Biochemistry, Biophysics, and Structural Biology Commons^™

Role Of Host Immune Response And Bacterial Autolysin Atl In Human Nasal Colonization By Staphylococcus Aureus, Vanathy Paramanandam

Electronic Theses and Dissertations

Staphylococcus aureus (SA) is a major human pathogen that colonizes the anterior nares in 30% of the human population. Though nasal carriage of SA is a known risk factor for the subsequent spread of SA infections, the dynamics of SA nasal colonization is poorly understood. Our research focuses on understanding the host and bacterial factors that might contribute to the human nasal colonization by SA. In an attempt to elucidate the host response to SA, we performed an autologous human in vivo nasal colonization study, which showed decreased survival rates of SA in hosts who elicited a robust immune response. …

Expression Of Rna Nanoparticles Based On Bacteriophage Phi29 Prna In Escherichia Coli And Bacillus Subtilis, Le Zhang

Theses and Dissertations--Pharmacy

Currently, most of the RNAs used in lab research are prepared by in vitro transcription or chemical synthesis, which can be costly. In vivo expression in bacterial cells is another approach to RNA preparation that allows large scale production at a lower cost. However, there are some obstacles in bacterial expression, including RNA degradation in host cell, as well as RNA extraction and purification. tRNA and 5S RNA have been reported as scaffolds to circumvent the degradation problem. These scaffolds can not only make the RNA product survive in the cell but also increase the stability after extraction.

The packaging …

Towards Elucidation Of A Viral Dna Packaging Motor, Chad T. Schwartz

Theses and Dissertations--Pharmacy

Previously, gp16, the ATPase protein of phi29 DNA packaging motor, was an enigma due to its tendency to form multiple oligomeric states. Recently we employed new methodologies to decipher both its stoichiometry and also the mechanism in which the protein functions to hydrolyze ATP and provide the driving force for DNA packaging. The oligomeric states were determined by biochemical and biophysical approaches. Contrary to many reported intriguing models of viral DNA packaging, it was found that phi29 DNA packaging motor permits the translocation of DNA unidirectionally and driven cooperatively by three rings of defined shape. The mechanism for the generation …

Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard

Theses and Dissertations--Pharmacy

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading …

Regulation Of 7-Dehydrocholesterol Reductase By Vitamin D3, Ling Zou

Theses and Dissertations--Pharmacy

7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D₃ synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D₃ resulted …

Overexpression/Silencing Of Selected Soybean Genes Alters Resistance To Pathogens, Mohamed H. El-Habbak

Theses and Dissertations--Plant Pathology

Plant diseases remain a major obstruction to meeting the world’s increased demand for soybean oil and protein. Reducing the losses caused by diseases in order to improve crop production is a high priority for agricultural research. The need for novel strategies for plant disease control cannot be overstated. In the present study, selected defense-related genes were silenced and/or overexpressed in soybean using a virus-based vector and the resultant plants were tested for their responses to pathogens. The first part of the study focused on Rps1k (Resistance to Phytophthora sojae) gene. The two conserved domains encoding ‘P-Loop NTPase’ and ‘PLN03210’ …

Synthesis Of 4-Azidocoumarins And Their Use In Copper-Catalyzed Azide-Alkyne Cycloaddition Reactions, Anthony J. Netsuri

Dissertations and Theses

Triazole-containing compounds have shown great biological activity ranging from antiviral, antibacterial, to anticancer, to name a few. Coumarin derivatives have also shown interesting biological activities. The combination of these bioactive compounds appears to have great promise for new and future medicines. In this work, various 4-azido-coumarins were synthesized via the transformation of the 4-hydroxy derivatives to 4-benzotriazolyloxy coumarins by reaction with the peptide coupling agent (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,8-diazabicycloundec-7-ene (DBU) as the base, in tetrahydrofuran (THF) solvent. The 4-benzotriazolyloxy coumarins were converted to the 4-azidocoumarins by reaction with sodium azide (NaN3), and the overall process was simplified to a …

Epigenetic Regulation Of Oxysterol Formation, Steve Meaney

Articles

Oxysterols are oxygenated derivatives of cholesterol that may be formed by either enzymatic or non-enzymatic mechanisms. Expression of the genes responsible oxysterol synthesis (GROS) is known to be restricted across different tissues and cell types. Regulation of the transcription of GROS and the activity of their enzyme transcripts has been the subject of intense activity for many years. Recent studies have sought to decipher the mechanism(s) that underpin the restricted expression of the GROS. Available data indicates that epigenetic mechanisms have an important role to play in the control of the expression of GROS. In the current review we summarize …

Combined Treatment With Mild Heat, Manothermosonication And Pulsed Electric Fields Reduces Microbial Growth In Milk., Rachel Halpin, O. Cregenzán-Alberti, P. Whyte, J.G Lyng, F. Noci

