Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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Item Investigating Copper Acquisition And Delivery via Transporters and a Pharmacological Chaperone in Copper-Deficient Worms and Mice(2019) Yuan, Sai; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Copper (Cu) is a key micronutrient required for a variety of essential biochemical pathways. Systemic or tissue-specific Cu-deficiencies, caused by insufficient dietary Cu uptake or mutations in Cu transporting genes, result in impaired growth, neuropathy, ataxia, hypopigmentation, osteoporosis and anemia-like symptoms in mammals. How organisms regulate Cu homeostasis at the systemic levels in response to Cu deficiencies remain elusive. In this study, we use Caenorhabditis elegans (C. elegans), a genetically tractable, multi-tissue metazoan to explore Cu homeostasis and investigate these unknowns. The high-affinity Cu transporters encoded by CTR family genes are required for dietary Cu uptake and maintaining systemic Cu balance from yeast to mammals. However, little is known about Cu acquisition mechanisms in C. elegans. We identified ten CTR ortholog genes in C. elegans; of these, chca-1 was functionally characterized. Cu availability regulates transcription of chca-1 in both the intestine and hypodermis, and chca-1 is essential for normal growth, and reproduction in the worm. Additionally, altered Cu balance caused by the loss of CHCA-1 results in defects in Cu-responsive avoidance behavior. Identification of this CTR-like gene in C. elegans, which appears to be essential for normal Cu homeostasis in the worm, illustrates the importance of Cu delivery via CHCA-1 for normal metazoan development and behavioral phenotypes. In addition, we show that a Cu-binding pharmacological chaperone, elesclomol (ES), fully restores the developmental defects and Cu deficiencies in chca-1-depleted worms, as well as the lethality in worms lacking cua-1 expression (Cu exporter ATP7A ortholog), suggesting ES is able to efficiently deliver Cu from dietary sources to peripheral tissues through the intestine in C. elegans. Our study was further expanded to mammalian models such as cardiac-specific Ctr1-depleted (Ctr1hrt/hrt) mice. We found that ES administration fully restores the postnatal lethality, developmental defects and cardiac hypertrophy found in Ctr1hrt/hrt mice, as well as rescuing the secondary systemic Cu homeostasis responses, including aberrant ATP7A protein levels in the liver and intestine. Moreover, ES shows the potential ability to transport Cu across the blood-brain-barrier in in vitro studies. These results illustrate the capability of ES to rescue systemic Cu deficiency in worms and mice, independent of the presence of functional Cu transporters, and shed light on the therapeutic usage of ES in Cu-deficient human diseases.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 CTL-derived exosomes enhance the activation of CTLs stimulated by low affinity peptides(2018) Wu, Shu-Wei; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cytotoxic T cell (CTL) is the key to induce an effective immune response against infections caused by intracellular pathogens, such as virus and cancer. CTLs with low affinity can induce and maintain a substantial population during an adaptive immune response, although CTLs with a highest-affinity receive competitive activation signals. Low-affinity CTLs are important to induce effector response and maintain a diverse memory repertoire. However, the mechanism of generating and maintaining the expansion of lower affinity CTLs is still unknown. Here, we showed that the presence of exosome (Exo) enhanced the CTL survival and increased the cell proliferation especially in CTLs treated with the low-affinity peptide. Exo together with peptides also improved the production of IFN-γ and GZB. The exosomal stimulation in CTLs was relative to up-regulation of CD25 expression, which enhanced the IL-2 survival signals. Moreover, Exo derived from an early stage of activation had a similar but weaker function comparing with Exo derived from a late stage of activation in activating CTLs. This study identified the important role of the exosome derived from CTLs stimulated by the highest-affinity peptide in activating the naïve T cells stimulated by the low-affinity peptide.Item The Role of Adipocyte Lipid Droplet Lipolysis in Thermogenesis and Metabolic Health(2017) Shin, Hyunsu; Yu, Liqing; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)My thesis was focused on the role of Comparative Gene Identification-58 (CGI-58)-mediated adipocyte lipid droplet (LD) lipolysis in thermogenesis and metabolic health. LD lipolysis in energy-dissipating brown adipose tissue (BAT) was believed to play a central role in cold-induced non-shivering thermogenesis, but this concept has not been tested in whole animal in vivo. We created a mouse line that lacks BAT CGI-58, a coactivator of Adipose Triglyceride Lipase (ATGL) that initiates the first step of cytosolic LD lipolysis by cleaving a fatty acyl chain from a triglyceride (TG) molecule. We found that BAT-specific CGI-58 knockout (BAT-KO) mice defend against the cold normally when food is absent, despite a defect in BAT LD lipolysis. Interestingly, BAT-KO versus control mice display higher