Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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Item MODULATING KEY GENES INVOLVED IN PANCREAS FORMATION AND INSULIN SIGNALING USING CRISPR/CAS9 IN THE PIG(2019) Sheets, Timothy P; Telugu, Bhanu P; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Among the metabolic diseases, diabetes remains a “pressing problem” as recognized by World Health Organization, not only due to the impact on individuals’ lives, but also because of the rapid increase in newly diagnosed patients. To better understand the mechanisms of diabetes, this dissertation investigates the role of NGN3 in pancreas development using CRISPR/Cas9 gene targeting in the pig model. NGN3 was selected for study because of its critical role in endocrine pancreas formation. Our research demonstrates that the targeted ablation of NGN3 blocks development of the endocrine pancreas, a finding supported through gene expression analysis. Furthermore, follow-up studies show that clonal piglets derived from NGN3-ablated animals lack the major endocrine islet cell types and subsequent expression of key endocrine hormones. This porcine model provides valuable insights into the study of type 1 diabetes in early post-natal life and future applications of human-to-pig chimeric organ development for transplant surgery. Expanding upon this porcine model for diabetes, we sought to apply this approach to the study of type 2 diabetes using a novel pig model, thus bridging the gap between mouse and human. For this endeavor, we identified GRB10 as a potential critical mediator in insulin signaling, development, and growth potential following an extensive literature review. The potential for dual applications in both agriculture and medicine was also identified as an objective. Analysis of qPCR data from in vitro overexpression studies supports that GRB10 modulates insulin signaling through the canonical insulin pathway. Additional data from two in vivo gene editing trials targeting the GRB10 locus in both Ossabaw and domestic pig breeds show a supportive qualitative trend towards growth regulation in the Ossabaw pig breed. Further evidence is required to determine whether GRB10 plays the same role in the domestic pig, as a limited cohort size of mutants precluded an extensive analysis of phenotypes. Together, our assessment of NGN3 and GRB10 offer significant potential for modeling of both type 1 and type 2 diabetes as well as modeling of growth traits in the pig through application of advanced genome engineering technology.Item Genetic Architecture of Complex Traits and Accuracy of Genomic Selection in Dairy Cattle(2018) Jiang, Jicai; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genomic selection has emerged as an effective approach in dairy cattle breeding, in which the key is prediction of genetic merit using dense SNP genotypes, i.e., genomic prediction. To improve the accuracy of genomic prediction, we need better understanding of the genetic architecture of complex traits and more sophisticated statistical modeling. In this dissertation, I developed several computing tools and performed a series of studies to investigate the genetic architecture of complex traits in dairy cattle and to improve genomic prediction models. First, we dissected additive, dominance, and imprinting effects for production, reproduction and health traits in dairy cattle. We found that non-additive effects contributed a non-negligible amount (more for reproduction traits) to the total genetic variance of complex traits in cattle. We also identified a dominant quantitative trait locus (QTL) for milk yield, revealing that detection of QTLs with non-additive effect is possible in genome-wide association studies (GWAS) using a large dataset. Second, we developed a powerful Bayesian method and a fast software tool (BFMAP) for SNP-set association and fine-mapping. We demonstrated that BFMAP achieves a power similar to or higher than existing software tools but is at least a few times faster for association tests. We also showed that BFMAP performs well for fine-mapping and can efficiently integrate fine-mapping with functional enrichment analysis. Third, we performed large-scale GWAS and fine-mapped 35 production, reproduction, and body conformation traits to single-gene resolution. We identified many novel association signals and many promising candidate genes. We also characterized causal effect enrichment patterns for a few functional annotations in dairy cattle genome and showed that our fine-mapping result can be readily used for future functional studies. Fourth, we developed an efficient Bayesian method and a fast computing tool (SSGP) for using functional annotations in genomic prediction. We demonstrated that the method and software have great potential to