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Saxena, Manvendra, and s3031657@student rmit edu au. "Utilising salmonella to deliver heterologous vaccine antigen." RMIT University. Applied Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080522.095907.
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Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.
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Pinel, Karine. "Imagerie in vivo du contrôle de l’inhibition génique et de l’électroporation d’ARN." Thesis, Bordeaux 2, 2012. http://www.theses.fr/2012BOR22004/document.
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Ces travaux de thèse d’imagerie moléculaire et translationnelle proposent, sur des modèles murins, deux approches innovantes pour les thérapies géniques. La plupart des cancers sont associés à des dérégulations de l’expression génique et certains gènes sont surexprimés. L’utilisation de microARN (miARN) permet d’envisager une réduction de l’expression d’un gène spécifique mais il est nécessaire de limiter cette inhibition au tissu pathologique. L’utilisation des promoteurs thermo-inductibles couplés à un dépôt local de chaleur autorise un contrôle spatial et temporel de l’expression génique in vivo. Notre projet a été de coupler le contrôle spatio-temporel et l’inhibition d’un gène cible. A cette fin, un miARN synthétique a été placé sous contrôle du promoteur thermo-inductible Hsp70B pour induire l’inhibition d’un gène d’imagerie (luciférase firefly) surexprimé dans une tumeur. L’étude a été menée in vitro sur des lignées cellulaires génétiquement modifiées puis in vivo sur un modèle de xénogreffes chez la souris grâce au suivi en imagerie optique de bioluminescence (BLI). Nos résultats montrent la faisabilité d’induire transitoirement l’inhibition génique au sein d’une tumeur. L’induction est modulable par la température. Cette stratégie peut être couplée à des méthodes couramment utilisées en clinique et ouvre des perspectives thérapeutiques intéressantes. Notre travail de thèse s’intéresse également à l’utilisation d’ARN comme molécule thérapeutique pour la thérapie génique. L’électroporation intra-dermique d’ARN codant pour la luciférase permet de suivre et de quantifier in vivo par BLI l’expression génique. Plusieurs types d’ARN ont été utilisés pour comparer les efficacités respectives des différentes voies traductionnelles. Notre travail démontre que les ARN permettent l’expression transitoire, sans risque d’insertion génomique, d’un gène in vivo. Nous montrons ainsi tout le potentiel de l’utilisation des ARN en thérapie géniqueThe present thesis work in molecular and translational imaging establishes two innovative approaches for gene therapy in mouse models. Abnormal regulation of gene expression is the hallmark of cancer, and some of them are overexpressed. MicroRNA (miRNA) can be used as tools to reduce specific gene expression but requires inhibition to be limited to the pathological tissue. Thermo-inducibles promoters associated with local hyperthermia allow for spatial and temporal control of gene expression in vivo. The goal of the present study was to achieve gene inhibition with spatio-temporal control of miRNA expression to inhibit a target gene. In our strategy, a synthetic miRNA was placed under transcriptional control of the heat-inducible promoter Hsp70B to induce inhibition of the imaging reporter gene firefly luciferase overexpressed in a tumor. The study was conducted both in vitro using genetically modified cells lines and in vivo using a xenograft model in mice monitored by optical bioluminescence imaging (BLI). Our data show the feasibility of transient induction and heat-modulation of gene inhibition within a tumor. This strategy can be performed with currently clinically available methods and thus, offers interesting therapeutics prospects. Our work also includes a study on RNA as therapeutic vector for gene therapy. The intradermic electroporation of RNA encoding the imaging reporter gene firefly luciferase allows to monitor and quantify gene expression by BLI in vivo. Several types of RNA have been used to investigate efficiency of the different translational mechanisms. Our data clearly demonstrate that RNA allows for transient gene expression in vivo without any risk of insertion into the target cell’s genome. Altogether, our data highlight the potential use of RNA in gene therapy
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Yan, Shao-feng. "Development of an inducible promoter system in Leishmania donovani /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/9306.
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Ali, N. A. "Investigations into the regulation of the thiostrepton inducible promoter of Streptomyces lividans." Thesis, Swansea University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.635755.
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The thiostrepton inducible P-tipA promoter of Streptomyces is widely used as a tool for gene cloning and over expression studies. This thesis describes the experiments carried out to investigate the regulation of P-tipA in S. lividans 1326 in order to improve expression of the promoter. Regulation of the promoter was studied using three different reporter genes, aphII, luxAB and egfp in both plasmid borne and chromosomally integrative forms. The promoter was found to be induced by soya flour as well as thiostrepton (Tsr). Expression of the promoter was determined to be growth-phase development and sensitive to extracellular osmolarity. The expression of the promoter was increased and prolonged in high osmolarity media in the presence and absence of Tsr. The transcriptional activator protein TipAL, which is required for thiostrepton induction was also shown to be required for induction by soya flour and increased extracellular osmolarity. P-tipA has several features, which suggest that it may be responsive to DNA superhelicity. These include a sub-optimally spaced promoter, which is contained within a palindromic sequence with the potential to form a cruciform secondary structure in vivo. The possible role of DNA superhelicity in the regulation of P-tipA was analysed using the gyrase inhibitors, novobiocin and coumermycin. These had little effect on P-tipA expression in the presence of high extracellular osmolarity. The promoter was also exploited for the production of transposon Tn1798, which carries an inducible outward reading P-tipA. The purpose of this transposon is to produce conditional lethal mutations for the study of essential genes. The construction and application of this transposon is described.
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Gross, Tiffany Lauren. "Aedes aegypti Heat Shock 70 Genes and their Inducible Promoters." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/28305.
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Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. In depth genetic studies of vector species have been made possible due to the availability of genome sequences and techniques for producing stably transformed mosquitoes. These resources have also contributed to the establishment of new genetics-based approaches to the control of vector borne disease.
Genetic studies of Ae. aegypti have benefited from the ability to drive targeted transgene expression, however a ubiquitous inducible promoter has not been identified in this mosquito. The Drosophila melanogaster heat shock 70 promoter has been shown to drive inducible expression in heterologous systems; however, DmHsp70 possesses significant basal activity in Aedes aegypti.
This study characterized the sequence and expression of the heat shock 70 genes of Aedes aegypti. AaHsp70 genes were found to be organized in two clusters, each comprised of three divergent pairs. AaHsp70 genes exhibited robust expression upon heat shock in larvae, pupae, and adults as well as in heads, salivary glands, midguts and ovaries.
Genomic regions upstream of AaHsp70 genes were found to drive heat-inducible expression of a reporter in both cell and embryo assays. Deletion analysis of AaHsp70-derived promoters yielded two ~1.5 kb genomic fragments that maintained robust heat inducibility in these systems.
Aedes aegypti were transformed with AaHsp70-luciferase gene cassettes using the transposable element Mos1. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Heat-induced expression of luciferase was observed in transgenic larvae, pupae and adults as well as heads, midguts and ovaries but not salivary glands, with levels varying between transgenic strains.
The effect of heat shock on the endogenous RNAi pathway as well as the effect of blood feeding on the expression of AaHsp70 genes was investigated, though reproducible results could not be obtained using the assays employed.
In conclusion, the heat shock 70 gene family of Aedes aegypti was identified and characterized. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules.Ph. D.
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Oduor, Okoth Richard. "Functional Analysis of the Novel Stress- Inducible XVPSAP promoter isolated from Xerophya Viscosa." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/4314.
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Winston, Eugenia Michele. "The Utilization of the Hmg2 Inducible Promoter to Genetically Engineer Parasite Resistance in Tobacco." Diss., Virginia Tech, 2003. http://hdl.handle.net/10919/27200.
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The cyst nematode, Globodera tabacum tabacum Behrens, and the parasitic angiosperm, Egyptian broomrape, Orobanche aegyptiaca Pers., are obligate root parasites that cause severe yield and quality loss of many important crop hosts. Although these represent two diverse classes of parasites, they have significant similarities in the modes of parasitism and complex interactions with their hosts. Conventional control methods have had limited success in controlling these parasites. The overall objective of this research was to engineer resistance to the cyst nematode and Egyptian broomrape by expressing genes encoding parasite specific toxins under the control of parasite-responsive promoters using tobacco (Nicotiana tabacum L. cv. Xanthi). For nematode resistance, an anti-feeding strategy was employed utilizing the tomato proteinase inhibitor I (PI-I) gene as a nematode specific toxin. Transgenic tobacco plants were generated that expressed genes encoding an intracellarly retained or secreted form of tomato PI-I under the control of the nematode-inducible promoter, derived from tomato (Lycopersicon esculentum L.) Hmg2 gene. Our goals were to determine the effectiveness of local PI-I expression on nematode resistance and to determine if intracellular or extracellular PI-I deposition enhances resistance. Two constructs were generated that contained either the coding region of the tomato PI-I gene, lacking the signal sequence (EM1), or the coding region of PI-I including the signal sequence (EM2), fused to the nematode-responsive Hmg2 promoter. Transgenic PI-I plants were inoculated with G. t. tabacum cysts and evaluated for nematode interactions. Our results suggest that local expression of intercellular of PI-I significantly reduced cyst production when compared to the nontransformed controls. For broomrape resistance, a well characterized R/avr gene pair, the tobacco N resistance gene and the tobacco mosaic virus replicase (TMV) gene, was utilized to create novel gene-for-gene resistance via a N gene-mediated hypersensitive response (HR) to limit broomrape parasitism. The bean (Phaselous vulgaris L.) chalcone synthase 8 (CHS8) promoter has been characterized as a broomrapeâ responsive promoter. We introduced the CHS8:TMV replicase gene construct into tobacco plants that contains an endogenous N gene. Transgenic tobacco plants were inoculated with O. aegyptiaca seeds and monitored for parasite attachment and development. The expression of the TMV replicase leads to a significant reduction in broomrape parasitism. These genetic engineering strategies show promise in enhancing resistance to these destructive parasites.Ph. D.
