Thesis

The effects of thyroxine on gene expression in fibroblasts from a male affected with X-linked dyskeratosis congenita

Thyroid hormones have an overarching influence on cellular processes and directly affect gene expression. Thyroid hormones are involved in telomere maintenance, ribosomal pseudouridylation, and p53-mediated apoptosis. Males affected with X-linked Dyskeratosis Congenita (XDC), a deficiency in the dyskerin complex, experience problems with their hair, teeth, nails, and skin, and are susceptible to developing cancer. XDC fibroblasts from a seven-year-old boy were grown using conventional tissue culture methods to assess the effect of an elevated level of thyroxine (T4) on apoptosis and gene expression. Cells exposed to various levels of thyroxine over time periods up to 96 hours were examined by fluorescence microscopy for the detection of apoptosis. Purified RNA was assessed by quantitative PCR (qPCR) for expression of 11 genes and two reference (housekeeping) genes. These genes were c-myc, dkc1, thra, thra truncated isoform, thrb, dio3, 18S rRNA, 28S rRNA, rpl11, p53, and mdm2, genes purported to be involved in thyroxine's signaling cascade and apoptosis. The reference genes were ube2d2 and β-actin. Quantitative PCR showed that the vast majority of the genes studied were down-regulated by 96 hours in both low and high thyroxine. Exposure of cells to thyroxine generally resulted in reduced expression of the genes tested. In conclusion, the results of this investigation indicated that apoptosis is observed in response to high T4 exposure, as expected. However, it remains unclear if p53 mediates this response. This study offers insight into the specific effects of thyroid hormone in a rare disease and may contribute to a larger body of research investigating central cellular mechanisms. This study was limited by its focus on one particular pathway of the thyroid hormone and did not include a wild-type cell strain to compare results. A future study would consider more IRES-type transcripts, crosstalk into other pathways including Wnt/β-catenin and Sonic hedgehog. Future studies could also include an investigation into the significance of prenatal and postnatal gene regulation from thyroid response elements. This may offer new understanding of the effect of various interventions in premature neonate healthcare.

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