Life Sciences Commons^™

Characterization Of The Multifunctional Enzyme Proline Utilization A, Yizi Mao

Department of Biochemistry: Dissertations, Theses, and Student Research

Proline is a unique and important amino acid. Proline is a proteogenic amino acid and its metabolism is involved in many critical cellular functions. Therefore, proline metabolism is tightly regulated, and dysfunction of proline metabolism is related to human diseases. The first step of proline oxidization to ∆¹-pyrroline-5-carboxylic acid (P5C) is catalyzed by proline dehydrogenase (PRODH). P5C is then non-enzymatically hydrolyzed to glutamate-γ-semialdehyde (GSA), which can be further oxidized to glutamate by P5C dehydrogenase (P5CDH/GSALDH). In Gram-negative bacteria, the PRODH and P5CDH enzymes are expressed as one polypeptide called proline utilization A (PutA). In some Gram-negative bacteria an …

Mitochondrial Metal Homeostasis: A Look Into Iron And Copper Mobilization Within Mitochondria, Jonathan Dietz

Department of Biochemistry: Dissertations, Theses, and Student Research

Cellular iron and copper homeostasis is interdependent with mitochondrial iron and copper homeostasis. Mitochondria must import iron to form iron-sulfur clusters and heme, while it must import copper for usage and storage. These cofactors are incorporated into mitochondrial proteins that support essential functions, including cellular respiration and maintaining redox homeostasis. In turn, mitochondria also provide heme to the cell and enables the biogenesis of cytosolic iron-sulfur cluster containing proteins, while also providing copper when needed. Due to both metals (and their modified species) reactivity, iron and copper are stored and trafficked within the mitochondria carefully. Although these cofactors are crucial …

Development Of High Value Oil Traits Using The Model Oilseed Crop Camelina Sativa, Evan Updike

Department of Biochemistry: Dissertations, Theses, and Student Research

Plant oils are an important source of food, fuel, and feed in our society today. The oil found in the seeds of plants is composed almost entirely of triacylglycerol (TAG) molecules, which consist of three fatty acids esterified to a glycerol backbone. As crude oil supplies decline, vegetable oils are gaining traction as a renewable substitute to petroleum-based materials in fuels, lubricants, and specialty oleochemicals. However, as it currently stands vegetable oils do not possess the properties necessary to fill the void of a petroleum free world.

To address this problem, plant biotechnologists have done extensive work on genetic engineering …

Characterization Of Sphingolipid Biosynthesis And Modification In Plants, Dongdong Zhang

Department of Biochemistry: Dissertations, Theses, and Student Research

Sphingolipids are ubiquitous membrane lipids in eukaryotic cells. Ceramides, the backbone of sphingolipids, are synthesized by ceramide synthases (CerSs), which form amide bonds between long-chain bases (LCBs) and the acyl groups of acyl-CoAs. Class I CerS works predominantly towards C16-CoA, whereas Class II CerS works towards C24-CoA. Plant sphingolipids generally have 16-carbon and 24-carbon as the major acyl groups. However, the sphingolipids of Oryza sativa and Zea mays have 20-carbon and 24-carbon as the predominant acyl groups. This dissertation aims to understand the substrate preference of CerSs in monocots such as rice. Phylogenetic analysis shows the Class I CerSs from …

The Role Of Liver Sinusoidal Endothelial Cells In Liver Malady Homeostasis, Fatima Cabral

Department of Biochemistry: Dissertations, Theses, and Student Research

Current literature described techniques for the purification of liver cell types through text alone. The techniques described for the isolation of liver sinusoidal endothelial cells as well as hepatocytes described here are modified from a published article in the Journal of Visualized experiments. The video protocol allows for the user to successfully isolate cells as the most difficult parts of the procedure are demonstrated visually. The detection of liver maladies such as the non-alcoholic fatty liver disease, the stage if this disease and differentiation between non-alcoholic and alcoholic fatty liver disease is demonstrated in the development of a unique panel …

