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Dissertations / Theses on the topic 'Nuclear Reprogramming'
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Koziol, Magdalena Justyna. "Identification of nuclear reprogramming factors." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613159.
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McClellan, Michael J. "Cellular reprogramming by Epstein-Barr virus nuclear antigens." Thesis, University of Sussex, 2015. http://sro.sussex.ac.uk/id/eprint/54308/.
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Epstein-Barr virus (EBV) is a widespread human B cell virus that is linked to many malignancies. EBV modulates the transcriptome of B lymphocytes to drive immortalisation and viral persistence. This is primarily coordinated by the EBV nuclear antigens (EBNA) 2 and the EBNA 3 family (3A, 3B and 3C), which regulate overlapping sets of cellular genes. Using Chromatin immunoprecipitation (ChIP) coupled to next generation sequencing we found >21000 EBNA 2 and >7000 EBNA 3 binding sites in the human genome, providing the first evidence of EBNA 3 association with the human genome in vivo. Binding sites were predominantly distal to transcription start sites (TSS) indicating a key role in long-range gene control. This was especially pronounced for EBNA 3 proteins (84% of sites over 4kb from any TSS). 56% of genes previously reported to be regulated by these EBNA proteins in micro array experiments were bound by an EBNA. Using ChIP-QPCR we confirmed EBNA 3C bound to and promoted epigenetic silencing of a subset of integrin receptor signalling genes (ITGA4, ITGB1, ADAM28, ADAMDEC1). Indirect silencing of CXCL10 and CXCL11 chemokines by EBNA 3C was also demonstrated. 75% of sites bound by EBNA 3 were also bound by EBNA 2 implicating extensive interplay between EBNA proteins in gene regulation. By examining novel (WEE1, CTBP2) and known (BCL2L11, ITGAL) targets of EBNA 3 proteins bound at promoter-proximal or distal binding sites, we found both cell-type and locus-specific binding and transcriptional regulation. Importantly, genes differentially regulated by a subset EBNA 3 proteins were bound by the same subset, providing a mechanism for selective regulation of host genes by EBNA 3 proteins. In summary, this research demonstrates that EBNA proteins primarily act through long-range enhancer elements and regulate gene expression in a locus and gene-specific manner through differential binding.
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Greggains, Gareth David. "Cell cycle regulation and nuclear reprogramming in mammalian oocytes." Thesis, University of Newcastle Upon Tyne, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.538926.
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Eggan, Kevin C. (Kevin Carl) 1974. "Cloning, stem cells and epigenetic reprogramming after nuclear transfer." Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/29931.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.Includes bibliographical references (leaves 128-146).
The process by which a single totipotent cell becomes a complex organism is a unidirectional program, with each mitotic division generating new cells that gradually differentiate towards more specified fates and specialized functions. Nuclear transfer (NT) experiments have demonstrated the epigenetic nature of development and showed, that although differentiated cells have a very limited developmental potential, the nuclei of these cells retain the potency to direct embryogenesis after reintroduction into the unfertilized oocyte. Herein, we have used the mouse as a model system for understanding both the nature of epigenetic reprogramming that occurs after NT as well as the ramifications it has for the development of cloned animals. Specifically, we investigated how epigenetic states are reprogrammed after NT and demonstrated that the inactive X chromosome is reactivated in NT embryos, resulting in normal X inactivation in female clones. Additionally, investigations into the factors that influence the survival of cloned animals, indicate that there are considerable genetic influences on the cloning process. These genetic factors modify the survival of mice cloned from ES cells by influencing the developmental potential of the donor ES cells rather then the reprogramming process itself. This realization has subsequently led to the development of novel methods for the expedited production of complex mutant mice, which are also described. Finally, we have created cloned embryos by NT from both cortical and mature olfactory sensory neurons to address question of nuclear equivalence in the brain and to investigate whether generation of synaptic diversity or odorant receptor choice, are mediated by genetic as well as epigenetic events.
by Kevin C. Eggan.
Ph.D.
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Sullivan, Stephen. "Assessment of nuclear reprogramming activity in mammalian ES cells." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/27486.
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A murine cell hybrid system was established and optimised to study nuclear reprogramming of somatic cells via fusion with embryonic stem cells. The system generated hybrid clones which displayed an ES phenotype and in which a somatically derived transgenic Oct-4 promoter had been reactivated. Methods were also developed to quantify heterokaryon formation, so that effects on the fusion process could be distinguished from effects on nuclear reprogramming per se. Hybrid cell lines displayed high endogenous alkaline phosphatase activity and expressed undifferentiated cell marker SSEA-1 but not markers associated with differentiated cells (SSEA-4 and CD90). These lines were pluripotent, demonstrating the ability to form the three embryonic lineages both in vivo and in vitro. This system was used to investigate whether several treatments (all either known or expected to perturb global gene expression patterns) affected nuclear reprogramming. Moderate heat shocking of thymocytes prior to fusion with murine ES cells resulted in increased hybridisation frequencies but, as fusion was also increased, it was impossible to verify whether an increase in nuclear reprogramming was partly responsible. Serum starvation of primary embryonic fibroblasts significantly increased nuclear reprogramming, as did ES cell confluence. Murine ES cells were found to lose their capacity to reprogram as they reached high passage numbers. Constitutive or transient expression of nucleoplasmin in murine ES cells did not increase their capacity to reprogram but instead led to increased cell death. Attempts were made to generate hybrids from human ES cells, but no hybrids were successfully generated. This was at least partly due to human ES cells being more difficult to fuse with other cells even using a range of different fusagens. Finally it was found that treating human ES cells with hyaluronidase prior to electropulsing resulting in five times more heterokaryon formation indicating that the extracellular matrix of these cells had prevented fusion. The methods developed here provide the basis for further study of the mechanisms underpinning reprogramming.
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Byrne, J. A. "Nuclear transfer, nuclear reprogramming and the delivery of exogenous macromolecules into living amphibian cells." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597205.
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This thesis describes a number of foundation studies regarding Xenopus nuclear transfer, inter-species transcriptional reprogramming and a method for delivering exogenous macromolecules into living amphibian cells. I divided my research into four stages. First, I demonstrated that morphologically and reproductively normal cloned animals could be obtained following Xenopus nuclear transfer using streptolysin permeabilised donor cells. The use of streptolysin, as opposed to the traditional cell-squashing method, permits more consistent, more controlled and gentler donor cell permeabilisation, and makes nuclear transfer quicker and technically easier to perform. Second, I demonstrated that epigenetically aberrant and developmentally defective cloned Xenopus embryos possess cells that retain the capacity to differentiate into multiple cell types, survive for an extended period of time and exhibit a normal growth morphology. Third, I demonstrated that Xenopus oocytes could transcriptionally reprogram mammalian somatic cell nuclei to express Oct-4, a mammalian stem cell/pluripotency marker. Finally, I demonstrated that the reversible streptolysin permeabilisation technique, previously described for mammalian cell lines, could be modified slightly and then used with an amphibian cell line to deliver exogenous macromolecules into living amphibian cells. This modified cell delivery technique has a wide variety of potential reprogramming, transgenesis and differentiation applications for researchers working with amphibian cells. In my opinion, the long-term objectives of the reprogramming/cloning field are to identify the reprogramming molecular mechanisms, to improve the efficiency of nuclear transfer and to obtain therapeutically useful isogenic human embryonic stem cells. My research provides a number of foundation result on which these larger problems can be addressed in the future. First, my result with developmentally defective cloned Xenopus embryos provides a strong case for future research to investigate whether isogenic embryonic stem cells can be obtained from developmentally defective cloned primate/human embryos; stem cells that could potentially be used to treat various degenerative diseases.
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Pérez, Camps Mireia. "Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish." Doctoral thesis, Universitat Politècnica de València, 2010. http://hdl.handle.net/10251/6902.
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El estudio de los mecanismos de reprogramación nuclear tiene actualmente una notable importancia, dado que el dominio de estos procesos constituyen la clave para actuar eficazmente en cuestiones tan dispares como el cáncer o la medicina regenerativa. También resulta muy importante este tipo de estudios sobre reprogramación cuando se pretende la obtención de animales transgénicos múltiples y orientados.
