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 …

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 …

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 …