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Hartman, Andrea H. "Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens." Thesis, Virginia Tech, 2012. http://hdl.handle.net/10919/76874.
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Researchers of Clostridium perfringens, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the E. coli reporter GusA, we characterized its induction in three different strains of C. perfringens. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when C. perfringens was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in Bacillus subtilis. PilT's typical localization in Bacillus subtilis was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins.Master of Science
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Modica, Teresa Maria Elisa. "A mouse model to study inducible oncogene cooperation in vivo." Thesis, Open University, 2012. http://oro.open.ac.uk/54234/.
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The current model for cancer development envisions cells under going a series of genetic mutations and/or alterations which result in their inability to respond normally to intracellular and extracellular signals that control proliferation, differentiation and death. The number of required genetic alterations varies for different types of cancer and it is likely that further changes occur during its progression to increased malignancy. Thus, cancer is not a static disease but during the development and progression of tumour, multiple changes occur in two kinds of genes: oncogenes and tumour suppressor genes. Oncogene-products can be classified as growth factors, growth factor receptors, Ras oncoproteins, cytoplasmic protein kinases, transcription factors, anti-apoptotic proteins. In particular, the ras oncogene family includes three members: N-ras, K-ras, H-ras. In non-transformed cells, Ras protein, belonging to G-protein family, transduces growth signals from external to the internal environment. In fact, when activated, Ras exchanges GDP with GTP and this allosteric change allows binding of Ras effector molecules and transduction of signalling cascades. Ras activity is required for cell cycle progression. In cancer it has been observed that this oncogene is constitutively activated by mutations and induces the cell to enter into cell-cycle also in the absence of growth signals. Among the transcription factors, a gene involved in many tumours is myc. This transcription factor plays a key role in cell proliferation as its target proteins include many positive regulators of the cell-cycle. In tumour cells the protein product of this oncogene is overexpressed. The cooperationb etweenm ultiple oncogenesa nd/orl oss of tumour suppressors from different functional classes is necessary for transformation to proceed. In fact, it was observed that, although overexpression of a single oncogene does not transform wild-type mouse embryonic fibroblasts, combinations of myc and H-rasVAL12, can induce cellular transformation and the cells expressing both oncogenes displayeda markedp roliferative advantage. In thyroid, neoplastic transformation generates several different histotypes of tumours, ranging from poorly aggressive and well-differentiated, to highly malignant and undifferentiated anaplastic cancers. The aim of my thesis was to study the tumorigenesis induced by oncogenes and the oncogene cooperation in vivo during the gradual passage from a poorly aggressive to a much more aggressive tumour. To this end a mouse model expressing the two oncogenes H-rasVAL12 and c-myc (referred as ras and myc) in a tissue-specific as well as in a conditional manner was generated. For this purpose, the coding sequences of the two oncogenes were fused in a bicistronic construct and an IRES (Internal Ribosome Entry Sequence or Site) was inserted between them, to ensure the expression of the second oncogene. The construct was inserted under the control of the promoter of the ubiquitously expressed genes ROSA26 and Eeflal. In order to express these oncogenes in a tissue-specific manner, the transcription of the two oncogenesis prevented by a STOP sequence flanked by two LoxP sites. Such a STOP sequence can be removed by Cre recombinase protein. The transgenic mice were crossed with mice expressing Cre in a tissue-specific manner. Two strains of transgenic mice expressing Cre in thyroid cells were used: 1. transgenic mice for TgCre, in which Cre is expressed under the control of Tg promoter after the development of the thyroid; 2. Pax8Cre, in which the Cre sequence is inserted in the Pax8 locus and is expressed during the early stages of the thyroid development. In such a manner the oncogenes were expressed only in thyroid cells, but were still inactive. In particular, ras was fused to the mutated ligand binding domain of the estradiol receptor that is sensitive to tamoxifen and not to endogenous estradiol; while mycwas fused to the mutated ligand binding domain of the progesterone receptor (hPR891) that is sensitive to RU486 and not to endogenous progesterone. With these fused oncogenes it is possible to activate only Ras (with tamoxifen) or only Myc (with RU486) or both (providing both tamoxifen and RU486). Moreover the activity of two oncogenes might be used to immortalize mouse cell lines in culture.
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Giesel, Christian. "Transformation of tobacco with a lupin chitinase gene under control of a stress inducible promoter." Diss., Pretoria : [s.n.], 2007. http://upetd.up.ac.za/thesis/available/etd-07082008-135301.
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Lo, Cascio Leandro. "In vivo assessment of class-specific inducible inhibitors of metalloproteinases in osteoarthritis." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/30768.
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The role of articular cartilage is providing a painless and attrition-free surface for joints movements. Osteoarthritis (OA) is a debilitating multifactorial pathology characterized by progressive articular cartilage loss, exposition of the bone surface accompanied by pain, and final loss of joint functions. To date there is not an effective alternative to surgical replacement of damaged joints. Metalloproteinases (MPs) play an important role in cartilage remodelling both in physiological and pathological conditions. Among the metalloproteinases, the aggrecanase family was shown to be the major family responsible for aggrecan breakdown, while matrix metalloproteinases (MMPs) target preferentially the collagen molecules. Tissue inhibitor of metalloproteinases (TIMPs) are the endogenous regulators of those enzymes and in particular TIMP-3 was found to possess the broadest activity. So far all synthetic MP inhibitors failed in trials for OA treatment, and TIMP-3 may prove to be a possible alternative. Notably several synthetic mutants were generated to restrict its activity towards metalloproteinases. In order to test the potential therapeutic effects of these proteins, I decided to employ transgenic overexpressing mice. However I appreciated that it is of paramount importance to restrict the exogenous expression just to cartilage. Moreover because it was previously observed that overexpression of TIMP-3 in cartilage severely affects bone development and caused embryonic lethality, I decided to generate tissue specific inducible transgenic mice. I focused on the Aggrecan promoter in order to achieve specific cartilage expression and the Cre/loxP system to obtain inducible expression. Here I show the design and generation of a new aggrecan-driven Cre deleter mouse line. These mice can effectively target floxed genes in cartilage. I also show the generation of inducible TIMP-expressing mouse lines for both a cartilage-specific and ubiquitous expression. I aim to induce OA in those transgenic mice, after induction of TIMPs expression, to evaluate the inhibitors role in OA treatment.
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Jo, Norihide. "Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling." Kyoto University, 2019. http://hdl.handle.net/2433/242397.
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Proença, João Tiago Alves Prior. "Historical analysis of Herpes Simplex Virus type 1 promoter activation in vivo." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611699.
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Vasina, Jess A. "Expression of recombinant proteins in Escherichia coli under the transcriptional control of the cold-shock inducible cspA promoter /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/9866.
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Cosimo, Emilio. "Evaluation of telomerase control elements and radiation-inducible WAF1 promoter for the enhancement of targeted radiotherapy in neuroblastoma cells." Thesis, University of Glasgow, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437995.
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Lagor, William Raymond. "Occupancy and function of the hepatic HMG-CoA reductase promoter." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001739.
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Mason, Bryan Patrick. "Adding Upstream Sequence and a Downstream Reporter to the Bile Acid Inducible Promoter of CLOSTRIDIUM scindens VPI 12708." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/99.
