Animal & Avian Sciences Theses and Dissertations
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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 DELINEATING THE ROLES OF C. ELEGANS HEME RESPONSIVE GENES HRG-2 AND HRG-3 IN HEME HOMEOSTASIS(2009) Chen, Caiyong; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential cofactor for diverse biological processes such as oxygen transport, xenobiotic detoxification, and circadian clock control. Since free heme is hydrophobic and cytotoxic, we hypothesize that within eukaryotic cells, specific trafficking pathways exist for the delivery of heme to different subcellular destinations where hemoproteins reside. To identify molecules that may be involved in heme homeostasis, we conducted a C. elegans microarray experiment on RNA extracted from worms grown at different concentrations of heme in axenic liquid medium. Analysis of the microarrays revealed that the mRNA levels of heme-responsive gene-2 (hrg-2) and hrg-3 increased more than 70 fold when worms were grown at 4 µM compared to 20 µM heme. hrg-2 is expressed in hypodermal tissues in the worm, and the protein localizes to the endoplasmic reticulum and the apical plasma membrane. In vitro hemin agarose pull-down experiments indicate that HRG-2 binds heme. Deletion of hrg-2 in C. elegans leads to reduced growth rate at low heme. Moreover, expression of HRG-2 in hem1δ, a heme-deficient yeast strain, results in growth rescue at submicromolar concentrations of exogenous heme. These results indicate that HRG-2 may either directly participate in heme uptake or facilitate heme delivery to another protein. Unlike hrg-2, hrg-3 is exclusively expressed in the worm intestine under heme deficiency. Following its synthesis, HRG-3 is secreted into the body cavity pseudocoelom. Deletion of hrg-3 results in increased heme levels in the worm intestine, suggesting that HRG-3 may function in intercellular heme transport in C. elegans. To identify the functional network or pathways for HRG-2 and HRG-3, we performed a genome-wide microarray analysis using RNA samples prepared from the worms grown at different concentrations of heme and oxygen. The results showed that a total of 446 genes were transcriptionally altered by heme and/or oxygen. Among them, 41 and 29 genes exhibited similar expression profiles to hrg-2 and hrg-3, respectively. We postulate that these genes may function in conjunction with hrg-2 and hrg-3. Taken together, we have identified two novel heme-responsive genes in metazoa that may play critical roles in modulating organismal heme homeostasis in C. elegans.Item Identification and characterization of a heme responsive element in the hrg-1 promoter(2008) Sinclair, Jason; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite its biological significance, little is known about how animals sense and respond to heme to maintain homeostasis. C. elegans is a heme auxotroph, which makes it an excellent model to identify and dissect heme homeostasis pathways. Using C. elegans we have identified HRG-1, a vesicular heme transporter that is transcriptionally upregulated when environmental heme is low. The current study seeks to address how hrg-1 is regulated by heme. Here, we show that a putative 23 base pair (bp) heme-responsive element (HRE) and GATA-binding motifs are necessary for heme-dependent regulation of hrg-1. The HRE comprises both enhancer and repressor elements and works in conjunction with ELT-2 to regulate hrg-1 expression. We propose that the HRE could be used as a molecular tool in C. elegans to tightly regulate internal gene expression by modulating environmental heme. Our ultimate goal is to identify the trans-acting factor to eventually create a whole animal sensor for monitoring organismal heme homeostasis.Item THE FUNCTIONAL REGULATION OF FCRN EXPRESSION AND FCRN-MEDIATED ANTIGEN PRESENTATION(2009) Liu, Xindong; Zhu, Xiaoping; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The neonatal Fc receptor for IgG (FcRn), a major histocompatibility complex (MHC) class I-related molecule, plays an important role in IgG transport and protection. The transport of IgG across epithelial and endothelial barriers and the IgG homeostasis maintained by FcRn contributes to the effective humoral immunity. Thus, the level of FcRn itself will affect the IgG-associated immune responses. