TOXOPLASMA DEVELOPMENT AND CELL CYCLE

描述(改编自申请人的摘要)：专有的细胞内 弓形虫寄生虫是一种重要的条件致病菌。 免疫功能受损的个人，如艾滋病患者。弓形虫还会导致 对新生儿造成毁灭性的出生缺陷。这些研究旨在 进一步了解弓形虫的致病机制 在其人类宿主内持续存在并引起疾病。工资支持 由K02奖提供的将使Kim博士有“保护时间” 专注于她努力采用其他公司的技术和专业知识 生物学研究领域，特别是真核细胞周期 对细胞周期和信号转导的研究 弓形虫发育。在人类宿主中，弓形虫以 速殖子，这是导致临床上明显疾病的原因，以及AS 缓殖子，这是一种潜在的囊状形式。随着免疫功能的减弱， 生长缓慢的缓殖子可以重新激活到快速增殖的 速殖子形成，导致弓形虫脑炎。考虑到被标记的 速殖子和缓殖子在增殖方面的差异 弓形虫不能在宿主细胞外复制，细胞周期 监管无疑是弓形虫发展和发展的关键 疾病的发病机制。从对其他真核生物的研究中可以清楚地看到， 对发育或分化信号的反应发生在特定的 细胞周期内的点，并导致坐标调制 阶段特异性基因表达与细胞周期进程。候选人 假设弓形虫细胞周期调控的独特方面 协调导致疾病发病机制和细胞内的过程 生死存亡。目的1：弓形虫细胞周期基因表达的调控 将重点分析核糖核苷酸还原酶(RNR)基因的表达。 目的2：弓形虫细胞周期蛋白依赖性蛋白激酶的表达和功能 (CDK)将使用免疫荧光、流式细胞术、Northern 和Western blotting。带有突变CDK的构建物将被转染到 速殖子以确定CDKs在细胞周期进程中的作用。 最后，在目标3中， 细胞周期调控与弓形虫缓殖子发育的关系 将会被研究。细胞周期标志物将被用来检查细胞周期 对缓殖子发育的依赖性。细胞周期与缓殖子报告 构建将被用来测试细胞周期抑制剂和 基因表达分化的潜在诱导剂或抑制物。 这些将是长期努力的初步实验，目的是定义 细胞周期调控在弓形虫速殖子-缓殖子中的作用 分化和致病力。

DESCRIPTION (adapted from applicant's abstract): The obligate intracellular parasite Toxoplasma gondii is an important opportunistic pathogen in immunocompromised individuals such as AIDS patients. T. gondii also causes devastating birth defects in neonates. These studies are designed to provide further understanding of the mechanisms by which T. gondii is able to persist and cause disease within its human host. The salary support provided by the K02 award will enable Dr. Kim to have "protected time" to concentrate upon her efforts to adapt technology and expertise from other areas of biological investigation, specifically eukaryotic cell cycle regulation and signal transduction, to the study of cell cycle and Toxoplasma development. Within human hosts, T. gondii exists as tachyzoites, which are responsible for clinically apparent disease, and as bradyzoites, which are latent encysted forms. With waning immune function, slow-growing bradyzoites can reactivate to the rapidly proliferating tachyzoite forms, leading to Toxoplasma encephalitis. Given the marked difference in proliferation of the tachyzoite compared to the bradyzoite and Toxoplasma's inability to replicate outside host cells, cell cycle regulation is undoubtedly critical in Toxoplasma development and pathogenesis of disease. It is clear from studies of other eukaryotes that responses to signals for development or differentiation occur at specific points within the cell cycle and result in a coordinate modulation of stage-specific gene expression and cell cycle progression. The candidate hypothesizes that unique aspects of T. gondii cell cycle regulation coordinate the processes leading to disease pathogenesis and intracellular survival. In Aim 1, regulation of cell cycle gene expression in T. gondii will be analyzed focusing on ribonucleotide reductase (RNR) gene expression. In Aim 2, expression and function of T. gondii cyclin dependent kinases (cdk) will be examined using immunofluorescence, flow cytometry, Northern and Western blotting. Constructs with mutant cdks will be transfected into tachyzoites to determine the role of cdks in cell cycle progression. Finally, in Aim 3, the relationship of cell cycle regulation and T. gondii bradyzoite development will be studied. Cell cycle markers will be used to examine the cell cycle dependence of bradyzoite development. Cell cycle and bradyzoite reporter constructs will be used to test the effects of cell cycle inhibitors and potential inducers or inhibitors of differentiation on gene expression. These will be the initial experiments in a long-term effort to define the role of cell cycle regulation in Toxoplasma tachyzoite-bradyzoite differentiation and virulence.