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Bile acids in the small intestines of animals serve to breakdown fats and fatsoluble vitamins. Most of the bile acids are reabsorbed into the enterohepatic circulation, but approximately five percent of these bile acids pass into the large intestine. These bile acids are swiftly deconjugated by the bacterial population, and then subjected to further intestinal bacterial chemical modifications. The most significant of these modifications are 7α-dehydroxylations which form secondary bile acids (deoxycholate and lithocholate). Much research has illuminated the 7α-dehydroxylation pathway: of particular interest is the bile acid inducible operon, for which Clostridium scindens VPI 12708 serves as the model organism. There is a lack of knowledge on how this operon is regulated, so the goal of this project was to create a genetic construct consisting of upstream regulatory elements, a bile acid inducible promoter, and a ϐ- glucuronidase reporter. Cloning strategies utilized PCR to amplify desired DNA fragments and sewing methodology to combine DNA fragments. DNA fragments were ligated into plasmids and transformed into competent E. coli. Transformants were evaluated for the desired reporter gene fusion by blue/white screening, additional PCR, and/or restriction digestion. The bile acid inducible promoter was successfully amplified, and the upstream sequence and uidA (ϐ- glucuronidase) reporter was demonstrated. However, no E. colitransformants were demonstrated to possess the baiP-uidA gene fusion. The project strategy is plausible and data regarding the bile acid inducible promoter are greatly needed.
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Sego, Ashley Diana. "A Doxycycline Inducible HEK-293 Model for the Characterization and Screening of ∂3β2 Nicotinic Acetylcholine Receptors." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/7576.
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Nicotinic acetylcholine receptors (nAChR) are found widely throughout the body. Like all members of the cys-loop family of receptors, nAChRs are composed of five protein subunits, each with a large extra-cellular domain and four transmembrane domains. Together these subunits form a binding domain, transmembrane pore, and selectivity filter. Neuronal nicotinic acetylcholine receptors, formed exclusively from α2-10 and β2-4 subunits, can form in many arrangements and stoichiometries. Each arrangement can have varying binding affinities and channel kinetics, resulting in great modulatory control. α3 and β2 subunit mRNA is found in CA1 interneurons in the stratum radiatum and stratum oriens of the rat hippocampus, and in surprising expression frequency and ratios. Further study of α3 and β2 subunit mRNA injected into Xenopus laevis oocytes yields interesting results about the potential for two α3β2 subtypes. These results were in intriguing, and prompted further study to better characterize and screen the α3β2 nAChR. In order to do so, a model was needed where the α3β2 nAChR could be studied in a more physiologically relevant mammalian environment, with consistent control over α3 and β2 subunit expression ratios, and sufficient protein expression and functionality. To this end, we created a doxycycline inducible HEK-293 cell line, stably transfected with the genetic sequences for the α3 and β2 subunits and NACHO, a transmembrane protein of the neuronal endoplasmic reticulum, which has been shown to mediate the assembly of α3β2 and other nAChRs. This new model is able to induce expression various ratios between α3 and β2 subunits in a consistent, manner, proving to be valuable tool in the characterization and screening of the α3β2 nAChR.
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Walker, David J. F. "Development of novel molecular tools for the identification of essential genes of Clostridium difficile and a Clostridium tetracycline inducible promoter system." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12528/.
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Clostridium difficile is the leading cause of nosocomial diarrhoea in the world, and a considerable burden to healthcare services. For colonisation of C. difficile to occur in the gut of an individual, the resident gut flora must first be quantitatively or qualitatively altered, normally through antibiotic treatment for an unrelated infection. At present, broad-spectrum antibiotics such as vancomycin and metronidazole, the frontline choices for the treatment of C. difficile infection; disturb the normal gut flora, suffer from poor recurrence rates, and have received reports of sporadic emergence of resistance. Development of novel narrow-spectrum antimicrobials would circumvent these problems but depend on the identification of novel essential genes. Molecular techniques available to identify and study essential genes in other organisms have not yet been applied to C. difficile. In this study, we identified 208 candidate essential genes via in silico analysis based upon similarity to known Bacillus subtilis essential genes. In order to provide experimental evidence of essentiality, we developed a novel method utilizing partial diploids and functionally characterised three C. difficile genes as essential; CD0274, metK, and trpS. This method not only identified CD0274, a candidate narrow-spectrum drug target and the first essential genes in C. difficile, but also provides a reliable method to identify further essential genes for novel antimicrobial targeting. In addition, we developed a lac repressor system, a rationally designed theophylline-responsive riboswitch, and most importantly, a Tet inducible promoter system able to conditionally control expression of a catP reporter through a wide dynamic range in both C. difficile and C. sporogenes, to maximum induction factors of 192.89 and 1,275.63, respectively. The combinations of these characteristics make this Tet system not only a powerful tool for identifying essential genes, but bestows a great potential for further analysing gene function far beyond the scope of this project.
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Ali-Adeeb, Rana. "«In vivo» promoter analysis in zebrafish of the «Fugu rubripes» NMDA receptor subunit 1 gene." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=32542.
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A 5kb fragment of the pufferfish Fugu rubripes NR1 promoter spanning nucleotides -2729 to + 2343 was shown to be sufficient to drive expression of the NR1 gene in zebrafish. I performed a cross-species sequence comparison of the 5kb Fugu NR1 promoter with homologous pufferfish (Tetraodon nigroviridis), zebrafish (Danio rerio) and stickleback (Gasterosteus aculeatus) genomes and identified five non-coding evolutionary conserved regions (ECRs) containing cis-elements that serve as putative binding sites for eleven different trans-acting factors. I performed 5' serial deletions of the 5kb NR1 fragment based on the location of the ECRs and subsequently injected the truncated promoter constructs into fertilized zebrafish embryos to assess the activity of the deletion constructs. Analysis of the NR1 promoter requires an investigation of development in an intact organism. Thus, the main aim of my thesis was to establish a transgenic line of zebrafish expressing the 5kb Fugu NR1 promoter. The transgenic data validated results obtained by in vitro methods regarding the activation of the NR1, which occurs via promoter de-repression, and the regulation of the NR1 gene, whereby tissue-specific trans-acting factors act on its cis-elements and determine its expression. The results suggest the presence of a putative fish NR1 gene control region that spans nucleotides -1360 to -194 and contains ECRs 1-3. The results also demonstrate that the minimal cis promoter spans nucleotides -149 to +131 and contains ECR4 and ECR5.Un fragment de 5kb du promoteur NR1 de Fugu rubripes comprenant les nucléotides -2729 à +2343 fut démontré à diriger l'expression du gène NR1 chez le poisson zébré. J'ai entrepris une comparaison entre espèces des séquences génomiques du promoteur 5kb du Fugu avec les séquences d'un poisson homologue (Tetraodon nigroviridis), du poisson zébré (Danio rerio) et du « stickleback» (Gasterosteus aculeatus). J'ai identifié cinq régions non-codantes conservées durant l'évolution (ECRs) qui servent comme sites de liaison putatives pour onze différents facteurs agissant en «trans». J'ai performé des délétions 5' du fragment NR1 5kb basé sur la localisation des ECRs et par conséquence j'ai injecté les construits de promoteurs tronqués dans des embryons de poissons zébré nouvellement fertilisés afin de déterminer l'activité des construits tronqués. Une analyse du promoteur NR1 nécessite une investigation du développement comme il se produit chez un organisme intacte. Ainsi, le but primaire de ma thèse fut d'établir une lignée transgénique chez le poisson zébré exprimant le promoteur 5kb NR1 du Fugu Les données en transgenèse ont validées les résultats obtenus par des méthodes in vitro concernant l'activation du NR1, qui se produit par la dé-répression du promoteur, et la régulation du gène NR1, par laquelle des facteurs agissant en trans de façon spécifique aux tissus agissent sur ses éléments en «cis» et déterminent son expression. Les résultats suggèrent la présence d'une région putative contrôlant le gène NR1 chez les poissons qui comprend les nucléotides -1360 à -194 et qui contient les ECRs 1-3. Les résulta
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Warshamana, Gnana Sakuntala. "Interactions of T7 RNA polymerase with its promoters : Part I: T7 promoter contacts essential for promoter activity in vivo ; Part II: Isolation and characterization of a mutant T7 RNA polymerase with altered promoter specificity." Diss., Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/26303.
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Harbourne, Bryant Thomas. "Effect of hypoxia-inducible secreted protein, tenascin c, on 4T1 tumour cells in vitro and in vivo." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/45992.
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Introduction: Metastatic cancer is responsible for 90% of cancer related deaths. Past research focused mainly on the primary tumour, leaving the process of metastasis poorly understood.
Poorly oxygenated (hypoxic) tumour cells express hypoxia inducible proteins and have a more aggressive and invasive phenotype correlated with poorer prognosis. Hypoxic tumour cells are responsible for increased angiogenesis, invasion, matrix deposition and remodelling along with many other functions.
We hypothesize hypoxic tumour secreted proteins are responsible for promoting metastasis. Our aim is to identify these hypoxia inducible secreted proteins and determine mechanisms promoting metastasis.
