Collagen X and heparan sulfate proteoglycan in the hematopoietic stem cell niche.

我在德雷克塞尔大学的本科学习期间对细胞生物学和免疫学的复杂性产生了兴趣，在本科学习之后，我继续培养这种兴趣，在那里我在宾夕法尼亚大学的传染病系医学工程工作，并描述了各种新型的牛痘病毒载体用作活疫苗载体。然后我继续在托马斯杰斐逊大学读研究生，在那里我扩大了我的研究范围，包括普通细胞生物学、发育生物学和免疫学，研究对B型肝炎病毒的体内和体外免疫反应。在我的博士后研究期间，我已经能够进一步表征胶原X转基因小鼠和裸小鼠中的淋巴细胞生成缺陷，以及在体外和体内寄生虫挑战的细胞水平上描述免疫应答缺陷。我的博士后工作成果是一个描述软骨-骨连接处造血生态位的拟议模型，我将在K 01奖中进一步测试。 这个建议将是我的博士后工作的延续，其特征是软骨内骨化（EO），其中肥大的软骨被骨骼和骨髓取代，以及造血生态位的建立之间的联系。为此，将使用胶原X转基因和无效、串珠素亚型和多配体蛋白聚糖-1无效小鼠，因为我们提出，与胶原X小鼠一样，所有这些小鼠的疾病表型可能涉及生长板缺陷、骨髓环境改变和异常造血。我们提出，胶原蛋白X和硫酸乙酰肝素蛋白聚糖，例如串珠素和多配体蛋白聚糖-1，提供了一个结构网络，在肥大的软骨中隔离细胞因子，网络破坏可能会导致软骨-骨连接（COJ）处的不适当信号传导，导致造血缺陷。为了测试这一点，1）骨髓移植、共培养和细胞因子分析将证实改变的骨髓环境并鉴定COJ驻留细胞类型（造口、肥大软骨细胞或成骨细胞）不能支持X型胶原小鼠的造血，2）异位骨形成试验将证实EO产生造血生态位，X型胶原小鼠在此过程中存在缺陷，和3）流式细胞术和共培养测定将证实并开始表征串珠素亚型和多配体蛋白聚糖-1缺失小鼠中的造血缺陷。这些数据是对授予该研究者的NRSA完成目标的扩展，并将为表征细胞外基质对发育和疾病中富含细胞因子的小生境的贡献的未来目标提供数据。 这些研究将在宾夕法尼亚大学进行，该大学是美国竞争性研究赠款的顶级获得者之一，每年获得超过5亿美元的外部资金。我将在兽医学院动物生物学系工作的具体研究环境。成员是一个专门的基础科学小组，由约20名常设（助理）教授和7名研究教授组成，他们都由NIH或NSF资助，并在生物化学，细胞，分子和发育生物学方面进行积极的研究。正在研究的主题包括骨骼发生、造血、转基因和精原细胞移植、克隆、线粒体、模式形成、染色质结构、血管生成、作为翻译后修饰的去乙酰化、分化的转录调节、生长激素、呼吸、细胞周期、细胞死亡、精子发生、肥胖、应激、免疫学、眼睛发育和恐龙古生物学。教师和博士后研究员在部门研讨会上互动，并有良好的思想交流，技术援助和设施共享。在兽医学院之外进行互动的机会是毫不费力的，因为我们在宾夕法尼亚大学校园内，其中包括医学和牙科学校，Wistar研究所以及费城儿童医院。此外，在宾夕法尼亚大学，我们有机会参加五月校际研讨会和座谈会与托马斯杰斐逊大学，福克斯大通，天普大学，以及在费城周边郊区的许多小学院。最后，宾夕法尼亚大学的教职员工将为我提供一个独特的机会，寻求骨骼/基质生物学，血液学和B细胞生物学方面的建议，包括：杰先科博士和爱默生博士（我的导师），汉肯森博士，索斯洛斯基博士和亚当斯博士（在我的指导委员会），博士。（我的合作者），以及众多的教师，我将在许多杂志俱乐部，研讨会和研讨会在宾夕法尼亚大学和周围的学校提供互动。

I developed an interest in the complexities of cell biology and immunology during my undergraduate studies at Drexel University, which I continued to foster after my undergraduate studies, where I worked at the University of Pennsylvania in the Department of Infectious Diseases in Medicine engineering and characterized various novel Vaccinia virus vectors to be used as live vaccine prophylactics. I then went on to graduate school at Thomas Jefferson University where I broadened my studies to include general cell biology, developmental biology and immunology studying in vivo and in vitro immune responses to Hepatits B virus. During my post doctoral studies, I have been able to further characterize the lymphopoietic defects in the collagen X transgenic and null mice, as well as describe the immune response defects at both a cellular level in vitro and in vivo with parasite challenges. The outcome of my postdoctoral work is a proposed model depicting the hematopoietic niche in the cartilage-bone junction, which I will test further in this K01 award. This proposal will be a continuation of my post doctoral work characterizing the link between endochondral ossification (EO), where hypertrophic cartilage is replaced by bone and marrow, and the establishment of a hematopoietic niche. To do this, collagen X transgenic and null, perlecan hypomorphic and syndecan-1 null mice will be used, since we propose that, as in collagen X mice, the disease phenotypes of all these mice may involve growth plate defects, an altered marrow environment and aberrant hematopoiesis. We proposed that collagen X and heparan sulfate proteoglycans, e.g. perlecan and syndecan-1, provides a structural network that