Aunque para ello se pueden utilizar muy diversos modelos animales, en nuestro caso, se ha optado por el pez cebra, por sus características en el desarrollo, como la brevedad en la embriogénesis y transparencia de los embriones, su capacidad de regeneración y el conocimiento de su genoma, entre otras. Bien es cierto que a estas ventajas le acompañan cierto inconvenientes tales como no disponer hasta este momento de técnicas tales como el transplante nuclear y, a otro nivel, el quimerismo. Técnicas cuyos desarrollo se pretende en esta tesis, lo que justifica los objetivos aquí planteados.
Para ello se han realizado diferentes trabajos experimentales titulados: "Ultraviolet radiation and handling medium osmolarity affect chimaerism success in zebrafish", "Evaluation of presumptive caudal fin blastema cells as candidate donors in intraspecies zebrafish (Danio rerio) chimaeras", "Definition of three somatic adult cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization", "Transplant of adult fibroblast into the central region of metaphase II eggs resulted in mid blastula transition (MBT) embryos", "Electroactivation of zebrafish (Danio rerio) eggs", "Comparison of different activating stimuli efficiency in zebrafish nuclear transplant", "Reconstruction of heteroparental gynogenetic diploid condition by nuclear transplant in zebrafish".
Los dos primeros relativos al quimerismo, su eficiencia final se optimizó mediante la penalización con radiación UV del embrión receptor.Pérez Camps, M. (2009). Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/6902
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Kuo, Yen-Hsi. "Using Xenopus oocyte as a model system for nuclear reprogramming studies." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612393.
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Köhler, Daniela. "Cloning in cattle : nuclear architecture and epigenetic status of chromatin during reprogramming of donor cell nuclei." kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/9915/.
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Pawlak, Mathias. "Stem cells, differentiation and nuclear reprogramming : the roles of Klf4 and geminin /." Heidelberg, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000259539.
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Pasque, Vincent. "Mechanisms that induce, promote, and restrict nuclear reprogramming of somatic cells by nuclear transfer to the Xenopus oocyte." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610168.
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Aston, Kenneth Ivan. "Indentification Of Factors Affecting Bovine Somatic Cell Nuclear Transfer Efficiency And Characterization Of Transciptional Profiles Of Nuclear Transfer Embyos and Cotyledons." DigitalCommons@USU, 2007. https://digitalcommons.usu.edu/etd/87.
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Since the production of the first sheep by somatic cell nuclear transfer a great deal of effort has been made to improve efficiency and to understand nuclear reprogramming mechanisms. Unfortunately efficiency remains low, and nuclear reprogramming mechanisms remain uncharacterized. The objectives of this research were to identify factors associated with somatic cell nuclear transfer efficiency and to analyze the transcriptome of blastocyst-stage clone and control embryos and cotyledonary tissue in an effort to elucidate mechanisms responsible for the low developmental efficiency and high post-implantation losses. The experiments reported here identify factors including oocyte source and timing of activation following nuclear transfer that yield improved efficiencies. It was determined the use of cow oocytes for somatic cell nuclear transfer results in improved in vitro development and increased pregnancy rates. These data further indicate prolonged exposure of the donor nucleus to pre-activated oocyte cytoplasm results in increased nuclear fragmentation and reduced developmental efficiency in vitro. Several aberrantly expressed genes were identified in nuclear transfer blastocysts and cotyledons that could impact cloning efficiency. Major histocompatibility complex I and down-regulator of transcription 1 were overexpressed in nuclear transfer blastocysts, and retinol binding protein 1 was overexpressed in nuclear transfer cotyledons. The functions of these genes in immune response, transcriptional regulation, and retinol binding and transport make them attractive candidates for further nuclear transfer research. Expression levels of six developmentally important genes were analyzed in various stages of preimplantation nuclear transfer embryos by real-time polymerase chain reaction to determine the timing of nuclear reprogramming following nuclear transfer. Five of the six genes were aberrantly expressed multiple developmental stages, however by the blastocyst stage only one gene was aberrantly expressed. These data indicate reprogramming is delayed in nuclear transfer embryos resulting in over- or under-expression of developmentally important genes during early embryogenesis. These experiments report factors associated with improved nuclear transfer efficiency; provide insight into potential mechanisms for low developmental rates, abnormal placentation, and fetal loss of clones; and characterize the timing of nuclear reprogramming following somatic cell nuclear transfer.
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Hao, Ru. "Reprogramming of mesenchymal stem cells and adult fibroblasts following nuclear transfer in rabbits." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-96652.
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Wen, Ming-Hsuan. "The role of transcription factors in somatic cell nuclear reprogramming by eggs and oocytes." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288437.
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Somatic cell nuclear reprogramming (SCNR) by eggs is a way to forcibly transform the nuclei of terminally differentiated somatic cells to an embryonic state and gain totipotency (Gurdon et al., 1958). Additionally, induced pluripotency is applied to transform identities of somatic cells to induced pluripotent stem cells by overexpression of combinatorial Yamanaka factors (iPS, Takahashi et al., 2006). Although both approaches aim to derive cells with highest plasticity, the mechanisms and differences between these procedures are not yet clear. In my thesis, I used quantitative polymerase chain reaction (QPCR) and RNAseq plus 5-bromouridine 5'-triphosphate (BrUTP) pulldown to evaluate the transcriptional reprogramming by maternal factors and overexpressed transcription factors during SCNR by Xenopus oocytes, which are inactive in DNA replication and cell division. QPCR measures changes in the steady-state levels of transcripts within 2 days of nuclear transfer to Xenopus oocytes (Oocyte-NT). Three pairs of Yamanaka factor homologs were tested by QPCR and Yamanaka factor homologues regulated similar sets of pluripotency genes in mouse embryonic fibroblasts (MEFs). Pioneer factor mFoxA1 could not up-regulate most pluripotency genes and their binding targets of neurogenic genes in MEFs while pioneer factors are proposed to bind to their targets even if they may reside in inaccessible chromatin. This shows that the existence of other factors is needed at specified developmental stages. Hence, gene activation by transcription factors in the Oocyte-NT system requires not only corresponding binding on regulatory elements of linked genes but transcription cooperators to exert effective gene activation. Additionally, RNA-seq plus BrUTP pulldown measures the extent to which oocytes change the transcriptional activity of nuclei transplanted to oocytes. Through RNA-seq plus BrUTP pulldown, I compared the reprogrammed transcriptomes of embryonic and somatic cells, including mouse embryonic stem cells, mouse embryonic fibroblasts and mouse myoblasts, to demonstrate the effects of maternal factors and overexpression of transcription factors on gene activities during SCNR by oocytes. Importantly, I find that maternal factors of oocytes and the overexpression of transcription factors exert different strategies to reprogram somatic cells. Oocyte factors reprogram the donor cell nuclei to an oocyte-steady state except for the SCNR resistance genes and xklf2-HA overexpression enhances expression of reprogrammable genes and activates SCNR resistance genes.
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Biddle, Adrian James. "The role of gene-specific transcription factors in nuclear reprogramming by Xenopus laevis oocytes." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612228.
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Yang, Chao-Shun. "Molecular Landscape of Induced Reprogramming: A Dissertation." eScholarship@UMMS, 2002. http://escholarship.umassmed.edu/gsbs_diss/698.
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Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming.
First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming.
Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells.
To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.
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Yang, Chao-Shun. "Molecular Landscape of Induced Reprogramming: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/698.
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Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming.
First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming.
Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells.
To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.
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Villafranca, Locher Maria Cristina. "Fusion of bovine fibroblasts to mouse embryonic stem cells: a model to study nuclear reprogramming." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/82864.
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The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates.
In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells.
Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome.Ph. D.
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Davis, Aaron Patrick. "Apoptotic and Epigenetic Induction of Embryo Failure Following Somatic Cell Nuclear Transfer." DigitalCommons@USU, 2013. https://digitalcommons.usu.edu/etd/1941.
