.To better understand how HR mechanism is involved in the archaeal genome maintenance process, a previous work proposed a protein-protein interaction network based on Pyrococcus abyssi proteins. Through the network, new interactions involving proteins from DNA replication and DNA recombination were highlighted. The targets of the study presented here for two protein interaction are: PCNA/Mre11-rad50 complex (MR complex) and Primase/RadA. For the first time in P. furiosus, we showed both physical and functional interactions between PCNA (Maestro in DNA replication) and MR complex (initiator of HR). We have identified a PCNA-interaction motif (PIP) located in the C-terminal of Mre11, and shown that PCNA stimulated MR complex endonuclease cleavage proximal to the s’ strand of DNA DSBs at physiological ionic strength. For the second interaction, we have purified the proteins PabRadA/PfuRadA, PabPrimase and PabP41, and confirmed its enzymatic functions. However, we were not able to characterize the function of Primase/RadA association
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Droney, Kelly Eileen. "The evolution of a bmp5 enhancer in primates." Kent State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=kent1310587653.
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Brockmann, William G. Eick J. David. "An investigation of a rapid fluorescence microtiter plate methodology for measuring chemically-induced DNA damage, suitable for use in development of a primary DNA damage database." Diss., UMK access, 2004.
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Thesis (Ph. D.)--School of Dentistry and School of Pharmacy. University of Missouri--Kansas City, 2004."A dissertation in oral biology and pharmacology." Advisor: J. David Eick. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Feb. 22, 2006. Includes bibliographical references (leaves 176-189). Online version of the print edition.
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Guillet-Renard, Claire. "Évolution des îlots CpG chez les primates." Thesis, Lyon 1, 2009. http://www.theses.fr/2009LYO10162.
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Cette thèse a pour l’objet l’étude des pressions de sélection qui s’appliquent sur les îlots CpG, courtes séquences génomiques qui échappent à la méthylation chez les mammifères. Nous avons tout d’abord étudié les caractéristiques génomiques des îlots CpG, notamment leurs liens avec l’initiation de transcription des gènes et les origines de réplication de l’ADN, en utilisant des jeux de données récemment publiés. Nous avons ensuite déterminé si les caractéristiques de séquence des îlots CpG (richesse en dinucléotides CpG et richesse en GC) étaient sous pression de sélection et pouvaient jouer un rôle dans les fonctions des îlots CpG. Nous avons montré que la richesse relative en dinucléotides CpG des îlots CpG résulte uniquement de la faible méthylation de ces séquences. De plus, la richesse en bases GC des îlots CpG n’est pas soumise à pression de sélection mais semble résulter d’un mécanisme neutre, la conversion génique biaisée vers GC. Nous discutons également du devenir des îlots CpG chez les primates, qui et avons montré que si le taux de GC de ces séquences est en train de diminuer, la richesse relative en CpG quant à elle reste stableThis thesis analyses selective pressures applying on CpG islands, short sequences which escape methylation in mammalian genomes. We first studied genomic characteristics of CpG islands. We namely studied their relationships with gene transcription start, and with DNA replication origins, using recently published data. We then determined wether base peculiar composition of CpG islands (high number of CpG dinucleotides, high GC content) may be under (negative or positive) selective pressures, and thus play a role in their function, or not. We showed that the relative CpG-richness of CpG islands is the mere consequence of the low methylation of these genomic regions. Moreover, we showed that the high GC content of CpG islands is not under selective pressures, and seem to result from a neutral mechanism, biased gene conversion toward GC. We also discussed the future of CpG islands and primates. We showed that the GC content of CpG islands is decreasing, while the relative CpG content remains constant
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Kransdorf, Evan Paul. "Purification and Characterization of a Methyl-DNA Binding Protein Complex from Primary Erythroid Cells." VCU Scholars Compass, 2004. http://hdl.handle.net/10156/1685.
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Martin, Paul Ryan. "Studies on the Primary Mechanisms of (6-4) photolyase : Photoactivation and DNA Repair." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066375/document.
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Ce travail concerne les mécanismes photoinduits d’une flavoprotéine appartenant à la famille des cryptochromes et photolyases (CPF) : la photolyase (6-4). En utilisant de la lumière bleue cette protéine répare un dommage de l’ADN induit par les UV, le photoproduit (6-4). Nous avons étudié cette photoreparation ainsi qu’une autre réaction photoactivée que la photolyase utilise pour réduire son cofacteur flavine, la photoactivation. Nous avons faits nos études en utilisant la photolyase (6-4) de Xenopus laevis. Nous avons étudié la photoréduction du chromophore FADox de l’enzyme par spectroscopie d’absorption transitoire femtoseconde polarisée. Nous avons observé un transfert d’électron ultrarapide (~400 fs) après excitation du chromophore FADox. Nous avons caractérisés un résidu tryptophane comme réducteur. Nous avons cherché de distinguer entre les différents tryptophanes présents dans le site de photoactivation par des mesures d’anisotropie. Les résultats obtenus suggèrent que le mécanisme de transfert d’électron dans la photolyase n’est pas compatible avec le mécanisme supposé chez les photolyases qui consiste des transferts d’électron de long d’une chaine de trois résidus tryptophane. Grâce à des trains d’impulsions courtes, nous avons démontré que la photolyase (6-4) répare l’ADN par un mécanisme à deux photons successif. Le premier photon sert à convertir le défaut (6-4) en un intermédiaire métastable, X, qui a une durée de vie de ~2 min. Un second photon absorbé pendant cette durée de vie permet d’achever le cycle de réparationWe studied the light-induced reactions of the (6-4) photolyase, a flavoenzyme of the cryptochrome/photolyase family that repairs the UV-induced (6-4) photodamage in DNA with the aid of blue light. We studied this photorepair reaction as well as the light-induced cofactor reduction called photoactivation that the enzyme uses to bring itself to a repair-active state in the (6-4) photolyase from Xenopus laevis. We have studied the photoactivation of the FADox cofactor of the enzyme using femtosecond polarised transient absorption spectroscopy. We observed a sub-picosecond electron transfer (~400 fs) after excitation of the FADox cofactor. We were able to characterise a tryptophan residue as the electron donor. We sought to differentiate the spectroscopically identical but differently oriented tryptophan residues within the protein’s photoactivation site by transient anisotropy measurements. Our results suggest that the photoactivation mechanism is not fully compatible with the mechanism thought to be conserved among photolyases: an electron transfer mechanism via electron hopping along a chain of three highly conserved tryptophan residues.Using series of single turnover flashes, we have found that the repair reaction proceeds by a successive two-photon mechanism. The first photon converts the (6-4) lesion into a metastable intermediate X, the lifetime of which is ~2 min. Absorption of a second photon within the lifetime of X results to the restoration of intact nucleobases. In light of our findings, the reaction was also studied by femtosecond transient absorption spectroscopy
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Gallego, Jiménez Alicia 1985. "Addressing functional and evolutionary implications of microRNA variation at the DNA and RNA levels in primates." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/585943.
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microRNAs are small non-coding RNAs with crucial roles in gene regulation and whose contribution to animal evolution has been largely demonstrated. In this thesis we explored some functional consequences of microRNA sequence variation and their possible effects in primate evolution. We first evaluated microRNA nucleotide variation at the genomic level in great apes. Taking advantage of recently published whole-genome sequencing data from 82 individuals including oragutans, gorillas, bonobos, chimpanzees and humans, we analyzed microRNA sequence conservation patterns, both among and within populations. We observed that the entire microRNA mature region was significantly conserved, suggesting its central role for the microRNA regulatory function. We additionally observed that more conserved microRNAs tend to be older, duplicated, clustered, highly associated with disease and show higher expression levels. Further functional analyses revealed that lineage-specific changes in the microRNA mature sequences and/or in the length of the precursor molecules of mir-299, mir-503, mir-508 and mir-541 altered their expression levels and redirected the spectrum of target genes and regulatory networks, some of them linked to neuronal functions. We secondly investigated microRNA sequence variation generated by RNA editing. Focusing on mir-376a1 we studied the RNA editing patterns among different primate individuals including human placenta and macaque, gorilla, chimpanzee and human brain cortex samples. Although mir-376a1 editing showed high inter-individual variation, it was more frequently detected in brain than in placenta and in one particular site. This highly edited site conferred the highest stability to the hairpin molecule, revealing the important contribution of RNA editing to the stability of the transcripts. In summary, we provide evidence on how DNA and RNA nucleotide changes may drive microRNA diversification and redefine new regulatory functions, which could have importantly contributed to primate phenotypic diversification processes and to the recent evolution of our species.Los microARNs son ARNs de pequeño tamaño no codificantes para proteína, con un papel crucial en la regulación genética y cuya contribución en la evolución animal ha sido ampliamente demostrada. En esta tesis exploramos algunas consecuencias funcionales asociadas a la variación en las secuencias de los microARNs y sus posibles implicaciones en la evolución de los primates. En primer lugar evaluamos la variación nucleotídica de los microARNs a nivel del genoma en los grandes simios. Aprovechando datos de secuenciación de genomas completos recientemente publicados provenientes de 82 individuos orangutanes, gorilas, bonobos, chimpancés y humanos, analizamos los patrones de conservación de las secuencias de microARNs, entre y dentro de las poblaciones. Observamos que la región madura del microARN aparecía significativamente conservada, sugiriendo su papel central en la función reguladora del microARN. Vimos además que los microARNs más conservados tienden a ser más antiguos, estar duplicados, agrupados, asociados a enfermedad y más altamente expresados. Análisis funcionales posteriores revelaron que cambios específicos de linaje en las secuencias maduras y/o en la longitud de las secuencias precursoras de mir-299, mir-503, mir-508 and mir-541 alteraron sus niveles de expresión y redefinieron el conjunto de genes dianas y redes reguladoras, algunas de ellas implicadas en funciones neuronales. En segundo lugar, investigamos la variación de la secuencia de los microARNs generada por la edición del ARN. Centrándonos en mir-376a1 estudiamos los patrones de edición en diferentes individuos de primates incluyendo muestras de placenta humana y cortex cerebral de macaco, gorila, chimpancé y humano. Aunque la edición de mir-376a1 mostró gran variación entre individuos, se detectó en mayor frecuencia en cerebro que en placenta y en un sitio concreto. Este sitio altamente editado confería mayor estabilidad a la molécula precursora, indicando la importancia de la edición del ARN en la estabilidad de los transcritos. En resumen, esta tesis muestra evidencias sobre cómo los cambios nucleotídicos en el ADN o ARN podrían conducir a la diversificación de los microARNs y definir nuevas funciones reguladoras, las cuales podrían haber contribuido de manera importante en los procesos de diversificación fenotípica en los primates y en la evolución reciente de nuestra especie.
