Дисертації з теми "Recombinant lines"
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Anderson, Amy D. "The genetic structure of related recombinant lines /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8935.
Повний текст джерелаKim, Sumee M. "Expression of a recombinant NMDA R1 cDNA in mammalian cell lines." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq21068.pdf.
Повний текст джерелаKeith, Michelle Barbara Ann. "Screening of stably transformed insect cell lines for recombinant protein production." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ38634.pdf.
Повний текст джерелаCastilho, Alexandra Marina Machado Ferreira. "Molecular cytogenic analysis of recombinant chromosomes in wheat - Aegilops umbellulata lines." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296341.
Повний текст джерелаGuess, Adam Joseph. "QTL analysis of ray pattern in Caenorhabditis elegans recombinant inbred lines." Wright State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=wright1205197070.
Повний текст джерелаLarge, Charles Henry. "Characterisation of dopamine D3 receptors in recombinant cell lines and rat brain." Thesis, University of Bristol, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388037.
Повний текст джерелаBello, Halima Thelma. "Phenotypic and genotypic evaluation of generations and recombinant inbred lines for response to aflatoxin." [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1359.
Повний текст джерелаSteinmetz, Ralf Dirk. "Functional expression of recombinant N-methyl-D-aspartate (NMDA) receptors in eukaryotic cell lines." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=961238070.
Повний текст джерелаPatokar, Chetan. "Molecular cytogenetics and genomics of novel wheat-Thinopyrum bessarabicum recombinant lines carrying intercalary translocations." Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/36195.
Повний текст джерелаVoulgaropoulou, Frosso. "Construction and characterization of stable cell lines that generate recombinant adeno-associated virus (rAAV) /." The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487942476408687.
Повний текст джерелаAnderson, James Arthur. "EVALUATION OF SOYBEAN RECOMBINANT INBRED LINES FOR YIELD POTENTIAL AND RESISTANCE TO SUDDEN DEATH SYNDROME." OpenSIUC, 2012. https://opensiuc.lib.siu.edu/theses/837.
Повний текст джерелаPearce, Alison. "Characterisation of the endoplasmic reticulum stress proteins GRP78 and GRP94 and their interaction with a recombinant antibody." Thesis, Liverpool John Moores University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343132.
Повний текст джерелаPorter, Alison J. "Analysis of the efficiency of selecting GS-CHO cell lines for cGMP manufacture of recombinant proteins." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.692544.
Повний текст джерелаTakaluoma, K. (Kati). "Lysyl hydroxylases:studies on recombinant lysyl hydroxylases and mouse lines lacking lysyl hydroxylase 1 or lysyl hydroxylase 3." Doctoral thesis, University of Oulu, 2007. http://urn.fi/urn:isbn:9789514284281.
Повний текст джерелаMoretti, Pierre [Verfasser]. "Establishment of recombinant cell lines and characterization of primary cells for stem cell technology applications / Pierre Moretti." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover, 2010. http://d-nb.info/1004967861/34.
Повний текст джерелаDriscoll, Jeremy Neal. "The Establishment and Characterization of Recombinant Human Embryonic Kidney Cell Lines Stably Expressing the Human CB2 Receptor." Thesis, The University of Arizona, 2011. http://hdl.handle.net/10150/144336.
Повний текст джерелаKAMMILI, RAMANA. "GENERATION OF RECOMBINANT MOUSE EMBRYONIC STEM CELL LINES AND THEIRAPPLICATION FOR IN VIVO BIOLUMINISCENCE IMAGING IN THE HEART." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2171.
Повний текст джерелаM.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
Crowell, Christopher Kenyon. "Depleted amino acids and sodium butyate [sic] alter the phenotype and genotype of cell lines expressing rHuEPO /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2006.
Знайти повний текст джерелаTypescript. Includes bibliographical references (leaves 133-142). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
Anhalt, Ulrike C. M. "Characterisation of the initial generations of recombinant inbred lines in perennial ryegrass (Lolium perenne L.) using molecular markers and cytogenetics." Thesis, University of Leicester, 2009. http://hdl.handle.net/2381/7495.
Повний текст джерелаBaser, Bahar [Verfasser], and Wulf [Akademischer Betreuer] Blankenfeldt. "New Strategies to Improve the Expression of Recombinant Mammalian Proteins in Engineered Animal Cell Lines / Bahar Baser ; Betreuer: Wulf Blankenfeldt." Braunschweig : Technische Universität Braunschweig, 2015. http://d-nb.info/1175819409/34.
Повний текст джерелаAvalos, Melva Nidia. "Partial agonist interactions with dopamine in clonal cell lines expressing recombinant receptors : towards a molecular model of antipsychotic drug action /." Digital version accessible at:, 1999. http://wwwlib.umi.com/cr/utexas/main.
Повний текст джерелаClark, William Daniel. "EVALUATION OF RECOMBINANT INBRED LINE POPULATION AND ADVANCED BREEDING LINES AGAINST SUDDEN DEATH SYNDROME IN SOYBEAN [GLYCINE MAX (L.) MERR.]." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/theses/1424.
Повний текст джерелаCotsapas, Chris Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "The genetics of variation in gene expression." Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2005. http://handle.unsw.edu.au/1959.4/30204.
Повний текст джерелаBomfim, Aline de Sousa. "Clonagem e expressão de fator IX recombinante em células 293T e SK-Hep-1 e caracterização das células produtoras." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-13122013-111826/.
