Biochemistry Commons^™

Utilization Of Ferrioxamine Microarrays For The Rapid Detection Of Pathogenic Bacteria, Nigam Bir Arora

Open Access Dissertations

Siderophores are low-molecular weight species utilized by bacteria for the sequestration of iron, an essential nutrient. Siderophores and their cognate receptors are considered to be virulence factors, due to their prominent role in pathogenicity. The work presented here focuses on ferrioxamine (FOx) as an “immutable” ligand for pathogen detection. A number of bacterial strains expressing high-affinity FOx receptors were identified by a proteomic BLAST search, and screened against microarrays patterned with FOx conjugates for detection using label-free optical imaging. Aspects such as inkjet printing and surface chemistry, iron-limiting conditions and bacterial selection protocols, and linker conjugate design were addressed and …

Functional And Structural Characterization Of The Mevalonate Diphosphate Decarboxylase And The Isopentenyl Diphosphate Isomerase From Enterococcus Faecalis, Chun-Liang Chen

Open Access Dissertations

Enterococcus faecalis causes a diverse range of nosocomial infections (in wounds, the gastrointestinal tract, the blood stream and the endocardium), and multidrug-resistant strains have become a serious issue across countries. Vancomycin, a FDA-approved drug for the disruption of the bacterial cell wall biosynthesis, has been utilized to treat infectious diseases caused by Enterococci; however, the prevalence of vancomycin-resistant enterococci (VRE) threatens communities all over the world. We aim at developing novel therapeutic strategies to control bacterial growth of Enterococci, and we focus on targeting two essential enzymes involved in poly-isoprenoid biosynthesis in Enterococcus faecalis; one is the mevalonate diphosphate decarboxylase …

Sugarcane Bagasse Hydrolysis Enhancement Using Bsa, Antonio Carlos Freitas Dos Santos

Open Access Theses

Lignocellulose is composed of polysaccharides linked to lignin and other aromatic compounds, making the sugars not readily available to fermentation. This entails that biomass must go through the unit operations of pretreatment and enzyme hydrolysis. Pretreatment opens the structure to allow the enzymes to act on and hydrolyze cellulose and hemicellulose to glucose and/or xylose which in turn are fermented to ethanol. Concomitantly, the enzymes interact with soluble phenols and insoluble solids derived from lignin that inhibit hydrolysis. This leads to high enzyme loadings and higher production costs. Soluble phenols can be eliminated through washing. Insoluble lignin, however, demands another …

Antibacterial Activity Of Essential Oil Encapsulated Sodium Iota-Carrageenan Fibers, Carlos D. Carter

Open Access Theses

Spoilage microorganisms cause food waste and loss of quality. While the foodborne pathogen outbreaks lead to thousands of hospitalizations and deaths. Essential oils (EOs), plant extracts, possess the required antimicrobial activities and thus their usage stands out as a feasible approach for controlling the undesirable bacterial growth in food systems. However, EOs are highly volatile and lose their activity upon exposure to environmental conditions. In this regard, their encapsulation in Generally Recognized As Safe (GRAS) matrices such as food grade polysaccharides especially iota-carrageenan could be one of the viable alternatives. Iota-carrageenan, sulfated polysaccharide from marine algae, is being used in …

The Role Of Hif1alpha And Hif2alpha In Muscle Development And Satellite Cell Function, Shiqi Yang

Open Access Theses

Hypoxia inducible factors (HIFs) are central mediators of cellular responses to fluctuations of oxygen, an environmental regulator of stem cell activity. Muscle satellite cells are myogenic stem cells whose quiescence, activation, self-renewal and differentiation are influenced by microenvironment oxygen levels. However, the in vivo roles of HIFs in quiescent satellite cells and activated satellite cells (myoblasts) are poorly understood. Expression analyses indicate that HIF1α and HIF2α are preferentially expressed in pre- and post-differentiation myoblasts, respectively. Interestingly, double knockout of HIF1α and HIF2α (HIF1α/2α dKO) in embryonic myoblasts results in apparently normal muscle development and growth. However, HIF1α/2α dKO in postnatal …

Influence Of Ph And Acidic Side Chain Charges On The Behavior Of Designed Model Peptides In Lipid Bilayer Membranes, Venkatesan Rajagopalan

