Biochemistry Commons^™

Characterization Of Arabidopsis Eukaryotic Translation Initiation Factor 2Α (Eif2Α) Mutants, Mark Edens

Masters Theses

Plants are stationary organisms that are charged with overcoming a multitude of biotic and abiotic stresses. The eukaryotic translation initiation factor 2 (eIF2) is responsible for charging the P-site of the forming 80S ribosome with the initiator methionyl-tRNA. Thus, eIF2 is a protein of utmost consequence in the growth and development of organisms. The eIF2 protein is also a mediator of global translational regulation in the eukaryotic integrated stress response (ISR), where the α-subunit is phosphorylated by protein kinases, such as the kinase GCN2. Here, five eIF2α allele mutants in Arabidopsis were found to be deleterious to plant growth, development, …

Influence Of Physical Variability Of Highly Weathered Sedimentary Rock On Nitrate In Area 3 Of The Enigma Field Research Site At Y-12, Erin Kelly

Masters Theses

Uranium processing and waste storage in unlined waste ponds leached contaminants into the groundwater at Y-12, Oak Ridge, Tennessee, from the 1950s to 1980s. Groundwater wells near the S-3 ponds have had the highest nitrate concentrations of groundwater anywhere in the world (>10,000 mg/L). For reference, the maximum contaminant level for nitrate in drinking water set by the U.S. Environmental Protection Agency is 10 mg/L. Since 2012, the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) group has been characterizing, monitoring, and conducting field experiments to understand the interactions between contaminants, microbes, and the subsurface. The goals …

Comparative Chemical Characterization Of Lunasin-Enriched Preparations And Modifications Of The Inflammasomes In Vitro, Samuel James Price

Masters Theses

Soybean (Glycine max) is one of the most cultivated crops in the world providing the population with large amounts of protein and oil. In addition to its nutritional composition, soybean also contains biologically active compounds with potential health-promoting properties. The presence of these bioactives may be responsible for the lower incidence of chronic diseases in populations that consume a significant portion of soybeans in their diet. One group of soybeanderived bioactives are bioactive peptides and proteins including lunasin, Bowman-Birk inhibitor (BBI) and Kunitz-type trypsin inhibitor (KTI). The overall objective of this research was to develop a method of …

Lignin Maximization: Analyzing The Impact Of Different Feedstocks And Feedstock Ratios Using Organosolv Fractionation, Marc Banholzer

Masters Theses

Over-exploitation of fossil fuels coupled with increasing pressure to reduce carbon emissions are prompting a transition from conventional petrochemical feedstocks to sustainable and renewable sourced carbon. The use of lignocellulosic biomass as a feedstock for integrated biorefining is of current high interest, as separation into its component parts affords process streams of cellulose, hemicellulose and lignin, each of which can serve as a starting point for the production of biobased chemicals and fuels. Given the large number of potential sources of lignocellulosic feedstocks, the biorefinery will need to adapt to the supplies available over a normal growing season. Of particular …

Switchgrass Extractives Have Potential As A Value-Added Antimicrobial Against Plant Pathogens And Foodborne Pathogens, Alexander Ian Bruce

Masters Theses

Panicum virgatum (switchgrass), a perennial grass native to North America, is a leading biomass feedstock candidate for the manufacture of cellulosic ethanol. Switchgrass is considered a viable option for biofuel production due to its cheap production cost and ability to grow on marginal land. Biofuel derived from switchgrass has been shown to be very energy efficient, producing 540% more renewable energy versus nonrenewable energy expended. Switchgrass-derived biofuel is also estimated to have greenhouse gas emissions that are 94% lower than emissions from gasoline (Schmer et al 2008). Biofuels are created through biochemical processes that utilize various enzymes and microorganisms for …

Effect Of Varying Rumen Degradable And Undegradable Protein On Milk Production And Nitrogen Efficiency In Lactating Dairy Cows Under Summer Conditions, Jeffrey D. Kaufman

Masters Theses

The objective is to determine the effect of reducing nitrogen input through feeding low rumen degradable protein (RDP) and rumen undegradable protein (RUP) proportions on milk production, nitrogen efficiency and metabolism in heat-stressed cows. Forty-eight mid-lactating, Holstein cows were assigned to treatments using a randomized block design in a 2x2 factorial arrangement of treatments (n = 12/treatment). Treatments included two levels of RDP (10 and 8%) and two levels of RUP (8 and 6%). From d 1 to 21, a common diet (10% RDP-8% RUP) was fed to cows followed with their respective treatment diets fed from d 22 to …

Manipulating Adipose Tissue Fatty Acid Oxidation To Reduce Fatness In Broiler Chickens, Tania Emmanuelle Torchon