Повний текст джерелаBlood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the treatment of Hemophilia B which is based on the plasma-derived coagulation factors or recombinant protein produced in murine cells. Coagulation therapy based on these approaches has high costs and is closely associated with prion and virus contamination besides the FIX inhibitors development. These effects increase the risk for bleeding-related morbidity and mortality. The purpose of this study was to clone hFIX into a lentiviral vector and evaluate the expression of the recombinant protein in two human cell lines. The cloning of the hFIX cDNA into 1054 lentiviral expression vector was confirmed by enzymatic restriction obtaining the expected 1407 bp and 10054 bp bands in agarose gel. The 293T and SK-Hep-1 cell lines have been stable transduced with 1054-FIX lentiviral vector generated in our laboratory and the cells with higher expression of EGFP were selected and separated by flow cytometry. The quantification of the expression of rhFIX was performed by ELISA and chromogenic assays. The concentration of total rhFIX was 500 ng/106 cells in 293T cell line and 803 ng/106 cells in SK-Hep-1 cell line. The biological activity of FIX secreted by 293T and SK-Hep-1 was 0,047 UI/106 cells and 0,186 UI/106 cells, respectively. In order to evaluate the active rhFIX production profile over time, we conducted a monitoring of 180 days, which was noted that the SK-Hep-1 cell line ceased FIX expression, while 293T cells maintained the expression during this period. rhFIX was characterized by western blot analysis confirming the presence of a expected 57 kDa immunereactive band. The 293T and SK-Hep-1 cell lines showed 7.67 and 17 integrated vector copies/cell, respectively. Considering the importance of the ?-carboxylation process, we performed a gene expression analysis of genes involved in this process, such as VKORC1, ?-carboxylase and calumenin, in cell lines. The results showed high ratios among the genes VKORC1 and calumenin and among VKORC1 and ?-carboxylase in both cell lines. The cell growth kinetics was performed by a 7-day period, showed significant differences between SK-Hep-1 transduced cells and non-transduced cells, whereas 293T cells showed no difference in cell growth. Enrichment of culture medium with Ca +2 and Mg +2 ions was tested to evaluate its influence on the expression of active FIX. 293T cells showed better performance in 0.5 mmol/L Ca+2 and 1.0 mmol/L Mg +2 concentrations and SK-Hep-1 cells in culture medium control. Our data indicate that transduced SK-Hep-1 cells are the best producer of functional rhFIX, and comparisons between these two cell lines are important in characterizing the behavior of genetically modified cell lines focused on the heterologous expression of recombinant proteins and opens new avenues for future studies aimed at improving the production of this type of protein.
Jayatilake, Dimanthi. "A novel quantitative trait loci for fusarium head blight resistance in wheat chromosome 7A." Thesis, Manhattan, Kan. : Kansas State University, 2010. http://hdl.handle.net/2097/4265.
Повний текст джерелаKansu, Cigdem. "Characterization Of Yellow Rust And Stem Rust Resistant And Sensitive Durum Wheat Lines At Molecular Level By Using Biophysical Methods." Master's thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12613677/index.pdf.
Повний текст джерелаShiringani, Amukelani Lacrecia [Verfasser]. "Identification of genomic regions of Sorghum bicolor (L.) Moench linked to biofuel-related traits in grain x sweet sorghum recombinant inbred lines / Amukelani Lacrecia Shiringani." Gießen : Universitätsbibliothek, 2011. http://d-nb.info/1061195546/34.
Повний текст джерелаHenriksson, Sara. "Helicobacter pylori : multitalented adaptation of binding properties." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-60751.
Повний текст джерелаBriñez, Rodriguez Boris 1975. "Desenvolvimento da plataforma DART e mapeamento de locos associados com tolerância à seca em feijão (Phaseolus vulgaris L.)." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316986.
Повний текст джерелаTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O feijão comum (Phaseolus vulgaris L.) é uma cultura importante economicamente tanto para o consumo nacional como para a exportação. A seca é um dos principais estresses abióticos em todo o mundo e afeta cerca de 60% da área de cultivo de feijão. O avanço nas tecnologias de marcadores moleculares oferecem poderosos métodos para examinar as relações entre as características, gerando um grande volume de informações potencialmente úteis para assessorar os programas de melhoramento. O presente projeto teve como objetivo o desenvolvimento da Plataforma DArT para feijão comum junto à empresa DArT Pty Ltd, e o mapeamento destes marcadores juntamente com microssatélites e SNPs na população AND 277 x SEA 5 proveniente do CIAT (Colômbia), a fim de localizar os QTLs associados à tolerância à seca. O genitor SEA 5 é uma linhagem avançada do BAT 477, é tolerante à seca e de origem Mesoamericano e o genitor AND 277 é um genótipo resistente à mancha angular e antracnose e de origem Andina. Um total de 4.468 marcadores DArTs, 288 marcadores SNPs e 180 marcadores microssatélites polimórficos foram identificados na população e utilizados na genotipagem para construir um mapa genético saturado. A fenotipagem das 105 linhagens endogâmicas recombinantes (RILs) na geração F8 mais os dois genitores foi realizada avaliando 18 características associadas à tolerância a seca utilizando um delineamento inteiramente casualizado com quatro repetições, aplicando um estresse terminal na fase vegetativa V3/V4. Dois mapas foram construídos, um integrando 80 SSR e 251 SNPs e outro com cinco SSR, 91 SNPs e 4.468 DArTs. A identificação dos QTLs foi realizada através da análise de mapeamento por intervalo composto (CIM) para o mapa SSR - SNPs e mapeamento de precisão (SML) para o mapa SSR-SNPs-DArT. Um total de 12 QTLs foram identificados para o tratamento não irrigado e 29 QTLs para o tratamento irrigado pela análise CIM. Para as análises SML, 23 QTLs foram identificados para o tratamento não irrigado e 11 QTLs para o irrigado. QTLs de maior efeito foram encontrados para clorofila, biomassa fresca do caule e da folha, Massa seco da folia, temperatura da folha, número de vagens, número de sementes, massa de sementes, dias para florescimento, massa seca das vagens e produtividade nos dois tratamentos. Todos os QTLs detectados sob condições de seca apresentaram o alelo do genitor SEA 5. Este estudo é importante para o melhoramento genético não só para entender melhor a herança genética de uma característica tão complexa como a tolerância à seca, bem como para encontrar ferramentas moleculares a serem utilizados para a seleção assistida por marcadores
Abstract: Common bean (Phaseolus vulgaris L.) is the most important food legume for consumption and for exportation. Drought is one of the main abiotic stresses in the world and affects about 60% of bean growing area across the world. The advance in technologies of molecular markers provide a powerful method to examine the relationships between traits, generating large amount of potentially useful information to assist the breeding programs. The objective of this project was the development of DArT platform for common beans with DArT Pty Ltd and the mapping of these markers with microsatellites and SNPs in the population AND 277 x SEA 5 from CIAT (Colombia), in order to locate the QTLs associated with drought tolerance. The SEA 5 parent is a drought tolerant advanced line (Mesoamerican) and the AND 277 is resistant to the angular leaf spot and antracnose (Andean). A total of 4.468 DArT markers, 288 SNP and 180 SSR polymorphic markers were identified in the population and used in genotyping to constructed a saturated genetic map. Phenotyping of 105 recombinant inbred lines (RILs) in F8 generation plus the genitors were performed evaluating 18 traits associated with drought tolerance using a completely randomized design with four replicates, applying terminal stress at vegetative phase V3/V4. Two maps were constructed, one integrating 80 SSR and 251 SNPs and another with five SSR, 91 SNPs and 4,468 DArTs. The identification of QTL analysis was performed by composite interval mapping (CIM) for the SSR - SNPs map and the precision mapping (SML) to map DArT-SSR-SNPs. A total of 12 QTLs were identified for the non-irrigated treatment and 29 QTLs for the irrigated treatment by CIM analysis. For SML analysis, 23 QTLs were identified for the non-irrigated and 11 QTLs for irrigated treatment. QTLs of major effect was found for chlorophyll, fresh biomass of stem and leaf dry weight, leaf temperature, number of pods, number of seeds, seed weight, days to flowering, dry weight of pods and yield in both treatments. All QTLs detected under dry conditions showed the allele of parent SEA 5. This study is important for genetic improvement not only to better understand the genetic inheritance of a trait as complex as drought tolerance, as well as to find molecular tools to be used for marker assisted selection
Doutorado
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular
Kober, Lars [Verfasser], and Jürgen [Akademischer Betreuer] Bode. "Generation of high expressing CHO cell lines for the production of recombinant antibodies using optimized signal peptides and a novel ER stress based selection system / Lars Kober ; Betreuer: Jürgen Bode." Braunschweig : Technische Universität Braunschweig, 2012. http://d-nb.info/117582416X/34.
