Theses and Dissertations from UMD
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Item Ontogenic and glucocorticoid-regulated gene expression in the developing neuroendocrine system(2010) Ellestad, Laura Elizabeth; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The neuroendocrine system is a critical regulator of vertebrate homeostasis that includes five hypothalamic-pituitary axes which develop during embryogenesis. Adrenal glucocorticoids play an important role in functional maturation of the anterior pituitary through initiation of growth hormone (GH) production. These studies were aimed at characterizing ontogenic and glucocorticoid-regulated changes in gene expression during neuroendocrine system development in the chick. First, to ascertain timing of initiation and establishment of each neuroendocrine axis, we measured mRNA levels of hypothalamic regulatory factors, their pituitary receptors, and pituitary hormones from embryonic day (e) 10 through post-hatch day (d) 7. We found that the adrenocorticotropic axis is the first to be established (e12), followed by establishment of the thyrotropic (e18), somatotropic (e20), lactotropic (d1), and gonadotropic (d5) axes. Next, we examined in detail mechanisms through which glucocorticoids initiate pituitary GH expression during embryogenesis. We determined that glucocorticoids elevate GH mRNA levels on e11 by increasing transcriptional activity of the GH gene rather than enhancing mRNA stability, and protein synthesis, histone deacetylase activity, ras signaling, and ERK1/2 signaling are required for this activation. Conversely, sustained activation of ERK1/2 and p38MAPK pathways reduced glucocorticoid stimulation of GH expression, indicating the requirement for ERK1/2 activity is transitory. Finally, we identified ras-dva as a novel Pit-1 and glucocorticoid-regulated gene in the chicken embryonic pituitary gland. Pituitary ras-dva mRNA levels increased between e10 and e18, decreased just prior to hatch, and remained low or undetectable post-hatch. Ras-dva expression was highly enriched within the pituitary gland on e18, and glucocorticoids rapidly induced ras-dva mRNA in cultured pituitary cells through a mechanism involving transcriptional activation. Potential regulatory elements within the 5'-flanking region of chicken ras-dva responsible for pituitary-specific expression were identified, as was a 2 kb fragment necessary for its glucocorticoid induction in embryonic pituitary cells. These results enhance our understanding of neuroendocrine system development and establishment during embryogenesis, reveal mechanisms underlying glucocorticoid initiation of GH expression in somatotrophs, and identify a new Pit-1 and glucocorticoid target gene that may play an important role in pituitary development.Item COMPARATIVE STUDY OF LIPOPROTEIN METABOLISM IN MAREK'S DISEASE SUSCEPTIBLE AND RESISTANT LINES(2010) Yuan, Ping; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Marek's disease virus (MDV) infection causes atherosclerosis, and prior vaccination prevented the development of this disease. Two main strategies to resist Marek's disease (MD) have been demonstrated: vaccination and genetic resistance. However, little is known about the role of genetic resistance in the progression of MDV induced atherosclerosis. Atherosclerosis is primarily associated with lipoprotein metabolism. The purpose of this study was to investigate whether lipoprotein metabolisms are different in distinct MD susceptible and resistant chicken lines. Here, we studied different backgrounds of lipoprotein metabolism in the two lines and the changes of lipoprotein levels in response to MDV infection. The results showed that during chicken growth, the increase in total cholesterol was mostly due to the increasing (LDL+VLDL) in MD susceptible line, whereas it was mainly due to the elevating HDL in MD resistant line. These results suggested that different lipoprotein metabolisms exist in MD susceptible and resistant lines.Item IDENTIFICATION OF A NON-CLASSICAL GLUCOCORTICOID-RESPONSIVE ELEMENT IN THE 5'-FLANKING REGION OF THE CHICKEN GROWTH HORMONE GENE(2010) Knubel, Kristina Heuck; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) effects growth and contributes to a lean phenotype in broiler chickens. GH secretion by the anterior pituitary begins on embryonic day (e) 14, concomitantly with a rise in adrenal glucocorticoids (GC) or corticosterone (CORT) secretion. CORT treatment of chicken embryonic pituitary (CEP) cells induces GH secretion prematurely. GC induction of the GH gene requires on-going protein synthesis or an intermediary protein, but the gene lacks a classical GC-response element. We hypothesized that a GC-responsive intermediary protein is necessary for the CORT induced increase in GH. Characterization of the upstream region of the gene may help identify such a protein. To this end, a fragment of the GH gene (-1727/+48) was cloned into a luciferase reporter and characterized in e11 CEP cells. CORT treatment increased luciferase activity and mRNA. Inclusion of CHX blocked CORT induction of luciferase mRNA. Through deletion analysis, we found that a GC-responsive region (GCRR) is located at -1045 to -954. By defining the GC-responsive region and cis-acting elements located within, trans-acting proteins involved in GC induction of the GH gene may be identified. The GCRR is CORT-responsive in either orientation, but it is context-dependent. Potential transcription factor motifs in the GCRR include ETS-1 and a degenerate GRE (GREF). Nuclear proteins bound to a GCRR probe in a CORT-regulated manner and unlabeled competitor DNA competed off binding. Mutation of the central portion of the DNA probe resulted in a significant decrease in protein binding. Mutation of the ETS-1 site or GREF site in the -1045/+48 GH construct resulted in ablation of luciferase activity. ETS-1 and GR are associated with the endogenous gene under basal and 1.5 h CORT-treated conditions, while GR recruitment increased after CORT treatment. GC regulation of the GH gene during chicken embryonic development requires cis-acting elements located 1 kb upstream from the transcription start site and includes recruitment of ETS-1 and GR. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development. Characterization of GC regulation of the GH gene during embryonic development enhances our understanding of growth regulation in vertebrates.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 CAN CHOLINE SPARE METHIOININE FROM CATABOLISM IN LACTATING MICE AND DAIRY COWS?(2009) Benoit, Sarah Lee Ann; Erdman, Richard A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Several studies have demonstrated that supplementation of rumen-protected choline (RPC) improves milk production in the lactating dairy cow; however, there are an equal number of studies failing to observe production responses. To date, there are only three studies that provide quantitative information in ruminants on the metabolic fates of methyl groups derived from choline and Methionine (Met). This has limited the ability to predict when, and under what conditions, RPC supplementation will be beneficial. The objectives of this thesis were to determine the interaction of choline and Met methyl group metabolism and the extent of methyl group transfer during lactation, and define what role, if any, is there for RPC in remethylation of homocysteine and in the sparing of Met in lactating animals. A preliminary study with lactating mice consuming a low protein basal diet (10%) was conducted. From 11 to 15 d postpartum, stable isotopes of [methyl,2H3] choline and [methyl,2H3] Met replaced the unlabeled choline and Met in the basal diet to measure the metabolic fates of choline and Met including Met remethylation and sources of Met methyl in the mammary gland. Isotopic analysis revealed that the liver is a major site of Met remethylation from dietary choline with a minimum choline methyl group contribution to Met remethylation of 35%. Mammary tissue was not a major site of Met remethylation from dietary choline (< 10% of Met methyl) as measure by Met methyl in mammary tissue and milk casein. However, there was a significant unlabeled source of methyl groups contributing at a minimum of 45% Met remethylation in the mammary tissue, presumably by de novo synthesis. This suggested that in addition to the liver, the mammary gland is an active site of methyl group transactions. In a subsequent study, lactating dairy cows were fed a total mixed ration formulated to meet the nutrient requirements with exception of metabolizable Met that was restricted to 1.49 % of metabolizable protein. Treatments included a Control (basal diet) and RPC supplemented diet where the basal diet was top dressed with 15g/d RPC, diets were fed in a single reversal design with 2 week experimental periods. Stable isotopes of Met, [1-13C] Met, [13CH3] Met, and [methyl-2CH3] choline were continuously infused on d 14 of each period to determine the metabolic fate and methyl transactions of Met methyl as measured in blood and milk casein. Treatment had no effect on milk production or composition. However, plasma free Met from choline transmethylation was shown to act as a significant contributor to casein synthesis. Fractional Met remethylation rates in the control and RPC treatments were 26 and 23%, respectively. Methionine Met methyl loss within the mammary tissue appears to be minimal. Based on casein Met enrichment, upwards of 40% of Met present in casein had undergone transmethylation with choline serving as the ultimate methyl donor. Furthermore, plasma versus casein Met methyl enrichment data suggested that a significant amount of de novo methyl group synthesis occurs in the dairy cow's mammary gland with choline serving as a major methyl donor.Item KINEMATICS OF THE MAYFLY NYMPH GILL ARRAY: AN INTERMEDIATE REYNOLDS NUMBER VENTILATION PUMP(2009) Sensenig, Andrew T.; Shultz, Jeffrey W.; Kiger, Ken T.; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Comparative studies encompassing a wide range of aquatic animals have shown that rowing is exclusively used at low Reynolds numbers (Re <1) while flapping is predominantly used at Re > 100, but few studies have been undertaken to document the transition in individual species that traverse the intermediate Re regime using a single set of appendages. Thus, it is not generally known whether a gradual increase in Re within a system results in a gradual or sudden shift between rowing and flapping. Here I document both the kinematics of the appendages and the surrounding fluid of a nymphal mayfly Centroptilum triangulifer that uses a serial array of seven pairs of abdominal gill plates and operates at Reynolds numbers ranging from 2-22 during ontogeny. I found that some kinematic variables (stroke frequency and metachronal phase lag) did not change during ontogeny but that others changed substantially. Specifically, gills in small instars used strokes with large