increase accuracy in genomic prediction and the capability to handle very large data. Collectively, these studies advance our understanding of the genetic architecture of complex traits in dairy cattle and provide fast computing tools for analyzing complex traits and improving genomic prediction.Item DYNAMIC ANALYSIS OF CD4+ T CELL EPIGENETIC STATUS IN CHICKENS FOLLOWING MDV INFECTION AND DURING DIFFERENTIATION IN MICE(2018) Ding, Yi; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Epigenetic modifications constitute a complicated regulatory network controlling various biological processes from cell development to immune responses. The mechanisms through which CD4+ T cells react to environmental stimuli, including virus intrusion and differentiation signals, represent the fundamental cell biological question of how the external microenvironment influences intrinsic transcriptional networks. This dissertation investigates the epigenetic status changes in CD4+ T cells induced by Marek’s disease virus (MDV) infection in chickens and during differentiation in mice. First, a genome-wide gene expression analysis in the immune organs from resistant line 63 and susceptible line 72 chickens was performed to explore Marek’s disease (MD) resistance mechanisms. MDV infection influences both cytokine-cytokine receptor interaction and cellular development in resistant and susceptible chickens. Second, we examined the epigenetic status of CD4+ T cells induced by MDV infection, including chromatin accessibility and chromosome organization. Our results revealed extensive epigenetic modification changes caused by MDV infection. Only resistant line 63 chickens could initiate robust adaptive immune responses at the transcription level, and the increase in chromatin accessibility and chromosome reorganization represented by A/B compartment flipping were related to up-regulated genes induced by MDV infection at 10 days post-infection in line 63 chickens. Finally, we investigated CD4+ T cells plasticity during Th1 helper cell differentiation. We showed “early” (48 hours) CD4+ T cells were plastic for cellular reprogramming while “late” (72 hours) cells lost reprogram plasticity and became committed to Th1 cell fate. T-bet, the Th1 cell master regulator, was not the direct determinant of Th1 cell plasticity. Our integrative analysis of multiple “omics” datasets revealed dynamic and genome-wide changes of chromatin accessibility associated with the process of cellular differentiation and commitment. We predicted that several candidate regulators could contribute to cellular plasticity, including Mxi1, JunB, BATF, IRF4, and Hif-1α. We observed that substantial alterations of chromatin interactions occurred at the IRF4 locus across differentiation time. Conditional deletion of IRF4 in CD4+ T cells impacted the expression of T cell activation and differentiation genes, including T-bet, and extended Th1 cell plasticity during the differentiation process. Our findings provided deeper understanding of CD4+ T cell commitment and responses toward viral infection.Item FUNCTIONAL CHARACTERIZATION OF HEME TRANSPORTERS IN ZEBRAFISH(2017) Zhang, Jianbing; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hrg1 and Mrp5 are identified as eukaryotic heme importer and exporter, respectively. Two Hrg1 paralogs have been annotated in zebrafish genome, Hrg1a (Slc48a1b) and Hrg1b (Slc48a1a) with 84% homology in protein sequences. Hrg1a and hrg1b are widely expressed in embryonic and adult zebrafish. Yeast growth assays reveal that zebrafish Hrg1a and Hrg1b are both capable of heme import. However, hrg1a and hrg1b double knockout (hrg1 DKO) zebrafish generated by CRISPR/Cas9 has no overt defects in differentiation and maturation of erythroid cells. Knockdown of hrg1a in hrg1b mutants or vice versa does not impair erythroid lineage in zebrafish embryos. These genetic results suggest that Hrg1 is not required for maturation and hemoglobinization of primitive erythroid cells. Hrg1a and hrg1b mRNA are upregulated in adult kidneys and spleens upon PHZ-induced hemolysis, together with hmox1, a downstream heme degrading enzyme, suggesting that Hrg1 is involved in adult heme-iron recycling during erythrophagcytosis in kidney and spleen of adult zebrafish. DAB-enhanced Perl’s iron staining reveals that iron is accumulated in macrophages in the kidney and spleen in adult wild-type zebrafish. However, macrophages with positive Perl’s staining are rarely found in the kidney of hrg1 DKO and instead large amount of iron is deposited in renal tubules, suggesting defects in heme-iron recycling by kidney macrophages in hrg1 DKO under PHZ-induced hemolysis. Whole transcriptome sequencing of mRNA extracted from spleens and kidneys reveals massive differentially expressed genes in hrg1 DKO involved in