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Caird, Mandy Ruth. "Molecular cloning studies on DNA polymerase alpha." Thesis, University of Aberdeen, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292388.
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The DNA polymerase α assay systems and partial purification techniques already in use in the laboratory were improved by isolating better-activated DNA and freezing cells prior to sonication. The suitability of monoclonal antibodies against Xenopus laevis and human KB cell (Tanaka et al., 1982) DNA polymerase α was established as a prelude to screening expression libraries. All were found to inhibit polymerase activity by approximately 50%. Two out of three anti-Xenopus laevis DNA polymerase α antibodies and three out of four anti-human DNA polymerase α antibodies bound antigen in HeLa cell cytoplasmic extracts. Human hepatoma, human foetal liver, human fibroblast and mouse bone marrow lambda gt11 expression libraries were screened with the above antibodies using the method of Young and Davis (1983) and various horseradish peroxidase and alkaline phosphatase conjugated second antibody systems in an attempt to isolate a human or mouse DNA polymerase α gene. A 1.9kb cDNA clone from a calf thymus expression library (Foster et al., 1986) was characterised. The molecular weight of 165-175kDa and polymerase activity of the β-galactosidase-calf fusion protein was confirmed but the fusion protein could not be detected with any of the antibodies mentioned above. The 1.9kb cDNA clone was used to show the regulation of DNA polymerase α activity in growth-regulated bovine kidney cell to be at the transcriptional level. The DNA sequence of the calf thymus cDNA fragment was determined. The DNA sequence of 2042 bases and all predicted open reading frames were compared with sequences contained in the GenBank, EMBL and NBRF (nucleic acid and protein) databanks of the University of Wisconsin Genetics Computer Group molecular biology package. No homology to any DNA polymerase α nucleic acid or protein sequence was found, suggesting that the sequence of Foster et al. (1986) might not code for a DNA polymerase α. In searches of international sequence databanks the 1.9kb DNA fragment shows the greatest similarity to human, bovine and rodent sequences. However, the similarity scores are very low suggesting that this sequence is unique and will require the determination of a longer sequence before location of an identity.
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Montgomery, Douglas S. "An investigation of rat DNA polymerase alpha." Thesis, University of Aberdeen, 1985. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU362670.
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The aim of this project was to clone the gene encoding the catalytic subunit of the rat DNA replication enzyme, DNA polymerase alpha. A strategy was adopted in which cDNA clones expressing the catalytic subunit sequences would be identified using anti-DNA polymerase antibodies. DNA polymerase alpha was partially purified from regenerating rat liver and exponentially growing rat Y3 myeloma cells. The catalytic subunit was identified as a 170-180kD polypeptide by activity gel analysis of partially purified Y3 cell fractions. The catalytic subunit was found to be susceptible to degradation but without loss of polymerase activity. Glycerol gradient analysis indicated a two stage degradation of DNA polymerase in vivo. Sera were collected from mice immunised with partially purified DNA polymerase alpha from regenerated rat liver. These sera cross-reacted with Western-blotted Y3 cell fractions; removed polymerase activity from solution in plate binding assays and bound alpha polymerase activity (140-180kD) on an immuno-adsorption column cDNA was synthesised using size selected mRNA from exponentially growing Y3 cells and cloned into the expression vectors pUC8 and ?gtll, both of which utilise the lac Z gene to express cloned DNA sequences. Immunoscreening of the ?gtll library was frustrated by non-specific binding of the serum. This non-specific binding was overcome by pre-adsorbing the serum against a lysate of E. coli JM 83. Screening of the pUC8 library revealled 27 out of 2.25x104 colonies which bound pre-adsorbed anti-DNA polymerase alpha serum.
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Sutton, A. D. "An investigation of DNA polymerase alpha from regenerating rat liver." Thesis, University of Aberdeen, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370114.
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Mello, Richard. "IDENTIFICATION OF DQ ALPHA POLYMORPHISM USING THE POLYMERASE CHAIN REACTION." VCU Scholars Compass, 1991. https://scholarscompass.vcu.edu/etd/5213.
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This was a study of detection systems for DQ alpha HLA polymorphism that could be exploited for the demonstration of simulated chimerism. Polymorphic segments of DO alpha DNA were amplified by the polymerase chain reaction (PCR). Simulated chimerism was represented by a mixture of minor and major component DNA. The goal was to detect minor component DNA in the presence of major component DNA utilizing various laboratory techniques. Techniques studied included probe strip typing with the AmpliType HLA-DO Alpha test kit, allele-specific amplification, polyacrylamide gel electrophoresis, restriction enzymes, and Southern transfer combined with a peroxidase detection system.
The AmpliType HLA-DO Alpha test kit had a detection sensitivity of at least 0.2%. This is much better than the 10% detection sensitivity in non-PCR techniques. When the 3.0 DC alpha type was mixed as the minor component with undiluted 1.1 DQ alpha type, the detection sensitivity for the 3.0 DC alpha type increased to a detection level of 0.1%.
The allele-specific primers were able to specifically amplify the minor component DNA in the presence of major component DNA. Major component DNA did not amplify and thus did not compete with the minor component DNA for Taq polymerase. The allele-specific primers provided an overall detection sensitivity of 0.2%.
Background interference prevented detection of minor component bands on both polyacrylamide gels stained with ethidium bromide and on Southern blots reacted with the peroxidase detection system.
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Wolter, Gwen Annette. "Charakterisierung der Strahlen-induzierten Komplexbildung von DNA-Polymerase [alpha]-Primase [Alpha-Primase] mit dem Tumorsuppressorprotein p53." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=967492394.
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Krawczynska, Karolina. "An investigation of the interaction between MDM2 and DNA polymerase alpha." Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533989.
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Burton, Sara Katherine. "DNA-binding proteins associated with DNA polymerase alpha in pea (Pisum sativum)." Thesis, University of Exeter, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357961.
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Liku, Muluye E. "CDK regulation of replication proteins: Mcm2-7 and DNA polymerase alpha primase." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324598.
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Höpfner, Marco. "Veränderungen des alpha-beta-T-Zell-Rezeptor-Repertoires der zytotoxischen T-Lymphozyten im peripheren Blut bei akuter Hepatitis C-Infektion." Ulm : Universität Ulm, Medizinische Fakultät, 2000. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9918621.
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Hurt, Nicholas S. "Electronic detection of DNA polymerase complex formation and dissociation using an alpha-hemolysin nanopore /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2009. http://uclibs.org/PID/11984.
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DONALDSON, ROBERT WILLIAM. "PHOSPHORYLATION OF DNA POLYMERASE ALPHA IN NORMAL AND ROUS SARCOMA VIRUS TRANSFORMED RAT FIBROBLASTS." Diss., The University of Arizona, 1987. http://hdl.handle.net/10150/184054.
