Biochemistry, Biophysics, and Structural Biology Commons^™

Oma1 Links Mitochondrial Protein Quality Control And Tor Signaling To Modulate Physiological Plasticity And Cellular Stress Responses, Iryna Bohovych, Stavroula Kastora, Sara Christianson, Danelle Topil, Heejeong Kim, Teresa Fangman, You J. Zhou, Antoni Barrientos, Jaekwon Lee, Alistair J.P. Brown, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

A network of conserved proteases known as the intramitochondrial quality control (IMQC) system is central to mitochondrial protein homeostasis and cellular health. IMQC proteases also appear to participate in establishment of signaling cues for mitochondrion- to-nucleus communication. However, little is known about this process. Here, we show that in Saccharomyces cerevisiae, inactivation of the membrane-bound IMQC protease Oma1 interferes with oxidative-stress responses through enhanced production of reactive oxygen species (ROS) during logarithmic growth and reduced stress signaling via the TORC1-Rim15- Msn2/Msn4 axis. Pharmacological or genetic prevention of ROS accumulation in Oma1-deficient cells restores this defective TOR signaling. Additionally, inactivation …

Regulation Of Dj-1 By Glutaredoxin 1 In Vivo – Implications For Parkinson’S Disease, William M. Johnson, Marcin Golczak, Kyonghwan Choe, Pierce L. Curran, Olga Gorelenkova Miller, Chen Yao, Wenzhang Wang, Jiusheng Lin, Nicole M. Milkovic, Ajit Ray, Vijayalakshmi Ravindranath, Xiongwei Zhu, Mark A. Wilson, Amy L. Wilson-Delfosse, Shu G. Chen, John J. Mieyal

Department of Biochemistry: Faculty Publications

Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH; and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has …

Endoplasmic Reticulum-Associated Degradation Of Pca1p, A Polytopic Protein, Via Interaction With The Proteasome At The Membrane, Nathan Smith, David J. Adle, Miaoyun Zhao, Xiaojuan Qin, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Endoplasmic reticulum-associated degradation (ERAD) plays a critical role in the destruction of terminally misfolded proteins at the secretory pathway. The system also regulates expression levels of several proteins such as Pca1p, a cadmium exporter in yeast. To gain better insight into the mechanisms underlying ERAD of Pca1p and other polytopic proteins by the proteasome in the cytosol, our study determined the roles for the molecular factors of ERAD in dislodging Pca1p from the endoplasmic reticulum (ER). Inactivation of the 20S proteasome leads to accumulation of ubiquitinated Pca1p in the ER membrane, suggesting a role for the proteasome in extraction of …

Caloric Restriction Of Db/Db Mice Reverts Hepatic Steatosis And Body Weight With Divergent Hepatic Metabolism, Kyung Eun Kim, Youngae Jung, Soonki Min, Miso Nam, Rok Won Heo, Byeong Tak Jeon, Dae Hyun Song, Chin-Ok Yi, Eun Ae Jeong, Hwajin Kim, Jeonghyun Kim, Seon-Yong Jeong, Woori Kwak, Do Hyun Ryu, Tamas L. Horvath, Gu Seob Roh, Geum-Sook Hwang

Department of Biochemistry: Faculty Publications

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of liver disease and its prevalence is a serious and growing clinical problem. Caloric restriction (CR) is commonly recommended for improvement of obesity-related diseases such as NAFLD. However, the effects of CR on hepatic metabolism remain unknown. We investigated the effects of CR on metabolic dysfunction in the liver of obese diabetic db/db mice. We found that CR of db/db mice reverted insulin resistance, hepatic steatosis, body weight and adiposity to those of db/m mice. H-NMR- and UPLC-QTOF-MS-based metabolite profiling data showed significant metabolic alterations related to lipogenesis, …

Inhibiting Hexamer Disassembly Of Human Udp-Glucose Dehydrogenase By Photoactivated Amino Acid Crosslinking, George Grady, Ashley Thelen, Jaleen Albers, Tong Ju, Jiantao Guo, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD⁺-dependent oxidation steps. Human UGDH apoprotein purifies as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking was achieved by encoding p-benzoyl-L-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing to long wavelength …