immune response, lipid transport, oxidation-reduction process and proteolysis. These indicate that hrg1 DKO are deficient in recycling heme-iron derived from damaged RBCs in the absence of functional Hrg1. Phylogenetic analysis reveals that Mrp5 and Mrp9 are closed homologs in the zebrafish genome. Yeast growth assays reveal that both zebrafish Mrp5 and Mrp9 are capable of heme export. Morpholino knockdown of mrp5 and mrp9 in zebrafish showed severe anemia in developing embryos indicating their involvements in erythropoietic development. Subsequent generation and characterization of mrp5 and mrp9 mutants by CRISPR/Cas9 will further define the function of Mrp5 and Mrp9 during zebrafish development.Item Integrative genomic, epigenetic and metabolomic characterization of beef from grass-fed Angus steers(2016) Carrillo Tabakman, Jose Adrian; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Beef constitutes a main component of the American diet and still represent the principal source of protein in many parts of the world. Currently, the meat market is experiencing an important transformation; consumers are increasingly switching from consuming traditional beef to grass-fed beef. People recognized products obtained from grass-fed animals as more natural and healthy. However, the true variations between these two production systems regarding various aspects remain unclear. This dissertation provides information from closely genetically related animals, in order to decrease confounding factors, to explain several confused divergences between grain-fed and grass-fed beef. First, we examined the growth curve, important economic traits and quality carcass characteristics over four consecutive years in grain-fed and grass-fed animals, generating valuable information for management decisions and economic evaluation for grass-fed cattle operations. Second, we performed the first integrated transcriptomic and metabolomic analysis in grass-fed beef, detecting alterations in glucose metabolism, divergences in free fatty acids and carnitine conjugated lipid levels, and altered β-oxidation. Results suggest that grass finished beef could possibly benefit consumer health from having lower total fat content and better lipid profile than grain-fed beef. Regarding animal welfare, grass-fed animals may experience less stress than grain-fed individuals as well. Finally, we contrasted the genome-wide DNA methylation of grass-fed beef against grain-fed beef using the methyl-CpG binding domain sequencing (MBD-Seq) method, identifying 60 differentially methylated regions (DMRs). Most of DMRs were located inside or upstream of genes and displayed increased levels of methylation in grass-fed individuals, implying a global DNA methylation increment in this group. Interestingly, chromosome 14, which has been associated with large effects on ADG, marbling, back fat, ribeye area and hot carcass weight in beef cattle, allocated the largest number of DMRs (12/60). The pathway analysis identified skeletal and muscular system as the preeminent physiological system and function, and recognized carbohydrates metabolism, lipid metabolism and tissue morphology among the highest ranked networks. Therefore, although we recognize some limitations and assume that additional examination is still required, this project provides the first integrative genomic, epigenetic and metabolomics characterization of beef produced under grass-fed regimen.Item Genetic Suppressors of mrp-5 Lethality in C. elegans(2016) Beardsley, Simon; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential cofactor in numerous proteins, but is also cytotoxic. Thus, directed pathways must exist for regulating heme homeostasis. C. elegans is a powerful genetic animal model for elucidating these pathways because it is a heme auxotroph. Worms acquire dietary heme though HRG-1-related importers, and intestinal export was demonstrated to be mediated by the ABC transporter MRP-5. Loss of mrp-5 results in embryonic lethality. Although heme transporters have been identified, there are significant gaps in our understanding for the heme trafficking beyond HRG-1 and MRP-5. To identify additional components, we conducted a forward genetic screen utilizing the null allele mrp-5(ok2067). Screening of 160,000 haploid genomes yielded thirty-two mrp-5(ok2067) suppressor mutants. Deep-sequencing variant analysis revealed three of the suppressors subunits of adapter protein complex 3 (AP-3). We now seek to identify mechanisms for how adaptor protein deficiencies bypass a defect in MRP-5-mediated heme export.Item Population Declines and Genetic Variation: Effects of Serial Bottlenecks(2015) Callicrate, Taylor Eilers; Song, Jiuzhou; Fleischer, Robert C; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Islands foster unique