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Immunochemical and immunohistochemical techniques were used to determine the role of post-translational modifications in the regulation of DNA polymerase α in Rat-1(tsLA24/RSV) cells. Immunoaffinity purification following sucrose gradient fractionation showed two immunospecific polypeptides of Mᵣ ≃ 185,000 and 220,000 only in those fractions exhibiting DNA polymerase α activity. The Mᵣ ≃ 220,000 polypeptide was shown to be phosphorylated, primarily at serine residues. Incubation of cell lysates with immobilized alkaline phosphatase reduced enzyme activity and subsequent readdition of ATP, but not ATP-γ-S, restored activity suggesting the involvement of an endogenous serine protein kinase. This kinase may be a cAMP dependent protein kinase because prior incubation of the catalytic subunit stimulated DNA polymerase α activity 3-4 fold. In the absence of serum growth factors or pp60ˢʳᶜ, DNA polymerase α activity and semi-conservative DNA replication rates in growth arrested cells were severely depressed. However, both polymerase activity and DNA synthetic rates were subsequently restored by either activation of pp60ˢʳᶜ by temperature shift or by serum addition. DNA polymerase α protein was found primarily in the nucleus of all cells in log phase, growth arrested or subsequently stimulated cultures, independent of whether the cells were replicating DNA. Stimulation by either pp60ˢʳᶜ or serum did not alter DNA polymerase α localization within the cell nor lead to a preferential synthesis of Mᵣ ≃ 220,000 peptide or proteolytic conversion of the Mᵣ ≃ 220,000 peptide to smaller peptides, but did result in phosphorylation of the Mᵣ ≃ 220,000 polypeptide. This phosphorylation was not apparent in serum deprived, growth arrested cells. It is suggested that pp60ˢʳᶜ acts to initiate DNA synthesis through the temporal activation of DNA polymerase α through a mechanism similar to that used by serum growth factors and that phosphorylation by a serine protein kinase serves an important function.
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Brückner, Florian. "Molecular basis of translocation, alpha-amanitin inhibition, and CPD damage recognition by RNA polymerase II." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-86461.
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Brückner, Florian. "Molecular basis of translocation, alpha-amanitin inhibition, and CPD damage recognition by RNA polymerase II." kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/8646/.
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Mehrpouyan, Majid. "A Temperature-sensitive Mutant of Escherichia Coli Affected in the Alpha Subunit of RNA Polymerase." Digital Commons @ East Tennessee State University, 1990. https://dc.etsu.edu/etd/2728.
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A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44$\sp\circ$C. The thermostability at 44$\sp\circ$C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about four fold at 37$\sp\circ$C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed. In addition during the course of this study a new procedure for the purification of E. coli RNA polymerase was developed. This method is rapid, convenient, and useful for the preparation of enzyme from 1-5 grams of cells in two days. The ease and speed of this method allowed the rapid characterization of the mutant enzyme. This system should also find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.
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Hill, Donna. "Isolation & Characterization of DNA Polymerase Alpha and Gamma from Turnips (Brassica rapa) and Etiolated Soybeans (Glycine max)." TopSCHOLAR®, 1988. https://digitalcommons.wku.edu/theses/2483.
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DNA polymerase alpha, the enzyme involved in nuclear DMA replication, and DNA polymerase gamma, the enzyme involved in organellular DNA replication, were isolated and purified from soybean and turnip. The enzymes were characterized following ammonium sulfate precipitation, DEAF-cellulcse, phosphocelluloca, and hydroxylapatite chromatography, and by non-denaturing polyacrylamide gel electrophoresis. Protein banct were electropluted and the enzymes characterized using kinetic studies and sensitivity to divalent cations and inhibitors. Molecular weight and subunit composition studies indicated a molecular weight for the catalytic subunit of DNA polymerase alpha in soybean and turnip to be 46kDa. DNA polymeraqe gamma was composed of a catalytic subunit with a molecular weight of 66kDa. Although the two enzymes appear to share common subunits, characterization of their genetic origin remains to be determined before alpha and gamma can be classified as isoenzymes.
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EBERLE, FREDERIC. "Peut-on guerir la leucemie myeloide chronique par l'interferon-alpha ? recherche de la maladie residuelle par une technique d'amplification genique (polymerase chain reaction)." Aix-Marseille 2, 1993. http://www.theses.fr/1993AIX20903.
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Chen, Shuhua. "Multiple mechanisms regulate the human replication factors : replication protein A and DNA polymerase alpha-during DNA replication and DNA repair /." [S.l. : s.n.], 2003.
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DAMAGNEZ, VERONIQUE, and Paul R. Cohen. "La sous-unite catalytique de l'adn polymerase alpha de la levure schizosaccharomyces pombe : structure et regulation au cours du cycle cellulaire." Paris 6, 1992. http://www.theses.fr/1992PA066105.
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Pour etudier la replication de l'adn eucaryote et sa regulation au cours du cycle cellulaire, nous avons choisi comme systeme modele la levure schizosaccharomyces pombe. Nous avons sequence le gene poli de s. Pombe qui code pour la sous-unite catalytique de l'adn polymerase alpha. Ce gene, unique, est transcrit en un arn messager d'environ 4500 nucleotides. Il code pour une proteine de 1405 acides amines, de poids moleculaire calcule 160 kda, qui presente 7 blocs d'acides amines caracteristiques de l'une des grandes familles d'adn polymerases et 5 blocs specifiques des seules adn polymerases alpha eucaryotes. Des anticorps polyclonaux diriges contre cette proteine inhibent une partie de l'activite adn polymerase totale d'un extrait cellulaire de s. Pombe. Ils reconnaissent specifiquement un peptide de 170 kda present tout au long du cycle cellulaire, avec une augmentation de 3 a 4 fois en phases g1-s, et une forme modifiee: p165, presente uniquement en phases g1-s. Nous avons mis en evidence un gene situe en 5 et transcrit de facon divergente par rapport au gene poli. Ce gene: rpl7b, code pour un peptide predit qui presente de fortes homologies avec differentes proteines ribosomiques l7. Le gene rpl7b est fortement et constitutivement exprime. L'arn messager de poli est faiblement exprime et presente une augmentation de 3 a 4 fois en phases g1-s. Les 320 paires de bases qui separent les atg de rpl7b et poli doivent contenir les signaux responsables de la transcription de ces genes. Nos donnees ne permettent pas encore de trancher entre l'existence d'un promoteur bidirectionnel ou de deux promoteurs divergents. Cette etude devrait deboucher sur l'identification des signaux responsables de la regulation de la transcription du gene poli et sur la caracterisation des facteurs se fixant a ces signaux chez s. Pombe
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KOLINJIVADI, CHANDRA MOULI ARUM KUMAR. "DISSECTING THE ROLE OF BRCA2, RAD51 AND SMARCAL1 IN VERTEBRATE CHROMOSOMAL DNA REPLICATION." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/470900.