Повний текст джерелаPassos, Ana Laura Pereira. "Mapeamento de locos de resistência ao crestamento bacteriano comum do feijoeiro (Phaseolus vulgaris L.)." Universidade Federal de Goiás, 2016. http://repositorio.bc.ufg.br/tede/handle/tede/7319.
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The common bean (Phaseolus vulgaris) is grown in Brazil in various locations, soil and climatic conditions. The diseases are among the leading causes of losses in productivity of this legume, and the common bacterial blight (CBB) is the most important bacterioses that affects the culture. The resistance of CBB in common bean is a complex quantitative trait that results from the interaction of several genes. Genetic maps are tools that optimize the search for loci associated with this type of feature, and the most commonly used molecular markers available for this type of study are the SNPs (Single Nucleotide Polymorphism). In this sense, this study aimed to: (i) develop a robust genetic map for common bean using SNP markers and the RIL (Recombinant Inbreed Lines) mapping population derived from Ruda × AND 277; (ii) characterize this RIL population and their parents about the reaction to common bacterial blight in field and greenhouse; and (iii) identifying genomic regions (major genes and/or QTL) that control the bacterial blight in this population. We used 393 individuals of the Ruda × AND 277 RIL population, evaluated for reaction to CBB in two field trials in Ponta Grossa - PR, in the rain growing season of 2012 and 2014 and in an inoculation test at the greenhouse, in Santo Antônio de Goiás - GO. The population was genotyped with 5,398 SNP markers and mapping was performed using the R-OneMap and MapDisto programs. Statistical analyzes were performed in the Genes program, and the Scott-Knott method was used for averages groupingin R platform. The QTL analysis was conducted in QTLCartographer program. Using the chi-square test (1:1), 2,062 markers were selected for mapping. Three genetic maps with high strengt, saturation and resolution were built. Statistical analysis showed that there is genetic variability for the CBB resistance in the population of RILs. The QTL analysis identified 10 QTLs linked to resistance of CBB in the Ruda × AND 277 RIL mapping population, in the chromosomes PV01, PV02, Pv07, Pv09 and PV11, based on results from evaluations carried out in the field and greenhouse. The maps constructed for this population have high strength and resolution and may be used for future work on integrative mapping. The statistical analysis evidenced the quantitative character of resistance to CBB in common bean and showed that the parent Rudá has the CBB resistance alleles. It is expected that the markers linked to these QTLs identified can be used in future studies of marker assisted selection.
O feijoeiro-comum (Phaseolus vulgaris) é cultivado no Brasil em vários locais e diversas condições edafoclimáticas. As doenças estão entre as principais causas de prejuízos na produtividade dessa leguminosa, sendo o crestamento bacteriano comum (CBC) a principal bacteriose que afeta essa cultura. A resistência ao CBC no feijoeiro-comum é uma característica complexa, quantitativa, que resulta da interação de vários genes. Os mapas Genéticos são ferramentas que otimizam a busca de locos associados a esse tipo de característica, e os marcadores moleculares mais utilizados disponíveis para esse tipo de estudo são os SNPs (Single Nucleotide Polymorphism). Neste sentido, o presente trabalho teve como objetivos: (i) construir um mapa genético robusto para o feijoeiro-comum, utilizando marcadores SNP e a população de RILs (Recombinant Inbred Lines, ou linhagens endogâmicas recombinantes) derivada do cruzamento Rudá × AND 277; (ii) caracterizar esta população de RILs e seus genitores quanto à reação ao crestamento bacteriano comum, em campo e em casa de vegetação; e (iii) identificar regiões genômicas (genes de efeito principal e/ou QTLs) que controlam a reação ao crestamento bacteriano comum nesta população. Foram utilizados 393 indivíduos da população de RILs Rudá × AND 277, avaliados quanto à reação ao CBC em dois ensaios de campo em Ponta Grossa – PR, nas águas de 2012 e 2014, e em um ensaio de inoculação em casa de vegetação, em Santo Antônio de Goiás - GO. A população foi genotipada com 5.398 marcadores SNP e o mapeamento das RILs foi realizado utilizando os programas R-OneMap e MapDisto. As análises estatísticas foram realizadas no programa Genes, sendo o agrupamento de médias de Scott-knott realizado na plataforma R. A análise de QTL foi realizada no programa QTLCartographer. Por meio do teste de quiquadrado (1:1) foram selecionados 2.062 marcadores para o mapeamento. Foram construídos três mapas genéticos com elevada robustez, saturação e resolução. As análises estatísticas evidenciaram que há variabilidade genética para a característica de resistência ao CBC na população de RILs. A análise de QTL identificou 10 QTLs ligados à resistência ao CBC na população de RILs Rudá × AND 277 nos cromossomos Pv01, Pv02, Pv07, Pv09 e PV11 com base em dados obtidos a partir de avaliações em campo e casa de vegetação. Os mapas construídos para essa população apresentam elevada robustez e resolução e poderão ser utilizados para futuros trabalhos de mapeamento integrativo. As análises estatísticas evidenciaram o caráter quantitativo da resistência ao CBC em feijoeiro-comum e mostraram que o genitor Rudá possui alelos de resistência ao CBC. Espera-se que os marcadores ligados a esses QTLs identificados possam ser utilizados em futuros trabalhos de seleção assistida por marcadores.