body temperature when food is present during cold exposure. This cold adaptation in BAT-KO mice is associated with increases in BAT glucose uptake, insulin sensitivity, white adipose tissue (WAT) browning, energy expenditure, and sympathetic innervation. To identify the sources of fuels for thermogenesis of BAT-KO mice in the fasted state, we hypothesized that WAT lipolysis is a major source of thermogenic fuels during fasting. To test this hypothesis, we genetically inactivated CGI-58 expression in the whole fat tissues (both BAT and WAT) of mice (FAT-KO mice). We observed that FAT-KO mice are cold sensitive when food is absent, but tolerate cold normally when food is present, demonstrating that WAT lipolysis is essential for cold-induced thermogenesis during fasting and that dietary nutrients can substitute WAT lipolysis for fueling whole-body thermogenesis. Intriguingly, FAT-KO mice display a dramatic increase in cardiac glucose uptake under both basal and insulin-stimulated conditions, which is associated with significant increases in glucose tolerance, insulin sensitivity, and cardiac expression levels of natriuretic peptides. In conclusion, our studies demonstrate that 1) BAT LD lipolysis is not essential for cold-induced whole-body thermogenesis due to increased BAT uptake of circulating fuels and WAT browning; 2) WAT lipolysis is required for fueling thermogenesis during fasting; and 3) Adipose lipolysis is critically implicated in whole-body energy metabolism and cardiac function.Item ROLE OF ANNEXIN A6 IN SENSORY NEURONS DURING EARLY CHICK CRANIAL GANGLIA DEVELOPMENT(2017) Shah, Ankita; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The cranial sensory ganglia are created from neural crest cells and placode cell-derived neurons. Defects in the migration and interaction of these cells can cause abnormalities in craniofacial development and the sensory nervous system. To this end, we are using the embryonic chick cranial ganglia to elucidate the signaling mechanisms underlying cellular interactions. The Annexin protein superfamily has an evolutionarily conserved role in the development of the sensory ganglia. Our lab previously identified a function for chick Annexin A6 in modulating early NCC migration, but a later role for Annexin A6 in cranial ganglia assembly has not been investigated. We hypothesize that Annexin A6 acts a core cytoskeletal scaffold in cranial ganglia neurons to facilitate cranial ganglia formation. In support of this, our results show that placode cell-derived neurons express Annexin A6 during cranial ganglia assembly, and that expression is maintained throughout cranial gangliogenesis. Annexin A6 is also observed in neurons within the dorsal root ganglia and ventral neural tube, suggesting that Annexin A6 may be a specific neuronal marker. To investigate the function of Annexin A6 within the placode cells of the assembling cranial ganglia, we used a gene perturbation approach. Annexin A6 depletion from developing placode cells does not affect placode cell-derived neurons’ position within the ganglionic anlage nor disturb the surrounding neural crest cell corridors. Annexin A6 knockdown in placode cells results in neurons that produce very few short and/or no axonal projections instead of the normal bipolar morphology observed in the presence of Annexin A6. Placode cell-derived neurons with reduced level of Annexin A6 still express mature neuronal markers, they do not possess two long processes, which are characteristic morphological features of mature neurons, and fail to innervate their designated targets due to the absence of this bipolar morphology. In keeping with these results, Annexin A6 overexpression causes some placode cell-derived neurons to form extra protrusions alongside these bipolar processes. These data demonstrate that the molecular program associated with neuronal maturation is distinct from that orchestrating changes in neuronal morphology, and, importantly, reveal Annexin A6 to be a key membrane scaffolding protein during neuron membrane biogenesis.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 CELLULAR PATHWAYS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITIONS IN NEURAL CREST CELLS(2013) Li, Shen; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neural crest cells are a population of multi-potent progenitor cells in the developing vertebrate embryo that undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively to generate diverse derivatives. As such, abnormal development of neural crest cells can lead to human congenital and hereditary malformations, diseases and cancers. Both internal molecular signals and external mechanical factors play essential roles in facilitating neural crest cell EMT. How cells modulate their adhesion machinery and dynamically reorganize their actin cytoskeleton to respond to the mechanical features of their external environment during EMT is not well understood. To evaluate the role of the actomyosin cytoskeleton during neural crest cell EMT and migration, midbrain neural folds that contain premigratory neural crest cells were dissected out from chick embryos, explanted into chamber slides, and incubated to allow for the formation of migratory neural crest cells. Time-lapse imaging technique was used to record cell behaviors. To