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Somatic cell nuclear transfer (SCNT) is a useful tool for selective breeding, conservation, and production of transgenic animals. Despite the successful cloning of several species, high rates of embryo failure following SCNT prevent the wide-scale use of the technique. Embryos produced through cloning have a higher incidence of developmental arrest, decreased developmental potential, frequent implantation failures, and increased incidence of abortion. The objective of this dissertation research was to characterize the factors that lead to SCNT failures by examining epigenetic and apoptotic pathways that can negatively influence the development of cloned preimplantation embryos. Aberrant genome reprogramming is generally considered to be a key factor in the failure of SCNT embryo development. Therefore, we used bisulfite pyrosequencing technology to compare DNA methylation patterns of several genes critical for embryonic development (POU5F1, NANOG, SOX2, and KLF4) in SCNT and in vitro fertilized (IVF) blastocyst stage embryos. The methylation profiles obtained from these experiments indicate that methylation patterns of the POU5F1 gene were undermethylated compared to IVF embryos, suggesting reprogramming did occur, but that the reduced methylation was inappropriate for the blastocyst stage. Furthermore, aberrant methylation profiles were detected for SOX2 and NANOG, suggesting that problems of genome reprogramming following SCNT can be gene-specific or localized. Because high rates of apoptosis are associated with failure of preimplantation embryos, we compared the activation of the P53-mediated apoptosis pathway in individual IVF and SCNT preimplantation embryos at multiple developmental stages. This pathway is activated in response to cell stress and genomic instability, and in response to the expression of genes associated with somatic cell reprogramming. Evidence from gene expression and immunohistochemistry analyses suggests that the P53 pathway is frequently active in SCNT embryos. Also, we detected expression of several factors known to induce apoptosis more frequently and at higher levels in SCNT embryos. Collectively, the work presented here illuminates some of the molecular consequences of incomplete or inappropriate genome reprogramming in cloned embryos. The identification of these factors may lead to interventions that target the apoptosis pathway during preimplantation development and increase SCNT success rates.
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Chang, Chia-Wei. "Polycistronic lentiviral vector for hit and run reprogramming of mouse and human somatic cells to induced pluripotent stem cell." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2009p/changc.pdf.
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Sampaio, Rafael Vilar. "Modificações epigenéticas da cromatina e sua relação com a reprogramação nuclear de bovinos." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10132/tde-27082015-114515/.
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A reprogramação nuclear de uma célula somática a um estado embrionário tem diversas aplicações, como pesquisas básicas na biologia do desenvolvimento, terapia celular, melhoramento genético em animais de produção e conservação de espécies. As principais técnicas utilizadas para a reprogramação nuclear são a transferência nuclear de células somáticas (TNCS) e a geração de células tronco pluripotente induzidas (iPS). Muitos trabalhos têm mostrado uma baixa eficiência no processo de reprogramação nuclear nas duas técnicas, além disso, modificações epigenéticas tem sido apontada como a principal barreira para uma reprogramação nuclear eficiente. Por esse motivo, medidas como a utilização de células menos diferenciadas e/ou alteração do perfil epigenético das células somáticas podem aumentar a eficiência destas técnicas. Por isso, o objetivo deste trabalho foi investigar a influência de marcas epigenéticas em células bovinas utilizadas na reprogramação nuclear mediada por TNCS ou superexpressão de genes relacionados a pluripotêcia (iPS). Para isso, utilizamos 3 abordagens. Primeiro, analisamos marcações epigenéticas relacionadas ao desenvolvimento embrionário e pluripotência (H3K9me2, H3K9me3, H3K9ac, 5mC e 5hmC) em diferentes tipos celulares, analisamos a expressão gênica de genes responsáveis por essas marcações em células de diferentes tecidos (ex. células tronco mesenquimais (MSC) e fibroblastos) e as utilizamos como doadoras de núcleo na TNCS. Na segunda e a terceira abordagem, utilizamos células com menores níveis de H3K9me2 para a geração de iPS e na TNCS, respectivamente. Além disso, por se mostrar eficiente na TNCS, analisamos o efeito da sincronização do ciclo celular por privação de soro fetal bovino (SFB) na geração de células iPS. Com o intuito de diminuir os níveis de H3K9me2, as células foram tratadas com UNC0638, um inibidor especifico das metiltransferases de histona G9a/GLP. Nossos resultados do primeiro experimento mostraram que as MSC podem ser utilizadas como doadoras de núcleo na TNCS, no entanto, mesmo com algumas diferenças na expressão gênica em relação aos fibroblastos, a produção de blastocistos não foi diferente entre as duas células. No segundo experimento, as células privadas de SFB geraram mais colônias que as células controle, enquanto que as células tratadas não apresentaram diferença. Por último, as células tratadas com o UNC0638 apresentaram um menor nível de metilação no DNA em zigotos em relação às células controle. Os resultados encontrados neste trabalho podem contribuir para o melhor entendimento dos mecanismos epigenéticos envolvidos na reprogramação nuclear de bovinosNuclear reprogramming of somatic cells to embryonic state has several aplications, such as basic research on developmental biology, cell therapy, genetic improvement in livestock animals and preservation of endangered species. The principal techniques utilized to achieve nuclear reprogramming are Somatic Cell Nuclear Transfer (SCNT) and induced pluripotency. Several works has reported low efficiency rates of nuclear reprogramming when these techniques are used to reprogram somatic cells. Moreover, epigenetic modifications acquired during development act as epigenetic barrier to the complete reprogramming process. For this reason, strategies such as use of less differentiated cells and/or modification of epigenetic profile of somatic cells might increase the efficiency these techniques. The objective of this work was investigate the influence of epigenetic marks in bovine cells utilized on nuclear reprogramming experiments mediated by SCNT or induced pluripotency. To investigate it, we used three approaches. First, we analyzed the epigenetic marks related to the embryonic development and pluripotency (e.g H3K9me2, H3K9me3, H3K9ac, 5mC and 5hmC), gene expression of genes involved in these epigenetic marks in different tissues (i.e. mesenchymal stem cells (MSC) and fibroblasts) and their use as nuclear donor cells on SCNT procedure. Regarding the second and the third approach, we utilized cells with reduced levels of H3K9me2 to generate iPS cells and cloned embryos, respectively. Furthermore, since serum starvation has been demonstrated increase SCNT developmental rates, we assessed the effect of cell cycle synchronization mediated by serum starvation on nuclear reprogramming using iPS cells. Aiming decrease the levels of H3K9me2, cells were treated with UNC0638, a chemical probe that works as a specific inhibitor of the histone methyltransferases G9a and its counterpartner GLP. Our results showed that MSC are suitable to be used as nuclear donors on SCNT procedures, however, in spite of differences on gene expression comparing with fibroblasts, the embryonic developmental rates were not improved. On the second experiment, cells privated of fetal calf serum produced more iPS cells colonies than control cells, whereas cells treated with UNC did not show differences when compared with untreated cells. Lastly, UNC treated donor cells treated produced cloned zygotes with lower levels of DNA methylation compared to zygotes derivated from untreated cells. The results presented here will contribute to the better understanding of the epigenetic mechanisms involved on bovine nuclear reprogramming
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Santos, Diogo Gonçalves Biagi dos. "Uso de células-tronco pluripotentes induzidas para compreensão de alterações em cardiomiócitos de pacientes com cardiomiopatias de base-genética." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5166/tde-12082015-155410/.
