Biochemistry, Biophysics, and Structural Biology Commons^™

Fabrication Of Responsive Polymer Brushes For Patterned Cell Growth And Detachment, Ashley B. Sutherland

Browse all Theses and Dissertations

Patterned polymer brushes are used in biological applications to enhance cell function and to achieve selective cell growth. These patterned surfaces have a variety of applications, including cell sheet harvesting and tissue engineering. This work describes creation of chemical, topological, and responsive patterns on glass by sequential surface-initiated polymerization of poly(N-isopropylacrylamide)-co-(2-hydroxyethyl methacrylate) (pNIPAM-co-HEMA) and subsequent use of UV lithography to pattern the surface with pNIPAM. The cell adhesion peptide, RGD, is then immobilized on the surface of the copolymer pHEMA via DSC-coupling to spatially enhance cell adhesion properties. By culturing cells at 37 degrees celsius, cells selectively grow on DSC-activated …

Structural And Functional Study Of Multidrug Membrane Transporters, Feng Ding

Theses and Dissertations in Biomedical Sciences

Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for ineffective treatment of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding of their structures and functions for design of effective therapies. Despite extensive studies over decades, their underlying molecular mechanisms remain largely unknown. In this dissertation, we focus on the study of structures and functions of multidrug membrane transporters, including ATP-binding cassette transporter (BmrA) and Resistance-Nodulation-Cell Division transporter (MexA,B-OprM) in gram-positive and gram-negative bacteria ( Bacillus subtilis and Pseudomonas aeruginosa), respectively. We fused EGFP with N-terminus and C-terminus of …

Attachment Ability And Melanoma Inhibitory Activity Mrna Expression Level Changes In Murine B16-F10 Melanoma Cells Post Nanosecond Electric Pulses, Hongxia Jia

Theses and Dissertations in Biomedical Sciences

The effects of high-voltage nanosecond electric pulses (nsEPs) on metastatic melanoma are still unclear. Hence, we applied one, two, three, and four 300 ns 40 kV/cm pulses to murine B16-F10 melanoma cells. Cell attachment ability was determined by comparing the number of floating cells and the percentage of attached cells. Melanoma inhibitory activity (MIA) is a secretory protein that is highly correlated with the malignancy and metastasis of malignant melanomas. We used MIA as our target to evaluate the effect of nsEPs on metastasis. Pulsed (experimental) and unpulsed (control) cells were incubated at 37°C under a 5% CO₂ atmosphere. …

New Tools For Real-Time Study Of Embryonic Development, Lauren M. Browning

Theses and Dissertations in Biomedical Sciences

Embryonic development represents one of the most complex and dynamic cellular processes in biology, and plays vital roles in understanding of functions of embryonic stem cells (ESCs) and design of ESC-based therapy. Conventional assays and fluorescence-based imaging methods have been widely used for the study of embryonic development. These conventional methods cannot effectively provide spatial and temporal resolutions with sufficient sensitivity and selectivity that are required to depict embryonic development in vivo in real-time at single-cell and single-molecule resolutions. In this dissertation, we have developed a wide range of innovative tools for real-time study of embryonic development. These new tools …

The Cellular Nucleic Acid Binding Protein In Aging And Disease, Robin Webb

Theses and Dissertations--Molecular and Cellular Biochemistry

The ZNF9 gene on chromosome 3 encodes the cellular nucleic acid binding protein (CNBP), a ubiquitously expressed, 177 amino acid (≈19.5kDa) protein that is highly conserved among vertebrates. The function of the protein is largely unknown, however an expansion in the first intron of the protein results in myotonic dystrophy type 2 (DM2), a multisystemic disease featuring cardiac arrhythmia, muscle wasting, cataracts, and a range of neuropathologies. Remarkably, we recently discovered that CNBP is involved in regulating the activity of β-secretase, the enzyme that produces the first cleavage event in the generation of the amyloid-β peptide (Aβ). The progressive fibrillization …

The Disordered Regulation Of Calcineurin: How Calmodulin-Induced Regulatory Domain Structural Changes Lead To The Activation Of Calcineurin, Victoria B. Dunlap

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has …

Characterization Of G-Patch Motif Contribution To Prp43 Function In The Pre-Messenger Rna Splicing And Ribosomal Rna Biogenesis Pathways, Daipayan Banerjee

Theses and Dissertations--Biology

The DExD/H-box protein Prp43 is essential for two biological processes: nucleoplasmic pre-mRNA splicing and nucleolar rRNA maturation. The biological basis for the temporal and spatial regulation of Prp43 remains elusive. The Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 G-patch proteins bind to and activate the Prp43 DExD/H box-helicase in pre-mRNA splicing (Spp382) and rRNA processing (Sqs1, Pxr1). These Prp43-interacting proteins each contain the G-patch domain, a conserved sequence of ~48 amino acids that includes 6 highly conserved glycine (G) residues. Five annotated G-patch proteins in baker’s yeast (i.e., Spp382, Pxr1, Spp2, Sqs1 and Ylr271) and with the possible exception of the uncharacterized Ylr271 …