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DNA replication is a fundamental macromolecular event that is essential for cell division. During each cell cycle the entire genome has to be precisely duplicated to ensure genome integrity and stability. In the process of DNA replication, replication forks encounter endogenous and exogenous lesions and these lesions have to be rectified to transfer stable genetic material to daughter cells. Emerging evidences connected a role for Homologous Recombination (HR) proteins to replication fork protection during unperturbed DNA replication. Since HR protein, BRCA2 and Rad51 are essential for cell survival we used Xenopus laevis cell-free extract to dissect the function of BRCA2 and Rad51 during chromosomal DNA replication. Using Electron Microscopy (EM) we show that BRCA2 and Rad51 function together to prevent single-stranded DNA (ssDNA) accumulation at and behind the forks during unperturbed DNA replication. We further discovered that BRCA2 mediates interaction between Rad51, Polymerase alpha (α) and Polymerase delta (δ) and this interaction likely promote efficient re-priming and polymerising activity at stalled replication forks to prevent ssDNA accumulation. Moreover we show that inhibition of replicative polymerases in the absence of Rad51 results in increased frequency of replication fork reversal activity. We further found that replication fork reversal is predominantly induced by an annealing helicase SMARCAL1 in the absence of Rad51. Collectively our findings indicate that, to prevent ssDNA accumulation and aberrant replication fork architecture, timely re-priming of Polymerase alpha mediated by Rad51 is essential. Hence, loss of Rad51 impact replication fork architecture, eventually resulting in chromosomal abnormalities.
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Finney, Angela H. "Role of the C-terminal domain of the a subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/36285.
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Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, Vibrio fischeri. In V. fischeri, the luminescence or lux genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the lux operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the aCTD of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effect of seventy alanine substitution variants of the a subunit was determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by in vitro assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxRDN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDN, and fourteen residues in the aCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxRDN-dependent activation in vivo and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type aCTD is capable of interacting with the lux DNA fragment tested, all five of the variant forms of the aCTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that aCTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the lux operon during quorum sensing.Master of Science
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Liska, Olga. "Effect of CTCF and Cohesin on the dynamics of RNA polymerase II transcription and coupled pre-messenger RNA processing." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:ba9454b8-4498-42c8-bc4c-16dd971af164.
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The CCCTC-binding factor (CTCF) is a versatile, multifunctional zinc-finger protein involved in a broad spectrum of cellular functions. In mammalian cells, CTCF functions together with the Cohesin complex, an essential regulator of sister chromatid cohesion. Together, CTCF and Cohesin have been shown to regulate gene expression at a genome-wide level in mammalian cells. In the yeast Saccharomyces pombe, Cohesin has been implicated in transcription termination of convergently transcribed genes, in a cell cycle dependent manner. The aim of this thesis was to investigate the possibility of direct transcriptional involvement of CTCF and Cohesin in human cells. The first model system applied for this experimental purpose was the β-globin gene with introduced canonical CTCF-binding sites replacing the endogenous Co- Transcriptional Cleavage (CoTC) element downstream of β-globin. The results obtained indicate that recruitment of CTCF to the β-globin 3` flanking region does not prevent read-through transcription. However, CTCF-binding does mediate RNA Polymerase II (Pol II) pausing at the site of recruited CTCF. This results in more efficient pre-mRNA 3` end processing and therefore rescues β-globin mRNA to wild type levels. Cohesin was not detected at the introduced CTCF-binding sites. These results are a contribution to our understanding of the spatio-temporal requirements for cotranscriptional events like 3` end pre-mRNA processing and Pol II kinetics. The second part of my thesis presents an investigation on the involvement of CTCF and Cohesin in lipopolysaccharide (LPS)-induced Tumor Necrosis Factor α (TNFα) gene expression regulation in human monocytes and differentiated M1- and M2-type macrophages. These studies provide first evidence of Cohesin recruitment to the TNFα gene body and its regulatory NFκB-binding sites. Differences in the recruitment profiles obtained indicate potential regulatory differences of TNFα among the three cell types. Preliminary data provide an insight into the effects on TNFα mRNA levels upon down-regulation of Cohesin subunits.
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Esipenko, Nina A. "Design, Synthesis, and Application of Sensors for Biologically Relevant Molecules." Bowling Green State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1395589938.
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Attey, Adrian Gavin. "The direct observation and characterisation of the interaction between the #alpha#-subunit of the RNA-polymerase and the catabolite receptor protein in E. coli and its role in transcription activation." Thesis, University of York, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297310.
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Amaral, Paulo de Paiva Rosa. "Estudo da biossíntese e regulação de RNAs não-codificadores intrônicos em células humanas." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-03012007-000256/.
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Recentemente, tem sido demonstrado que a maioria dos RNAs transcritos em células humanas são RNAs não-codificadores de proteínas (ncRNAs) originados de íntrons ou regiões intergênicas. Em trabalhos anteriores realizados por nosso grupo, foram descritos longos ncRNAs transcritos de regiões intrônicas de genes codificadores e cuja expressão foi correlacionada ao grau de diferenciação de tumores de próstata, apontando para a relevância fisiológica desta classe de transcritos. Apesar de sua abundância, as propriedades, funções e regulação da grande maioria dos ncRNAs ainda não foram elucidadas. O objetivo do presente trabalho foi investigar a biossíntese de ncRNAs intrônicos em células humanas, primordialmente a contribuição da RNA Polimerase II (RNAP II), bem como aspectos de sua regulação. Primeiramente, o modelo de regulação da expressão gênica por hormônio andrógeno foi utilizado para avaliação da participação direta de um fator de transcrição de RNAP II, o Receptor de Andrógeno (AR), na modulação da transcrição de ncRNAs intrônicos. Utilizando-se a técnica de imunoprecipitação da cromatina, foi detectada a ligação do AR ao elemento de resposta a andrógeno (ARE) presente em um possível promotor de um transcrito intrônico antisenso (derivado do locus Myo5A), cuja expressão é aumentada em células da linhagem LNCaP tratadas com o hormônio. A ligação ao ARE foi induzida pelo tratamento, sugerindo que o efeito do andrógeno na expressão do ncRNA é mediado pelo AR. Em uma segunda abordagem, o efeito da inibição da transcrição por RNAP II com α-amanitina por 24 h em células LNCaP foi avaliado com o uso de microarranjos de oligonucleotídeos representando transcritos total ou parcialmente intrônicos, além de éxons de genes codificadores. A expressão de menos de 20 % dos transcritos intrônicos foi afetada, fração significativamente menor que a observada para os transcritos exônicos (40 %). Ainda que a maioria dos ncRNAs intrônicos diferencialmente expressos tenha sua abundância diminuída, interessantemente, 13 a 16 % foram aumentados, contrastando com aproximadamente 2 a 3 % de exônicos que aumentaram. Os resultados obtidos neste trabalho indicam que a RNAP II atua na transcrição de ncRNAs intrônicos, mas que uma fração considerável pode ser transcrita por outra RNA Polimerase.It has been recently shown that the bulk of the transcription in human cells is comprised of non-protein-coding RNAs (or noncoding RNAs - ncRNAs) transcribed from introns and intergenic regions of the genome. Previous work from our group has demonstrated that expression of long intronic ncRNAs can be correlated to the degree of prostate tumor differentiation, underscoring the physiological relevance of these transcripts. However, the properties, functions, and regulation of this huge population of ncRNAs remain largely unknown. The present work aimed to investigate the biosynthesis of intronic ncRNAs and aspects of its regulation in human cells, focusing on the contribution of RNA Polymerase II (RNAP II). Initially, the model of regulation of gene expression by androgen hormone was used in order to evaluate the participation of the RNAP II transcription factor Androgen Receptor (AR) in the transcriptional regulation of intronic ncRNAs. Chromatin immunoprecipitation experiments revealed the binding of the AR in an androgen response element (ARE) present in a putative promoter driving the expression of an antisense intronic transcript in Myo5A locus in LNCaP cells. The interaction occurred in an androgen-inducible fashion, along with the up-regulation of the transcript, suggesting that hormone activation occurred in a direct manner mediated by the AR. In a different approach, the effect of RNAP II inhibition with α-amanitin for 24 h in LNCaP cells was analyzed using an oligoarray representing totally and partially intronic transcripts, as well as exons of proteincoding genes. The expression of less than 20 % of the intronic transcripts was affected by the treatment, contrasting to a significantly higher fraction observed for exonic messages (40 %). Moreover, most differentially expressed intronic transcripts were down-regulated, but strikingly 13 to 16 % were up-regulated in cells with blocked RNAP II, while this fraction for exonic transcripts was about 2 %. The results described here demonstrate that RNAP II in fact plays a role in intronic transcription in human cells, but also highlight that another transcriptional system may account for the biogenesis of a fraction of intronic ncRNAs.
