Chemicals and Drugs Commons^™

Characterizing And Overcoming Resistance To Aminomethylspectinomycins In Gram-Negative Bacteria, Nisha Das

Theses and Dissertations (ETD)

Spectinomycin (SPC) is a broad-spectrum aminocyclitol antibiotic. Its use in agriculture has led to widespread resistance in enteric bacteria, necessitating the development of more effective analogs. Aminomethyl spectinomycins (amSPC) are modified spectinomycins with increased potency against many bacterial species. These species include Legionella pneumophila, which harbors a chromosomally encoded aminoglycoside modifying enzyme (AME). In this study, we follow up on this observation and examine the extent to which the amSPCs are substrates for AMEs through adenylation (ANTs) and phosphorylation (APH). APH(9)-Ia and ANT(3")(9) were expressed in E. coli BL21(DE3) and purified using the Ni-affinity chromatography. The ability of AMEs to …

Investigating The Role Of Znf384 Rearrangements In Acute Leukemia, Kirsten Dickerson

Theses and Dissertations (ETD)

Chromosomal rearrangements involving ZNF384 are the defining lesion in 5% of pediatric and adult B-cell acute lymphoblastic leukemia and tumors are characterized by aberrant myeloid marker expression. Additionally, ZNF384 rearrangements are the defining lesion in nearly half of pediatric B/myeloid mixed phenotype acute leukemia. These fusions juxtapose full-length ZNF384 to the N terminal portion of a diverse range of partners, most often, transcription factors or epigenetic modifiers. It has been shown that ZNF384-rearranged tumors have a distinct gene expression profile that is consistent between disease groups and N terminal partners. Genomic analyses of patient tumors has shown that ZNF384 fusions …

Genetic Variation And Sex Mediate Differential Responses To ∆-9-Tetrahydrocannabinol Among Inbred Mice, Cory Parks

Theses and Dissertations (ETD)

The plant Cannabis sativa has been used by people for both recreational and medicinal use for thousands of years, but scientific investigation of the plant and its components didn’t begin until the early nineteen hundreds when Cannabis components known as phytocannabinoids were characterized and later isolated. In the 1970’s, ∆9-tetrahydrocannabinol (THC) was isolated and recognized as the major constituent responsible for the psychoactive and intoxicating effects associated with consumption of cannabis. This opened the door for intensive research in the field that lead to the discovery of the endogenous cannabinoid system and its associated receptors, effectors of signaling, and biosynthetic …

Validation And Application Of A Novel Target-Based Whole-Cell Screen To Identify Antifungal Compounds, Christian Alexander Dejarnette

Theses and Dissertations (ETD)

Traditional approaches to drug discovery are inefficient and have several key limitations that constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically-active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein were constructed, tagged with spectrally distinct fluorescent proteins (FPs), and mixed together. The pooled strains were then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture …

Characterization Of The Hepatotoxicity Of Rifampicin And Isoniazid, Christopher T. Brewer

Theses and Dissertations (ETD)

In a mouse model, rifampicin and isoniazid combination treatment results in cholestatic liver injury that is associated with an increase of protoporphyrin ix (PPIX), the penultimate heme precursor. Excess PPIX is believed to bind to bile acids, precipitate in bile canaliculi, and form bile plugs leading to cholestasis fol owed by liver injury. Both ferrochelatase (FECH/Fech) and aminolevulinic acid synthase 1 (ALAS1/Alas1) are crucial enzymes in regulating heme biosynthesis. Isoniazid has recently been reported to up-regulate Alas1 but down-regulate Fech protein levels in mice; however the mechanism of isoniazid mediated heme synthesis …

Habituation And Dishabituation In The Olfactory Bulb: From Neural Responses To Behavior, Mary Cameron Ogg

Theses and Dissertations (ETD)

Habituation and dishabituation modulate the neural resources and behavioral significance allocated to incoming stimuli across the sensory systems. The purpose of the research presented in this dissertation was to characterize these processes in the mouse olfactory bulb (OB) and to determine if OB acetylcholine (ACh) has a role in physiological and behavioral olfactory dishabituation. Calcium imaging was used to determine the timecourse and magnitude of habituation in different parts of the OB during and after a prolonged odor presentation. Widefield imaging of the dendritic, or glomerular, response of OB output cells demonstrated that prolonged odor input habituates glomerular responses during …

Investigating The Role Of Dispatched In Hedgehog Ligand Transport And Delivery, William Bodeen

Theses and Dissertations (ETD)