Valdo, Stella Cristina Dias. "Estudo de resistência à murcha-de-fusarium e identificação de QTLs em feijeiro-comum." Universidade Federal de Goiás, 2017. http://repositorio.bc.ufg.br/tede/handle/tede/8960.
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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The common bean (Phaseolus vulgaris) crop plays an important role in the culture and economy of Brazil. It is cultivated in all Brazilian regions and is affected by several diseases like fusarium wilt which is caused by Fusarium oxysporum f. sp. phaseoli (soil-born fungus). This disease brings significant losses in common bean culture and genetic resistance is the primary form of control. One of the core goals of breeding programs is the development of resistant cultivars, therefore the objectives of this work are: i) To select F. oxysporum f. sp. phaseoli resistant F5:7 lines resulted from the crossing between Ouro Branco X CNFP10132, under controlled field and environment conditions ii) To identify SSR markers and QTL-linked SNPs associated with the resistance of common bean to fusarium wilt using 92 recombinat inbred lines(RILs) resulted from the crossing between Ouro Branco x CNFP10132. In the first study, 140 lines, the breeders Ouro Branco and CNFP10132, BRS Esplendor (resistant) and BRS Supremo (susceptible) as controls were evaluated. Field trials were conducted in a center pivot area where natural infestation of the pathogen occurs. The treatments were evaluated in summer and winter crop and the experimental design used was 12x12 triple lattice. The two controlled environment trials were conducted in a completely randomized design. The treatments were inoculated by cutting and immersing the roots in a conidial suspension, which was adjusted to 1x106 conidia/ml for five minutes. The evaluation was performed using a scale of nine grades that represent the severity of the disease: 1 – absence of symptoms and 9 – over 75% of foliage with wilt symptoms. Data were submitted to analysis of variance and Scott-Knott test for both environments. The area under the disease progress curve (AUDPC) and genetic parameters were estimated for controlled environment tests. Significant differences were observed for crops and for controlled environment trials, indicating that environment influences directly the severity of the disease. Highly significant differences were found for lines in all environments evaluated, demonstrating the existence of genetic variability, which allows the selection of resistant lines resistant to fusarium wilt. Treatments were classified in different groups according to the Scott-knott test. When considering the lowest averages in field, controlled environment and AUDPC, the strains Ouro Branco x CNFP 10132.140, Ouro Branco x CNFP 10132.49, Ouro Branco x CNFP 10132.12, Ouro Branco x CNFP 10132.90 and Ouro Branco x CNFP 10132.48 were prominent and are candidates to produce a breeding program. Heritability estimates were high for all environments, mean of 85.48% for field and 95.47% for controlled environment. Therefore, selection for resistance to F. oxysporum f. sp. phaseoli of these lines, will be successful. In the second study it was extracted DNA from 92 lines and from genitors for genotyping with SSRs and SNPs. In order to obtain the localization of these markers, sequences of the primers were aligned to the andean genome of the common bean. The method of single marker (analysis of QTLs based on linear regression) was used to identify QTLs associated with fusarium wilt resistance. These markers were considered significant when brought up p-value <0.05. Ninety-three markers were linked to 104 QTLs associated with fusarium wilt resistance and among these, were considered significant in more than one environment PV 115, PV 251, BARC-PV-0004089, BARC-PV-0004548, BARC-PV-0003450, BARC-PV-0006051, BARC-PV-0003368 , BARC-PV-0005477 and BARC-PV-0004897. However only the BARC-PV-0003450 marker was highly significant in the two environment controled trials (p <0.001) and winter crop (p <0.01) and explained up to 21.5% of the phenotypic variance. Subsequently, the gene annotation was made considering the location of all markers that were significant at p <0.01 comprising 500 kb before and after the localization. 960 coded transcripts were annotated. It was observed in gene annotation that BARC-PV-0003450 marker is located on the chromosome 8, 338.54 kb distant of the gene Phvul.008G014700 which is associated with the putative protein RPP13 related to disease resistance, identified in Arabidopsis thaliana. This protein belongs to the third class of resistance genes that encloses the domain called Leucine-Rich Repeats (LRR). This domain is involved in the recognition of the pathogen by the host during the infection process. Therefore, this marker is suitable for marker- assisted selection aiming the development of cultivars resistant to fusarium wilt.
