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ItemEnrichment and Isolation of Iron-Oxidizing Bacteria from an Ancient Earth Analogue(Georgia Institute of Technology, 2019-08) Ghazi, LaylaFe2+ was an abundant component of ancient anoxic oceans and could have acted as a respiratory electron donor. The overall goals of this study were to test whether anaerobic microbial growth could occur with Fe2+ as the electron donor in Fe2+-rich sediments from an ancient ocean analogue (Lake Matano, Indonesia) and to determine the taxonomic identity of the bacteria. Sediments were incubated with Fe2+ sulfide as the electron donor in a nitrogen:carbon dioxide (90/10%) atmosphere. Manganese (III), nitrate, nitrite, and oxygen were provided as electron acceptors. With Mn3+ as the electron acceptor, cultures showed some evidence of growth near the middle of the gradient tube. However, orange Fe3+ oxides were absent, suggesting that anaerobic Fe2+ oxidation had not occurred. Ferric oxides were also absent in tubes containing nitrate and nitrite. A white precipitate was present in cultures with Mn3+, which indicated that the microbes reduced Mn3+ to Mn2+. The precipitate was not present in uninoculated controls. With oxygen as the electron donor, a layer of orange Fe3+ oxide minerals formed near the water-air interface, indicative of growth of microaerophilic Fe2+-oxidizing bacteria. This layer did not form in uninoculated controls. Our preliminary results suggest that anaerobic Lake Matano enrichments are capable of Fe2+ oxidation using oxygen but not alternative electron acceptors. After subsequent transfers of the enrichments that showed growth of microaerophilic Fe2+-oxidizing bacteria, the bacteria were isolated and their 16S rRNA gene was sequenced. Sequences were most similar to the Betaproteobacteria genus Comamonas and the Alphaproteobacteria genus Skermanella. Some species of Comamonas are known to oxidize Fe2+, while the exact mechanism of the metabolism of Skermanella are not well known. The presence of microaerophilic Fe2+ oxidizing bacteria from Lake Matano, Indonesia serves as a link between understanding the transition from an anoxic to an oxic world.
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ItemExpansion of Mitochondrial and Nuclear Heme Sensor Library(Georgia Institute of Technology, 2019-05) Atuluru, PranushaThe long-term objective of the work in the lab is to determine the mechanisms by which cells sense and respond to the utilization of heme, an essential nutrient. Heme is an iron-containing compound of the porphyrin class that enables proteins to carry out an array of functions. Heme-dependent processes require that heme be dynamically mobilized to hemoproteins in almost every subcellular compartment. Although it is understood that the cytotoxicity and hydrophobicity of heme requires heme be tightly regulated by the cell, the method by which this is done is unknown [1]. The primary factor that limits the understanding of heme mobilization and trafficking is the lack of tools available to sense heme, more specifically labile heme. The Reddi lab is working to develop ratiometric fluorescent sensors to offer better insight into subcellular labile heme pools relevant for heme trafficking and signaling. HS1 (Heme Sensor 1) is mutated at either the His or Met in the heme-binding coordinating bundle of cytochrome to create sensors of different affinity. Ten new mutant sensors were created from the original HS1 and HS1-M7A, and it is seen that two sensors, H102C and H102C-M7H, are the most suitable sensors to be used in the mitochondria, nucleus and cytosol. With the use of these sensors, different pathways of heme trafficking and signaling can be studied in the cell.
