Дисертації з теми "Genome cloning"
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1
Wain, Hester Mary. "Targeted mapping of the chicken genome." Thesis, University of Hertfordshire, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338594.
2
Jones, C. Peter. "Application of large insert cloning technology to genome analysis." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306253.
3
Swan, Daniel. "Cloning and characterisation of arkadia, a recessive, lethal, gene trap mutation." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324521.
4
Moore, Catherine Samantha. "The use of repetitive DNA sequences, in particular retrotransposons, in the genetic analysis of oil palm (Elaeis guineensis)." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340012.
5
Shull, Bruce Colin. "Molecular cloning and analysis of the genome of bovine parvovirus." Diss., Virginia Polytechnic Institute and State University, 1987. http://hdl.handle.net/10919/49895.
Анотація:
The genome of bovine parvovirus (BPV) has been cloned by blunt end ligation of double-stranded virion DNA into the plasmid pUC8. The resulting genomic clones were infectious after transfection into bovine fetal lung (BFL) cells. Sequencing of the plasmids demonstrated that deletions were common at both ends of the cloned BPV genome. Deletions of up to 34 bases at the 3’ end lowered but did not abolish infectivity, while a deletion of 52 bases eliminated infectivity, End label analysis demonstrated the repair of deletions of up to 34 bases at the 3’ end or 35 bases at the 5’ end to the wild type length. Mutually inverted sequence orientations of the palindromic termini, known as the flip and flop forms, can occur during replication of parvovirus DNA. Cloning of BPV terminal sequences permitted the identification of the 3’ flop sequence inversion as a natural component of BPV DNA. This is the first report of sequence inversions within the 3’ end of an autonomous parvovirus. Clones with the 3’ flop or flip conformations were equally infectious. Wild type virion DNA was shown to have predominantly the 3’ flip conformation but a significant amount of 3’ flop was also detected. At the 5’ end, both the flip and flop sequence conformations were identified in nearly equal amounts. The progeny virion DNA from transfection of genomic clones had the same ratio of flip to flop as did wild type at both the 3’ and 5’ ends, regardless of the starting terminal conformations of the genomic clone. These data suggest that, while sequence inversion occurs at both termini during BPV DNA replication, some mechanism exists for the preferential replication of the 3’ flip conformation. Replicative form DNA from BPV infected cells had the same ratio of flip and flop at each end and the same termini as virion DNA. A set of deletion and frameshift mutants affecting each of the coding regions of BPV was constructed using one of the genomic clones. None of these mutants was infectious when transfected into BFL cells, which demonstrates that all three of the major open reading frames are essential for the production of infectious virus.Ph. D.
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6
Moore, Karen Anne. "Cloning and expression of MCM3 genes in plants." Thesis, University of Exeter, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312072.
7
Bernard, Emmanuelle Alexa. "Cloning and characterisation of the Xenopus laevis bloom's protein." Thesis, University of Sussex, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367351.
8
Bigger, Brian William. "Adaptation of the mitochondrial genome as a vehicle for gene delivery." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325568.
9
Curran, Martin David. "Cloning and characterization of the 5' end of the measles virus genome." Thesis, Queen's University Belfast, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336037.
10
Deng, Ruitang. "Molecular cloning, nucleotide sequencing and genome replication of bovine viral diarrhea virus /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487779914825349.
11
Gutekunst, Karen Ann. "Molecular cloning and characterization of the region of the bacteriophage T4 genome coding for thymidine kinase." Diss., Georgia Institute of Technology, 1987. http://hdl.handle.net/1853/25387.
12
Anderson, Marie June. "Complementary DNA cloning, sequencing, and analysis of the Pelargonium flower-break virus genome /." The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487842372897839.
13
Pearson, Isobel V. "Cloning, mutagenesis and expression studies of the pazS gene, encoding pseudoazurin from Paracoccus denitrificans." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326025.
14
Cheung, Yee-lam Elim, and 張以琳. "Cloning and characterization of putative molecular targets of Penicillium marneffei identified by random genome exploration." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B26642608.
15
Lubbe, Lizel. "Cloning and Expression of the M-Gene from the Human Coronavirus NL-63 in Different Expression Systems." Thesis, University of the Western Cape, 2008. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_2721_1266364969.
Анотація:
In this study, the HCoV-NL63 genome was transcribed from RNA to DNA from which the M gene was amplified with various primers designed for use in specific expression systems. The various genes were cloned into the pGEM vector and confirmed by sequencing. The genes were now expressed in cloning vectors suited for each expression system (pFastBac for baculovirus expression, pFlexi for bacterial expression and pCMV for mammalian expression). Clones were sequenced for a second time. The recombinant clone in pFlexi was expressed in KRX cells and a 36hr time course was performed. The recombinant pFastBac clone was used to infect Sf9 insect cells and P1 and P2 viral stocks were obtained. The recombinant pCMV clone was used to transfect Cos1 mammalian cells.
16
Morris, Viktoriya. "Map-based cloning of the NIP gene in model legume Medicago truncatula." Thesis, University of North Texas, 2007. https://digital.library.unt.edu/ark:/67531/metadc3638/.
Анотація:
Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP at the top of linkage group 1 of the M. truncatula genome. A NIP mapping population was established with the purpose of performing fine mapping in the region containing NIP. DNA from two M. truncatula ecotypes A17 and A20 can be distinguished through polymorphisms. Positional mapping of the NIP gene is based on the A17/A20 genetic map of M. truncatula. The NIP mapping population of 2277 plants was scored for their nodulation phenotype and genotyped with flanking molecular genetic markers 146o17 and 23c16d, which are located ~1.5 cM apart and on either side of NIP. This resulted in the identification of 170 recombinant plants, These plants' DNAs were tested further with different available genetic markers located in the region of interest, to narrow the genetic interval that contains the NIP gene. Segregation data from genotyping analysis of recombinant plants placed NIP in the region between 4L4 and 807 genetic markers.
17
Coffey, Alison Jane. "Physical mapping on the human X chromosome and its application to the positional cloning of the XLP gene." Thesis, King's College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327181.
18
Hayes, Alec J. "Characterization of the soybean genome in regions surrounding two loci for resistance to soybean mosaic virus." Diss., Virginia Tech, 1998. http://hdl.handle.net/10919/11275.
Анотація:
Soybean mosaic virus (SMV), has been the cause of numerous and often devastating disease epidemics, causing reduction in both the quality and quantity of soybeans worldwide. Two important genes for resistance to SMV are Rsv1 and Rsv4. Alleles at the Rsv1 locus have been shown to control resistance to all but the most virulent strain of SMV. This locus has been mapped previously to the soybean F linkage group. Rsv4 is an SMV resistance locus independent of Rsv1 and confers resistance to all strains of SMV. This locus has not been mapped previously. The purpose of this study is to investigate the two genomic regions that contain these vitally important resistance genes.
A population of 281 F2 individuals that had previously been genotyped for reaction to SMV was evaluated in a mapping study which combined bulk segregant analysis with Amplified Fragment Length Polymorphism (AFLP). A Rsv4-linked marker, R4-1, was identified that mapped to soybean linkage group D1b using a reference mapping population. More than 40 markers were mapped in the Rsv4 segregating population including eleven markers surrounding Rsv4. This will provide the necessary framework for the fine mapping of this important genetic locus.