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ROSSIELLO, FRANCESCA. "PERSISTENT DNA DAMAGE AT TELOMERES, CAUSED BY TRF2-MEDIATED DNA REPAIR INHIBITION, TRIGGERS CELLULAR SENESCENCE AND IS ASSOCIATED WITH PRIMATES AGEING." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234141.
Full textAbstract:
The DNA damage response (DDR) coordinates DNA repair events and transiently arrests cell-cycle progression until DNA damage has been removed. If the damage is not resolved, cells can enter an irreversible cell cycle arrest called cellular senescence. In irradiation-induced senescent cells a large fraction of persistent DDR markers are associated with telomeric DNA, both in cultured cells and in in vivo tissues.
The aim of my PhD project was to investigate the mechanism underlying this phenomenon. I showed that persistent DDR activation has a causative role for the senescence-associated cell cycle exit and that a double-strand break (DSB) within telomeric repeats is inducing a more protracted DDR activation compared with a non-telomeric one in human cells. The DDR persistency at telomeres is neither dependent on their heterochromatic state nor on TRF2 loss from telomeres during senescence establishment. Rather, TRF2 recruitment next to a DSB, in the absence of telomeric DNA, is sufficient to induce a more protracted site-specific DDR focus and to impair DSB repair in mouse cells. Ageing is associated with accumulation of markers of DDR activation. In terminally differentiated brain neurons from old primates, I observed DDR activation at telomeres that were not critically short.
Taken together, these results strongly suggest that TRF2 inhibits DNA repair at broken telomeres, contributing to the accumulation of unrepaired, DDR-positive telomeres during ageing. This can in turn trigger cellular senescence and impair tissue homeostasis providing a mechanism for ageing also in non-proliferating tissues.
Finally, I focused my attention on DICER and DROSHA-dependent DNA damage response RNAs (DDRNAs), novel components of the DDR machinery, which have been described to be necessary for DDR activation at DSBs. I showed that RNase A treatment as well as DICER or DROSHA down-regulation impair DDR activation at uncapped telomeres and that DICER and DROSHA may have a role in chromosomal fusions. Furthermore, in cells with dysfunctional telomeres, the inhibition of telomeric DDRNAs using inhibitory oligonucleotide molecules with a complementary sequence can prevent DDR activation and senescent-associated cell cycle arrest.
These data indicate that at uncapped telomeres, DDRNAs with telomeric sequences are generated and that they are necessary for DDR activation and chromosomal fusions.
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Esbenshade, Timothy A. "Characterization of [alpha]₁-adrenergic receptor mediated DNA and protein syntheses in primary cultured rat hepatocytes /." The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487687115925499.
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Burger, Katharina [Verfasser]. "Influence of different noxa on DNA repair capacity of primary human skin cells / Katharina Burger." Konstanz : Bibliothek der Universität Konstanz, 2011. http://d-nb.info/1017360456/34.
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Valle, Rodrigo Del Rio do. "Colheita, análise e criopreservação de sêmen de uma espécie modelo de primata neotropical, Sagui-de-Tufo-Branco (Callithrix jacchus)." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-21052007-103859/.
Full textAbstract:
A espécie Callithrix jacchus pode ser utilizada como modelo experimental para outras espécies de primatas neotropicais, principalmente Callithriquídeos. Colheu-se sêmen desta espécie com os objetivos de otimizar a colheita de sêmen por vibro estimulação peniana, validar técnicas de avaliação espermática que possam ser utilizadas a campo, comparar as características seminais de indivíduos de duas colônias (Brasil e Alemanha), testar diferentes protocolos de congelação espermática e avaliar a atividade citoquímica mitocondrial (DAB) e a fragmentação de DNA de espermatozóides congelados. As técnicas de coloração para avaliação espermática utilizadas foram: eosina-nigrosina, coloração dupla trypan-blue giemsa, coloração simples para acrossoma, FITC-PSA, SpermOscan®, Spermac®, coloração com 3,3'-diaminobenzidina (DAB) e ensaio Cometa alcalino. A congelação foi realizada com 100 µL de sêmen com meio TEST-Gema de ovo clarificado com glicerol 4% em palhetas 0,25 mL, e os protocolos de congelação testados foram: 1) fase de equilíbrio com curva de resfriamento (25°C-4°C) em 2 horas, 10 minutos no vapor de nitrogênio e finalmente imersão em nitrogênio líquido; 2) como Protocolo 1, mas sem fase de equilíbrio/resfriamento; e 3) diretamente no nitrogênio líquido. As palhetas foram descongeladas em água a 37°C por 5-10 segundos. A técnica de colheita resultou em 83,33% dos procedimentos com ejaculação. A coloração simples para acrossomo foi eficiente e pôde ser validada para utilização a campo. Não houve diferença significante (p<0,05) nas características seminais avaliadas entre as colônias do Brasil e da Alemanha. As análises pós-descongelação demonstraram queda nas variáveis motilidade e integridade da membrana plasmática com discreta superioridade no Protocolo 1, não houve diferença significante (p<0,05) entre os protocolos para a atividade citoquímica mitocondrial e o Protocolo 2 apresentou a menor taxa de fragmentação de DNA.The Callithrix jacchus can be used as a model species for other Neotropical primates species, mainly Callithriquids, for reproductive studies. Semen samples were collected with the following objectives: to improve the penile vibrostimulation technique, validate sperm evaluation techniques for field work, analyse the semen characteristics from animals at 2 colonies (Brazil and Germany), use 3 different freezing protocols in common marmosets sperms, evaluate a cytochemical technique for mitochondrial activity demonstration and evaluate DNA damage in frozen-thawed sperms. The staining techniques used in this work were: Eosin-nigrosin, Trypan-blue Giemsa, simple staining for acrosome, FITC-PSA, SpermOscan®, Spermac®, demonstration of cytochrome c oxidase activity in situ by the oxidation of 3,3'-diaminobenzidina (DAB) and the Comet assay. Freezing was done in 100 µL semen with TESt-Yolk clarified medium plus 4% glycerol using 0.25 mL straws. The freezing protocols tested were: 1) straws at equilibrium phase with a 25°C to 4°C curve in 2 hours, 10 minutes at nitrogen vapour and finally into liquid nitrogen; 2) same as Protocol 1 without the equilibrium phase; and 3) straws directly into liquid nitrogen. All attempts resulted in 83,33% ejaculates. The simple staining technique for acrosome was validated and resulted in an obvious differentiation of the intact acrosome. Semen characteristics evaluation showed no difference between the 2 colonies. Post-thaw evaluation showed all protocols had negative effects on motility and plasmatic membrane integrity with a slightly superiority on Protocol 1. There was no difference between protocols in the mitochondrial activity demonstration and Protocol 2 presented the lowest DNA damage.
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Xu, Ke. "Comparative genomic and epigenomic analyses of human and non-human primate evolution." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/52935.
Full textAbstract:
Primates are one of the best characterized phylogenies with vast amounts of comparative data available, including genomic sequences, gene expression, and epigenetic modifications. Thus, they provide an ideal system to study sequence evolution, regulatory evolution, epigenetic evolution as well as their interplays. Comparative studies of primate genomes can also shed light on molecular basis of human-specific traits. This dissertation is mainly composed of three chapters studying human and non-human primate evolution. The first study investigated evolutionary rate difference between sex chromosome and autosomes across diverse primate species. The second study developed an unbiased approach without the need of prior information to identify genomic segments under accelerated evolution. The third study investigated interplay between genomic and epigenomic evolution of humans and chimpanzees.
Research advance 1: evolutionary rates of the X chromosome are predicted to be different from those of autosomes. A theory based on neutral mutation predicts that the X chromosome evolves slower than autosomes (slow-X evolution) because the numbers of cell division differ between spermatogenesis and oogenesis. A theory based on natural selection predicts an opposite direction (fast-X evolution) because newly arising beneficial mutations on the autosomes are usually recessive or partially recessive and not exposed to natural selection. A strong slow-X evolution is also predicted to counteract the effect of fast-X evolution. In our research, we simultaneously studied slow-X evolution, fast-X evolution as well as their interaction in a phylogeny of diverse primates. We showed that slow-X evolution exists in all the examined species, although their degrees differ, possibly due to their different life history traits such as generation times. We showed that fast-X evolution is lineage-specific and provided evidences that fast-X evolution is more evident in species with relatively weak slow-X evolution. We discussed potential contribution of various degrees of slow-X evolution on the conflicting population genetic inferences about human demography.
Research advance 2: human-specific traits have long been considered to reside in the genome. There has been a surge of interest to identify genomic regions with accelerated evolution rate in the human genome. However, these studies either rely on a priori knowledge or sliding windows of arbitrary sizes. My research provided an unbiased approach based on previously developed “maximal segment” algorithm to identify genomic segments with accelerated lineage-specific substitution rate. Under this framework, we identified a large number of human genomic segments with clustered human-specific substitutions (named “maximal segments” after the algorithm). Our identified human maximal segments cover a significant amount of previously identified human accelerated regions and overlap with genes enriched in developmental processes. We demonstrated that the underlying evolutionary forces driving the maximal segments included regionally increased mutation rate, biased gene conversion and positive selection.