A cultura do feijoeiro-comum (Phaseolus vulgaris) tem importância cultural e econômica no Brasil. O feijoeiro-comum é cultivado em todas as regiões brasileiras e é acometido por várias doenças, como a murcha-de-fusarium, causada pelo fungo habitante de solo Fusarium oxysporum f. sp. phaseoli. Esta doença causa significativas perdas na cultura e a principal forma de controle é a resistência genética. Desenvolver cultivares resistentes é um dos alvos dos programas de melhoramento, portanto os objetivos deste trabalho foram: i) selecionar linhagens resistentes obtidas de população F5:7 oriunda do cruzamento entre Ouro Branco e CNFP10132 para F. oxysporum f. sp. phaseoli, em condições de campo e de ambiente controlado e ii) identificar marcadores SSR e SNP's ligados a QTLs associados à resistência do feijoeiro-comum à murcha-de-fusarium utilizando 92 linhagens recombinantes endogâmicas (RILs) derivadas do cruzamento Ouro Branco x CNFP10132. No primeiro estudo 140 linhagens, os genitores Ouro Branco e CNFP10132, duas testemunhas BRS Esplendor (resistente) e BRS Supremo (suscetível) foram avaliados. Os ensaios de campo foram conduzidos em área de pivô central onde ocorre infestação natural do patógeno. Os tratamentos foram avaliados em duas safras (safra das águas e de inverno) em delineamento de látice triplo 12x12. Os dois ensaios em ambiente controlado foram conduzidos em delineamento inteiramente causalizado. As plantas foram inoculadas utilizando o método de corte de raiz e imersão destas na suspensão de conídios, que foi ajustada para 1x106 conídeos/mL durante cinco minutos. A avaliação foi feita utilizando uma escala de notas de nove graus que representam a severidade da doença: sendo 1 - ausência de sintomas e 9 - acima 75% da folhagem com sintomas de murcha. Os dados foram submetidos à análise de variância e teste de Scott-Knott para os ambos ambientes. Para os ensaios em ambiente controlado foram estimados área abaixo da curva do progresso da doença (AACPD) e parâmetros genéticos. Foram observadas diferenças significativas para safras e para ensaios de ambiente controlado, indicativo de que o ambiente influencia diretamente na severidade da doença. Foram encontradas diferenças altamente significativas para linhagens em todos os ambientes avaliados, evidenciando a existência de variabilidade genética, o que possibilita seleção de linhagens resistentes à murcha-de-fusarium. Ao considerar as menores médias em campo, ambiente controlado e ACCPD as linhagens Ouro Branco x CNFP 10132.140, Ouro Branco x CNFP 10132.49, Ouro Branco x CNFP 10132.12, Ouro Branco x CNFP 10132.90 e Ouro Branco x CNFP 10132.48 se destacaram e são candidatas para compor o programa de melhoramento. As estimativas de herdabilidade foram altas para todos os ambientes, média de 85,48% para campo e 95,47% para ambiente controlado. Portanto, a seleção para resistência à F. oxysporum f. sp. phaseoli dentre estas linhagens, será bem sucedida. No segundo estudo foi extraído o DNA de 92 linhagens e dos genitores para genotipagem com marcadores SSRs e SNPs. Para obtenção da localização destes marcadores as sequências dos primers foram alinhadas no genoma andino do feijoeiro-comum. O método de mapeamento por marcas simples (análise de QTLs por meio da regressão linear) foi utilizado para identificar QTLs associados à resistência à murcha-de-fusarium. Foram considerados marcadores significativos os que apresentaram p-valor<0,05. Noventa e três marcadores foram identificados ligados a 104 QTLs associados à resistência à murcha-de-fusarium. Dentre estes marcadores destaca-se os que foram significativos em mais de um ambiente PV 115, PV 251, BARC-PV-0004089, BARC-PV-0004548, BARC-PV-0003450, BARC-PV-0006051, BARC-PV-0003368, BARC-PV-0005477 e BARC-PV-0004897. Dentre os marcadores, somente o marcador BARC-PV-0003450 foi altamente significativo nos dois ensaios, em ambiente controlado (p<0,001) e na safra de inverno (p<0,01), e explicou até 21,5% da variância fenotípica. Foi feita a anotação gênica considerando a localização de todos os marcadores que foram significativos à p<0,01 e abrangeu 500 kb anterior e posterior à localização. Foram anotados 960 transcritos codificados. Ainda observou-se que o marcador BARC-PV-0003450 está localizado no cromossomo 8 distante 338,54 kb do gene Phvul.008G014700 o qual está associado à proteína putativa RPP13 relacionada com resistência à doenças, identificada em Arabidopsis thaliana. Esta proteína pertence à terceira classe de genes de resistência que engloba o domínio denominado de Repetições Ricas em Leucina (LRR; Leucine Rich Repeats). Este domínio está envolvido no reconhecimento do patógeno pelo hospedeiro durante o processo de infecção. Portanto há a possibilidade de selecionar linhagens resistentes à murcha-de-fusarium e identificar QTLs que possivelmente estão ligados aos marcadores utilizados
Dahl, Mads Ronald. "Mannan-binding lectin (MBL) associated serine protease-3 (MASP-3) : complex formation in serum and plasma, conditions required for the conversion of the zymogen form into a two-chain serin protease, and a search for substrates using recombinant material produced by stable expression in eukaryotic cell lines." Thesis, University of Leicester, 2004. http://hdl.handle.net/2381/29483.
Повний текст джерелаBASÍLIO, João Paulo Santana. "Genetic engineering of human cell lines for the improvement of viral vector production for gene therapy." Master's thesis, Instituto de Higiene e Medicina Tropical, 2018. http://hdl.handle.net/10362/61545.
Повний текст джерелаGene therapy using viral vectors harnesses naturally occurring viral biological mechanisms to deliver therapeutic genes and control their expression in patient target cells. Several viral gene therapy products have already reached the market, with nearly half being based on recombinant viruses belonging to the Retroviridae family. These are a frequent option since they have large genetic payload capacity, high transduction efficiency, stable genome integration in transcriptionally active loci of dividing and non-dividing cells and sustained long-term expression of the delivered gene. Revenue predictions for viral gene therapy market in 2020 surpass 200 million US dollars, with no decline perspective until at least 2026. Yet, recombinant retroviral synthesis faces several challenges. High non-infective particle concentration – roughly 1 in every 1000 produced particles are infective – and low yields of current production platforms, both of which impose high production costs, present the hardest barriers to overcome in clinical to market transition. Previously, metabolic pathways recruited in recombinant retroviral production were identified. In this work, five target genes were overexpressed in a stable recombinant retrovirus producer cell line through lentiviral vector transduction with incremental target gene expression. The five considered genes – BCL2, GSR, HSPA5, PDIA and XBP1 – belong to pathways involved in anti-apoptosis, glutathione metabolism and endoplasmic reticulum protein synthesis. Resulting populations were characterized for cell growth, recombinant retrovirus productivity, viral components and metabolic gene expression. Increase in productivity was associated to overexpression of genes intervenient in endoplasmic reticulum protein synthesis. Increases of 140% were obtained with XBP1 gene – which is associated to unfolded protein response and thus, correct protein folding. More modest increases of 63% were attributed to PDIA2 gene – which is associated to disulfide bond catalysis. Lastly, increases of 73% can be observed with HSPA5 – which is associated to a wide range of protein synthesis processes within the endoplasmic reticulum. Improvements in productivity were not observed with anti-apoptotic nor glutathione associated genes, namely BCL2 and GSR. The results herein obtained demonstrate cell metabolic engineering as a valuable strategy to improve recombinant retroviral production. Three of the five targeted genes resulted in higher recombinant retroviral production supporting protein synthesis as powerful targets for debottlenecking recombinant retroviral production. This work contributes for the viral gene therapy field. The knowledge generated in this work is relevant to other viral vectors and for metabolic engineering of human derived cell lines.
