Dissertations / Theses on the topic 'Recombinant lines'
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1
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.
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Kim, 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.
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Keith, 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.
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Castilho, 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.
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Guess, 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.
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Large, 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.
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Bello, 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.
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Steinmetz, 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.
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Patokar, 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.
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The diploid wild grass Thinopyrum bessarabicum (2n = 2x = 14, JJ or EbEb) is a rich source of important genes for bread wheat (2n = 6x = 42) improvement because of its salinity tolerance and disease resistance. Development of wheat–Th. bessarabicum translocation lines by backcrossing amphiploids in the absence of the Ph1 gene (allowing intergenomic recombination) enables its practical utilization in wheat improvement. Using genomic in situ hybridization (GISH) and repetitive probes for fluorescent in situ hybridization (FISH), six novel wheat–Th. bessarabicum translocation lines involving different chromosome segments (T4BS.4BL-4JL, T6BS.6BL-6JL, T5AS.5AL-5JL, T5DL.5DS-5JS, T2BS.2BL-2JL, and the whole arm translocation T1AL.1JS) were identified and characterized in this study. No background translocations between wheat genomes were observed. The involvement of 5 of the 7 chromosomes, and small terminal segments of the Th. bessarabicum chromosome arm were important, contributing to both reduced linkage drag of the derived lines by minimizing agronomically deleterious genes from the alien species, and high stability including transmission of the alien segment. All three wheat genomes were involved in the translocations with the alien chromosome, and GISH showed the Th. bessarabicum genome was more closely related to the D genome in wheat. All the introgression lines were disomic, stable and with good morphological characters. The work also generated a high-resolution karyotype of two accessions of Th. bessarabicum using multiple repetitive DNA probes for chromosome identification. A complete CS-Th. bessarabicum amphiploid (2n=8x=56, AABBDDJJ) was used and each individual Jgenome unambiguously identified. The established karyotype will be useful for the rapid identification of potential donor chromosomes in wheat improvement programs, allowing appropriate alien-chromosome transfer. Genotyping-by-sequencing (GBS) data was collected from the wheat-Th. bessarabicum introgression lines, but the complexity of the wheat genome and need for further development of data analysis pathways limited interpretation.
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Voulgaropoulou, 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.
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Anderson, 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.
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Evaluation of soybean recombinant inbred lines for seed weight yield, agronomic traits, and resistance to sudden death syndrome Sudden death syndrome (SDS) caused by Fusarium virguliforme is a devastating disease in soybean (Glycine max (L.) Merr.) that causes up to 70% of yield losses depending on the developmental stage when the plant become infected. The characterization of resistance is greatly significant for disease management. Therefore, three populations were developed by crossing three resistant lines, `Hamilton', LS90-1920 and LS97-1610 with a susceptible line to SDS, `Spencer'. Ninety-four F5:6 recombinant inbred lines from each population (Hamilton x Spencer, LS90-1920 x Spencer, and LS97-1610 x Spencer) were evaluated for two years (2009 and 2010) at two locations (Carbondale and Valmeyer) in southern Illinois. Population statistics, genotype x environment interaction, and broad-sense heritability were used to reveal any major resistance genes. Genetic correlation coefficients of SDS resistance with important agronomic traits such as lodging, pubescence, growth habit, and plant height were also calculated. The information from this study will be helpful to breeders in developing populations for genetic analyses and enforcing selection practices.
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Pearce, 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.
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Porter, 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.
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The market size for biotherapeutics is growing rapidly and the hunt is always on for the next blockbuster drug. A wide range of cell phenotypes are obtained during the process of generating mammalian cell lines suitable for the production of a biotherapeutic such as a monoclonal antibody. A strategy is therefore required for the selection of a cell line with ’desirable’ characteristics, from a population of closely related cell lines, for the successful manufacture of the biotherapeutic. As this process is included in the sequence of activities which determines the minimum time to start cGMP manufacturing of the biotherapeutic, companies are actively seeking to improve its efficiency. Stringent examination of a typical selection strategy revealed that it was capable of identifying high producing cell lines. However, it only isolated a fraction of the highest producing cell lines and ranking positions between assessment stages that form the selection strategy were not consistent. These results suggested that there was potential to improve selection strategies. Models of alternative selection strategies, built by in silico analysis of data typically collected during cell line construction, were not able to identify ’better’ cell lines. Alternative markers for cell lines capable of achieving high product concentrations could be used to improve selection strategies. In this study, parameters that showed a relationship with recombinant product concentration were identified, suggesting the possibility for development of more predictive assessment criteria. However, results also showed that it was unlikely that a single predictive parameter would be identified. The study also highlighted the affect the environment, which may change between assessment stages, had on cell lines. Closer matching of the cellular environment was therefore also suggested as a means to improve selection strategies. This study has highlighted the importance of designing a selection strategy that is highly predictive of the final production process. In the future, cell line selection strategies are likely to require the incorporation of closer matching of the cellular environment and the use of multiple predictive markers which form profiles of cell lines capable of producing the highest product concentrations.