Synthesis And Transfer Of Galactolipids In The Chloroplast Envelope Membranes Of Arabidopsis Thaliana, Amélie Kelly, Barbara Kalisch, Georg Hölzl, Sandra Schulze, Juliane Thiele, Michael Melzer, Rebecca L. Roston, Christoph Benning, Peter Dörmann

Department of Biochemistry: Faculty Publications

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol

(DGDG)] are the hallmark lipids of photosynthetic

membranes. The galactolipid synthases MGD1 and DGD1

catalyze consecutive galactosyltransfer reactions but localize to the

inner and outer chloroplast envelopes, respectively, necessitating

intermembrane lipid transfer. Here we show that the N-terminal

sequence of DGD1 (NDGD1) is required for galactolipid transfer

between the envelopes. Different diglycosyllipid synthases (DGD1,

DGD2, and Chloroflexus glucosyltransferase) were introduced into

the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions

(NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction

of DGDG synthesis in the outer envelope membrane was

observed only with diglycosyllipid synthase fusion …

Evidence For Pipecolate Oxidase In Mediating Protection Against Hydrogen Peroxide Stress, Sathish Kumar Natarajan, Ezhumalai Muthukrishnan, Oleh Khalimonchuk, Justin L. Mott, Donald F. Becker

Department of Biochemistry: Faculty Publications

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ¹-piperideine-6-carboxylate (P6C) by the flavoenzyme lpipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 …

Ceramide Induces Human Hepcidin Gene Transcription Through Jak/Stat3 Pathway, Sizhao Lu, Sathish Kumar Natarajan, Justin L. Mott, Kusum K. Kharbanda, Duygu Dee Harrison-Findik

Department of Biochemistry: Faculty Publications

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown.We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked …

Engineering A Trifunctional Proline Utilization A Chimaera By Fusing A Dna-Binding Domain To A Bifunctional Puta, Benjamin W. Arentson, Erin L. Hayes, Weidong Zhu, John J. Tanner, Donald F. Becker

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ¹-pyrroline-5- carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon–helix–helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the …

Mechanistic Nanotherapeutic Approach Based On Sirna-Mediated Dj-1 Protein Suppression For Platinum-Resistant Ovarian Cancer, Canan Schumann, Stephanie Chan, Oleh Khalimonchuk, Shannon Khal, Vitaliya Moskal, Vidhi Shah, Adam W. G. Alani, Olena Taratula, Oleh Taratula

Department of Biochemistry: Faculty Publications

We report an efficient therapeutic modality for platinum resistant ovarian cancer based on siRNAmediated suppression of a multifunctional DJ-1 protein that is responsible for the proliferation, growth, invasion, oxidative stress and overall survival of various cancers. The developed therapeutic strategy can work alone or in concert with a low dose of the first line chemotherapeutic agent cisplatin, to elicit a maximal therapeutic response. To achieve an efficient DJ-1 knockdown, we constructed the polypropylenimine dendrimer-based nanoplatform targeted to LHRH receptors overexpressed on ovarian cancer cells. The quantitative PCR and western immunoblotting analysis, revealed that the delivered DJ-1 siRNA downregulated the expression …

Antisense Oligonucleotides: Treatment Strategies And Cellular Internalization, Colton M. Miller, Ed Harris

Department of Biochemistry: Faculty Publications

The clinical application of antisense oligonucleotides (ASOs) is becoming more of a reality as several drugs have been approved for the treatment of human disorders and many others are in various phases in development and clinical trials. ASOs are short DNA/RNA oligos which are heavily modified to increase their stability in biological fluids and retain the properties of creating RNA-RNA and DNA-RNA duplexes that knock-down or correct genetic expression. This review outlines several strategies that ASOs utilize for the treatment of various congenital diseases and syndromes that develop with aging. In addition, we discuss some of the mechanisms for specific …