Research advance 3: DNA methylation is one of the most common epigenetic modifications and plays a significant role in gene regulation. How DNA methylation status varies on the evolutionary timescale is not well understood. In this study, we investigated the role of genetic changes in shaping DNA methylation divergence between humans and chimpanzees in their sperm and brain, separately. We find that for orthologous promoter regions, CpG dinucleotide content difference is negatively correlated with DNA methylation level difference in the sperm but not in the brain, which may be explained by the fact that CpG depleting mutations better reflect germline DNA methylation levels. For the aligned sites of orthologous promoter regions, sequence divergence is positively correlated with methylation divergence for both tissues. We showed that the evolution of DNA methylation can be affected by various genetic factors including transposable element insertions, CpG depleting mutations and CpG generating mutations.
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James, Samantha. "Herpèsvirus de primates et de chauves-souris du nouveau monde : modèles d'étude des relations évolutives hôtes-virus DNA polymerase sequences of New World Monkey Cytomegaloviruses : another molecular marker with which to infer Platyrrhini systematics Novel herpesviruses of neotropical bats and their relationships with other members of the Herpesviridae family." Thesis, Guyane, 2019. http://www.theses.fr/2019YANE0004.
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Les virus appartenant à la famille Herpesviridae (ordre Herpesvirales) sont répartis au sein de trois sous-familles : Alpha-, Beta- et Gamma-herpesvirinae. Ils ont été identifiés à partir d’un large spectre d’espèces hôtes, allant des mammifères aux reptiles en passant par les oiseaux et ont la capacité de persister toute au long de la vie de l’hôte. La plupart de ces virus sont par ailleurs spécifiques d’une espèce hôte. La large distribution des herpèsvirus, associée à une infection généralement asymptomatique chez l’hôte naturel suggèrent que ces virus auraient co-évolué avec leurs hôtes.Mon premier axe de travail a porté sur l’identification de cytomégalovirus (CMV) chez les primates non-humains du Nouveau Monde (PNHNM) afin de voir si la diversification de ces virus suivait celle de leurs hôtes. De fait, une étude préalable conduite sur les primates de l’Ancien Monde avait démontré que les CMV les infectant présentaient un fort signal de co-divergence avec leurs hôtes. Or, parmi les différentes espèces de primates testées, seules quelques-unes provenaient du Nouveau Monde. Afin de répondre à cette question, nous avons effectué un criblage moléculaire de 244 échantillons d'ADN sanguin provenant de 20 espèces d'Amérique centrale et du sud. Par une approche de PCR utilisant des amorces consensus dégénérées ciblant des motifs hautement conservés du gène de l'ADN polymérase des herpèsvirus, nous avons caractérisé de nouvelles séquences virales de sept genres représentatifs des trois familles de PNHNM. Ces résultats démontrent ainsi que la plupart des espèces de PNHNM peuvent être infectées par un virus appartenant au genre Cytomegalovirus. Par ailleurs, les analyses phylogénétiques effectuées, couplées à la datation moléculaire des séquences obtenues, soutiennent cette hypothèse co-évolutive.Mon second axe visait à identifier des herpèsvirus de chauves-souris du Nouveau Monde afin d’en étudier la distribution et la diversité. La recherche d’herpèsvirus chez les chauves-souris est plus récente. Elle a bénéficié de l’intérêt porté à ces mammifères au début des années 2000 du fait de leur rôle de réservoirs de virus potentiellement zoonotiques. C’est à partir de 2007 que les premières séquences d’herpèsvirus de chauves-souris ont été décrites. Il s’en est suivi la description de dizaines de nouvelles séquences identifiées de différentes espèces d’Afrique, d’Asie, d’Océanie, d’Europe et d’Amérique. Néanmoins, la distribution des espèces testées était géographiquement inégale et seules quelques-unes provenaient du Nouveau Monde. Nous avons effectué un criblage moléculaire de 195 échantillons d’ADN sanguin provenant de 11 espèces appartenant à trois familles (Phyllostomidae, Mormoopidae et Molossidae). En utilisant la même approche que celle appliquée au PNHNM, nous avons obtenu des séquences virales (ADN polymérase et/ou Glycoprotéine B) de toutes les espèces testées. Celles-ci se répartissent au sein des sous familles Beta- et Gamma-herpesvirinae. Quatorze séquences partielles du gène de l'ADN polymérase, correspondant à trois beta- et onze gamma-herpèsvirus, ont été identifiées. Douze séquences partielles du gène de la glycoprotéine B, toutes de gamma-herpèsvirus, ont été caractérisées. Chaque séquence était spécifique à une espèce de chauve-souris et, chez certaines espèces, de multiples virus ont été identifiés. Les analyses phylogénétiques de ces séquences ont permis d'identifier des clades spécifiques aux virus de chauve-souris. Ceux composés de séquences obtenues à partir de différentes espèces appartenant à des sous-familles distinctes suivent la taxonomie des chauves-souris. Cette étude confirme l'étonnante diversité des herpèsvirus de chauve-souris et élargit nos connaissances sur leur spectre d'hôtesViruses belonging to the Herpesviridae family (order Herpesvirales) are enveloped double-stranded DNA viruses distributed into three subfamilies: Alpha-, Beta- and Gamma-herpesvirinae. These viruses have been identified from a wide range of host species, ranging from mammals to reptiles to birds. They have the ability to persist throughout the life of the host in a latent form and to reactivate. Most of these viruses are specific to a host. The wide distribution of herpesviruses, generally associated with an asymptomatic infection in their natural host, suggests that these viruses have co-evolved with their hosts.The first part of this work was dedicated to the identification of cytomegaloviruses (CMV, genus Cytomegalovirus) in New World non-human primates (NWNHP) to see if the diversification of these viruses followed that of their hosts. A previous study on Old World primates had demonstrated that CMVs infecting them showed a strong signal of co-divergence with their hosts. Nevertheless, among the different species of primates tested, only a few were from the New World. To test this hypothesis, we performed a molecular screening of 244 blood DNA samples from 20 Central and South American species. Through a PCR approach using degenerate consensus primers targeting highly conserved motifs of the DNA polymerase gene of herpesviruses, we characterized new viral sequences from 12 species belonging to seven representative genera of the three families of NWNHP. These results demonstrate that most species of NWNHP can be infected with a virus belonging to the Cytomegalovirus genus. In addition, phylogenetic analyzes of the obtained sequences combined with their molecular dating support a co-evolutionary scenario. This study led us to propose that CMVs sequences of NWNHP could serve as a molecular marker with which to infer the not yet fully resolved systematics of these primates.The second part of this work was on identifying herpesviruses from New World bats to study their distribution and diversity. The search for herpesviruses in bats is more recent. In the early 2000s it benefited from studies dedicated to other viral families given their role as reservoirs of potentially zoonotic viruses. The first description of bat herpesvirus sequences dated back from 2007. Over the past decade, dozens of herpesvirus sequences have been described from different bat species on every continent. Nevertheless, the distribution of the tested species was geographically uneven and only a few were from the New World. Molecular screening of 195 blood DNA samples from 11 species belonging to three families (Phyllostomidae, Mormoopidae and Molossidae) was performed. Using the same approach as applied to NWNHP, we obtained viral sequences (DNA polymerase and/or glycoprotein B) from all tested species. These are distributed within the Beta- and Gamma-herpesvirinae subfamilies. Fourteen partial sequences of the DNA polymerase gene, corresponding to three beta- and eleven gamma-herpesviruses, have been identified. Twelve partial sequences of the glycoprotein B gene, all gamma-herpesviruses, have been characterized. Each sequence was specific to a bat species and in some species multiple viruses were identified. Phylogenetic analyzes of these sequences have identified clades specific to bat viruses. Those composed of sequences obtained from different species belonging to distinct subfamilies follow the taxonomy of bats. This study confirms the astonishing diversity of bat herpesviruses and broadens our knowledge on their host spectrum.This work is the largest conducted to date in terms of species diversity of non-human primates and bats from the Neotropical realm. Nevertheless, the samples tested represent only a tiny part of this diversity. Further analyzes, on a broader panel of representative species from other geographic areas will increase our understanding of the evolutionary history of these viruses
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DANCKAERT, PERRUCHOT ANNE. "Traitement informatique des autoradiographies de gels de sequences : conception et mise en oeuvre d'un systeme automatique d'interpretation d'autoradiographies, elaboration d'un algorithme de construction de la sequence consensus a partir de fragments." Paris 7, 1988. http://www.theses.fr/1988PA077045.
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Laubscher, Maxine. "Genetic and morphological comparisons within the orthopteran family Pneumoridae." University of the Western Cape, 2019. http://hdl.handle.net/11394/7949.
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>Magister Scientiae - MScBladder grasshoppers belong to the order Orthoptera, ancient family Pneumoridae and Superfamily Pneumoroidea. This small group of grasshoppers are sound producing, nocturnal, herbivorous grasshoppers endemic to the coastal regions of southern Africa. Very little genetic work has been done on these grasshoppers, and there is some taxonomic confusion regarding the validity of some species descriptions. The aim of this study was to provide much needed clarity on the true taxonomic diversity and polymorphic attributes within the Pneumoridae, focusing on selected taxa of uncertain status. Bladder grasshoppers show distinct discontinuous polymorphism, resulting in two clearly different male morphs utilizing two different mating strategies. Primary males make use of acoustic communication for mate location. Secondary males (alternate males) are significantly smaller and employ a “sneaker” or satellite strategy where they exploit the calling between duetting couples to locate the females before the primary male. Three species of bladder grasshoppers have been described (Parabullacris vansoni, Paraphysemacris spinosus and Pneumoracris browni) that only have an alternate male morph. The validity of these species descriptions has come into question with the discovery of alternate male morphs in at least three other species (Bullacris discolor, B. membracioides and B. obliqua). Thus, the species described by Dirsh (1963) may simply be alternate males of existing species. However, to date there have been no studies looking at the genetics of alternate males, which would definitively establish whether they are conspecific with primary males.
