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Jibin Zhang - PhD Dissertation.pdf (3.79 MB)

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Identification of Important Cell Cycle Regulators and Novel Genes in Specific Tissues using Microarray Analysis, Bioinformatics and Molecular Tools

Zhang, Jibin
http://rave.ohiolink.edu/etdc/view?acc_num=osu1429637289

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Animal Sciences.
Study of cell cycle activity is critical to understand development of adipose tissue, because the switch on and off of cell cycle activity is tightly related to hyperplasia and hypotrophy of the fat cells. Three cell cycle inhibitors - CCNG2, CDKN2C and PMP22, and three cell cycle activators for adipose tissue - CCNA1, CCND3 and ANAPC5, were selected as important cell cycle regulators for adipogenesis based on analyses of Gene Expression Omnibus (GEO) DataSets (GDSs) in the website of the National Center for Biotechnology Information (NCBI). The three important cell cycle inhibitors showed higher expression in fat cells than in preadipocytes and increased exprerssion during primary cell culture and development of adipose tissue in quantitative real-time PCR (qPCR) or western blotting. The results suggest that expression of these cell cycle inhibitors generally increases with the differentiation of fat cells and the growth of pig adipose tissue. In broiler chickens, expression of CCNG2, CDKN2C, CCND3 and ANAPC5 reaches a peak at post-hatch day 5 and decreases in the later stages, suggesting that day 5 may be an important time with active proliferation and differentiation of preadipocytes. The result of PCNA immunostaining suggests that proliferation activity of fat cells keeps decreasing from embryo day 15 to post-hatch day 33. The result of H&E staining suggests that fat cell size keeps increasing until post-hatch day 11. Therefore, both hypertrophic and hyperplastic growth is active before post-hatch day 5. From post-hatch day 5 to day 11, as proliferation activity decreases and most preadipocytes become differentiated, hypertrophic growth gradually becomes dominant. In the third experiment, some tissue specific secretory factors in mouse were selected based on GEO data analysis and bioinformatic tools. The tissue specific expressions of the selected genes were confirmed using semi-quantitative PCR. Secretion of five novel genes were then validated through expression vector construction, subsequent transfection to HEK293 cells and protein detection using western blotting with cell lysate and medium. Finally, their functions in the specific tissues were further predicted by integrating microarray data from different GEO profiles. In addition, alternative splicing inhibiting secretion was also identified in CTLA2A and SERPINA1F. In the fourth study, a novel adipose specific gene HPS4 was revealed by DNA microarray analysis. Its higher expression in adipose tissue than in other tissues and in fat cells than in stromal vascular cells was detected by qPCR or western blotting. In addition, the increasing expression of HPS4 during primary cell culture in vitro and adipose development in vivo also suggests its important function in adipocytes. Finally, alternative splicing was identified and two different proteins were also detected in embryos and chickens in early ages in western blotting. However, the associations of the transcripts and proteins still need to be clarified and their functions still need to be studied. In general, our studies not only developed a strategy to identify genes with important effects and novel genes in adipose and other tissues, but also increased our understanding about adipose tissue and provided some guidance to studies in other tissues.
Michael Davis, E. (Advisor)
Kichoon Lee (Committee Member)
Macdonald Wick (Committee Member)
Harald Vaessin (Committee Member)
191 p.
Animal Sciences; Animals; Bioinformatics; Biology
adipocytes, cellularity, differentiation, cell cycle, gene expression, secretory factors, GEO datasets, microarray, CCNG2, CDKN2C, PMP22, CCNA1, CCND3, ANAPC5, CTLA2A, SERPINA1F, MUP19, WFDC15B, DEFB29, HPS4, Chicken, Pig, Mouse

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Citations

  • Zhang, J. (2015). Identification of Important Cell Cycle Regulators and Novel Genes in Specific Tissues using Microarray Analysis, Bioinformatics and Molecular Tools [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429637289

    APA Style (7th edition)

  • Zhang, Jibin. Identification of Important Cell Cycle Regulators and Novel Genes in Specific Tissues using Microarray Analysis, Bioinformatics and Molecular Tools. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1429637289.

    MLA Style (8th edition)

  • Zhang, Jibin. "Identification of Important Cell Cycle Regulators and Novel Genes in Specific Tissues using Microarray Analysis, Bioinformatics and Molecular Tools." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429637289

    Chicago Manual of Style (17th edition)

osu1429637289
324
© 2015, some rights reserved.Creative Commons License Identification of Important Cell Cycle Regulators and Novel Genes in Specific Tissues using Microarray Analysis, Bioinformatics and Molecular Tools by Jibin Zhang is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.