MOLECULAR & BIOCHEMICAL ANALYSIS OF MUTANT VWF RECEPTORS

私家侦探一直专注于在学术界发展事业 从她早年作为一名非全日制的研究生到现在的医学 她的家乡波多黎各。在过去的几年里，私家侦探已经做了 赞助商乔纳森·米勒博士的研究。私家侦探已经 对血小板膜糖蛋白Ib/Ix缺陷患者的研究 血管性血友病因子受体复合体(VWF)这种受体 由GPIB-Alpha、GPIB-beta和Gpix组成。在她成长的过程中 经过研究训练，P.I.已经学到了许多分子生物学 技术，包括：分离和纯化DNA和RNA， Southern和Northern杂交，RNA斑点杂交，反向 转录和聚合酶链式反应技术，竞争性寡核苷酸 启动、DNA克隆和测序、定点突变和 人糖蛋白在昆虫细胞中的体外表达。这个 P.I.将应用这些和其他较新的技术来完成 拟开展的研究工作。拟议工作的目标之一是 突变型GPIB-α在稳定细胞表面膜上的表达 表达GPIB-β和Gpix的哺乳动物细胞系。最初， 工作将集中在受体复合体的表达上 含有突变的缬氨酸GPIB-α或突变的苯丙氨酸 我们的血板型von患者中的GPIB-β Willebrand病和变异的Bernard-Soulier病， 分别进行了分析。P.I.S的研究方法将是表达 纯合子(仅含突变型GPIB-β)或杂合子 (含突变型GPIB-α和野生型GPIB-α)突变体vWF 感受器。然后，私家侦探将研究每个人的功能活动 很复杂。为了实现这一目标，私家侦探将研究 RWF对这些突变受体的作用 其他调制器。预计这些研究将 揭示特定的氨基酸序列在细胞内的作用 成熟的GPIB-α在调节vWF结合中起重要作用。 最后，是私家侦探S想要识别分子 导致典型伯纳德-苏利尔病的异常 GPIB-α基因正常患者的表型。这部作品 应提供有关 GPIB/IX受体的组件，以及这两个 受体及其配体vWF。为了实现这些目标， 机构，通过P.I.‘董事长S致力于提供 充足的实验室空间和必要的资源，以创建 为P.I.创造了富有成效的研究环境。

The P.I. has been focused upon developing a career in academic medicine since her early years as a part-time research student in her native Puerto Rico. For the past few years, the P.I. has done research with the sponsor, Dr. Jonathan Miller. The P.I. has studied patients with defective platelet glycoprotein (GP) Ib/IX receptor complexes for von Willebrand factor (vWF). This receptor consists of GPIb-alpha, GPIb-beta, and GPIX. In the course of her research training, the P.I. has learned numerous molecular biology techniques, including: isolation and purification of DNA and RNA, Southern and Northern blot hybridization, RNA dot blot, reverse transcription and PCR techniques, competitive oligonucleotide priming, DNA cloning and sequencing, site-directed mutagenesis, and in vitro expression of human glycoproteins in insect cells. The P.I. will apply these and other newer techniques to accomplish the proposed research work. One of the goals of the proposed work is to express mutant GPIb-alpha on the surface membrane of stable mammalian cell lines expressing GPIb-beta and GPIX. Initially, the work will be focused in the expression of receptor complexes containing the mutant valine GPIb-alpha or the mutant phenylalanine GPIb-beta described in our patients with platelet-type von Willebrand disease and variant Bernard-Soulier disease, respectively. The P.I.'s research approach will be to express homozygous (containing mutant GPIb-beta alone) or heterozygous (containing mutant GPIb-alpha and wild-type GPIb-alpha) mutant vWF receptors. The P.I. will then study the functional activity of each complex. To accomplish this goal, the P.I. will study the binding of vWF to these mutant receptors in the presence of ristocetin or of other modulators. It is anticipated that these studies will reveal the roles of particular amino acid sequences within the mature GPIb-alpha that are important in modulating vWF binding. Finally, it is the P.I.'s intent to identify the molecular abnormality responsible for the classic Bernard-Soulier disease phenotype in patients with normal GPIb-alpha genes. This work should yield relevant information about the interaction among the components of the GPIb/IX receptor, as well as between this receptor and its ligand, vWF. To accomplish these goals, the institution, through the P.I.'s chairman, is committed to providing adequate laboratory space and the necessary resources to create a productive research environment for the P.I.