Regulation of Nuclear Receptor Expression and Function

最新进展。我的研究计划的总体目标是增加我们对化学生物学以及控制核受体超家族成员表达和功能的细胞和分子因素的复杂网络的了解，如孕烷X受体(PXR)、构成雄烷受体(CAR)和维生素D受体(VDR)。这些受体调节与许多化学物质的运输、生物激活和解毒有关的基因的表达，这些化学物质包括药物、环境化学物质以及那些存在于我们体内的化学物质，如类固醇激素和维生素。在过去的五年里，我的研究主要集中在研究PXR、CAR和VDR的化学调控，阐明PXR表达的转录后机制，并发现PXR和CAR的新的生物学作用。这些研究的结果为我的NSERC发现补助金续签申请提供了科学基础。目标。其目的是通过研究参与调控PXR和VDR表达和功能的特定microRNAs的作用来研究PXR和VDR的共同调控，识别和描述共激活因子在PXR和CAR调控中的作用，并研究其他受体对PXR和CAR的调控。我的研究计划的长期目标是加强我们对核受体系统生物学的基础知识。方法论。人肝细胞原代培养、人肠道细胞原代培养和LS180人腺癌细胞将作为实验模型。实验方法将包括使用microRNA的模拟和抑制剂、药理拮抗和反向激动剂、基因编辑、基于细胞的荧光素酶报告基因分析、时间分辨荧光共振能量转移竞争配体结合分析、荧光显微镜、免疫共沉淀、下拉试验、哺乳动物双杂交分析、染色质免疫沉淀分析、实时聚合酶链式反应和免疫印迹分析。HQP。我建议的研究计划将为2名硕士学生、2名博士生和5名本科生提供学习机会。这项研究将构成每个研究生论文的基础，并为本科生提供研究经验。冲击力。产生的基础科学知识将有助于增加我们对影响PXR和CAR表达和功能的各种因素的理解。这些信息将预测和解释我们对内源性物质、药物和其他化学物质的反应方式的个体差异。我提出的研究计划将为受体生物学领域的下一代科学家提供培训。我的HQP具备生物化学、细胞生物学和药理学方面的技能和经验，将为加拿大生命科学/生物技术/制药部门的研究和开发工作贡献他们的专业知识。

Recent Progress. The overall goal of my research program is to increase our understanding of the chemical biology and the intricate network of cellular and molecular factors that govern the expression and function of members of the superfamily of nuclear receptors, such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), and vitamin D receptor (VDR). These receptors regulate the expression of genes involved in the transport, bioactivation, and detoxification of many chemicals, including drugs, environmental chemicals, and those present in our body, such as steroid hormones and vitamins. My studies in the past five years have focused on studying chemical regulation of PXR, CAR, and VDR, elucidating post-transcriptional mechanism of PXR expression, and discovering novel biological actions of PXR and CAR. Results from these studies provide a scientific foundation for my NSERC Discovery Grant renewal application. Objectives. The objectives will be to study co-regulation of PXR and VDR by investigating the role of specific microRNAs involved in controlling the expression and function of PXR and VDR, identify and delineate the contribution of coactivators to the regulation of PXR and CAR, and investigate the regulation of PXR and CAR by other receptors. The longer-term objective of my research program is to enhance our fundamental knowledge of the systems biology of nuclear receptors. Methodology. Primary cultures of human hepatocytes, primary cultures of human intestinal cells, and LS180 human adenocarcinoma cells will be the experimental models. Experimental approaches will include the use of a mimic and an inhibitor of a microRNA, pharmacological antagonism and inverse agonism, gene editing, cell-based luciferase reporter gene assays, time-resolved fluorescence resonance energy transfer competitive ligand binding assay, fluorescence microscopy, co-immunoprecipitation, pull down assays, mammalian two-hybrid assay, chromatin immunoprecipitation assay, real-time polymerase chain reaction, and immunoblot analyses. HQP. My proposed research program will provide learning opportunities for 2 MSc students, 2 PhD students, and 5 undergraduate summer students. The research will form the basis of each graduate student's thesis and provide research experience to undergraduate students. Impact. The fundamental scientific knowledge generated will help increase our understanding of the various factors that influence PXR and CAR expression and function. The information will predict and explain inter-individual differences in how we respond to endogenous substances, drugs, and other chemicals. My proposed research program will provide training to the next generation of scientists in the field of receptor biology. Equipped with skills and experiences in biochemistry, cell biology, and pharmacology, my HQP will contribute their expertise to the research and development efforts in the life sciences/biotechnology/pharmaceutical sectors in Canada.