Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis

项目概要/摘要 该多样性补充奖R 01 AI 156382，“在动质体中开发多重测定法” 寄生虫识别糖酵解探针”将支持克莱姆森大学Sabrina Pizarro的培训 遗传学和生物化学系的研究生来自卡托巴印第安民族， 在科学领域代表性不足的群体。这个项目的目标是双重的。首先，该项目将支持 萨布丽娜的研究工作是开发和验证用于非洲锥虫的新分子工具， 布氏锥虫Sabrina将产生前循环形式（PF）和血流形式（BF）T。布鲁氏菌寄生虫， 表达基于pHluorin 2的蛋白质pH生物传感器。pHluorin 2是比率式pHluorin变体，其显示pH 可以通过流式细胞术和显微术容易地监测（酸）依赖性激发。PF和BF寄生虫 表达这些传感器将用于解决酸化的细胞机制或亚细胞器 它负责糖酵解，也就是糖体。pH敏感细胞系将用于另外的目的，在以下方面发挥作用： 主提案中描述的工作。pHluorin 2线将用作反屏幕，使我们能够 区分通过破坏细胞或糖体pH影响糖酵解酶活性的小分子， 这些抑制剂更直接地抑制途径中的酶。该项目的第二个目标是支持 塞布丽娜的职业发展，因为她的工作，以实现她的职业目标。通过与PI的互动 和共同导师，沿着参与各种专业发展机会，我们预计， 塞布丽娜将完全有能力成功地追求她的职业道路的下一步。

Project Summary/Abstract This Diversity Supplement to the award R01AI156382, "Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis” will support the training of Sabrina Pizarro, a Clemson University graduate student in the Department of Genetics and Biochemistry that is from the Catawba Indian Nation, an under-represented group in the sciences. The goal of this project is two-fold. First, the project will support Sabrina’s research effort to develop and validate new molecular tools for use in the African trypanosome, Trypanosoma brucei. Sabrina will generate procyclic form (PF) and bloodstream form (BF) T. brucei parasites that express a protein pH biosensor based on pHluorin2. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid) dependent excitation that can be readily monitored by flow cytometry and microscopy. PF and BF parasites expressing these sensors will be used to resolve cellular mechanisms of acidification or the subcellular organelle that houses glycolysis, the glycosome. The pH sensing cell lines will serve an additional purpose, playing a role in the work described in the parent proposal. The pHluorin2 lines will be used as a counterscreen, allowing us to discriminate small molecules that influence glycolytic enzyme activity by disrupting cellular or glycosomal pH from those inhibitors that more directly inhibit enzymes in the pathway. The second goal of the project is to support Sabrina’s professional development as she works to achieve her career goals. Through interactions with the PI and co-mentor, along with participation in a variety of professional development opportunities, we anticipate that Sabrina will be fully positioned to successfully pursue the next step in her career path.