Articles

In recent years, there has been considerable interest in non-thermal milk processing. The objective of the present study was to assess the efficacy of two non-thermal technologies (manothermosonication; MTS, and pulsed electric fields; PEF) in comparison to thermal pasteurisation, by assessing the microbial levels of each of these milk samples post-processing. Homogenised milk was subjected to MTS (frequency; 20 kHz, amplitude; 27.9 μm, pressure; 225 kPa) at two temperatures (37 °C or 55 °C), before being immediately treated with PEF (electric field strength; 32 kV/cm, pulse width; 10 μs, frequency; 320 Hz). Thermal pasteurisation (72 °C, 20 s) was included …

Osteogenic And Antimicrobial Nanoparticulate Calcium Phosphate And Poly-(D, L-Lactide-Co-Glycolide) Powders For The Treatment Of Osteomyelitis, Vuk Uskoković, Charles Hoover, Marija Vukomanović, Dragan P. Uskoković, Tejal A. Dasai, Charles Hoover, Marija Vukomanović, Dragan P. Uskoković, Tejal A. Dasai

Pharmacy Faculty Articles and Research

Development of a material for simultaneous sustained and localized delivery of antibiotics and induction of spontaneous regeneration of hard tissues affected by osteomyelitis stands for an important clinical need. In this work, a comparative analysis of the bacterial and osteoblastic cell response to two different nanoparticulate carriers of clindamycin, an antibiotic commonly prescribed in the treatment of bone infection, one composed of calcium phosphate and the other comprising poly-(D,L-lactide-co-glycolide)-coated calcium phosphate, was carried out. Three different non-cytotoxic phases of calcium phosphate, exhibiting dissolution and drug release profiles in the range of one week to two months to one year, respectively, …

Synthesis And Biochemical Activities Of Antiproliferative Amino Acid And Phosphate Derivatives Of Microtubule-Disrupting Beta-Lactam Combretastatins, Niamh M. O'Boyle, Lisa M. Greene, Niall O. Keely, Shu Wang, Tadhg S. Cotter, Daniela M. Zisterer, Mary J. Meegan

Articles

The synthesis and biochemical activities of novel water-soluble β-lactam analogues of combretastatin A-4 are described. The first series of compounds investigated, β-lactam phosphate esters 7a, 8a and 9a, exhibited potent antiproliferative activity and caused microtubule disruption in human breast carcinoma-derived MCF-7 cells. They did not inhibit tubulin polymerisation in vitro, indicating that biotransformation was necessary for their antiproliferative and tubulin binding effects in MCF-7 cells. The second series of compounds, β-lactam amino acid amides (including 10k and 11l) displayed potent antiproliferative activity in MCF-7 cells, disrupted microtubules in MCF-7 cells and also inhibited the polymerisation of …

Novel Cis-Restricted Β-Lactam Combretastatin A-4 Analogues Display Anti-Vascular And Anti-Metastatic Properties In Vitro, Seema M. Nathwani, Lisa M. Greene, Linda Hughes, Miriam Carr, Niamh O'Boyle, Susan Mcdonnell, Mary J. Meegan, Daniela M. Zisterer

Articles

No abstract provided.

A Bacterial Symbiont Is Converted From An Inedible Producer Of Beneficial Molecules Into Food By A Single Mutation In The Gaca Gene, Pierre Stallforth, Debra A. Brock, Alexandra M. Cantley, Xiangjun Tian, David C. Queller, Joan E. Strassmann, Jon Clardy

Biology Faculty Research

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria's host is a "farmer" clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer's spore production and …

Nanoparticles Of Cobalt-Substituted Hydroxyapatite In Regeneration Of Mandibular Osteoporotic Bones, Nenad Ignjatović, Zorica Ajduković, Vojin Savić, Stevo Najman, Dragan Mihailović, Perica Vasilijević, Zoran Stojanović, Vuk Uskoković, Dragab Uskoković

Pharmacy Faculty Articles and Research

Indications exist that paramagnetic calcium phosphates may be able to promote regeneration of bone faster than their regular, diamagnetic counterparts. In this study, analyzed was the influence of paramagnetic cobalt-substituted hydroxyapatite nanoparticles on osteoporotic alveolar bone regeneration in rats. Simultaneously, biocompatibility of the material was tested in vitro, on osteoblastic MC3T3-E1 and epithelial Caco-2 cells in culture. The material was shown to be biocompatible and nontoxic when added to epithelial monolayers in vitro, while it caused a substantial decrease in the cell viability as well as deformation of the cytoskeleton and cell morphology when incubated with the osteoblastic …

Periplasmic Binding Proteins In Thermophiles: Characterization And Potential Application Of An Arginine-Binding Protein From Thermotoga Maritima: A Brief Thermo-Story, Alessio Ausili, Maria Staiano, Jonathan D. Dattelbaum, Antonio Varriale, Alessandro Capo, Sabato D'Auria