biodiversity, yet also present biogeographic limitations that impose increased risk for population extinction through demographic and genetic constraints and decreased probability of surviving a catastrophe. Of particular interest, especially with regard to endangered species, is the genetic response of insular species to severe population declines or translocations. Both types of events, considered population bottlenecks, are expected to reduce genetic variation, and correspondingly, adaptive potential. For these reasons, it is important to understand how bottlenecks interact with insular population dynamics to affect genetic diversity. I used a combination of a laboratory model experiment and population genetics study of an in situ bottleneck in an endangered species to investigate how quantitative and molecular genetic variation are affected during bottlenecks. I used a laboratory animal model (red flour beetle, Tribolium castaneum) to compare how quantitative genetic variation is affected if a serial bottleneck occurs in a novel versus familiar environment. The experiment was designed to model a founder event or translocation to a new island with a novel environment. I found that phenotypic and additive variance for a quantitative trait were larger following a bottleneck occurring in the novel environment, suggesting that the novel environment could improve adaptive potential in bottlenecked populations. Next, I used molecular genetic markers to assess variation and signatures of selection in the Laysan finch (Telespiza cantans), a Hawaiian honeycreeper endemic to a small Northwestern Hawaiian island. Laysan finches experienced a major bottleneck on Laysan in the early 20th century, followed by a translocation and series of founder events as populations were established on the islets of Pearl and Hermes Reef (PHR) in the 1960s – 70s. I found that, contrary to expectation, bottlenecked Laysan finch populations did not show declines in genetic variation and were not differentiated as a result of genetic drift. These results are potentially caused by insular demographic dynamics. I identified loci with extreme differentiation between modern populations, potentially indicating genomic signals of selection. These regions could be important for adaptation to the novel environment on PHR and are candidates for future study.Item LONG-RANGE SIGNALING AT THE INTESTINAL-NEURAL AXIS PROMOTES ORGANISMAL HEME HOMEOSTASIS IN C. ELEGANS(2014) Sinclair, Jason Wallace; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metazoans synthesize and regulate intracellular heme in a cell autonomous manner although genetic evidence in vertebrates suggests that cell non-autonomous mechanisms may exist at the organismal level. In C. elegans, a heme auxotroph, extraintestinal tissues are intrinsically dependent on the intestine, which acquires dietary heme for sustenance, supporting the concept that intestinal heme status must be coordinated at the systemic level to regulate whole-organism heme homeostasis. Here we show, by conducting a functional genome-wide RNAi screen in an intestinal-restricted heme sensor worm, that an interorgan heme signaling pathway exists and that >30% of the genes identified from the RNAi screen altered heme homeostasis in the intestine even though these genes are not expressed in the intestine. The biological basis for this signaling is underscored by HRG-7, a cathepsin protease-like protein secreted by the intestine and internalized by distally-located neurons. HRG-7 is specifically secreted from the intestine during heme limitation and hrg-7 depletion causes embryonic lethality concomitant with a heme deficiency response. Reciprocally, neuron-to-intestine heme signaling is mediated by the bone morphogenic protein homolog DBL-1, which recapitulates hrg-7 deficiency when depleted. Remarkably, depletion of both genes simultaneously results in markedly enhanced growth and heme deficiency phenotypes, suggesting that bidirectional signaling between the intestine and neurons mediates systemic heme homeostasis. Our results have uncovered an unexpected role for a protease family member in long-range communication between organs at the intestinal-neural axis to regulate systemic heme homeostasis in metazoa. As humans have over thirty cathepsin and cathepsin-like proteases, several of which are secreted, we anticipate that these proteins may play analogous roles in mammalian biology.Item Investigating genetic and health factors related to AA amyloidosis prevalence in captive cheetahs (Acinonyx jubatus): implications for population management(2014) Franklin, Ashley Danielle; Porter, Tom E; Crosier, Adrienne E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Systemic amyloid A (AA) amyloidosis is an increasingly important cause of morbidity and mortality