During the development of all metazoans, the Hedgehog (Hh) signaling pathway provides instructional cues influencing a variety of cellular processes. The pathway ligand, Hh, is dually lipidated by cholesterol and palmitate, which effectively anchors the molecule to the lipid bilayer of the signal producing cell. To complicate the Hh pathway induction process, the Hh ligand is often produced at a significant distance from the cells it influences. Only one known conserved molecule, Dispatched (Disp), can alleviate the membrane tethering imparted by Hh lipidation. Underscoring the importance of Disp protein during development, knockout animals succumb to lethality at E9.5, an exact …

Antibodies To Heterogenous Nuclear Ribonucleoprotein A1 Penetrate Neurons Leading To Multiple Downstream Effects Resulting In Neurodegeneration, Joshua Nathan Douglas

Theses and Dissertations (ETD)

Multiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system. MS is believed to occur in genetically susceptible individuals due to an unknown environmental stimulus. MS patients produce autoantibodies to heterogenous nuclear ribonuclearprotein A1 (hnRNP A1), an RNA binding protein (RBP) highly expressed in neurons. hnRNP A1 functions in pre-mRNA splicing, mRNA trafficking, and translation. Furthermore, the anti-hnRNP A1 antibodies are specific to a N-terminal region termed ‘M9’ which serves as a nuclear export sequence/nuclear localization sequence (NES/NLS) responsible for nuclear/cytoplasmic transport of the protein. In this manuscript we will provide data revealing that anti-hnRNP A1 …

Interaction Between Two E3 Ligases, Nedd8ylated Cullin And Hhari, Kheewoong Baek

Theses and Dissertations (ETD)

RBR (RING1-in between RING-RING2) is a special type of E3 ubiquitin ligase containing three zinc-binding RING (Really Interesting New Gene) domains, while adopting mechanisms of HECT (Homologous to E6-AP Carboxyl Terminus) for substrate ubiquitination. Most well known RBRs include Parkin and HOIP, which are associated with Parkinson’s disease and innate immune deficiency. However, it is not well known how the RBR proteins gain activity, as they are known to be autoinhibited. Here I show that a specific F430A, E431A, E503A triple mutation of RBR protein HHARI (Human homologue of Ariadne) and its interaction with NEDD8ylated cullin RING ligase can both …

Mrp4-Dependent Regulation Of Fibroblast Migration, Chandrima Sinha

Theses and Dissertations (ETD)

Roles of cyclic nucleotides and cyclic nucleotide-dependent signaling molecules in regulating several signaling pathways including cell migration have long been known. However, the new and revolutionary concept is that it is not just the absence or presence of cyclic nucleotides, but a highly coordinated balance between these molecules regulates cell migration. Multi-drug resistance protein 4 (MRP4), is a member of the large family of ATP binding cassette (ABC) transporter proteins, that localizes to the plasma membrane and functions as a nucleotide efflux transporter and thus plays a pivotal role in the regulation of intracellular cyclic nucleotide dynamics. In our study …

Strengths And Weaknesses Of Hybrid Tpr Technology For Obtaining Structural And Mechanistic Insights Into Tpr Proteins, Shanshan Yu

Theses and Dissertations (ETD)

Tetratricopeptide (TPR) repeats are a 34-residue helix-turn-helix motif that when repeated pack into a superhelical structure. TPR domains are frequently found mediating protein-protein interactions, often through a central groove. One protein complex bearing numerous TPR repeats is the Anaphase Promoting Complex (APC). The anaphase-promoting complex (APC) is a multi-subunit complex, which orchestrates mitotic cell cycles. APC is an E3 ligase in the ubiquitin cascade, and directs the 26S proteosome degradation of cell-cycle regulators. Throughout mitotic progression, proteins that are key regulators of the cell cycle are assembled with polyubiquitin chains by APC.

One domain of the human APC is comprised …

Erg11-Mediated Azole Resistance In Candida Albicans, Stephanie Ann Flowers

Theses and Dissertations (ETD)

Although many medically important Candida species are commensal to the gut or colonizers of the skin, these organisms have the propensity to cause disease in the event of a waning immune system. Clinical manifestations of infections with Candida species can range from superficial mucosal infections to deep organ involvement usually resulting from haematogenous spread of infection. Despite significant progress made in the management of patients with fungal infections, the emergence of antifungal resistant clinical isolates creates significant problem in regards to antifungal prophylaxis and empirical treatment strategies. Antifungal resistance is associated with high mortality rates and hefty medical costs. The …

Full-Gestational Exposure To Nicotine And Ethanol Results In Long-Term Dysregulation In The Ventral Tegmental Area, Emily Elizabeth Roguski

Theses and Dissertations (ETD)

Nicotine is considered a gateway drug for other drugs of abuse. The majority of smokers that begin smoking in adolescence, continue into adulthood. Multiple factors have been linked with smoking initiation, including maternal smoking. Drug exposure during pregnancy has long been suspected to exert deleterious effects on the fetal brain. Concurrent use of smoking and drinking alcohol throughout pregnancy is an all too frequent occurrence in the human population especially in disadvantaged and younger women.