Selkirk, Julie Victoria. "An investigation into the binding and signalling properties of group I metabotropic glutamate receptors expressed recombinantly in mammalian cell lines." Thesis, University of Leicester, 2001. http://hdl.handle.net/2381/29929.
Повний текст джерелаCortijo, Sandra. "Etude des variations épigénétiques liées aux séquences répétées comme source de changements phénotypiques héritables chez Arabidopsis thaliana." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00742834.
Повний текст джерелаMartin-Vandelet, Nathalie. "Inhibiteur inter-alpha de la trypsine : assemblage et sécrétion des chaînes recombinantes dans les cellules COS." Rouen, 1998. http://www.theses.fr/1998ROUES009.
Повний текст джерелаCaron, Angelo Luis. "Estratégias para a produção de fator VIII recombinante (FVIIIr) em uma linhagem humana em condições de cultivo livres de soro e em suspensão." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/60/60138/tde-11042017-160340/.
Повний текст джерелаHemophilia A is a genetic X-linked disorder caused by the coagulation factor VIII (FVIII) deficiency. The current treatment is the replacement therapy with plasma derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) products. Nowadays, of the seven products available in the market, six are produced in rodent expression systems, which can result in a rFVIII molecule with different post-translational modifications and may lead to immune responses to non-human epitopes. Therefore, new production strategies have been evaluated, as the use of more efficient hosts in terms of protein expression potential. Among potential cell lines, the hepatic SK-HEP-1 cell line features high levels of rFVIII production and potential for serum-free suspension culture. In face of the exposed above, the goal of this study was to evaluate rFVIII production in the SK-HEP-1 human cell line comparing two strategies for the establishment of production process in a suspension serum-free condition: strategy 1 - adaptation to these conditions of a genetic modified cell line; strategy 2 - genetic modification of an already adapted cell line to rFVIII protein expression. For strategy 1, two adherent rFVIII producer cell lines were established in serum containing medium, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1. Characterization of cell growth and rFVIII production showed a maximum specific growth rate (?max) of 0.064 and 0.00311h-1 with rFVIII production of 1.0 and 0.78UI/mL, respectively. Different adaptation protocols were used; however, it was not possible to adapt the recombinant cell lines to growth in suspension serum-free conditions. For strategy 2, the wildtype SK-HEP-1 cell line adapted growth in SFMII BSF medium, showed a ?max of 0.0186h-1 and a maximum cell concentration (Xmax) of 1.9x106cells/mL. For the genetic modification, it were employed the same lentiviral vectors used for the recombinant adherent cells generation, pLVmpsvFVIII?B-Neo and pLVCMVFVIII?B-GFP. For the first, no attempts were successful. For the second, it was possible to generate two rFVIII producer populations with 0.14 and 0.12IU/mL activity, measured by chromogenic assay. These results demonstrate that the SK-HEP-1 cell line is appropriate for the production of high levels of rFVIII. Nevertheless, efforts should be made in developing specific medium to support efficient rFVIII production in suspension and suspension serum-free conditions.
Di, Guilmi Anne-Marie. "Étude de l'interaction de l'adénovirus humain de sérotype 3 avec les cellules HeLa." Université Joseph Fourier (Grenoble ; 1971-2015), 1994. http://www.theses.fr/1994GRE10058.
Повний текст джерелаPerot, Eloïse. "Production et caractérisation de nouveaux facteurs IX recombinants améliorés dans le cadre du traitement de l'hémophilie B." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10342/document.
Повний текст джерелаIntroduction: Hemophilia B (HB) is an inherited X-linked recessive bleeding disorder, due to a defect in human factor IX (FIX). Replacement therapy, in severe HB is very effective but is limited by FIX concentrates injections frequency and cost issues. Production of a recombinant FIX with enhanced clotting activity and prolonged half-life is one of the current challenges for HB treatment. Materials and Methods: To improve activity, we focused on an important residue known to be involved in the interaction of activated FIX with its cofactor, activated factor VIII (FVIIIa), and four mutated FIX-E410 were developed. To prolong stability, a new chimeric FIX cDNA was constructed too. Recombinant FIX molecules were produced by the human hepatoma cell line Huh-7. Results: The in-vitro clotting activity of FIX-E410 was 3 to 5-fold higher than wild-type FIX (FIX-WT) and this improvement was confirmed using thrombin generation assay. FIX-E410H induced 5.2-fold higher thrombin generation than FIX-WT. In HB mice, we observed significantly higher in-vivo clotting activity and thrombin generating capacity with FIX-E410H compared to FIX-WT, mainly explained by 2.5-fold enhanced affinity of the mutant for FVIIIa. Chimeric FIX showed a 10-fold increase in the in-vitro molar specific activity and a significantly increased half-life in mice (up to 2.8-fold), compared to FIX-WT. Conclusion: We have engineered and characterized four improved FIX proteins with enhanced in- vitro and in-vivo activity, and a new chimeric FIX with in-vivo increased activity and prolonged half- life. These results suggest that these new molecules could optimize protein replacement therapy forHB treatment
Maldaner, Fernanda Pavani Stamm. "Avaliação da potência do hormônio da paratireóide humano recombinante por bioensaio, métodos cromatográficos e eletroforético." Universidade Federal de Santa Maria, 2017. http://repositorio.ufsm.br/handle/1/13313.