Previous work has located Rsv1 to a genomic region containing several important resistance genes including Rps3, Rpg1, and Rpv. An RFLP probe, NBS5, whose sequence closely resembles that of several cloned plant disease resistance genes has been mapped to this chromosomal region. The efficacy of using this sequence to identify potential disease resistance genes was assessed by screening a cDNA library to uncover a candidate disease resistance gene which corresponds to this NBS5 sequence. Two related sequence classes were identified that correspond to NBS5. Interestingly, one class corresponds to a full length gene closely resembling other previously cloned disease resistance genes offering evidence that this NBS5-derived clone is a candidate disease resistance gene.
A new marker technique was developed by combining the speed and efficiency of AFLP with DNA sequence information from cloned disease resistance genes. Using this strategy, three new markers tightly linked to Rsv1 were identified. One of these markers, which maps 0.6 cM away from Rsv1, has motifs consistent with other cloned disease resistance genes, providing evidence that this approach is an efficient method for targeting genomic regions where disease resistance genes are located.Ph. D.
19
Li, Sheping. "Molecular cloning and physical mapping of bertha armyworm, Mamestra configurata, nuclear polyhedrosis virus genome and preliminary study of geographic isolates." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1996. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq24029.pdf.
20
Marty, DeeMarie. "Characterization of Lab and Novel Agrobacterium Species for Development of New Tools for Plant Transformations." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1406138595.
21
Kemdirim, Stella C. "Molecular cloning of the polymerase genes of influenza B virus : complete nucleotide sequence of the virus genome RNA segment encoding the PBI protein." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=65397.
22
Pantoja, Morales Carlos Roberto. "Hepatopancreatic parvovirus of penaeid shrimp (HPV): Partial cloning and genome characterization, in situ hybridizationat the ultrastructural level, geographic diversity and non-invasive detection." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/284064.
Анотація:
The genome of a Korean isolate of Hepatopancreatic parvovirus (HPV) was partially cloned, sequenced and characterized. Random PCR amplification of viral DNA was combined with conventional cloning methods to generate three clones named HPV8 (2,136 bp insert), HPV3 (1,560 bp insert), and CP1139 (413 bp insert). DNA sequencing demonstrated overlapping regions between HPV8 and HPV3 and between HPV3 and CPII39. The combined sequence of these three clones encompass approximately 3,350 bp of the total 5,000 bp estimated for the HPV genome. A large open reading frame (1,692 bp) was found within clones HPV3/CPII39 encoding a polypeptide of 549 residues (∼60 kDa) whose amino terminus shows 100% homology with the first 12 residues sequenced from an apparently single 54 kDa (by SDS-PAGE) structural protein found in a Korean isolate of HPV. Two new gene probes EC.592 (592 bp) and EC.350 (350 bp) were developed by PCR amplification of previously constructed HPV (Korean) clones and labeled with DIG11-dUTP. These probes recognize different regions of the HPV genome. The specificity of both probes was confirmed by in situ hybridization using HPV-infected Penaeus chinensis (Korean) as a positive control and specific-pathogen free P. vannamei and IHHNV-infected P. stylirostris, as negative controls. Both probes were used in in situ hybridization to compare their reaction to HPV-type lesions detected by conventional H&E histology in 7 species of HPV-infected shrimp, and one of freshwater prawn, from 13 countries. The results of this comparison strongly suggest the existence of genomic differences among these geographic isolates. A post-embedding in situ hybridization assay at the electron microscope level was developed to detect HPV nucleic acids on HPV-infected hepatopancreata from P. chinensis and P. monodon . Hybridized probe (EC.592) was detected with an anti-DIG sheep antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal was observed within HPV-infected hepatopancreatic cells, which was specifically located within intranuclear viral inclusions, cytoplasm, microvillous border, and associated to necrotic debris within the lumen of hepatopancreatic tubules. A non-destructive method, based on the PCR, was developed to detect HPV by examination of crude fecal samples from HPV-infected shrimp.
23
Ringholm, Aneta I. "Cloning, Expression, Pharmacological Characterization and Anatomical Distribution of Melanocortin Receptors in an Evolutionary Perspective." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4149.
24
Guesdon, Gabrielle. "Développement de méthodes de clonage de génomes entiers chez la levure pour la construction de souches châssis semi-synthétiques de Bacillus subtilis." Thesis, Bordeaux, 2022. http://www.theses.fr/2022BORD0204.
Анотація:
Un des défis de la biologie de synthèse (BS), est d’apporter des solutions nouvelles à des enjeux globaux majeurs (thérapeutiques/sanitaires, climatiques), en particulier via la construction de souches de production utiles, performantes et respectueuses de l’environnement.Bacillus subtilis (Bsu) est une bactérie Gram+ non pathogène utilisée en biotechnologie comme plateforme de production de molécules d’intérêt. Or, des études récentes ont établi que des souches de Bsu génétiquement modifiées permettaient d’obtenir une plus grande production de protéines recombinantes. Cela suggère que la production de châssis Bsu réduits pourrait être une étape importante dans l’amélioration des souches à visée industrielle.Le projet ANR Bacillus 2.0 dans lequel s’inscrit cette thèse, vise à transposer à Bsu des outils récents du domaine de la BS, et à mettre en place un pipeline efficace pour construire à haut débit des châssis modulables selon l’application biotechnologique. Ce pipeline rassemble les technologies suivantes : (i) design d’un génome synthétique (ii) assemblage de fragments d’ADN chevauchants au sein de la levure Saccharomyces cerevisiae (iii) isolement du génome et transplantation dans une cellule receveuse bactérienne et (iv) sélection et caractérisation des souches mutantes.Les objectifs de cette thèse étaient d’attester la faisabilité de ces méthodes, en tentant de cloner et maintenir le génome réduit de Bsu MPG192 (2,86 Mb) dans la levure, puis de le modifier avec les outils d’ingénierie disponibles chez S. cerevisiae. Dans un premier temps des stratégies déjà décrites dans la littérature ont été déployées afin de cloner le génome entier de Bsu, mais sont restées infructueuses. En nous basant sur une approche de TAR-Cloning, nous avons tenté de cloner plusieurs fragments obtenus par restriction du génome de Bsu. Initialement, seuls cinq fragments sur sept ont été clonés. L’incapacité à cloner le plus grand de ces fragments (1,50 Mpb) est vraisemblablement due à un manque d’ARS et/ou à sa taille. Concernant le second fragment, un ensemble de facteurs peuvent expliquer notre échec : à nouveau le manque d’ARS mais aussi, la présence de nombreux éléments répétés (7 opérons ribosomiques) ou l’expression délétère de ces gènes. Finalement, d’autres expérimentations ont permis de découper le génome en sous-fragments de tailles variables (6kb à 515kb) et ainsi de cloner en 21 morceaux la totalité du génome de Bsu MGP 192. Le TAR-Cloning imposant des contraintes dans le choix des sites de restriction, une nouvelle méthode de clonage, appelée CReasPy-Fusion, a été développée. Elle combine le système d’édition CRISPR-Cas9 et la fusion entre des sphéroplastes de levure et des cellules bactériennes. Comme preuve de concept, nous avons d’abord travaillé avec six espèces de mycoplasmes, et démontré qu’il est possible de cloner et modifier des génomes entiers. Cette approche a ensuite été transposée à Bsu, validant pour la première fois la fusion entre des sphéroplastes de levure et des protoplastes de bactéries Gram+. Elle a permis la capture précise d’un