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Penha, Marcelo De Luca. "Detecção dos genes das toxinas alfa, beta e épsilon de Clostridium perfringens isolados a partir de amostras clínicas de bovinos pela reação em cadeia da polimerase." Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-06072005-101119/.
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O Clostridium perfringens é um microrganismo anaeróbio que está presente no solo e no trato intestinal dos mamíferos. Provoca intoxicação alimentar nos seres humanos, doenças enterotoxêmicas nos animais domésticos e gangrena gasosa em ambos os grupos. O C. perfringens é classificado em cinco tipos (A, B, C, D e E) mediante a produção de quatro toxinas principais (alfa, beta, épsilon e iota). Neste trabalho foi possível padronizar a técnica de PCR para detectar a presença dos genes cpa, cpb e etx a partir de culturas de C. perfringens. A sensibilidade analítica da técnica de PCR a partir de culturas de C. perfringens foi de 2,27 ng/µL para o gene cpa, 22,7 pg/µL para o gene cpb e 22,7 pg/µL para o gene etx. A pesquisa dos genes cpa, cpb e etx partir de 35 amostras de C. perfringens isoladas de bovinos revelou que 16 (45,7%) eram do tipo A; 18 (51,4%) eram do tipo C e 1 (2,9%) era do tipo B. Não foi observada nenhuma amostra do tipo D. A metodologia de PCR revelou-se útil na tipificação de amostras de C. perfringens isoladas de bovinos, contribuindo para o diagnóstico dessa bacteriose neste país, eliminando as dificuldades de tipificação oriundas do alto custo e da indisponibilidade de anti-soros para a tipificação pela reação de soroneutralização e evitando a utilização de animais de laboratório.Clostridium perfringens is an anaerobic micro-organism that is present in the soil and gastrointestinal tract of mammals. It causes food poisoning in humans, enterotoxemic diseases in domestic animals and gas gangrene in both. C. perfringens is classified into five types (A, B, C, D and E) according to the production of four major toxins (alpha, beta, epsilon and iota). In this trial was possible to standardize the PCR?s technique to detect cpa, cpb and etx genes from cultures of C. perfringens. PCR?s analythical sensibility was 2.27 ng/µL for cpa gene, 22.7 pg/µL for cpb gene and 22.7 pg/µL for etx gene. The research of cpa, cpb and etx genes from 35 samples of C. perfringens isolated from cattle reveals that 16 (45.7%) were classified as type A, 18 (51.4%) as type C and 1 (2.9%) as type B. No sample of type D was observed. PCR?s technique reveals to be usefull to typify samples of C. perfringens isolated from cattle, contributing to diagnose of this bacterial disease in this country and solving typifing problems represented by the high costs of the process and by the lack of antiserum that is required to typify the micro-organism by seroneutralization. PCR?s technique avoid the use of laboratory animals, too.
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Wongsriraksa, Anong. "Characterisation of nicotine binding sites on human blood lymphocytes." Thesis, University of Hertfordshire, 2008. http://hdl.handle.net/2299/1960.
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Nicotine exerts a therapeutic effect in ulcerative colitis (UC) but the mechanism underlying this effect, is not clear. However, this effect may imply that nicotine has some, as yet to be discovered, effect on the immune system. The aim of the work described in this thesis was to characterise the nicotinic acetylcholine receptors (nAChRs) on human peripheral blood lymphocytes in term of receptor subtype. To achieve this, a combination of radioligand binding assays, pharmacological and molecular biological techniques were used. The data obtained from the binding studies suggested that the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes with a Kd 15 ± 5.759 nM (1.5 ± 5.759 x 10-8 M) and Bmax 2253 ± 409 sites/cell. The competition studies showed that ligands competing with [3H]-(-)-nicotine were (-)-nicotine, epibatidine and α-bungarotoxin, while others ligands for nAChRs displaced radiolabelled nicotine in insignificant quantities. Thus, radioligand-binding experiments suggest that the binding site for nicotine on human peripheral blood lymphocytes is a nAChR containing α7 and possibly α4 or/and b2 containing nAChR subunits. No evidence was obtained to suggest the presence of a non-cholinergic nicotine receptor. Furthermore, considerable subject to subject variation in the specific binding of radiolabelled nicotine was observed. Because of this only tentative conclusions could be drawn from radioligand binding data. Polymerase chain reaction (RT-PCR) was then used to demonstrate mRNA for the subunits of nAChRs suggested by radioligand binding studies. Data obtained show that the human peripheral blood lymphocytes tested, expressed mRNAs for α4, α5, α7, β2 neuronal nAChRs subunits and β1 muscle nAChR subunit. Expression of the α5 mRNA subunit of nAChR was observed in the lymphocytes in each sample of lymphocytes tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between individuals. Finally, Western blot analysis was used to confirm that mRNA expression resulted in the expression of protein for nAChR subunits in human peripheral lymphocytes using monoclonal antibodies against α4, α5, α7, and β2 nAChR subunits, which had been detected by RT-PCR. The results obtained from the Western blot analysis show that protein for α4, α5, and α7 nAChR subunits was expressed in most, but not all of the human peripheral blood lymphocyte samples tested and some of the bands obtained were faint. In contrast, protein for the β2 nAChR subunit was observed in a few samples tested and the bands were faint. From the results obtained in this study, it is possible to conclude that human peripheral blood lymphocytes may contain nAChRs with subunit compositions of α4β2, α4β2α5, and/or α7. However, further studies are necessary to show whether or not the single binding site for nicotine demonstrated by radioligand binding experiments is due to one or all of these nAChRs. Thus, the findings of the present study suggest the presence of nAChR on human peripheral blood lymphocytes. Nicotine and its effect may occur through these non- neuronal nAChRs mechanisms. Such a mechanism of action could account for the beneficial of nicotine in ulcerative colitis. Furthermore, a compound that acts on these receptors, but not on nAChRs found on other cells may have therapeutic utility in the treatment of inflammation.