Methods: Mammary carcinoma cells (4T1 – metastatic and 67NR – non-metastatic) were placed in 1% O₂ (hypoxic) or 21% O₂ (normoxic) for 24 hours. Stable Isotope Labelling of Amino acids in Cell culture (SILAC) and mass spectrometry were used to perform a quantitative proteomic screen of conditioned medium. Proteins in 4T1 conditioned media, up-regulated in hypoxia and absent from the 67NR results represented candidate secreted proteins in metastasis.
Tenascin C (TNC), a candidate protein identified from the proteomic screen was stably knocked down and over-expressed. In vitro, the Boyden chamber and wound healing assay were used to study invasion and migration. In vivo, metastasis was assessed using flow cytometry-based quantification of metastasized tumour cells in the murine lungs.
Results: (TNC) was identified as a secreted protein with a role promoting metastasis in vivo through enhanced migratory ability. In vitro, knockdown of TNC in 4T1 enhanced migratory ability whereas over-expression decreased migratory ability. These results were contradictory to the expected results based on the hypothesized in vivo role. However, in vivo knockdown of TNC in 4T1 tumour cells resulted in a significant decrease in lung metastases. These results are consistent with the expected role of TNC in vivo.
Conclusions: Despite the contradictory results in vitro, TNC had a positive metastatic role potentially through a migratory mechanism. TNC represents a potential new therapeutic drug target.
Given the 4T1 cell line results, these data support further examination of the migratory role of TNC and how it promotes metastasis. In addition, TNC expression in other tumour cell lines including human breast cancer should be examined.
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Besse, Sébastien Jules. "In vivo promoter analysis of the Vf cluster genes by Agrobacterium-mediated transient transformation of tobacco leaves /." Zürich : Swiss Federal Institute of Technology Zürich, 2003. http://e-collection.ethbib.ethz.ch/show?type=dipl&nr=161.
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Martin, Teresa M. "The ahp promoter of Salmonella enterica sv. typhimurium : regulation and merits for heterologous antigen expression in vivo." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/12587.
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In this thesis, regulation of wild type and Mudlux-tagged ahp expression was examined by reverse transcription and the polymerase chain reaction (RT-PCR). Maximum induction of ahpC in MPG203 was seen in LSLB and minimal media, after treatment with both hydrogen peroxide and sodium chloride in combination, which is in agreement with the luminometer data. However, ‘alpC in MPG203 was induced to a greater degree after treatment with hydrogen peroxide compared with sodium chloride, which is in maximum induction of alpC in LSLB was recorded after treatment with salt, and after treatment with a combination of salt and hydrogen peroxide in minimal media. The basis for this aberrant osmoregulation of ahp is still not fully resolved, as to whether it is under transcriptional or post-transcriptional control. Because of its mode of activation of the ahp promoter as a tool for in vivo expression of foreign antigens (Francis et al., 1993; Taylor et al.,1998) was also examined. Two other known in vivo expressed promoters, PpagC and PspvA were included in the study for comparison. The antigens encoded on the plasmids used for immunisation, were gene fragments cloned from listeriolysin (LLO) of Listeria monocytogenes and glycoprotein 63 (Gp63) of Leishmania major which contained T cell epitopes> GFP, was used as a stable protein control was also expressed from a plasmid construct and used for immunisations. Salmonella typhimurium strain MPG424 was utilised as a vector for immunisation. It contained both a purA and an aroA mutation, leaving the strain hyper-attenuated. However, the Escherichia coli purA promoter and gene were supplied in trans on plasmids encoding antigens of interest, to ensure plasmid maintenance and to moderate attenuation by complementation (Nakayama et al. 1988; Curtiss et al. 1990).
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Zhang, Tingdi [Verfasser]. "Identification of a New Marine Steroid-degrading Bacterium S19-1 and Isolation of Estradiol-inducible Genes and a Novel Promoter from this Bacterium / Tingdi Zhang." Kiel : Universitätsbibliothek Kiel, 2012. http://d-nb.info/1020283513/34.
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Molin, Magnus. "Adenovirus vector systems permitting regulated protein expression and their use for in vivo splicing studies." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-4926-3/.
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Zhang, Xiao-Qun. "Functional Studies on the PDGFR α gene promoter and effects of autocrine PDGF-A stimulation in vivo." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-1455.
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Platelet-derived growth factor receptor α (PDGFRα) plays an important role during embryogenesis. After implantation, the patterns of expression of Pdgfrα and its ligand Pdgf-A undergo an "autocrine-paracrine transition", in that Pdgf-A becomes expressed in the ectoderm and epithelia, while Pdgfrα is expressed in the adjacent mesenchymal tissue. In human tumors, such as malignant glioma, both PDGF and PDGFRα are overexpressed within the same tissue, indicating that an autocrine PDGF loop is generated in the tumors. This thesis is focused on the in vivo functionality of the PDGFRα gene (PDGFRA) promoter, arid on the effect of autocrine PDGF-A stimulation in transgenic n-iice during embryogenesis. To test the in vivo promoter function of a human PDGFRA 2.2 kb 5' flanking fragment, we generated transgenic mouse lines and found that the 2.2 kb fragment was able to promote lacZ reporter gene expression in most of the endogenous Pdgfra expressing tissues. Absence of expression and "ectopic" expression of the transgenic lacZ were also observed. To investigate the autocrine PDGF effect, we produced autocrine PDGF-As (A short-chain) transient transgenic embryos. These transgenic embryos carried a 6 kb mouse Pdgfra 5' flanking sequence linked to a human PDGF-As cDNA. The pattern of expression of the PDGF-As transgene mRNA was similar to that of lacZ. Some of the transgenic embryos exhibited severe abnormal phenotypes, such as midline fusion defects in the cephalic and craniofacial region and small body size, and these embryos die at mid-gestation stage. These findings indicate that a paracrine pattern of expression and the dosage of PDGF are important for sustaining normal embryo development, especially with regard to the middline fusion in craniofacial regions. The possible signaling pathways that may be involved in regulating Pdgfra activity were also studied by comparison of patterns of mRNA expression of Gli, Ptc, and Paxl with that of Pdgfra. The results pointed to the possibility that the Shh signaling pathway may be involved in the regulation of Pdgfra expression for example during early bone and foregut development. The specific regulatory mechanisms may vary for different tissues.
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Messina, Julia Antoinette. "Molecular Localization of Hypoxia Inducible Factor-1-Alpha in Post-Ischemic Myocardium Following in Vivo Prolyl-4 Hydroxylase-2 Gene Silencing." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/2197.
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Leung, Tung-ming. "An in vivo study on the distinctive role of inducible and endothelial nitric oxide synthase in carbon tetrachloride-induced liver injury." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36846806.
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Leung, Tung-ming, and 梁東明. "An in vivo study on the distinctive role of inducible and endothelial nitric oxide synthase in carbon tetrachloride-induced liver injury." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B36846806.
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Wanken, Amy Elizabeth. "Helicobacter pylori colonization of the mouse gastric mucosa: the entner-doudoroff pathway and development of a promoter-trapping system." The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1059079727.
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Barjona, do Nascimento Coutinho Paula [Verfasser], Rainer [Akademischer Betreuer] Bucholz, Rainer [Gutachter] Buchholz, and Barbara [Gutachter] Kappes. "Evaluating inducible promoter systems for controlled nuclear transgene expression in the green alga Chlamydomonas reinhardtii / Paula Barjona do Nascimento Coutinho ; Gutachter: Rainer Buchholz, Barbara Kappes ; Betreuer: Rainer Bucholz." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2018. http://d-nb.info/117297246X/34.
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Kollert, Leonie [Verfasser], Jürgen [Gutachter] Deckert, Katharina [Gutachter] Domschke, and Charlotte [Gutachter] Förster. "Epigenetics of anxiety and depression – a differential role of TGFB-Inducible Early Growth Response Protein 2 gene promoter methylation / Leonie Kollert ; Gutachter: Jürgen Deckert, Katharina Domschke, Charlotte Förster." Würzburg : Universität Würzburg, 2021. http://d-nb.info/1240614721/34.
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Rodriguez, Flores Juan Lorenzo. "In silico, in vitro, in vivo and in populo regulatory genetics of single nucleotide polymorphisms in the phenylethanolamine N-methyltransferase promoter /." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3356146.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed June 15, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 72-75).
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Thomson, Helen. "An in vitro and in vivo study of the mechanical stress-controlling region of the extA extensin gene promoter from Brassica napus." Thesis, Bangor University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401900.
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Arbeiter, Andreas [Verfasser], Bernhard [Akademischer Betreuer] Küster, and Dieter K. M. [Akademischer Betreuer] Saur. "Generation of a spatiotemporally inducible reporter mouse model for in vivo imaging of pancreatic cancer therapy / Andreas Arbeiter. Gutachter: Bernhard Küster ; Dieter K. M. Saur. Betreuer: Bernhard Küster." München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/1061126021/34.