fragment d’environ 150kb.Bien que le génome entier de Bsu n’ait pas été cloné dans la levure à ce jour, plusieurs éléments critiques ont pu être identifiés. Tout d’abord, ces travaux soulignent l’importance de la méthode de clonage à adopter en fonction de l’organisme avec lequel on travaille. Ensuite, ils mettent en exergue l’existence de facteurs biologiques et techniques qui expliquent les difficultés actuelles et qui devront être pris en compte dans la suite des expérimentations. Enfin, ils ont permis le développement d’une nouvelle technique de clonage appelée CReasPy-Fusion, qui vient étoffer le catalogue des méthodes déjà décrites. Par sa versatilité, elle ouvre des perspectives pour la capture de grands fragments de génome, pour l’élimination de loci problématiques, ou encore, en appui à l’assemblage de fragments synthétiquesOne of the major challenges in the synthetic biology (BS) field, is to provide new solutions to global issues (therapeutic/sanitary or climatic), in particular through the construction of useful, efficient and environmentally friendly production strains.The well-characterized, non-pathogenic, Gram+ bacterium Bacillus subtilis (Bsu), is widely used in industry as a biotechnological workhorse. Recent studies have established that mutant strains with modified genomes are able to produce larger amounts of recombinant proteins. This suggests that the production of rationally designed Bsu chassis could be an important step in the improvement of valuable strains for industrial purposes.This work was performed within the Bacillus 2.0's ANR project, which aims at applying SB tools for Bsu, and at developing an effective pipeline for the high-throughput construction of versatile Bsu chassis strains. Selected SB technologies for the pipeline include (i) the synthetic genome design, (ii) the in-yeast DNA assembly methods using Saccharomyces cerevisiae, (iii) the from-yeast whole genome isolation and transplantation (GT) to a recipient bacteria cell and, (iv) the characterization of recombinant strains.The objectives of this thesis were to ensure the feasibility of these methods using a Gram+ bacterium, by showing, in particular, that it was possible to clone and maintain in S. cerevisiae the genome of a minimal Bsu strain, MPG192 (2.86 Mbp) and to modify it using the large repertoire of yeast genetic tools. Our first attempts to clone the entire Bsu genome into yeast using already described methods failed. Using a TAR-Cloning approach, we then attempted to clone large DNA fragments obtained by restriction of the Bsu genome. In a first experiment, five out of seven fragments were cloned. Difficulties to clone the largest fragment (1.50 Mbp), are presumably related to its size, and/or the lack of ARS elements. Concerning the other fragment, several factors have been proposed to explain the cloning failure: again, an insufficient number of ARS elements, but also, the presence of many repeated sequences (7 ribosomal operons), and/or the deleterious expression of these genes. Finally with other experiments, the whole 2.86 Mb genome was cloned in 21 pieces ranging from 6 kbp to 515 kbp. As TAR-Cloning imposes constraints in the choice of restriction sites, a new cloning method, called CReasPy-Fusion, was developed. This method allows the simultaneous cloning and engineering of mega-sized genome in yeast using the CRISPR-Cas9 system, after direct bacterial cell to yeast spheroplast cell fusion. As a proof of concept, we demonstrated that the method can be used to capture a piece of genome, or to clone and edit the whole genome from six different Mycoplasma species. This method was then adapted to Bsu, showing for the first-time yeast spheroplast and Gram+ protoplast cell fusion. A fragment of ~150 kb has been successfully cloned in yeast.Even if, the entire Bsu genome has not yet been cloned in yeast, several critical elements have been identified. First of all, this work underlines the importance of the cloning method to be adopted depending on the organism of interest. Then, it emphasizes the existence of both biological and technical factors that explain current difficulties and that will have to be taken into account in subsequent experiments. Finally, it enabled the development of the new in-yeast cloning method called CReasPy-Fusion which expands the catalog of technics already described. Through its versatility, it opens up prospects for the capture of large genome fragments, the suppression of problematic loci, and to support the assembly of synthetic fragments
25
Desprès, Philippe. "Le virus de la fievre jaune : clonage du genome amaril et expression de la proteine d'enveloppe (e)." Paris 7, 1988. http://www.theses.fr/1988PA077049.
26
Beaudenon, Sylvie. "Clonage moleculaire et caracterisation du genome de quatre papillomavirus humains associes a des lesions benignes ou a des neoplasies des muqueuses." Paris 6, 1988. http://www.theses.fr/1988PA066048.
27
Khan, Daulat Raheem. "Reprogrammation embryonnaire et somatique au moment de la mise en route du génome dans l’embryon bovin." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA11T060.
Анотація:
Lors de la fécondation, le sperme et l'ovule s'unissent pour former un zygote totipotent. Initialement, le zygote est transcriptionnellement inactif. Au cours des premiers clivages a lieu la mise en route du génome embryonnaire (EGA) et le développement passe alors sous le contrôle de l’information embryonnaire (au stade 8-16-cellules chez le bovin). Cette transition d’un contrôle maternel à un contrôle embryonnaire est appelée « maternal to embryonic transition (MET) ». De la même façon, lors du transfert nucléaire (clonage), un noyau de cellule somatique placé dans un ovocyte énucléé devient totipotent. Ce processus est appelé «reprogrammation nucléaire somatique?». En fait, la reprogrammation nucléaire lors du clonage est équivalente à la MET, toutefois, le clonage est très peu efficace. Les objectifs de cette étude chez les bovins sont a) d'explorer le processus de reprogrammation lors de la MET dans des embryons fécondés in vitro (FIV) et b) d’estimer l'efficacité de la reprogrammation génique après le transfert nucléaire lors du clonage. Nous émettons l'hypothèse que l'acquisition d'un profil d'expression génique correct pourrait être prédictif d’un potentiel de développement à terme de l'embryon, et pourrait être évalué dès juste après l'activation du génome embryonnaire (EGA) chez les bovins. Nous avons développé notre travail selon deux axes a) des analyses globales d'expression génique utilisant une puce dédiée à l’EGA et b) l’analyse du profil d'expression de gènes candidats par qRT-PCR dans les embryons fécondés et clonés. Dans un premier temps nous avons optimisé le protocole d'amplification d'ARNm pour l'analyse du transcriptome de matériels rares. Puis nous avons fait l'analyse du transcriptome avant et après EGA d’embryons issus d’ovocytes prélevés sur des vaches phénotypées comme « bonnes » ou « mauvaises » donneuses d’embryons. En outre, ces ovocytes ont été maturés soit in vivo soit in vitro. Nos analyses montrent que l'effet individuel est plus important que l'effet « bonne ou mauvaise donneuse » ou même que l’effet « conditions de maturation ». Nous avons ensuite analysé les expressions géniques de 5 types d'embryons clonés ayant différents potentiels de développement à terme en fonction de la lignée cellulaire utilisée comme source de cellules donneuses. Globalement, leur expression génique est proche de celle de morulae FIV, mais quelques gènes présentent une expression différente. Ces gènes varient avec la lignée de cellules donneuses et leur nombre n’est pas lié à l’aptitude au développement à terme. L’analyse d’un lien éventuel entre leur nature et cette aptitude devra être poursuivie. Dans un deuxième temps, nous avons analysé les profils d'expression spatio-temporelle des transcrits et des protéines des gènes de pluripotence (OCT4, SOX2 et NANOG) et les niveaux d'ARNm de certains de leurs cibles dans les ovocytes et les embryons précoces chez le bovin. Les profils d'expression de ces gènes ont aussi été analysés dans des embryons clonés présentant