Dissecting The Regulatory Network Of Sphingolipid Biosynthesis In Plants, Ariadna Gonzalez-Solis

Department of Biochemistry: Dissertations, Theses, and Student Research

Sphingolipids are a diverse group of lipids recognized as important components of cellular membranes and regulators of processes during development and in response to environmental stresses. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is a primary regulatory point for homeostasis. ORM proteins have been identified as negative regulators of SPT activity, however the mechanistic details of the regulation are only beginning to be understood. In this work, we show that ORM1 and ORM2 are essential for life cycle completion in Arabidopsis thaliana. Furthermore, the study of a structural ORM1 variant provided information about a transmembrane …

Characterization Of Human Pyrroline-5-Carboxylate Reductase Enzymes Responsible For L-Proline Biosynthesis, Sagar Patel

Department of Biochemistry: Dissertations, Theses, and Student Research

Pyrroline-5-carboxylate reductases (EC 1.5.1.2) are important housekeeping enzymes of L-proline biosynthesis, which generate L-proline and influence redox cycling of NAD(P)H/NAD(P)⁺ to support cellular growth in all domains of life. Structural evidence from X-ray crystal structures of HsPYCR1 (PDB codes 5UAT, 5UAU, and 5UAV) shows both NADPH bound in the N-terminal Rao-Rossmann fold motif and an important hydrogen bond or proton donor role for Thr238 with L-P5C. The Thr238Ala mutation results in 10-fold loss in catalytic efficiency with varied L-P5C relative to the wild-type enzyme, thus indicating Thr238’s potential hydrogen bond and proton donation to L-P5C is critical for catalysis. …

Protease Oma1 Modulates Mitochondrial Bioenergetics And Ultrastructure Through Dynamic Association With Micos Complex, Martonio P. Viana

Department of Biochemistry: Dissertations, Theses, and Student Research

Remodeling of mitochondrial ultrastructure is a complex dynamic process that is critical for a variety of mitochondrial functions and apoptosis. Although the key regulators of this process - mitochondrial contact site and cristae junction organizing system (MICOS) and GTPase Optic Atrophy 1 (OPA1) have been characterized, the mechanisms behind this regulation remain incompletely defined. Here, we found that in addition to its role in mitochondrial division, metallopeptidase OMA1 is required for maintenance of contacts between the inner and outer membranes through a dynamic association with MICOS. This association is independent of OPA1, appears to be mediated via the MIC60 MICOS …

The Role Of Conformational Dynamics In Isocyanide Hydratase Catalysis, Medhanjali Dasgupta

Department of Biochemistry: Dissertations, Theses, and Student Research

Post-translational modification of cysteine residues can regulate protein function and is essential for catalysis by cysteine-dependent enzymes. Covalent modifications neutralize charge on the reactive cysteine thiolate anion and thus alter the active site electrostatic environment. Although a vast number of enzymes rely on cysteine modification for function, precisely how altered structural and electrostatic states of cysteine affect protein dynamics, which in turn, affects catalysis, remains poorly understood.

Here we use X-ray crystallography, computer simulations, site directed mutagenesis and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in the enzyme, isocyanide hydratase (ICH), affects the protein …

Xenobiotic Exposure Requires Mitochondrial Metabolism For Redox Homeostasis And Survival In Astrocytes, Jordan Rose

Department of Biochemistry: Dissertations, Theses, and Student Research

Astrocytes are integral components of glutamatergic neurotransmission, providing essential metabolic processes for neuronal homeostasis and acting as the first line of defense against xenobiotics crossing the blood brain barrier. Arsenic is a xenobiotic with widespread natural and industrial prevalence, and has been linked to impaired neurodevelopment and neuronal death. Given the integrated metabolic nature of astrocytes and neurons, we sought to explore how arsenic impacts astrocyte metabolism in order to better understand the mechanisms of xenobiotic toxicity in the mammalian brain.

