Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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Item MEDIATION OF CORTICOSTERONE-INDUCED GROWTH HORMONE GENE EXPRESSION IN CHICKEN EMBRYONIC PITUITARY CELLS: IDENTIFICATION OF TRANS-ACTING FACTORS AND A NOVEL PITUITARY CELL TYPE(2024) Liu, Kuan Ling; Porter, Tom E.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) is responsible for up to 30% of growth in broiler chickens. Somatotrophs, or GH secreting cells, begin to differentiate around embryonic day (e)14, in conjunction with an increase in the primary plasma glucocorticoid (GC) corticosterone (CORT). CORT treatment of e11 chicken embryonic pituitary (CEP) cells induces premature GH secretion. This GC-induced process involves trans-acting factors because the GH gene lacks a canonical GC response element (GRE). In addition to the binding of ETS1 and the GC receptor (GR) to the GC-responsive region (GCRR; 1045/ 964), we hypothesize that there are other regulatory factors necessary for CORT responsiveness. By modifying the pGL3_-1742/+25 GH-luciferase reporter, we have constructed various other GH-luciferase reporters and analyzed them for promoter activity in response to CORT treatment. We identified a putative distal (d) ETS-Like 1 (ELK1) binding site that is necessary. The proximal (p)PIT1 site and pTATA box were also identified to be critical for CORT induction of the GH gene. Interestingly, cloning multiple copies of the extended GCRR (eGCRR; -1067/-900) further increased promoter activity in an additive manner under both basal and CORT treated conditions. Through single-cell RNA sequencing (scRNAseq), 8 members of the ETS family of transcription factors were identified in > 5% of the somatotroph population. Commercial antibodies were validated, and human (h)ETV1, hELF2, hELK3, and hETV6 antibodies were confirmed to recognize their recombinant chicken ortholog and to identify their corresponding protein in e11 CEP cells. Results from chromatin immunoprecipitation quantitative PCR suggest that multiple ETS members are involved in CORT induction of the GH gene with more evidence pointing towards ELF2 and ELK3. Identifying trans-acting factors for the GH gene inducible by CORT allows for better understanding of endogenous GH regulation in chickens. Further analysis of the scRNAseq data from e11 CEP cells revealed a cluster of cells expressing genes for more than one hormone-producing cell type (“premature nebulous” cluster). Within the premature nebulous cluster, a large population (~30%) was co-expressing proopiomelanocortin (POMC) and growth hormone (GH). We named this novel cell population the cortico-somatotrophs. Through RNA fluorescent in-situ hybridization (RNA-FISH) and dual label immunofluorescence, we verified the existence of the cortico-somatotrophs at both the mRNA and protein level, respectively. Cortico-somatotrophs were also shown to share genes for receptors normally specific to both corticotrophs (CRH-R1) and somatotrophs (GHRHR). Additionally, in response to CORT treatment, the cortico somatotrophs showed an increase in GH as well as a decrease in POMC mRNA levels. The discovery of the cortico-somatotrophs suggests a modification to the current dogma on pituitary cell lineages, where corticotrophs and somatotrophs may have overlapping developmental pathways. In conclusion, our discovery of the cortico somatotrophs has furthered our understanding of CEP development and opened the door for further exploration of the cell lineages during pituitary development.Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.Item ANTAGONISTIC MECHANISM OF METABOLITES FROM LACTOBACILLUS CASEI AGAINST FOODBORNE ENTEROHEMORRHAGIC ESCHERICHIA COLI(2022) Aditya, Arpita; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Enterohemorrhagic Escherichia coli O157: H7 (EHEC), a foodborne enteropathogen, remains a significant public health concern since its discovery in 1982. With an incredibly low infectious dose (10-100 bacteria), this pathogen can cause self-limiting diarrhea, vomiting, and abdominal cramps. However, more complicated disease conditions such as bloody diarrhea or hemolytic colitis have been known to develop depending on the serotype involved in the infection, and on immune status and/or age of the patients. Due to its Shiga toxin (Stx) production ability, EHEC infection may lead to a kidney-related problem known as hemolytic uremic syndrome (HUS), which requires advanced medical care. Unlike other bacterial illnesses, therapeutic administration of antibiotics to treat EHEC infections is not recommended due to their controversial association with Stx production. As a result, only preventative/prophylactic and immune-supportive strategies are followed for EHEC infections. Using the antibacterial properties of probiotic bacteria and the metabolites they produce are promising alternative strategies for preventing EHEC infections. We have targeted the probiotic bacteria Lactobacillus casei to determine the mechanism of this alternative strategy. In our study, we have executed microbiological, molecular, chromatographic, and metagenomic approaches to determine the antagonistic mechanisms of action of their metabolites, specifically conjugated linoleic acid (CLA) produced by Lactobacillus casei, against the growth and metabolism of EHEC. The metabolites of wild-type L. casei (LCwt) were augmented by supplementing it with a prebiotic-like dietary component, namely peanut flour (PF) (LCwt+PF), while another LCwt was also genetically engineered (LCCLA) to over convert CLA from linoleic acid (LA). These modifications showed effective results in controlling EHEC both in vitro and in ex vivo conditions. Total metabolites present in cell-free culture supernatant (CFCS) of LCwt, LCwt+PF, and LCCLA were able to control the growth of EHEC without negatively hampering the relative abundance of Firmicutes and Bacteroidetes present in rumen fluid (RF). Among these CFCSs, CFCSCLA exerted the most desirable outcome by eliminating EHEC. In vitro studies demonstrated that, a lower concentration of purified CLA worked synergistically with other metabolites of LCwt and augmented their inhibitory activity against EHEC. The orchestrated effect of metabolites has been observed to downregulate the virulence genes, disrupt the cell membrane, interfere with cell division, and damage their genomic DNA. The probable effect of these metabolites, specifically CLA, on Stx production and neutralization was also investigated by assessing host cell cytotoxicity. Total metabolites of Lactobacillus spp. as well as CLA itself, showed improvement in cell survivability when exposed to Stx. Our findings established a ground to explore the effect of specific metabolites obtained from probiotic bacteria in control and prevention of EHEC. The findings also showed a promising association of purified CLA in neutralizing Stx which can be further explored to use it in therapeutic purposes.