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Takaluoma, 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.
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Abstract
Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures.
In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend.
In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos.
The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.
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Moretti, 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.
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Driscoll, 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.
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KAMMILI, 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.
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ABSTRACT Cardiovascular disease is the major cause of death in the United States, with 80 million people suffering from some form of heart disease each year. One major limitation is the inability of the heart to repair the damaged tissue. Stem cell therapy holds enormous promise to repair and regenerate the damaged myocardium, but there are many technical difficulties that must first be overcome. One such difficulty is the present lack of ability to track and assess transplanted stem cells over time in vivo. The central hypothesis of this thesis is that in vivo bioluminescence imaging is a safe and useful method for monitoring transplanted stem cells in mouse hearts. To evaluate this hypothesis, two aims were performed. In aim 1, stable recombinant mES cell lines expressing the firefly luciferase (fLUC) reporter gene under the control of constitutive and cardiac-specific promoters were generated and characterized in vitro. In aim 2, these fLUC-expressing recombinant cell lines were evaluated following transplantation into neonatal mouse hearts. The major findings are: (1) Novel stable recombinant mES reporter cell lines were developed for in vivo bioluminescence imaging; (2) One of these cell lines was created using the glyceraldehyde 3-phosphodehydrogenase (GAPDH) promoter fused to the fLUC reporter and it showed similar levels of fLUC expression in undifferentiated (pluripotent) compared to cardiac-differentiated mES cells; (3) Another cell line was produced using the cardiac-specific sodium-calcium exchanger 1 (NCX1) promoter fused to the fLUC reporter and this cell line showed markedly increased fLUC expression following induction of cardiac differentiation in culture when compared to the pluripotent cells. (4) Transplantation of the recombinant fLUC-expressing cells into neonatal mouse hearts produced bioluminescent signals that persisted for at least 24 days, the maximum timepoint analyzed in this study; (5) Transplantation of 100,000 or more mES cells to the heart consistently produced teratoma and tumor formations, regardless of which recombinant clone was used or whether the mES cells were injected as pluripotent or cardiac-differentiated cells, (6) Transplantation of between 10,000 and 50,000 cardiac-differentiated NCX1-fLUC mES cells(containing mixed population of other cells) per heart resulted in measurable bioluminescent image signals in vivo with low incidence of tumor formation, and (7) Some of the transplanted NCX1-fLUC mES cells were identified in ventricular muscle tissue in postmortem histological sections where it was found that they had developed cardiomyocyte characteristics. In summary, I developed stable recombinant mES cell lines suitable for non-invasive bioluminescence imaging to study the survival and proliferation of the cells in vivo. These results demonstrate that bioluminescence imaging in the neonatal mouse heart model is an effective strategy for non-invasive monitoring of transplanted stem cells over time in vivo, and minimizes animal usage through elimination of the need for animal sacrifice at multiple timepoints.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
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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.
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Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 133-142). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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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.
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Baser, 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.
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Avalos, 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.
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Clark, 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.
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Soybeans [Glycine max (L.) Merrill] are a major commercial crop that originated in Eastern Asia, which date back 5,000 years ago in China and are still used worldwide today. Soybeans are considered an oil seed crop that averages twenty percent oil content and consists of thirty-five to forty percent protein. Soybeans are used in most aspects of the modern world as a source of protein for humans and animals alike. It is also used for its oils, which can be found in food, consumables, and plastic. Soybean production came about in the 18th Century in the United States as a hay crop and in some regions as an ornamental plant, but did not start being grown in large-scale production until the early 19th Century. Seed producing companies did not take interest in the plant until 1970, when Congress established the United States Plant Variety Act. This Act allows protection for companies against unauthorized use of proprietary material. Plant breeders focused on improving yield, drought tolerance, and disease resistance. Sudden Death Syndrome (SDS) is a disease of soybeans that affect soybean populations in the Western Hemisphere. SDS is a seedling disease in which a soil-borne fungal infection attacks the roots of a young soybean plant. This infection is more severe in soils highly saturated with water early in the planting year and then followed by cool weather before the soybean plant flowers in late summer. Yield losses commonly do not exceed ten to fifteen percent of a crop, but cases have occurred where yield was reduced over seventy percent due to SDS. Three species that affect the Western Hemisphere; Fusarium virguliforme (FV), formally know as Fusarium solani f. sp. Glycines (FSG); which mainly affects soybean production in the North American continent, Fusarium phaseoli and Fusarium tucumaniae, which affect the South American continent. SDS in the United States can account for yield losses occurring in primarily Arkansas, Iowa, Illinois, Indiana, Kentucky, Missouri, and Tennessee during 1999 to 2002 time period, with Iowa, Illinois, and Indiana having the most severe effects. SDS has rapidly spread throughout the United States and it was estimated to suppress the soybean yield in 2002, with damage that was valued at $157.4 million. There is not a 100 percent proven agronomic practice for controlling SDS, so the identification of host resistant genes are required in order to develop different varieties that will offer the producer the most economically efficient way to manage the disease.
