Physiology Core

生理学核心

• 批准号: 9983064
• 负责人: Marcelo Daniel Carattino
• 金额: $ 14.52万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9983064
• 关键词: Address; Animal Model; Animals; Biochemical; Biological Assay; Biological Models; Biological Process; Carrier Proteins; Cell Line; Cell Separation; Cells; Characteristics; Collaborations; Complement; Data; Development; Disease; Electric Capacitance; Electrophysiology (science); Enzymes; Epithelial; Epithelial Cells; Epithelium; Equipment; Fluorescence; Generations; Genetic Transcription; Goals; Health; Imaging technology; Immunoblotting; In Vitro; Individual; Instruction; International; Ions; Kidney; Libraries; Light; Measurement; Mediating; Membrane; Modeling; Molecular; Mutate; Nephrons; Organ; Phenotype; Phosphorylation; Physiology; Post Translational Modification Analysis; Post-Translational Protein Processing; Proteins; RNA analysis; Regulation; Regulatory Pathway; Renal function; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Series; System; Technical Expertise; Techniques; Technology; Therapeutic; Tissues; Training; Transcript; Ubiquitination; design; genetic regulatory protein; glycosylation; imaging system; kidney imaging; mutant; palmitoylation; protein expression; single molecule; solute; tool; transcriptome sequencing

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.

摘要 生理学核心提供了一系列分级的体外技术来研究功能和调节 转运蛋白和其他膜驻留蛋白的复杂性从单一 分子来模拟自然上皮细胞的系统。为了了解生物过程中 健康和疾病状态下的肾功能，鉴于其表型多样性和功能复杂性 器官，使用允许特定片段/细胞/蛋白质在定义的条件下进行分离研究的策略 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 以及涉及肾脏健康(包括发育)和疾病的关键蛋白的调节。要完成 为了实现这一目标，Core将为研究人员提供：a)用于分析RNA表达的显微解剖的小管 和/或丰度、蛋白质表达、免疫定位和酶/转运体微量分析，b)功能 分离肾小管上皮细胞通道/转运蛋白功能的荧光分析 体外微灌流，c)跨离体肾小管跨上皮离子/溶质通量的测量 体外微灌流，d)转运蛋白功能分析的电生理学检测 异源表达系统，无论是野生型还是突变型，单独或与假定的调控相结合 蛋白质，e)模型和天然上皮跨细胞和旁细胞转运的分析 钱伯斯，f)分析蛋白质翻译后修饰的技术，包括磷酸化， 泛素化、棕榈酰化和糖基化，以及g)研究成分、动力学和 通过评估分离的单细胞中的转录丰度来调节机制。一个中环 核心的功能是提供核心执行的所有技术的指导，并使用 核心设备。生理学核心将与其他核心协同作用，以：a)扩展研究 在异源表达系统或分离的小管到动物模型中进行，b)识别 潜在的针对转运蛋白和相关调控途径的治疗化合物，以及c)执行 利用成像技术对转运和调控表达进行详细和定量的分析 异源表达系统中的蛋白质，模型和天然上皮，以及分离的小管。