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Conroy, M. "Synthesis of #alpha#, #omega# functionalised oligomers." Thesis, Lancaster University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240508.
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Zhang, Fan. "Synthesis of organometallic foldamers and cyclopropene alpha-amino acids." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 211 p, 2006. http://proquest.umi.com/pqdweb?did=1172109511&sid=6&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Laguerre, Albert. "Oligomerisation et fonctionnalisation en serie methacrylique : greffage d'acides alpha amines." Le Mans, 1987. http://www.theses.fr/1987LEMA1017.
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Oligomerisation anionique du methacrylate de methyle. Modification des fonctions ester en fonctions plus reactions (alcool, chloroformiate, acide). Greffage d'aminoacides dont une seule fonction est libre pour eviter leur oligocondensation. Caracterisations des produits obtenus par rmn**(13)c, **(1)h et ir. Utilisation de ces produits pour la synthese de polyamides et de polyurethannes optiquement actifs
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Mela, Marianna. "A pathogenic role for alpha-1-antitrypsin polymers in liver injury." Thesis, University of Cambridge, 2016. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709473.
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Kluzek, Monika. "Lipid membrane alteration under exposure to alpha-cyclodextrins and pH-responsive pseudopeptide polymers." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAE045/document.
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Le développement de nanotransporteurs basés sur des lipides, des polymères et des nanoparticules avec des propriétés «sur mesure» pour augmenter l’efficacité de médicaments, fait l’objet de recherches intensives. Toutefois, la physico-chimie subtile des intéractions polymères-lipides and nanoparticules-lipides présente encore de larges domaines mal compris et de nombreuses questions sans réponse. Ce projet de recherche doctoral utilise des techniques de visualisation (Cryo-MET, LSCM), et de caractérisation (ITC, DSC, SAXS, SANS, QCM-D) avancées pour obtenir des informations nouvelles sur les mécanismes d’interaction entre des Cyclodextrines-α d’autre part, des polymères sensibles au pH d’autre part, et des bicouches modèle de DOPC. La forte influence de ces deux composés sur ces systèmes modèle élucide certains aspects relatifs à la toxicité vis-à-vis des membranes biologiques et suggère de nouvelles approches pour des applications pharmaceutiquesThe primary goal of nanomedicine is to improve clinical outcomes. To this end, the development of nanocarriers based on lipids, polymers and nanoparticles with tailor-made properties that enhance the in vivo potency of drugs is a subject of intense research. However, the subtle physical-chemistry of the polymer-lipid and nanoparticle-lipid interactions still present many poorly understood fields of investigation as well as unanswered questions. This doctoral research project utilizes state-of-the-art visualization (Cryo-TEM, LSCM) and characterization (ITC, DSC, SAXS, SANS, QCM-D) techniques to gain novel insights into the interaction between α-Cyclodextrins in the first hand, a pH-responsive polymer in the other hand, and model DOPC bilayers. The strong influence of both compounds on these model systems elucidate some aspects regarding biological membrane toxicity and suggests novel strategies for pharmaceutical applications
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Kaufmann, Martin. "Lipid Bilayers Supported by Multi-Stimuli Responsive Polymers." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-106231.
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Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding.
The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length.
These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin.
The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions.
The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer.
On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.
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Boury, Frank. "Influence des propriétés de surface de microsphères de poly(alpha-hydroxyacides) sur l'adsorption de la BSA." Paris 11, 1995. http://www.theses.fr/1995PA114801.
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Sormani, Patricia M. "The ring-opening polymerization of cyclosiloxanes in the presence of bis ([alpha], [omega]-aminopropyl)-1,3-tetramethyldisiloxane." Diss., Virginia Polytechnic Institute and State University, 1986. http://hdl.handle.net/10919/71193.
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The ring-opening polymerization of octamethylcyclotetrasiloxane, D₄ , in the presence of bis(α,ω-aminopropyl)-1,3-tetramethyldisiloxane using potassium siloxanolate and tetramethylammonium siloxanolate catalysts has been investigated. The use of reversed-phase high performance liquid chromatography (HPLC) and capillary gas chromatography (GC) allowed the disappearance of the starting materials to be monitored as a function of reaction temperature, time, targeted molecular weight, catalyst type and concentration. Due to electronegativity differences, the cyclic tetramer was found to react more quickly than the disiloxane under all conditions studied. This work was extended to the study of polydimethyl-co-diphenylsiloxane oligomers, prepared by the ring-opening copolymerization of D₄ with octaphenylcyclotetrasiloxane, D₄". Reversed-phase HPLC was used to study the disappearance of the cyclic starting materials. Due to volatility considerations these oligomers were not analyzed by capillary GC. ²⁹Si NMR was used to determine the number-average sequence length of each type of siloxane unit as a function of reaction conditions. The co-oligomer composition played the greatest role in determining the average sequence lengths. Oligomers with close to a 50/50 molar composition of dimethyl and diphenyl units showed a tendency towards an alternating distribution, while oligomers with an ≃27/83 molar composition displayed a tendency towards blockiness.
A series of polyester-siloxanes was prepared using both bulk and solution polycondensation techniques. Copolymers based on polybutylene terephthalate were highly insoluble, due to the presence of crystallinity in the systems. The incorporation of some isophthalate linkages increased the solubility of the polymer products, making the solution polymerization technique possible.Ph. D.
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Roth, Peter J. [Verfasser]. "Alpha, Omega end group functionalization of RAFT polymers based on pentafluorophenyl esters and methane thiosulfonates / Peter J. Roth." Mainz : Universitätsbibliothek Mainz, 2011. http://d-nb.info/1025062914/34.
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Gonçalves, Josiane de Oliveira. "Comparação entre os resultados da expressão gênica da desmina, alfa-actina e TGF-beta1 obtidos a partir dos métodos da reação em cadeia de polimerase via transcriptase reversa (RT-PCR) semiquantitativa e em tempo real (qRT-PCR) no modelo." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5141/tde-15082014-163708/.
