Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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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 INVESTIGATING GENE REGULATORY ARCHITECTURES THAT DICTATE TRANSGENERATIONAL EPIGENETIC EFFECTS IN C. ELEGANS(2023) Chey , Mary Somontha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The form and function of an organism rely on the recreation of similar gene expression patterns in every generation. The information for these expression patterns is stored in a single cell (e.g., zygote) in two forms – the genome sequence and the spatial arrangements of gene regulators. The interactions of regulators and the genome form intricate networks with different regulatory architectures. However, a change in the environment can impact gene expression by disrupting the physical and/or chemical properties of regulators or interactions without mutations in DNA sequence. Such epigenetic information can be transmitted across generations, but how long these effects can last is unclear. Here, we investigate the regulatory elements that promote transient or permanent epigenetic effects by analyzing the properties of a recombinant two-gene operon that expresses the fluorescent proteins mCherry and GFP and is susceptible to long-term RNA silencing in the nematode C. elegans. We reveal that 1) multiple mechanisms regulate transgenerational gene silencing and 2) the presence of the mCherry sequence can perturb RNA regulation within the germline to facilitate heritable epigenetic changes. Previous studies showed that the Argonaute protein HRDE-1 is required for the maintenance of silencing in the germline initiated by double-stranded (ds)RNAs and that poly-UG (pUG)-RNAs are key intermediates generated from the target mRNA. We found that loss of HRDE-1 can selectively rescue the expression of one cistron in a two-gene operon, suggesting that the two cistrons are not regulated by the same silencing pathway, but rather by a chromatin-independent mechanism that requires an unknown regulator. Surprisingly, we detected distinct populations of pUG-RNAs associated with expressed and silenced genes, suggesting that pUG-RNAs could potentially prime expressed genes for long-term silencing. Consistently, total RNA sequencing revealed trace amounts of anti-sense RNAs against mCherry and gfp that could trigger the production of pUG-RNAs. Examining the endogenous genes perturbed by the presence of mCherry suggests that long-term RNA silencing relies on the synergy between the sensing and processing of dsRNAs. Together, our results provide insights into the regulatory architectures and mechanisms of heritable gene silencing that occur without genetic mutations.