The present study investigated the potential contributing factors for the enhanced nicotine selfadministration as seen in offspring with gestational exposure to nicotine and ethanol (Nic+EtOH).* …

The Protumorigenic Role Of Caspase-8 In Neuroblastoma, Devin Drew Twitchell

Theses and Dissertations (ETD)

Neuroblastoma (NB), the most common extracranial solid tumor in children, accounts for 15% of cancer-related deaths in pediatric patients. Caspase-8 (casp8), a proapoptotic protein, is silenced in approximately, 50-70% of neuroblastoma patient samples. Loss of casp8 has been suggested to increase NB metastasis and correlated, in some studies, with advanced-stage NB. Furthermore, decreased casp8 expression may facilitate neuroblastoma tumorigenesis by protecting cells from cell death mediated by either integrins or chemotherapeutics. Paradoxically, casp8 expression is maintained in 30-50% of NB patient samples giving rise to the possibility that casp8 may provide selective advantages for NB tumorigenesis. Caspase-8 is shown to …

Mrp4 Is A Crucial Regulator Of Testosterone Biosynthesis, Jessica Ann Morgan

Theses and Dissertations (ETD)

The physiological role of multidrug resistance protein 4 (Mrp4) in the testes is unknown. It was discovered that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone biosynthesis. The role of Mrp4 was investigated in Leydig cells where testosterone production is regulated by cAMP, an intracellular second messenger formed when the luteinizing hormone (LH) receptor (Lhr) is activated. As Mrp4 regulates cAMP, we compared testosterone levels in our Mrp4 WT and KO mice. Prepubertal KO mice had significantly reduced testicular testosterone, impaired gametogenesis, and disrupted cAMP homeostasis, resulting in …

Discovery Of Dihydroartemisinin And Dasatinib Drug Combination To Cure Pooroutcome Bcr-Abl+ Acute Lymphoblastic Leukemia, Harpreet Singh

Theses and Dissertations (ETD)

Oncogenic signaling by the Philadelphia chromosome-encoded BCR-ABL fusion kinase initiates and drives both Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myelogenous leukemia (CML). Food and Drug Administration (FDA)- approved BCR-ABL-specific kinase inhibitors (BCR-ABL–KIs) imatinib, dasatinib and nilotinib induce prolonged remissions in CML but poor leukemia-reduction and relapse-control in Ph+ ALL. The relative primary BCR-ABL–KI-resistance in Ph+ ALL patients carrying predominantly BCR-ABLWT disease cannot be attributed to drug-resistant BCR-ABL mutations (BCR-ABLMUTANTS), and remains poorly understood.

We established a cell-based platform to evaluate the modulation of anti-Ph+ ALL activity of drugs by both tumor-extrinsic cytokines normally present in the leukemia …

The Molecular Basis Of Fitness And Transmissibility Of Neuraminidase Inhibitor Resistant Influenza A Viruses, Susu Duan

Theses and Dissertations (ETD)

Neuraminidase (NA) inhibitors including oral oseltamivir and inhaled zanamivir are among the first line of defense against influenza virus infection. Development of resistance to NA inhibitors is a huge drawback for limited options for the control of influenza. During the first decade of NA inhibitor use, the detection rates of resistance to both NA inhibitors had remained low in circulating influenza viruses. However, the 2008~2009 season was marked by a radical increase of prevalence of oseltamvir resistance from <1% to >90% in worldwide surveillance in less than a year. The resistance was solely linked to NA H275Y variants of seasonal H1N1 viruses, …

Characterization Of The Mechanism Of Pparγ-Mediated Neointima Formation In Rodents, Ryoko Tsukahara

Theses and Dissertations (ETD)

Lysophosphatidic acid (LPA) and its ether analog alkyl glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for …

Novel Insights Into Ubiquitin-Like Protein E1-E2 Interactions, Asad Taherbhoy

Theses and Dissertations (ETD)

Posttranslational modification of macromolecules by ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo and NEDD8 regulate a vast array of processes in the cell. Transfer of UBLs to their target generally occurs by a series of molecular handoffs down an E1‑E2‑E3 cascade. We are interested in understanding how E1‑E2 pairs interact and mediate UBL transfer. To this effect, we studied two E1‑E2 pairs: the Sumo pathway (Sumo utilizes a canonical E1 and E2) and the Atg8 pathway (Atg8 is a UBL involved in autophagy that utilizes a non-canonical E1‑E2 pair).