We demonstrate that astrocyte viability depends upon de novoglutathione (GSH) synthesis during arsenic exposure, and sub-lethal arsenic exposure …

Transcriptomic Analyses And Computational Modeling Reveal That Fatty Acid Transport Protein 2 (Fatp2) Impacts The Transcriptional Activity Of Pparα Thus Altering The Lipid Metabolic Landscape, Vincent M. Perez

Department of Biochemistry: Dissertations, Theses, and Student Research

Fatty acid transport protein 2 (FATP2) is highly expressed in liver, small intestine, and kidney where it functions in both the uptake of exogenous long chain fatty acids (LCFAs) and in the activation of very long chain fatty acids (VLCFAs). Here we address the phenotypic impacts of deleting FATP2 with the following three separate approaches: [1] Utilizing an unbiased next-generation sequencing analysis of FATP2-null (fatp2-/-) mice fed a standard chow diet; [2] Utilizing an unbiased next-generation sequencing analysis of fatp2-null (fatp2-/-) mice fed a high-fat ketogenic diet (KD) and fasted for 24-hours [3] Building dynamic computer models built with data …

Defining The Roles Of Serine Palmitoyltransferase-Interacting Proteins In The Regulation Of Sphingolipid Homeostasis, Athen N. Kimberlin

Department of Biochemistry: Dissertations, Theses, and Student Research

Sphingolipids are major structural components of the plasma membrane and endomembrane system. Research suggests that sphingolipids are involved with the formation of lipid microdomains, also known as lipid rafts, which may help to organize proteins within the membrane and may be important for membrane trafficking. Aside from their structural roles in membranes, sphingolipids and their metabolic products have been implicated in several cellular signaling responses like programmed cell death (PCD). Because of this, maintenance of sphingolipid homeostasis is critical for eukaryotic cell growth and development. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is the primary regulatory …

Reactive Oxygen Species Homeostasis And Proline Catabolism, Lu Zhang

Department of Biochemistry: Dissertations, Theses, and Student Research

The role of proline metabolism in regulating cellular redox status was first proposed three decades ago. Proline catabolism was then later found to induce programmed cell death and cell apoptosis by regulating ROS signaling. Proline oxidation was also found to promote cell survival under oxidative stress. Proline catabolism-mediated reactive oxygen species (ROS) were suggested to be involved in both cases by serving as a regulatory signal. In this work, the sources of proline oxidation-induced ROS production were explored in both bacteria and animal cells. Proline oxidation-induced ROS was found to be shared by bacteria (Escherichia coli) and animals …

Investigation Of Pathways For Complex Sphingolipid Biosynthesis In Arabidopsis Thaliana (L.) Heynh, Kyle Luttgeharm

Department of Biochemistry: Dissertations, Theses, and Student Research

Sphingolipids are essential components of eukaryote membranes. The ceramide backbone of complex sphingolipids is composed of an 18 carbon Long Chain Base (LCB) bound to a 16-26 carbon fatty acid (FA) through an amide linkage. Ceramides are synthesized de novo from a free LCB and fatty acyl coA by ceramide synthase (sphingosine N-acyl transferase, EC 2.3.1.24) which can be inhibited by the fungal mycotoxin Fumonisin B₁. Arabidopsis thaliana contains three ceramide synthases denoted LOH1, LOH2, and LOH3 that have previously been hypothesized to have unique substrate preferences that control the final sphingolipid composition, different susceptibilities to …

Characterization Of Inhibitors Of Fatty Acid Transport Protein-2 In Cell And Animal Models, Nipun Saini

Department of Biochemistry: Dissertations, Theses, and Student Research

Obesity is correlated with insulin resistance and elevated levels of glucose, triglycerides and free fatty acids in blood. This affects overall metabolism and leads to disease. In the obese state, fat also accumulates in non-adipose tissue including liver, muscle and pancreas, where it can lead to cellular dysfunction and death. Currently, only a limited number of drugs are available to combat obesity and it is clear that new drugs, which more narrowly target the metabolic pathways involved, are required. Fatty Acid Transport Proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme …

Functional Genomic Analyses Of Switchgrass Developmental Processes, Nathan Palmer

Department of Biochemistry: Dissertations, Theses, and Student Research

Switchgrass (Panicum virgatum L.), a C_4-perennial grass species, is being developed as a bioenergy crop. Although much is known from a breeding perspective, there is limited information on the functional genomics of this crop, specifically regarding molecular mechanisms controlling aerial senescence, winter dormancy, and traits that confer winter hardiness. Using functional genomics to generate a transcriptional roadmap underpinning senescence and winter dormancy will provide researchers with a molecular understanding that can be applied to improve switchgrass germplasm.

In an initial study, a de novo assembly of the crown and rhizome transcriptome from an upland cultivar Summer was …

Er-Associated Degradation And Cadmium Dependent Rescue Of Pca1, Nathan Smith

Department of Biochemistry: Dissertations, Theses, and Student Research

Protein synthesis and proper folding is an essential process for all organisms. In eukaryotes proteins of the secretory pathway are synthesized and inserted into the lumen or membrane of the endoplasmic reticulum. Eukaryotic cells maintain a mechanism for removal of proteins unable to fold properly. This process is known as ER-associated degradation (ERAD). A poorly functioning ERAD can lead to a build-up of misfolded proteins which has been implicated in several degenerative diseases such as Alzheimer’s, Amyotrophic lateral sclerosis, and Parkinson’s. Thus, the study of how proteins are recognized, extracted from the ER, and degraded is essential for determining methods …

Single Tube, Multiple Enzyme Reaction For Detection Of Uv And Oxidative Damage In Forensic Physiological Stains, Nicholas J. Eurek

Department of Biochemistry: Dissertations, Theses, and Student Research

For decades, the use of DNA as a biological tool has revolutionized forensic investigations. The primary use of this genetic evidence is for identification of a victim or suspect through short tandem repeat (STR) profiling. However, the usefulness of this evidence can be compromised through inhibition of PCR, damage to the DNA, or low copy number. Here, we investigate damage induced to DNA by environmental factors. UV light is known to damage DNA by the formation of cyclobutane pyrimidine dimers, 6-4 photoproducts, and strand breaks. These lesions can stall polymerase action or misincorporate bases during extension. Oxidative damage is also …

Redox Regulation Of Protein Translation In Eukaryotes, Maxim Gerashchenko

Department of Biochemistry: Dissertations, Theses, and Student Research

Gene expression may be controlled at multiple levels, e.g., through genomic architecture, transcription and translation. In the current work, we focused on regulation of protein synthesis. Historically, the investigation of the regulation of gene expression at the level of translation lagged behind the transcriptional control because of the lack of accessible high-throughput methods. Our research has begun with the finding of the use of alternative non-AUG start codon in thioredoxin-glutathione reductase (TGR), a selenoprotein involved in redox control during male reproduction. The use of this codon, CUG, relies on the Kozak consensus sequence and ribosomal scanning mechanism. However, the CUG …

Making Chlamydomonas Reinhardtii A Better Model Organism: Tackling The Inefficiency Of Nuclear Transgene Expression And Improving Methods For The Generation And Characterization Of Insertional Mutant Libraries, Thomas M. Plucinak

Department of Biochemistry: Dissertations, Theses, and Student Research

The green algal species Chlamydomonas reinhardtii possesses many beneficial features that have made it a useful model organism for many decades. Many types of experimentation however are difficult to conduct with this organism due to the relative under-development of genetic tools available for use. Tasks such as transgene expression, overexpression of proteins of interest (POIs) or site specific genomic modification that are routine in other more facile microbial model organisms such as Escherichia coli and yeast are difficult to accomplish in C. reinhardtii. The second chapter of this thesis describes the development of a novel nuclear transgene expression system …

Transcriptomic Analyses Of The Co2-Concentrating Mechanisms And Development Of Molecular Tools For Chlamydomonas Reinhardtii, Andrew J. Brueggeman