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown.I showed here that FcRn was up-regulated by the stimulation of inflammatory cytokines or Toll-like receptor ligands in human peripheral blood mononuclear cell (PBMC) and THP-1 cell line. By chromatin immunoprecipitation, I identified three NF-κB binding sites within introns 2 and 4 of the human FcRn gene. These intronic binding sites boost FcRn transcription activities through looping with the promoter region. In contrast, FcRn expression was down-regulated by Th1 cytokine IFN-γ, and the down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. It has been demonstrated that IFN-γ activated STAT1 bound with GAS sequence in human FcRn promoter, and which blocked the transcriptional machinery. Fc gamma receptors (FcγRs) expressed in macrophages (MФ) and dendritic cells (DCs) can mediate antigen presentation in both MHC class II and MHC class I pathways. We tested here the role for FcRn in antigen presentation of IgG-restricted Immune complexes (ICs). It was observed that the expression of FcRn in MФ, but not in DC enhanced the phagosomal ICs antigen presentation to CD4 T cells. A low pH value in phgosome of MФ facilitated FcRn binding to ICs, stabilizing the antigens and promoting the efficient MHC II-peptide assembly. However, the alkalized phagosomes in DC failed FcRn to enhance the antigen presentation of ICs.Item Regulation of milk fat synthesis by dietary fatty acids(2008-08-04) Kadegowda, Anil Kumar Gopalapura; Erdman, Richard A.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objectives of this thesis research were to determine the molecular mechanisms by which dietary fatty acids (FA) regulate lipogenic gene expression and milk fat synthesis. Principal component and multivariate analyses were conducted to establish the relationship between milk fat and FA concentrations in lactating cows fed milk fat depressing (MFD) diets. This analysis showed that in addition to the established inhibitory effect of t10c12 CLA, t7-18:1 and t7c9-CLA isomers might be involved in MFD. Lactating mice were used to test the effects of several individual trans-18:1 isomers and t10c12-CLA on milk fat synthesis, lipogenic genes in liver and mammary tissues. Both MFD and extensive conversion of t7-18:1 to t7c9-CLA in mammary and liver tissues were shown in mice fed the t7-18:1. As expected, t10c12-CLA feeding caused MFD and reduced the expression of lipogenic transcription factor (TF) SREBP-1C. Potential roles of the TF ChREBP, PPARG, and INSIG1 were also established. A subsequent study aimed to establish whether these mechanisms operated in lactating dairy cows. Compared with un-infused controls and a t10c12 CLA negative control, post-ruminal infusion of butterfat which contains all fatty acids in the same proportion to those found in milk fat to a mixture of fats containing only the long chain fatty acids (LCFA) were examined. Milk fat content, milk yield and mammary lipogenic gene expression were increased by butterfat but not by the LCFA mixture. This suggested that rates of short and medium chain fatty acid synthesis might be limiting for milk fat production. The effects of individual FA and a PPARγ-specific agonist (Rosiglitazone) on mRNA expression via qPCR of 19 genes with roles in de novo synthesis, FA uptake and transport, desaturation, triacylglycerol synthesis, transcriptional regulation, and nuclear receptor signaling in a MACT cell culture system were examined. The FA regulated mammary lipogenic gene expression to different extents. PPAR-γ activation of de novo lipogenesis coupled with exogenous FA availability might play a role in regulating milk fat synthesis. These experiments demonstrate the role of FA in regulating mammary lipogenic pathways, highlighting the complexity and multiple transcriptional factor involvement in milk fat synthesis.Item Regulation of factors contributing to virulence in Escherichia coli(2008-04-25) Mitra, Arindam; Mukhopadhyay, Suman; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Extraintestinal pathogenic strains of Escherichia coli cause a wide range of diseases including colibacillosis in chickens and urinary tract infections in humans. Persistent infections in E. coli and other gram-negative species are associated with population-dependent physiological processes such as cell-cell signaling and biofilm formation. Such social behaviors require careful coordination and modulation of gene expression in response to environmental cues. Adaptive response of bacteria in new environment is predominantly achieved through a signaling cascade called two-component regulatory systems. The function of the BarA/UvrY two-component regulatory system and its downstream factors in controlling virulence associated processes, specifically regulation of AI-2 based signaling and biofilm formation was investigated. In E. coli, a type of cell-cell signaling termed Quorum Sensing involves release, detection, and response to small molecule called autoinducer (AI-2), synthesis of which is dependent on luxS gene products via methyl cycle. The BarA-UvrY and Csr system displayed dual regulation on luxS expression at the level of transcription and post-transcription. The uptake of AI-2 by the Lsr transporter is also modulated by the signaling cascade suggested a balance between AI-2 synthesis and uptake in the cell. The role of transcriptional regulator uvrY in biofilm formation in Uropathogenic Escherichia coli was also studied. Mutation of uvrY reduced expression of fimA and papA, major fimbrial subunit of Type 1 and Pap pilus respectively. Acidic exopolysaccharide accumulation and the ability to swarm are also being impaired. Finally, uvrY mutants demonstrated poor colonization in kidneys and bladders in an ascending model of UTI. Overall, the effect of uvrY on biofilm formation