Physiology Core

摘要 生理学核心提供了一系列分级的体外技术，用于研究功能和调节 运输和其他膜居民蛋白质的复杂程度从单一的 分子来模拟天然上皮细胞的系统。为了了解生物过程， 健康和疾病状态下的肾功能，鉴于这一疾病的表型多样性和功能复杂性， 器官，使用允许在定义的条件下分离研究特定片段/细胞/蛋白质的策略， 条件是必需的。这个核心的总体目标是在分子和细胞水平上阐明 以及调节与肾脏健康（包括发育）和疾病有关的关键蛋白质。完成 为了实现这一目标，核心项目将为研究人员提供：a）用于分析RNA表达的显微解剖小管 和/或丰度、蛋白质表达、免疫定位和酶/转运蛋白微量测定，B）功能性 在分离的小管中单独鉴定的细胞中通道/转运蛋白功能的荧光测定 c）测量跨离体小管的跨上皮离子/溶质通量 d）用于体外微灌注的转运蛋白的功能分析的电生理学测定， 野生型或突变的异源表达系统，单独或与推定的调控基因组合， 蛋白质，e）在Ussing中模型和天然上皮中的跨细胞和细胞旁转运的分析 f）用于分析蛋白质的翻译后修饰（包括磷酸化）的技术， 泛素化、棕榈酰化和糖基化，以及g）研究组成、动力学和 通过评估分离的单细胞中的转录本丰度来研究调节机制。中央 核心的功能是提供核心执行的所有技术以及核心的使用的指导 核心设备。生理学核心将与其他核心协同互动，以：a）扩展研究 在异源表达系统或分离的小管中对动物模型进行，B）鉴定 靶向转运蛋白和相关调节途径的潜在治疗化合物，和c）执行 使用成像技术对运输和调节的表达进行详细的定量分析， 异源表达系统、模型和天然上皮细胞以及分离的小管中的蛋白质。

Abstract The Physiology Core provides a series of graded in vitro technologies for studying the function and regulation of transport and other membrane resident proteins with progressive degrees of complexity from single molecules to model systems to native epithelia. To gain understanding of biological processes that mediate kidney function in health and disease states, in light of the phenotypic diversity and functional complexity of this organ, the use of strategies that allow specific segment/cell/protein to be studied in isolation under defined conditions is required. The overall goal of this core is to elucidate at a molecular and cellular level the function and regulation of key proteins involved in kidney health (including development) and disease. To accomplish this goal, the Core will provide investigators with: a) microdissected tubules for analysis of RNA expression and/or abundance, protein expression, immunolocalization, and enzyme/transporter microassays, b) functional fluorescence assays of channel/transporter function in individually identified cells in isolated tubules microperfused in vitro, c) measurements of transepithelial ion/solute fluxes across isolated tubules microperfused in vitro, d) electrophysiological assays for functional analysis of transport proteins in heterologous expression systems, either wild-type or mutated, alone or in combination with putative regulatory proteins, e) analyses of transcellular and paracellular transport in model and native epithelia in Ussing chambers, f) technologies for analysis of post-translational modification of proteins including phosphorylation, ubiquitination, palmitoylation and glycosylation, and g) technologies to study composition, dynamics, and regulatory mechanisms through the assessment of transcript abundance in isolated single cells. A central function of the Core is to provide instruction in all the techniques performed by the Core and the use of the Core equipment. The Physiology Core will interact synergistically with the other Cores to: a) extend the studies conducted in either heterologous expression systems or isolated tubules to animals models, b) identify potential therapeutic compounds that target transporters and associated regulatory pathways, and c) perform detailed and quantitative analysis using imaging technologies of the expression of transport and regulatory proteins in heterologous expression systems, model and native epithelia, and isolated tubules.