Understanding cellular and transcriptional regulatory changes in human aging.

摘要 在这个项目中，我们建议增加Marliette Rodriguez Matos女士作为我们项目的核心成员，研究 CD 4 + T淋巴细胞老化。 亲本R 01代表了一个雄心勃勃的项目，研究一种原代纯化的人类细胞类型， 与年龄相关的疾病有关，并已被证明表现出DNA的系统性变化， 甲基化在多种组织类型和物种中随年龄发生。我们的目标是了解 随着年龄的增长，分子基因组特性的变化谱是由于细胞亚型的变化，有多少是 细胞自主性，这些如何反映细胞信号传导对转录因子生物学的影响，以及个体间 DNA序列变异与这些分子和细胞表型相互作用。 为了实现这些目标，我们正在进行基因分型，T细胞受体扩增子测序，和染色质 使用ATAC-seq对样品进行可及性研究。我们最初的计划是加入大量的RNA-seq和DNA 甲基化研究，但我们现在正在探索一种单细胞RNA-seq方法， 他们担心DNA甲基化研究将是没有信息的，因为最近的数据表明， 这些变化代表了随着年龄的增长，组织中多能干细胞比例的下降，这可能解释了为什么 DNA甲基化时钟尚未被证明在纯化的细胞中起作用。我们已经用 CD 4+表面标记物，其耗尽所有前体细胞。我们正在进行的工作由凯瑟琳克罗克博士领导， 旨在阐明这个问题，以便我们明智地进行实验投资。 女士Rodriguez Matos将带头对队列进行所选的一组分子基因组检测 400个样本。她来到我们的小组与强大的湿板凳经验，并已证明自己是 基因组检测技术卓越。鉴于COVID-19造成的大规模项目延误， 罗德里格斯·马托斯女士补充说，这是基于实际考虑，这将使我们能够提供 该项目最后分析阶段所需的基因组数据。 我们将发展Rodriguez Matos女士对该项目的参与，作为结构化培训经验的一部分。我们 在本建议书中描述她的项目活动将如何涉及旨在促进可转移性的活动 skills.这些技能不仅与老龄化领域有关，还包括项目管理、沟通、 领导才能和监督技能，以及道德操守和廉正培训。她的分析能力将通过 与纽约基因组中心的联合PI Tuuli Lappalainen博士一起工作，完善了她的技能。 女士罗德里格斯·马托斯已经明确将她的职业目标定为未来的独立调查员。我们的目标在 R 01项目是给她的技能和出版物，这将使基础所需的结果。 我们将欢迎有机会培养一位西班牙裔美国妇女成为人类基因组学的未来领导者 尤其是在老龄化研究领域。

ABSTRACT In this project, we propose to add Ms. Marliette Rodriguez Matos as a central member in our project to study the aging of CD4+ T lymphocytes. The parent R01 represents an ambitious project to study a primary, purified human cell type that has been associated with age-related diseases and has been shown to manifest the systematic changes in DNA methylation that occur with age in multiple tissue types and species. Our goal is to understand how much of the spectrum of changes of molecular genomic properties with age are due to cell subtype changes, how many are cell-autonomous, how these reflect cell signalling effects on transcription factor biology, and how inter-individual DNA sequence variation interacts with these molecular and cellular phenotypes. Towards these goals we are performing genotyping, T cell receptor amplicon sequencing, and chromatin accessibility studies using ATAC-seq on the samples. Our original plan was to add bulk RNA-seq and DNA methylation studies, but we are now exploring instead a single cell RNA-seq approach with pseudobulking, and are concerned that the DNA methylation studies will be uninformative given more recent data that indicates these changes represent a decrease in multipotent stem cell proportions in tissues with age, which may explain why the DNA methylation clock has not been demonstrated to work in purified cells. We have purified our cells with the CD4+ surface marker, which depletes all precursor cells. Our ongoing work led by Dr. Katherine Crocker is designed to shed light on this issue so that we make our experimental investments wisely. Ms. Rodriguez Matos will take the lead in performing the chosen set of molecular genomic assays on the cohort of 400 samples. She comes to our group with strong wet bench experience, and has proven herself to be exceptional technically with genomic assays. Given the extensive project delays enforced by the COVID-19 pandemic, adding Ms. Rodriguez Matos is based on the practical consideration that this will allow us to deliver the genomic data needed for the final, analytical stage of the project. We will develop Ms. Rodriguez Matos’ participation in this project as part of a structured training experience. We describe in this proposal how her project activities will involve activities that are designed to foster transferrable skills. These skills will not only be related to the field of aging but also project management, communication, leadership, and supervisory skills, and training in ethics and integrity. Her analytical skills will be fostered by working with the group of co-PI Dr. Tuuli Lappalainen at the New York Genome Center, rounding out her skills. Ms. Rodriguez Matos’ has defined her career goal explicitly as a future independent investigator. Our goal during the R01 project is to give her the skills and publications that will give the foundation needed for that outcome. We would welcome the opportunity to train a Hispanic-American woman as a future leader in human genomics and in the field of aging research in particular.