seems to be multi-factorial and might play a critical role in adaptation and colonization of UPEC. The fine tuning of processes associated with cell-cell communication and biofilm formation at the level of transcription and post-transcription by the BarA/UvrY/CsrA signaling cascade indicated that this system might be crucial for quick adaptation, social behavior, colonization and virulence attributes in Escherichia coli.Item Evaluation of Lef1 Transcription Factor in Mammalian Preimplantation Embryos(2007-09-14) Meece, Ashley Elaine; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The first lineage differentiation of cells during pre-implantation embryo development is critical for continued embryonic growth. Though several transcription factors (TFs) have been identified that are involved in this transition, a specific mechanism of regulation has yet to be determined. Previous studies in our laboratory have shown the TF Lef1 is involved in differentiation of mouse embryonic stem cells into trophoblastic stem cells through the Wnt signaling pathway. Lef1 is known to be involved in lineage determination of adult skin stem cells. As we observed that two isoforms of Lef1 were expressed at different stages of pre-implantation development, we hypothesized that Lef1 was interacting with the other well-established lineage differentiation TFs, Nanog, Oct4, and Cdx2, in these early embryos. At the blastocyst stage, no significant changes in mRNA expression were seen when siRNAs, specifically designed to knockdown Lef1 expression, were injected in early embryos; however, knockdown of Lef1 did not interfere with blastocyst formation.Item Characterization of Glucocorticoid-Induced Changes in Gene Expression in the Embryonic Pituitary Gland(2006-04-27) Jenkins, Sultan Ali; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We have previously shown that corticosterone (CORT) can induce premature differentiation of somatotrophs in the chicken embryo. CORT induction of GH mRNA is indirect, in that protein synthesis inhibition blocks its inducing effect. In this study, we used a custom chicken microarray to analyze global gene expression in the embryonic pituitary gland and to identify potential direct targets of CORT that may regulate its induction of somatotroph differentiation. Dispersed embryonic (e) day 11 pituitary cells were pretreated with cycloheximide then with CORT or treated with CORT alone for 24 hrs. Amplified RNA from these samples was then used on our microarray to analyze gene expression and in quantitative real-time PCR (qRT-PCR) analysis to determine relative gene expression levels. qRT-PCR from these samples showed that GH was induced within 1.5 hrs and continued to significantly increase until 3 hrs. Our microarray analysis revealed 25 direct early targets of CORT. From these 25 we chose 3 genes, Dexras1, Ras-dva, and Prostaglandin-D Synthase to transfect into e11 pituitary cells and measure their effect on GH mRNA. Neither of these genes had a direct effect on GH mRNA; however Dexras1 acted synergistically with CORT to induce GH mRNA. Dexras1 was discovered in murine AtT-20 corticotroph cells because its expression was rapidly induced in response to glucocorticoids. We report here a chicken Dexras1 cDNA that is 1631 bp in length and encodes a protein of 278 amino acid residues. Comparison of the consensus chicken Dexras1 amino and nucleic acid sequence with those of human, mouse, and rat Dexras1 showed high homology among the species. Expression of Dexras1 mRNA was detected in various tissues by Northern analysis, but was highest in the pituitary. RT-PCR analysis showed expression of Dexras1 only in the pituitary. The precise role of DexRas1 is unidentified at the present time; however, its distribution in a range of tissues suggests a possible role in glucocorticoid action within a variety of systems.Item Adaptation of A/Mallard/Potsdam/178-4/83 (H2N2) in Japanese quail leads to Replication and Transmission in Chickens(2005-05-02) Sorrell, Erin Maureen; Perez, Daniel R; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza is a single stranded, negative-sense RNA virus with a segmented genome that can infect avian and mammalian species. Influenza viruses from the avian reservoir do not seem to replicate efficiently in humans upon direct transmission. Therefore, an intermediate host is involved in generating mutations to create a more transmissible or an avian-human reassortant virus. Quail have been highlighted as a potential reservoir and intermediate host of avian influenza. To assess the potential of quail as an intermediate host, A/Mallard/Postdam/178-4/83, H2N2 was tested to determine if through adaptation in quail a mallard strain can replicate and transmit in quail, as well as other avian species. After six serial passages of lung homogenate a virus arose, which replicated and transmitted directly to contact quail. When chickens were infected with this quail-adapted virus replication and transmission were observed, while no replication was noted in the chickens infected with wild type H2N2 virus.