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Ikemori, Rafael Yamashita. "Análise da expressão de galectina-3 em células de glioma expostas a condições hipóxicas e seu papel no desenvolvimento de tumores in vivo." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-14082014-105241/.
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A galectina-3 (gal-3) pertence a uma família de proteínas com domínios de ligação a beta-galactosídeos e está relacionada com diversos aspectos tumorais, como proliferação e adesão celular, angiogênese e proteção contra morte celular. Estudos mostram sua relação com o fenômeno da hipóxia, característica de diversos tumores sólidos que apresentam altas taxas de proliferação celular. A adaptação à hipóxia é mediada principalmente pelo Fator Induzido por Hipóxia (HIF-1), a qual atua na indução de diversos genes de sobrevivência em ambientes com baixas concentrações de oxigênio. Além de HIF, outros fatores são importantes nesse processo, como NF-kB, por exemplo, sendo um fator de transcrição responsivo a diversos estresses celulares, entre eles, a hipóxia. Alguns modelos tumorais apresentam-se ideais para o estudo dos efeitos da hipóxia no microambiente tumoral, como os glioblastomas. Estes são tumores do sistema nervoso central com altas taxas de letalidade, são refratários aos principais métodos de tratamento por sua plasticidade, crescimento infiltrativo e heterogeneidade. Histologicamente, estes tumores apresentam atipia nuclear, altas taxas de mitose e áreas de pseudopaliçada. Postula-se que estas áreas sejam compostas por células migrantes de ambientes necróticos, os quais são também hipóxicos devido a sua distância de vasos sanguíneos e é demonstrado que estas células expressam tanto HIF-1alfa quanto gal-3. Ensaios in vitro realizados por nosso grupo demonstraram que a gal-3 é positivamente regulada pela hipóxia em uma linhagem de glioma híbrido, NG97ht, além de demonstrar que esta proteína é um fator chave na proteção destas células contra a morte celular induzida pela privação de oxigênio e nutrientes, mimetizando condições necróticas de pseudopaliçada in vivo, destacando-se as habilidades antiapoptóticas desta proteína. Embora uma de suas possíveis funções tenha sido elucidada, os mecanismos de atuação e de indução da gal-3 ainda são obscuros. Deste modo, este projeto visa explorar os papéis pró-tumorais da gal-3, podendo torná-la um possível alvo em terapias anti-neoplásicas, entendendo melhor seus mecanismos de proteção contra a morte celular e controle de expressão em ambientes hipóxicos, além de estudar suas possíveis funções in vivo no desenvolvimento de tumores, e também estendendo seus estudos para outras linhagens de glioblastoma. Nossos resultados demonstraram que a gal-3 está co-localizada com mitocôndrias nestas linhagens de glioma, podendo sofrer alterações pós-traducionais em hipóxia, como a fosforilação e que houve acúmulo de HIF-1alfa nuclear nestas células em hipóxia. Vimos também que a gal-3 na linhagem NG97ht apresentou-se proveniente de dois alelos diferentes e que fatores intermediários deveriam ser expressos previamente pela célula antes da indução de gal-3 em hipóxia. Também demonstramos que houve dependência de NF-kB na indução transcricional de gal-3 nestas condições. Estes experimentos também demonstraram que a exposição de células à hipóxia e privação de nutrientes é capaz de induzir tanto espécies reativas de oxigênio como o aumento da autofagia nestas células, fatores importantes na indução da morte celular, além de demonstrar que na linhagem NG97ht a indução da morte nestas condições ocorreu por necrose, sem apresentar apoptose celular. Expandimos esta teoria da participação da gal-3 como molécula protetora contra a morte em hipóxia e privação de nutrientes para outra linhagem de glioma humano, a T98G. E finalmente, demonstramos que a diminuição da expressão de gal-3 em células tumorais da linhagem U87MG levou a diminuição das taxas de estabelecimento e crescimento tumoral in vivoGalectin-3 (gal-3) belongs to a family of proteins with beta-galactoside binding domains and is related to various tumoral aspects, such as cell proliferation and adhesion, angiogenesis and protection against cell death. Studies show its relationship with the hypoxia phenomenon, a characteristic of many solid tumors that have high cell proliferation rates. The adaptation to hypoxia is mainly mediated by Hypoxia Induced Factor (HIF-1), which acts in the induction of several survival genes in environments with low oxygen concentrations. In addition to HIF, other factors are important in this process, such as NF-kB, for example, which is a transcription factor responsive to various cellular stresses, including hypoxia. Some tumor models are ideal for studying the effects of hypoxia in the tumor microenvironment, e.g. glioblastomas. These central nervous system tumors with high mortality rates are refractory to the main treatment methods due to their plasticity, heterogeneity and infiltrative growth. Histologically, these tumors exhibit nuclear atypia, high mitotic rates and pseudopalisading areas. It is postulated that these areas are composed of migrating cells out of necrotic microenvironments, which are also hypoxic due to their distance from the blood vessels and it is shown that these cells express both HIF-1alfa and gal-3. In vitro assays performed by our group demonstrated that gal-3 is positively regulated by hypoxia in a hybrid glioma cell line, NG97ht, and demonstrated that this protein is a key factor in protecting these cells against cell death induced by oxygen and nutrient deprivation conditions mimicking necrotic pseudopalisading areas in vivo, highlighting the pro-survival abilities of this protein. Although one of its possible functions has been elucidated, gal-3 mechanisms of action and induction are still unclear. Thus, this project aims to explore the gal-3 pro-tumoral effects, which may make it a possible target for anti-neoplastic therapies, better understanding the mechanisms of protection against cell death and expression in hypoxic environments, and also study its possible functions in vivo, extending these studies to other glioma cell lines. Our results demonstrated that gal-3 is located within the mitochondria in these glioma cell lines and may undergo posttranslational modifications in hypoxia, such as phosphorylation and that there is accumulation of nuclear HIF-1alfa in these cells under hypoxia. We have also seen that gal-3 in the NG97ht cell line presents two different alleles and that intermediate factors must be expressed previously by the cell before gal-3 induction in hypoxia. We also demonstrated that there is dependence on the NF-kB transcriptional factor for the gal-3 induction under these conditions. These experiments also demonstrated that exposure of cells to hypoxia and nutrient deprivation is capable of inducing reactive oxygen species and increased autophagy in these cells, which are important factors in the induction of cell death. In addition, we demonstrated that the induction of the NG97ht cell death in these conditions is due to necrosis. We expanded this theory of the participation of gal-3 as a protective molecule against cell death in hypoxia and nutrient deprivation to another human glioma cell line, T98G. And finally, we demonstrated that decreased expression of gal-3 in the U87MG glioma cell line leads to lower tumor establishment rates and decreased growth in vivo
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Jokela, T. (Tiina). "Analyses of kidney organogenesis through in vitro and in vivo approaches:generation of conditional Wnt4 mouse models and a method for applying inducible Cre-recombination for kidney organ culture." Doctoral thesis, Oulun yliopisto, 2013. http://urn.fi/urn:isbn:9789526201559.