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ItemProbing Heme Trafficking Factors via Organellar Contact Points Using Genetically Encoded Fluorescent Heme Sensors(Georgia Institute of Technology, 2019-05) Saini, ArushiHeme is an important protein cofactor and signaling molecule that plays diverse roles in biological systems. The hydrophobicity and cytotoxicity of heme necessitates that it is transported and trafficked in a regulated manner. However, the molecules and mechanisms responsible for mediating heme trafficking remain poorly understood. Until recently, the tools to study heme in vivo did not exist, but the emergence of genetically encoded fluorescent sensors has enabled comprehensive real time analysis of heme in model organisms such as Saccharomyces cerevisiae. This study showcases a new a protocol that allows investigation of heme trafficking from its site of synthesis in the matrix side of the mitochondrial inner membrane to the outer matrix, cytosol, and nucleus over time. The method allows for the simultaneous examination of heme re-population in three cellular compartments after chemically depleting it. The study revealed that mitochondrial contact points play central roles in regulating heme availability and illuminates novel approaches to heme trafficking. These methods have the potential to be adapted to more inclusive compartmental analyses and enable a better understanding of heme trafficking which can empower innovative approaches to study infectious diseases, neurodegenerative disorders, and anemias associated with perturbations in heme cellular dynamics.
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ItemCarrier protein and halogenase selectivity in the biosynthesis of halogenated pyrroles(Georgia Institute of Technology, 2019-05) Lail, Andrew J.Natural product biosynthetic pathways often share similar architecture even when they lead to different final products. In polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) enzymatic pathways, the substrate is attached to a carrier protein (CP) while the tailoring enzymes make modifications to yield a final product. The CP may therefore have a role in determining what enzymes act on the substrate, influencing the final product’s chemistry. In this study, pyrrole halogenases from several different bacterial species were characterized in vitro to test their ability to halogenate pyrrolyl CPs from four different natural product biosynthetic pathways. The reactions were analyzed via mass spectrometry to determine the halogenation state of the products formed. This study concludes that only some halogenases can act promiscuously on CPs from other pathways. Additionally, there is some modulation in the number of halogenation events between certain CP and halogenase pairs. The selectivity of these halogenase and CP interactions is likely caused by protein-protein interactions, and the structure of the CP/halogenase complex may provide new insights into such interaction.
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ItemAntibody targeted delivery of prodrug converting enzymes using protein nanoparticle platform for HER2-positive breast cancer therapy(Georgia Institute of Technology, 2019-05) Guldberg, SophiaApproximately 1 in 8 women in the United States will be diagnosed with breast cancer. Among these women, 25-30% will have breast cancer where the HER2 gene is overexpressed and human epidermal growth factor receptor 2 (HER2) is overexpressed, which increases the aggressiveness of the cancer. The aggressiveness of HER2-positive breast cancer results in decreased long-term survival. For this reason, new HER-2 targeted therapies need to be developed to increase remission and survival of HER2-postive breast cancer patients. A notable success in this field has been the use of Genentech’s anti-HER2 antibody (Herceptin, trastuzumab), but this is an expensive option that not all patients can access and must still be frequently combined with chemotherapy drugs. Protein nanoparticles (PNPs) are increasingly used in a wide variety of biotechnology settings due to their low toxicity, high potential specificity deriving from their polyvalent nature, and low cost. This project focuses on the modification of PNPs to target HER2-positive breast cancer cells for drug delivery. The relative success of this project was determined by flow cytometry and fluorescence microscopy, which confirmed the binding of targeted PNPs and a lack of nonspecific binding. While further experimentation in cytotoxicity and in vivo studies is needed, this project presents a novel and successful method of targeting HER2-positive breast cancer cells with PNPs.
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ItemUtilization of Cytochrome b562 as a Localized Labile Heme Chelator(Georgia Institute of Technology, 2019-05) Jenkin, BryanHeme is an essential, but toxic cofactor required for virtually all aerobic life. As a consequence, cells are challenged to safely traffic heme to hemoproteins that reside in every subcellular compartment. However, the mechanisms underlying heme transport and trafficking are largely unknown. Moreover, it is unclear how various subcellular compartments communicate their requirement for heme to the mitochondria, where heme is synthesized. In order to determine how different subcellular compartments sense and respond to heme deficiency, I have been developing a heme chelator to induce local heme deficiencies. Once this is achieved, we can employ transcriptome and proteome profiling to determine pathways that enable various organelles to adapt to heme deficiency. Altogether, we seek to better understand how cells appropriate and distribute heme to diverse compartments that require this essential nutrient.