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Cotsapas, 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.
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The majority of genetic differences between species and individuals have been hypothesised to impact on the regulation, rather than the structure, of genes. As the details of genetic variation are uncovered by the various genome sequencing projects, understanding the functional effects on gene regulation will be key to uncovering the molecular mechanisms underying the genesis and inheritance of common phenotypes, such as complex human disease and commercially important traits in plants and animals. Unlike coding sequence polymorphisms, genetic variants affecting gene expression will reside in the transcriptional machinery and its regulatory inputs. As these are largely specific to cell- or tissue-types, we would expect that regulatory variants will also affect final mRNA levels in a tissue specific manner. Genetic variation between individuals may therefore be more complex than the sum total of sequence differences between them. Demonstrating this hypothesis is the main focus of this thesis. We use microarrays to measure mRNA levels of approximately 22,000 transcripts in inbred and recombinant inbred strains of mice, and present compelling evidence that the genetic influences on these levels are tissue-specific in at least 85% of cases. We uncover two loci which apparently influence transcript levels of multiple genes in a tissue-specific manner. We also present evidence that failure of microarray data normalisation may cause spurious linkage of expression phenotypes leading to erroneous biological conclusions, and detail a novel, extensible mathematical framework for performing tailored normalisation which can remove such systematic bias. The wider context of these results is then discussed.
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Bomfim, 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/.
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O fator IX (FIX) da coagulação sanguínea é uma proteína dependente de vitamina K de grande valor farmacêutico no tratamento da Hemofilia B, o qual é baseado na administração do fator de coagulação derivado de plasma humano ou da proteína recombinante produzida em células murinas. A terapia baseada nestas abordagens apresenta alto custo e está associada às contaminações com vírus e príons, além do desenvolvimento de inibidores de FIX. Esses efeitos aumentam o risco de morbidade e mortalidade relacionadas às hemorragias. Neste trabalho, clonamos o cDNA do FIX em um vetor lentiviral e avaliamos a expressão da proteína recombinante em duas linhagens celulares humanas. A clonagem do cDNA do FIXh no vetor de expressão lentiviral 1054 foi confirmada através da análise com enzimas de restrição específicas obtendo-se as bandas esperadas de 1407 pb e 10054 pb visualizadas em gel de agarose. As linhagens celulares 293T e SK-Hep-1 foram transduzidas com o vetor lentiviral 1054-FIX gerado em nosso laboratório e as células que apresentaram maior expressão de EGFP foram selecionadas e separadas por citometria de fluxo. A quantificação da expressão de FIXrh foi realizada por ensaios de ELISA e cromogênico. A quantificação de FIXrh total foi de 500 ng/106 células para a linhagem 293T e 803 ng/106 células para a linhagem SK-Hep-1. A atividade biológica específica de FIXh nas células 293T e SK-Hep-1 foi 0,047 UI/106 células e 0,186 UI/106 células, respectivamente. Com o intuito de avaliar o perfil de produção de FIXrh ativo ao longo do tempo, foi realizado um acompanhamento de 180 dias, no qual foi observado que a linhagem SK-Hep-1 cessou a expressão de FIX, enquanto as células 293T mantiveram a expressão durante o período. O FIXrh foi caracterizado por western blot confirmando a presença de uma banda imunoreativa esperada de 57 kDa. As linhagens 293T e SK-Hep-1 apresentaram 7,67 e 17 cópias do vetor inserido/célula, respectivamente. Considerando a importância do processo de ?-carboxilação, foi realizada uma análise da expressão gênica dos genes envolvidos neste processo, tais como o VKORC1, ?-carboxilase e o inibidor calumenina, nas linhagens celulares. Os resultados demonstraram razões elevadas entre os genes VKORC1 e calumenina e VKORC1 e ?-carboxilase nas duas linhagens. A cinética de crescimento das células foi realizada por um período de 7 dias apresentando diferenças significativas entre as células SK-Hep-1 transduzidas e não transduzidas, enquanto que as células 293T não presentaram diferenças estatísticas no crescimento celular. A suplementação do meio de cultura com íons Ca+2 e Mg+2 foi testada para avaliar sua influência na expressão de FIXrh ativo. As células 293T apresentaram melhor desempenho nas concentrações de 0,5 mmol/L de Ca+2 e 1,0 mmol/L de Mg+2 e as células SK-Hep-1 no meio de cultura não suplementado. Nossos dados indicam que a linhagem hepática SK-Hep-1 é a melhor produtora de FIXrh funcional e as comparações realizadas entre os dois tipos celulares são importantes na caracterização do comportamento de linhagens geneticamente modificadas voltadas para a expressão de proteínas recombinantes heterólogas e abre novos caminhos para futuros estudos que visam o melhoramento da produção desse tipo de proteína.Blood 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.