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O estudo de mutações genéticas como causa das cardiomiopatias teve início com a descoberta de mutações em proteínas sarcoméricas que levavam à Cardiomiopatia Hipertrófica, desde então, alterações em diversos genes, de proteínas contráteis ou não, foram descobertas e listadas como a responsável pelo desenvolvimento de diferentes cardiomiopatias. Estudar o efeito destas mutações nos cardiomiócitos destes pacientes permanecia um desafio devido ao difícil acesso às células cardíacas. Em 2007, a técnica de reprogramação de células somáticas em células-tronco pluripotentes foi descoberta. Pelo fato das células-tronco pluripotentes serem capazes de ser diferenciadas em cardiomiócitos, surgiu-se a possibilidade de se estudar essas células de indivíduos portadores das mutações genéticas. Esta tese teve como objetivo a criação de um modelo celular para estudar a Cardiomiopatia Hipertrófica causada por mutações genéticas. Inicialmente foi estabelecido um protocolo de reprogramação celular para se estabelecer linhagens celulares das células-tronco induzidas de um paciente com mutação no gene MYH7. Tendo as células caracterizadas, elas foram diferenciadas em cardiomiócitos através de um protocolo adaptado de protocolos de diferenciação direta em cardiomiócitos. Os cardiomiócitos gerados apresentaram características moleculares e funcionais semelhantes à cardiomiócitos primários humanos e foi visualizado, através de microscopia eletrônica de transmissão, que os cardiomiócitos do paciente com alteração genética possuíam grande proporção de sarcômeros desorganizados em comparação a cardiomiócitos de indivíduos saudáveis. Em conclusão, o modelo celular desenvolvido sugere ser possível o estudo do efeito de mutações genéticas em Cardiomiopatia Hipertrófica.The study of genetic mutations as the cause of cardiomyopathies initiates with the discovery of mutations in sarcomeric proteins genes that lead to Hypertrophic Cardiomyopathy. Since then, mutations in several genes, coding to sarcomeric proteins or not, were discovered and listed as the reason to the cardiomyopathies. To study the effect of these mutations was a challenge due the difficulty to accesses cardiac cells. In 2007, the technique of reprogramming somatic cells into pluripotent stem cells was discovered. The fact that the pluripotent stem cells are capable of differentiating into cardiomyocytes opened the opportunity to study these cells from individuals with genetic mutations. This thesis aimed to create a cellular model to study Hypertrophic Cardiomyopathy caused by genetic mutations. Initially we established a cell reprogramming protocol to establish induced stem cells lines from a patient with mutation in MYH7 gene. Having characterized the cells, they were differentiated into cardiomyocytes using an adapted protocol from direct differentiation protocols. Cardiomyocytes generated showed molecular and functional characteristics similar to human primary cardiomyocytes and were visualized by means of transmission electron microscopy. The patient\'s cardiomyocytes had a large proportion of disorganized sarcomeres compared to cardiomyocytes from healthy individuals. In conclusion, the cell model developed suggests that it is possible to study the effect of genetic mutation in Hypertrophic Cardiomyopathy using induced pluripotent stem cells derived cardiomyocytes.
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Koch, Katharina [Verfasser], Jaroslaw [Gutachter] Maciaczyk, and Dieter [Gutachter] Willbold. "Analysis of metabolic reprogramming during the enrichment of glioblastoma stem-like cells in glioblastoma via high resolution proton nuclear magnetic resonance (HR 1H-NMR) spectroscopy / Katharina Koch ; Gutachter: Jaroslaw Maciaczyk, Dieter Willbold." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2020. http://d-nb.info/1223705242/34.
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Tiwari, Vijay Kumar. "The Epigenetics of Gene Transcription and Higher Order Chromatin Conformation." Doctoral thesis, Uppsala universitet, Zoologisk utvecklingsbiologi, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6302.
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It is becoming increasingly clear that long-range control of gene expression is mediated through direct physical interactions between genes and regulatory elements, either intra- or interchromosomally. In addition to transcriptional initiation, formation of active chromatin hubs seem to be crucial for increased transcriptional efficiency as well as insulation from neighbouring heterochromatic environment. Regulatory factors apparently have an important role in organization of such functional modules in a development and differentiated- dependent fashion. The relevance of trans-acting factors in the ‘choice’ process of X-Chromosome Inactivation (XCI) was highlighted by our observations where CTCF was shown to occupy a homologous position on the active mouse and human Xist/XIST promoters and its binding affinity was altered in familial cases of opposite skewed X-inactivation patterns. The paradigm of genomic imprinting, i.e. the Igf2-H19 locus, manifests its imprinted states through the H19 Imprinting Control Region (ICR). The repression of the maternal Igf2 allele depends on the insulator properties of the H19 ICR when this interacts with CTCF. The studies here detected a novel kind of CTCF-dependent tightly closed pocket- like higher order structure exclusively on maternal allele which was found to be essential for imprinted Igf2 expression as well as maintenance of precise epigenetic marks at various Differentially Methylated Regions (DMRs) across this locus. Despite the highly condensed state of the mitotic chromosome, the insulator protein CTCF was found to constitutively occupy its known target sites. Furthermore, pivotal CTCF-dependent long-range regulatory loops within Igf2-H19 locus were found to survive mitotic compaction and such mechanisms might serve as a novel kind of epigenetic memory to minimize transcriptional chaos and to reset proper expression domains in the daughter cells as soon as cells exit mitosis. Our observations also suggest that the epigenetic reprogramming of H19 ICR during spermatogenesis is initiated by a CTCF-dependent recruitment of chromatin remodeling factor Lsh to the H19 ICR followed by completion of the imprint acquisition process by a replacement of CTCF with its closely related paralogue termed BORIS. Overall, this thesis unravels the novel roles for CTCF as an architectural factor in the organization of higher order chromatin conformations and transcriptional regulation.
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Acquistapace, Adrien. "Reprogrammation nucléaire de cardiomyocytes vers un stade progéniteur par fusion partielle avec des cellules souches adultes." Thesis, Paris Est, 2011. http://www.theses.fr/2011PEST0078/document.
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La thérapie cellulaire régénératrice offre des perspectives d'applications dans de nombreuses pathologies entraînant une perte cellulaire. Cependant, suite à un infarctus du myocarde et donc une diminution importante du nombre de cardiomyocytes, l'injection de cellules souches n'a permis de mettre en évidence qu'une amélioration légère et transitoire de la fonction cardiaque. Ces résultats suggèrent qu'il est nécessaire d'améliorer l'efficacité des protocoles de thérapie cellulaire cardiaque. Cette amélioration passe par une meilleure compréhension des mécanismes mis en jeu par les cellules souches dans la régénération myocardique. Parmi les hypothèses soulevées, la fusion entre les cellules souches et les cardiomyocytes a été décrite dans plusieurs études mais le rôle physiologique de ce phénomène reste inconnu. Mon travail de thèse a consisté à étudier ce mécanisme in vitro au sein de cocultures entres des cellules souches adultes humaines (cellules hMADS pour human multipotent adipose derived stem cells) et des cardiomyocytes murins adultes. Nous avons pu mettre en évidence un processus de fusion hétérologue entre ces deux types cellulaires, aboutissant à la reprogrammation du cardiomyocyte vers un stade de progéniteur. Les cellules hybrides résultant de cette fusion ont exprimé des marqueurs cardiomyogéniques précoces et de prolifération et ont été montrées comme ayant un génotype exclusivement murin. Ces cellules hybrides ou progéniteurs cardiaques se sont formés préférentiellement par un mécanisme de fusion partielle par l'intermédiaire de structures intercellulaires appelées nanotubes composés de f-actine et de microtubules. En outre, nous avons montré que le transfert de mitochondries des cellules souches vers les cardiomyocytes était indispensable pour la reprogrammation des cardiomyocytes. En conclusion, nos résultats apportent de nouveaux éléments dans la compréhension des mécanismes de régénération médiés par les cellules souches qui est un pré-requis pour optimiser les protocoles de thérapie cellulaire cardiaqueRegenerative cell therapy offers potential applications in many diseases involving cell loss. However, following myocardial infarction and the dramatic decrease in the number of cardiomyocytes, the injection of stem cells led to a poor and transient improvement of cardiac function. Therefore stem cell-based therapy to treat myocardial infarction requires a better understanding of the mechanisms brought into play by stem cells in heart regeneration. Among the different hypothesis raised, cell fusion between stem cells and cardiomyocytes has been described in several studies. However, the respective physiological impact of cell fusion remains unknown. During my thesis, I investigated this cell fusion mechanism in vitro in a coculture model between human multipotent adipose-derived stem cells (hMADS) and murine fully differentiated cardiomyocytes. We showed intercellular exchanges of cytoplasmic and nuclear material between both cell types, followed by a heterologous cell fusion process promoting cardiomyocyte reprogramming back to a progenitor-like state. The resulting hybrid cells expressed early cardiac commitment and proliferation markers and exhibited a mouse genotype. We provided evidence that cardiac hybrid cells were preferentially generated through partial cell fusion mediated by intercellular structures composed of f-actin and microtubule filaments. Furthermore, we showed that stem cell mitochondria were transferred into cardiomyocytes and were required for somatic cell reprogramming. In conclusion, by providing new insights into previously reported cell fusion processes, our results might contribute to a better understanding of stem cell-mediated regenerative mechanisms and thus, the development of more efficient stem cell-based heart therapies
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Bassaneze, Vinícius. "Reprogramação de células mesenquimais de tecido adiposo em células-tronco pluripotentes por meio de proteína de fusão TAT." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5166/tde-11052012-093336/.
