Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item 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.Item Behavioral and Neuroendocrine Correlates of Sex Change in the Gilthead Seabream (Sparus aurata)(2009) Reyes-Tomassini, Jose J.; Zohar, Yonathan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sequential hermaphroditism is the most radical form of environmental sex determination observed in fish: functional adult males or females retain their ability to change sex even as adults. Among the factors that affect sex change in these species, the least understood is the social environment. Here, I studied the influences of social context on sex change in the Gilthead Seabream, Sparus aurata, by using the individual‟s dominance rank as an indicator of social status. To understand the role that the brain might play in sex change, I also studied the two main neuroendocrine factors that serve as the sexually differentiated axes of neural plasticity in most teleost species: AVT and GnRH. To do this, I first developed a set of tools designed to address the challenges associated with observing the behavior of aquacultured species. Using these tools, I provide the first in-depth study of seabream captive behavior, including the results of size-matched and sex-matched paired encounters. I found that females are more aggressive than males, but this difference is influenced by gonadal developmental status. I also showed that small but young males are more aggressive than bigger but older females. I cloned the AVT mRNA in seabream, and validated a quantitative assay to measure total brain AVT levels together with GnRH-1, GnRH-2, and GnRH-3 levels. I found that AVT and GnRH-3 levels rise during the onset of the hypothesized sex-change window, and drop to pre-quiescent levels until spawning, when all of these factors seem to increase their expression levels again. I also show for the first time, that GnRH-2 and dominance rank are strongly correlated in seabream during the spawning season but not during quiescence. GnRH-1 was strongly correlated to rank during quiescence but not during spawning. Finally, neither dominance rank nor size were a good predictor of the outcome of sex change, which seems to contradict what has been documented in sequential hermaphrodite reef fishes. I provide a model that accounts for this apparent contradiction and conclude that the Gilthead seabream remains true to the size-advantage hypothesis of sex allocation theory, if size and dominance are seen as proximate, rather than ultimate, factors.Item Reproductive Physiology of the Female Blue Crab, Callinectes sapidus: Spawning Induction and Vitellogenesis(2009) Bembe, Sarah Elizabeth; Chung, J. Sook; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In aquaculture, spawning is the baseline for production; therefore, the optimization of spawning conditions will directly increase production. The current study aims to optimize spawning conditions for Callinectes sapidus using environmental manipulations of photoperiod and temperature for induction while monitoring the physiological vitellogenin (VtG) levels during ovarian development and maturation. The photothermal manipulations for this study resulted in increased spawning events in 21°C temperatures (compared to 11°C and 15°C) and complete darkness (0L:24D; compared to 8L:16D, 16L:8D, and 24L:0D) while 24L:0D and 11°C suppressed spawning. When assessing the VtG levels in the hemolymph prior to, during, and after all spawning events, the VtG showed a decrease prior to spawning, and significant VtG activity was seen in 21°C for all photoperiods. Overall, spawning and vitellogenesis are temperature dependent events with 67% of the females spawning in 21°C. Photoperiod also has an effect on spawning, but not on vitellogenesis.Item Comparative effects of embryonic exposure to polybrominated diphenyl ethers in domestic and wild birds(2008-11-21) McKernan, Moira A; Ottinger, Mary Ann; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Concentrations of polybrominated diphenyl ethers (PBDEs) in eggs of wild birds have increased dramatically over the past 25 years, yet only limited data are available to interpret toxicological consequences of exposure. Embryonic survival, pipping and hatching success, and sublethal biochemical, endocrine, and histological endpoints were examined in chicken (Gallus gallus), mallard (Anas platyrhynchos), and American kestrel (Falco sparverius) hatchlings following in ovo (air cell) administration of the commercial penta-BDE mixture DE-71 (0.01-20 µg DE-71/g egg). Environmentally realistic concentrations of DE-71 induced ethoxyresorufin-O-dealkylase activity, and reduced bursa of Fabricius follicle size and number in chicken hatchlings, but not in other species. Pipping and hatching success decreased in American kestrels receiving 10 and 20 µg DE-71/g egg, but these endpoints were unaffected in other species. Absorption of air cell administered DE-71 (dose = 11.1 µg/g egg) into the contents of eggs varied among species and uptake rate tended to increase during the later half of development (dose absorbed by pipping: chicken 29.6%, kestrel 18.8%). At least six PBDE congeners and two metabolites were detected in DE-71 dosed eggs that were not present in the dosing solution, suggesting evidence of debromination and methoxylation. Uptake of the commercial octa-BDE mixture DE-79 (dose = 15.4 µg /g egg) following air cell administration to avian eggs was found to be low (dose absorbed <6.5%). Based on the DE-71 uptake rate in kestrels, the lowest observable effect level on pipping and hatching success may be 1.8 µg total PBDE/g egg, which approaches concentrations detected in eggs of free-ranging birds. As some PBDE congeners are still increasing in the environment, the embryotoxic effects observed following DE-71 administration are cause for concern.Item A Comparison of the Biochemistry and Physiology of the Eastern Oyster, Crassostrea virginica, and the Asian Oyster, Crassostrea ariakensis(2007-08-13) Harlan, Nicole Porter; Paynter, Kennedy T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Chesapeake Bay Foundation, US Environmental