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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.
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Kansu, 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.
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Stem rust and Yellow rust diseases are the two major wheat fungal diseases causing
considerable yield losses in Turkey and all around the world. There are studies which
are carried out to identify and utilize resistance sources in order to obtain resistant
lines of wheat. However, virulent pathotypes are continuously being important
threats to wheat production and yield. For that reason, new approaches for rapid
identification are needed.
The aim of this study was to investigate and to understand the structural and
functional differences between the resistant and sensitive durum wheat cultivars to
the plant fungal diseases of stem and yellow (stripe) rusts. To aim this, forty durum
wheat recombinant inbred lines (RILs), which were previously determined to be
resistant or sensitive to stem and yellow rust diseases, were investigated by the noninvasive
Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR)
Spectroscopy. Also, classification of the resistant and sensitive lines depending on
the structural and functional differences has been attempted. The FTIR spectra for stem rust disease showed that, resistant durum wheat lines had
a significant increase in the population of unsaturation in acyl chains of lipid
molecules, an increase in lipid and in total protein content and also an increase in
carboxylic acids and alcohols. For yellow rust disease, resistant lines had a
significant increase in hydrogen bonding and they had also a more ordered
membrane structure.
In Principal Component Analysis for stem rust disease, according to 3700-650 cm-1
region, amide III band (1213-1273 cm-1 region) and C-H stretching region (3020-
2800 cm-1), the resistant and sensitive groups were separated successfully. For
yellow rust disease, according to 3700-650 cm-1 region, Amide A and Amide III
bands, the resistant and sensitive lines were grouped distinctly.
FTIR spectroscopy provides a useful approach to determine the differences in
molecular structure of durum wheat RILs regarding resistance of lines to fungal
diseases. However, further research is still needed to ensure if the structural and
functional differences in biomolecules of the samples could be used as molecular
markers for discrimination of rust resistant materials from rust sensitive ones.
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Shiringani, 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.
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Henriksson, 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.
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Helicobacter pylori infects and persistently colonizes the stomach, which results in gastritis and in some individuals peptic ulcer disease or gastric cancer. Adherence of H. pylori to the epithelium is an important factor for development of disease. Attachment is mediated by the adhesins BabA and SabA that binds the ABO/Leb blood group antigens and sialylated glycoconjugates respectively. High-affinity attachment could be anticipated to be of disadvantage for H. pylori because epithelial cells have a fast turnover rate and the dislocated and shed epithelial cells would carry attached bacteria to the acidic gastric juice in the lumen. However, here we describe that H. pylori manage to adapt to this innate clearance mechanism by unique acid regulatory binding properties of its adhesins. We propose that pH regulated binding properties enable bacteria to detachment from host cells for chemotactic guided motility and successful return to the more neutral epithelium for a fresh restart of the infectious cycle. By comparison of BabA from different stomach loci we identified amino acid key position for acid regulated binding activity. Previous studies found lower prevalence of Leb-binding among H. pylori isolates from southern Europe compared to Sweden. Here we tested if the reduced prevalence of Leb-binding could be explained by a novel binding mode; in among Spanish strains, we identified S812 that demonstrates preference for multivalent binding to ABO antigens in glycolipids; we found that 812 BabA had drifted in its preferred binding epitope away from the consensus a1,2fucosylation and towards the blood group A and B derivatives. Such epitope drift might in particular optimize binding to ABO antigens in densely packed lipid rafts. In parallel, we studied the influence of BabA for disease progression by an inventory of gastric biopsies. BabA correlated both with the oncoprotein CagA, the VacAs1 toxin and, in addition, to severe disease progression. We further correlate BabA expression with positive secretor phenotype and stronger adhesion of H. pylori in vitro. For functional adherence studies in vitro, we constructed a recombinant Leb-expressing cell lineage that supports BabA mediated H. pylori attachment.
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Briñez, 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.
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Orientadores: Luciana Lasry Benchimol Reis, Matthew Ward BlairTese (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
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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.
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Passos, 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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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
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.
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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
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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.