sequesters cytokines in hypertrophic cartilage, and network disruption may cause inappropriate signaling at the chondro-osseous junction (COJ) leading to defective hematopoiesis. To test this, 1) bone marrow transplants, co-cultures and cytokine analyses will confirm the altered marrow environment and identify the COJ resident cell type (stomal, hypertrophic chondrocyte, or osteoblast) unable to support hematopoiesis in the collagen X mouse, 2) ectopic bone forming assays will confirm EO generates the hematopoietic niche and that collagen X mice are defective in this process, and 3) flow cytometry and co-culture assays will confirm and begin to characterize the hematopoietic defects in the perlecan hypomorphic and syndecan-1 null mice. These data are an extension to the completed aims of a NRSA awarded to this investigator, and will provide data towards future goals of characterizing the extracellular matrix contribution to cytokine rich niches in development and disease. These studies will be performed at the University of Pennsylvania, which is ranked among America's top recipients of competitive research grants, earning more than $500 million in external funding annually. The specific research environment that I will be working is in the Department of Animal Biology at the School of Veterinary Medicine. The members are an exclusively basic science group, consisting of ~20 standing (Assistant to Full) professors, and 7 research professors, all of who are funded by NIH or NSF, and are conducting active research in biochemistry, cell, molecular, and developmental biology. Subjects under investigation include skeletogenesis, hematopoiesis, transgenesis and spermatogonial transplanation, cloning, mitochondria, pattern formation, chromatin structure, angiogenesis, arginylation as a posttranslational modification, transcriptional regulation of differentiation, growth hormones, respiration, cell cycle, cell death, spermatogenesis, obesity, stress, immunology, eye development, and dinosaur paleobiology. The faculty and postdoctoral fellows interact at department seminars, and there is good exchange of ideas, technical assistance, and sharing of facilities. Opportunities for interactions outside of the Veterinary School are effortless, since we are within the University of Pennsylvania campus, which includes the Medical and Dental Schools, Wistar Institute, as well as Children's Hospital of Philadelphia. Further, at the University of Pennsylvania we have the opportunity to participate in may intercollegiate seminars and symposiums with Thomas Jefferson University, Fox Chase, Temple University, and the many small collages in the surrounding suburbs of Philadelphia. Finally, the Penn faculty members that will provide me with a unique opportunity to seek advise in skeletal/matrix biology, hematology, and B cell biology, including: Drs. Jacenko and Emerson (my mentors), Drs. Hankenson, Soslowsky, and Adams (on my mentoring committee), Drs. Cancro and Akintoe (my collaborators), and the numerous faculty members that I will interact with at the many journal club, seminar, and symposiums offered at Penn and the surrounding schools.