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Os vírus são eficazes na transferência de genes em células devido aos seus mecanismos especializados. No entanto, vírus como veículos de entrega de genes podem acarretar em problemas, particularmente quando proposto para reprogramar células somáticas em células-tronco pluripotentes induzidas (iPS) visando utilização terapêutica. No presente estudo, procurou-se desenvolver um sistema alternativo para entregar diretamente proteínas nucleares (Oct4, Sox2, KLF4, e c-Myc) fusionadas com o domínio de transdução de proteína TAT, para promover a reprogramação de fibroblastos embrionários de camundongos (MEF) ou células mesenquimais derivadas de tecido adiposo humano (hASC) em células iPS. Primeiramente o PTD TAT ou TAT- foi fundido a proteína verde fluorescente (GFP) como modelo para prova de princípio e padronização detalhada. Inesperadamente, TAT-GFP produzido e secretado pelas células NIH-3T3 produtora não foi capaz de ser detectado no meio de cultura por verificação quantitativa fluorimétrica, nem foi capaz de ser detectada em células-alvo, por citometria de fluxo, depois de co-cultura em transwells. Essa observação pode ser explicada por: (1) ineficiência desse tipo de célula em secretar proteínas e (2) falta de resistência à clivagem por endoproteases furinas. Para contornar esses fatores limitantes usou-se citometria de fluxo para avaliar as melhores condições para a transfecção por seis diferentes tipos de células (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t e COS-7) com TAT (modificada para ser resistente à furinas) fundido a GFP. Células 293t-TAT-GFP exibiram a maior eficiência de transfecção e também de secreção. O mesmo pôde ser observado para as seis linhagens celulares expressando fatores de transcrição nucleares TAT, determinados por ELISA. Em seguida, diferentes estratégias de entrega foram testadas. A primeira foi baseada na co-cultura de uma mistura de células produtoras com MEF ou hASC. No entanto, não foi possível observar a reprogramação devido à morte celular. A segunda foi baseada na concentração de meio condicionado de cultura de células por centrifugação usando colunas Amicon, trocando o meio a cada 24h, em quatro ciclos. No entanto, apesar da presença de algumas colônias após 20-30 dias, nenhuma colônia verdadeira iPS foi obtida. Na sequência, as células foram tratadas com cada proteína de forma independente, e as demais foram substituídas pelo retrovírus correspondente, trocando meio a cada 72h, em quatro ciclos. Essa estratégia, apesar de permitir verificar a função de cada proteína, também não resultou em reprogramação. Este achado pode ser explicado pela diferenciação celular induzida por BCS, que também é concentrado no processo. Assim, passou-se a adaptação de \"células produtoras\" em condições de cultura livre de soro, para enriquecer a produção dos fatores nucleares individuais, necessários para a reprogramação. A otimização sistematizada deste processo está sendo realizada em parceria com o IPT e deve resultar em quantidades de proteína de fusão suficientes para o teste final da hipótese proposta. Em conjunto, são apresentados os dados da geração de linhagens celulares expressando estavelmente os vários fatores de transcrição e estratégias para melhorar a eficiência necessária para a produção iPS. Esta nova estratégia garante uma produção eficiente de TAT fundida a fatores nucleares de reprogramação e sua eficácia para promover a reprogramação de células somáticas de maneira livre de vírus merece ser investigado futuramenteViruses are effective at transferring genes into cells by its specialized mechanisms. However, viruses as gene delivery vehicles entail problems, particularly when proposed to reprogram somatic cells into induced pluripotent stem cells (iPS) for therapeutic uses. In the present study, we aimed to develop an alternative system for directly delivering nuclear proteins (Oct4, Sox2, Klf4, and c-Myc) fused with TAT protein transduction domain to promote reprogramming of mouse embryonic fibroblasts (MEF) or human adipose tissue derived mesenchymal cells (hASC) into iPS cells. First TAT- or TAT- PTD was fused to green fluorescent protein (GFP) as a proof of principle model and for detailed standardization. Unexpectedly, TAT-GFP produced and secreted by NIH-3T3 producer cells was not detected in the culture medium by quantitative fluorimetric verification, nor detected on target cells, by flow cytometry, after being co-cultured using transwells. This observation maybe explained by: (1) inefficiency of this cell type to be transfected and to secrete proteins and (2) lack of resistance to furin endoproteases cleavage on Golgi of TAT sequence. To circumvent these limiting factors we used flow cytometer to assess the best conditions for transfection in six different cell types (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t and COS-7) with TAT- (a modified PTD to be resistant to furin endoproteases) fused to GFP. 293t-TAT-GFP cells displayed the highest transfection efficiency and secretion levels. The same could be observed for the six cell lineages expressing TAT- nuclear transcription factors, determined by ELISA.Next, different delivery strategies were tested for TAT- nuclear transcription factor system. Co-culturing a mix of producer cells with MEF or hASC resulted in not reprogramming and this was associated with cell death. The second was based on the use of microconcentrated conditioned cell culture medium, changed every 24h, in four cycles. However, despite the presence of some emerging colonies after 20-30 days, no true iPS colonies were obtained. Then, cells were treated with each protein independently, and the others were replaced by the corresponding retrovirus, changing cell medium every 72h, in four cycles. We verified the reprogramming potential of each protein, but no true colonies were obtained.One possibility for this finding is that BCS is also concentrated by centrifugation and may induce cell differentiation. To circumvent these problems, we have started the adaptation of producer cells in a serum-free culture condition to enrich the production of the individual factors required for reprogramming. This optimization process is taking place in collaboration with the IPT and shall result in large amounts of the fusion protein to finally test the proposed hypothesis. Altogether, we presented the generation of several cell lines stably expressing the transcription factors and strategies to improve the efficiency required for iPS production. This novel strategy guarantees efficient production of TAT-fused reprogramming nuclear factors and its efficacy to promote somatic cells reprogramming in a virus-free manner deserves to be further investigated
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Bressan, Fabiana Fernandes. "Geração de células pluripotentes através da indução gênica e transferência de núcleo: modelo bovino de aquisição de pluripotência." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/74/74131/tde-04072013-151314/.