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The complement system is part of the innate immune system and is crucial for identifying invading microorganisms. The lectin pathway of complement activation is initiated through multimeric macromolecules which recognise pathogen-associated patterns and translate binding through activation of associated serine proteases that start a cascade of proteolytic events leading to bactericidal, opsonising and proinflammatory responses. Mannan-binding lectin (MBL) is one of the macromolecules mediating binding to specific carbohydrate structures common to a range of microorganisms. Three different mannan-binding lectin associated serine proteases (MASP-1/-2/-3) and a non-enzymatic protein of 19 kDa (MApl9) have been described. MASP-2 appears to mediate all processes required for complement activation while little or no complement-related functional activity was found to be mediated by MASP-1, MASP-3 or MApl9. Functional and biophysical studies of MASP-3 relied on continuous and reliable supply of recombinant MASP-3. In this work production of recombinant MASP-3 in mammalian cells with subsequent affinity purification and characterisation of the recombinant MASP-3 was performed to obtain large quantities of homogeneous enzyme. The development of screening assays and assays for quantitative determination of MASP-3 levels were two other tools essential for the development of this thesis. As a result of this thesis MASP-3 levels in different body fluids were determined in healthy individuals using a quantitative assay. The assays were used to analyse the MASP-3 level in sample collections from patients suffering from Alzheimer disease and type-1 diabetes. The correlation of MASP-3 level and MBL genotype, H-/L-Ficolin concentration, age, BMI, acute phase and time of year were analysed. The enzymatic activity of MASP-3 was analysed on chromogenic substrates and the results permitted a study of MASP-3 inhibition. Attempts were made to affinity purify potential MASP-3 substrates and ligands using beads coupled with recombinant MASP-3 and anti- MASP-3 antibodies. The influence of calcium on MASP-3 complex formation, dissociation, activation and stability was analysed.
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BASÍLIO, 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.
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A terapia génica baseada em vetores virais toma partido dos mecanismos biológicos naturais para entregar genes terapêuticos e controlar a sua expressão nas células alvo do paciente. Vários produtos de terapia génica viral estão já no mercado, com cerca de metade destes utilizando vírus recombinantes da família Retroviridae. Estes são uma opção frequente devido à sua elevada capacidade de transporte de carga genética, elevada eficiência de transdução, integração estável de genoma em loci transcricionalmente ativos, quer as células estejam em divisão ou não e devido à expressão a longo termo do gene entregue. As previsões de receita no mercado de terapia génica viral até 2020 ultrapassam os 200 milhões de dólares, sem perspetivas de declínio pelo menos até 2026. No entanto, a produção de retrovírus recombinantes depara-se com vários desafios. A elevada produção de partículas não-infecciosas – aproximadamente 1 em cada 1000 partículas produzidas são infecciosas – e baixo rendimento das plataformas de produção, dois fatores que impõem custos de produção altos, são as barreiras mais difíceis de ultrapassar na transição clinico-mercado.
Vias metabólicas recrutadas em produção de retrovírus recombinantes foram previamente identificadas. Neste trabalho, cinco genes-alvo foram alvo de estudo numa linha celular produtora de retrovírus recombinantes. A sua sobre-expressão foi realizada através de transdução com vetores lentivirais. Os cinco genes considerados – BCL2, GSR, HSPA5, PDIA e XBP1 – pertencem a vias envolvidas em anti-apoptose, metabolismo de glutationa e síntese proteica no retículo endoplasmático. As populações resultantes foram caracterizadas quanto a crescimento celular, produtividade viral, expressão dos componentes virais e dos genes metabólicos.
Verificou-se um aumento de produtividade associada à sobre-expressão dos genes intervenientes na síntese proteica no retículo endoplasmático. Aumentos de 140% foram obtidos com o gene XBP1 – que está associado à resposta de proteínas mal conformacionadas. Aumentos mais modestos, na ordem dos 63% foram obtidos com o gene PDIA2 – que está associado à catálise de pontes dissulfureto. Por último, aumentos de 76% foram observados com HSPA5 – que está associado a um grande leque de processos envolvidos em síntese proteica no retículo endoplasmático. Não foram observados aumentos de produção com genes associados à anti-apoptose nem ao metabolismo de glutationa, nomeadamente BCL2 e GSR.
Os resultados aqui obtidos demonstram que engenharia metabólica é uma estratégia valiosa para o melhoramento de produção de retrovírus recombinantes. Três dos cinco genes abordados levaram ao aumento de produção de retrovírus recombinantes, o que apoia a síntese proteica como um alvo valioso na resolução dos constrangimentos encontrados na produção de retrovírus recombinantes. Este trabalho contribui para o campo da terapia génica baseada em vetores virais. O conhecimento gerado neste trabalho é relevante para outros vetores virais e para engenharia metabólica de linhas celulares humanas.Gene 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.
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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.