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Marcaud, Hélène. "Etude sur les transitions coopératives et les cinétiques de transconformation associées dans les acides nucléiques." Paris 6, 1986. http://www.theses.fr/1986PA066514.
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Jones, Christine. "A candidate gene based investigation of aberrant DNA methylation in the pathogenesis of primary cutaneous T-cell lymphoma." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/a-candidate-gene-based-investigation-of-aberrant-dna-methylation-in-the-pathogenesis-of-primary-cutaneous-tcell-lymphoma(dacab6e6-a162-4ac7-9cca-fd77cd3ae576).html.
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Primary cutaneous T-cell lymphoma (CTCL) is a clinically heterogeneous malignancy of mature skin-homing T-cells. Mycosis fungoides (MF) is an indo¬lent subtype of CTCL whilst Sezary syndrome (SS) is an aggressive leukaemic variant characterised by the presence of malignant Sezary cells in the periph¬eral blood. A role for epigenetic mechanisms in the pathogenesis of CTCL has been proposed but not extensively investigated. In this thesis, the contribution of DNA methylation to the regulation of SHP-1, Fas and PLS3, which have been implicated in the pathogenesis of CTCL, was examined. Gene expression was assessed on the mRNA and protein levels using qPCR and flow cytometry re¬spectively whilst DNA methylation was evaluated using bisulphite conversion and Pyrosequencing. Contrary to results in CTCL cell lines, primary malignant cells from CTCL patients did not display aberrant DNA methylation or dysregu-lated expression of SHP-1, a negative regulator of STATS signalling. Dysregula-tion of Fas, a key regulator of apoptosis, was observed in 34/47 SS patients with 13/47 showing over-expression compared to healthy controls and 21/47 showing under-expression, attributed to the positional hypermethylation of five CpG din-ucleotides in the Fas CpG island. PLS3, an actin-binding protein not normally expressed in any haematopoietic cell, was aberrantly expressed in malignant cells from 21/35 SS patients and 3/8 MF patients with demonstrated clonal blood in¬volvement. CpG dinucleotides 95-99 in the PLS3 CpG island were differentially methylated between healthy lymphocytes and keratinocytes, and hypomethyla-tion of these CpG dinucleotides was observed in SS patients expressing PLS3. To investigate expression of PLS3 on the protein level a novel anti-PLS3 anti¬body was raised and optimised for use in Western blotting, flow cytometry and immunofluorescence. In conclusion, these data demonstrate that SHP-1 is not regulated by methylation in primary CTCL cells whilst Fas is dysregulated by hypermethylation of five key CpG dinucleotides and PLS3 is dysregulated by hy-pomethylation of CpG dinucleotides 95-99.
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Goidts, Violaine. "Identification of large-scale DNA copy number differences between human and non-human primate genomes and their role in mediating evolutionary rearrangements." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-56317.
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Cangiano, daniela. "Detection of KCNJ5 mutations in the APA tissues and cell-free DNA with a novel Taqman-based approach." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3424711.
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Background: Primary aldosteronism (PA) is the most common endocrine form of secondary arterial hypertension with an estimated prevalence of ~ 11.2% in patients referred to specialized centers and 4% in primary care, but as high as 50% in patients with resistant hypertension. The two main forms of PA are: aldosterone-producing adenoma (APA), surgically curable with the removal of adenoma that is able to overproduce aldosterone, and bilateral adrenocortical hyperplasia (BAH), which needs drug therapy. About ~ 30% APAs have somatic mutations in KCNJ5 gene that encodes for the potassium channel (KIR3.4), which plays a crucial role in the maintenance of the cell membrane by pumping potassium (K+) out of the cells, thereby producing a negative membrane potential. The KCNJ5 channels that contain G151R, T158A, or L168R mutations cause membrane permeability to Na+, resulting in Na+ entry, cell depolarization, constitutive aldosterone production, and cell proliferation.
The standard approach used to detect the mutations in KCNJ5 gene is sequencing of DNA extracted from adrenal tissue of hypertensive patients with lateralized excess in aldosterone production. However, DNA sequencing is a time consuming and rather expensive technique not feasible in all standard laboratories.
Aims of our study were 1) to develop a strategy for genotyping DNA extracted from the adrenal tissue, which exploited a TAQ-MAN based PCR technology; 2) to evaluate if such Taq-Man-base technology allows detection of KCNJ5 mutations in the adrenal tissue and cf-DNA isolated from peripheral blood.
Methodologic Approach:
1. Development of a novel strategy based on Taq-man probe to detect KCNJ5 mutations
2. Development of a protocol to isolate cf-DNA from blood collected in the inferior vena cava and adrenal veins.
3. Measurement of cf-DNA concentration and assessment of its fragmentation.
4. Identification of KCNJ5 mutation in cf-DNA
Results: By applying the novel technology based on Taq-man probe we correctly identified 30 mutated patients in a cohort of 50 consecutive APA patients, with no misclassification.
After isolating cf-DNA from the adrenal veins and inferior cava blood, and measuring its concentration, we evaluated cf-DNA fragmentation with the integrity index in 24 samples. Integrity index < 1 suggested that cf-DNA was released from the adrenal tissue through an apoptotic mechanism. HRM analysis of cf-DNA isolated from adrenal blood allowed us to identify the KCNJ5 mutation in the APA side.
Conclusions and perspectives: The novel technology based on Taq-man probe allowed detection of all mutated patients in the examined cohort of APA patients, thus proving that this strategy could be used as an alternative to DNA sequencing. The results of our study also showed the feasibility to isolate cf-DNA from small amount of blood collected from the adrenal veins, and suggested that KCNJ5 mutations may be detected in cf-DNA. However, this approach needs to be verified for a larger population to determine feasibility and accuracy.
If confirmed, analysis of cf-DNA isolated from the peripheral venous blood could be helpful for an early detection of KCNJ5 mutations and therefore for the selection of PA patients to be submitted to adrenal vein sampling. Furthermore, this strategy could be also useful for detecting KCNJ5 germline mutations responsible for the rare hereditary form of hyperaldosteronism FH-3.Backgouund. L’Iperaldosteronismo primario (PA) è la forma endocrina più comune d’ipertensione arteriosa secondaria con una prevalenza stimata di circa il 4% nella popolazione generale e dell’11% nei pazienti che afferiscono ai centri di riferimento per l’ipertensione. Nei pazienti con ipertensione resistente la prevalenza del PA è stimata pari al 50%, mostrando che tale patologia non è così rara come ritenuto in passato. Le due forme principali di PA sono l’adenoma producente aldosterone (APA), caratterizzato da iperproduzione lateralizzata di aldosterone, e l’iperplasia surrenalica bilaterale (BAH). La distinzione tra le due forme è di cruciale importanza poiché la prima richiede terapia chirurgica, mentre la seconda terapia medica. Considerando che la rimozione dell’APA determina la correzione del quadro biochimico-clinico di PA e la cura o il miglioramento dell’ipertensione, il riconoscimento dell’APA è fondamentale per offrire una chance di guarigione dell’ipertensione o di miglioramento del controllo dei valori pressori ai pazienti che ne sono affetti. Il 40% degli APA presenta mutazioni somatiche nel gene KCNJ5 che codifica per il canale del potassio KIR 3.4. Questo canale gioca un ruolo fondamentale nel mantenimento del potenziale di membrana pompando il K+ al di fuori della cellula, provocando in tal modo un potenziale di membrana negativo. Allorquando il gene KCNJ5 contiene le mutazioni G151R, T158A o L168R il canale KIR 3.4 acquisisce capacità di condurre Na+ all’interno della cellula. Gli effetti della mutazione a livello della cellula sono depolarizzazione cronica, produzione costitutiva di aldosterone e proliferazione cellulare. L’approccio attualmente in uso per identificare tali mutazioni è il sequenziamento, secondo Sanger, del DNA estratto dal tessuto surrenalico rimosso durante surrenectomia nei pazienti con PA e iperproduzione lateralizzata di aldosterone. Il sequenziamento del DNA, tuttavia, è costosa richiede tempo e non è disponibile di routine in tutti i laboratori. Scopo generale dello studio è stato quello di sviluppare una strategia alternativa al sequenziamento che preveda l’uso delle sonde Taq-man in Real Time PCR (Q-PCR) per il rilevamento di mutazioni KCNJ5 nel tessuto surrenalico e nel DNA circolante (cell-free DNA, cf-DNA) isolato da sangue periferico. Lo sviluppo di questa metodologia potrebbe semplificare notevolmente l’identificazione delle mutazioni KCNJ5 negli APA e, infine, permetterne la detenzione nel DNA del sangue circolante. In sintesi, l’approccio metodologico include: sviluppo di una nuova strategia basata sull’utilizzo delle sonde Taq-man per rilevare le mutazioni nel gene KCNJ5. Sviluppo di un protocollo per isolare il cf-DNA da sangue delle vene surrenaliche e dalla vena cava inferiore. Misurazione della concentrazione del cf-DNA valutandone la sua frammentazione. Identificazione di mutazioni nel gene KCNJ5 a partire dal cf-DNA Risultati. Applicando la tecnologia sviluppata nel nostro laboratorio, basata sulle sonde Taq-man, sono stati identificati correttamente 30 pazienti mutati in una coorte di 50 pazienti APA consecutivi, senza errori di classificazione. Dopo aver isolato il cf-DNA dal sangue delle vene surrenaliche e dalla vena cava inferiore, e misurato la sua concentrazione, abbiamo valutato la frammentazione del cf-DNA in 24 campioni con l'indice di integrità. I bassi valori dell’indice d’integrità riscontrati nei cf-DNA isolati da sangue venoso surrenalico suggeriscono che la ghiandola surrenalica rilasci per apoptosi frammenti di DNA. L’analisi HRM dei cf-DNA isolati dal sangue delle vene surrenaliche di un paziente con un APA sinistro contenente la mutazione L168R ha permesso d’identificare correttamente la mutazione nel cf-DNA isolato dalla vena surrenalica sinistra. Conclusioni e prospettive. La tecnologia basata sulle sonde Taq-man ha permesso d’identificare, senza errori di misclassificazione, in una coorte di 50 pazienti con APA, tutti i 30 pazienti che presentano una mutazione del gene KCNJ5. Tale strategia, pertanto, potrebbe rappresentare un’alternativa alla ben più lunga e complessa tecnica basata sul sequenziamento del DNA. I risultati del nostro studio hanno anche mostrato che è possibile isolare il cf-DNA da esigue quantità di sangue raccolto dalle vene surrenaliche permettendo l’identificazione delle mutazioni KCNJ5 usando il cf-DNA tramite approccio combinato sonde Taq-man e analisi HRM. Quest’ultimo approccio, che prevede l’uso del cf-DNA richiede, tuttavia, conferma in un ampio numero di soggetti. La stessa strategia potrebbe anche essere impiegata in futuro per la rilevazione di mutazioni germinali KCNJ5 responsabili della nota forma ereditaria d’iperaldosteronismo FH-3.