Повний текст джерелаThe human parathyroid hormone (hPTH) is a polypeptide secreted by the parathyroid glands that is essential for the maintenance of the calcium ion homeostasis in the blood. The recombinant DNA technology has enabled the expression of hPTH gene in Escherichia coli, and thus the large-scale production of recombinant human parathyroid hormone (rhPTH 1-34), teriparatide, which contain the active amino-terminal fragment of the full length hPTH. The rhPTH is clinically used to treat osteoporosis at high risk of fractures in postmenopausal women, men with osteoporosis primary or hypogonadal and adults with glucocorticoid-induced osteoporosis (GIO). Cappilary zone electrophoresis (CZE) method was developed and validated for the assessment of rhPTH in biopharmaceutical formulations. The analysis for CZE method was performed on a fused-silica capillary (effective length, 40 cm; 50 μm i.d.), using electrolyte solution consisted of 50 mM dihydrogen phosphate solution at pH 3.0. The capillary was maintained at 25º C, the applied voltage was 20 kV. Injections were performed using a pressure mode at 50 mbar for 45 s, with detection by photodiode array detector set at 200 nm. Separation was obtained with a migration time of 5.3 min, and was linear over the concentration range of 0.25-250 μg mL-1 (r2 = 0.9992). The limits of detection and quantitation were 0.12 and 0.40 μg/mL, respectively. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.28% with bias lower than 0.85%. Moreover, the in vitro cytotoxicity test of acidic, photolytic and thermal degradated forms showed significant differences (p<0.05) compared to intact molecule. The cell proliferation and alkaline phosphatase activity bioassays in UMR-106 cells were developed and applied to assess the biological activity of rhPTH in biopharmaceutical formulations The results of content/potency were correlated to those of the validated reversed-phase liquid chromatography (RP-LC), size-exclusion liquid chromatography (SE-LC) and CZE methods, showing significant correlation (p> 0.05) Thus, the application of the validated physico-chemical methods together with in vitro bioassays, was suggested to improve quality control of rhPTH biotechnology-derived product and to support studies of biosimilars.
O hormônio da paratireóide humano (hPTH) é um polipeptídeo produzido e secretado pelas glândulas paratireóides, e é fundamental para a manutenção da homeostase dos íons cálcio no sangue. A tecnologia do DNA recombinante possibilitou a expressão do gene do hPTH em Escherichia coli, e a produção em grande escala do hormônio da paratireóide humano recombinante (rhPTH 1-34), também denominado Teriparatida, o qual apresenta a sequência de aminoácidos responsável pela porção biologicamente ativa do paratôrmonio natural. O rhPTH é clinicamente indicado para o tratamento da osteoporose de alto risco de fraturas em mulheres pós-menopausa, de homens com osteoporose primária ou hipogonadal, e da osteoporose associada à terapia sistêmica com glicocorticóides. Neste trabalho foi desenvolvido e validado método por eletroforese capilar de zona (ECZ) para a avaliação de rhPTH em produtos biofarmacêuticos. No método por ECZ, utilizou-se capilar de sílica fundida (40 cm de comprimento efetivo x 50 μm d.i.) e solução eletrolítica composta de fosfato de sódio dihidrogenado 50 mM, pH 3,0. O capilar foi mantido a temperatura de 25ºC, e a tensão aplicada foi de 20 kV. O tempo de injeção foi de 45 s, com pressão de 50 mBar, e detecção por arranjo de diodos (DAD), em 200 nm. A separação eletroforética foi obtida com tempo de migração de 5,3 min, sendo linear na faixa de concentração de 0,25-250 μg/mL (r2 = 0,9992). Os limites de detecção e quantificação foram de 0,12 e 0,40 μg/mL, respectivamente. A especificidade foi avaliada através de análises com os excipientes da formulação biofarmacêutica e estudos de degradação, demonstrando a seletividade do método. A exatidão foi 100,28% com bias inferior a 0,85%. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, apresentando, para as amostras submetidas às condições ácida, fotolítica e térmica, diferença significativa (p< 0,05) em relação à molécula íntegra. Os bioensaios de proliferação celular e da atividade da fosfatase alcalina em células UMR-106 foram desenvolvidos e aplicados para avaliação da atividade biológica de rhPTH em formulações biofarmacêuticas. Os resultados de teor/potência foram correlacionados com os métodos já validados por cromatografia líquida em fase reversa (CL-FR), cromatografia líquida por exclusão molecular (CL-EM) e ECZ, apresentando correlação significativa (p> 0,05). Assim, sugere-se que o métodos físico-químicos validados sejam aplicados paralelamente aos bioensaios in vitro para aprimorar o controle da qualidade do produto biotecnológico de rhPTH, e para avaliação da biossimilaridade de rhPTH.
Chen, Xiaomi. "Aberrant DNA Replication at an Ectopic Chromosomal Site in Human Cells." Wright State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=wright1302884072.
Повний текст джерелаGrimsley, Philip George Medical Sciences Faculty of Medicine UNSW. "Receptor mediated catabolism of plasminogen activators." Awarded By:University of New South Wales. Medical Sciences, 2009. http://handle.unsw.edu.au/1959.4/44489.
Повний текст джерелаChen, Hsing-Liang, and 陳薪喨. "Genetic Recombination Analysis of Two Recombinant Inbred Lines Populations in Rice." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/68124054195660577564.
Повний текст джерела國立臺灣大學
農藝學研究所
103
In this study comparative maps of rice were constructed with a reference physical map (Nippobare) using SSR DNA markers and two recombinant inbred lines (RILs) populations, which were generated by a modified Single Seed Descendent (SSD) method. One population including 121 RILs was generated from an inter-subspecific cross between cultivars Taichung Sen No.10 (TCS10) and Koshihikari (KH). While the other population including 146 RILs was generated from an intra-subspecific cross between cultivar Koshihikari and Tai Nung No.67 (TNG67) with the same method.More misposition and conversion events were found in the linkage groups of the population TCS10 × KH. Besides, the genetic contribution of indica variety TCS10 is larger than that of japonica variety KH in the populations TCS10 × KH, and japonica TNG67 also has larger genetic contribution than KH. In addition, TCS10 × KH has more obvious segregation distortion (SD) were observed in the population TCS10 × KH than in the population KH × TNG67. There were several SD events at the corresponding positions on most chromosomes of the two populations, suggesting that SD events were not taken place in random. The rates of recombination event or double crossing over event are higher in the population KH × TNG67. Also, linkage disequilibrium (LD) decay was slower in the population TCS10 × KH than KH × TNG67. According to LD decay data, the amount of SSR molecular markers were sufficient in both populations; these markers were not sufficiently distributed evenly. To sum up, more projenies and selfing generations in a population developed from a cross between indica and japonica varieties. The results of this study could provide informations for rice breeding.
Moon, Hyeon Gui. "Quantitative genetic analysis of recombinant inbred lines (RIL) from tropical maize singlecrosses." Thesis, 1995. http://hdl.handle.net/10125/9269.