Graduate Theses and Dissertations

The molecular properties of transmembrane proteins and their interactions with lipids regulate biological function. Of particular interest are interfacial aromatic residues and charged residues in the core helix whose functions range from stabilizing the native structure to regulating ion channels. This dissertation addresses the pH dependence and influence of potentially negatively charged tyrosine, glutamic acid or aspartic acid side chains. We have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as favorable host peptide framework. We have substituted W5 with Tyr (Y5GWALP23) and Leu residues with Glu (L12E, L14E or L16E) or Asp (L14D or L16D), and have incorporated specific 2H-labeled alanine residues within the …

Membrane Chromatography For Bioseparations: Ligand Design And Optimization, Zizhao Liu

Graduate Theses and Dissertations

Membrane chromatography, or membrane adsorber, represents an attractive alternative to conventional packed bed chromatography used in downstream processing. Membrane chromatography has many advantages, including high productivity, low buffer consumption and ease to scale up. This doctoral dissertation focuses on developing novel polymeric ligands for protein separations using membrane chromatography. Atom transfer radical polymerization (ATRP), known as a controlled radical polymerization technique, has been used to control the architecture of grafted polymeric ligands. The center theme of this dissertation is to develop new polymeric ligands and investigate how the polymer’s property (e.g. flexibility, hydrophobicity) and architecture (e.g. chain density, chain length) …

Bioorthogonal Reactions: Synthesis And Evaluation Of Different Ligands In Copper Catalyzed Azide-Alkyne1,3-Dipolar Cycloaddition (Cuaac), Zainab Abdullah Almansaf

Graduate Theses and Dissertations

The Copper CatalyzedAzide-Alkyne1,3-Dipolar Cycloaddition (CuAAC) reaction has unique features that qualify it to be one of the best click reactions. Its applications have been shown in different aspects and for multiple purposes. The oxidative degradation of biological systems (labile proteins and live cells) is, however, generally recognized as the major problem when using this reaction in living systems. Reactive oxidation species can be easily produced in the presence of copper(II), ascorbate and air, and this is the main cause of toxicity. However, the uses of ligands have shown a major impact on reducing copper toxicity, protecting Cu(I) from the redox …

Intestinal Cytoplasmic Lipid Droplets, Associated Proteins, And The Regulation Of Dietary Fat Absorption, Theresa M. D'Aquila

Open Access Dissertations

Dietary fat provides essential nutrients, contributes to energy balance, and regulates blood lipid concentrations. These functions are important to health, but can also become dysregulated and contribute to diseases such as obesity, diabetes, and cardiovascular disease. The small intestine absorbs dietary fat through an efficient multi step process of digestion, uptake, metabolism, and secretion or storage. When dietary fat is taken up by the absorptive cells of the small intestine, enterocytes, it can be secreted into circulation where it contributes to blood lipid levels or temporarily stored in cytoplasmic lipid droplets (CLDs). The objective of this dissertation is to investigate …

Biochemical Changes In Animal Models Of Fetal Alcohol Spectrum Disorder, Christine E. M. Keller

Open Access Dissertations

Fetal alcohol spectrum disorder (FASD) is a completely preventable disease, that has profound effects on life-long health and function of the affected individual. Prevalence estimates of FASD in the United States indicate 33.5 per 1,000 live births are affected with this disorder (Roozen, 2016). FASD is caused by maternal ethanol intake during pregnancy. However, recommendations of the amounts of alcohol safe to drink during pregnancy are not established. Further, we lack a comprehensive understanding of the biochemical pathways modified in prenatal ethanol exposure. Biomarkers are also lacking. Our results demonstrate the vast array of biochemical pathways modified in the chronic …

Investigation Of An Energetic Coupling Between Ligand Binding And Protein Folding, Nathan W. Gardner

Open Access Dissertations

The cellular environment presents a protein with many small molecules with which it may interact. Many novel interactions between proteins and non-substrate metabolites are being uncovered through proteome-wide screens. The homodimeric Escherichia coli cofactor-dependant phosphoglycerate mutase (dPGM) was identified as an ATP binding protein in a proteome-wide screen, but dPGM does not use ATP for catalysis. This dissertation elucidates the effect of ATP and other non-substrate metabolites on dPGM. Initial investigations revealed a partially unfolded, monomeric intermediate of dPGM that forms during equilibrium unfolding. ATP binding was found to occur at the active site of dPGM and to be energetically …