différents potentiels de développement à terme. Nos résultats montrent que (1) la triade de gènes de pluripotence n'est probablement pas impliquée dans l’EGA bovine. (2) les transcrits et protéines de SOX2 et de NANOG sont restreints au lignage pluripotent plus tôt que ceux de OCT4, (3) les embryons à faible taux de développement à terme ont un taux de transcription plus élevé, néanmoins, l’équilibre précaire entre les gènes de pluripotence est maintenue. Cet équilibre pourrait permettre un développement normal in vitro, mais le taux de transcription plus élevé pourrait avoir des conséquences délétères sur le développement ultérieurIn natural fertilization, sperm and ovum unite to form a totipotent zygote. Initially, the zygote is transcriptionally inactive and after few cleavages (8-16-cell stage in bovine) embryonic genome activation (EGA) takes place and embryo shifts from maternal to embryonic control, the process called maternal to embryonic transition (MET). Likewise, in nuclear transplantation (cloning) a somatic cell nucleus achieves totipotency when placed in an enucleated oocyte, the process called “nuclear reprogramming”. In fact, nuclear reprogramming in cloning experiments is equivalent to MET; however, this process is afflicted with low efficiency. The objectives of this study in bovine were a) to explore the process of MET reprogramming of in vitro fertilized (IVF) embryos and b) to estimate the efficiency of gene reprogramming after nuclear transfer in animal cloning. We hypothesized that the acquisition of a proper gene expression pattern could herald development potential of the embryos, which could be assessed as early as morula stage or after embryonic genome activation (EGA) in bovine. Here, we opted for a study plan consisting of two axes a) global gene expression analysis using an EGA-dedicated microarray and b) candidate gene expression profiling through qRT-PCR in the fertilized and cloned bovine embryos. Firstly, we optimized the protocol of mRNA amplification for transcriptome analysis which generates antisens-RNA (aRNA). Then we did transcriptomic analysis of the 4-cell and morulae derived from two genotypes having better and two genotypes having poorer in vitro embryonic development potentials. In addition, these oocytes were either matured in vivo or in vitro. We observed that the effect of individual genotype was more important than the effect of the phenotypic category (poorer or better) or conditions of oocyte maturation. Furthermore, we explored the expression patterns of 5 types of cloned embryos having different full term developmental potentials depending upon the donor cell line used. Their genes expression patterns closely resembled to the IVF morulae, except for few genes which present differences. These genes vary with the cell line used as somatic cell donor for SCNT and the number of these deregulated genes did not increase with the poorer developmental potential of the cloned embryos. The analysis of an eventual correlation between the potential for embryonic development to term and nature of the deregulated genes should be addressed. Secondly, we charted quantitative and/or qualitative spatio-temporal expression patterns of transcripts and proteins of pluripotency genes (OCT4, SOX2 and NANOG) and mRNA levels of some of their downstream targets in bovine oocytes and early embryos. Furthermore, to correlate expression patterns of these genes with term developmental potential, we used cloned embryos, instead of gene ablation, having similar in vitro but different full term development rates. We chose these genes to be analysed since pluripotency genes are implicated in mouse embryonic genome activation (EGA) and pluripotent lineage specification. Moreover, their expression levels have been correlated with embryonic term development. Our findings affirm: first, the core triad of pluripotency genes probably is not implicated in bovine EGA since their proteins were not detected during pre-EGA phase, despite the transcripts for OCT4 and SOX2 were present. Second, an earlier ICM specification of SOX2 and NANOG makes them better candidates of bovine pluripotent lineage specification than OCT4. Third, embryos with low term development potential have higher transcription rates; nevertheless, precarious balance between pluripotency genes is maintained. This balance presages normal in vitro development but, probably higher transcription rate disturbs it at later stage that abrogates term development
28
Bouzoubaa, Salah Eddine. "Structure du genome du virus des nervures jaunes et necrotiques de la betterave (bnyvv), agent responsable de la rhizomanie de la betterave sucriere." Université Louis Pasteur (Strasbourg) (1971-2008), 1986. http://www.theses.fr/1986STR13193.
Анотація:
Le bnyvv est un virus a composants multiples a symetrie helicoidale dont les arn sont cappes en 5' et polyadenyles a l'extremite 3'. Les arn 1 et 2 ont des longueurs constantes. Les arn 3 et 4 ont des longueurs variables, ne sont pas des arn subgenomiques et peuvent meme etre absents dans certains isolats du bnyvv. En appliquant les techniques classiques de clonage et de sequencage, les structures primaires completes des quatre arn ont ete obtenues
29
Kuroki, Mayra Akemi. "Desenvolvimento de uma estratégia de clonagem customizada de regiões promotoras do genoma da cana-de-açúcar." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-05022013-082130/.
Анотація:
O objetivo deste trabalho foi desenvolver uma metodologia para identificação de regiões promotoras funcionais a partir de segmentos de um genoma qualquer. O genoma da cana-de-açúcar foi escolhido para o desenvolvimento desta estratégia na qual envolve a obtenção de fragmentos de DNA os quais foram clonados em vetores de expressão. A triagem destes fragmentos foi realizada através de biobalística e resultou no isolamento de quatro clones. Um ensaio de transformação permanente em arroz com três clones gerou 12 plantas. Foi detectada expressão do marcador GUS em calos, folhas e raízes, comprovando sua funcionalidade. Desta maneira, o presente trabalho permitiu estabelecer uma metodologia de recuperação de sequências regulatórias funcionais com ampla possibilidade de serem explorados biotecnologicamente.The aim of this work is develop a strategy to identify functional promoter regions from any genome. The modern sugarcane genome was chosen as a model for the development of this strategy that involves the generation of fragments of DNA and cloning them into expression vectors. These fragments were then screened by a transient expression assay using biolistic particle delivery resulting in the isolation of four clones. Three clones were permanently transformed in rice, and 12 plants were obtained. GUS expression was detected in the callus, leaves and roots of the rice plants thus confirming the functionality of sequences in these clones. The present work has established a strategy to identify and extract functional regulatory sequences containing functional regulatory regions which show great potential of being useful in both the biotechnology field and in the field of basic science.
30
Potgieter, Abraham Christiaan. "Cloning viral dsRNA genomes : analysis and application / A.C. Potgieter." Thesis, North-West University, 2004. http://hdl.handle.net/10394/334.
Анотація:
Double-stranded RNA viruses occur in a large number of hosts in nature ranging from
bacteria to mammals. Molecular studies of the double-stranded RNA viruses have greatly
enhanced man's understanding of this large group of viruses as far as structure and function
of their genes and epidemiology is concerned. However, one of the major prerequisites of
obtaining this information is the ability to clone the genomes of these viruses for nucleotide
sequencing and recombinant protein expression studies. In the dsRNA field, cloning viral
genomes has historically been difficult and time consuming and created a bottleneck that
hampered molecular studies. The main aim of this investigation was to optimise a method for
cloning viral dsRNA genomes to the extent that it would be easy and fast as well as
applicable to most dsRNA viruses.
