The immediate goal of the applicant is to devote 75% of his time during the five-year support period of this K02 award to the exploration of novel approaches to subcellular biology. The time off from teaching and service provided by the award would allow him to focus more intensely on the development of new strategies based on individual organelle analysis that would then be used to better understand disease and aging. These strategies are being developed as part of two ongoing R01 projects in his laboratory that investigate (i) the role of mitochondrial DNA (mtDNA) mutations in aging and age-related diseases, and (ii) subcellular drug metabolism. This award would also allow the candidate to devote more time to (i) integrating robotics, cybrid technology, and proteomics expertise into his laboratory; (ii) training scientists, (iii) establishing a solid network of collaborations, and (iv) providing cohesive leadership to a multi-disciplinary research team. In the research plan of this application, the candidate proposes to study the distributions of mtDNA mutations based on individual mitochondrion measurements. While the accumulation of these mutations has been implicated in the aging process and age-related diseases, the link between mutation levels and age-related phenotypes or disease symptoms is not known. The hypothesis of this application is that individual mitochondria contain both wild-type and mutated DNA, a condition known as heteroplasmy, which determines how mutations are distributed and propagated. Two models will be used to test this hypothesis: cybrid cell lines harboring large mtDNA deletions, and skeletal muscle tissue from aged Fisher 344 rats that is expected to have accumulated similar deletions with age. The three goals of the study are: (i) establish the existence of heteroplasmy within individual mitochondria, (ii) monitor changes in heteroplasmy following cybrid fusion, and (iii) measure heteroplasmy along skeletal muscle fibers. Since no technology exists to directly test this hypothesis, this application will require the further development of the applicant's strategies initially on based capillary electrophoresis with laser-induced fluorescence detection for characterizing mtDNA and peptide expression in individual organelles. Upon the completion of this K02 award, the applicant is expected to have provided the scientific community with new technologies and to be directing a widely recognized research program. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Scientist Development Award - Research (K02)
Project #
5K02AG021453-04
Application #
7025013
Study Section
National Institute on Aging Initial Review Group (NIA)
Program Officer
Finkelstein, David B
Project Start
2003-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
4
Fiscal Year
2006
Total Cost
$108,135
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Xu, Xin; Arriaga, Edgar A (2009) Qualitative determination of superoxide release at both sides of the mitochondrial inner membrane by capillary electrophoretic analysis of the oxidation products of triphenylphosphonium hydroethidine. Free Radic Biol Med 46:905-13
Feng, Juan; Navratil, Marian; Thompson, LaDora V et al. (2008) Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging. Anal Bioanal Chem 391:2591-8
Kostal, Vratislav; Katzenmeyer, Joseph; Arriaga, Edgar A (2008) Capillary electrophoresis in bioanalysis. Anal Chem 80:4533-50
Feng, Juan; Arriaga, Edgar A (2008) Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detection. Electrophoresis 29:475-82
Kostal, Vratislav; Arriaga, Edgar A (2008) Recent advances in the analysis of biological particles by capillary electrophoresis. Electrophoresis 29:2578-86
Feng, Juan; Navratil, Marian; Thompson, Ladora V et al. (2008) Principal component analysis reveals age-related and muscle-type-related differences in protein carbonyl profiles of muscle mitochondria. J Gerontol A Biol Sci Med Sci 63:1277-88
Whiting, Christofer E; Dua, Rajat A; Duffy, Ciaran F et al. (2008) Determining under- and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser-induced fluorescence detection. Electrophoresis 29:1431-40
Navratil, Marian; Terman, Alexei; Arriaga, Edgar A (2008) Giant mitochondria do not fuse and exchange their contents with normal mitochondria. Exp Cell Res 314:164-72
Andreyev, Dmitry; Arriaga, Edgar A (2007) Simultaneous laser-induced fluorescence and scattering detection of individual particles separated by capillary electrophoresis. Anal Chem 79:5474-8
Chen, Yun; Xiong, Guohua; Arriaga, Edgar A (2007) CE analysis of the acidic organelles of a single cell. Electrophoresis 28:2406-15

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