이교수님의 제안에 착안하여 저의 2004년도 AHA Predoctoral Fellowship Grant Proposal의 Specific Aims부분을 올려드립니다.
본 연구과제는 2004년 7월 부터 수행되어 지난달 (June, 2006) 완료되었습니다.
미국에서 박사과정 중에 계신 선생님들에게 도움이 되셨으면 하는 바램으로 올립니다.
연구서 작성에서 필요한 구성 요소들이 있습니다. 일반적으로 연구내용의 독창성, 일반성, 임상적의의, 파급효과 등을 이야기 하지요, 하지만 Training grant에서는 이것들 만큼이나 지원자의 장기목표와 본 연구과제가 지원자의 미래 연구방향에 어떠한 도움을 줄것 인가를 명백히 서술하는 것이 중요합니다. 구체적인 내용 보다는 전반적인 흐름과 구성요소들에 촛점을 맞추시면 전공 분야와 관계없이 많은 도움이 되실거라 생각됩니다.
참고로 본 연구과제의 Average merit score: 1.39, Percentile rank: 3.49% 였습니다.
Title: Functional Analysis of NFKB1 Gene Variation in Hypertensives: Exercise Training Intervention and Human Endothelial Cell Shear Studies
(P.I. Joon Young Park)
The inter-individual variability in responses to exercise training has been described as a normal biological phenomenon that may reflect genetic diversity. As a non-pharmacological intervention to improve cardiovascular disease (CVD) risk factors including hypertension, the beneficial effects of exercise training are highly variable among individuals. However, few studies have examined the role of gene variation in the intracellular mechanisms underlying the responses to exercise training. By understanding the role of genes that code for components involved in intracellular signal transduction pathways, researchers will gain a better understanding of how exercise training affects CVD risk factors. Furthermore, this knowledge may help to identify individuals who will benefit the most from exercise training.
Hypertension affects as many as 50 million people in the U.S. More than $34 billion is spent annually for medications, office visits, and laboratory tests related to the treatment of hypertension. It is thought that endothelium-dependent vasodilation is an important component of the pathophysiology of hypertension. Exercise training improves the vasodilatory capacity in hypertensives, in part, through increasing antioxidant enzyme levels and consequently, decreased oxidative stress levels in the vasculature. With respect to the vasculature, the exercise stimulus can be defined as wall shear stress. Acute exercise eliminates flow oscillations present at rest and promotes laminar shear stress which may activate various biological defense processes in endothelial cells. There is evidence that laminar shear stress induces gene expression of antioxidant enzymes, such as copper-zinc superoxide dismutase (SOD1) and manganese superoxide dismutase (SOD2) in endothelial cells, through the nuclear factor – kappa B (NF-kB) pathway. NF-kB family is a ubiquitous transcription factor that regulates various biological defense processes. The NFKB1 gene encodes important NF-kB subunits, p50/p105. The recently described NFKB1 -94 insertion/deletion ATTG (NFKB1 I/D) polymorphism affects de novo synthesis of NF-kB at the transcriptional level. It is thought that the NFKB1 I/D polymorphism may be an important genetic factor involved in the intracellular signal transduction pathways that are associated with vasodilatory capacity. Therefore, it is likely that the NFKB1 I/D polymorphism plays a role in the differential changes in vasodilatory capacity after exercise training in hypertensives by modulating downstream antioxidant enzyme gene expression.
The applicant’s long-term research goal is to identify genetic factors and understand their roles in the adaptations of the antioxidant system when exercise training is used to prevent and treat CVD. The objective of this proposed study is to investigate how the NFKB1 I/D polymorphism affects vascular adaptations to exercise training in hypertensive individuals. The central hypothesis is that the NFKB1 I/D polymorphism has functional characteristics that respond to laminar shear stress in endothelial cells, and that the polymorphism is associated with differential changes in vasodilatory capacity following exercise training in hypertensive subjects. This hypothesis has been formulated based on the applicant’s strong preliminary data showing that exercise-induced NF-kB activation and sequence analysis for NF-kB binding cis-element on antioxidant enzyme gene promoters. The applicant is uniquely qualified to undertake the proposed research based on his previous research experience in this area and based on his recently acquired skills in genetics. The work will be conducted in a research environment under the guidance of experts in exercise physiology and hypertension, vascular cell biology, and molecular genetics. The applicant will test the hypothesis and accomplish the objectives of this proposal by conducting an in vivo exercise intervention and an in vitro laminar shear stress study on Human Umbilical Vein Endothelial Cells (HUVECs) in hypertensives.
1.Determine whether the NFKB1 I/D polymorphism is associated with changes in vasodilatory capacity measured by forearm plethysmography during reactive hyperemia before and after exercise training in hypertensives. The working hypothesis is that the NFKB1 I homozygote subjects will have a greater increase in vasodilatory function following a standardized exercise training intervention than NFKB1 D allele carriers.
2.Determine whether the NFKB1 I/D polymorphism has functional characteristics that respond to in vitro physiological levels of laminar shear stress in HUVECs. Based on previous research on this polymorphism, the working hypothesis is that NFKB1 I homozygotes will have higher promoter activities, binding activities, and SOD isoform protein levels than NFKB1 D allele carriers after application of in vitro shear stress to HUVECs.
2.1 Determine whether the NFKB1 I/D polymorphism affects promoter activity following in vitro laminar shear stress in HUVECs.
2.2 Determine whether the NFKB1 I/D polymorphism affects NF-kB binding activity following in vitro laminar shear stress in HUVECs.
2.3 Determine whether the NFKB1 I/D polymorphism affects intracellular SOD isoform (SOD 1, SOD2, and SOD3) protein levels following in vitro laminar shear stress in HUVECs.
The proposed study is innovative because, to our knowledge, it will be one of few studies to investigate a genetic factor directly involved in a signaling pathway that is activated by exercise. In addition, the complementary in vivo and in vitro experiments are a novel study design to assess physiological and molecular adaptations to exercise training in hypertensive subjects. These results will have clinical significance because they will provide new insights into the genetic factors involved in the beneficial effects of exercise training in hypertensives. Also, this predoctoral fellowship will allow the applicant to gain valuable research training in his area of research interest and will be the initial step in launching his independent research career investigating gene-exercise interactions linked to the antioxidant system.