Enhancing Silymarin Fractionation Via Molecular Modeling Using The Conductor-Like Screening Model For Real Solvents, Emma C. Brace

Open Access Theses

The market for bio-based products from plant sources is on the rise. There is a global challenge to implement environmentally clean practices for the production of fuels and pharmaceuticals from sustainable resources. A significant hurdle for discovery of comparable plant-derived products is the extensive volume of trial-and-error experimentation required. To alleviate the experimental burden, a quantum mechanics based molecular modeling approach known as the COnductor-like Screening Model for Real Solvents (COSMO-RS) was used to predict the best biphasic solvent system to purify silymarins from an aqueous mixture. Silymarins are a class of flavonolignans present in milk thistle ( Silybum marianum …

Applications Of The Gst- Affinity Tag In The Purification And Characterization Of Proteins, Wibke Beatrice Kachel

Graduate Theses and Dissertations

With the latest innovations in biological sciences, large quantities of biologically active polypeptides as well as high throughput screening methods to quickly evaluate if these biomolecules potentially have therapeutic, diagnostic, or industrial purposes are required. The synthesis and purification of peptides and small proteins continue to be demanding as the production of high yields through chemical synthesis can involve large costs. On the other hand, there are only few examples of acquiring those biomolecules through cloning and expression in bacterial systems in form of recombinant fusion proteins. Glutathione S-Transferase (GST) is not only a very commonly used affinity tag to …

Investigation Of The Interaction Of Dimeric Ruthenium Complexes With Cytochrome B5, Christopher Dain Rupar

Graduate Theses and Dissertations

Photoreactive complexes to study the kinetics of electron transfer of proteins have been in use for a long time. It has always been speculated that complexes bind near the heme or the electron transfer reaction would not occur. But it is unkown exactly how the complex interacts with the protein. The structural, thermodynamic, and kinetic properties of rat liver microsomal cytochrome b5 were investigated when bound to ruthenium dimer complexes. Heteronuclear Single Quantum Coherence studies support a dynamic binding model of a dimer Ru complex bound near the protein’s heme involving residues H39, E44, G42, V61, G62, and H63. The …

Two-Electron Quenching Of Dinuclear Ruthenium(Ii) Polypyridyl Complexes, Yinling Zhang

Graduate Theses and Dissertations

A bridging ligand 5,5’-Bi- 1,10-phenanthroline, diphen, was prepared using dichlorobis(triphenylphosphine)Ni(II), Ni(PPh3)2Cl2 as catalyst with a yield of 40%. Yellow cubic crystals were able to obtain from the good purity product for single crystal analysis. The torsion angle between the planes of the subunit phenanthrolines is about 66 degrees.

A dinuclear ruthenium (II) polypyridyl complex, (phen)2Ru(diphen)Ru(phen)24+, was synthesized by using polymeric ruthenium carbonyl compound as the entry point, diphen as the bridging ligand and 1,10-phenanthroline, phen, as the terminal legand. Brown needlelike crystals were precipitated from acetonitrile that were not suitable for single crystal diffraction.

The photochemistry of the dimer was …

Mechanism Of Rapid Electron Transfer Reactions Involving Cytochrome Bc1, Cytochrome C And Cytochrome Oxidase, Jeremy Erik Durchman

Graduate Theses and Dissertations

Electron transfer between mitochondrial proteins complexes represents the primary means by which living things acquire the requisite energy for survival. The coupling of electron transfer to proton translocation creates an electrochemical gradient that drives the synthesis of highly energetic compounds such as ATP. The purpose of these studies is to measure rates of electron transfer and elucidate the important governing factors in the redox events involving cytochrome bc1, cytochrome c and cytochrome oxidase. Using rapid initiation of redox events triggered by laser flash excitation of ruthenium compounds, and strategically monitoring unique spectral properties of these proteins in the visible region …

Biophysical Studies Of The Allosteric Regulatory Mechanism Of Syk Tandem Sh2 Domains Interacting With Immunoreceptor Tyrosine-Based Activation Motifs, Chao Feng

Open Access Dissertations

The non-receptor spleen tyrosine kinase (Syk) is an important player in signal transduction from immunoreceptors to various downstream targets. It is widely expressed in both haematopoietic and epithelial cells. Syk disorder is closely related with many inflammatory and autoimmune diseases, as well as cancers.