Повний текст джерелаLIN, TA-WEI, and 林大惟. "Attachment of Recombinant Infectious Bursal Disease Virus Subvirus Particle to Susceptible Cell Lines." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/09544670773171662611.
Повний текст джерела國立中興大學
生物科技學研究所
93
Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. The outer capsid protein VP2 of IBDV has been suggested to play an important role in virus binding and cell recognition. VP2 can form a particle called subvirus particle (SVP) of 25 nm in diameter, when it was expressed in insect cells. This study attempts to identify the cellular receptors of IBDV susceptible cell lines using SVP. Inhibition infection assay with VP2-formed SVPs supports that SVPs competition with IBDV virons to the attachment of CEF and partially inhibit the replication of IBDV in CEF. Then, DF1 and Vero cells were used for study as host cell lines. Binding of VP2 particles to either DF1 or Vero cells was observed using various biochemical assays. The localization of the VP2 particles on Vero and DF1 cells was also confirmed by immunofluorescence microscope. The binding of the VP2-formed SVPs to Vero and DF1 cell surfaces was specific and occurred in a dose-dependent manner. Furthermore, the neutralizing monoclonal antibody against IBDV inhibits the attachment of SVPs particles to Vero and DF1 cells. The results suggest that the attachment of IBDV to susceptible cell is mediated by VP2.
Fernandes, Fabiana Carreira. "Establishment and evaluation of flexible insect cell lines for rapid production of recombinant proteins." Doctoral thesis, 2015. http://hdl.handle.net/10362/15270.
Повний текст джерелаChang, Yuan-Chen, and 張淵琛. "Effect of RNAi of argininosuccinate synthetase on recombinant arginine deiminase (rADI)-resistant cancer cell lines." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/62267342417420295505.
Повний текст джерела國立臺灣大學
藥學研究所
96
L-arginine is not only one of the essential amino acids for protein synthesis, but also the substrate for the conversion of other amino acids, and several non-protein compounds relating to the biochemical functions of cells, such as polyamines and nitric oxide. It has been demonstrated that recombinant arginine deiminase (rADI), a protein starving arginine-auxotrophic malignant cells by the degradation of arginine to citrulline in vitro and in vivo as well, has anti-tumoral activity. rADI is currently in clinical trials, used in patients with unresectable hepatocellular carcinoma and metastatic melanoma. However, not all malignant cells are sensitive to rADI. Endogenous argininosuccinate synthetase (AS), a rate-limiting enzyme in the arginine regeneration from citrulline, has been reported playing a crucial role in the resistance of malignant cells to rADI. Therefore, we would like to use RNA interference (RNAi) to down-regulate the expression level of AS gene and it combines with rADI to increase the sensitivity of resistant cells to rADI-treatment. Human breast cancer cell line MCF-7 and cervical cancer cell line HeLa, both of the resistant cancer cell lines to rADI-treatment, were used in our experiments. Firstly, the 21-nucleotide sequences of small interference RNA (siRNA) of AS gene and negative control (NC) were designed. MCF-7 and HeLa cells were transfected AS-siRNA and NC-siRNA, respectively, with lipofectamineTM 2000. Subsequently, cells were transfected AS-siRNA / NC-siRNA with lipofectamineTM 2000 and treated with rADI concurrently in this in vitro model. After 24-96 hours treatment, the cell viability and cell cycle distribution were analyzed by MTT assay and flow cytometry. Additionally, MCF-7 cells were incubated in L-arginine-free medium with 10% dialyzed FBS for 1-7 days to measure their cell viability by using MTT assay. The designed AS-siRNA significantly down-regulated AS gene in mRNA and protein levels in both cell lines, but not NC-siRNA. Four days after the treatment of AS-siRNA and NC-siRNA, the AS protein expression level in MCF-7 and HeLa cells were 37.8±7.2% and 0.2±0.3%, respectively, compared to each control group by Western blotting. We also measured the AS mRNA expression level in MCF-7 and HeLa cells after the treatment of AS-siRNA and NC-siRNA at day 4, they were 22.3±2.9% and 49.3±5.2%, respectively, compared to each control group. After 24-96 hours treatment of the combination of AS-siRNA and rADI in MCF-7, the cell viability was not significantly affected by MTT assay. On the contrary, the percentage of cell viability in HeLa were 90.1±5.0%, 64.9±0.4%, 13.1±1.4%, and 7.7±0.2%, respectively, after 24, 48, 72, 96 hr treatment of the combination. Four days after the combination of AS-siRNA and rADI, the percentage of apoptosis in MCF-7 and HeLa cells were 8.1±3.4% and 63.4±4.7%, respectively, by the flow cytometry. In addition, when MCF-7 cells were cultured in L-arginine-free medium, the cell viability was not affected by the absence of L-arginine. From our results, although the combination of AS-siRNA and rADI decreased the AS protein expression and AS mRNA level in both HeLa and MCF-7 cell lines, only HeLa cells were sensitive to the combination treatment via the apoptotic pathway. In addition, the MCF-7 can survive and proliferate in the L-arginine depletion medium. It may indicate the L-arginine is not the essential amino acid for MCF-7 cells. In our study, it is known that the endogenous AS protein expression level are different between HeLa cells and MCF-7 cells. For HeLa cells, their endogenous AS protein expression level is low, but it is induced to 5 fold of the AS expression in the control group after 4 days treatment of rADI. For MCF-7 cells, the induction of AS protein expression level is only minimal (1.1 fold) of it in the control after 4 days treatment of rADI. Therefore, down-regulation of AS gene by RNAi could be a strategy to overcome the resistance of rADI in some malignant cells, such as HeLa cells. However, it may need further studies to understand the mechanism of the resistance of the combination treatment of AS-siRNA and rADI in other cells, such as MCF-7 cells.
Barbosa, Taylor Marcelo Correa. "Molecular characterization of natural recombinant ALVS and their use as vectors for gene delivery into stable cell lines." 2010. http://purl.galileo.usg.edu/uga%5Fetd/barbosa%5Ftaylor%5Fm%5F201005%5Fphd.
Повний текст джерелаSteinmetz, Ralf Dirk [Verfasser]. "Functional expression of recombinant N-methyl-D-aspartate (NMDA) receptors in eukaryotic cell lines / by Ralf Dirk Steinmetz." 2000. http://d-nb.info/961238070/34.
Повний текст джерела