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Contri, Daniela Gazoto. "Detecção de resíduos de DNA em alimentos: avaliação da qualidade, da quantidade e da capacidade de amplificação por PCR de DNA extraído de matérias-primas e produtos acabados para fins de análise de transgenia." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/9/9131/tde-20122006-143753/.
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O objetivo do trabalho foi avaliar a qualidade, a quantidade e a capacidade de amplificação por PCR de DNA extraído de grãos de soja e milho, seus derivados e produtos acabados contendo como ingredientes obtidos desses grãos, com vistas à detecção de resíduos de organismos geneticamente modificados em alimentos. Para a amplificação de DNA pela PCR convencional, não houve melhor adequação de um protocolo de extração. Ambos métodos, CTAB e coluna de sílica tiveram desempenho comparável para as 32 matrizes avaliadas. A técnica de PCR em tempo real se mostrou mais sensível à qualidade do DNA testado e nesse contexto, o método CTAB se mostrou mais eficiente do que o método de coluna de sílica. Independentemente do método de extração utilizado não foi possível detectar DNA em óleos de soja e milho e em alguns derivados de amido, sugerindo que a aplicabilidade da lei de rotulagem pode esbarrar num entrave técnico no caso de algumas matrizes alimentares altamente processadas.The aim of the study was to evaluate the quality, the quantity and the amplifiability by PCR of DNA isolated from soybean and maize grains and their by-products targeting the detection of genetically modified organisms in food. PCR amplification of DNA samples isolated either from CTAB and silica-column extraction methods achieved comparable performances. Both extraction methods showed similar results for the 32 tested matrices. The DNA amplification by real time PCR appeared to be affected by the quality of the isolated DNA. In this context, the CTAB extraction method showed to be more suitable when compared to the silica-column method. No DNA was amplified from soy and maize oils, as well as from some starch by-products, regardless the DNA extraction method used. It suggests that, the labeling requirement may rely on technical issues considering some high processed foodstuffs.
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Cabuy, Erik. "Investigations of telomere maintenance in DNA damage response defective cells and telomerase in brain tumours." Thesis, Brunel University, 2005. http://bura.brunel.ac.uk/handle/2438/5157.
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Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.
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Soto-Calderon, Ivan D. "Evolution of Nuclear Integrations of the Mitochondrial Genome in Great Apes and their Potential as Molecular Markers." ScholarWorks@UNO, 2012. http://scholarworks.uno.edu/td/1510.
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The mitochondrial control region (MCR) has played an important role as a population genetic marker in many taxa but sequencing of complete eukaryotic genomes has revealed that nuclear integrations of mitochondrial DNA (numts) are abundant and widespread across many taxa. If left undetected, numts can inflate mitochondrial diversity and mislead interpretation of phylogenetic relationships. Comparative analyses of complete genomes in humans, orangutans and chimpanzees, and preliminary studies in gorillas have revealed high numt prevalence in great apes, but rigorous comparative analyses across taxa have been lacking.
The present study aimed to systematically compare the evolutionary dynamics of MCR numts in great apes. Firstly, an inventory numts derived from the region containing the MCR subdomains was carried out by genomic BLAST searches. Secondly, presence/absence of each candidate numt was determined in great ape taxa to estimate numt insertion rate. Thirdly, alternative mechanisms of numt insertion, either through direct mitochondrial integration or post-insertional duplications, were also assessed. Fourthly, the effect of nuclear and mitochondrial environment on patterns of nucleotide composition and substitution was assessed through sequence comparisons of nuclear and mitochondrial paralogous sequences. Finally, numts in the gorilla genome were identified through two experimental methods and their use as polymorphic genetic markers was then evaluated in a sample of captive gorillas from U.S. zoos.
A deficit of MCR numts covering two particular mitochondrial subdomains was detected in all three apes examined, and is largely attributed to rapid loss of mitochondrial and nuclear sequence identity in the mitochondrial genome. Insertion rates have varied during the great ape evolution and exhibit substantial differences even between related taxa. The most likely mechanism of numt insertion is direct mitochondrial integration through Non-Homologous-End-Joining Repair. Transition/transversion ratios differed significantly between both mitochondrial and nuclear sequences and between numts from coding and non-coding mitochondrial regions. A previously documented upward bias in the GC content of the primate mitochondrial genome was confirmed and the extent of this bias relative to the corresponding numt sequences increased with numt age. Five gorilla-specific numts were isolated, including three exhibiting insertional polymorphisms that will be used in future population genetic studies in free-range gorilla.
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Reis, Leonardo Mariano. "Glaucoma primário de ângulo aberto e DNA mitocondrial: uma análise da produção científica." Pontifícia Universidade Católica de Goiás, 2013. http://localhost:8080/tede/handle/tede/2368.
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Previous issue date: 2013-03-18Abstract of the First Chapter Objective: The objective was to make a wide review of the literature about the genetic aspects of glaucoma, with a highlight at the works dealing with mitochondrial DNA. Methods: Bibliographical research was carried out accessing the sites Scopus, Science Direct, PubMed, Scirus, Scielo and Google. Books, theses and dissertations were also used at the research. Results: The works brought to evidence that there is a relation between certain polymorphisms in mitochondrial DNA and a higher loss of retinal ganglion cells, which leads to the development of Glaucoma, especially Primary Open Angle Glaucoma. Conclusion: Higher incidence of Primary Open Angle Glaucoma (POAG) in afro-descendants is justified by the mitochondrial haplotypes, since it was observed a higher susceptibility of POAG in individuals of the L haplogroups, who are exactly the ones of African origin. It could also justify, based on mitochondrial inheritance, the major incidence of POAG in the maternal lineages, more frequent than paternal inheritance. Abstract of the Second Chapter Objective: To quantify the volume of the scientific production about Primary Open Angle Glaucoma and Mitocohndrial DNA and assess which are the main institutions of research; verify the contribution of the authors that most published about the subject; list the main journals with their Impact Factor; compare the countries and other bibliometric results. Methods: Bibliographical research about mitochondrial DNA and Primary Open Angle Glaucoma was carried out accessing the site Scopus. Then, search with the keywords mitochondrial DNA and glaucoma in all fields for publications until august of 2012 was performed. Finally, data was tabulated and the descriptive statistics was done, with the principal data, as: authors that published about mitochondrial DNA and Primary Open Angle Glaucoma; journals that had works about the subject; Research Centers and Universities that most published; countries where the researchs were performed. Results: The works brought to evidence the influences of certain polymorphisms in mitochondrial DNA at the development of Primary Open Angle Glaucoma. Statistics showed that theese articles have increased in the last few years, though yet confined, mostly, to some centers of research and concentrated in selected researchers in ophthalmology of developed countries. In Brazil, we do not have any article published about the issue, yet. Conclusion: 98 works were listed, until august of 2012, carried out in 30 countries, with higher concentration in the United States and England, but also with contributions of the Arabic world: Qatar and Saudi Arabia. There were more publications in the following journals: Molecular Vision, Investigative Ophthalmology and Visual Science, Archives of Ophthalmology, British Journal of Ophthalmology, Experimental Eye Research, Journal of Glaucoma and Progress in Retinal and Eye Research. More studies about the subject must be encouraged, for they are extremely important to the elucidation of the genetic causes of glaucoma and for the development of new therapies aiming not only the reduction of intraocular pressure.