Sumo conjugation is initiated by the heterodimeric Aos1‑Uba2 E1 enzyme (in …

Contribution Of Organic Cation Transporter 2 (Oct2) To Cisplatin-Induced Nephrotoxicity, Kelly K. Filipski

Theses and Dissertations (ETD)

Cisplatin is the most widely used anticancer agent; however, the cellular pharmacokinetics are poorly understood. Cisplatin is predominantly eliminated through the urine via active secretion and is associated with nephrotoxicity. Currently, prehydration therapy is employed to prevent toxic renal side effects; however it has not been completely ameliorated. The studies described herein aim to determine the mechanism in which cisplatin enters the kidney cell from the blood and how it is subsequently secreted into the urine. Organic cation transporter 2 (OCT2) and ABCC2 are highly expressed in the kidney on the basolateral and apical membrane, respectively. We determined the contribution …

Insights Into Btb-Cul3 Ubiquitin Ligases From The Structures Of Spop-Substrate Complexes, Min Zhuang

Theses and Dissertations (ETD)

Cullin-Ring ubiquitin ligases (CRLs) are E3 complexes that specifically recognize substrates through substrate adaptors. In the largest CRL subfamily, Cul3 binds a BTB domain, and a protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH and BTB domain containing protein, SPOP, which regulates diverse signaling pathways. First, we identified a conserved SPOP Binding Consensus (SBC) motif in the transcriptional regulator Ci, the protein phosphatase Puc, and the chromatin component MacroH2A. The SBC motif specifically binds the MATH domain of SPOP, and is required for Puc ubiquitination in vitro and in …

Abcb6 Is A Porphyrin Transporter With A Novel Trafficking Signal That Is Conserved In Other Abc Transporters, Yu Fukuda

Theses and Dissertations (ETD)

The ATP-binding cassette (ABC) transporters play an important role as a barrier to protect cells from the accumulation of toxic xenobiotics and metabolites due to their ability to translocate a wide array of compounds across lipid bilayers. However, many ABC transporters, especially the ones localized in the intracellular organelles, are involved in critical biological processes such as antigen presentation. The core unit of ABC transporters contains two functional domains: the membrane spanning domain (MSD) and the nucleotide binding domain. The full transporters contain two of these units in tandem in a single polypeptide, whereas the half transporters only contain one …

Regulation Of The Human Parainfluenza Virus (Hpiv3) Fusion Protein, Amanda Ruth Chapman

Theses and Dissertations (ETD)

Paramyxoviruses include a number of important human pathogens, including measles virus, mumps virus, and the human parainfluenza viruses (hPIV) 1-4, as well as several animal pathogens, such as Sendai virus, Nipah virus and Hendra virus. The creation of effective drugs and vaccines against this family of viruses would play an important role in decreasing the prevalence of these viruses and contributing to the health of both humans and animals worldwide. The purpose of this work was to determine how the fusion (F) protein is regulated with a focus on the heptad repeat B (HRB) region of the F protein located …

The Structural And Functional Study Of Git1 Paxillin Binding Domain, Ziwei Zhang

Theses and Dissertations (ETD)

The G protein coupled receptor (GPCR)-kinase (GRK) interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of p21-activated kinase (PAK) and PAK-interactive exchange factor (PIX) to focal adhesions, and its activation is regulated by the interaction between its C terminal paxillin-binding domain (PBD) and the LD motifs of paxillin.