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Abstract In mice, gene targeting has become a useful tool for resolving the functions of proteins and for creating new animal models. Cre/loxP technology has been used broadly for generation of genetically modified mice. The Cre recombinase recognizes a specific DNA sequence, called loxP, and removes any DNA fragment between two loxP-sites. The activity of the Cre recombinase can be controlled spatially and temporally with cell- or tissue-specific promoters and synthetic inducing agents, such as tamoxifen or tetracycline. In this thesis, we employed tamoxifen-induced Cre recombination in vitro. Cre-ERTM mice were crossed to ROSA26LacZ reporters and Cre-recombination induced by 4OH-TM was monitored by LacZ staining. 0.5 μM 4OH-TM treatment was competent for tamoxifen-induced Cre-mediated activation of LacZ both in kidney cultures and in experimentally induced kidney mesenchymes. Wnt4 is a secreted signaling molecule, which is necessary for the development of several organs including kidney, ovary, adrenal gland, mammary and pituitary glands. Wnt4 is crucial for kidney development and conventional Wnt4-/- mice die soon after birth, likely due to renal failure. In this thesis, two different Wnt4 alleles, Wnt4EGFPCre and floxed Wnt4, were generated and analyzed to learn more about the Wnt4 functions and to apply these mouse lines to renal functional genomics. In the Wnt4EGFPCre, the EGFPCre fusion cDNA was targeted into exon I of the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates from E12.5 embryonic kidney onwards. Further characterization by crossing with the floxed ROSA26LacZ and yellow fluorescent protein (YFP) reporter lines demonstrated that in addition to the kidney, reporter expression was observed in the gonad, spinal cord, lung and the adrenal gland. The expression pattern of the Cre activity recapitulates well the known pattern of the Wnt4 gene. Time-lapse analysis in organ culture settings showed that the Wnt4 expressing cells contributed to the nephrons, some cells near the stalk of the developing ureter, as well as a number of positive supposed medullary stromal cells. In the conditional Wnt4 knock-out, loxP sites were placed to flank exons 3 to 5. The Wnt4 gene was specifically inactivated with CAGCre and Wnt4EGFPCre lines. In both of these crosses deletion of Wnt4 gene function led to impaired kidney development. In conclusion, we identified the culture conditions that can be used for the tamoxifen-induced conditional mutagenesis in tissue cultures. In addition, the created Wnt4 mouse lines serve as new useful tools for addressing the roles of Wnt4 function in diverse tissues and in different stages of developmentTiivistelmä Hiirillä geenikohdennuksesta on muodostunut hyödyllinen väline proteiinien tehtävien selvittämisessä ja uusien eläinmallien luomisessa. Cre/loxP -tekniikkaa on käytetty laajasti muuntogeenisten hiirien tuottamisessa. Cre-rekombinaasi tunnistaa spesifisen DNA-jakson, niin kutsutun loxP:n, ja poistaa kaikki DNA-jaksot kahden loxP-sekvenssin väliltä. Cre-rekombinaasin aktiivisuutta voidaan säädellä paikallisesti ja ajallisesti solu- tai kudosspesifisillä promoottoreilla ja synteettisillä indusoivilla kemikaaleilla, kuten tamoksifeenillä tai tetrasykliinillä. Tässä väitöskirjassa hyödynsimme tamoksifeenin aiheuttamaa Cre-rekombinaatiota in vitro -kudosviljelmissä. Cre-ERTM-hiirilinja risteytettiin ROSA26LacZ-reportterilinjan kanssa, ja 4-hydroksitamoksifeenin indusoima Cre-rekombinaasin aktiivisuutta monitoroitiin LacZ–värjäyksellä. 0.5 µM:n 4OH-TM konsentraatiolla LacZ-reportterigeeni saatiin aktivoitua tehokkaasti Cre-rekombinaasin avulla sekä munuaisviljelmissä että munuaismesenkyymiviljelmissä. Wnt4 on erittyvä signalointimolekyyli, jolla on keskeinen rooli useiden elinten, kuten munuaisen, munasarjan, lisämunuaisen, rintarauhasen ja aivolisäkkeen kehittymisessä. Wnt4-geenillä on ratkaisevan tärkeä rooli munuaisen kehityksessä, ja poistogeeninen Wnt4-/-hiiri kuolee pian syntymän jälkeen, todennäköisesti munuaisen vajaatoimintaan. Tässä väitöskirjatyössä tuotettiin kaksi eri Wnt4 alleelia, Wnt4EGFPCre ja konditionaalinen Wnt4. Nämä hiirilinjat analysoitiin, jotta saisimme lisää tietoa Wnt4-geenin toiminnasta ja pystyisimme soveltamaan kyseisiä hiirikantoja munuaisten toiminnan selvittämisessä. Wnt4EGFPCre-alleelissa EGFPCre-fuusio -cDNA kohdennettiin osaksi endogeenisen Wnt4-geenin ykköseksonia. Vihreän fluoresoivan proteiinin (EGFP) aktiivisuus havaittiin varhaisen munuaisen kehityksen aikana. Wnt4EGFPCre-alleelin lisäkarakterisointi reportterilinjoilla (Rosa26LacZ ja Rosa26YFP) osoitti, että Wnt4-geenin ilmentyminen havaittiin munuaisen lisäksi sukurauhasissa, selkäytimessä, keuhkoissa sekä lisämunuaisessa. Wnt4EGFPCre-alleeli ilmentyi niissä kudoksissa, joissa endogeenisen Wnt4-geenin tiedetään olevan aktiivinen. Time-lapse -analyysin avulla osoitettiin, että Wnt4-geeniä ilmentävät solut muodostavat tiettyjä rakenteita munuaisen kehityksen aikana. Wnt4-geeni ilmentyi nefroneissa, kehittyvän virtsajohtimen soluissa sekä useissa medullaarisissa stroomasoluissa. Konditionaalisessa (ehdollisessa) Wnt4 knock-out-hiirilinjassa loxP-sekvenssit sijoitettiin eksonien kolme sekä viisi ympärille. Wnt4-geenin toiminta inaktivoitiin CAGCre- ja Wnt4EGFPCre-hiirilinjojen avulla. Näissä molemmissa tapauksissa Wnt4-geenin toiminnan poistaminen johti munuaisen kehityshäiriöön. Yhteenvetona voimme todeta, että olemme tunnistaneet ne kasvatusolosuhteet, joita voidaan hyödyntää, kun halutaan aktivoida reportterigeenejä tai kehityksen kannalta tärkeitä geenejä tamoksifeenin aiheuttamaa Cre/loxP -rekombinaatiota hyväksikäyttäen kudosviljelmissä. Samoja olosuhteita ja menetelmää käyttäen voidaan myös poistaa jonkun kehityksen kannalta tärkeän geenin toiminta ja tutkia sitä kudosviljelmässä. Tuotetut Wnt4-hiirikannat ovat lisäksi uusia hyödyllisiä työkaluja, kun halutaan tutkia Wnt4-geenin toimintaa erilaisissa kudoksissa ja eri kehitysvaiheiden aikana
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Hyvärinen, J. (Jaana). "Enzymes involved in hypoxia response:characterization of the in vivo role of HIF-P4H-2 in mouse heart, of a novel P4H in human and zebrafish and of the catalytic properties of FIH." Doctoral thesis, University of Oulu, 2010. http://urn.fi/urn:isbn:9789514261947.
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Abstract Oxygen homeostasis is critical to all animals, as both excess (hyperoxia) and reduced (hypoxia) levels of oxygen can result in pathological changes and ultimately in the loss of cellular and organismal viability. Complex systems have evolved to sense and adapt to changes in cellular oxygen availability, and the hypoxia-inducible factor HIF plays a pivotal role in this elaborate molecular network. In normoxic conditions the α-subunit of HIF becomes hydroxylated by HIF prolyl 4-hydroxylases (HIF-P4Hs 1-3), earmarking HIF-α for proteasomal degradation. Additionally, in the presence of oxygen the hydroxylation of an asparagine residue by the HIF asparaginyl hydroxylase FIH inhibits the transactivation of HIF-target genes by blocking the interaction of HIF-α with a transcriptional coactivator. In addition to being a feature of an organism’s normal life, hypoxia is also characteristic of many common diseases such as severe anemia and myocardial infarction, and it notably decreases these hydroxylation reactions, as HIF-P4Hs and FIH have an absolute requirement for oxygen as a cosubstrate. HIF-α thus escapes degradation and translocates into the nucleus, where it dimerizes with HIF-β and recruits transcriptional coactivators to the hypoxia-response elements of target genes, inducing their transcription and triggering the hypoxia response aimed at restoring cellular oxygen homeostasis. In this study we generated a genetically modified HIF-P4H-2 hypomorphic mouse line that expresses only 8% of the wild-type HIF-P4H-2 mRNA in the heart. We showed that chronic cardiac HIF-P4H-2 deficiency leads to stabilization of HIF-1α and HIF-2α and protects the heart against acute ischemia-reperfusion injury without causing any adverse effects. Furthermore, we identified and cloned a novel human transmembrane prolyl 4-hydroxylase P4H-TM and showed that it regulates HIF-1α protein levels in cellulo and hydroxylates HIF-1α in vitro similarly to the HIF-P4Hs, but may also have other physiological substrates. Using forward genetic tools we showed that lack of P4H-TM during development leads to basement membrane defects and compromised kidney function in zebrafish embryos. Finally, we demonstrated that FIH displays substrate selectivity in terms of hydroxylation and binding of HIF-1α and novel substrates Notch1-3. We showed that FIH has higher affinity for oxygen with Notch1 than with HIF-1α as a substrate, implying that FIH-mediated hydroxylation of Notch can continue in oxygen concentrations where HIF-1α hydroxylation would be markedly reducedTiivistelmä Happitasapainon ylläpito on edellytys elimistön normaalille toiminnalle, koska sekä liian korkea (hyperoksia) että liian matala (hypoksia) happipitoisuus ovat elimistölle stressitiloja ja johtavat pitkittyessään haitallisiin seurauksiin. Happipitoisuuden muutosten havaitsemiseksi ja niihin reagoimiseksi onkin elimistössä kehittynyt monimutkainen säätelyjärjestelmä, jossa avainasemassa on hypoksia-indusoituva tekijä HIF. Solun happipitoisuuden ollessa normaali yksi kolmesta HIF prolyyli 4-hydroksylaasi-isoentsyymistä (HIF-P4Ht 1-3) katalysoi kahden proliinitähteen hydroksylaation HIF-α-alayksikössä. 