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Os mecanismos responsáveis pela fibrose hepática na infância são pouco conhecidos. Crianças com atresia das vias biliares, quando submetidas a portoenterostomia a Kasai com sucesso, se tornam anictéricas mas mesmo assim desenvolvem cirrose a longo prazo. Da mesma forma, a ocorrência de estenoses biliares segmentares intra-hepáticas no pós-operatório de transplante hepático podem levar ao desenvolvimento de cirrose em todo o órgão. Tais fatos sugerem que mecanismos endócrinos ou parácrinos estejam envolvidos na fibrogênese hepática. Para elucidar este processo o modelo de ligadura seletiva das vias biliares em ratos jovens foi desenvolvido em nosso laboratório. Usando este modelo, identificamos mudanças na expressão do gene da alfa-actina de músculo liso, tanto no parênquima hepático obstruído como no parênquima hepático adjacente à obstrução. No entanto, o perfil de expressão gênica da desmina, uma proteína presente em níveis elevados durante a ativação das células estreladas hepáticas e o TGF-beta1, principal citocina pró-fibrogênica, não demostraram diferenças significantes quando analisados pelo método do RT-PCR semiquantitativo. Assim, os mecanismos moleculares envolvidos na modulação da fibrogênese hepática nesse modelo experimental não estão totalmente compreendidos. A metodologia do qRT-PCR (PCR em tempo real), têm sido previamente descrita como um método mais preciso e sensível, possibilitando a detecção do aumento do número de cópias do gene à medida que ocorre a amplificação, enquanto que o método do RT-PCR semiquantitativo a análise dos transcritos só é realizada após a etapa da amplificação. O objetivo deste estudo foi avaliar as alterações moleculares no modelo experimental de ligadura seletiva das vias biliares e comparar os resultados obtidos entre os métodos do RT-PCR semiquantitativo e do qRT-PCR em tempo real. Foi realizada a ligadura seletiva do ducto biliar em ratos Wistar com 21 dias de vida, os grupos foram separados de acordo com o momento da morte: 7 ou 60 dias após a cirurgia. A expressão da desmina, alfa actina de músculo liso e TGF-beta1 foi avaliada no tecido do parênquima hepático com obstrução biliar (ducto ligado - DL) e no parênquima hepático adjacente à obstrução biliar (ducto não ligado - DNL) usando o RT-PCR semiquantitativo e o qRT-PCR em tempo real. A metodologia do qRT-PCR em tempo real permitiu identificar mudanças no perfil de expressão gênica que não foram demonstrados pelo método semiquantitativo. O parênquima DL mostrou reação fibrogênica mais intensa, com aumento na expressão da alfa actina de músculo liso e TGF-beta1 após 7 dias. O parênquima DNL apresentou resposta fibrogênica tardia, com aumento da expressão da desmina 7 dias e 60 dias após a cirurgia, além de aumento da alfa-actina de músculo liso 60 dias após a cirurgia. O qRT-PCR em tempo real apresentou maior sensibilidade para identificar mudanças no perfil de expressão gênica em relação ao método convencional do RT-PCR semiquantitativo. Nossos resultados ajudam a esclarecer a dinâmica das alterações moleculares envolvidas na modulação da fibrogênese hepática no modelo experimental de ligadura seletiva do ducto biliar e pode ser diretamente aplicado para o estudo de estenoses biliares intra-hepáticas e atresia das vias biliaresThe mechanisms responsible for liver fibrosis in childhood are poorly understood. Children suffering from biliary atresia, when submitted to the successful Kasai portoentostomy, become anicteric, but nonetheless develop cirrhosis in the long term. Similarly, the occurrence of intrahepatic biliary stenosis in the postoperative period of liver transplantation may lead to cirrhosis of the whole organ. Such facts suggest that endocrine or paracrine mechanisms are involved in hepatic fibrogenesis. To elucidate this process, the selective bile duct ligation model in young rats was developed in our laboratory. Using this model, we identified changes in the expression of smooth muscle alpha-actin both in the obstructed parenchyma and the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin, a protein present at high levels during activation of hepatic stellate cells and TGF-beta1, the main pro-fibrogenic cytokine, were unchanged when analyzed with semiquantitative RT-PCR. Thus, the molecular mechanisms involved in the modulation of hepatic fibrogenesis in this experimental model are not fully understood. The methodology of qRT-PCR (real time PCR) has previously been described as a more precise and sensitive method, allowing the detection of increased copy number of the gene while amplification occurs, whereas by semiquantitative RT-PCR analysis transcripts is only perfomed after the amplification step. This study aimed to evaluate the molecular changes in experimental model of selective bile duct ligation and compare the results between semiquantitative RT-PCR and real-time qRT-PCR methods. Selective biliary duct ligation was performed on Wistar rats with 21 days of life, the groups were separated according to the moment of death: 7 or 60 days after surgery. The expression of desmin, alpha-actin smooth muscle and TGF-beta1 was examined in tissue from hepatic parenchyma with biliary obstruction (duct ligation - DL) and in the adjacent hepatic parenchyma (duct non-ligated - DNL) using semiquantitative RT-PCR and real-time qRT-PCR. The methodology of the real-time qRT-PCR allowed to identify changes in gene expression profile that were not shown by semiquantitative method. The DL parenchyma showed a more severe fibrogenic reaction, with increased alpha-actin smooth muscle and TGF-beta1 expression after 7 days. The DNL parenchyma presented a later fibrotic response, with increased desmin expression 7 and 60 days after surgery, besides of increased alpha-actin smooth muscle 60 days after surgery. Real-time qRT-PCR was more sensitive to identify changes in gene expression profile comparated to the semiquantitative method. Our results help to clarify the dynamic of molecular changes involved in the modulation of hepatic fibrogenesis in an experimental model of selective bile duct ligation and can be directly applied to the study of intrahepatic biliary stenosis and biliary atresia
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GHARBI, RI DHA. "Synthese et caracterisation de polymeres vinyliques porteurs de groupes lateraux alpha-amines et carboxyliques libres derives d'azalactones et de l(+)-lysine." Paris 6, 1990. http://www.theses.fr/1990PA066522.
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Deux voies d'acces a de nouveaux polymeres vinyliques fonctionnels porteurs de groupements lateraux libres amines et acides carboxyliques en position alpha ont ete explorees. Dans une premiere voie, la glycine aux fonctions protegees sous la forme azalactone, est condensee avec le formyl-4 styrene. La polymerisation radicalaire des monomeres derives; la phenyl-2 (a)/ou methyl-2 (b) (vinyl-4 benzylidene)-4 oxazolone-5 (4h) et l'acide vinyl-4 alpha-acetaminocinnamique (c), a donne de faibles rendements sans doute a cause de la grande conjugaison de leurs systemes. Cependant les solutions du poly(c) donne les meilleurs dpn. Au cours de la copolymerisation avec le mma et le styrene une bonne incorporation de nos monomeres est observee. Une polymerisation non desiree des liaisons cinnamique et benzylidene est soupconnee etre a l'origine de la mauvaise solubilite de ses polymeres. Par une deuxieme voie de synthese, nous avons fixe un motif l-lysine. La n epsilon-(vinyle-4 benzyl)l-lysine (d) et la n epsilon-(vinyl-4 benzoyl)-l-lysine (e), obtenues respectivement par condensation de la l-lysine (protegee sous sa forme complexee par le cuivre) avec le formyl-4 styrene et le chlorure de l'acide vinyl-4 benzoique, sont polymerises radicalairement a leur tour. La condensation des polymeres obtenus met en evidence sur une chaine vinylique la presence des motifs aminoacides libres pendants sous forme neutre et/ou ionisee. Ces polymeres sont cristallins et optiquement actifs
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Subedi, Prabal [Verfasser], Katrin [Gutachter] Marcus, and Dirk [Gutachter] Wolters. "Use of molecularly imprinted polymers for enrichment of phosphopeptides and alpha-Synuclein, and the mass spectrometric analyses of alpha-Synuclein in \(\it post-mortem\) human brains / Prabal Subedi ; Gutachter: Katrin Marcus, Dirk Wolters." Bochum : Ruhr-Universität Bochum, 2016. http://d-nb.info/1123283117/34.
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Baki, Mert. "Bone Marrow Targeted Liposomal Drug Delivery Systems." Master's thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12613251/index.pdf.