Manipulation Of Cellular Processing Bodies And Their Constituents By Viruses, Asit K. Pattnaik, Phat X. Dinh

School of Veterinary and Biomedical Sciences: Faculty Publications

The processing bodies (PBs) are a form of cytoplasmic aggregates that house the cellular RNA decay machinery as well as many RNA-binding proteins and mRNAs. The PBs are constitutively present in eukaryotic cells and are involved in maintaining cellular homeostasis by regulating RNA metabolism, cell signaling, and survival. Virus infections result in modification of the PBs and their constituents. Many viruses induce compositionally altered PBs, while many others use specific components of the PBs for their replication. PB constituents are also known to restrict virus replication by a variety of mechanisms. Further, continuing studies in this rapidly emerging field of …

Deep Sequencing And Genome-Wide Analysis Reveals The Expansion Of Microrna Genes In The Gall Midge Mayetiola Destructor, Chitvan Khajuria, Christie E. Williams, Mustapha El Bouhssini, R. Jeff Whitworth, Stephen Richards, Jeffrey J. Stuart, Ming-Shun Chen

School of Veterinary and Biomedical Sciences: Faculty Publications

Background: MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating post transcriptional gene expression. Gall midges encompass a large group of insects that are of economic importance and also possess fascinating biological traits. The gall midge Mayetiola destructor, commonly known as the Hessian fly, is a destructive pest of wheat and model organism for studying gall midge biology and insect – host plant interactions.

Results: In this study, we systematically analyzed miRNAs from the Hessian fly. Deep-sequencing a Hessian fly larval transcriptome led to the identification of 89 miRNA species that are either identical or very …

Functional Convergence Of Structurally Distinct Thioesterases From Cyanobacteria And Plants Involved In Phylloquinone Biosynthesis, Fabienne Furt, William J. Allen, Joshua R. Widhalm, Peter Madzelan, Robert C. Rizzo, Gilles J. Basset, Mark A. Wilson

Department of Biochemistry: Faculty Publications

The synthesis of phylloquinone (vitamin K₁) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy- 2-naphthoyl-CoA (DHNA-CoA) to release 1,4- dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacterium Synechocystis (Slr0204) and the flowering plant Arabidopsis thaliana (AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNACoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases from Pseudomonas and Arthrobacter. Like the 4-HBA-CoA …

Rapid Detection And Quantification Of Triacylglycerol By Hplc–Elsd In Chlamydomonas Reinhardtii And Chlorella Strains, Naoko Kobayashi, Eric A. Noel, Austin Barnes, Julian Rosenberg, Concetta C. Dirusso, Paul N. Black, George A. Oyler

Department of Biochemistry: Faculty Publications

Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thinlayer chromatography (TLC), and then analyzing the fatty

acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor

intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC–ELSD) was developed and compared to the conventional TLC/GC method. TAG …

The Basis For Limited Specificity And Mhc Restriction In A T Cell Receptor Interface, Kurt H. Piepenbrink, Sydney J. Blevins, Daniel R. Scott, Brian M. Baker

Department of Biochemistry: Faculty Publications

αβ Tcell receptors (TCRs) recognize peptides presented by major histocompatibility complex (MHC) proteins using multiple complementarity-determining region (CDR) loops. TCRs display an array of poorly understood recognition properties, including specificity, crossreactivity and MHC restriction. Here we report a comprehensive thermodynamic deconstruction of the interaction between the A6 TCR and the Tax peptide presented by the class I MHC HLA-A*0201, uncovering the physical basis for the receptor’s recognition properties. Broadly, our findings are in conflict with widely held generalities regarding TCR recognition, such as the relative contributions of central and peripheral peptide residues and the roles of the hypervariable and germline …

Effect Of Estrogen On Manganese-Induced Toxicity On Embryonic Astrocytes, Tyler T. Huynh, Kimberly J. Baker, Harold L. Komiskey

PCOM Scholarly Works

Manganese (Mn) is a natural trace metal that is essential for many physiological functions in the human body. Astrocytes in the central nervous system are susceptible reservoirs for Mn accumulation. Estrogen, a steroidal hormone, has been shown to mitigate Mn-induced toxicity in cultures of postnatal astrocytes. However, differences in expression/inducibility of glutamate transporters and glutamine synthetase, transmitters, and the natural gonadal steroids and their receptors are known to occur in astrocyte cultures derived from various stages of fetal and postnatal development. Cultures of embryonic (E18) hippocampal astrocytes were examined in this study for the ability of 17 β-estradiol (E2) to …

