Biology Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/2749

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    IDENTIFICATION OF THE MOLECULAR MECHANISMS OF ZEBRAFISH INNER EAR HAIR CELL REGENERATION USING HIGHTHROUGHPUT GENE EXPRESSION PROFILING
    (2010) Liang, Jin; Popper, Arthur N.; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    All nonmammalian vertebrates studied can regenerate inner ear mechanosensory receptors, i.e. hair cells, but mammals only possess a very limited capacity for regeneration after birth. As a result, mammals suffer from permanent deficiencies in hearing and balance once their inner ear hair cells are lost. The mechanisms of hair cell regeneration are poorly understood. Because the inner ear sensory epithelium is highly conserved in all vertebrates, we chose to study the hair cell regeneration mechanism in adult zebrafish, hoping the results would be transferrable to inducing hair cell regeneration in mammals. We defined the comprehensive network of genes involved in hair cell regeneration in the inner ear of adult zebrafish with the powerful transcriptional profiling technique, Digital Gene Expression (DGE), which leverages the power of next-generation sequencing. We also identified a key pathway, stat3/socs3, and demonstrated its role in promoting hair cell regeneration through stem cell activation, cell division, and differentiation. In addition, transient pharmacological up-regulation of stat3 signaling accelerated hair cell regeneration without over-producing cells. Taking other published datasets into account, we propose that the stat3/socs3 pathway is a key response in all tissue regeneration and thus an important therapeutic target not only for hair cell regeneration, but also for a much broader application in tissue repair and injury healing. The dissertation contains four supplemental files. Supplemental file 1 contains raw data of five expression profiles generated by DGE. It is a tab-delimited text file with six columns. The first column contains the sequences of the tags and the second to sixth columns contain the count of the corresponding tags in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 2 contains UniGene clusters identified from unambiguously mapped tags. It is a tab-delimited text file with six columns. The first column contains the UniGene IDs. The second to sixth columns contain the count of the corresponding UniGene clusters in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 3 contains candidate genes identified by comparison of the expression profiles during regeneration to the control profiles. It is a tab-delimited text file with 19 columns. The contents in each column are specified in the header. Supplemental file 4 contains a list of the candidate genes known to be expressed in the inner ear and/or the lateral line system during development. It is a tab-delimited text file with four columns which contain UniGene IDs, ZFIN IDs, Entrez Gene IDs, and gene symbols respectively.
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    DECIPHERING THE SECRET OF SARCOMERE ASSEMBLY AND DISEASES USING THE ZEBRAFISH MODEL SYSTEM: REGULATION OF MYOFIBRILLOGENESIS BY SMYD1B AND ITS PARTNERS
    (2009) Li, Huiqing; Du, Shao Jun; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Myofibrillogenesis is a process of precise assembly of sarcomeric proteins into the highly organized sarcomeres which are essential for muscle cell differentiation and function. Myofibrillogenesis requires proper folding and assembly of newly synthesized sarcomeric proteins. Mutations of the sarcomeric proteins are known to cause skeletal and cardiac muscle diseases. smyd1b is a skeletal and cardiac muscle-specific gene which encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2. Knockdown of smyd1b (tv1 and tv2) expression resulted in zebrafish larvae without locomotion and heart contraction. Thick filament assembly was significantly disrupted in smyd1b knockdown embryos. Yeast Two-Hybrid study showed that Smyd1 associates with another muscle-specific protein--skNAC, however, skNAC function in muscle cells is unknown. In order to expand the understanding of smyd1b function and study the working mechanism, I further characterized the function of Smyd1b and its partners including skNAC and Hsp90&alpha1 during muscle development, and carried out mechanistic studies using zebrafish as a model system. Our findings show that: 1) In addition to the thick filament, smyd1b plays an important role in the assembly of thin and titin filaments, as well as Z-line and M-line. 2) Knockdown of smyd1b has no effect for heart tube formation; however, it disrupts the myofibril assembly of the cardiac muscle that causes the heart defect. 3) Smyd1b_tv1, but not Smyd1b_tv2 can be localized on the M-line of sarcomeres. 4) Ser225 on Smyd1b_tv1, which is a potential phosphorylation site, is important for the M-line localization of Smyd1b_tv1. 5) Knockdown of smyd1b causes the upregulation of hsp90&alpha1 and unc45b gene expression. 6) hsp90&alpha1 plays an important role for myofibril assembly. 6) Knockdown of smyd1b or hsp90&alpha1 causes the reduction of myosin protein accumulation. 7) Smyd1b_tv1, but not Smyd1b_tv2 associates with the myosin chaperones Hsp90&alpha1 and Unc45b. 8) sknac is required for the thick and thin filaments assembly. 9) Knockdown of sknac causes the reduction of myosin protein accumulation. These studies provide us an in-depth characterization of smyd1b and its partners' function and expands the mechanistic understanding of how smyd1b fulfils its vital role in myofibrillogenesis. Most importantly, this study provides new insights to help us understand the complex process of myofibrillogenesis and sarcomere diseases.