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Homing is the process that stem cells move to their own stem cell niches under the influence of chemokines like stromal-derived factor-1&alpha(SDF-1&alpha
) upon bone marrow transplantation (BMT). There is a need for increasing homing efficiency after BMT since only 10-15% of the transplanted cells can home to their own niches and a limited amount of donor marrow can be transplanted. In this study, we aimed to develop and characterize bone marrow targeted liposomal SDF-1&alpha
delivery system prepared by extrusion method. Alendronate conjugation was chosen to target the liposomes to bone marrow microenvironment, particularly the endosteal niche. Optimization studies were conducted with the model protein (
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Holden, Gavin. "Cross-linking of polyethylene and ethylene/ #alpha#-olefin copolymers, initiated by dicumyl peroxide : a study of new polymers and the effects of molecular structure." Thesis, Lancaster University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322818.
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Garcia, Claudio J. "Investigation of single molecule and monolayer properties with Monte Carlo simulations of a coarse-grained model for alpha-sexithiophene." University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron1511314081783002.
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EL, MOUSTAFID TOURIYA. "Synthese electrochimique, proprietes physico-chimiques et caracterisation spectroscopique de nouveaux polymeres conducteurs solubles : poly(dialcoxy-2,5 phenylenes) et poly(alpha et beta-adamantyl-oxo-ethoxy naphtalenes)." Paris 7, 1991. http://www.theses.fr/1991PA077230.
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Nous avons effectue dans ce travail la synthese electrochimique, l'etude des proprietes physico-chimiques et la caracterisation de nouveaux polymeres conducteurs solubles: les poly(dialcoxy-2,5 phenylenes) (pdap) et les poly(alpha ou beta adamantyl-1(oxo-2ethoxy) naphtalene (padn). Ces polymeres ont ete prepares par oxydation electrochimique des monomeres correspondants. Leur solubilite augmente avec la longueur de la chaine alcoylee du groupe alcoxy porte par des noyaux benzeniques ou naphtaleniques et elle est plus elevee quand le substituant comprend un groupe adamantyle. Le degre de polymerisation qui a ete determine par chromatographie par permeation de gel est de 10 a 12 motifs pour les pdap et il est inferieur a 8 motifs pour les padn. La conductivite de ces polymeres ne depasse pas 10##3 s/cm malgre la regularite des enchainements. L'application de diverses methodes spectroscopiques (ir, uv, fluorescence, rmn) a l'etude de ces composes a permis de montrer que l'enchainement etait de type para-para dans les pdap. Pour le poly(beta-adn) on a mis en evidence un enchainement en 1,4 tandis que pour le poly(alpha-adn) on a trouve un melange de structures sur le plan mecanistique, on a pu montrer que la reaction de couplage necessitait la formation d'un intermediaire dicationique
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Oberholzer, Ian Dewald. "Evaluation of the enzyme inhibitory effect of carboxymethylated chitosan / Ian Dewald Oberholzer." Thesis, North-West University, 2003. http://hdl.handle.net/10394/245.
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Degradation of peroral administered drugs by various enzymes in the gastrointestinal
tract has proven to be troublesome for the absorption' and bioavailability of protein and
peptide drugs. Mucoadhesive polymers such as poly(acrylates) have proven to inhibit
protease enzymes responsible for initiating digestion of peptide drugs. Enzyme inhibitors
have unique chemical properties enabling it to interact with enzymes to form complexes
with such enzymes prohibiting it from functioning properly. Anionic carboxymethylated
chitosan derivatives such as N,N-dicarboxymethyl chitosan and N, O-carboxymethyl
chitosan display unique structural similarities to enzyme inhibitors being anionic
polymers that may interact with bi-valent cations...Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.
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Piras, Antioco. "Etude par résonnance magnétique nucléaire haute résolution en solution de la structure des polyesters insaturés." Paris 6, 1988. http://www.theses.fr/1988PA066483.
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Preparation et caracterisation par rmn a haute resolution d'alpha -omega -dihydroxy polyesters insatures modeles par rmn bidimensionnelle: mise en evidence de 3 types de branchements: cours, longs, cycliques. Preparation de polyesters lineaires par synthese en solution ou a partir de diacetates, reactions non adaptables industriellement sans modification des procedes; mise au point d'une technique de dosage de la reaction de saturation des produits industriels et etude des parametres influant sur cette reaction
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Rose, Marcus, Andreas Notzon, Maja Heitbaum, Georg Nickerl, Silvia Paasch, Eike Brunner, Frank Glorius, and Stefan Kaskel. "N-Heterocyclic carbene containing element organic frameworks as heterogeneous organocatalysts." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-138636.
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A bifunctional imidazolium linker was used for the production of highly crosslinked element organic frameworks by Suzuki-coupling with tetrafunctional boronic acids. The resulting porous materials are good heterogeneous organocatalysts in the N-heterocyclic carbene-catalyzed conjugated umpolung of α,β-unsaturated cinnamaldehydeDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Rose, Marcus, Andreas Notzon, Maja Heitbaum, Georg Nickerl, Silvia Paasch, Eike Brunner, Frank Glorius, and Stefan Kaskel. "N-Heterocyclic carbene containing element organic frameworks as heterogeneous organocatalysts." Royal Society of Chemistry, 2011. https://tud.qucosa.de/id/qucosa%3A27766.
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A bifunctional imidazolium linker was used for the production of highly crosslinked element organic frameworks by Suzuki-coupling with tetrafunctional boronic acids. The resulting porous materials are good heterogeneous organocatalysts in the N-heterocyclic carbene-catalyzed conjugated umpolung of α,β-unsaturated cinnamaldehyde.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Bianco, André de Macedo. "Expressão de CXCR7 e CXCR4 em em astrocitomas iniltrativos em relação ao tecido cerebral não neoplásico e sua interação com HIF1alfa e IDH1." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/5/5138/tde-01112013-122908/.