RESUMO DO CAPÍTULO I Objetivo: O objetivo foi fazer uma ampla revisão de toda a literatura sobre os aspectos genéticos do glaucoma, com enfoque nos trabalhos que envolvem pesquisa com DNA mitocondrial. Métodos: A pesquisa bibliográfica foi realizada por meio das bases de dados: Scopus, Science Direct, PubMed, Scirus, Scielo e pelo sítio de busca Google. Foi feito também levantamento através de livros, dissertações e teses. Resultados: Os trabalhos evidenciaram que há alguma correlação de determinadas linhagens de DNA mitocondrial com maior perda de células ganglionares da retina, o que acaba levando ao desenvolvimento do glaucoma propriamente dito, sobretudo do glaucoma primário de ângulo aberto. Conclusão: Explica-se a maior incidência de glaucoma primário de ângulo aberto em indivíduos afrodescendentes a partir de haplótipos mitocondriais, uma vez que houve maior susceptibilidade ao glaucoma primário de ângulo aberto em haplótipos pertencentes aos grupos L, que são justamente os de origem africana. Também poder-se-ia justificar, a partir da herança mitocondrial, a maior incidência de glaucoma primário de ângulo aberto quando a linhagem materna apresenta a doença, bem mais frequente do que a herança paterna. RESUMO DO CAPÍTULO II Objetivo: Quantificar o volume da produção científica que relaciona o DNA mitocondrial com o Glaucoma Primário de Ângulo Aberto e avaliar quais são as principais instituições; verificar a contribuição dos autores que mais produziram publicações sobre o assunto; listar os principais veículos com os respectivos Fatores de Impacto; comparar os países que mais fizeram trabalhos sobre o tema e outros resultados bibliométricos. Métodos: A pesquisa bibliográfica de trabalhos envolvendo DNA mitocondrial e Glaucoma Primário de Ângulo Aberto foi realizada por meio da base de dados do sítio Scopus. Foi feito o levantamento a partir das palavras-chaves Mitochondrial DNA e Glaucoma em todos os campos para publicações até agosto de 2012. Finalmente, realizada a tabulação dos dados obtidos e a estatística descritiva, com o principais dados como: autores que publicaram sobre o assunto DNA mitocondrial e glaucoma; revistas e outras publicações que tiveram trabalhos relacionados com o tema; Centros de Pesquisa e Universidades que mais publicaram e países onde foram realizados os estudos sobre DNA mitocondrial e glaucoma. Resultados: Os trabalhos evidenciaram a influência de determinados polimorfismos de DNA mitocondrial no desenvolvimento do Glaucoma Primário de Ângulo Aberto. A estatística mostrou que esses estudos têm aumentado sobremaneira ao longo dos últimos anos, mas ainda se encontram confinados, na maior parte, a alguns centros de pesquisa e concentrados em seletos grupos de autores na área da oftalmologia em países desenvolvidos. No Brasil, ainda não temos pesquisas publicadas sobre o assunto até o momento. Conclusão: Foram listados 98 trabalhos até agosto de 2012, realizados em 30 países, com maior concentração nos Estados Unidos e Inglaterra; mas também com contribuições do mundo árabe: Arábia Saudita e Catar. Esses estudos foram mais publicados nas revistas: Molecular Vision, Investigative Ophthalmology and Visual Science, Archives of Ophthalmology, British Journal of Ophthalmology, Experimental Eye Research, Journal of Glaucoma e Progress in Retinal and Eye Research. Mais estudos sobre o assunto devem ser encorajados pois são de fundamental importância para a elucidação das causas genéticas do glaucoma e para o desenvolvimento de novas terapias que não visem tão somente a diminuição da pressão intraocular.
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Han, Thomas. "Development of a dna vaccine against _streptococcus mutans_: A novel approach to immunization against dental caries." Scholar Commons, 2005. http://scholarcommons.usf.edu/etd/2912.
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Streptococcus mutans is the main causative agent of dental caries, which is a widespread infectious disease. A number of surface molecules are involved in the pathogenicity of this organism, including adherence and aggregation factors. The wall-associated protein A (WapA) of Streptococcus mutans GS-5 was previously demonstrated to be a sucrose-dependent adherence and aggregation factor, and is a larger precursor to extracellular antigen A (AgA), a candidate antigen for a dental caries vaccine.The full-length wapA gene and a C-terminal truncated version agA encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The above constructs were mixed with a cationic lipid and used to transfect Chinese hamster ovary (CHO) cells. Transient expression of the wapA and agA genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis.
In CHO, cells WapA containing the membrane and wall-spanning region was found in apoptotic bodies, whereas the soluble AgA, which lacked the hydrophobic region, was found in extracellular medium. A higher salivary IgA level was observed in mice immunized with the pcDNA-wapA vaccine as compared to those immunized with the pcDNA-agA vaccine. Furthermore, the anti-WapA antibody inhibited S. mutans sucrose-dependent adherence, suggesting potential protection of the tooth against S. mutans colonization, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs that are absent from AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not.
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Oliveira, Thaís Larré. "Vacinas contra leptospirose: potencial imunoprotetor do antígeno OmpL37." Universidade Federal de Pelotas, 2014. http://repositorio.ufpel.edu.br:8080/handle/prefix/3703.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
A leptospirose é uma doença infecciosa emergente causada por espiroquetas patogênicas do gênero Leptospira, com complicações humana e veterinária. Essa doença é transmitida através do contato direto com um animal reservatório ou com o ambiente contaminado com a urina destes animais. O desenvolvimento de vacinas seguras e multivalentes contra leptospirose, que substituam as bacterinas existentes, permanece um desafio. Bacterinas são reatogênicas e conferem imunidade sorovar-específica e de curta duração. Esforços para o desenvolvimento de vacinas recombinantes tem focado em proteínas de membrana externa (OMPs). A proteína de membrana externa OmpL37 é conservada entre diferentes sorovares de Leptospira e atua na adesão aos tecidos do hospedeiro. Neste estudo, nós relatamos pela primeira vez, a avaliação do potencial imunoprotetor de OmpL37 como antígeno vacinal em hamsters, sob diferentes estratégias: vacina de subunidade, vacina de DNA e prime-boost. A caracterização da resposta imune através de ELISA indireto e qRT-PCR demonstrou que os maiores níveis de IgG foram estimulados pela vacina de subunidade, a qual também induziu resposta inflamatória. A vacina de DNA falhou em induzir imunidade humoral, porém também estimulou TNF-α. Apesar da resposta induzida, nenhuma formulação protegeu significativamente os animais contra a doença.
Leptospirosis is an emerging infectious disease caused by pathogenic spirochetes of Leptospira genus, of human and veterinary concern. This infectious disease is transmitted through direct contact with an animal reservoir or an environmental contaminated with their urine. The development of safe and multivalent vaccines against leptospirosis, which replace existing bacterins, remains a challenge. Bacterins are reactogenic and afford serovar specific and short-term immunity. Efforts to develop recombinant vaccines against leptospirosis have focused on outer membrane proteins (OMPs). The outer membrane protein OmpL37 is conserved among different Leptospira serovars and plays a role in adherence to host tissues. In this study we report for the first time, the evaluation of OmpL37 immunoprotective potential as a vaccine antigen in hamsters, under different strategies: subunit vaccine, DNA vaccine and prime-boost. The characterization of the immune response by indirect ELISA and qRT-PCR showed that higher levels of IgG were stimulated by the vaccine subunit, which also induced an inflammatory response. The DNA vaccine failed to induce humoral immunity, but also stimulated TNF-α. Despite the induced response, no formulation significantly protected the animals against the disease.
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Adam, Lucille. "Dynamique de la réponse immunitaire précoce mise en place localement suite à l’injection d’un vaccin ADN associée à une électroporation chez le macaque cynomolgus." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA114812/document.
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La compréhension des mécanismes immunologiques précoces mis en place suite à l’administration de vaccins est encore de nos jours largement méconnue. Pourtant de plus en plus d’études démontrent l’importance de ces mécanismes très précoces faisant intervenir les acteurs de l’immunité innée dans la génération d’une réponse spécifique efficace après vaccination. La peau est un organe intéressant pour l'administration de vaccins du fait de sa richesse en cellules présentatrices d'antigènes (APC), qui sont des cellules essentielles dans la mise en place de la réponse immunitaire. L'administration par voie intradermique du vaccin ADN de type auxoGTU induit des réponses immunitaires fortes et persistantes, en particulier en association avec une électroporation (EP) locale chez le macaque cynomolgus. Le but de ce travail de thèse fut de caractériser les réponses immunitaires locales précocement mises en place suite à l’administration par voie intradermique du vaccin ADN auxo-GTU en association avec une EP. Dans un premier temps, nous avons décrit les populations de cellules immunitaires présentes dans la peau normale chez le macaque cynomolgus.L'épiderme contient des cellules de Langerhans (LC) qui sont : CD1a+ CD1c- et des lymphocytes T caractérisés par l’expression du CD3. Le derme contient des cellules CD1a+CD1c-, qui présentent des similitudes avec les LC et correspondent donc probablement à des LC en migration à travers le derme. Il contient également des cellules dendritiques dermales (DDC) CD1a+CD1c+, des macrophages résidants CD163highCD11b+ et les lymphocytes T CD3+. Chez certains animaux, nous avons mis en évidence la présence de granulocytes CD66+ dans le derme sain. Les populations de cellules immunitaires identifiées chez le macaque sont similaires à celles identifiées chez l’homme malgré de légères différences phénotypiques. Cette caractérisation nous a ensuite permis d'étudier l’impact de la vaccination sur les populations immunitaires de la peau.Nous avons démontré que la vaccination induit le recrutement de granulocytes et de monocytes/macrophages inflammatoires dans l'épiderme et dans le derme, ainsi qu’un recrutement plus tardif de cellules dendritiques inflammatoires épithéliales (IDEC) dans l'épiderme. Dans l'épiderme, 24h après immunisation, nous avons observé une augmentation transitoire des LC accompagnée d’une surexpression de HLA-DR, de CD86 et de CD83, ce qui démontre leur maturation. Entre 24h et 72h, le nombre de LC diminue, ce qui suggère que les LC matures quittent l’épiderme pour migrer vers les nœuds lymphatiques. Ces événements cellulaires sont majoritairement dus à l’EP, indépendamment de la présence du vaccin à ADN. L’analyse du microenvironnement mis en place dans la peau suite à la vaccination révèle une libération de facteurs solubles pro-inflammatoires, comme MCP-1, IL-18 , IL-15, IL-8 et de facteurs solubles anti- inflammatoires comme IL-1RA et sCD40L dès 24h, dont certains sont considérablement augmentés par la présence de l’ADN vaccinal. Nos résultats suggèrent que l’EP, indépendamment de la présence de l'ADN, est suffisante pour induire la mobilisation des cellules et la maturation des DC au niveau du site de vaccination, ce qui montre un important rôle adjuvant de l’EP. Cependant, il semble que l'ADN soit nécessaire pour générer un microenvironnement favorable à l'activation optimale des APC. Ce travail fournit des éléments importants sur les mécanismes de l'inflammation locale et ouvre de nouvelles possibilités pour les stratégies vaccinalesMechanisms involved in early vaccine response are poorly understood. However, more and more studies show the importance of innate immunity in the very early times following vaccine administration in the generation of an optimal specific immune response. Skin is an interesting target for vaccine delivery because of its richness in antigen presenting cells (APC) which are essential cells in immune responses. The intradermal delivery of auxoGTU DNA vaccine was shown to induce strong and persistent immune responses, especially in association with electroporation in cynomolgus macaque. The aim of this work was to characterize the early local immune responses followed intradermal auxoGTU DNA vaccination in association with EP in cynomolgus macaque. In a first step, we have described immune cell populations present in the normal skin in the cynomolgus macaques. The epidermis contains CD1a+CD1c- Langerhans cells (LCs), and CD3+ T cells. The dermis contains CD1a+CD1c- cells, which present similarities with LCs and probably correspond to LC in migration through dermis. It also contains CD1a+CD1c+ dermal dendritic cells (DDCs), CD163highCD11b+ resident macrophages, and CD3+ T cells. We found CD66+ polymorphonuclear cells in healthy dermis in some of the animals. Immune cell populations in the macaque are similar to those in humans despite moderate differences in phenotype. This characterization has allowed us to study the impact of vaccination on immune populations of the skin. We have demonstrated a recruitment of granulocytes and inflammatory monocytes/macrophages in epidermis and dermis, as well as a population of inflammatory dendritic epithelial cell (IDEC) in epidermis after vaccination. In epidermis, 24h after treatment, we have observed an initial increase of LC with an up-regulation of HLA-DR, CD86 and CD83, demonstrating their maturation. Between 24h and 72h, LC number decreased, suggesting that mature LC has leaved epidermis to migrate to skin draining lymph node. All these cellular events were almost due to EP process, independently of DNA vaccine presence. The skin microenvironment reveals a release of pro-inflammatory soluble factors, as MCP-1, IL-18, IL-15, IL-8 and anti-inflammatory mediators as IL-1RA and sCD40L by 24h, all considerably enhanced in the presence of DNA.Our results suggest that EP, independently of the presence of DNA, is sufficient to induce cells mobilization and DC maturation at the vaccinated site, suggesting an important adjuvant effect of EP. However, it seems that DNA is required to generate a favorable microenvironment essential for correct APC activation. This work provides important clues to local inflammation mechanisms and opens up new possibilities for vaccine strategies
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Trinade, Rhaysa Avila. "Hibrida??o e introgress?o em zona de contato entre Alouatta guariba clamitans e Alouatta caraya (Primates) no sul do Brasil estudadas com dados gen?micos." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2016. http://tede2.pucrs.br/tede2/handle/tede/7752.