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This Thesis investigates the G protein coupling profile of group I mGluRs using a novel 35S -GTPyS binding with subsequent Gcc subunit-specific immunoprecipitation protocol. This approach allows the quantitation of activation of individual G proteins or groups of G proteins to be assessed. Using this assay, it was revealed that mGlulct receptors couple to different endogenous sub-populations of G proteins when expressed recombinantly in model cell backgrounds. Thus mGlula receptors expressed stably in BHK cells couple to both PTx-sensitive and PTx-resistant G protein sub-types, yet when expressed inducibly in CHO cells couple solely to PTx-resistant Gctq/n G proteins. One consequence of the PTx-sensitive component of signalling by mGluRlot in BHK cells is to regulate PLC in a negative manner, the potency and efficacy of agonists increasing following pre-treatment of the cells with this toxin. Sequestration of GPy subunits using a PARK C-terminal minigene had no effect on mGluRla-mediated responses and thus the negative regulation of PLC appears to be mediated by the Get subunit of Gj/0 G proteins. The basis for this additional coupling of mGluRla in BHK cells could not conclusively be attributed to differences in receptor or G protein expression levels. Using the same approach, an interaction of mGluR5a could not be observed with either Gi/0 or Gq/ii G proteins when expressed stably in HEK-293 cells or inducibly in CHO cells. Despite this apparent lack of G protein coupling however, an accumulation of inositol phosphates was measurable, as was an elevation of Ca2+ j. The binding characteristics of the novel group I mGluR radioligand 3H -quisqualic acid were assessed at both mGlul and mGlu5 receptors, and also with respect to a soluble form of human mGluRl, truncated just prior to the first transmembrane domain, and secreted by CHO cells. The noticeably higher affinity of 3H -quisqualic acid for this truncated receptor compared to that reported for the rat receptor produced in insect Sf9 cells, prompted studies to investigate whether TV-linked glycosylation was important for agonist binding and subsequent signalling. Prevention of TV-linked glycosylation either by treatment with tunicamycin, or by site-directed mutagenesis, resulted in reduced specific 3H -quisqualic acid binding to mGlula receptors, and also in a reduction of cell surface receptor expression. This suggests that TV-linked glycosylation of mGlula is important for receptor expression and function.
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Cortijo, 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.
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Des changements de méthylation de l'ADN peuvent affecter l'expression des gènes et pour certains être transmis au travers des générations. De telles " épimutations " qui concernent des groupes de cytosines à proximité ou dans les gènes sont donc une source potentielle de variation phénotypique héritable en absence de changements de la séquence de l'ADN. Chez les plantes la méthylation de l'ADN est cependant principalement observée au niveau des séquences répétées. Il reste à déterminer dans quelle mesure les changements de méthylation au niveau de ce type de séquences peuvent être héritées et affecter les phénotypes. Afin de répondre à ces questions, plus de 500 épiRIL (epigenetic Recombinant Inbred Lines) quasi-isogéniques a été générée chez Arabidopsis thaliana. Cette population a été obtenue par le croisement d'un parent sauvage et d'un parent mutant pour le gène DDM1 présentant une très forte réduction du taux de méthylation de l'ADN. Après un rétrocroisement de la F1 avec une plante sauvage, les individus sauvages pour le gène DDM1 ont été sélectionnés et propagées sur 6 générations par autofécondation. Nous avons montré par l'analyse du méthylome de plus de 100 épiRIL que l'hypométhylation induite par ddm1 présente selon les séquences affectées différents degrés de transmission au travers des générations. La réversion de l'hypométhylation concerne des régions associées à une abondance élevée en sRNA de 24 nt. Nous avons utilisé l'hypométhylation stablement transmise dans les épiRIL induite par ddm1 afin de détecter des QTL (Quantitative Trait Loci) affectant le temps de floraison et la longueur de la racine primaire, deux caractères pour lesquels les variations observées dans les épiRIL présentent une héritabilité importante. En dernier lieu, nous avons recherché par différentes approches les variations causales de ces QTL.
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Martin-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.