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Estratégias como a transferência nuclear e a reprogramação induzida vêm sendo empregadas com o objetivo de induzir células somáticas a um estado pluripotente similar ao embrionário. O processo de reprogramação nuclear e extremamente desejável e possui importantes contribuições tanto no estudo da ciência básica como aplicada, como por exemplo, no aumento da eficiência das biotécnicas de produção animal ou na medicina, com a possibilidade de terapia celular autóloga. Uma série de estudos, porem, ainda são necessários para que tais aplicações sejam viáveis, uma vez que os mecanismos fundamentais das técnicas empregadas ainda não estão totalmente elucidados. Esta proposta teve como objetivo gerar células bovinas pluripotentes através da reprogramação direta e utilizá-las na transferência de núcleo para a produção animal visando o aumento da eficiência da reprogramação celular. Para tal, foi analisada a capacidade de indução e manutenção da pluripotência em células somáticas bovinas comparando-as com células humanas e equinas (células pluripotentes induzidas - iPSC), assim como a capacidade de desenvolvimento de embriões produzidos através da combinação das técnicas em bovinos. As células iPS derivadas neste estudo foram produzidas mediante transdução lentiviral de fatores de transcrição (OSKM) murinos, caracterizadas e utilizadas como doadoras de núcleo na clonagem. Resumidamente, oócitos bovinos obtidos de ovários provenientes de abatedouros foram maturados in vitro por 18h, enucleados e reconstruídos com células iPS (n=203 ou fibroblastos fetais bovinos (bFF, n=153), em cinco repetições. Após reconstrução os embriões foram ativados com ionomicina e 6-DMAP e cultivados in vitro até o estágio de blastocisto. Foram avaliadas as taxas de fusão, clivagem (48h após ativação) e desenvolvimento a blastocisto (192h após ativação) e os resultados foram submetidos ao teste de Qui-quadrado a 5% de significância. Foi possível a produção de embriões a partir de biPS, entretanto, este estudo evidenciou a necessidade de otimização da sincronização do ciclo celular em células iPS. Não foram encontradas diferenças entre os grupos quanto à capacidade de produção a blastocisto ou clivagem, porém o grupo reconstruído com células iPS apresentou uma menor taxa de fusão. Com a finalidade de entender a influência de fatores de transcrição específicos na reprogramação nuclear, bFF expressando OCT4 humano (hOCT4) e hSOX2 combinados com as proteínas repórteres fluorescentes vexGFP e mCitrine, respectivamente, foram submetidas à separação celular por citometria de fluxo e utilizados como doadores de núcleo. Foram utilizados bFF expressando OCT4-vexGFP (n=182, quadruplicata), SOX2-mCitrine (n=203, quadruplicata) ou células controle (não transduzidas, n=178 e n=149, em quadruplicata para grupos OCT4 e SOX2, respectivamente). Não foram encontradas diferenças entre os grupos nas características de capacidade de desenvolvimento in vitro estudadas. Em conclusão, este estudo relata a possibilidade de produção de células bovinas reprogramadas, além de também mostrar que a transferência de núcleo utilizando células expressando hSOX2 ou hOCT4, ou já reprogramadas, resulta em taxas similares de produção embrionária quando comparadas à utilização de células controle. O conhecimento da contribuição de cada fator utilizado na reprogramação induzida, aliado a estudos de comparação com a capacidade de desenvolvimento in vitro de organismos derivados de células reprogramadas deverá contribuir para o aumento da eficiência da clonagem e produção animal in vitro como para a medicina regenerativa.Nuclear transfer and induced reprogramming are technologies usually used for the induction of somatic cells into an embryonic-like pluripotent status. The knowledgment of nuclear reprogramming process is highly desirable, leading to important contributions for both basic and applied sciences; for example, resulting in the increase in the efficiency of several animal biotechnologies, or else enabling autologous cellular therapy for medical purposes. However, basic studies are still needed in order to enable such applications, once the mechanisms controlling in vitro reprogramming are yet to be unraveled. This study aims to generate induced pluripotent bovine stem cells through direct reprogramming and its use in nuclear transfer in order to enhance the cellular reprogramming efficiency, For that, the potential of pluripotency induction and maintenance was analyzed in bovine somatic cells, comparing those with human and equine cells, as well as the potential of embryonic development after combining direct and nuclear reprogramming. iPS cells derived in this study were produced trought lentivirus transduction of mouse transcription factors (OSKM), further characterized and used as nuclei donors for cloning. In summary, bovine oocytes were obtained from slaughterhouse ovaries, in vitro matured for 18h, enucleated and reconstructed with iPS cells (n=203) or fetal fibroblasts (bFF, n=153), in five replicates. Embryos were reconstructed, chemically activated with ionomycin and 6-DMAP and cultured in vitro until blastocyst stage. Fusion, cleavage (48h post activation) and blastocyst developmental rates (192h post activation) were analyzed and result submitted to Chi-square test at 5% significance. biPS enabled embryo production, however further optimization on cell cycle synchronization still needs to be accomplished. No difference was observed between groups regarding cleavage or blastocyst developmental rates, however iPS group presented a reduced fusion rate when compared to control. For a better understanding on how reprogramming associated transcription factors could influence on nuclear reprogramming, bFF expressing human OCT4 (hOCT4) or hSOX2 combined with the fluorescent protein reporters vexGFP and mCitrine, respectively, were submitted to flow citometry cell sorting and used as nuclei donors. bFF expressing OCT4-vexGFP (n=182, quadruplicate), SOX2-mCitrine (n=203, quadruplicate) or control cells (non transduced, n=178 and n=149, in quadruplicate for OCT4 and SOX2, respectively) were used. No difference was observed between groups regarding the in vitro developmental potential rates. In conclusion, the present study reports the generation of reprogrammed bovine cells, and its use the nuclear transfer. Donor cells expressing hOCT4, hSOX2 or reprogrammed cells resulted in similar developmental in vitro rates when compared to controls. The knowledge of each reprogramming factor influence on in vitro reprogramming, together with comparison studies on in vitro developmental potential of organisms derived from reprogrammed cells should help enhancing not only the cloning efficiency and in vitro animal production, but also the regenerative medicine.
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Mao, Weiming. "The role of bHLH gene ash1 in the developing chick eye." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2009r/mao.pdf.
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Halley-Stott, Richard Paul. "Quantitative transcriptional reprogramming of somatic cell nuclei with Xenopus leavis oocytes." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610108.
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Murata, Kazutaka. "Histone modifications and transcriptional reprogramming in mouse somatic cell nuclei transplanted to Xenopus laevis oocytes." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611676.
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Li, Zhonghan. "Dissecting Somatic Cell Reprogramming by MicroRNAs and Small Molecules: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/607.
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Somatic cells could be reprogrammed into an ES-like state called induced pluripotent stem cells (iPSCs) by expression of four transcriptional factors: Oct4, Sox2, Klf4 and cMyc. iPSCs have full potentials to generate cells of all lineages and have become a valuable tool to understand human development and disease pathogenesis. However, reprogramming process suffers from extremely low efficiency and the molecular mechanism remains poorly understood.
This dissertation is focused on studying the role of small non-coding RNAs (microRNAs) and kinases during the reprogramming process in order to understand how it is regulated and why only a small percentage of cells could achieve fully reprogrammed state. We demonstrate that loss of microRNA biogenesis pathway abolished the potential of mouse embryonic fibroblasts (MEFs) to be reprogrammed and revealed that several clusters of mES-specific microRNAs were highly induced by four factors during early stage of reprogramming. Among them, miR-93 and 106b were further confirmed to enhance iPSC generation by promoting mesenchymal-to-epithelial transition (MET) and targeting key p53 and TGFβ pathway components: p21 and Tgfbr2, which are important barrier genes to the process.
To expand our view of microRNAs function during reprogramming, a systematic approach was used to analyze microRNA expression profile in iPSC-enriched early cell population. From a list of candiate microRNAs, miR-135b was found to be most highly induced and promoted reprogramming. Subsequent analysis revealed that it targeted an extracellular matrix network by directly modulating key regulator Wisp1. By regulating several downstream ECM genes including Tgfbi, Nov, Dkk2 and Igfbp5, Wisp1 coordinated IGF, TGFβ and Wnt signaling pathways, all of which were strongly involved in the reprogramming process. Therefore, we have identified a microRNA-regulated network that modulates somatic cell reprogramming, involving both intracellular and extracellular networks.
In addition to microRNAs, in order to identify new regulators and signaling pathways of reprogramming, we utilized small molecule kinase inhibitors. A collection of 244 kinase inhibitors were screened for both enhancers and inhibitors of the process. We identified that inhibition of several novel kinases including p38, IP3K and Aurora kinase could significantly enhance iPSC generation, the effects of which were also confirmed by RNAi of specific target genes. Further characterization revealed that inhibition of Aurora A kinase enhanced phosphorylation and inactivation of GSK3β, a process mediated by Akt kinase. All together, in this dissertation, we have identified novel role of both small non-coding RNAs and kinases in regulating the reprogramming of MEFs to iPSCs.