Department of Biochemistry: Dissertations, Theses, and Student Research

Microalgae, such as Chlamydomonas reinhardtii, account for a large percentage of photosynthesis that occurs on the planet. Many algae possess a Carbon-Concentrating Mechanism, or CCM, that actively transports inorganic carbon (C_i) into the cell to create artificially high internal levels of CO₂, enhancing their rate of carbon fixation. The production of biofuels from algal sources can serve as both a renewable and carbon-neutral energy source. This thesis details research in Chlamydomonas, in the effort to both better understand the CCM in algae and improve laboratory and industrial manipulations with algae.

In the first chapter of this …

Investigation Of Proline Utilization A: Kinetic Analysis Of Substrate Channel-Blocking Mutants And Creation Of A Trifunctional Chimera Enzyme, Benjamin W. Arentson

Department of Biochemistry: Dissertations, Theses, and Student Research

Proline metabolism is known to be involved in many cellular processes such as cell signaling, cellular redox balance, and cell survival. One of the enzymes involved in proline catabolism, proline utilization A, plays a role in oxidizing proline to glutamate in a two-step oxidation pathway involving enzymes proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH).

Intermediate P5C/GSA has been shown to use an intramolecular channel to move from the PRODH active site to the P5CDH active site in a phenomenon called substrate channeling. In this work, one of the main objectives was to learn more about the channel usage. …

Investigations Of Substrate Channeling In The Proline Oxidative Pathway, Nikhilesh Sanyal

Department of Biochemistry: Dissertations, Theses, and Student Research

In cell metabolism, substrate channeling is a phenomenon where the product of one reaction is transported to a second enzyme active site without equilibrating into bulk solvent. Chapter 1 reviews the rationale and evidence for substrate channeling with the specific example of proline metabolism. Oxidation of proline to glutamate is catalyzed in consecutive reactions by proline dehydrogenase (PRODH) and pyrroline-5-carboxylate dehydrogenase (P5CDH). The intermediate Δ¹-pyrroline-5-carboxylate reportedly tends to be labile and inhibitory towards several metabolic pathways.

One of the main objectives of this dissertation was to investigate substrate channeling between independent proline oxidative enzymes from Thermus thermophilus- …

Sinusoidal Endothelial Dysfunction In Non-Alcoholic Fatty Liver Disease., Sandhya Lakshmi Gopalakrishnan

Department of Biochemistry: Dissertations, Theses, and Student Research

Non-alcoholic fatty liver disease (NAFLD) is an asymptomatic increasingly common disorder that affects liver metabolism and is often the precursor for liver pathologies such as fibrosis, cirrhosis and hepato-cellular carcinoma. The liver sinusoidal endothelial cells act as a liver sieve by allowing macromolecules and chylomicrons to traverse through their fenestrations (sieve plates) to hepatocytes. Since liver sinusoidal endothelial cells (LSEC) regulate serum derived macromolecular exposure to hepatocytes, we asked what role LSEC could play in the pathogenesis of NAFLD. To investigate the early events of NAFLD we used a rat model (Sprague-Dawley) in which animals were maintained on standard and …

Phylogenetic Engineering Of The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large Subunit In Chlamydomonas Reinhardtii, Boon Hoe Lim

Department of Biochemistry: Dissertations, Theses, and Student Research

Thirty-four residues in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) may account for the kinetic differences between Rubisco enzyme from green algae and land plants. By substituting these "phylogenetic residues" as groups and combinations of groups in the large subunit of the green alga Chlamydomonas reinhardtii with those of land-plant Rubisco, the functions and relationships of these "phylogenetic groups" were determined.