In this study a sequence-independent, oligo-ligation mediated dsRNA cloning procedure for
large genes (up to 6.8 kb) was perfected and tailored for routine use to amplify and clone
complete genome sets or individual genes. Complete genome sets could be amplified and
cloned from as little as 1 ng dsRNA. The method was shown to be simple and efficient
compared to other methods and is currently the only method that allows the amplification of
complete genomes in a single PCR reaction.
Complete gene sets of seven genomes from the Reovirus family, one from the Cystovirus
family and one mycovirus, have been amplified and cloned. The full-length VP2 genes of all
9 AHSV and 24 BTV serotypes were also cloned. Phylogenetic analysis of VP2-genes
revealed the same grouping of AHSVs and BTVs as serology. Several cloned genes of
AHSV, rotavirus and EEV have been utilised for recombinant protein production establishing
that the cloned cDNAs have full open reading frames. The nine AHSV VP2 genes have been
developed as serotype-specific probes which allowed serotyping of AHSV isolates within 4
days compared to 2-4 weeks needed with the traditional serological serotyping.
The new cloning procedure finally opens the bottleneck that hamstrung the development of
complete repertoires of recombinant vaccines, molecular diagnostics and epidemiology to
combat dsRNA viral diseases. It should now be possible to deliver on many of the
expectations that were envisaged for dsRNA virus research and biotechnology since the
advent of recombinant DNA technology.Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.
31
Mugnier, Marie-Ange. "Rna 3 du virus de la mosaique de la luzerne (almv) : obtention d'une copie cdna complete et etude conformationnelle de la region 5' du rna 3 de differentes souches." Université Louis Pasteur (Strasbourg) (1971-2008), 1986. http://www.theses.fr/1986STR13160.
Анотація:
Synthese d'une copie complete d'adn complementaire qui est ensuite clone dans un vecteur d'expression pgemi. La comparaison de la sequence des cdna avec celle de l'arn 3 met en evidence une duplication dans la region 5' non codante, d'une sequence de 56 nucleotides qui constitue la difference majeure entre ces 2 sequences. La structure primaire de la region 5' non codante a ete examinee dans l'arn 3 de 3 souches du virus. Cette etude est completee par une analyse conformationnelle, en utilisant des sondes chimiques (dms) et enzymatique (v1 et s1)
32
Bulman, S. R. "Testing the effect of in planta RNA silencing on Plasmodiophora brassicae infection." Lincoln University, 2006. http://hdl.handle.net/10182/1856.
Анотація:
In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.
33
Hui, Chi-chung. "Cloning and characterization of a genomic fragment encoding abundant transcripts in anemic rat /." Hong Kong : University of Hong Kong, 1986. http://sunzi.lib.hku.hk/hkuto/record.jsp?B12324838.
34
Millar, N. S. "Molecular cloning and sequence analysis of Newcastle disease virus." Thesis, University of Newcastle Upon Tyne, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380750.
35
馬忠華 and Chung-wah Ma. "Genomic isolation and molecular analysis of a rat [alpha]-globin gene cluster." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1998. http://hub.hku.hk/bib/B31237514.
36
Ma, Chung-wah. "Genomic isolation and molecular analysis of a rat [alpha]-globin gene cluster /." Hong Kong : University of Hong Kong, 1998. http://sunzi.lib.hku.hk/hkuto/record.jsp?B19473047.
37
Heng, Yee M. "Genomic cloning and identification of a novel murine Cyp4a gene." Thesis, University of Nottingham, 1997. http://eprints.nottingham.ac.uk/10399/.
Анотація:
The mouse cytochrome P450 4a family of genes is poorly characterised. This thesis describes a detailed study of these genes. Using primers derived from exon 1 of the mouse cyp3a10 cDNA sequence, a 1.4kb genomic fragment was cloned which contained the promoter region of the gene. Additionally, a lambda genomic library was screened with probes derived from the partial cDNA clones of the mouse cyp4a10 and cyp4a12. Originally, two classes of phage lambda clones were isolated. One clone, lambda 16, was partially sequenced and was found to contain the 3' end of the cyp4a12 gene. The second clone, lambda 4, was subcloned and found to be distinct from previously known murine cyp4a genes. This novel gene has been designated cyp4a14. To obtain the full cyp4a14 gene, the genomic library was rescreened. Lambda clone 12 was subsequently isolated from the library, which was found to contain exons 1 to 5 of Cyp4a14. In order to study the promoter region of cyp4a14, a pcr-based approach was undertaken to clone 4.4 kb upstream of exon 1 of the gene and a 1.2kb fragment has been sequenced. Cyp4a14 RNA was also found to be highly induced by a peroxisome proliferator, methylclofenapate. Primer extension analyses were performed to determine the start of transcription of Cyp4a14, which was mapped to a single T nucleotide 26bp upstream of the putative start site of protein translation. The promoter region of Cyp4a14 is highly similar to the corresponding regions of the rat CYP4A2 genes. however, similarity is dramatically reduced about 350bp upstream of the start of transcription, which coincides with the presence of two 358bp repeats in teh CYP4A2 gene. The promoter also contains a highly conserved 19bp element which is also found in the promoter regions of the CYP4A1 and CYP4A2 genes. Cyp4a14 is a novel member of the murine Cyp4a subfamily. It contains 12 exons and is highly similar to the rat CYP4A2/3 genes. However, there is high conservation in exon 3 between Cyp4a14 and cyp4a3, which makes it marginally more related to this gene. In addition, cyp4a14 shows very high similarity in exons 4,8, 11 and 12 with other known CYP4A genes. Correspondingly, the peptide sequences encoded by these exons are highly conserved. Exon 8 encodes a 16 amino acid motif, LRAEVDTFMGEGHDTT, which is highly conserved in the CYP4 family. Exons 22 and 12 encode the well known RNCIG motif, containing the haem-binding domain, which is the proposed signature for a P450 protein. Exon 4 in the CYP4A genes encodes for a highly conserved, but previously unreported motif, HRRMLLTPGFHYDIL. Primary sequence alignments indicate that these motifs map very close to or within predicted substrate recognition sites and thus could have an important role in the enzyme activities of the CYP4A enzyme. The identification of Cyp4a14 also enables the analysis of the evolution of the murine Cyp4a subfamily. The mouse has 4 genes in the Cyp4a subfamily; however, the closely related rat has four CYP4A members. As the rat CYP4A2, which is very similar to CYP4AA3, was not to have a homologue in the mouse, it must have arisen after the mouse lineage had diverged from the rat in evolutionary history. As all three mouse Cyp4a genes are significantly more similar to their equivalents in the rat than to other murine Cyp4a genes, the gene duplication events giving rise to these three genes must have occurred before the mouse had diverged from the rat. Phylogenetic analyses of the CYP4A, CYP4B and CYp4F subfamilies demonstrate that CYP4A genes are more similar to the CYP4B family than to the CYP4F subfamily. This suggests that the gene duplication event giving rise to the CYP4A and CYP4B genes must be a more recent event than that giving rise to the CYP4F subfamily. In addition, the CYP4A subfamily of genes is more divergent than the CYP4B subfamily across species.
38
許志忠 and Chi-chung Hui. "Cloning and characterization of a genomic fragment encoding abundant transcripts in anemic rat." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1986. http://hub.hku.hk/bib/B31207613.