Masters Theses

Compared to rodents, broiler chickens, those reared for meat, are an attractive model for studies of adipose biology, and obesity development in children. The broiler chicken lacks the gene for uncoupling protein 1, the hallmark for brown adipose tissue making them a useful model to study lipid metabolism in white adipocytes. Two studies were performed to investigate if white adipose tissue had the metabolic ability for fatty acid oxidation (FAO), and to investigate the effects of dietary fatty acids on abdominal fat development of young broiler chickens as a model for childhood obesity. In study one, chickens were fasted for …

Conformational Dynamics Of Cytochrome P450cam Upon Ligand Binding, Ana Virginia Bernal Gomez

Masters Theses

Cytochrome P450s are a superfamily of metalloenzymes that are responsible for the monooxygenation of their hydrophobic substrates. P450’s retain the same general structural scaffold, however are able to bind promiscuously to substrates with distinct physico-chemical properties. It is believed that they possess considerable flexibility in the substrate binding regions as well as their active site to accomplish the diverse binding and catalytic chemistry with high regio- and stereo-specificity. In order to investigate the conformational dynamics inherent in these enzymes, especially in context of binding different ligands, we carried out amide proton exchange studies via NMR spectroscopy on a model P450 …

Comparison Of Activation Enthalpies For Aminoglycoside Modification Reactions, Brittany Sterling Soto

Masters Theses

At highly elevated temperatures, many biological reactions can proceed spontaneously from the ground state to the transition state. However, due to the long half-life of these reactions, catalysts are required to catalyze these reactions at modern day temperatures by lowering the activation energy. Wolfenden et al. has previously shown that catalysts enhance the rate of the reaction by reducing the enthalpy of activation. Therefore, the activation energies have been determined for three aminoglycoside modifying enzymes, APH(3’)-IIIa, AAC(3)-IIIb, and AAC(3)-VIa, to determine whether these three enzymes distinguish between the two classes of aminoglycoside antibiotics by reducing the enthalpy of activation during …

Localization Of Chemoreceptors In Azospirillum Brasilense., Anastasia Aksenova

Masters Theses

In order to ensure their survival, bacteria must sense and adapt to a variety of environmental signals. Motile bacteria are able to orient their movement in a chemical gradient by chemotaxis. During chemotaxis, environmental signals are detected by chemotaxis receptors and are propagated via a signal transduction cascade to affect bacterial motility. In a model organism Escherichia coli, chemotaxis receptors, also called MCPs (for methyl-accepting chemotaxis proteins) sense changes in concentration gradients by making temporal comparisons about the chemical composition of their surroundings. Decreased attractant concentration or increased repellant concentration results in conformational changes in the MCPs that culminate …

Examining The Functional Consequences Of The Flexibility Of Aminoglycoside Phosphotransferase (3’)-Iiia, Katelyn Dawn Rosendall

Masters Theses

The use of aminoglycoside antibiotics began in 1940 with the discovery of streptomycin. The overuse and misuse of antibiotics has resulted in prevalent cases of antibiotic resistance. The most common source of aminoglycoside resistance is the presence of enzymes that covalently modify the antibiotics at specific locations. One such enzyme, APH(3′)-IIIa [the aminoglycoside phosphotransferase three prime three a] conveys resistance by transferring the γ-phosphate [gamma phosphate] from ATP [adenosine triphosphate] onto the 3′ [three prime] carbon of the aminoglycoside antibiotic sugar ring. APH(3′)-IIIa has been shown to be flexible in solution and this flexibility is proposed to be responsible for …

Engineering Of Photosystem I Attachment To Titanium Oxide Nanostructures Via Ferredoxin-Fusion Proteins, Tuo Zhu

Masters Theses

Ferredoxins (Fds) are iron-sulfur proteins that mediate electron transfer in a range of metabolic reactions. In the thylakoid membrane of photosynthetic organisms, Fd facilitates electron transfer from the stromal surface of photosystem I (PSI) to the ferredoxin Nicotinamide adenine dinucleotide phosphate oxidoreductase (FNR), which requires that Fd is capable of docking and transferring electrons between these two complexes. In applied photosynthesis, many efforts have been devoted towards re-directing these electrons into either a hydrogen-evolving catalyst or an electron-conducting semiconductor. In this study, the electrons from the PSI complex are directed to a titanium oxide (TiO₂) electrode, and Fd …

Value-Added Lignin Based Carbon Fiber From Organosolv Fractionation Of Poplar And Switchgrass, Andreas Attwenger

Masters Theses

Carbon fibers have unique properties that include high strength, low density and excellent chemical and thermal resistance. However, they have a low level of utilization because of their high price; typically around $30/kg for an entry level polyacrylonitrile (PAN) based carbon fiber. Low-cost carbon fibers derived from lignin are currently being investigated at the University of Tennessee, because using lignin as a precursor could significantly reduce production costs. Lignins obtained from the pulp and paper and the emerging biofuel industries have the potential to be used for carbon fiber production, however, they are typically unsuitable because of the high levels …

Angiotensinogen Gene Silencing Reduces Lipid Accumulation And Inflammation In Cultured 3t3-L1 Adipocytes, Wenting Xin Carroll

Masters Theses

Obesity is characterized by metabolic complications which are related to several life-threatening diseases. Dysregulated inflammatory adipokines secretion from adipose tissue is believed as the major contributor to obesity-associated local and systemic inflammation, insulin resistance, and other metabolic dysfunctions.