among captive cheetahs, yet wild cheetahs are virtually unaffected, suggesting the phenomenon is a result of the captive condition. The self-aggregating AA protein responsible for this disease, is a byproduct of serum amyloid A (SAA) protein degradation, an acute phase protein highly upregulated during inflammation. The objective of this study was to identify the relationship between genetics, stress, and inflammation with serum concentrations of the SAA protein and the incidence of AA amyloidosis in captive cheetahs. Fecal and serum samples collected from cheetahs held at the Smithsonian (NZP-SCBI) and Cheetah Conservation Fund (CCF) facilities, as well as wild, free-ranging cheetahs, were examined. Enzyme-linked immunosorbent assays were used to measure SAA protein and proinflammatory cytokine concentrations in serum samples and cortisol concentrations in feces. Additionally, cheetahs were genotyped for the SAA1A-97delG single nucleotide polymorphism (SNP) in the promoter region of the SAA1 gene. This study was the first to demonstrate that serum concentrations of the SAA protein in cheetahs are affected by the SAA1A-97delG SNP (P=0.0453). However, the high prevalence of AA amyloidosis observed among captive cheetahs is not attributable to genetic differences at this locus, but rather appears to be related to stress and/or inflammation, as captive cheetahs at NZP-SCBI have significantly higher SAA protein concentrations in serum compared to captive cheetahs at CCF, regardless of genotype (P=0.0003). Captive cheetahs at NZP-SCBI show levels of stress (fecal cortisol concentrations) greater than their captive counterparts at CCF in Namibia. Interestingly, wild cheetahs and captive cheetahs at CCF in Namibia had significantly higher proinflammatory cytokine concentrations (TNF-α and IL-1β) in serum compared to cheetahs at NZP-SCBI (P<0.0001). It is possible that chronic stress may be suppressing the production of proinflammatory cytokines in the NZP-SCBI cheetah population. Controlling the currently high SAA protein concentrations associated with AA amyloidosis is the best strategy to decreasing the diseases prevalence among captive cheetahs. Promoting management practices that reduce stress could help re-establish proper immune system homeostasis and mitigate the overproduction of SAA protein, decreasing the probability of developing AA amyloidosis.Item FUNCTIONAL INSIGHTS INTO HRG-1-MEDIATED HEME TRANSPORT(2012) Yuan, Xiaojing; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential cofactor involved in various biological processes including oxygen transport, xenobiotic detoxification, oxidative metabolism, gas sensing, circadian rhythm, signal transduction, microRNA processing and thyroid hormone synthesis. Heme is also an essential nutrient for parasites and is the major dietary iron source for humans. Despite our extensive understanding of the mechanisms of heme synthesis and degradation in eukaryotes, little is known as to how heme is transported and trafficked in eukaryotes. Recently, CeHRG-1 and CeHRG-4 were identified as the first bona fide heme importers/transporters using the heme auxotroph, Caenorhabditis elegans. To gain mechanistic insights into the heme transport function of HRG-1-related proteins, we conducted a structure-function analysis of CeHRG-1 and CeHRG-4 by exploiting yeast mutants that are genetically defective in heme synthesis. Our studies reveal that HRG-1-related proteins transport heme across membranes through the coordinated actions of strategically placed amino acids that are topologically conserved in both, the worm and human proteins. To further dissect the functional elements that dictate their intracellular localization, we generated a series of chimeras by swapping the amino and carboxy terminal segments of CeHRG-1 and CeHRG-4. Our analysis in yeast and mammalian cells demonstrate that the C-terminal domains are essential for membrane localization of the protein, while the N-terminal domains are important for proper function, and plausibly multimerization of HRG-1-related proteins. Currently, there are no pharmacological means to aid in the study of the cellular and physiological roles of eukaryotic heme transporters. We, for the first time, developed and executed a high-throughput screen of 233,360 compounds, to identify potential antagonists of HRG-1-related proteins by utilizing parasite heme transporters as the primary screening bait. Subsequent study in parasites will provide novel drug candidates against helminths that infect humans, livestock, and plants, as well as against genetic disorders of heme and iron metabolism in humans. Taken together, results from our studies will significantly advance novel functional and therapeutic insights into HRG-1 mediated heme transport in health and disease.