Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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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 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 QUALITY ASSESSMENT OF ATLANTIC STURGEON (ACIPENSER OXYRINCHUS) SPERMATOZOA UNDER CONDITIONS OF SHORT-TERM STORAGE(2009) Dorsey, Kathryn Michelle; Woods, Curry; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Short-term storage trials were conducted in 2008 and 2009 on Atlantic sturgeon semen obtained from captive males, held at the U.S. Fish and Wildlife Service, Northeast Fish Technology Center, that were hormonally induced to spermiate; and wild males collected during the spawning season from the Hudson River. Samples were stored under refrigeration (4 + 1°C) in treatments consisting of different gaseous environments (oxygen, nitrogen or air) and experimental diluents (Modified Tsvetkova extender, Park & Chapman extender and neat, i.e. undiluted). Analyses of gamete quality were performed on day 0 (pre-treatment), and then every other day for 7 days in 2008 and for 21 days in 2009. Sperm quality parameters evaluated included: viability, motion analysis, curvilinear velocity and cellular ATP levels. Higher gamete quality was maintained when spermatozoa were diluted in the PC extender and stored in the presence of oxygen.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 The potential role of butyrophilin and xanthine oxidoreductase in controlling the amount and size of milk-fat droplets(2008-08-05) Jacob, Jaison; Mather, Ian H; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The secretion of fat droplets from mammary epithelial cells requires the expression of two major proteins, butyrophilin1A1 (BTN) and xanthine oxidoreductase (XOR). Ablation of the BTN or XOR gene in mice results in the accumulation of large fat droplets suggesting a reciprocal relationship between BTN/XOR concentration and milk-fat droplet size. We tested this hypothesis by correlating BTN/XOR concentrations in cow and mouse with their fat droplet size. The amount of BTN in mouse was 75 times less than in bovine samples. The size of fat droplets in mice was larger than in cow, but no correlation was found between fat-droplet size and the amount of BTN/XOR. Experimental reduction in fat-droplet size in mice did not change the concentration of BTN. We propose that a low amount of BTN is sufficient to mediate its role in milk-fat secretion and that it may have additional functions to its potential role as a structural protein.Item Characterization of Chicken CAT-2 Isoforms(2007-08-20) Kirsch, Sandra B; Hamza, Iqbal; Humphrey, Brooke D; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lysine and arginine transport is primarily mediated by cationic amino acid transporters (CATs) in cells. The chicken CAT-2 (cCAT-2) transcript is alternatively spliced to three isoforms. Transcriptional and cellular localization experiments were utilized to study their regulation. The mRNA abundance of cCAT-2 isoforms was estimated in body tissues, and although differentially expressed, all tissues expressed each cCAT-2 isoform gene, indicating that alternative splicing was not tissue-specific. Both cCAT-2A and cCAT-2B proteins localized to the plasma membrane and cCAT-2C protein was retained in the cytosol. Chicken CAT-2A functions as a low affinity transporter with specificity for lysine and arginine. Chicken CAT-2B and cCAT-2C transporter functions were not detectable. Our data indicates that CAT-2 transporters are conserved in non-mammalian vertebrates, but cCAT-2 isoforms differ in their tissue distribution and transporter function from previously characterized CAT-2 transporters. These results also indicate a mechanism by which additional dietary lysine and arginine contribute to increased protein accretion in muscle tissue.Item Cytological localization of heme in Caenorhabditis elegans using microscopy.(2007-05-07) Kelley, Cornelia Ellefson; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This study was designed to develop an in situ histochemical heme staining method for an intact animal using the free-living nematode Caenorhabditis elegans. Although, heme is vital to many biological processes and synthesized by most known free-living organisms, C. elegans is a natural heme auxotroph. Using the substrate 3-3' diaminobenzidine and hydrogen peroxide, we used C. elegans to study the fate of heme. We found a direct correlation between heme in the growth medium and the organismal heme content. In addition, our studies confirmed that parents exposed to different heme levels contribute varying maternal heme to their progeny. Moreover, this methodology detected differences in heme levels between wild-type and mutants in heme homeostasis. Finally, we provide preliminary evidence that the technique can be applied to analyze heme-based structures at the electron microscopy level. Our studies described herein will aid in the characterization of heme transport pathways in eukaryotes.Item THE INTERELATIONSHIP BETWEEN CD14 AND LPS DURING MASTITIS: RELEASE OF SOLUBLE CD14 AND CYTOKINES BY BOVINE PMN FOLLOWING ACTIVATION WITH LPS(2005-08-11) sohn, eun j; Peters, Robert R; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Understanding the immune defense of the mammary gland is important in devising and developing measures to control mastitis. Lipopolysaccharide (LPS) is the predominant factor causing pathogenesis in Gram-negative bacterial infections. The cluster of differential antigen14 (CD14), which is located on the surface of leukocytes, is the receptor for LPS. The binding of LPS to CD14 results in release of pro-inflammatory cytokines that recruit polymorphonuclear neutrophil leukocytes (PMNs), to the site of inflammation, allowing them to kill bacteria through the process of phagocytosis. This research was designed to produce and characterize monoclonal antibodies (mAb) against recombinant bovine soluble (rbos) CD14, express rbosCD14 in plants and characterize the biological activity of plant-derived rbosCD14 in vitro and in vivo. The release of inflammatory cytokines and expression of CD14 on bovine PMN and the secretion of IL-8 by PMN in response to LPS was also invesitigated. A panel of ten murine mAb reactive with rbosCD14 were produced and a sandwich ELISA was developed using the mAbs and rabbit polyclonal antibodies. The mAbs recognized rbosCD14 (40 kDa), solubleCD14 (sCD14) (53 and 58 kDa) in milk and blood, and a 47 kDa mCD14 in lysates of macrophages obtained from involuted bovine mammary gland secretions by Western blot analysis. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions. The addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced TNF-alpha production in vitro. The anti-rbosCD14 mAbs generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretion produced during mastitis. This study demonstrates the functional activity of a recombinant animal receptor protein made in plants, and the use of a plant-derived protein as a potential animal therapeutic for bacterial infections. A truncated form of sCD14, carrying a histidine residue affinity tag was incorporated with potato virus X for transient expression in Nicotiana benthamiana. CD14 from crude plant extracts was recognized by Western blot analysis. Biological activities of plant-derived rbosCD14 (prbosCD14) were characterized in vitro and in vivo. Biological activity of prbosCD14 demonstrated in vitro by LPS-induced apoptosis, an increase of caspase activity and IL-8 production by bovine endothelial cells. In vivo, prbosCD14 enhanced LPS-induced PMN recruitment and in bovine mammary quarters challenged with Escherichia coli that resulted in decreased bacterial counts and elimination of clinical symptoms. In this disseration, shedding of sCD14 from the surface of PMN in response to LPS was negatively correlated with IL-8 release. Shedding of sCD14 from the surface of PMN increased in the absence of serum and at high concentrations of LPS. The use of real time RT-PCR showed that CD14 gene expression was not different between control and the LPS stimulated cells. This demonstrates that shedding of sCD14 comes from membrane-boundCD14 (mCD14). However, in contrast to the shedding of sCD14, IL-8 secretion by PMN was decreased at high concentrations of LPS in the absence of serum. Moreover, sCD14 secretion from PMN stimulated with LPS was increased in parallel with the decrease of IL-8 secretion at varying PMN cell densities. This result suggests that the release of CD14 leads to the down-regulation of IL-8 secretion by PMN in response to LPS. Bovine PMN produced different types of cytokines in response to LPS in this disseration. The secretion of TNF-alpha, IL-1beta, IL-8 and INF-gamma by PMN increased in a dose-dependent manner, but IL-12 secretion by PMN was decreased with increasing concentrations of LPS. Co-incubation of LPS with either TNF-alpha or IL-1beta increased secretion of IL-8 when compared to LPS alone. These studies contribute to a better understanding of the interrelationship between bovine CD14 and LPS during mastitis and provide new insights into events that resolve inflammation following bovine PMN activation.Item SOMATOTROPIN RESPONSE TO CORTICOSTERONE AND THE THYROID HORMONE T3 DURING CHICK EMBRYONIC DEVELOPMENT: INVOLVEMENT OF TYPE I AND TYPE II GLUCOCORTICOID RECEPTORS(2004-05-17) Heuck, Kristina; Porter, Tom E; Animal SciencesCorticosterone (CORT) can stimulate growth hormone (GH) cell (somatotroph) differentiation and GH secretion on embryonic day (e) 12 but not e20 in the chicken. GH induction involves both glucocorticoid receptors (GR) and mineralocorticoid receptors (MR); however, this finding has been characterized only on e12. To further define changes in somatotroph responsiveness to CORT, pituitary cells obtained on e12-e20 were cultured with CORT alone and in combination with T3. GH secretion increased over basal with CORT treatment on e12, e14, e16, and e18, but not e20. Contributions of GR and MR in CORT responses were evaluated using GR and MR antagonists. Blocking both receptors was required to abolish the CORT response by e12 cells. The same treatment on e20 decreased GH secretion relative to basal. We conclude that positive somatotroph responses to CORT are lost during embryonic development and that both GR and MR mediate CORT-induced GH secretion by cultured somatotrophs.