Numerous studies in our lab and others pointed to the role of local adipose tissue renin-angiotensin system (RAS) in the pathogenesis of obesity, inflammation and insulin resistance. We hypothesized that adipocytes-derived angiotensinogen (Agt) played a critical role in adipogenesis and/or lipogenesis as well as adipose inflammation. To test, we established 3T3-L1 preadipocytes stably transfected with Agt-shRNA or scrambled sequence (Sc-shRNA). Transfected preadipocytes …

Design And Synthesis Of Boronic Acid-Based Sensors For Microarray Analysis And Fret-Detection To Study Carbohydrates, Yilin Wang

Masters Theses

Carbohydrates play significant roles in various biological and pathological processes such as cancer metastasis and inflammation. They participate in various signaling pathways and are able to bind to a litany of biological receptors such as proteins and viruses. Biologists have known for decades that the structure of glycans changes with the onset of cancer and inflammation. Due to these important activities, carbohydrate sensing has long been a focus of research. One example of such a carbohydrate sensor is boronic acid-based sensors. Boronic acid-based sensors are of particular interest due to their selectivity for 1,2- or 1,3-diols. A variety of techniques …

Elucidating The Effect Of Silver On Ethylene Signaling In Arabidopsis Thaliana, Brittany Kathleen Mcdaniel

Masters Theses

Ethylene, a gaseous plant hormone, is involved in numerous plant developmental processes such as seed germination, senescence, and fruit ripening. In Arabidopsis thaliana, ethylene is perceived by a family of five membrane-bound receptors, which upon binding ethylene trigger downstream effects. At the receptor level, it is known that the coordination of a copper ion is necessary for ethylene to bind, resulting in a conformational change of the receptor and the initiation of the ethylene signal transduction pathway. Interestingly, silver ions are also able to support binding of ethylene but ethylene responses are blocked in the presence of silver. When …

Capturing More Light: Phycobilisome Characterization For Increased Hydrogen Production Efficiency, Paul Abraham Willard

Masters Theses

Alternative energy and biofuels are a growing area of research. The demand for more and clean energy is ever increasing, but the current technology is inefficient, expensive, and incapable of meeting the demands of the current market. Hydrogen is a potential future fuel, as it is both clean and renewable, but its formation through conventional means is costly and inefficient. Photosynthesis can be utilized for the formation of hydrogen, which can then serve as a convenient and renewable biofuel. Photosynthetic hydrogen evolution is observed in vitro, but the current photosystem design is not very versatile and optimized to use …

Getting Heavy: An Exploration Into The Effects Of D2o And High Hydrostatic Pressure On R67 Dihydrofolate Reductase, Mary Jane Timson

Masters Theses

Chromosomal dihydrofolate reductase (DHFR) enzymatically reduces dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. R67 DHFR is an R-plasmid encoded enzyme that confers resistance to trimethoprim (TMP), an antibacterial drug. It shares no structural homology with TMP targeted, chromosomal DHFRs.

Previous osmolyte studies in our lab have indicated that DHF binding to R67 DHFR is accompanied by water uptake and NADPH binding is accompanied by water release. These data suggest that water plays a role in balancing the binding affinity. This may happen as R67 DHFR has a generalized binding surface and may need differential water effects to …

Non-Thermal Plasma Inactivation Of Bacillus Amyloliquefaciens Spores, Yaohua Huang

Masters Theses

Bacterial spores have remarkable resistance to a variety of harsh conditions, causing spoilage in food industry and becoming the primary bacterial agent in biowarfare and bioterrorism. In this study, inactivation mechanisms of Bacillus amyloliquefaciens (BA) spores by non-thermal plasma (NTP) were investigated by using Fourier-transform infrared spectroscopy (FTIR) as a major tool to exam spores after NTP treatment. Chemometric techniques, such as multivariate classification models based on soft independent modeling of Class Analogy (SIMCA) and Principal Component Analysis (PCA), were employed to identify functional group changes in FTIR spectra. The IR absorbance bands correlated to dipicolinic acid (DPA) decreased after …

Examining The Roles Of Pstoc75 Potra Domains In Chloroplast Protein Import, Richard Franklin Simmerman