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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
Contact zones between Alouatta caraya and A. guariba clamitans with mixed groups has been recently documented and hybridization was inferred by the presence of individuals with mixed morphological patterns. Here we sequenced thousands of ultraconserved elements in noninvasive fecal samples within a hybrid zone and from adjacent populations from both species and characterize 3302 polymorphic sites. We found several F1 hybrids in a narrow contact zones, derived from both combinations of parents, but also found evidence of hybrids in the A. g. clamitans zone that likely originated from serial backcrosses of hybrid males with purebred A. g. clamitans females. There is no evidence of any maternal (mtDNA) introgression, and only two female F1 hybrids were found, therefore female F1 hybrids seem inviable or infertile. On the other hand, we found bidirectional introgression of the Y-chromosomes at least tens of kilometers away from both sides of the contact zone, in otherwise apparently purebred individuals, indicating a not recent introgression mediated by males.
Zonas de contato, com presen?a de bandos mistos, entre Alouatta caraya e Alouatta guariba clamitans, tem sido recentemente documentada e a hibrida??o foi inferida pela presen?a de indiv?duos com padr?es morfol?gicos mistos. Neste estudo n?s sequenciamos milhares de regi?es ultra conservadas em amostras n?o-invasivas de fezes dentro de uma zona hibrida e de popula??es adjacentes das duas esp?cies e caracterizamos 3302 s?tios polim?rficos. N?s encontramos h?bridos F1 limitados a zona de contato, derivados das duas combina??es de parentesco, mas tamb?m encontramos evid?ncia de h?bridos na zona de A. g. clamitans originados de retrocruzamentos por v?rias gera??es de h?bridos com indiv?duos provavelmente puros de A. g. clamitans. N?o existe evid?ncia de nenhum introgress?o maternal (mtDNA), e somente duas f?meas hibridas F1, ent?o f?meas hibridas F1 parecem ser invi?veis ou inf?rteis. Por outro lado, encontramos introgress?o bidirecional do cromossomo Y a pelo menos dezenas de quil?metros al?m da zona de contato em ambos os lados, em indiv?duos sem outro sinal de introgress?o, sinalizando uma introgress?o antiga mediada pelos machos.
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Andersson, Maria. "Chemically Induced DNA Damage in Extended-term Cultures of Human Lymphocytes." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7179.
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Jäder, Klara. "Optimization of a multiplex ARMS-PCR for detection of the primary mutations causing Leber’s hereditary optic neuropath." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-413665.
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Leber’s hereditary optic neuropathy (LHON) is a genetic disease that causes the patients to become blind, first in one eye and then the other, around the ages of 10-75 years. The disease is caused by mutations in the mitochondrial DNA, which disturbs the respiratory chain leading to the deterioration of the retinal ganglion cells. This study’s aim is to optimize a multiplex amplification-refractory mutation system PCR for detection of three primary mutations causing LHON. This was done through a series of PCRs, including PCR aimed at the ß-globin gene, conventional simplex PCR and a simplex ARMS-PCR aimed at the three primary mutations causing LHON. This study was, however, terminated prematurely due the Covid-19 outbreak and the optimization of the ARMS-PCR could therefore not be done. This study’s aim was adapted to the new circumstances to instead provide guidance on how to perform the optimization using the results from the PCRs that were done before the termination. The results found that for the ARMS-PCR 2 mM of magnesium would suffice as a start point overall and the need to solve the problems with the two 14484 plasmids was evident. The ARMS-PCR is one of many methods that can be used to the detect single nucleotide polymorphism, but its availability and robustness makes this a method worth optimizing. To continue with the optimization of the ARMS-PCR several factors would have to be tested, including annealing temperature, primer concentrations and magnesium concentration.
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Imgenberg-Kreuz, Juliana. "Epigenetic and Gene Expression Signatures in Systemic Inflammatory Autoimmune Diseases." Doctoral thesis, Uppsala universitet, Molekylär medicin, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-310388.
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Autoimmune diseases are clinical manifestations of a loss-of-tolerance of the immune system against the body’s own substances and healthy tissues. Primary Sjögren’s syndrome (pSS) and systemic lupus erythematosus (SLE) are two chronic inflammatory autoimmune diseases characterized by autoantibody production and an activated type I interferon system. Although the precise mechanisms leading to autoimmune processes are not well defined, recent studies suggest that aberrant DNA methylation and gene expression patterns may play a central role in the pathogenesis of these disorders. The aim of this thesis was to investigate DNA methylation and gene expression in pSS and SLE on a genome-wide scale to advance our understanding of how these factors contribute to the diseases and to identify potential biomarkers and novel treatment targets. In study I, differential DNA methylation was analyzed in multiple tissues from pSS patients and healthy controls. We identified thousands of CpG sites with perturbed methylation; the most prominent finding was a profound hypomethylation at regulatory regions of type I interferon induced genes in pSS. In study II, a cases-case study comparing DNA methylation in pSS patients with high fatigue to patients with low fatigue, we found methylation patterns associated to the degree of fatigue. In study III, RNA-sequencing was applied to investigate the transcriptome of B cells in pSS in comparison to controls. Increased expression of type I and type II interferon regulated genes in pSS was observed, indicating ongoing immune activation in B cells. In study IV, the impact of DNA methylation on disease susceptibility and phenotypic variability in SLE was investigated. We identified DNA methylation patterns associated to disease susceptibility, SLE manifestations and different treatments. In addition, we mapped methylation quantitative trait loci and observed evidence for genetic regulation of DNA methylation in SLE. In conclusion, the results presented in this thesis provide new insights into the molecular mechanisms underlying autoimmunity in pSS and SLE. The studies confirm the central role of the interferon system in pSS and SLE and further suggest novel genes and mechanisms to be involved in the pathogenesis these autoimmune diseases.
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Milella, Maria Serena. "Study of molecular mechanisms and pharmacological approaches of Alkaptonuria's disease." Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1204563.
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Alkaptonuria (AKU) is an autosomal recessive disorder, called also Black Bone Disease, for the characteristic dark coloration that some tissues and parts of the body assumed. The pathology is caused by the failure of the enzyme homogentisate 1,2- dioxygenase (HGD), that leads the accumulation of the metabolic intermediate homogentisic acid (HGA), derived by tyrosine. HGA highly reactivity triggers the formation of HGA-derived oxidized products, that react with cellular macromolecules, causing a significant generation of ROS and occurrence of oxidative stress. The ongoing oxidative stress status induces the expression of pro-inflammatory cytokines and the activation of immune cell system, with the consequently occurrence in patients of chronic inflammation and secondary AA amyloidosis. Moreover, HGA molecules bonds generate a dark polymer, called ochronotic pigment, that sticks on several organs, particularly on articular joints. Ochronosis triggers detrimental effects on tissues, as cellular death, extracellular matrix destruction, collagen fibrils rupture, since organs lose their function. Tyrosine and phenylalanine are daily taken by the body with diet, and catabolized by the HGD pathway. Thus, in AKU patients, HGA production is constant, since HGA levels in blood and urine are always elevated. Differently, the ochronotic pigment formation and deposit require more time, indeed symptoms in patients generally appear after the fourth decade of life.