A phylogenetic-group substitution at the base of catalytic loop 6 of the large subunit decreases the CO₂/O₂ specificity of the enzyme, but function is restored by a further phylogenetic-group substitution at the carboxy-terminal tail. Therefore, these …

Developing A High Throughput Protocol For Using Soil Molecular Biology As Trace Evidence, Sabreena A. Larson

Department of Biochemistry: Dissertations, Theses, and Student Research

The use of soil as trace evidence has changed significantly with the addition of new techniques. These techniques include using the biochemical molecules from soil microbial communities to make a fingerprint of the specific soil. This research examines the changes to the microbial community profile that take place during storage of a soil sample. To observe such changes both the DNA and fatty acid profiles will be examined.

The DNA profiles were made with capillary electrophoresis-single stranded conformation polymorphism (CE-SSCP). After statistical analysis using Bray-Curtis distances and ANOSIM (analysis of similarity) it was shown that storage of soil does not …

Studies On The Small Ubiquitin-Like Modifier (Sumo) E2 Conjugases Of The Sumoylation System In Chlamydomonas Reinhardtii And Their Role In Stress Physiology, Amy R. Knobbe

Department of Biochemistry: Dissertations, Theses, and Student Research

The eukaryotic protein post-translational modification by SUMOylation is involved in a diverse array of cellular processes, including various stress responses. A fully functional SUMOylation system is present in the unicellular green alga Chlamydomonas reinhardtii, and SUMOylation of multiple high molecular weight proteins is induced in response to abiotic stress in this organism. We report here the characterization of a SUMO E2 conjugase deletion mutant in C. reinhardtii, mut5. SUMO E2 conjugase enzymes are responsible for the conjugation of the protein SUMO to a lysine residue within a target protein. C. reinhardtii mutants in which the SUMO E2 …

The Crosstalk Between Human Fatty Acid Transport Protein 1 And Fatty Acid Transport Protein 4, Zhe Yuan

Department of Biochemistry: Dissertations, Theses, and Student Research

Fatty acid transport proteins (FATPs) provide pivotal roles in fatty acid transport and activation and thus are crucial for overall fatty acid homeostasis. Peroxisome Proliferators Activated Receptors (PPARs) are important transcription factors, which control many genes that govern lipid metabolism.

Using 293 T-REx cell lines that stably express FATP1 or FATP4 from a tetracycline-inducible promoter, this work evaluated gene expression of key genes involved in fatty acid metabolism using QPCR, protein expression of FATP1 and FATP4 using Western blots, and fatty acid transport to address the roles of these two FATP isoforms in fatty acid homeostasis. This work found that …

Functional Studies Of Human Cellular Detoxification Enzymes, Melanie Neely Willis

Department of Biochemistry: Dissertations, Theses, and Student Research

Cellular detoxification allows for the maintenance of cellular homeostasis and prevention of abnormal cell growth by clearing harmful xenobiotics and endobiotics. After oxygenation by phase I enzymes, phase II enzymes such as glucuronosyltransferases and glutathione-s-transferases conjugate a small molecule to the compound, marking it for subsequent export. Many up-stream enzymes are also essential to cellular detoxification by supplying the small compounds for conjugation. These up-stream enzymes include UDP-glucose dehydrogenase, which synthesizes UDP-glucuronate, and glutamate cysteine ligase, which catalyzes the first and rate-limiting step in the synthesis of glutathione.
UDP-glucose dehydrogenase (UGDH) is an important enzyme in human development and in …

The In Situ Function Of A Microbial Community Profiled By Ft-Ir: A Snapshot In Time, Ryan Roberts

Department of Biochemistry: Dissertations, Theses, and Student Research

Photographs of an ecosystem are an important tool in macro-community ecology. The photograph is a permanent record of species phenotype. In microbiology, biochemical activity provides the most descriptive information of an organism’s phenotype. A method for fingerprinting all biochemical activities occurring within a microbial community is analogous to a photograph. Infrared spectroscopy in the region between wavelengths 2500 to 20,000 nm (mid-IR) is a well established instrumental method for fingerprinting the total biochemical profile of axenic cultures. Spectra are complex and sensitive as demonstrated by the ability to discriminate between strains of bacteria, fungi, and algae. This thesis develops the …