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Goel, Vritti R. "A Proposal to Test the Effects of Factor ECAT1 on Pluripotency, from Reprogramming to Differentiation of Human Somatic Cells." Scholarship @ Claremont, 2012. http://scholarship.claremont.edu/cmc_theses/470.
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The field of stem cell research has been growing more because of the interest in using stem cells to cure diseases and heal injuries. Human embryonic stem cells, because of the controversy surrounding them—and subsequently the difficulties in acquiring samples of the existing aging cell lines—can only be used in limited capacities. While the development of induced pluripotent stem cells in the last decade has allowed the field to progress closer to medical treatments, the low efficiency of reprogramming a somatic cell to a pluripotent state, and the vast molecular and genomic differences between human embryonic stem cells and human induced pluripotent stem cells is still an issue. Therefore, the goal is to discover methods, chemicals, and factors that can reduce these differences and increase the efficiency of inducing pluripotency.
This proposal aims to look at the effects of the protein ECAT1 in inducing pluripotency in human somatic cells. Little is known about ECAT1, otherwise known as Embryonic Stem Cell-Associated Transcript 1, beyond its presence in human embryonic stem cells and oocytes and its absence in differentiated cells. While originally considered by scientists during the development of the reprogramming technique, ECAT1's effects have not been tested in humans. Therefore, a series of experiments will be performed in which ECAT1 will be used in conjunction with OSKM to induce pluripotency in adult human dermal fibroblasts, which will then be differentiated into spinal motor neurons. The three stages of this proposal--inducing pluripotency, comparing pluripotencies in the reprogrammed cells and embryonic stem cells, and differentiating the stem cells--should answer questions about ECAT1 and the reprogramming process. It is predicted that ECAT1 should reduce the genomic and molecular differences between embryonic stem cells and induced pluripotent stem cells. ECAT1's presence should also increase the efficiency of reprogramming as well as successful differentiation to other cell types.
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Beyhan, Zeki. "Cellular and genomic reprogramming of donor nuclei in bovine nuclear transfer embryos." 2003. http://www.library.wisc.edu/databases/connect/dissertations.html.
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Moreira, Pedro N. "Molecular markers of somatic cell reprogramming by nuclear transplantation." 2002. https://scholarworks.umass.edu/dissertations/AAI3056261.
Full textAbstract:
Cloning of animals by nuclear transplantation has demonstrated that reprogramming of nuclear function is possible. However, low pregnancy rates, elevated pregnancy losses and lethal abnormalities in most cloned animals born argue that somatic cell reprogramming by nuclear transplantation is not always complete. Here, we report the identification of four nuclear markers of incomplete reprogramming in nuclear transplant mouse embryos. Nuclear transplant embryos exhibit (i) pronucleur assembly of A-type lamins, (ii) increased NuMA content, (iii) stronger anchoring of AKAP95 and (iv) a greater proportion of heterochromatin, compared to fertilized embryos. We propose that deficiencies in reprogramming through nuclear transplantation result from failure to morphologically remodel the somatic donor nucleus into a normal, fully functional pronucleus.
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Cheng, Dai-Han, and 鄭岱瀚. "The Effect of Dnmt3L and Trichostatin A Treatment in Reprogramming of Mouse Somatic Nuclei following Nuclear Transfer." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/23870036349068993474.
Full textAbstract:
碩士臺灣大學
動物科學技術學研究所
98
It has been more than a decade since “Dolly” the sheep was produced via somatic cell nuclear transfer (SCNT). Successful reprogramming of the differentiated cell by SCNT has been reported for more than 15 mammalian species that resulted in the live clones. Therefore, SCNT technology has been recognized as a powerful tool to insight into various fundamental studies in cellular, developmental, and molecular biology, as well as hold great promise in applications of agriculture and regenerative medicine. However, the cloning efficiency has remained extremely low, and was believed due to incomplete reprogramming of the somatic nuclei that contributed by insufficient epigenetic modifications. DNA methylation plays an important epigenetic modification of gene expression in early development. It has been demonstrated that DNA methyltransferase (Dnmt) activity, including Dnmt3L, interacted with histone deacetylase (HDAC), was involved in histone deacetylation, chromatin remodeling and transcription repression. On the other hand, Trichostatin A (TSA), a histone deacetylase inhibitor (HADCi) has a function of increase histone acetylation and DNA demethylation, has been demonstrated improve the efficiency of nuclear cloning in numerous of species. If knock out Dnmt3L (Dnmt3L-KO), will it affect histone acetylation or methylation of reconstructed embryos following SCNT? Any beneficial effect during reprogramming process when combine Dnmt3L-KO nuclei with TSA treatment after SCNT? Therefore, the object of this study is to determine: 1) the role of Dnmt3L during nuclear reprogramming by SCNT, and 2) the reprogramming potential of Dnmt3L-KO fibroblast cells by nuclear transfer following TSA treatment. We compare cloning efficiency by using mouse embryonic fibroblasts derived from wild type or Dnmt3L-KO mice as donor cells for SCNT and reconstructed embryos were treated with 10 nM TSA for10 h immediately after activation. We found that cloning efficiency from both wild type and Dnmt3L-KO groups were significantly increased following TSA treatment. The development rates of cloned blastocysts compared with those observed in non-treated control groups were: 32% vs. 4% in wild type, 63% vs. 8% in D3L-KO- derived embryos. In addition, we use antibodies to detect the distribution of Oct-4/Cdx2 and divided them to Grade 1 (good) to Grade 4 (bad) together with total cell number to classify quality of cloned embryos. Combination of Dnmt3L and TSA increased not only in good quality of Grade 1 embryos but also in total cell number of cloned blastocyst. Our data indicate that TSA treatment significantly increased the developmental potential of cloned embryos and there seems a synergistic effect between Dnmt3L-KO and TSA treatment during nuclear reprogramming by SCNT.
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Köhler, Daniela [Verfasser]. "Cloning in cattle : nuclear architecture and epigenetic status of chromatin during reprogramming of donor cell nuclei / Daniela Köhler." 2009. http://d-nb.info/993853102/34.
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Mendes, Luís Filipe Figueiredo. "Metabolomics study of flavonoid-induced metabolic reprogramming in human macrophages." Master's thesis, 2018. http://hdl.handle.net/10773/25305.