elucidate cellular pathways controlling EMT and migration, chemical inhibitors (blebbistatin, Y-27632, latrunculin-A, and nocodazole) that perturb molecular cascades regulating cellular structures were employed. Effects of these perturbations on neural crest cell EMT and migration were quantified in terms of the spreading rate of the explants, and vorticity of collectively moving cell groups. We observed that blebbistatin treatment reduced the overall velocity of migratory neural crest cells to negligible levels. Moreover, migratory neural cells developed rounder cell bodies, and lamellipodia were transformed into filopodia at the periphery of the extract. Y-27632 treatment led to more neural crest cells coming out from these explants within a shorter time period compared to control. Nocodazole treatment blocked neural crest cell EMT and the resumption was dose-dependent. Latrunculin-A caused cell death at a very low concentration. These results implicate roles for non-muscle myosin II, the target of blebbistatin, and ROCK, the target of Y-27632, as well as microtubules and actin filaments, in chick midbrain neural crest cell EMT and migration. Actin crosslinkers such as α-actinin and actin-associated proteins like palladin also participate in pathways affected by these cytoskeletal inhibitors through their regulation of focal adhesion formation and cytoskeletal organization, thereby modulating cell stiffness and migration. We are also documented the distribution of α-actinin and palladin in migratory neural crest cells in vivo. Collectively, our studies have provided insight into specific cellular pathways regulating neural crest cell EMT and migration and the impact on various biophysical parameters upon perturbation of these pathways.Item THE IMPACT OF CULTURE MEDIA ON THE IN VITRO PRODUCTION OF CAT BLASTOCYSTS AND EXPLANT QUALITY(2012) Nestle, Emily; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Continued improvements in embryo culture media composition allow for the growth of high quality blastocysts, which can be used to derive embryonic stem cells (ESCs). ESCs are capable of becoming any cell type in the body making them a valuable research tool for therapeutic and regenerative research, while furthering our understanding of embryonic development and cell differentiation. The domestic cat is an important model species for both human medicine and wild felids. Cat embryo culture produces blastocysts at a rate far below that of the mouse and initial attempts at deriving cat ESCs have resulted in embryonic stem-like cells, which cannot be maintained indefinitely. In this study we assessed and compared the quality of cat blastocysts produced in vitro using two commercial human blastocyst growth media, and the maintenance of pluripotency markers OCT-4 and NANOG in inner cell mass explants from in vitro produced blastocysts over 14 days.Item The Potential Role of Milk-Fat-Globule Membrane (MFGM) Proteins in Regulating the Size of Milk-Lipid Droplets(2012) Shin, Hyunsu; Mather, Ian H; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The aim of this thesis was to identify protein factors that may regulate the size of lipid droplets in milk. To address this hypothesis, the relative amounts of specific MFGM proteins on lipid droplets fractionated according to size were measured. Protein amounts were estimated by quantitative western blotting and confocal microscopy. By quantitative confocal microscopy, small lipid droplets (<1.26 um) contained more XOR, BTN, adipophilin (ADPH) and fatty acid binding protein (FABP) per surface area than medium (>1.26 to <2.8 um) or large (>2.8 um) sized droplets. Milk-fat-globule-EFG-8 (MFG-E8) protein was more evenly distributed on small, medium and large droplets. In contrast CD36, in both cow and mouse milk, was concentrated on small droplets and absent from large droplets. Based on these data, we postulate that CD36 possibly association with FABP may have a function in small lipid droplet secretion by localizing excessively on the small lipid droplets.Item Generating memory cytotoxic T lymphocytes through repetitive peptide boosting(2012) Smyth, Kendra; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cytotoxic T lymphocytes (CTLs) play a critical role in controlling intracellular pathogens and cancer cells, and induction of memory CTLs holds promise for developing effective vaccines against critical virus infections. However, generating memory CTLs remains a major challenge for conventional vector-based, prime-boost vaccinations. Thus, it is imperative that we explore nonconventional alternatives, such as boosting without vectors. We show here that repetitive intravenous boosting with peptide and adjuvant generates memory CD8 T cells of sufficient quality and quantity to protect against infection in mice. The resulting memory CTLs possess a unique and long-lasting effector memory phenotype, characterized by decreased interferon-gamma but increased granzyme B production. These results are independent of the specific adjuvant applied and are observed in both transgenic and endogenous models. Overall, our findings have important implications for future vaccine development, as they suggest that intravenous peptide boosting with adjuvant following priming can induce long-term functional memory CTLs.