Animal & Avian Sciences Theses and Dissertations
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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 The study of hyperketonemia in the dairy cow.(2023) Barrientos-Blanco, Mario Alberto; Rico, Eduardo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The metabolic phenomenon of ketosis in dairy cows has remained ambiguous, casting uncertainty over our understanding and its real implications. Ketosis, commonly defined as blood β-hydroxy-butyrate (BHB) ≥ 1.2 mM (i.e., hyperketonemia), has been observationally connected to the onset of peripartal metabolic disorders (e.g., infectious diseases, fatty liver), and reduced milk yield in dairy cows. Although BHB is currently used as standard biomarker for the prediction of negative health and performance outcomes during the peripartum, the nature of this relationship is ambiguous. In contraposition, recent discoveries in mammalian biology indicate BHB as therapeutic metabolite (e.g., alleviation of inflammation and oxidative stress). Our overreaching goal was to study the effects of BHB on dairy cow metabolism and health. In our first study, 6 multiparous (parity = 2.8 ± 0.9) Holstein mid-lactation dairy cows (128 ± 52 days in milk; DIM), were enrolled in a study to evaluate a ketogenic diet using calcium butyrate (—CaBu—; a ruminal ketone precursor) against an un-supplemented control (Control) in a crossover arrangement of treatments. The CaBu resulted in nutritional ketosis (P < 0.05) with blood BHB levels of 0.2 mM higher relative to Control. Although CaBu resulted in reduced dry matter intake (DMI; P < 0.05), milk production was not affected (P > 0.40), and feed efficiencies were improved (P < 0.05) relative to Control. No differences in glucose, NEFA, respiration rates, pain scores, or rectal temperatures were observed between treatments. In the second experiment, 8 multiparous Holstein (2.75 ± 0.89) mid-lactation dairy cows (140 ± 48 DIM), feed ad libitum, were enrolled in a in a crossover arrangement of treatments. The aim of the study was to evaluate the effect of ketones by intravenous infusion of either Na-BHB solution (2.5mM; EK) to sustain hyperketonemia —BHB > 1.2 mM and < 3.0 mM—, or NaCl as a control (2.5mM; Control) over a 72h period. A systemic lipopolysaccharide (LPS) challenge (E. coli 055:B5; 0,085 g/kg BW,) was intravenously administered at h 60 from infusion start. Cows sustained hyperketonemia throughout the 72h experimental period (1.4 BHB mM vs. 0.7 BHB mM in EK vs. Control, respectively). While DMI and milk production were not affected by the BHB infusion, the combination with the LPS challenge resulted in reductions of 20.8% (P < 0.05) and 40.1%, (P = 0.14) for both measurements in EK vs. Control, respectively. No differences were detected in the glucose and NEFA concentrations, but insulin was higher 46.6% (P < 0.05) in EK group. Among the immune markers, IL-1 was 30.8% higher (P < 0.05) in the EK group, and not differences were detected in TNF, IL-10, CRP, and caspase-1. As expected, the LPS challenge induced increased respiration rates, temperature, and pain scores over the time course of the evaluation (P < 0.001); however, respiration rates tended to be reduced in 8.4% (P < 0.1) and rectal temperature increased in 0.3% (P < 0.05) by the BHB treatment (P < 0.05). Our results are indicative that, in the absence of an immune challenge, hyperketonemia results in no negative impact on cow productivity and health. These data add support to our hypothesis that cofactors other than ketones may be necessary for the development of negative trajectories of health and performance of lactating dairy cows. Future studies will be required to confirm that BHB hyperketonemia metabolic effects could differ from ketosis disorder in dairy cows.Item The Importance of Transfer Receptor 1 in Adipose Tissue(2021) Mejia-Guevara, Yasmin; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Iron homeostasis is essential for maintaining cellular function in a variety of cell types. Transferrin receptor 1 (TfR1), which is expressed ubiquitously, facilitates cellular iron (Fe) uptake through receptor-mediated endocytosis of Fe-loaded transferrin. This study was undertaken to evaluate the importance of TfR1-mediated Fe import into adipose tissues for thermogenesis and systemic metabolism. We found that adipose-specific TfR1 knockout mice exhibited severe cold susceptibility upon acute cold exposure, leading to death of the mutant mice within hours. This phenotype was exacerbated by dietary Fe limitation and partially rescued by Fe administration. Knockout mice showed marked defects in oxidative phosphorylation components and lipid droplet homeostasis in adipose tissues. Furthermore, elevated levels of plasma glucose and insulin in the mutant’s hint at an unexpected connection between adipocyte Fe deficiency and diabetes. Altogether, our results suggest that TfR1-mediated Fe uptake is critical for multiple aspects of adipose function and systemic energy metabolism.Item MRP5 AND MRP9 PLAY A CONCERTED ROLE IN MALE REPRODUCTION AND MITOCHONDRIAL FUNCTION(2021) Chambers, Ian George; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels are significantly lower in the DKO testes. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating normal male reproductive functions and mitochondrial sufficiency.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.