Chemistry Faculty Publications

Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima …

Structurally Diverse Hamigerans From The New Zealand Marine Sponge Hamigera Tarangaensis: Nmr-Directed Isolation, Structure Elucidation And Antifungal Activity, A. Jonathan Singh, Jonathan D. Dattelbaum, Jessica J. Field, Zlatka Smart, Ethan F. Woolly, Jacqueline M. Barber, Rosemary Heathcott, John H. Miller, Peter T. Northcote

Chemistry Faculty Publications

The NMR-directed investigation of the New Zealand marine sponge Hamigera tarangaensis has afforded ten new compounds of the hamigeran family, and a new 13-epi-verrucosane congener. Notably, hamigeran F (6) possesses an unusual carbon–carbon bond between C-12 and C-13, creating an unprecedented skeleton within this class. In particular, the structural features of 6, hamigeran H (10) and hamigeran J (12) imply a diterpenoid origin, which has allowed the putative biogenesis of three hamigeran carbon skeletons to be proposed based on geranyl geranyl pyrophosphate. All new hamigerans exhibited micromolar activity towards the HL-60 …

Executing And Teaching Science--The Breast Cancer Genetics And Technology-Rich Curriculum Professional Development Studies Of A Science Educator, Regina Evarn Wragg

Theses and Dissertations

This dissertation presents my explorations in both molecular biology and science education research. In study one, we determined the ADIPOQ and ADIPORI genotypes of 364 White and 148 Black BrCa patients and used dominant model univariate logistic regression analyses to determine individual SNP and haplotype associations with tumor or patient characteristics in a case-case comparison. We found twelve associations between individual SNPs and patient or tumor characteristics that impact BrCa prognosis. For example, the ADIPOQ rs1501299 C allele was associated with ER+ tumors (OR=4.73, p=0.001) among White women >50 years of age at their time of diagnosis. Also, the A …

A Comprehensive, Multi-Scale Dynamical Model Of Erbb Receptor Signal Transduction In Human Mammary Epithelial Cells, Tomáš Helikar, Naomi Kochi, Bryan Kowal, Manjari Dimri, Mayumi Naramura, Srikumar M. Raja, Vimla Band, Hamid Band, Jim A. Rogers

Department of Biochemistry: Faculty Publications

The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins …

Glutaredoxin-2 Is Required To Control Proton Leak Through Uncoupling Protein-3, Ryan J. Mailloux, Jian Ying Xuan, Brittany Beauchamp, Marjorie F. Lou, Mary-Ellen Harper

School of Veterinary and Biomedical Sciences: Faculty Publications

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle …

Biochemical And Cellular Mechanisms Of Retina And Retinal Pigment Epithelium Apoptosis, Srinivasa Rao Sripathi

Dissertations, Master's Theses and Master's Reports - Open

Oxidative stress, intense light exposure and oxygen imbalances such as hypoxic or hyperoxic conditions perturb mitochondria, nuclear function and further lead to cellular damage of retina and retinal pigment epithelial (RPE) cells. Our major aim is to understand the various biochemical and proteomic events that occur during the progression of retina and RPE cell death. The comprehensive objectives of this dissertation are to understand the functional aspects of protein expression, posttranslational modifications, protein or lipid binding changes, phenotypic, morphological alterations and their regulation during the retina and RPE apoptosis under oxidative stress. The entire study is divided into four chapters …

Oligodeoxynucleotide Synthesis Using Protecting Groups And A Linker Cleavable Under Non-Nucleophilic Conditions, Xi Lin

Dissertations, Master's Theses and Master's Reports - Open

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process.

To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and solid support. …

Determination Of Dimer Dissociation Constant Of Neural-Cadherin By Fluorescence Resonance Energy Transfer, Xiaoyun Z. Howard

Electronic Theses and Dissertations

No abstract provided.

The Characterization Of The Interplay Between Sumoylation And Influenza A Virus, Andres Santos

Open Access Theses & Dissertations

Influenza A virus is a negative sense single stranded RNA virus responsible for multiple devastating pandemics in the past. This virus is capable of entering multiple eukaryotic hosts, sequestering, and using their cellular machinery for its own replicative purposes. This plethora of molecular events is all orchestrated through a shockingly limited repertoire of only 11 viral proteins. All of which are all strictly dependent on cellular processes such as post-transcriptional regulation, protein translation, post-translational modifications, etc. This study is focused on the characterization of the interplay between Influenza A virus and the cellular SUMOylation system. The first part of this …

Electroporation-Mediated Gene Transfer Directly To The Swine Heart, Barbara Hargrave, Harre Downey, Cathryn Lundberg, Annelise Israel, Yeong-Jer Chen, Richard Heller