Logical Modeling And Dynamical Analysis Of Cellular Networks, Wassim Abou-Jaoudé, Pauline Traynard, Pedro T. Monteiro, Julio Saez-Rodriguez, Tomáš Helikar, Denis Thieffry, Claudine Chaouiya

Department of Biochemistry: Faculty Publications

The logical (or logic) formalism is increasingly used to model regulatory and signaling networks. Complementing these applications, several groups contributed various methods and tools to support the definition and analysis of logical models. After an introduction to the logical modeling framework and to several of its variants, we review here a number of recent methodological advances to ease the analysis of large and intricate networks. In particular, we survey approaches to determine model attractors and their reachability properties, to assess the dynamical impact of variations of external signals, and to consistently reduce large models. To illustrate these developments, we further …

Antisense Oligonucleotides: Treatment Strategies And Cellular Internalization, Colton M. Miller, Edward N. Harris

Department of Biochemistry: Faculty Publications

The clinical application of antisense oligonucleotides (ASOs) is becoming more of a reality as several drugs have been approved for the treatment of human disorders and many others are in various phases in development and clinical trials. ASOs are short DNA/RNA oligos which are heavily modified to increase their stability in biological fluids and retain the properties of creating RNA-RNA and DNA-RNA duplexes that knock-down or correct genetic expression. This review outlines several strategies that ASOs utilize for the treatment of various congenital diseases and syndromes that develop with aging. In addition, we discuss some of the mechanisms for specific …

Polyglycine Acts As A Rejection Signal For Protein Transport At The Chloroplast Envelope, Joshua K. Endow, Agostinho Gomes Rocha, Amy J. Baldwin, Rebecca L. Roston, Toshio Yamaguchi, Hironari Kamikubo, Kentaro Inoue

Department of Biochemistry: Faculty Publications

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion …

Mutation In Atg5 Reduces Autophagy And Leads To Ataxia With Developmental Delay, Myungjin Kim, Erin Sandford, Damian Gatica, Yu Qiu, Xu Liu, Yumei Zheng, Brenda A. Schulman, Jishu Xu, Ian Semple, Seung-Hyun Ro, Boyoung Kim, R. Nehir Mavioglu, Aslihan Tolun, Andras Jipa, Szabolcs Takats, Manuela Karpati, Jun Z. Li, Zuhal Yapici, Gabor Juhasz, Jun Hee Lee, Daniel J. Klionsky, Margit Burmeister

Department of Biochemistry: Faculty Publications

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects’ cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast …

Can Chilling Tolerance Of C4 Photosynthesis In Miscanthus Be Transferred To Sugarcane?, Katarzyna Glowacka, Aasifuddin Ahmed, Shailendra Sharma, Tom Abbott, Jack C. Comstock, Stephen P. Long, Erik J. Sacks

Department of Biochemistry: Faculty Publications

The goal of this study was to investigate whether chilling tolerance of C₄ photosynthesis in Miscanthus can be transferred to sugarcane by hybridization. Net leaf CO₂ uptake (A_sat) and the maximum operating efficiency of photosystem II (Ф_PSII) were measured in warm conditions (25 °C/20 °C), and then during and following a chilling treatment of 10 °C/5 °C for 11 day in controlled environment chambers. Two of three hybrids (miscanes), ‘US 84-1058’ and ‘US 87-1019’, did not differ significantly from the chilling tolerant M. xgiganteus ‘Illinois’ (Mxg), for A_sat, and Φ …

Novel Acyltranserases And Methods Of Using, Edgar B. Cahoon, Umidjon Iskandarov, Hae Jin Kim, Jillian Collins-Silva

Department of Biochemistry: Faculty Publications

Provided herein are novel acyltransferases and methods of using Such novel acyltransferases in making medium-chain fatty acids.