39
EFSTATHIOU, MOJAISKY IRENE. "Etude physiologique et genetique de clostridium acetobutylicum souche abkn8." Paris 6, 1987. http://www.theses.fr/1987PA066355.
40
Dindot, Scott Victor. "The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses." Texas A&M University, 2003. http://hdl.handle.net/1969.1/1161.
Анотація:
The advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that possess precise genetic modifications for agriculture and biomedical purposes. However, in spite of the revolutionary impact this technology presents, problems remain which hinder the production of healthy animals on a consistent basis. Research on cloned mice implicates improper reprogramming of epigenetic modifications and genomic imprinting for the low pregnancy rates and high incidence of abnormalities that are manifested in cloned animals; however, a systematic and comprehensive analysis of nuclear reprogramming in cloned cattle remains undone.
The purpose of this research is to assess and characterize the patterns of genomic imprinting in normal and nuclear transfer derived bovine fetuses. To facilitate the identification of imprinted genes in the bovine, a Bos gaurus/Bos taurus interspecies model has been incorporated to maximize the genetic heterozygosity that exists between the alleles of putative imprinted genes for allelic discrimination and parental inheritance.
The sequence of twenty-six genes, previously reported as imprinted in mice and humans, was analyzed in Bos gaurus (Gaur) and Bos taurus (Angus) cattle for the presence of single nucleotide polymorphisms (SNP). SNPs were detected in the Gene trap locus 2 (GTL2), Insulin like growth factor 2 (IGF2), Wilms tumor 1 (WT1) and the X chromosome inactivation specific transcript (XIST). Allelic expression analysis in interspecies hybrids indicated maternal genomic imprinting at the IGF2 and XIST loci, paternal genomic imprinting at the GTL2 locus and no imprinting at the WT1 locus. Analysis in cloned hybrids indicated fidelity of allelic expression at the IGF2 and GTL2 loci, however disruption of imprinting was observed at the XIST locus in the placenta of clones. These results are the largest identification of imprinted genes in the bovine and the first identification of the disruption of an imprinted gene in an animal derived from somatic cell nuclear transfer.
41
Wood, Katherine Anne. "A preproabrin : genomic cloning and expression of the constituent polypeptides in heterologous systems." Thesis, University of Warwick, 1991. http://wrap.warwick.ac.uk/108820/.
Анотація:
Synthetic oligonucleotides representing all possible sequences of an N-terminal and an internal region of the A- chain of abrin C were used to generate a probe specific for abrin-related sequences using the polymerase chain reaction on Abrus precatorius genomic DNA. A lambda phage library constructed from genomic DNA isolated from leaf tissue of A. precatorius was screened and positive hybridising clones were characterised by restriction enzyme analysis. The coding regions of unique clones were characterised by DNA sequencing. One clone encodes a preproprotein closely related to abrin C with 83% similarity between the A-chain sequences. Based on the similarity with the ricin toxins and Ricinus communis agglutinin, the preproprotein consists of an A-chain of 251 amino acids, preceded by 34 amino acids containing an N- terminal signal peptide, followed by a 14 amino acid linker and a B-chain of 263 amino acids. The mature A-chain of the preproabrin has been expressed cytoplasmically in Escherichia coli and the soluble recombinant protein was produced at levels exceeding 6% of total cell protein. The recombinant A-chain has been purified to homogeneity and its ability to depurinate 28S rRNA in eukaryotic ribosomes has been demonstrated in vitro. The mature B-chain of the preproabrin has been expressed in Xenopus laevis oocytes and was found to be unstable and lacking in lectin activity. The non-lectin activity of the protein was confirmed by expression and analysis of the preproabrin in X. laevis oocytes.
42
RABELO, FILHO Francisco de Assis Câmara. "Caracterização de um isolado de Yam mild mosaic virus (YMMV) obtido de Dioscorea trifida, sequenciamento do genoma e produção de antissoro policlonal." Universidade Federal Rural de Pernambuco, 2013. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/6595.
Анотація:
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The yam (Dioscorea spp.) has an important economic and social role in the Northeast of Brazil, providing food with high value to the human diet. It is also a source of income to poor family, employing large number of manpower. Among the phytosanitary problems of this crop, the fungal and nematode diseases are the most important ones. The virus diseases also have high importance, especially for causing cultivar degeneracy, with reduction of plant vigor and consequent qualitative and quantitative production losses. Three viruses stand out in Brazil with occurrence on main cultivars used in Northeast, Yam mosaic virus (YMV) and Yam mild mosaic virus (YMMV), belonging to the genus Potyvirus, family Potyviridae and one virus of the genus Badnavirus, family Caulimoviridae. Recently, two more viruses from the genus Begomovirus and Curtovirus genus, family Geminiviridae were detected. In the present work, initially, the detection and characterization of an YMMV isolate obtained from D. trifida were undertaken by electronic microscope analysis including visualization of cytoplasmatic inclusions typical of the genus Potyvirus. In addition, molecular studies with specific primers for YMMV in RT–PCR, followed by RT–PCR with degenerate primers to viruses of the Potyvirus genus, cloning the product corresponding to the coat protein and phylogenetic analysis including sequences available at the GenBank were done. Sequencing of the complete genome of a YMMV isolate and a detailed study on the nucleotide sequence of this virus by cDNA amplification and cloning in plasmids pGEM-T Easy and the pCR 4 Topo were also realized, and the data deposited in GenBank. The diagnosis of plant virus diseases can be realized by serological and molecular techniques. In yam crop, molecular tools normally are used due to difficulties in acquiring specific antisera, making more expense the identification of viral agents. Polyclonal antiserum was produced in rabbit for YMMV, using the coat protein of an isolate of YMMV expressed in vitro using Escherichia coli system.
O inhame (Dioscorea spp.) apresenta importante papel socioeconômico na região Nordeste do Brasil. Além de fornecer alimento de alto valor nutritivo para a dieta humana, é fonte de renda para famílias de baixo poder aquisitivo, empregando grande contingente de mão de obra. Dentre os problemas fitossanitários desta cultura, destacam-se as doenças fúngicas e aquelas causadas por nematoides. As viroses apresentam também elevada importância, principalmente por ocasionarem degenerescência de cultivares, com diminuição do vigor das plantas e, consequentemente perdas qualitativas e quantitativas da produção. Três vírus se destacam, em nível de Brasil, com registros nas principais cultivares empregadas no Nordeste, Yam mosaic virus (YMV) e Yam mild mosaic virus (YMMV), pertencentes ao gênero Potyvirus, família Potyviridae e um vírus do gênero Badnavirus, família Caulimoviridae. Recentemente, foram detectados mais dois vírus, pertencentes aos gêneros Begomovirus e Curtovirus, família Geminiviridae. No presente trabalho, inicialmente foram efetuadas a detecção e caracterização de um isolado de YMMV, obtido de D. trifida, por meio de análise eletromicroscópica, incluindo a visualização de inclusões citoplasmáticas típicas do gênero Potyvirus. Em seguida, foram realizados estudos moleculares com primers específicos para o YMMV em RT–PCR, seguido de RT–PCR com primers degenerados para vírus do gênero Potyvirus, clonagem do produto correspondente à capa proteica e análise filogenética, com os dados de sequências disponíveis no GenBank. Também foi realizado o sequenciamento do genoma completo de um isolado de YMMV e estudo detalhado da sequência de nucleotídeo, mediante amplificação do cDNA e clonagem em plasmídeos pGEM-T Easy e do pCR 4 Topo, cujos dados foram depositados no GenBank. A diagnose das fitoviroses pode ser efetuada por técnicas sorológicas e moleculares. Na cultura do inhame, usa-se, em geral, ferramentas moleculares, pela dificuldade na aquisição de antissoros específicos, o que encarece a identificação dos agentes virais. Antissoro policlonal para YMMV foi desenvolvido em coelho usando a proteína capsidial de um isolado do vírus expressa in vitro no sistema Escherichia coli.