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Introdução: Existem dados suficientes disponíveis demonstrando a importância da quimiocina CXCL12 e seu receptor CXCR4 na progressão tumoral e angiogênese dos gliomas. O CXCR4 é regulado positivamente pelo HIF1alfa. Recentemente um novo receptor com maior afinidade à CXCL12 foi identificado, o receptor órfão RDC1, agora denominado CXCR7. O objetivo deste estudo é investigar a expressão de mRNA CXCR7 em tecidos astrocitomas difusos e avaliar suas interações com expressão CXCR4 e HIF1alfa, bem como analisar sua relação com mutação do IDH1. Métodos: A expressão do CXCR7, CXCR4, IDH1 e HIF1alfa foram avaliadas por PCR quantitativo em tempo real (qRT-PCR) em 129 amostras congeladas de astrocitomas (25 astrocitoma difuso - AGII, 18 de astrocitoma anaplásico - AGIII e 86 glioblastoma - GBM) e 22 amostras de tecido cerebral não neoplásico (NN) obtidos de cirurgia de epilepsia. A mutação do IDH1 previamente determinada foi analisada em relação aos níveis de expressões de mRNA do CXCR7, CXCR4 e HIF1alfa, combinado com os parâmetros clínico-patológicos e sobrevida global. Adicionalmente, a expressão proteica do CXCR7 foi analisada por imuno-histoquímica em astrocitomas de diferentes graus e em linhagem celular de glioma (U87MG) por microscopia confocal. Resultados: Houve diferença significativa nos níveis de expressão dos genes estudados entre astrocitomas e NN (p < 0,001). Na análise da expressão gênica associada nos AGII não se observou correlação entre os níveis de expressão de CXCR7/HIF1alfa (p = 0,548); observou-se correlação significativa entre CXCR7/IDH1 (p < 0,001) e CXCR7/CXCR4 (p = 0,042). Nos GBM houve correlação significativa entre CXCR7/CXCR4 (p = 0,002), CXCR7/IDH1 (p < 0,001) e CXCR7/HIF1alfa (p = 0,008). Hiperexpressão do HIF1alfa foi associado com maior expressão do CXCR7 e CXCR4 (p = 0,001), enquanto a presença de IDH1 mutado foi associada a menor expressão de mRNA do CXCR7 e CXCR4 (p = 0,009). A expressão proteica de CXCR7 foi identificada em todas as amostras estudadas, e aumentou com malignidade. A proteína CXCR7, na linha celular U87MG, foi localizada principalmente na membrana celular. Conclusão: O CXCR7 é um gene diferencialmente expresso em astrocitomas difusamente infiltrativos em relação tecido cerebral não neoplásico. O nível de expressão do CXCR7 correlacionou-se significativamente com os níveis de expressão do CXCR4 e IDH1 nos AGII e com CXCR4, IDH1 e HIF-1alfa nos GBM. O nível de expressão elevado do CXCR7 e CXCR4 correlacionou-se com nível elevado de expressão de HIF-1a, enquanto a presença da mutação do IDH1 associou-se a níveis reduzidos de CXCR7 e CXCR4. Não se observou associação significativa entre os níveis de expressão de CXCR7 e CXCR4 com os dados de sobrevidaIntroduction: There is abundant evidence showing that chemokine CXCL12 and its receptor CXCR4 are involved in glioma progression and angiogenesis. CXCR4 is upregulated by HIF1alfa. The CXCR7, a recent additional receptor for CXCL12 with higher affinity than CXCR4 has raised key issues on glioma cell migration. The aim of this study is to investigate the CXCR7 mRNA expression in diffuse astrocytoma tissues and to evaluate its interactions with CXCR4 and HIF1alfa expression and IDH1 mutation. Methods: CXCR7, CXCR4, IDH1 and HIF1alfa expressions were evaluated by quantitative real-time PCR (qRT-PCR) in 129 frozen samples of astrocytoma (25 diffuse astrocytomas - AGII, 18 anaplastic astrocytomas - AGIII and 86 glioblastomas - GBM) and 22 samples of non-neoplastic tissue cerebral (NN) from epilepsy surgery. IDH1 mutation status was analyzed with CXCR7, CXCR4 e HIF1alfa mRNA expressions, matched with clinicopathological parameters and overall survival time. Furthermore, CXCR7 protein expression was analyzed by immunohistochemistry in different grades of astrocytoma and in glioma cell line (U87MG) by confocal microscopy. Results: There was significant difference in the expression levels of the genes studied between astrocytomas and NN (p < 0.001). The analysis of associated gene expressions in AGII showed no significant correlation between CXCR7/HIF1alfa (p = 0.548); there was significant correlation between CXCR7/CXCR4 (p = 0.042) and CXCR7/IDH1 (p = 0.008). In GBM, there were significant correlations between CXCR7/CXCR4 (p = 0.002), CXCR7/IDH1 (p < 0.001) and CXCR7/HIF1alfa (p = 0.008). HIF1alfa overexpression was associated with higher expressions of CXCR7 and CXCR4 (p = 0.001), while presence of IDH1 mutation was associated with lower CXCR7 and CXCR4 mRNA expressions (p = 0.009). Protein expression was identified in all samples studied, and it increased with malignancy. CXCR7 protein, in U87MG cell line, was mainly localized in the cellular membrane. Conclusion: CXCR7 was overexpressed in astrocytoma of different grades of malignancy compared to non-neoplastic brain tissue. CXCR7 expression levels correlates with CXCR4 and IDH1 in AGII and CXCR4, IDH1 and HIF1alfa in GBM. Overexpression HIF1alfa was related with higher expressions of CXCR7 and CXCR4, otherwise presence of IDH1 mutation related with lower expression of both genes. Protein expression level was associated with the degree of malignancy. The results revealed no significant association between CXCR7 and CXCR4 expression and survival data
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Leblanc, Jean-Pierre. "Synthese et caracterisation de polycondensats renfermant des motifs stilbeniques." Paris 6, 1988. http://www.theses.fr/1988PA066350.
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Polycondensation de l'acide trans stilbenedicarboxylique-4,4' (soa) avec des alpha ,omega -diamino alcanes et des alpha ,omega -diamino oligoethers. Etude de l'incorporation du soa dans des copolyarylates comme le poly(isophtalate de methyl phenylene) en vue d'obtenir des polymeres cristaux liquides, et des polyesters obtenus a partir d'autres monomeres (acide terephtalique). Amelioration des proprietes mecaniques des polycondensats par incorporation d'unites stilbeniques et d'acide p-acetoxybenzoique
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Karaderi, Tugce. "Genetics of ankylosing spondylitis." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:8c0e848a-e712-4603-b923-a96a2f1644ac.
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Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/TH17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.
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Al-Amodi, Amani. "Expression, Purification and Characterization of Human DNA Polymerase Alpha." Thesis, 2021. http://hdl.handle.net/10754/669018.
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DNA replication is a fundamental process in all living organisms. It is a semi- discontinuous process in which the leading strand is synthesized continuously and the lagging strand is synthesized discontinuously as short Okazaki fragments (OF). The initiation of DNA synthesis requires DNA polymerase α (Pol α/primase) in complex with the primase to form a complex of four subunits. Pol α/primase is the only enzyme that can perform de novo DNA synthesis on single-stranded DNA. The catalytic subunit of the primase (PRIM1) synthesizes RNA primers that are approximately nine nucleotides long. The synthesized RNA primers are then passed intramolecularly to the polymerase active site (POLA1), which is thought to be mediated by the C-terminal domain of the primase large subunit (PRIM2-C) to synthesize dNTPs of approximately 20 nucleotides. The aim of this project was to optimize the expression and purification of Pol α/primase. The insect codon optimized POLA1 was C-terminally Strep tagged and transposed into the baculovirus genome. The other subunits of Pol α/primase, POLA2, PRIM1 and PRIM2 were cloned and expressed in E. coli cells. The cell lysates from Sf9 insect cells and E. coli cells were then mixed and purified by immunoaffinity chromatography and size-exclusion chromatography. This helped us achieve a pure Pol α/primase containing all the four subunits with a good total yield. The identity of all the protein bands were verified by mass spectroscopy. Furthermore, the protein demonstrated primer extension activity on multiple primer/template substrates. We also characterized the effect of the human replication protein A (RPA) on the DNA polymerization activity of Pol α/primase.