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Les protéines de la famille de l'inhibiteur inter-α de la trypsine (ITI) humain sont synthétisées et structurées dans le foie à partir de 4 précurseurs protéiques : 3 chaînes lourdes H1, H2, H3 et une chaîne légère L. Ces précurseurs maturent et donnent naissance aux formes HC et à la bikunine. Trois protéines de cette famille ont été identifiées dans le plasma humain : l'ITI, l'inhibiteur pré-α de la trypsine (Pαl) et la protéine HC2-bikunine. Les chaînes constitutives de ces protéines sont assemblées par une structure covalente particuliére, de nature glycosidique, composée de glycosaminoglycanne. Cette liaison, une fois constituée, est intitulée Protéine-Glycosaminoglycanne-Protéine ou PGP. Compte-tenu de la complexité de la structure de ces protéines, nous avons mis en oeuvre un système original de transfection permettant l'expression des chaînes recombinantes H1, H2, H3 et L dans les cellules COS. La première partie de notre étude porte sur la biosynthèse de chaque chaîne constitutive des protéines de la famille de l'ITI en utilisant le modèle de transfection transitoire dans les cellules COS. Chaque chaîne possède une synthèse et une maturation indépendantes des autres chaînes (à l'exception de H2). La seconde partie de notre étude tend à mettre en évidence les mécanismes d'assemblages des chaînes H1, H2, H3 et L dans le but de générer les protéines de la famille de l'ITI. Dans notre système, quelle que soit la combinaison engageant la chaîne H1, la forme HC1 n'est jamais observée dans une protéine pluricaténaire. La maturation de H1 en HC1 et l'assemblage de HC1 au sein des protéines pluricaténaires se feraient selon un mécanisme différent de ceux des chaînes H2 et H3. Les chaînes H2 et H3 possèdent des comportements opposés au cours de l'assemblage. Le clivage de la chaîne H3 précéderait l'assemblage, alors que celui de H2 serait concomitant ou postérieur à son assemblage au sein des protéines pluricaténaires. D'autre part, la protéine Pαl observée dans notre modèle apparaît être un produit de synthèse alors que la protéine HC2-bikunine qui n'est jamais observée dans notre système représenterait un produit de dégradation de la forme ITI-like. La réalisation de la liaison originale PGP commune aux protéines pluricaténaires de l'ITI, ne nécessite pas la machinerie cellulaire de l'hépatocyte.
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Caron, 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/.
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A hemofilia A é uma doença ligada ao cromossomo X causada pela deficiência do fator VIII da coagulação sanguínea (FVIII). O tratamento disponível consiste na terapia de reposição da proteína do fator VIII derivada do plasma (FVIIIdp) ou recombinante (FVIIIr). Atualmente, dos 7 produtos recombinantes disponíveis no mercado, 6 são produzidos em linhagens celulares de roedores. A expressão dessa proteína em sistemas celulares não-humanos pode gerar uma molécula com perfil de modificações diferente do endógeno, podendo levar a reações imunogênicas e geração de inibidores anti-FVIIIr. Em função disso, novas estratégias de produção têm sido avaliadas, como a utilização de células hospedeiras mais eficientes no que diz respeito ao potencial de expressão da proteína de interesse. Dentre as linhagens de interesse, a linhagem hepática SK-HEP-1 tem se destacado por apresentar altos níveis de expressão do FVIIIr e potencial para o cultivo em suspensão em meios livres de soro fetal bovino (SFB). Dessa forma, o objetivo deste trabalho foi avaliar a produção de FVIIIr na linhagem celular humana SK-Hep-1 comparando duas estratégias para o estabelecimento de processos de produção em condições livres de soro e em suspensão: Estratégia 1 - adaptação para tais condições da linhagem já modificada geneticamente e Estratégia 2 - modificação gênica para a expressão da proteína já em células previamente adaptadas à tais condições. Para a estratégia 1, foram geradas duas linhagens recombinantes produtoras de FVIIIr, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1 aderentes e em cultivos suplementados com SFB. Na caracterização da cinética e produção do FVIIIr as linhagens apresentaram taxas específicas máxima de crescimento (?max) de 0,064 e 0,0031h-1 produzindo 1,0 e 0,78UI/mL de FVIIIr, respectivamente. Diversos protocolos de adaptação foram empregados, entretanto, não foi possível obter sucesso na adaptação das linhagens recombinantes para condições livres de soro e em suspensão. Para a estratégia 2, as células SK-HEP-1 selvagens adaptadas ao meio de cultura livre de SFB SFMII apresentaram um valor de ?max de 0,0186h-1 e Xmax de 1,9x106cels/mL. Para as etapas de modificação gênica da linhagem selvagem foram utilizados os mesmos vetores lentivirais empregados para a geração das células recombinantes aderentes, pLVmpsvFVIII?B-Neo e pLVCMVFVIII?B-GFP. Para o primeiro, não foi possível gerar uma linhagem produtora do FVIIIr. Para o segundo, foi possível obter duas linhagens produtoras do FVIIIr com 0.14 e 0.12IU/mL de atividade pelo ensaio cromogênico. O presente trabalho mostrou que a linhagem humana Sk-Hep-1 é apropriada para a produção de altos níveis de FVIIIr. No entanto, maiores esforços devem ser voltados ao desenvolvimento de meios de cultura livres de soro específicos para a linhagem para possibilitar a produção eficiente do FVIIIr em suspensão em meios livre de soro.Hemophilia 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.
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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.