The rarity of AKU implies important challenges for its study. Actually, some aspects of the disease are still unexplored, despite it represents the first genetic disorder discovered that follows the principles of Mendelian recessive inheritance. In particular, one of the principal obstacles is the retrieval of AKU samples, that are scarce and generally in bad condition, for the nature of the disease. For this reason, the first step of this thesis project, described in Chapter 3, focused on the set up of in vitro model that allowed to reproduce, in a simple and cheap manner, all the characteristics of pathology. Specifically, it was set up an AKU model based on human primary chondrocytes, that allowed to study the most affected compartment in the disorder. Moreover, it was showed a preliminary study on the development of an in vivo Zebrafish model, with the objective of overcome the numerous limitations related to AKU mouse model.
The aim of the present thesis work was dual: explore the molecular characteristics still unknown in AKU, and propose an innovative therapeutic approach, that could be extended to all the chronic inflammatory pathologies (Fig.1).
In our lab, it was already showed that HGA administration to cells led the activation of autophagic process. Following this observation, it was explored in Chapter 4 the role of lysosomes in AKU. Indeed, is known that a dysregulation of these organelles frequently occurs in different kind of pathologies, as autoimmune or neurodegenerative disorders. Actually, an increase in lysosomes’ number had been detected in both AKU samples and model. Moreover, AKU lysosomes were localized in the periphery of cells, that represent a not physiologic conformation suggesting a decrease in their activity. Despite the ochronotic pigment deposition on cartilage and collagen fibrils was deeply studied, its formation and intracellular localization was never explicated. Thus, considering lysosomes’ role in the storage and degradation of toxic compounds, it was demonstrated in this work, for the first time, that, when cells were exposed to HGA, the ochronotic pigment was developed intracellularly and concentrated in lysosomes. Obviously, this observation could have enormous impact for the treatment of the disease and the counteraction of ochronotic pigment accumulation.
Oxidative detrimental effects of HGA had been already described. Cellular macromolecules, as proteins and lipids, but also organelles, as mitochondria, undergo oxidative reactions, with the occurrence of damages often irreversible. Is known that oxidative stress and ROS target also DNA, with possible deleterious effects for cells and for all the body, considering the potential development of mutations that lead tumors onset. Thus, this aspect needs to be monitored in the progression of disorders. Therefore, the effect of HGA on the genome integrity was studied for the first time, as shown in Chapter 5. It was highlighted that HGA indirectly affected DNA, causing strand breaks and nucleolar stress. This induced the activation of repair mechanisms, on which depended cells destiny.
In addition, Chapter 8 was dedicated to the study of inflammatory signal activated in AKU, a crucial characteristic of the disease. For the first time, the disorder was modeled on immune cells, in order to analyze the pattern of cytokines stimulated by HGA. It was demonstrated that the molecule was able to directly induce pro-inflammatory cytokines expression in different immune cell types. This preliminary study provides the basis for deeply understand the key issue of inflammation and immune cells activation in AKU patients.
In scientific research, the molecular understanding of biological mechanisms and pathways involved in disorders results fundamental to improve their knowledge. This, beside its crucial significance, provides also the theoretical starting point for the research of possible therapeutic cure. Therefore, frequently these approaches are developed together and strictly connected. Until few years ago, AKU patients were treated only with palliative cure. Recently, EMA (European Medicines Agency) extended the application of the drug nitisinone for the treatment of AKU in adult patients. Despite this represents an important progress for the patients’ care, the drug carries some collateral effects, due to the induction of tyrosinemia, and its inability to counteract inflammation. Hence, in the present project it was studied a new therapeutic approach, described in Chapters 6 and 7, based on the combination of low-doses methotrexate (MTX), a widely used anti-inflammatory drug, with antioxidant molecules. In this way, it was obtained a formulation that combined anti-inflammatory and antioxidants properties, with a stronger effect in the counteraction of these conditions, compared to the effect of single treatments. Moreover, it was proved that the co-administration of drugs allowed to use a low concentration of MTX, with the consequent decrease of its adverse effects, and beneficial impact on patients’ health. The effectiveness of the proposed treatment was tested against typical markers of inflammation, oxidative stress and amyloidosis, proving that its application could be extended to different kind of inflammatory disorders (Chapter 6). It was also studied specifically its effect on AKU model, and demonstrated that the combination of MTX and antioxidants successfully reduced ochronotic pigment and amyloid fibrils (Chapter 7).
In summary, the present thesis work gives new insight into molecular mechanisms of AKU and presented a new potential formulate for its treatment.
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Sarkar, Purbasha. "Cell death mechanisms leading to vascular cavity formation in pea (Pisum sativum) L. ‘Alaska’) primary roots." Miami University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=miami1218090008.
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ALVARENGA, C. S. "Diversidade e estruturação genética de Brachyteles hypoxanthus (Primates: Atelidae) em um ambiente fragmentado no município de Santa Maria de Jetibá (ES) usando DNA mitocondrial e nuclear." Universidade Federal do Espírito Santo, 2010. http://repositorio.ufes.br/handle/10/3817.
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Previous issue date: 2010-02-26Brachyteles hypoxanthus, muriqui-do-norte, encontra-se ameaçado pela redução e fragmentação do habitat, com 12 populações remanescentes isoladas, sendo classificado como criticamente em perigo de extinção. Estudos preliminares com DNA mitocondrial (DNAmt) revelaram a população de Santa Maria de Jetibá (SMJ) como uma Unidade de Manejo, apesar do habitat menor e altamente fragmentado. O principal objetivo desse estudo foi avaliar se as populações de cada fragmento de SMJ também formam unidades diferenciadas utilizando o DNA nuclear (DNAn) e DNAmt. Analisamos 43 indivíduos de seis áreas do município utilizando-se cinco locos de microssatélites e 366 pb da região hipervariável I (D-loop-DNAmt). Observou-se alta diversidade genética (Dg=0,74) e heterozigosidade (Hobs=0,60) com DNAn, mas duas populações, São Sebastião Belém (SSB) e Córrego do Ouro 1 (CO1), apresentaram desvios de HWE e valores significativos de coeficiente de endocruzamento (Fis=0,259 e 0,206, respectivamente). Foram detectados sete haplótipos (DNAmt), com diversidade haplotípica alta (h=0,7540), mas diversidade nucleotídica baixa (n=0,010683). O haplótipo H1 foi exclusivo para SSB, enquanto as demais populações compartilharam três haplótipos (H2, H3 e H5). Não foram observados sinais genéticos de gargalo e expansão populacional para a maioria das populações. Observou-se fraca estruturação genética para DNAn (Fst=0,0768), porém forte estruturação para DNAmt (Φst=0,58013), com SSB distinta das demais populações (0,65256≤Φst≤0,94310). Alta diversidade genética em SMJ deve-se provavelmente ao longo tempo das gerações, não ocorrendo tempo suficiente para afetar dramaticamente a diversidade genética, visto que as populações apresentaram fraca estruturação genética entre si apesar da maior taxa de evolução dos microssatélites. No entanto, desvios de HWE e a predominância de um haplótipo para SSB podem ser sinais sutis da perda de diversidade devido aos efeitos da deriva genética, dada à ausência de evidências genéticas de gargalo e expansão populacional e ao longo tempo das gerações. Tais efeitos são congruentes com a proposta de que a deriva genética tende a ser mais intensa em populações insulares, uma analogia aos fragmentos de mata de SMJ. Visto a baixa diferenciação na freqüência de alelos e o compartilhamento da maioria dos haplótipos, nossos dados fornecem suporte às conclusões prévias de que a população total de SMJ deve ser vista como uma unidade genética distinta, mas suas populações em particular não devem ser tratadas como unidades diferenciadas. Baseado nisso, simulações da diversidade genética mostram que o aumento da conectividade dos fragmentos florestais em SMJ a médio e longo prazo pode ser uma medida essencial para a recuperação, manutenção e conservação do muriqui-do-norte.
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Todorova, Biliana. "Imagerie in vivo de la réponse immune locale à la vaccination par voie intradermique à l’aide d’un ADN plasmidique associée à l’électroporation chez le macaque cynomolgus." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA114837.
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L’électroporation (EP) in vivo est utilisée comme stratégie d’amélioration de la réponse immune induite par les vaccins ADN. Cependant son effet sur les acteurs du système immunitaire inné reste méconnu. Dans l’objectif de mettre en évidence le comportement cellulaire sur le site de la vaccination, nous avons développé des approches d’imagerie par fluorescence in vivo chez le macaque. Nos résultats montrent que l’EP locale, augmente non seulement la quantité et la distribution de l’antigène vaccinal, mais induit également la mobilisation et la migration des cellules de Langerhans. De plus, l’EP cause un recrutement de leucocytes dans la peau et le tissu sous-cutané et favorise la production de cytokines pro-inflammatoires dans la peau. Ces évènements précoces, qui résultent de l’utilisation de l’EP en tant que système de délivrance des vaccins ADN, mettent en évidence le potentiel de l’EP en tant qu’adjuvant vaccinalIn vivo electroporation (EP) is used as a strategy to improve the immune response induced by DNA vaccines. However, its local effect on the innate immune cells has not been fully described. We developed in vivo fluorescence imaging approaches to highlight the cell behavior in the site of vaccination in macaques. Our results show that the local EP not only increases the amount and the distribution of the vaccine antigen, but also induces the mobilization and migration of Langerhans cells. Furthermore, EP causes the recruitment of leukocytes into the skin and subcutaneous tissue and promotes the production of pro-inflammatory cytokines. These early events that result from the use of the EP as a delivery system for DNA vaccines, highlight its potential as a vaccine adjuvant