Studie av retinoid X reseptorer (Rxr) og Rxr -signalveier i Atlanterhavstorsk (Gadus morhua) ved bruk av immunokjemiske of ex vivo teknikker

• Main Author: Christiansen, Emily Marie
• Format: Master Thesis
• Language: Old English
• Published: The University of Bergen 2023
• Subjects: 759929; atlanterhavstorsk; atlantic cod; Gadus morhua
• Online Access: https://hdl.handle.net/11250/3091221

Nuclear receptors (NRs) comprise a large superfamily of ligand-activated transcription factors. Many NRs facilitate gene expression through heterodimeric binding to retinoid X receptor (RXR). RXR is thus part of a vast array of signaling pathways. Pollutants from anthropogenic origin are a concern as many have been found to cause endocrine disruption by acting as exogenous ligands for NRs. Organotin compounds, in particular, tributyltin (TBT) has been found to cause endocrine disruption through binding to RXR. TBT exposure remains a concern in aquatic environments due to its long half-life in sediments, giving rise to hotspots in areas of former high use such as shipyards and marinas. Atlantic cod (Gadus morhua) has become an important bioindicator species, however, only recently have we begun to unravel the properties of Rxr in Atlantic cod. Previously, our research group has established that there are four subtypes of Rxr in Atlantic cod, Rxra, Rxrb1, Rxrb2 and Rxrg. The focus of this thesis was to study signaling pathways governed by Rxr that may be affected by TBT exposure of Atlantic cod liver. To do so, the binding properties of custom-made antibodies, designed against a peptide sequence of gmRxra, were characterized, to determine if these antibodies could be used in downstream analysis with immunoassays, such as chromatin immunoprecipitation (ChIP). Importantly, the antibodies were found to bind Rxrb1 in liver tissues by using two-dimensional polyacrylamide gel electrophoresis in combination with immunoblotting and mass spectrometry analysis. The molecular weight (MW) of the immunoreacting band/spot identified as Rxrb1 was estimated to be 35 kDa, which is lower than its theoretical MW close to 50 kDa. This could be due to a truncation of Rxrb1 in Atlantic cod. Also, the antibodies were found to bind the Rxrb1 while denatured, but they were not able to capture this protein under the standard non-denaturing immunoprecipitation (IP) technique used in this thesis. A hydropathy plot and structural analyses ...