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Now showing 1 - 10 of 39

Enrichment and Isolation of Iron-Oxidizing Bacteria from an Ancient Earth Analogue

(Georgia Institute of Technology, 2019-08) Ghazi, Layla

Fe2+ 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.
Expansion of Mitochondrial and Nuclear Heme Sensor Library

(Georgia Institute of Technology, 2019-05) Atuluru, Pranusha

The 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.
Probing Heme Trafficking Factors via Organellar Contact Points Using Genetically Encoded Fluorescent Heme Sensors

(Georgia Institute of Technology, 2019-05) Saini, Arushi

Heme 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.
Carrier 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.
Antibody targeted delivery of prodrug converting enzymes using protein nanoparticle platform for HER2-positive breast cancer therapy

(Georgia Institute of Technology, 2019-05) Guldberg, Sophia

Approximately 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.
Utilization of Cytochrome b562 as a Localized Labile Heme Chelator

(Georgia Institute of Technology, 2019-05) Jenkin, Bryan

Heme 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.
Towards More Efficient ab initio Computation of Physical Properties

(Georgia Institute of Technology, 2018-05) Zott, Michael D.

The introduction of the modern computer has been a boon to myriad scientiﬁc communities. Scientiﬁc experiment can be categorized into the categories of physical experiment and thought experiment. In the chemical arena, these thought experiments are now able to be tested for validity through advanced semi-empirical and ab initio computational methods. Theoretical chemistry continues to increase in efﬁcacy, and the spread of classical, wavefunction, and density functional methods into experimental communities is now undeniable. An aspiration of computational chemistry is to provide predictive power to lower the number of physical experiments that need to be performed. This is especially important when systems arise that are difﬁcult to study experimentally. This has the possibility to lower ﬁnancial and environmental costs, in addition to reducing the time needed to perform physical experiments. Here, methods to computationally study solvent effects and crystal lattice energies are reported on. Both of these physical properties have substantial relevance to human-focused enterprises such as targeted drug design. For example, drugs are often delivered in solid, crystalline form and must dissolve into molecular form prior to being pharmaceutically active. Although the speciﬁc research reported on here does not use systems directly related to such applications, it is posited that fundamental advances in computational methods for computing physical properties for arbitrary systems will contribute to solving problems in drug design, material development, and biomolecule recognition.
Using Defects in Anion Excess Rhenium Trioxide Type Fluorides to Control Thermal Expansion; Ytterbium Zirconium Fluoride as a Case Study

(Georgia Institute of Technology, 2017-05) Ticknor, John Owen

The average structure, thermal expansion, behavior on compression, high temperature stability, and moisture sensitivity of YbZrF₇, an anion-excess ReO₃-type material, was explored. Cubic YbZrF₇ (a = 4.10 Å) was synthesized by a fast quench from 1000 °C. Powder x-ray diffraction and density measurements showed reasonable agreement with prior work by Poulain, et. al. Elemental analysis confirmed stoichiometry. Attempts to synthesize monoclinic YbZrF₇ by a slow-cool from 675 °C resulted in a mixed-phase product, suggesting an incomplete reaction. Variable temperature powder x-ray diffraction performed at the Advanced Photon Source (17-BM) identified anomalous volumetric expansion behavior in cubic YbZrF₇ including a dependence on thermal history. When the material is initially heated above 310 K, negative thermal expansion persists to approximately 350 K. This is probably associated with an irreversible change in local structure due to the migration of fluoride. Zero thermal expansion close to 300 K was consistently evident on cooling from 500 K, while negative thermal expansion was observed below room temperature. Cubic YbZrF₇ underwent an abrupt amorphization on compression to ~ 0.9 GPa. This was preceded by a pressure-induced softening. The latter has been previously observed for similar ReO₃-type fluoride materials that display negative thermal expansion. Cubic YbZrF₇ begins to thermally decompose above 700 °C in dry nitrogen, where ZrF₄ is sublimated. Under ambient conditions, cubic YbZrF₇ appears to be air stable for at least 24 hours. For temperatures above 300 °C, it is susceptible to hydrolysis when exposed to moist air exceeding 43 percent relative humidity.
Quantification of Lymphatic Vascular Permeability via Near-Infrared Imaging

(Georgia Institute of Technology, 2017-05) Ross, Mindy

Though the lymphatic system is involved in many essential bodily functions, little is known about its role in the progression of lymphatic diseases like lymphedema. Recently, inflammation has been implicated as the primary mediator of lymphatic pathologies, due to its ability to decrease lymphatic function and induce a mal-adaptive remodeling response (Aldrich & Sevick-Muraca, 2013). One of the failure modes that inflammation is hypothesized to influence is by increasing the permeability of the lymphatic vasculature (Scallan & Huxley, 2010). A minimally-invasive method of quantifying lymphatic vessel permeability was designed using near-infrared imaging, a fluorescent tracer, and applied pressure. The method partially occluded the lymphatic collecting vessels and was tested using IFN-γ as a positive control. The average apparent permeability for the control group was determined to be somewhat similar to a previous in vivo study of isolated vessels but had a wide range of values overall (Scallan & Huxley, 2010). Comparison of the IFN-γ treated group to the control group revealed no significant difference and therefore inconclusive results as to the accuracy of the method. Future work will include testing different positive controls to verify the method followed by application of the method on diet-induced obese mice for determination of the changes to vessel permeability as compared to the control group to understand possible causes that contribute to the development of lymphatic diseases like lymphedema.
Steps to Improving Stability of the β-propeller Structure of Myocilin's Olfactomedin Domain: Understanding the Evolution of the β-propeller

(Georgia Institute of Technology, 2017-05) Kwon, Michelle S.

Olfactomedin (OLF) domain-containing proteins, first identified in relation to bullfrog olfactory chemoreception, are part of a superfamily of proteins implicated in many important biological functions and human diseases. The myocilin OLF domain (mOLF), one of the best studied, is closely associated with the ocular disease glaucoma. Nearly 100 myocilin mutations have been reported in glaucoma patients; >90% are missense mutations within mOLF. Disease-associated mutant myocilins are destabilized and aggregation prone, leading to toxicity and death of cells that maintain the anatomical trabecular meshwork extracellular matrix in the eye. The Lieberman lab solved the crystal structures of OLF domains from myocilin and gliomedin (gOLF), a peripheral nervous system OLF domain. While both are similar five-bladed β-propellers, only mOLF contains a stabilizing calcium ion. Remarkably, gOLF is ~20 °C more stable than mOLF, even though it doesn't have a calcium ion and is phylogenetically more primitive. The goal of this project was to use insights from mOLF and gOLF to create a thermostable mOLF. Surprisingly, mutagenesis of a calcium-coordinating aspartate (D478) to alanine abolished calcium binding but increased mOLF thermal stability to near gOLF levels. Addition of D478A to the destabilized, glaucoma-associated variant D380A rescued thermal stability to that of wild-type (WT) mOLF. Structures of thermostable mOLF variants reveal unexpected changes in tertiary structure compared to WT mOLF, which were confirmed by solution biophysical measurements. The findings from this study expand our understanding of the structure-stability relationship of mOLF and provide further insight into the evolution of the OLF β-propeller.