Dna-Binding Site Recognition By Bhlh And Mads-Domain Transcription Factors, Joshua R. Werkman

Theses and Dissertations--Plant and Soil Sciences

Herewithin, two transcription factor (TF) regulatory complexes were investigated. A bHLH–MYB–WDR (BMW) DNA-binding complex from maize was the first complex to be studied. R, a maize bHLH involved in the activation of genes in the anthocyanin pathway, had been characterized to indirectly bind DNA despite the presence of a functional DNA-binding domain. Findings presented here reveal that this is only partially correct. Direct DNA-binding by R was found to be dependent upon two distinct dimerization domains that function as a switch. This switch-like mechanism allows R to be repurposed for the activation of promoters of differing cis-element structure.

The …

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 …

The Contribution Of Visceral Fat To Positive Insulin Signaling In Ames Dwarf Mice, Vinal Menon

Electronic Theses and Dissertations

Ames dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells – somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice also have high circulating levels of anti-inflammatory and antidiabetic adiponectin. Plasma levels of this adipokine usually decrease with an increase in accumulation of …

Cerium Oxide Nanoparticles Sensitize Pancreatic Cancer Cells To Radiation By Promoting Acidic Ph, Ros, And Jnk Dependent Apoptosis, Melissa Wason

Electronic Theses and Dissertations

Side effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl peroxide resulting in accumulation of the latter whereas in neutral pH CONPs scavenge both. CONP treatment prior to RT markedly potentiated the cancer cell apoptosis both in culture and in tumors and …

The Role Of Mitochondrial Omi/Htra2 Protease In Protein Quality Control And Mitophagy, Camilla Ambivero

Electronic Theses and Dissertations

Omi/HtrA2 is a mitochondrial serine protease with a dual and opposite function depending on its subcellular localization. Most of the previous studies focused on Omi/HtrA2’s pro-apoptotic function when the protein is released to the cytoplasm. It is becoming apparent that the main function of Omi/HtrA2 is within the mitochondria, where it has a pro-survival role. However, its mechanism is still poorly understood. To this end, we used the yeast two-hybrid system to dissect the Omi/HtrA2 pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a deubiquitinating (DUB) complex. In addition …

The Cytopathic Activity Of Cholera Toxin Requires A Threshold Quantity Of Cytosolic Toxin., Carly Bader

Electronic Theses and Dissertations

Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the …

Characterization Of Motility Alterations Caused By The Impairment Of Dynein/Dynactin Motor Protein Complex, Swaran Nandini

Electronic Theses and Dissertations

Transport of intracellular cargo is an important and dynamic process required for cell maintenance and survival. Dynein is the motor protein that carries organelles and vesicles from the cell periphery to the cell center along the microtubule network. Dynactin is a protein that activates dynein for this transport process. Together, dynein and dynactin forms a motor protein complex that is essential for transport processes in all the vertebrate cells. Using fluorescent microscope based live cell imaging techniques and kymograph analyses, I studied dynein/dynactin disruptions on the intracellular transport in two different cell systems. In one set of experiments, effects of …

Bone Morphogenetic Protein-7 (Bmp-7) Polarizes Monocytes Into M2 Macrophages, Crystal Rocher

Electronic Theses and Dissertations

Atherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however, its role in monocyte polarization is yet to be revealed. In the current study, we hypothesize that monocyte treatment with BMP-7 will significantly result in …

Delineating Key Genetic Components On Linear Plasmid 36 That Contribute To Its Essential Role In Borrelia Burgdorferi Mammalian Infectivity., Tisha Choudhury

Electronic Theses and Dissertations

The spirochete Borrelia burgdorferi is the etiologic agent of Lyme disease. This pathogen has a complex enzootic life cycle that involves passage between the tick vector (Ixodes scapularis) and various vertebrate hosts with humans being inadvertent hosts. There is a pressing need to study the genetic aspects of the B. burgdorferi infectious cycle and particularly spirochete genes involved in mammalian infectivity so as to develop novel therapeutic and diagnostic strategies to combat Lyme disease. The B. burgdorferi genome is fragmented and comprised of a single 900 kb linear chromosome and multiple linear and circular plasmids. It has been observed that …

Mixed Valence Copper(Cu)/Silica Nanocomposite: Synthesis, Characterization And Systematic Antimicrobial Studies, Mikaeel Young