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Les travaux decrits dans ce rapport concernent l'interaction de l'adenovirus humain de serotype 3 avec les cellules hela. Dans un premier temps, nous avons defini les caracteristiques de la liaison du virus ad3 avec les cellules hela a 37c et a 4c. Pour ces deux conditions de temperature, nous avons montre que le virus ad3 a la propriete de se lier avec deux affinites distinctes sur ses sites recepteurs. Nous avons demontre que les virus ad2 et ad3 (appartenant respectivement aux sous-groupes c et b) ne partagent pas les memes sites de liaison a la surface des cellules hela. La fibre est la proteine virale dont la fonction est de se lier au recepteur. Nous avons produit cette proteine dans le systeme de baculovirus. La fibre ad3 recombinante presente une morphologie et une structure correctes. L'activite fonctionnelle de la fibre a ete mise en evidence par l'efficacite de l'inhibition de la fixation du virus ad3, a la surface des cellules hela. Dans le but d'identifier le recepteur cellulaire de l'ad3 nous avons applique la technique du vopba en utilisant la fibre ad3 recombinante et le virus ad3. A partir des cellules hela, le virus et la fibre ad3 ont detecte une proteine membranaire de 130kda avec laquelle ils interagissent. Cette reconnaissance presente un caractere specifique. Des travaux similaires effectues sur des cellules a549, permissives a l'infection par le virus ad3, n'ont cependant pas permis de detecter cette proteine 130
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Perot, 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.
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Introduction : L'hémophilie B (HB) est une maladie hémorragique héréditaire due à un défaut en facteur IX (FIX) de la coagulation. Le traitement substitutif est efficace mais pose deux problèmes : la fréquence des injections intraveineuses de concentrés de FIX et leurs coûts. La production d'un FIX recombinant à activité améliorée et à demi-vie prolongée est un enjeu important du traitement. Matériel et méthodes : Afin d'améliorer l'activité du FIX, l'étude s'est portée sur un résidu impliqué dans l'interaction du FIX activé avec son cofacteur, le facteur VIII activé (FVIIIa). Quatre FIX mutés au niveau de l'acide aminé E410 ont été développés. Afin de prolonger la demi-vie, un ADN complémentaire de FIX chimérique a été généré de façon à produire la protéine correspondante par la lignée cellulaire humaine Huh-7. Résultats : L'activité coagulante in vitro des FIX-E410 était 3 à 5 fois plus élevée que celle du FIX wild-type (FIX-WT). De plus, le FIX-E410H induisait une génération de thrombine 5,2 fois plus élevée comparée à celle du FIX-WT. Chez la souris HB, l'activité coagulante et la capacité de génération de thrombine du FIX-E410H in vivo étaient significativement plus élevées que celles du FIX-WT. La protéine chimérique a présenté une activité molaire spécifique 10 fois augmentée in vitro et une demi-vie jusqu'à 2,8 fois plus allongée, par rapport à celles du FIX-WT. Conclusion : Nous avons développé et caractérisé quatre molécules de FIX ayant une activité améliorée in vitro et in vivo, ainsi qu'un FIX modifié à activité augmentée et à demi-vie prolongée in vivo. Ces nouvelles molécules pourraient ouvrir de nouvelles perspectives thérapeutiques de traitement de l'HBIntroduction: 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
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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.
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Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGSThe 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.
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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.
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Grimsley, 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.
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Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2 macroglobulin, apolipoproteins, β amyloid precursor protein, and a number of serpin-enzymes complexes, including PA??PAI-1 complexes. Further studies for the work presented here centred on this receptor. By using radiolabelled binding assays, ligand blots, and Western blots on cultured cells, the major findings are that: (1) basal LRP1 expression on HepG2 is low compared to a clone termed, HepG2a16, but appears to increase in long term culture; (2) a soluble form of LRP1, which retains ligand-binding capacity, is present in human circulation; (3) soluble LRP1 is also present in cerebral spinal fluid where its role in neurological disorders such as Alzheimer??s disease is a developing area of interest; and (4) the release of LRP1 is a mechanism conserved in evolution, possibly as distantly as molluscs. The discovery, identification, and characterisation of soluble LRP1 introduces this protein in the human circulation, and presents a possible further level of regulation for its associated receptor system.
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Chen, 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.
Full textAbstract:
碩士國立臺灣大學
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.
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Moon, Hyeon Gui. "Quantitative genetic analysis of recombinant inbred lines (RIL) from tropical maize singlecrosses." Thesis, 1995. http://hdl.handle.net/10125/9269.
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LIN, 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.
Full textAbstract:
碩士國立中興大學
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.
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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.
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Chang, 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.
Full textAbstract:
碩士國立臺灣大學
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.
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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.
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Steinmetz, 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.
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