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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Ubiquitous chromatin opening element (UCOE)-mediated human coagulationFactor IX secretion by lentiviral transduction of human mesenchymalstem cells

Wong, Chi-kin, Felix., 黃子鍵. January 2013 (has links)
Haemophilia B is a bleeding disorder caused by various mutations of the coagulation Factor IX gene (F9) resulting in qualitative or quantitative Factor IX protein (FIX) deficiency. Factor replacement therapy is the current standard of care. Cure may be possible in the near future by gene therapy — the transfer of normal copies of F9 to patients with haemophilia, causing establishment of FIX production and correction of the bleeding phenotype. Mesenchymal stem cells (MSC) are potential vehicles for gene delivery through ex vivo gene transfer and subsequent transplantation to the patient. Lentiviral vectors can transduce MSC effectively and mediate long term gene expression. However, gene expression may decline with time due to transgene silencing. Ubiquitous Chromatin Opening Element (UCOE) is a set of genetic sequences cloned from housekeeping genes that can maintain a transcriptionally competent, open chromatin structure and was shown to prevent gene silencing by resisting DNA methylation. We tested human F9 expression and FIX protein secretion by transducing MSC with lentiviral vectors that carry the FIX gene under the control of A2UCOE (A2UCOE-hF9). A2UCOE is a 2.2 kb sequence cloned from the HNRPA2B1–CBX3 gene loci that harbour UCOE function. A2UCOE-eGFP, an enhanced Green Fluorescent Protein (eGFP) gene expression construct, was used to assist in vector titration of A2UCOE-hF9 by flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MSC were transduced at various Multiplicities of Infection (MOIs) by A2UCOE-hF9 lentiviral vector. Upon transduction, F9 mRNA expression and FIX secretion were measured by qRT-PCR and ELISA respectively. Osteogenic and adipogenic differentiation assay were performed to compare differentiation potential before and after transduction at an MOI of 1. F9 mRNA expression and FIX secretion were both undetectable in untransduced MSC. Upon transduction, vector dose-dependent increase in F9 mRNA expression and FIX secretion were detected at MOIs of 1, 2, 4 and 8. The level of secreted FIX ranges from 20 to 150 μIU in 72 hours. Osteogenic and adipogenic differentiation were not affected post-transduction at an MOI of 1. In conclusion, FIX secretion by MSC was detected upon A2UCOE-hF9 lentiviral transduction. However, the level of FIX appeared to be low compared to published studies. Further studies are required to determine the cause of low FIX expression, develop methods to maximize FIX expression and confirm whether A2UCOE can prevent gene silencing and maintain sustainable gene expression. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine
2

PAX 23 in normal kidney development and as therapeutic targets in renal cancer

Hueber, Pierre-Alain. January 2007 (has links)
The PAX gene family of transcription factors plays a prominent role during embryogenesis however can be aberrantly re-activated during tumorigenesis and contributes to the malignant phenotype. / During embryonic kidney development, PAX2 exerts an anti-apoptotic function however its expression typically attenuates during the post-natal period. On the other hand, PAX2 aberrant expression is observed in the majority of Renal Cell Carcinomas (RCC). RCC is resistant to chemotherapy; up-regulation of anti-apoptotic genes is recognized to contribute to tumor resistance to chemotherapy. We hypothesized that the anti-apoptotic effect of the PAX2 gene that is expressed in RCC cells contributes to RCC and their resistance to chemotherapy-induced cell death. / Human embryonic kidney (HEK293) cells transfected with a PAX2 expression vector and exposed to cisplatin, were protected from apoptosis compared to control cells. Conversely, murine collecting duct cells stably transfected with PAX2 antisense cDNA had twofold increases in cisplatin-induced apoptosis. Similarly, PAX2 knockdown using PAX2 siRNA in RCC cells CAKI-1 and ACHN enhances cisplatin-induced apoptosis in vitro. / To test the combination of PAX2 expression silencing and cisplatin treatment in vivo we developed a model of renal tumors by injecting ACHN cells as a xenograft under the skin of nude mice. I showed that a PAX2 shRNA successfully knocks down PAX2 mRNA and protein levels in a RCC cell line (ACHN). ACHN cells stably transfected with shRNAs targeted against the PAX2 homeodomain, are more susceptible to cisplatin-induced caspase-3 activation than the control ACHN cell line. Furthermore, growth of subcutaneous ACHN/shPAX2 xenografts in nude mice is significantly more responsive to cisplatin therapy than control of ACHN cell tumors. This work proposes PAX2 as a potential therapeutic gene target in metastatic renal cell carcinoma and suggests that adjunctive PAX2 knockdown may enhance the efficacy of chemotherapeutic agents such as cisplatin. / Wilms tumor, the most common pediatric renal cancer, is thought to arise from a progenitor cell of the metanephric mesenchyme that fails to complete nephrogenesis. In addition to its characteristic triphasic histology, WT can exhibit myogenic differentiation. Myogenic programming during muscle development is controlled by a PAX3 transcription factor determinant for muscle development; unexpectedly PAX3 transcriptional activity has been recently identified in the embryonic mouse kidney. These observations led us to hypothesize that PAX3 plays a role during kidney development. Furthermore, we predict that if PAX3 expression is verified during renal development, PAX3 may also be expressed in Wilms tumor with a myogenic component. / I showed that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of the developing mouse kidney. In a panel of 20 Wilms tumors, PAX3 was identified in tumor samples with myogenic histopathology. Furthermore, mutations of WT1 were consistently associated with PAX3 expression in Wilms tumors and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. / This work supports a novel model of normal renal development in which progenitor cells of the metanephric blastema express PAX3 when targeted toward the stromal cell fate. Suppression of PAX3 is integral to the mesenchyme-to-epithelium transition, which defines the nephrogenic cell fate and may be accomplished, in part, by WT1. Conversely, failure to suppress PAX3 may account for the myogenic phenotype in a subset of WT1-negative Wilms tumors.
3

PAX 23 in normal kidney development and as therapeutic targets in renal cancer

Hueber, Pierre-Alain. January 2007 (has links)
No description available.

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