43
Muona, A. (Anu). "Type XV collagen:complete structures of the human COL15A1 and mouse Col15a1 genes, location of type XV collagen protein in mature and developing mouse tissues, and generation of mice expressing truncated type XV collagen." Doctoral thesis, University of Oulu, 2001. http://urn.fi/urn:isbn:9514265858.
Анотація:
Abstract
This study was initiated to elucidate the complete genomic structures of type XV
collagen in man and mouse and the functional properties of their promoters, as well as to
obtain knowledge of the biological role of type XV collagen during development and
maturity using immunofluorescence and transgenic techniques.
The cloning and characterization of genomic clones revealed that the human
COL15A1 gene is 145-kb in size and consists of 42 exons, and the
mouse Col15a1 gene is 110-kb with 40 exons. The genomic organization
of the two genes was found to be highly conserved, except for two regions of divergence.
The nuclease S1 protection analysis revealed multiple transcription initiation sites in
both genes, which is in accordance with the overall genomic structures of their
5'-flanking sequences. Transient cell transfection experiments with varying lengths of
5'-deletion constructs identified the fragments necessary for basic promoter activity in
both genes and those implicated in the positive and negative regulation of the mouse
Col15a1 gene. Furthermore, the involvement of transcription factor
Sp1 in the gene regulation of the human COL15A1 gene was demonstrated. A mouse specific
polyclonal antibody against type XV collagen was generated and utilized in the
localization of type XV collagen protein in developing and mature mouse tissues. Type XV
collagen was deposited early in the development and was particularly prominent in
capillaries. Spatio-temporal differences in the expression of type XV collagen in various
capillary types was demonstrated. Early expression was also detected in the skeletal
muscle and peripheral nerves, while expression in the heart, lung, and kidney appeared to
be developmentally regulated. Transgenic mice lines expressing truncated type XV collagen
driven by either short or long endogenous type XV collagen promoters were generated. The
two promoters conferred different tissue-specificities and expression levels, the longer
one resulting in more endogenous-like expression. Despite some expression at both mRNA
and protein levels, the truncated type XV collagen did not cause any obvious phenotypic
or histological changes in any of the lines driven by the shorter promoter fragment. In
heterozygote matings of one of the lines driven by the longer promoter fragment, however,
a portion of the transgene positive mice appeared to be lost prenatally. Furthermore,
pregnancy terminations in this line indicated a high number of abortions beginning at
about 11 days of development. Further studies are needed before detailed conclusions on
the consequences of the generated mutation can be drawn.
The elucidation of the genomic structure of the human
COL15A1 gene provides the necessary database for screening mutations
in patient samples for candidate diseases caused by this collagen. The genomic clones and
the mouse-specific antibody against type XV collagen are valuable tools also in future
projects. The knowledge of the developmental dynamics of type XV collagen is of great
value, as it helps to understand the physiological consequences that the as yet
unidentified mutations in type XV collagen may cause in humans.
44
Wang, Wen-Pin. "Cloning and characterization of the cDNA and genomic DNA coding for human class I heparin-binding growth factor /." The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487693923197075.
45
Morch, Marie-Dominique. "Organisation et expression genetiques du virus de la mosaique jaune du navet : contribution a l'etude des strategies d'expression des virus a arn, a la discussion de leur variabilite et a l'elaboration de strategies de protection." Paris 7, 1988. http://www.theses.fr/1988PA077124.
Анотація:
On procede au sequencage du genome complet du virus constitue d'un arn de polarite positive a 6318 nucleotides et contenant 3 cistrons. Parmi les strategies mises en oeuvre pour synthetiser ses proteines, on presente la maturation proteolytique de la proteine 206 k codee par ce virus. Une approche "arn-sens" pour la lutte anti-virale est testee experimentalement: des arn "pseudogenomiques" rentrent en competition avec l'arn du virus et sont capables d'inhiber sa replication in vitro
46
Xia, Yu. "Molecular cloning and characterization of the human [alpha]-synuclein (SNCA) gene : genomic structure, transcription start site, promoter region and polymorphisms /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9835292.
47
Guan, Ping. "Identification, genomic structure, and functional studies of the human novel CC chemokine MIP-4, and the cloning of the murine homologue /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu148795320428111.
48
Wang, Qiong. "Characterization of white spot syndrome virus of penaeid shrimp: Genomic cloning and sequencing, structural protein analyzing and sequencing, genetic diversity, pathology and virulence." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/284292.
Анотація:
The purpose of this dissertation was to characterize virulence, genomic and protein composition of a newly emerged virus of penaeid shrimp: white spot syndrome virus (WSSV). A partial genomic library, covering approximately 30-50% of the genome, of WSSV isolated from crayfish Orconectes punctimanus, was constructed by digesting viral DNA with endonuclease ClaI and cloning into the system pBluescript-JM109. Three viral inserts of approximately 2.2 kb, 2.8 kb and 6.3 kb, named as QW245, CR44, and QW237 respectively, were sequenced and analyzed. Six geographic isolates of WSSV, from China, India, Thailand, South Carolina, Texas, as well as from crayfish obtained from the US National Zoo in Washington D.C. were compared by electron microscopy (TEM) and SDS-PAGE. All viral isolates contained three major polypeptides of 25, 23 and 19 kDa. A fourth major polypeptide at the 14.5 kDa position was observed in four of the viral isolates. The 19 kDa polypeptide of the crayfish WSSV appeared larger in size than that of the other isolates. Amino acid composition of four of the major structural polypeptides of the South Carolina WSSV was analyzed. The NH₂ terminal amino acids of the 25, 23 and 14.5 kDa polypeptides of the SC WSSV were sequenced as MDLSFTLSVVTA, MEFGNLTNLDVA, and VARGGKTKGRRG, respectively. The genomic composition of the six geographic isolates of WSSV were compared by combining the methods of restriction analysis using nine endonucleases AccI, BglII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI, XhoI and Southern blot hybridization applying three digoxigenin-11-dUTP labeled WSSV genomic probes LN4, C42 and A6. No distinctive difference among five WSSV isolated from penaeid shrimp was detected; differences were observed in the crayfish isolate of WSSV. The virulence of the six geographic isolates of WSSV were compared by per os challenge of Litopenaeus vannamei postlarvae and juveniles, and Farfantepenaeus duorarum juveniles. The Texas WSSV caused higher and more rapid mortalities; the crayfish WSSV caused lower and less rapid mortalities. L. vannamei postlarvae and juveniles were very susceptible to WSSV infection, while Fa. duorarum juveniles showed moderate resistance.