Protection Agency, and the Chesapeake Bay Commission, requested research on the introduction of the Asian oyster, Crassostrea ariakensis, to help restore the fishery and ecosystem function of the native Eastern oyster, Crassostrea virginica. In order to augment the role of C. virginica in Chesapeake Bay, C. ariakensis will likely require tolerances to low dissolved oxygen similar to that of the native oyster. This research showed that triploid and diploid C. virginica lived significantly longer than C. ariakensis under anoxic conditions, although the oxygen consumption rates of diploid oysters did not differ between species. Free amino acid pools in the gill tissue of oysters exposed to normoxia or hypoxia were analyzed. Alanine increased in both species during hypoxia, indicating the use of alternative metabolic pathways. Aspartate was consumed by C. virginica during hypoxia, confirming the use of a pathway coupling glucose and aspartate fermentation. Differences in the free amino acid pools of these two species suggest an explanation for the disparity in anaerobic metabolism between C. ariakensis and C. virginica.Item Mycobacteriosis in Chesapeake Bay Striped Bass (Morone saxatilis): The Interaction of Nutrition and Disease(2007-05-17) Jacobs, John M.; Harrell, Reginal M.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Field and laboratory studies were conducted from 1998 - 2005 to examine the relationship between nutritional status and mycobacteriosis in Chesapeake Bay striped bass (Morone saxatilis). A review of DNA from archived tissue blocks indicated that the disease has been present since at least 1984. Field surveys and feeding trials were conducted from 1998-1999 to determine the nutritional condition of striped bass and the association with disease state. Proximate composition revealed elevated moisture (~ 80%) and low storage lipids (< 0.5% ww), characteristic of a poorly nourished population. These findings were not consistent with data collected in 1990-1991, or with experimentally fed fish. Mycobacteriosis explained little of the variance in chemical composition (p > 0.2); however elevated moisture and low lipid concentration were associated with fish with ulcerative lesions (p < 0.05). This suggests that age 3 and 4 striped bass were in poor nutritional health in 1998-1999, which may be independent from the disease process. Challenge studies were performed to address the hypothesis that disease progression and severity may be altered by nutritional status of the host. Intraperitoneal inoculation of 104 CFU M. marinum resulted in high mortality, elevated bacterial density, and poor granuloma formation in low ration (0.15% bw/d) groups while adequately fed fish (1% bw/d) followed a normal course of granulomatous inflammation with low mortality to a steady, equilibrium state. Further, we demonstrated that an active inflammatory state could be reactivated in fish through reductions in total diet. The energetic demand of mycobacteriosis, was insignificant in comparison to sham inoculated controls in adequately fed fish (p > 0.05). Declines in total body energy were only apparent during active, inflammatory stages of disease. Overall, these findings suggest that: 1) mycobacteriosis is not a new disease of Chesapeake Bay striped bass, 2) the disease has little energetic demand in the normal, chronic progression, and 3) poor nutritional health can greatly enhance the progression and severity, and reactivation of disease. The implications of this research are that management strategies focused on enhancing the nutritional state of striped bass could potentially alter the disease dynamics in Chesapeake Bay.Item Gonadal and steroid feedback regulation of the hypothalamus-pituitary axis in striped bass (Morone saxatilis)(2006-09-18) Klenke, Ulrike; Zohar, Yonathan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objective of the present study was to expand our understanding of the mechanisms of gonadal steroid feedback regulation of the hypothalamus-pituitary (HP) axis during several reproductive stages (juvenile, pubertal, adult) throughout the life cycle of the striped bass. Towards this end, we investigated effects of bilateral gonadectomy and steroid replacement on the endocrine correlates of the HP axis in vivo. We also developed a brain-slice culture method and utilized pituitary cell cultures to investigate direct effects of estrogen on these correlates at the level of the brain and the pituitary in vitro. Our findings indicate that: 1) During their development, the gonads play an important role in providing feedback to the HP axis. These feedback patterns change during the transformation from the juvenile to the adult and throughout the adult reproductive cycle. The pathways involved use both non-steroidal and steroidal pathways as regulatory mechanisms. 2) Gonads, through their steroids, become more involved in regulating the HP axis during reproductive development and their main feedback target appears to be gene transcription in the pituitary. 3) The observed changes in gonadal feedback throughout the adult reproductive cycle probably reflect the physiological requirements of gametogenesis. 4) The responsiveness of the HP axis towards steroids initially appears during puberty and further increases in adult females. In adults, steroids solely affect the pituitary in early stages of gametogenesis, while in later stages GnRH expression in the brain is also regulated by steroids. However, the nature of the feedback is dependent on estrogenic and/or androgenic pathways. 5) Our in vitro studies showed that estrogens act directly at the levels of the brain and the pituitary in female adult fish. Based on these findings, it appears that the activity along the endocrine reproductive web of striped bass intensifies with age, and that prior cycles of oocyte development may prime the HP axis to respond faster and more vigorously in subsequent years. This study has provided an improved resolution and a broader perspective on mechanisms involved in gonadal steroid feedback regulation of GnRH neural activity and its targets at the level of the pituitary in striped bass.