Syk associates with immunoreceptors through its tandem SH2 domains (tSH2), which contain two SH2 domains connected by interdomain A. The association of Syk with immunoreceptors is regulated by Y130 phosphorylation in interdomain A. The unphosphorylated tSH2 can bind with the doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the cytoplasmic domains of immunoreceptors with very high …

Mechanistic Characterization Of Acetic Acid Resistance Enzymes Of Acetobacer Aceti, Jesse R. Murphy

Open Access Dissertations

Acetobacter aceti (A. aceti) is a Gram-negative, acidophilic bacterium that is used for the industrial production of acetic acid from ethanol. Oxidation of ethanol by membrane-bound oxidoreductases provides energy for A. aceti and the production of high concentrations of acetic acid is an effective defense mechanism. Acetic acid diffuses through cell membranes at low pH and effectively kills many bacteria, including E. coli, at low millimolar concentrations. The ability of A. aceti to thrive in molar concentrations of acetic acid is partially due to the twin subjects of this thesis, the acetic acid resistance factors AarA (citrate synthase, …

Axonal Transport And Life Cycle Of Mitochondria In Parkinson's Disease Model, Hyun Sung

Open Access Dissertations

In neurons, normal distribution and selective removal of mitochondria are essential for preserving compartmentalized cellular function. Parkin, an E3 ubiquitin ligase associated with familial Parkinson’s disease, has been implicated in mitochondrial dynamics and removal. However, it is not clear how Parkin plays a role in mitochondrial turnover in vivo, and whether the mature neurons possess a compartmentalized Parkin-dependent mitochondrial life cycle. Using the live Drosophila nervous system, here, I investigate the involvement of Parkin in mitochondrial dynamics; organelle distribution, morphology and removal. Parkin deficient animals displayed less number of axonal mitochondria without disturbing organelle motility behaviors, morphology and metabolic state. …

Learning The Abc's Of Ribose Transport Using Biophysical Methods, Satchal K. Erramilli

Open Access Dissertations

ATP-binding cassette transporters comprise a large superfamily of proteins that are involved in a variety of biological phenomenon, from bacterial metabolism to cellular homeostasis, antigen-presentation, and drug resistance. These proteins are implicated in a variety of clinically relevant phenomenon, including the human diseases cystic fibrosis, macular degeneration, and cancer. Understanding their structure-function can guide therapeutics and contribute to our overall understanding of these biological phenomena.

This study focuses on understanding the motor protein of the bacterial ribose ABC transporter in the context of transport. This complex is required for the uptake of the nucleotide precursor, ribose. Using biophysical methods, we …

Characterization Of Cu-Rich Aggregates In Neurogenic Niches Of The Rodent Brain By X-Ray Fluorescence Microscopy, Brendan T. Sullivan

Open Access Dissertations

Copper is an essential element in the brain playing several critical roles ranging from neurotransmitter synthesis to ATP production. As Cu is typically present in micromolar concentrations and has a spatially capricious distribution in the brain, determining concentrations has historically been challenging. X-ray fluorescence microscopy (XRF) offers excellent spatial resolution (down to 30~nm) and detection limits (sub parts per million), making it an excellent tool for analyzing metal distributions in the brain. Using XRF, it is demonstrated that Cu-rich aggregates with concentrations in the hundreds of millimolar are present in the subventricular zone of rats and mice. As the subventricular …

Investigating And Expanding The Functionality Of Rna Catalysts: Studies Of The Hepatitis Delta Virus, The Hammerhead, And The Aminoacyl-Trna Synthetase-Like Ribozymes, Ji Chen

Open Access Dissertations

Ribozymes, just like protein enzymes, catalyze diverse chemical reactions. The first goal of this dissertation is to understand the mechanism of ribozyme-mediated phosphodiester cleavage reaction. Biochemical assays and X-ray crystallography were used for probing the active site of two small self-cleaving ribozymes, the hepatitis delta virus (HDV) ribozyme and the hammerhead ribozyme. Results presented here suggest that divalent metal ions play critical roles in the catalytic mechanisms of both the HDV and the hammerhead ribozymes. In the HDV ribozyme, the result is consistent with an active site Mg2+ being directly involved in catalysis. In the hammerhead ribozyme, however, Mg2+ ions …