Electronic Theses and Dissertations

Copper (Cu) compounds are widely used as effective agricultural bactericides. Continuous use of these materials has led to Cu accumulation in soil over time. The United States Environmental Protection Agency (US EPA) is concerned about potential Cu contamination in the environment. Improving biocidal efficacy of Cu is an attractive alternative, allowing reduction of Cu amount per application. In this research, we focused on making watersoluble mixed-valence Copper/Silica composite nanogel (CuSiNG) material. The objective is to improve the efficacy of Cu by manipulating Cu valence states. It has been shown in the literature that Cu (0) and Cu (I) states are …

Immuno-Pcr Detection Of Lyme Borreliosis, Micah Halpern

Electronic Theses and Dissertations

Lyme borreliosis, more commonly referred to as Lyme disease, is the fastest growing zoonotic disease in North America with approximately 30,000 confirmed cases and 300,000 estimated infections per year. In nature, the causative agent of Lyme disease, the bacterium Borrelia burgdorferi, cycles between Ixodes sp. ticks and small mammals. Humans become infected with Lyme disease after being bitten by an infected tick. The primary indicator of a Borrelia burgdorferi infection is a bull’s eye rash typically followed by flu-like symptoms with treatment consisting of a 2-4 week course of antibiotics. If not treated, later stages of the disease can result …

Doxorubicin-Induced, Tnf-Α-Mediated Brain Oxidative Stress, Neurochemical Alterations, And Cognitive Decline: Insights Into Mechanisms Of Chemotherapy Induced Cognitive Impairment And Its Prevention, Jeriel T. Keeney

Theses and Dissertations--Chemistry

The works presented in this dissertation provide insights into the mechanisms of chemotherapy-induced cognitive impairment (CICI or “ChemoBrain”) and take steps toward outlining a preventive strategy. CICI is now widely recognized as a complication of cancer chemotherapy experienced by a large percentage of cancer survivors. Approximately fifty percent of existing FDA-approved anti-cancer drugs generate reactive oxygen species (ROS). Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, produces the reactive superoxide radical anion (O₂^-•) in vivo. Dox treatment results in oxidation of plasma proteins, including ApoA-I, leading to TNF-α-mediated oxidative stress in plasma and brain. TNF-α elevation in brain …

Understanding Multidrug Resistance In Gram-Negative Bacteria -- A Study Of A Drug Efflux Pump Acrb And A Periplasmic Chaperone Sura, Meng Zhong

Theses and Dissertations--Chemistry

Multiple drug resistance (MDR) has been a severe issue in treatment and recovery from infection.Gram-negative bacteria intrinsically exhibit higher drug tolerance than Gram-positive microbes. In this thesis, two proteins involved in Gram-negative bacterial MDR were studied, AcrB and SurA.

Resistance-nodulation-cell division pump AcrAB-TolC is the major MDR efflux system in Gram-negative bacteria and efficiently extrudes a broad range of substances from the cells. To study subtle conformational changes of AcrB in vivo, a reporter platform was designed. Cysteine pairs were introduced into different regions in the periplasmic domain of the protein, and the extents of disulfide bond formation were …

Structural And Functional Characterization Of Mulitdrug Resistance Transporter And Regulator, Linliang Yu

Theses and Dissertations--Chemistry

Drug resistant bacteria pathogen poses a severe threat to human health. Bacterial drug efflux pumps are transporter proteins involved in the export of antibiotics out of cells. Efflux by transporters is one of the major drug resistant mechanisms. Multidrug efflux pumps can transport multiple classes of antibiotics and are associated with bacteria multiple drug resistance (MDR). Overproduction of these pumps reduces susceptibility of bacteria to a variety of antibiotics. MDR regulators are cytoplasmic proteins that control the expression level of MDR transporters in response to the cellular concentration of antibiotics. This thesis research focuses on three main directions in the …

Determination Of Structural Stabilities Of Metal-Free Porphyrin Cytochrome C, Zinc Cytochrome C, And Cobalt Cytochrome C, Usha K. Tottempudi

Masters Theses

No abstract provided.

Molecular Mechanisms Involved In Anhydrobiosis Of Insect Cells, Diyagama Arachchi Ralalage Dilini Sewwandi Samarajeewa

Masters Theses

Animals possessing tolerance to extreme water stress are termed anhydrobiotes. Many desiccation tolerant organisms respond to water stress by intracellular accumulation of selected sugars such as trehalose and larger macromolecules such as Late Embryogenesis Abundant (LEA) proteins and thereby maintain the cell viability. Evidence indicates that the presence of trehalose and Late Embryogenesis Abundant (LEA) proteins may work synergistically to confer cellular protection during drying in eukaryotic cells. We evaluated any increase in cellular desiccation tolerance by expressing different LEA proteins in a non-desiccation tolerant insect cell line Drosophila melanogaster (fruit fly) (Kc167 cells) in the presence of trehalose.

Transgenic …

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 …