4-hydroksiproliini toimii signaalina HIF-α:n nopealle proteasomaaliselle hajotukselle. Lisäksi HIF asparaginyyli hydroksylaasi FIH:n katalysoima HIF-α:n asparagiinitähteen hydroksylaatio estää transaktivaatiovaikutuksen. Koska HIF-P4Ht ja FIH tarvitsevat kosubstraatikseen happea, nämä hydroksylaatioreaktiot vähenevät happipitoisuuden laskiessa, jolloin HIF-α stabiloituu ja siirtyy solun tumaan, jossa se muodostaa kompleksin HIF-β-alayksikön kanssa ja houkuttelee paikalle tarvittavat kofaktorit. HIF-kompleksi tehostaa hypoksiavasteessa tarvittavien geenien luentaa sitoutumalla tumassa niiden promoottoreihin ja pyrkii näin palauttamaan solun happipitoisuuden normaaliksi. Tässä työssä luotiin geneettisesti muunneltu HIF-P4H-2 hypomorfi-hiirilinja, jonka sydämissä tuottuu vain 8 % normaalista HIF-P4H-2 lähetti-RNA:n määrästä. HIF-P4H-2:n puutoksen havaittiin johtavan HIF-1α:n ja HIF-2α:n stabiloitumiseen sydämessä ja suojaavan sydäntä kudosvaurioilta iskemian ja reperfuusion aikana aiheuttamatta haitallisia vaikutuksia. Tässä väitöskirjassa karakterisoitiin aiemmin tuntematon ihmisen transmembraaninen prolyyli 4-hydroksylaasi, P4H-TM. Sen osoitettiin säätelevän HIF-1α:n määrää soluissa ja katalysoivan HIF-1α:n kahden proliinitähteen hydroksylaatiota in vitro-olosuhteissa HIF-P4H-entsyymien tavoin. Seeprakalamallin avulla näytettiin, että P4H-TM:n puutos kalan kehityksen aikana aiheuttaa tyvikalvopoikkeavuuksia ja johtaa vakavaan munuaisen toiminnan häiriintymiseen seeprakalan poikasissa. FIH:n katalysoiman hydroksylaatioreaktion kineettisiä ominaisuuksia verrattiin tässä tutkimuksessa ensimmäistä kertaa aiemmin tunnetun HIF-α substraatin ja uusien Notch substraattien kesken. Tulokset osoittivat, että substraatin sitomisessa ja hydroksylaatiossa on merkittäviä eroja eri substraattien välillä
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Papadakis, Andreas. "Characterization of the eIF2alpha kinase PKR in regulating the Hypoxia Inducible Factor 1 alpha and the development of novel in vivo and in vitro experimental models to study the biological role of eIF2alpha phosphorylation." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107652.
Full textAbstract:
The procedure by which mRNA is translated to protein is an important and tightly regulated process in cellular biology. In mammalian cells, the phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2alpha) is an important mechanism of translational control. This event is mediated by a family of kinases which respond and are activated by distinct forms of cellular stress. The work described herein addresses novel roles of PKR in regulating the Hypoxia-inducible Factor 1-alpha (HIF-1alpha), a key factor that is activated by hypoxic conditions within the tumor microenvironment. Moreover, we developed novel in vivo and in vitro experimental models to better understand the role of the eIF2alpha phosphorylation pathway in cancer biology. More specifically we generated a transgenic mouse expressing a conditionally active eIF2alpha kinase and engineered a novel human cell culture model to dissect the biological functions of eIF2alpha phosphorylation in proliferation and the response to chemotherapeutic agents. Through this work we demonstrated that the eIF2alpha kinases can exert antitumor properties independently of eIF2alpha phosphorylation by inhibiting oncogenic signaling (Stat3), and tumor progression (HIF-1). Furthermore we demonstrate that eIF2alpha phosphorylation can act in a tumor promoting manner by being cytoprotective in response to chemotherapeutics. Our studies suggest that designing chemotherapeutic approaches that inhibit the cytoprotective arm of eIF2alpha phosphorylation under conditions where the kinases are activated may have important implications for impairing tumor progression not only by inhibiting hypoxic signaling but also by diminishing cell proliferation and enhancing efficacy of current chemotherapeutic drugs.La traduction des ARNm en protéines est un processus important et finement régulé dans la biologie de la cellule. Dans les cellules de mammifères, la phosphorylation de la sous unité alpha du facteur d'initiation de la traduction 2 (eIF2alpha) est un mécanisme important du contrôle de la traduction. Cet événement est induit par une famille de kinases activées par différentes formes de stress cellulaire. Notre travail s'intéresse aux nouveaux rôles de la voie de phosphorylation d'eIF2alpha dans la régulation du facteur induit par l'hypoxie 1-alpha (HIF-1alpha), un facteur clef activé par des conditions hypoxiques dans le microenvironnement de la tumeur. Nous avons développé des nouveaux modèles expérimentaux in vivo et in vitro pour mieux comprendre le rôle de la voie de phosphorylation d'eIF2alpha dans la biologie du cancer. Plus précisément, nous avons généré une souris transgénique qui exprime une kinase d'eIF2alpha active conditionnellement ainsi qu'un nouveau modèle de culture de cellule humaine pour caractériser les fonctions biologiques de la phosphorylation d'eIF2alpha dans la prolifération et la réponse aux agents chimiothérapeutiques. Nous avons établis que les kinases d'eIF2alpha peuvent exercer des propriétés antitumorales indépendamment de la phosphorylation d'eIF2alpha en inhibant la signalisation oncogénique, et la progression tumorale. De plus, nous avons démontré que la phosphorylation d'eIF2alpha peut agir de façon à promouvoir la tumeur en étant cytoprotective en réponse aux agents chimiothérapeutiques. Nos recherches suggèrent que des approches thérapeutiques conçus de façon à inhiber la voie cytoprotective de la phosphorylation d'eIF2alpha dans des conditions où les kinases sont activées peut avoir des implications importantes pour empêcher la progression des tumeurs pas seulement en inhibant la signalisation hypoxique mais aussi en diminuant la prolifération des cellules et en améliorant l'efficacité des médicaments chimiothérapeutiques courants.
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Chorobik, Paulina. "Opracowanie metody zwiększenia efektywności bakterii Salmonella typhimurium w terapii przeciwnowotworowej poprzez nadekspresję endogennego białka SipB." Praca doktorska, 2010. https://ruj.uj.edu.pl/xmlui/handle/item/274491.
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Tien-Lin, Chang. "Zebrafish HSC70 promoter is a novel cold-inducible promoter from vertebrate organism." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2807200620191200.
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Chang, Tien-Lin, and 張天麟. "Zebrafish HSC70 promoter is a novel cold-inducible promoter from vertebrate organism." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/19646658475033759313.
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碩士國立臺灣大學
漁業科學研究所
94
This study reports the in vivo expression research of hsc70 gene in zebrafish and the cloning of zebrafish hsc70 promoter for temperature induction study, trying to establish a cold-inducible gene expression system to replace the constitutive CMV promoter system, which we used before in our cold tolerance project. Heat shock proteins (Hsps) are well-known stress-inducible chaperons. It had been reported can be induced by cold shock in many species from plant to vertebrate. To understand the regulation of cold-inducible gene expression, we identified the expression of zebrafish hsc70 by semi-quantitative real time-PCR, isolated the 5’-flanking sequence of zebrafish hsc70 gene and used a green fluorescent protein (GFP) for in vivo assay the promoter activity. In basal expression pattern assay, cold shock treatment resulted in a 10 folds enhanced induction of zebrafish hsc70 gene. In in vivo promoter assay, transient zebrafish showed an 2-3 folds enhanced induction. It’s clearly that we’ve identified a novel cold-inducible promoter in vertebrate. In order to further finding out the promoter function, a series deletion of the promoter region had been preformed. After in vivo expression assay we’ve found that the shortest promoter still keeps the cold-inducibility.
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Wang, Wei-Shiuan, and 王偉璇. "Oncolytic adenovirus driven by hypoxia-inducible hTERT promoter for cancer therapy." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/47378699877191665993.
