THE PHYSIOLOGY OF DRUG RESISTANT MALARIA

为了迅速开发新的和更好的治疗方法来防治抗药性疟疾，我们必须充分阐明抗疟作用和抗疟药物抗药性的分子机制。 此外，迫切需要对各种抗疟药物耐药表型进行更快速和方便的生化和生理测定。 这两个问题都有效地解决了通过更详细的，单细胞水平的疟疾寄生虫生理学和生物物理学分析。 因此，我们提出了一个详细的分析运输，pH值和膜电位现象，在生活红细胞内的疟疾寄生虫，使用现代亚细胞成像和单细胞光度法。 具体而言，我们将：1）使用恒定灌注下细胞的单细胞光度法和激光共聚焦显微镜，测试活的红细胞内恶性疟原虫的胞质和空泡pH（pHcyt，pHvac）和膜电位与抗疟药物抗性之间的推定关系。2)阐明pHcyt、pHvac和膜电位调节的离子依赖性、抑制剂敏感性和动力学特征。 3)使用抗性和敏感寄生虫之间遗传杂交的后代，从1，2中得出测试结论。 4)确定抗疟药物和逆转剂对生物物理参数的影响。 5)将恶性疟原虫和间日疟原虫的实验室菌株与田间分离株的测量结果进行比较和对比。沿着定义耐药寄生虫的离子转运生理学，这些实验将着眼于发展有助于快速、“现场兼容”诊断耐药疟疾的原理。

To rapidly develop new and better therapies to combat drug resistant malaria, we must fully elucidate the molecular mechanisms of antimalarial action and antimalarial drug resistance. In addition, more rapid and convenient biochemical and physiological assays for various antimalarial drug resistance phenotypes are desperately needed. Both of these issues are effectively addressed via more detailed, single-cell level analysis of malarial parasite physiology and biophysics. We thus propose a detailed analysis of transport, pH, and membrane potential phenomena in living intraerythrocytic malarial parasites using modern subcellular imaging and single - cell photometry methods. Specifically, we will: 1) Test putative relationships between cytosolic and vacuolar pH (pHcyt, pHvac) and membrane potentials of living intraerythrocytic P. falciparum and resistance to antimalarial drugs using single-cell photometry of cells under constant perfusion and laser confocal microscopy. 2) Elucidate ion dependencies, inhibitor sensitivities, and kinetic characteristics of pHcyt, pHvac, and membrane potential regulation. 3) Test conclusions from 1,2 using progeny of genetic cross between resistant and sensitive parasites. 4) Define effects of antimalarial drugs and reversal agents on biophysical parameters. 5) Compare and contrast measurement with laboratory strains vs. field isolates of P. falciparum and P. vivax. Along with defining the ion - transport physiology of drug resistant parasites, these experiments will be performed with an eye towards development of principles that will aid in the rapid, "field compatible", diagnosis of drug resistant malaria.