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López, i. Ruiz Marina. "Perfil psicopatològic i perfil de la personalitat, segons el model psicobiològic de Cloninger, en pacients amb artrosi de genoll i fibromiàlgia, com a síndromes de sensibilització central al dolor." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/459081.
Анотація:
Les patologies musculoesquelètiques són una de les causes més comunes de discapacitat degut al dolor. Dues de les patologies on el dolor crònic (DC) està típicament present són l’artrosi (OA), de genoll com a més freqüent, i la fibromiàlgia (FM). És obvi que l'OA i la FM són diferents tot i que, a més del DC, pot haver-hi una característica comú, la sensibilització, en concret la del Sistema Nerviós Central (SC), definida com hipersensibilitat al dolor, alodínia tàctil, hiperalgèsia a pressió i sumació temporal. Diferents estudis mostren relació entre les característiques psicològiques i trastorns psicopatològics i les malalties com l’OA i la FM, centrant-se en ansietat i depressió. Els trets de personalitat també són rellevants en malalties cròniques, però no s’ha estudiat tant com a síndrome de SC, i menys en la OA. La percepció del dolor relacionada amb la personalitat, no s’ha estudiat profundament en presència de SC. Així, els objectius principals d’aquesta tesi són definir el perfil psicopatològic, el patró de personalitat i el lligam de la percepció del dolor amb la personalitat en un grup de pacients amb DC, amb i sense el fenomen de SC. Metodologia. L’estudi va ser dut a terme als Serveis de Reumatologia de l’Hospital del Mar i l’Hospital CIMA-Sanitas, a Barcelona, durant 18 mesos. La selecció dels pacients es va fer segons els criteris de l’American College of Rheumatology per FM i OA. Els grups van formar-se per pacients amb artrosi amb SC (OA-SC), sense ella (OA-noSC), amb FM i grup control (C). Es van emprar l’Inventari Clínic Multiaxial de Millon, l’Inventari de Temperament i Caràcter de Cloninger i el Qüestionari de Dolor McGill, en l’avaluació psicopatològica, de personalitat i percepció subjetiva del dolor, respectivament. Es van dur a terme anàlisis de la variància, de regressió logística, de correlacions i de regressió lineal. Resultats. Els resultats mostren que el perfil del grup OA respecte el grup C emfatitza en les escales Histriònica, Passiu-Agressiu, Antisocial, Compulsiva, Esquizotípica i Somatoforme. En el grup OA-SC versus OA-noSC ho són l’escala Límit i Depressió Major. En el grup FM respecte al grup OA-noSC destaquen les escales Esquizoide, Depressiva, Histriònica i Sàdica, Límit, Somatoforme, Trastorn per Estrès Post-traumàtic i Depressió Major. I, finalment, en el grup FM respecte l’OA-SC, grups que presenten SC al dolor, ho són les escales Somatoforme i Depressió Major. La dimensió de temperament d’ED és típicament característica de pacients amb DC, sempre i quan, aquests pacients presentin SC. Les dimensions d’Evitació del Dany (ED) i Auto-Direcció (AD) són les més rellevants en la diferenciació del grup de FM. La percepció del dolor es correlaciona amb Cerca de Novetat (CN) (positiva i negativament), ED, Dependència a la Recompensa (DR) i Persistència (P) (totes tres en sentit positiu), és a dir, totes les dimensions de temperament. La percepció de dolor es correlaciona amb les dimensions de caràcter d’AD i Cooperació (CO), en sentit negatiu i positiu, respectivament. Les dimensions ED i CO de la personalitat prediuen en un 37,2% la percepció del dolor espontàni. Conclusions. El perfil psicopatològic dels grups sensibilitzats se centra especialment en trets somatoformes i de depressió major. El fenomen de SC sembla determinar la importància de la dimensió ED en pacients amb DC. Els nivells de percepció del dolor referits es correlacionen amb diverses dimensions de la personalitat, destacant l’ED. Les dimensions d’ED i AD prediuen en un 37,2% els resultats en percepció subjetiva del dolor espontàni. Així, es continua augmentant el gruix de literatura a favor de la importància de la psicopatologia i la personalitat per tractar millor als pacients amb DC, i especialment, en pacients que presentin SC.Musculoskeletal pathologies are one of the most common causes of disability due to pain. Two pathologies where chronic pain (CP) is typically present are osteoarthritis (OA), of the knee as most common, and fibromyalgia (FM). Obviously, OA and FM are different, although, in addition to CP, there is a mutual characteristic, central sensitization (CS), defined as pain hypersensitivity, tactile allodynia, hyperalgesia by pressure and temporal summation. Research shows the relationship between psychological characteristics and diseases such as OA and FM, focusing on anxiety and depression. Personality traits are also relevant in chronic diseases, but have not been studied in patients with CS, nor in OA. Perception of pain related to personality has not been neither studied deeply in the presence of CS. Thus, the main objectives of this thesis are to define the psychopathological profile, the personality pattern and the link pain perception with personality in a group of patients with CP, of which 2 subgroups present the CS phenomenon. Methodology. The study was carried out at the Rheumatology Services of the Hospital del Mar and the Hospital CIMA-Sanitas in Barcelona, during 18 months. The patient selection was done according to the criteria of the American College of Rheumatology for FM and OA. The groups were formed by patients with osteoarthritis with CS (OA-CS), without it (OA-noCS), with FM and control group (C). The Millon Clinical Multiaxial Inventory, Temperament and Character Inventory of Cloninger, and the McGill Pain Questionnaire, were used in psychopathological and personality assessment. Analysis of the variance, logistic regression, correlation and linear regression were carried out. Results. The results show that the profile of OA group versus C group emphasizes the Histrionic, Passive-Aggressive, Antisocial, Compulsive, Schizotypal and Somatoform scales. In the OA-CS group versus OA-noCS highlights Limit and Major Depression scales. In the FM group versus OA-noCS group, the Schizoid, Depressive, Histrionic, Sadistic, Borderline, Somatoform, Posttraumatic Stress Disease and Major Depression scales are the most relevant. And, finally, in the FM group versus OA-CS, both sensitized, Somatoform and Major Depression scales are the most important. The temperament dimension of Harm Avoidance (HA) is typically characteristic of patients with CP, as long as these patients present CS. The dimensions of HA and Self-Directedness (SD) are the most relevant in the differentiation of the FM group. The perception of pain is correlated with Novelty Seeking (NS) (positively and negatively), HA, Reward Dependence (RD) and Persistence (P) (all three in the positive way), that is, all dimensions of temperament. The perception of pain is correlated with the character dimensions of SD and Cooperativeness (CO), in negative and positive sense, respectively. The HA and CO dimensions of the personality predict 37.2% of the perception of pain. Conclusions. The psychopathological profile of the sensitized groups focuses especially on somatoform and major depression. The CS phenomenon seems to determine the importance of the HA dimension in patients with CP. The levels of pain perception are correlated with different dimensions of personality, highlighting the HA dimension. The dimensions of HA and SD predict 37.2% of the results regarding subjective perception of spontaneuos pain. Thus, the amount of literature continues to increase in favor of the importance of psychopathology and personality to better treat patients with CP, and especially in patients who present CS.
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Kanasi, Eleni. "Molecular analysis of the oral microbiota of dental diseases." Doctoral thesis, Umeå : Oral Microbiology, Department of Odontology, Umeå University, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1911.