In this dissertation, we determined the solution structure of rat GIT1 PBD by nuclear magnetic resonance (NMR) spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting …

The N-Terminal Domain Of The Y-Box Binding Protein Yb-1 Plays A Major Role In Cell Proliferation And Apoptosis, Payal Khandelwal

Theses and Dissertations (ETD)

Y-box binding protein, YB-1, is a member of the cold shock domain superfamily of proteins. It is involved in a plethora of cellular functions, including cell proliferation. The molecular mechanisms governing the involvement of YB-1 in cell proliferation are still unclear. Earlier studies done in chicken pre-B lymphocyte DT-40 cells in our laboratory have shown that a targeted disruption in one allele of chicken YB-1

(Chk-Yb-1b) gene at its N-terminal domain resulted in multiple abnormalities in the heterozygous mutants, including slower growth rate, abnormal cell morphology, increased cell size, increased genomic DNA content and significant changes in levels of cell …

Tyrosine Phosphorylation Of Villin: Effects On Actin Dynamics, Cell Morphology And Cell Migration, Alok Tomar

Theses and Dissertations (ETD)

Cell migration is a key aspect of many normal and abnormal biological processes, including embryonic development, defense against infections, wound healing, and tumor cell metastasis. In this study we demonstrate that an epithelial cell actin-binding protein, villin, plays a crucial role in the process of cell migration. Overexpression of villin in doxycyline-regulated HeLa Tet-off and MDCK Tet-off cells enhanced cell migration. We further demonstrate that tyrosine phosphorylation of villin by c-src is required for villin-induced cell migration. Previously, we identified four tyrosine phosphorylation sites in the amino-terminal domain of villin. I further identified six new sites in the carboxylterminal region …

Developmental Expression Of Rat Target Of The Antiproliferative Antibody (Rtapa) Protein In The Brain, Clyde Dale Sullivan

Theses and Dissertations (ETD)

The present study defines the expression pattern of rTAPA (CD81) in the developing rat brain. rTAPA is a member of the tetramembrane spanning family of proteins, and like other members of this family, appears to be associated with the stabilization of cellular contacts. On immunoblots of the brain, rTAPA is present in higher levels than any other tissue examined: muscle, tendon, peripheral nerve, cartilage, liver, kidney, skin, and testicle. Immunohistochemical methods were used to define the distribution of rTAPA in the brain. This protein is expressed by ependyma, choroid plexus, astrocytes, and oligodendrocytes, and rTAPA is dramatically upregulated at the …

Regulation Of Luteinizing Hormone And Catecholamine Release, Benjamin Aaron Adler

Theses and Dissertations (ETD)

These studies tested the interrelated hypotheses that the ovarian hormones produce their positive feedback effects on luteinizing hormone (LH) secretion through activation of noradrenergic and adrenergic systems in specific hypothalamic regions. Furthermore, the ovarian hormones may alter the activity of opioid neuropeptide and Gamma-Aminobutyric Acid (GABA) systems to produce these alterations in catecholamine transmission and gonadotropin secretion. Radioimmunoassays were utilized to determine plasma LH and median eminence LHRH, and hypothalamic catecholamine concentrations were measured by radioenzymatic assay.

The first two studies tested whether epinephrine (EPI) synthesis inhibition blocks the accumulation of median eminence LHRH that precedes the ovarian hormone-induced LH …

Characteristics Of The Recovery Of The Coenzyme A-Synthesizing Protein Complex From Saccharomyces Cerevisiae, Stanley Joseph Tarnowski Jr.

Theses and Dissertations (ETD)

A multienzyme complex contained in Bakers' yeast (Saccharomyces cerevisiae) which synthesizes CoA has been named the coenzyme A-synthesizing protein complex (CoA-SPC). The CoA-SPC has been shown to be insoluble in the crude Bakers' yeast cell lysate formed by exposing the yeast cell to ether and dry ice. Only after solubilization has this multienzyme complex been shown to catalyze the formation of bound dephospho-CoA utilizing the substrates adenosine triphosphate, D-pantothenic acid and L-cysteine. A low molecular weight component or components of the soluble fraction of the yeast cell and chloride ion appears to be responsible for the solubilization of CoA-SPC. This …

The Purification, Properties And Subunit Structure Of Glycerol Dehydrogenase, Michael James Barrett

Theses and Dissertations (ETD)

The inducible, NAD-linked glycerol dehydrogenase (E.C. 1.1.1.6) of a guanine requiring mutant of A. aerogenes has been purified to homogeneity. The molecular weight of the pure enzyme was found to be 3.4 x 10⁵ daltons. The sedimentation coefficient of the enzyme was 10.7 x 10^-13 sec.^-1. The diffusion constant was 3.07 x 10^-7 cm²/sec.

The partial specific volume calculated from the amino acid composition was 0.72 ml/g. The following kinetic parameters were determined; K_m glycerol, 2.4 x 10^-3 m, K_m NAD, 2.8 x 10^-4 M, K_m dihydroxyacetone, 5.1 …