Full textAbstract:
The ability of macrophages to change between pro-inflammatory (M1-like) and anti-inflammatory (M2-like) phenotypes makes their modulation an attractive therapeutic strategy to mitigate excessive and/or chronic inflammation. Bioflavonoids are natural compounds with well documented anti-inflammatory activity. However, little is known about their molecular and metabolic effects on human macrophages. In this thesis, NMR metabolomics has been used to assess how different bioflavonoids (Quercetin, Naringenin and Naringin) modulate the metabolism of human macrophages, with a view to better understand their modes of action. In vitro-cultured macrophages differentiated from human THP-1 monocytes were treated with each flavonoid, both in the uncommitted state (M0) or after pre-polarization with LPS/IFN-γ (M1). Treatment with IL-4/IL-13 (M2) was also carried out for comparison. Cells were solvent-extracted to obtain the polar fractions and subsequently analysed by 1H NMR spectroscopy. Near 50 intracellular metabolites were detected (endometabolome), whereas analysis of cells-conditioned culture medium (exometabolome) was useful to assess consumption and excretion patterns. Multivariate analysis and discrete quantitative assessments of metabolic profiles revealed consistent variations upon macrophage exposure to the different stimuli. The three flavonoids produced pronounced metabolic alterations, with the strongest impact being observed for Quercetin and the mildest for Naringin. The main effects shared by all flavonoids, especially in M1 pre-polarized macrophages, comprised downregulation of glucose uptake and decreased energetic pools (ATP, UTP, phosphocreatine). Other effects were highly flavonoid-specific, namely TCA cycle modulation and amino acid metabolism, antioxidant response and variations in membrane-related metabolites. Overall, this work has shown that, although sharing similar ability to attenuate pro-inflammatory activity of macrophages, the three flavonoids affect macrophage metabolism in distinct ways. Future studies should entail assessment of key metabolic enzymes to further advance current understanding on flavonoid-mediated modulation of macrophage metabolism. This knowledge is expected to support the development of flavonoids as immunomodulatory drugs, especially aimed at attenuating and/or resolving inflammation, for instance, in the context of chronic inflammatory diseases or the rejection of biomaterial implants.A capacidade dos macrófagos alterarem o seu fenótipo entre estados pro-inflamatórios (tipo-M1) e anti-inflamatórios (tipo-M2), faz com que a sua modulação seja uma estratégia promissora para mitigar a inflamação excessiva e/ou crónica. Os bioflavonóides são compostos naturais com atividade anti-inflamatória bem documentada. No entanto, pouco se sabe acerca dos seus efeitos metabólicos em macrófagos humanos. Nesta tese, utilizou-se a metabolómica baseada em RMN para averiguar de que forma diferentes bioflavonóides (Quercetina, Naringenina e Naringina) modulam o metabolismo de macrófagos humanos, com vista a compreender melhor os seus mecanismos de ação. Macrófagos derivados de monócitos humanos THP-1 foram cultivados in vitro e tratados com cada flavonóide, tanto no estado não-polarizado (M0) como após uma pré-polarização com LPS/IFN-γ (M1). A estimulação com IL-4/IL-13 (M2) foi também empregue para comparação. As frações polares das células foram obtidas por extração com solventes orgânicos e subsequentemente analisadas por espetroscopia de RMN-1H. Perto de 50 metabolitos intracelulares foram identificados (endometaboloma), sendo que a análise dos meios de cultura (exometaboloma) foi útil para estudar padrões de consumo e excreção. O estudo dos perfis metabólicos através de análise multivariada e quantitativa revelou variações consistentes perante os diferentes estímulos fornecidos aos macrófagos. Os três flavonóides causaram diferentes alterações metabólicas, sendo que o maior impacto foi observado para a Quercetina e o menor para a Naringina. Os principais efeitos partilhados por todos os flavonóides, especialmente em macrófagos pré-polarizados para M1, incluíram diminuição do consumo de glucose e das reservas energéticas (ATP, UTP, fosfocreatina). Outros efeitos foram muito específicos dependendo do flavonóide, nomeadamente a modulação do ciclo TCA e do metabolismo de aminoácidos, a resposta anti-oxidante e variações em metabolitos associados às membranas. De forma geral, este estudo demonstrou que, apesar de apresentarem capacidade semelhante de atenuar a atividade pró-inflamatória de macrófagos, os três flavonóides afetaram o metabolismo celular de formas distintas. Em estudos futuros, será importante avaliar a expressão/atividade de enzimas metabólicas chave, de modo a incrementar o conhecimento atual sobre a modulação do metabolismo de macrófagos mediada por flavonóides. Este conhecimento deverá pois suportar o desenvolvimento de flavonóides como fármacos imunomodulatórios, especialmente direcionados para a atenuação e/ou resolução da inflamação, por exemplo, no contexto de doenças associadas a inflamação crónica ou da rejeição de biomateriais implantados.
Mestrado em Bioquímica
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Hao, Ru [Verfasser]. "Reprogramming of mesenchymal stem cells and adult fibroblasts following nuclear transfer in rabbits / by Ru Hao." 2008. http://d-nb.info/993277586/34.
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Liang, Yachun, and 梁雅淳. "Investigation of some selected anthraquinone derivatives as potential PARP inhibitors in lung cancer cells and nuclear reprogramming." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/03400329543827269587.
Full textAbstract:
碩士國防醫學院
藥學研究所
99
The abundant nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1), a multifunctional regulator of chromatin structure and DNA repair. High expression of PARP-1 has been indicated in several cancer types, so inhibitors of PARP-1 might be effective as therapeutic agents for the treatment of tumors. Germline mutations in the genes BRCA sensitize breast cancer cells to PARP inhibitors in a PARP-1-dependent manner. BRCA mutation also has been observed in non-small-cell lung cancers (NSCLCs). We screen and develop some lead compounds with anthraquinone-based pharmacophore, which have cytotoxicity to NSCLCs partly via inhibiting PARP activity. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient reprogramming clones. We found expression of PARP-1 is different between mouse embryonic fibroblast (MEFs) and induced pluripotent stem (iPS) cells. During reprogramming process, treating with PARP inhibitor and our found one putative PARP inhibitor of anthraquinone derivatives (NSC747854) might suppress the efficiency of colony formation and decrease secretion of alkaline phosphatase.
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Stroud, Todd. "Reprogramming DNA Methylation in Bovine Cells by Knocking Down DNA Methyltransferase-1 with RNA Interference." 2009. http://hdl.handle.net/1969.1/ETD-TAMU-2009-05-623.
Full textAbstract:
Embryos derived by somatic cell nuclear transfer (SCNT) produce few
pregnancies that result in a live, healthy offspring. This has largely been attributed to the
aberrant reprogramming of the somatic cell DNA used for cloning. In order to improve
the efficiency of cloning there is a great deal of research needed to determine the role of
proteins involved in early embryonic reprogramming. In addition, studies are needed to
determine effects on somatic and embryonic cell development as a result of altering
these proteins.
In this study we investigate the use of RNA interference in bovine somatic cells
and embryos to knock down the expression of DNA methyltransferase-1 (DNMT1), an
enzyme responsible for maintenance methylation in mammalian cells. We designed our
experiments to test whether or not knocking down the DNMT1 gene would lead to a
decrease in global methylation. It is our hypothesis that using somatic cells with reduced
methylation may be advantageous for increasing the efficiency of cloning via somatic
cell nuclear transfer. To accomplish this task, we have designed an infectious non-replicating lentiviral
vector capable of delivering a gene that produces a short hairpin RNA targeting the
mRNA of DNMT1. The construct included a sequence coding for green fluorescent
protein (GFP) that will allow us to identify cells expressing the hairpin as well as a
region coding for neomycin resistance so we could select for a pure population of
transgenic cells to use for analysis.
Infecting bovine fetal fibroblast cells with genes encoding shRNAs that target
DNMT1 was successful. Quantitative real time PCR analysis of DNMT1 mRNA
suggests that our shRNAs are capable of an 80% knockdown. The protein blot of
indicates up to 90% knockdown of DNMT1. Cells transduced twice with a high titer
virus showed the highest knockdown of both DNMT1 mRNA and the protein. Analysis
of immunolabeled cytosine methylaiton showed a global decrease in DNA methylation
as a result of the DNMT1 knockdown. However, double transduced cells with a high
knockdown percentage of DNMT1 mRNA and protein became hypermethylated.
The second experiment was conducted to determine the effect of injecting small
interfering RNAs (siRNAs) targeting DNMT1 into oocytes prior to parthenogenic
activation. This experiment was designed to give us information on the survivability and
epigenetic profile of early embryos with decreased DNMT1. Oocytes injected with
siRNA targeting DNMT1 had little development past the 8-cell stage as compared to the
sham injected oocytes. This treatment group also had decreased DNA methylation as
determined by immunolabeling of methylated cytosine residues.
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Swaminathan, Suresh Kumar [Verfasser]. "Screening for nuclear reprogramming factors and analysis of DNA demethylation during in vitro myoblasts differentiation / presented by Suresh Kumar Swaminathan." 2007. http://d-nb.info/98533407X/34.
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Adams, Allison M. "The knockdown of Dnmt1 using small inhibitory RNA a method to assist in the reprogramming of a donor genome during nuclear transfer /." 2004. http://purl.galileo.usg.edu/uga%5Fetd/adams%5Fallison%5Fm%5F200412%5Fms.
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