High-Throughput Mutation, Selection, And Phenotype Screening Of Mutant Methanogenic Archaea, Mary E. Walter, Alicia Ortiz, Casey Sondgeroth, Nathan M. Sindt, Nikolas Duszenko, Jennie L. Catlett, You Zhou, Shah R. Valloppilly, Christopher Anderson, Samodha Fernando, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or …

Mutual Information Upper Bound Of Molecular Communication Based On Cell Metabolism, Massimiliano Pierobon, Zahmeeth Sakkaff, Jennie L. Catlett, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Synthetic biology is providing novel tools to engineer cells and access the basis of their molecular information processing, including their communication channels based on chemical reactions and molecule exchange. Molecular communication is a discipline in communication engineering that studies these types of communications and ways to exploit them for novel purposes, such as the development of ubiquitous and heterogeneous communication networks to interconnect biological cells with nano and biotechnology-enabled devices, i.e., the Internet of Bio-Nano Things. One major problem in synthetic biology stands in the development of reliable techniques to control the engineered cells from the external environment. In this …

Transcriptional Regulation Of Specialized Metabolites In Arabidopsis Thaliana And Catharanthus Roseus, Craig M. Schluttenhofer

Theses and Dissertations--Plant and Soil Sciences

For millennia humans have utilized plant specialized metabolites for health benefits, fragrances, poisons, spices, and medicine. Valued metabolites are often produced in small quantities and may command high prices. Understanding when and how the plant synthesizes these compounds is important for improving their production. Phytohormone signaling cascades, such as jasmonate (JA) activate or repress transcription factors (TF) controlling expression of metabolite biosynthetic genes. TFs regulating specialized metabolite biosynthetic genes can be manipulated to engineer plants with increased metabolite production.

WRKY transcription factor are known components of both JA signaling cascades and regulation of specialized metabolism. The presence of WRKY binding …

Effect Of Quantum Dots, Novel Biological Imaging Agents, In Liver Cells In Vitro, Utshaha Maharjan

All Master's Theses

The use of Quantum dots (QDs) coated with polymer and functionalized with carboxylic acid groups in medical applications are explored. Their water solubility and exceptional stability in aqueous environments make them potentially useful for such applications as imaging and ligand attachments. However, there are concerns regarding the toxic effects of QDs and the minimal dose that can be used without producing any detrimental effects to organisms. In this study, QDs coated with the amphiphilic polymer coating tri-n-octylphosphine oxide and poly (maleic anhydride-alt-1-tetradecene (TOPO-PMAT)) which is functionalized with carboxylic acid groups were used to investigate their toxic effect in mouse liver …

Piperlongumine (Piplartine) And Analogues: Antiproliferative Microtubule-Destabilising Agents, Mary J. Meegan, Seema M. Nathwani, Brendan Twamley, Daniela M. Zisterer, Niamh O'Boyle

Articles

Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and Western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerised when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, …

A Fret Investigation Into Molecular Mechanisms Of Cardiac Troponin Activation In Reconstituted Thin Filaments, Maria Eleni Moutsoglou

Electronic Theses and Dissertations

Cardiomyopathies (CM) are the leading cause of death in America, and can develop from mutations in sarcomeric proteins, leading to altered protein structure and function. Current therapies target upstream signaling pathways to treat the symptoms of heart failure, but are associated with increased mortality by affecting downstream signaling pathways and other muscle types. Rational drug design can develop therapies to treat CM at the protein level. However, a detailed knowledge of how sarcomeric proteins regulate muscle contraction is required. Muscle contraction occurs through a cyclic interaction between actin thin and myosin thick filaments, regulated by intracellular Ca2+ concentration. Troponin (Tn), …

Novel Regulatory Mechanisms Of Inositol Biosynthesis In Saccharomyces Cerevisiae And Mammalian Cells, And Implications For The Mechanism Underlying Vpa-Induced Glucose 6-Phosphate Depletion, Wenxi Yu

Wayne State University Dissertations

Myo-inositol is the precursor of all inositol containing molecules, including inositol phosphates, phosphoinositides and glycosylphosphatidylinositols, which are signaling molecules involved in many critical cellular functions. Perturbation of inositol metabolism has been linked to neurological disorders. Although several widely-used anticonvulsants and mood-stabilizing drugs have been shown to exert inositol depletion effects, the mechanisms of action of the drugs and the role of inositol in these diseases are not understood. Elucidation of the molecular control of inositol synthesis will shed light on the pathologies of inositol related illnesses.