Full textAbstract:
碩士國立成功大學
生物化學研究所
93
Hypoxia, a condition that oxygen density is low at local areas, plays a critical role in tumor malignancy and is associated with resistance of cancer cells to conventional chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1) is stabilized and accumulated when tissues are exposed to hypoxia. HIF-1 is a heterodimeric transcription factor that regulates the physiologic reaction to hypoxia by binding to hypoxia response element (HRE) of target genes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase is transcriptionally upregulated in about 90% of cancers. Therefore, overexpression of hTERT is considered as a tumorigenesis marker. It has been suggested that hypoxia activates telomerase via transcriptional activation of hTERT, and that HIF-1 plays an important role as a transcription factor. Novel therapeutic strategies to target tumor cells in hypoxia regions with high TERT promoter activity to overcome their resistance to chemotherapy and radiotherapy are urgently needed. Therefore, we have exploited 6 copies of HRE ligated to hTERT promoter to modify the transcription activity of hTERT and constructed AdWiSh (Ad5-6xHRE-hTERT), an oncolytic adenovirus driven by this modified promoter. The transcription activity of the 6xHRE-hTERT promoter has been proved higher than that of the original promoter in hypoxia conditions. Similarly the oncolytic efficacy of AdWiSh under hypoxia is better than under normoxia. Intratumoral injection of AdWiSh resulted in suppressing of tumor growth and prolonging survival in mice bearing subcutaneous Lewis lung carcinoma. Cisplatin combined with hypoxia stimulated HIF-1α upregulation and enhanced 6xHRE-hTERT promoter activity, and Ad.WiSh could have better cytolytic efficacy in this condition. Combination of hypoxia-inducible adenovirus and chemotherapeutic drug cisplatin exhibited higher antitumor efficacy compared with either treatment alone. Taken together, these results suggest that AdWiSh, a 6xHRE-hTERT-driven oncolytic adenovirus may have therapeutic potential for solid tumors.
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Hughes, Erik Heller. "Metabolic engineering of Catharanthus roseus hairy roots using an inducible promoter system." Thesis, 2003. http://hdl.handle.net/1911/18541.
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Plant metabolic engineering is a developing field still in need of improved tools and an increased understanding of relevant pathways. This thesis addresses both those needs by testing a new tool for improved metabolic engineering studies and utilizing that tool for the exploration of monoterpenoid indole alkaloid biosynthetic pathways. Using GFP as a model protein in Catharanthus roseus hairy roots, we report that the glucocorticoid inducible promoter system is active and has a tightly controlled, reversible, and dosage-dependent response to dexamethasone. Furthermore, it provides an improved negative control useful for the study of genes affecting alkaloid synthesis.
The most exhaustive transgenic studies reported here focus on the indole pathway. Expressing a feedback-resistant Arabidopsis anthranilate synthase alpha subunit results in dramatic increases in tryptophan and tryptamine yields. On induction, tryptophan increases from undetectable levels to 2.5 mg/g DW, while tryptamine increases from 25 mug/g DW to 267 mug/g DW. Additionally, a transient improvement in lochnericine yield indicates a possible increase in alkaloid flux countered by tight regulation of alkaloid levels. In lines transgenic for inducible tryptophan decarboxylase, serpentine specific yields increased by as much as 129% on induction. The reported studies on the indole pathway demonstrate successful methods to improve indole flux and show that increased indole flux can lead to improvements in certain alkaloids.
Precursors from the complementing terpenoid pathway are also required for alkaloid synthesis. A feeding study utilizing an intermediate and a specific inhibitor of the nonmevalonate pathway validates the importance of this pathway for improved alkaloid yields and points to the potential success of upstream metabolic engineering efforts. In preliminary results, improved yields of certain alkaloids are reported for hairy root lines transgenic for 1-deoxy-D-xylulose-5-phosphate synthase and geraniol 10-hydroxylase, while an ORCA3 study highlights some potential problems with the use of transcriptional activators. In the last study, we focus on the engineering of a valuable alkaloid pathway and report a transgenic hairy root line overexpressing the full coding sequence of tabersonine 16-hydroxylase. On induction, the line produces 16-methoxytabersonine. Overall, a valuable new tool is introduced and subsequently used to manipulate the indole, terpenoid, and alkaloid pathways.
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Hui-Wen, Liu. "Expression of a heat inducible promoter of Arabidopsis in tobacco hairy roots." 2005. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-1207200512274800.
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Liu, Hui-Wen, and 劉慧雯. "Expression of a heat inducible promoter of Arabidopsis in tobacco hairy roots." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/68450343262570940897.
Full textAbstract:
碩士國立臺灣大學
微生物與生化學研究所
93
Plant cells, compared to microbes and mammalian cells, hold great promise as an excellent expression system of exogenous proteins. They possess the advantages of similar post-translational modification, safety, and low cost as compared to animal cells. However, over-expression of certain gene products may reduce cell vitality if the produced protein is harmful to the host. Therefore, it is ideal to control the expression of a gene via a highly specific mechanism such as an inducible promoter through a two-stage culture system to avoid the inhibition of cell growth. In this research, the expression of gusA gene encoding β-glucuronidase (GUS) fused to the Arabidopsis small heat shock protein 18.2 promoter (-870∼+120, 990 bp, HSP18.2 promoter) was investigated in liquid tobacco hairy root cultures. Cell line GD-3 was selected from 436 clones after antibiotic treatment, PCR confirmation, and GUS activity screening. Our results showed that the optimum heat inducible conditions were 42 °C for 2 hr, with a maximum (267.6 nmol MU/mg protein/min) at the 24th hr after recovering the culture at 27 °C. The GUS yield was up to 0.1 % of the total soluble protein in hairy roots. However, prolonging the heating time to 24 hr did not cause higher expression of GUS, and a phenomenon of delayed expression of GUS was observed. We also modified the promoter corresponding to internal deletions of -870 to +678 and +42 to +120 bp in the HSP18.2 promoter. The modified promoter contained the same HSP18.2 promoter sequence between -679 to +1 (transcription start) and the same 5’ UTR (+1∼+41) compared to the original promoter. After antibiotics screening, PCR confirmation and GUS productivity, 576 constructs were selected, and cell line 22-4 was finally selected for further investigation. We compared the heat shock response of the GD-3 and 22-4, and found that the transcription during heat-shock showed no significant difference between these two construct. However, these 77 nucleotides encoding extra amino acid sequences to the N-terminus of GUS were found to maintain the stability of GUS activity for a longer time (24 hr). In this study, a model system inducible for exogenous protein expression in hairy roots has been established. It is not only useful to extrogenous protein expression study, but also helpful for gene tagging and secondary metabolism research in plant biotechnology.
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CHEN, XIU-XLING, and 陳秀玲. "Molecular cloning and characterization of a cell cycle G2 phase inducible promoter." Thesis, 1991. http://ndltd.ncl.edu.tw/handle/85811975328331413529.
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Mlambo, Tafadzwa. "Expression of anti-HBV primary micro-RNA shuttles using an inducible promoter system." Thesis, 2014.
Find full textAbstract:
Hepatitis B virus (HBV) infection is an important global health concern and chronic carriers of the virus are at high risk of developing hepatocellular carcinoma (HCC) and cirrhosis. Current therapies are only partially effective, which emphasises the need for improved treatment strategies. Harnessing the RNA interference (RNAi) pathway as a treatment strategy against HBV has shown great promise. However, there are obstacles that need to be overcome before RNAi-based treatment of HBV infection is realised. These include problems of liver tissue targeting and dose regulation. This study investigated the use of a liver specific and mifepristone-inducible RNA polymerase (Pol) II promoter system for the specific and precise regulation of anti-HBV sequence expression. The inducible system used consists of two expression cassettes; one containing the
regulator/transactivator protein and another containing the transgene. Natural primary microRNA (pri-miR) mimics, pri-miR-31/5 and pri-miR-31/5/8/9, were used as anti-HBV sequences. Firefly luciferase gene expression was used to test modulation by the inducible system and to determine optimal induction conditions. The pri-miR-31/5, pri-miR-31/5/8/9 and luciferase encoding fragments were incorporated into the plasmid vector pRS17 that bears the inducible promoter, creating pRS-31/5, pRS-31/5/8/9 and pRS-Luc respectively. Firefly luciferase expression with this system was shown to be inducible and mifepristone dose-dependent. Effective knockdown of HBV gene expression was achieved with both pRS-31/5 and pRS-31/5/8/9 in vitro and in vivo. However, with high vector amounts, similar efficiency in silencing of HBV gene expression was observed in the presence and absence of the inducer mifepristone suggesting leaky expression of the pri-miRs. To confirm this, knockdown studies were carried out with the pri-miR-31/5/8/9-expressing cassette separated from the transactivator cassette. HBV gene expression knockdown was observed with the pri-miR-31/5/8/9 cassette alone confirming leaky expression from the inducible system. Leakiness appears to be as a result of the E1B promoter driving the expression of the pri-miRs in the absence of mifepristone. However, reducing the vector amounts decreased basal expression and improved the inducibility of the system in cell culture studies. Successful propagation of an inducible and liver-specific RNAi-activating expression system will address the difficulty of achieving dose control of RNAi effectors and contribute to advancing the use of RNAi for HBV treatment.
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Meng, Ching-Ting, and 孟慶庭. "MSC delivery system of oncolytic adenovirus harboring inducible promoter for pancreatic cancer microenvironments." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/a4m83q.
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