pitch and stroke-plane deviations, while larger instars used strokes with minimal pitch and minimal stroke-plane deviation. Gills in larger instars also acquired an intrinsic hinge that allowed passive asymmetric movement between half strokes. Net flow in small animals was directed ventrally and essentially parallel to the stroke plane (i.e. rowing), but net flow in large animals was directed dorsally and essentially transverse to the stroke plane (i.e. flapping). The metachronal phasing of the gills produced a time-dependent array of vortices associated with a net ventilatory current, a fluid kinematic pattern here termed a "phased vortex pump". Significantly, absolute vortex size did not change with increasing animal size or Re, indicating that the vortex diameter (Lv) decreased relative to intergill spacing (Lis) during mayfly growth. Given that effective flapping requires organized flow between adjacent appendages, I proposed the hypothesis that rowing should be favored when Lis / Lv < 1 and flapping should be favored when Lis / Lv > 1. Significantly, the rowing-to-flapping transition in Centroptilum triangulifer occurs at Re ~5, when maximum dynamic intergill distance equals vortex diameter. This result suggests that the Re-based rowing-flapping demarcation observed in a wide array of aquatic organisms is determined by the relative size of the propulsive mechanism and its self-generated vortices.Item Nutrient regulation by an omnivore and the effects on performance and distribution(2009) Pearson, Rachel Estelle Goeriz; Gruner, Daniel S; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Omnivores have a unique interaction with their nutritional environment because they have adapted to consume food from different trophic levels. To successfully navigate their variable resources, omnivores must maintain some level of nutrient regulation. To explore the effects of nutrient regulation by an omnivore, I used a salt marsh katydid, Conocephalus spartinae. To first address the ability of Conocephalus to perform on a wide range of diets and to regulate their nutritional intake, I used artificial diets that differed in relative amounts of protein and carbohydrate (Chapter 1). I found that Conocephalus survival decreased on a high protein diet due in part to a decrease in lipid stores but growth was not affected by diet. In a second experiment Conocephalus showed a degree of nutrient regulation as evidenced by the difference in what they actually ate and the predicted consumption if they had been feeding equally on the diets presented in each treatment. However, I did not find evidence for tight macronutrient regulation. Next I explored capacity of Conocephalus to regulate their nutrient intake (nitrogen and lipid) when fed naturally co-occurring prey (Chapter 2). I first established that the prey differed in their protein and lipid content and that these differences were related to the size of the prey species. When Conocephalus were fed different prey species individuals showed no differences in either growth or survival. In the final experiment, I found that Conocephalus did show evidence of a degree of nitrogen and lipid regulation because they did not feed equally on all of the prey species offered. Lastly, I documented the relationship between the ability of Conocephalus to locate plant and prey resources and the effect that these resources have on omnivore performance (Chapter 3). I found that Conocephalus aggregates in areas of high plant quality but that their numbers do not correspond to areas of high prey density. However, I found that katydid growth and survival was enhanced by prey availability but not plant quality. Overall, I documented how an organism like an omnivore relates to its nutritional environment and how nutrient regulation might affect performance and distribution. Last, I documented the relationship between the ability of katydids to locate plant and prey resources and the effect that these resources have on omnivore performance (Chapter 3). I found that katydids aggregate in areas of high plant quality but that their numbers do not correspond to areas of high prey density.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 The effect of sperm mobility phenotype on fertility persistence in layer and broiler hens(2008) Baczynski, Kathleen; Estevez, Inmaculada; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Domestic fowl (Gallus gallus domesticus) were studied to identify accurate predictors of potential fertility in two lines of broiler breeder males along with fertility persistency in layer and broiler hens. Sixty-four Hy-Line layer and thirty-seven broiler breeder hens were AI with identical amounts of high or low mobility sperm from FG males. Morphological measurements were taken to determine relationships of these with semen volume, concentration, and mobility. We hypothesized that 1) semen quality would decline as males aged, 2) morphology would be positively correlated with semen quality, and 3) females AI with high mobility sperm would have a longer duration of fertility. Results revealed a significant age*line interaction for semen volume (p=0.0307), sperm concentration (p=0.0003), and sperm mobility (p=0.0405). Morphological measurements were correlated with different semen parameters in both lines. Fertility was positively correlated with semen quality. Sperm mobility influenced fertility in layer hens but not in broiler breeders.
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