In Saccharomyces cerevisiae, deletion of the four glycogen synthase kinase-3 genes, MCK1, MRK1, MDS1, …

Cardiolipin Is Required For Optimal Acetyl-Coa Metabolism, Vaishnavi Raja

Wayne State University Dissertations

The phospholipid cardiolipin (CL) is crucial for many cellular functions and signaling pathways, both inside and outside of mitochondria. My thesis focuses on the role of CL in energy metabolism. Many reactions of electron transport and oxidative phosphorylation, the transport of metabolites needed for these processes, and the stabilization of electron transport chain supercomplexes, require CL. Recent studies indicate that CL is required for the synthesis of iron-sulfur (Fe-S) co-factors, which are essential for numerous metabolic pathways. Activation of carnitine-acetylcarnitine translocase, which transports acetyl-CoA into the mitochondria, is CL dependent. The presence of substantial amounts of CL in the peroxisomal …

Structural Characterization And Therapeutic Utility Of The Proton-Coupled Folate Transporter, Michael Roy Wilson

Wayne State University Dissertations

Folate is a B9 vitamin essential to DNA synthesis. The proton-coupled folate transporter (PCFT) is a newly discovered proton/folate symporter with an acidic pH optimum and broad expression across a variety of solid tumor types, with limited expression in normal tissues. Several antifolate molecules have been developed as cancer therapeutics, although these classical antifolates display numerous off-target effects due to transport by the ubiquitous reduced folate carrier (RFC). In this dissertation, we determine the roles of multiple PCFT structure/function domains, and develop PCFT-specific antifolates to target solid tumors. We utilize substituted cysteine accessibility methods (SCAM) to identify a novel reentrant …

From Seed To Sky: Impacts Of Explosive Compounds On Vegetation Across Spatial And Developmental Scales, Stephen M. Via

Theses and Dissertations

Explosive compounds are broadly distributed across the globe as a result of nearly two centuries of munitions use in warfare and military activities. Two explosive compounds have seen disproportionate use; RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and TNT (2-methyl- 1,3,5-trinitrobenzene), being the most commonly found explosives in the environment. The effects of explosives on biota have been studied in great detail; however, there is a general lack of understanding with regard to broader ecological impacts of these contaminants. My dissertation objective was to follow the impacts of explosive compounds on vegetation across scales. Impacts on vegetation at the species scale alter community composition via …

Understanding Dna Condensation By Low Generation (G0/G1) And Zwitterionic G4 Pamam Dendrimers, Min An

Theses and Dissertations--Chemistry

Cationic polymers have shown potential as gene delivery vectors due to their ability to condense DNA and protect it from cellular and restriction nucleases. Dendrimers are hyperbranched macromolecules with precisely defined molecular weights and highly symmetric branches stemming from a central core. The nanosize, tunable surface chemistries and ease of surface functionalization has made dendrimers an attractive alternative to conventional linear polymers for DNA delivery applications. The commercially available, cationic dendrimer poly(amidoamine) or PAMAM is the most widely studied dendrimer for use as a gene delivery vector. The aim of this dissertation is to provide an increased understanding of the …

The Development Of Colorimetric Assays To Determine The Identity And Frequency Of Specific Nucleobases In Dna Oligomers, Elizabeth Marie Thomas

Theses and Dissertations--Chemistry

Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this dissertation, …

Assembly And Degradation Of A Trimeric Membrane Protein Acrb, Qian Chai

Theses and Dissertations--Chemistry

Multidrug efflux pumps are membrane proteins that actively transport foreign objects out of cells. The active efflux of these pumps is a critical self-defense mechanism that enables the survival of bacteria under hostile environments. Efflux pump AcrB is a member of the Resistance-Nodulation-Division (RND) super family. In E. coli, it associates with periplasmic protein AcrA and outer membrane channel TolC to extrude a variety of noxious compounds out of cell from both the cytoplasm and the periplasm. My dissertation research focused on two aspects of this multidrug efflux pump: the oligomerization process during the biogenesis of AcrB and its …