Bioelectrics Publications

In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on …

Exploring The Importance Of Single Nucleotide Polymorphisms Of Hspa9 In Dna Of Sarcoma Patients, Christine M. Hebert

Honors Theses and Capstones

The aim of this project was to identify genetic variants that may influence the risk and progression of sarcoma through targeted genotyping of HSPA9 gene. It is important to look at genetic variants in DNA samples because if a variant is determined to be more likely than another, a screening for the particular variant can be done to identify a patient’s risk of sarcoma. The study population was sarcoma patients from the International Sarcoma Kindred Study. These patients had no mutations in p53 or MDM2. Genotyping data from the HapMap project (hapmap.org) for HSPA9 was used to identify the polymorphisms …

An Acid Catalyzed Reversible Ring-Opening/Ring-Closure Reaction Involving A Cyano-Rhodamine Spirolactam, H. Li, H. Guan, X. Duan, J. Hu, Guiren Wang, Qian Wang

Faculty Publications

Cyanamide was introduced into the rhodamine spirolactam framework to produce a colorless and non-fluorescent compound RBCN. It shows a reversible ring-opening/ring-closure process in response to the solution pH, which exhibits an “ON/OFF” switching in its fluorescence. Different from other rhodamine-type dyes, the ring-open form of RBCN is stable in protic solvents under neutral, near neutral and basic conditions, showing a pink color and very strong fluorescence. We also demonstrated the potential of RBCN in live cell imaging.

The Terrestrial Biogeochemical Cycle Of Barium: A Proposed Study To Examine Barium Flux In Mojave Desert Dust, Mehar Kaur

Scripps Senior Theses

Barium is a relatively abundant element in the crustal environments, Ba quantities can range from anywhere between 200ppm to 900ppm. Most common forms of Ba-minerals found in the environment are barite (BaSO₄), witherite (BaCO₃) and hollandite (Ba₂Mn₈O₁₆). Ba is a useful element; it is used in various industries as a component in drilling fluids, in medical research and in manufacturing of various substances such as glass, ceramics, printing paper etc. However high quantity of Ba can be potentially toxic for the human body and can impair plant growth. It is …

Exploitation Of The Bacteriophage-Derived Peptidase Chapk, Pierre-Mehdi Hadbi

Theses

This project investigated an anti-staphylococcal bacteriophage-derived peptidoglycan hydrolase enzyme, namely a cysteine-histidine amydohydrolase/peptidase (CHAP_k). The study focused firstly on optimizing the production of recombinant CHAP_k, which was previously cloned in an E. coli expression system, and sets out the resulting optimal Standard Operating Procedures (SOPs) for production, purification and determination of concentration and activity of the resulting protein stock. A typical yield resulting from the investigation was 300pg of CHAP_k from one litre of E.coli culture. The activity of the purified enzyme was typically in the region of 190U nmol ’. Secondly, the project investigated …

The Relative Contribution Of Methanotrophs To Microbial Communities And Carbon Cycling In Soil Overlying A Coal-Bed Methane Seep, Christopher T. Mills, Gregpry F. Slater, Robert F. Dias, Stephanie A. Carr, Christopher M. Reddy, Raleigh Schmidt, Kevin W. Mandernack

Chemistry & Biochemistry Faculty Publications

Seepage of coal-bed methane (CBM) through soils is a potential source of atmospheric CH₄ and also a likely source of ancient (i.e. ¹⁴C-dead) carbon to soil microbial communities. Natural abundance ¹³C and ¹⁴C compositions of bacterial membrane phospholipid fatty acids (PLFAs) and soil gas CO₂ and CH₄ were used to assess the incorporation of CBM-derived carbon into methanotrophs and other members of the soil microbial community. Concentrations of type I and type II methanotroph PLFA biomarkers (16:1ω8c and 18:1ω8c, respectively) were elevated in CBM-impacted soils compared with a control site. Comparison …

Real-Time In Vivo Imaging Of Size-Dependent Transport And Toxicity Of Gold Nanoparticles In Zebrafish Embryos Using Single Nanoparticle Plasmonic Spectroscopy, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu

Chemistry & Biochemistry Faculty Publications

Noble metal nanoparticles (NPs) show distinctive plasmonic optical properties and superior photostability, enabling them to serve as photostable multicoloured optical molecular probes and sensors for real-time in vivo imaging. To effectively study biological functions in vivo, it is essential that the NP probes are biocompatible and can be delivered into living organisms non-invasively. In this study, we have synthesized, purified and characterized stable (non-aggregated) gold (Au) NPs (86.2 +/- 10.8 nm). We have developed dark-field single NP plasmonic microscopy and spectroscopy to study their transport into early developing zebrafish embryos (cleavage stage) and their effects on embryonic development …