Masters Theses

During chloroplast formation via endosymbiosis most of the plastid genome was transferred to the host nuclear genome. Genomic and proteomic analysis suggests that >95% of the original plastid proteome is now encoded in the nucleus, and these now cytosolically fabricated proteins require a post-translational transport pathway back into the organelle. This process is not well understood, yet it has been shown to involve translocons at the outer and inner envelope of the chloroplast membranes (TOC & TIC). These translocons interact with a cleavable N-terminal extension of between 20 and 100 residues on chloroplast-bound precursor proteins known as the transit-peptide. Precursor …

Design And Synthesis Of Metabolically Stabilized Lipid Probes For The Investigation Of Protein–Lipid Binding Interactions, Ashdeep Kaur Rajpal

Masters Theses

Protein–lipid binding interactions play crucial roles in various physiological and pathological processes, making it very important to study these interactions at the molecular level. However, investigation of these interactions is complicated by several issues, including the inherent complexity of membranes as well as the diverse mechanisms by which proteins interact with the membrane surfaces. As a result, many of these interactions remain poorly characterized. Synthetic probes are useful tools employed for studying protein–lipid binding interactions. This thesis will detail the design and synthesis of metabolically stabilized analogues of various signaling lipids, which mimic the natural species and are not easily …

Structural And Functional Analysis Of Toc75, Ashita Mukul Dave

Masters Theses

The majority of chloroplast proteins are nuclear-encoded and post-translationally imported into the chloroplast. These newly imported proteins are translocated from the cytosolic compartment to the stroma by the Translocons of the Outer/Inner membranes of Chloroplast (TOC/TIC). In order to understand protein transport across the chloroplast outer membrane, it is crucial to investigate the structure and function of these complexes. The TOC complex is composed of the beta-barrel channel protein Toc75 and the GTPase receptors Toc34 and Toc159.

Toc75 is a member of the OMP85 (Outer Member Protein, 85 kDa) superfamily. Other proteins of the OMP85 superfamily also exist in Gram-negative …

Design And Synthesis Of A Boronic Acid Sensor To Study Carbohydrate Binding Using Sers, Paul Russell Petersen

Masters Theses

Carbohydrates are known to play a large number of significant roles in various biological and pathological processes such as cancer metastasis and cellular communication. This is because of their ability to bind a wide range of hosts within the human body such as proteins and viruses. Due to these important interactions, carbohydrate sensing has long been a main focus of research. These research strategies have included the use of aptamers, non-covalent interactions, and boronic acid-based receptors. Boronic acid-based sensors are of particular interest due to their selectivity for 1,2- or 1,3-diols. Within these boronic acid-based studies, a large variety of …

Applications Of Bret: The Detection And Visualization Of Protein Interactions And Intramolecular Conformational Changes, Lindsay Anne Staron

Masters Theses

Bioluminescence is a phenomenon in which chemical energy is converted into light energy. Here, the oxidation of a luciferin substrate, catalyzed by a luciferase enzyme, results in the emission of a photon. This biological process is exploited in a technology referred to as Bioluminescence Resonance Energy Transfer (BRET). As its name implies, BRET depends on a nonradiative energy transfer event that occurs between a donor luciferase and an acceptor fluorophore. Fusion of the donor and acceptor molecules to a protein(s) of-interest allows one to identify and monitor molecular events, such as protein interactions or hormone binding events, based solely on …

Studies On The Mechanisms Of Homolog Pairing And Sister Chromatid Cohesion During Drosophila Male Meiosis, Jian Ma

Masters Theses

Meiosis is a complex process involving one round of DNA replication followed by two rounds of cell divisions. The proper segregation of homologs at meiosis I and sister chromatids during meiosis II is essential for the survival of the offspring. Aberrant chromosome segregation at any stage of meiosis can lead to aneuploidy. Meiotic chromosome segregation without crossing over or chiasmata is a widespread but poorly understand chromosome segregation pathway. In male Drosophila meiosis the absence of recombination in chromosomes makes it easier to identify mutations which influence homologous chromosome pairing and segregation.

Modifier of Mdg4 in Meiosis (MNM), a protein …

The Effects Of Acute Ethanol Treatment On The Suprachiasmatic Nucleus In Adult Male Mice, Charles Andrew Mangrum

Masters Theses

Light is the primary entraining signal for the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Light entering the eye leads to release of glutamate directly onto SCN neurons where it binds to N-methyl D-aspartate (NMDA) receptors initiating a cascade of cellular processes that ultimately modulates clock phase. SCN neurons show a 24-hour rhythm in neuronal activity that peaks in the middle of the day when isolated in a brain slice preparation. Treatments that phase-shift the SCN clock in vivo have been shown similarly to shift this rhythm of neuronal activity in vitro. Here, I have investigated …