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Impact Of Sample Conditions On Dna Phosphodiester Backbone Bi/Bii Conformational Equilibrium Dynamics, Autumn C. Pilarski

MSU Graduate Theses

DNA damage, such as single base lesions and mismatches, is highly prevalent within cells. If these DNA damage events are not repaired, they could lead to mutations and thus disease and cancer. Intricate repair mechanisms are in place to fix these damage events, one such being Base Excision Repair (BER) and associated enzyme: Thymine DNA Glycosylase (TDG). The first step of this repair process, recognition of the lesion by TDG, is not well understood. The following thesis presents results to better understand the fundamental biophysical question of how a DNA lesion within a mismatch context is recognized in a million …

Tissue And Sex-Dependent Regulation Of Innate Immunity And Rna Editing In Mice, Kelsey R. Kendrick

MSU Graduate Theses

Inflammation occurs as a result of insult or infection within the body. Individual cells respond to inflammation by upregulating genes that help mediate the immune response, such as ADAR1. ADAR1 helps regulate the immune response but also catalyzes a process called RNA editing. RNA editing alters the sequence of select mRNAs to alter the encoded proteins. The result is altered function of the encoded protein, which is often beneficial for the cell. Our goal was to determine how inflammation affects the function of ADAR1. Since we know that the effects of inflammation vary between different organs and sexes, we examined …

Sex-Dependent Effects Of Induced Acute Inflammation On Glucose Homeostasis And Rna Editing Enzymes, Christian A. Rivas

MSU Graduate Theses

The first line of defense against bodily insults, like pathogen invasion, is the innate immune system. Innate immunity sets in motion countless cascades that result in inflammation. Inflammation simultaneously affects multiple biological processes like metabolism and gene expression. Males and females react differently to inflammation. To understand both molecular and physiological sex differences in inflammation, we examined how inflammation affects gene expression and glucose metabolism. Adenosine deaminase acting on RNA (ADAR1) is upregulated by inflammation and catalyzes RNA editing, a process where nucleotides encoded by the genome are modified. ADAR1 also controls the innate immune reaction by decreasing activity of …

Tissue And Sex-Specific Rna Editing During Induced Acute Inflammation, Claire E. Nichols

MSU Graduate Theses

A-to-I RNA editing is a process where select adenosine (A) nucleotides are deaminated by an editing enzyme, ADAR, to become inosines (I) in RNA transcripts. RNA editing can affect the sequence of the encoded protein and the regulation of the RNA. ADAR1 also plays a role in regulating innate immunity and its expression is upregulated during inflammation. Current data on the effects of increasing ADAR1 on RNA editing is limited, and most studies are completed only in male animals. We are interested in expanding RNA editing data to include female animals. Lipopolysaccharide (LPS) was used to induce acute inflammation and …

Further Investigation Of The Initiating Mechanism Of The Type I Collagen Glomerulopathy, Matthew James Freese

MSU Graduate Theses

The progressive accumulation of collagen and other extracellular matrix proteins in the renal mesangium results in fibrosis, glomerulosclerosis, and eventual renal failure. Mice deficient in integrating α2(I) collagen into the type I collagen structure, termed Col1a2-deficient mice, model kidney fibrosis through the condition Type I Collagen Glomerulopathy, because homotrimeric type I collagen accumulates extracellularly in the mesangium of renal glomeruli. Accumulation of homotrimeric type I collagen compresses blood vessels in glomeruli, which reduces filtration, increases pressure, and results in fibrosis. Picrosirius red (PSR) staining was used on Col1a2 deficient and wildtype mice to evaluate collagen deposition. Histological evaluation and …

A Study Of Cobalt (Iii) Oxide Nanoparticle Delivery Of Sirna Molecules Directed Against Signaling Intermediates Of The P2y2 Receptor, Rachel Blair Stroud

MSU Graduate Theses

G protein-coupled receptors are evolutionarily ubiquitous sensors of extracellular signals, propagating intracellular signal cascades through heterotrimeric G proteins. P2Y₂ receptors are GPCRs which are activated by extracellular nucleotides to mediate signaling cascades via Gαq coupling. Many GPCRs are subject to a common mechanism for signal termination involving phosphorylation of the C-terminal tail followed by β-arrestin binding and subsequent endocytic internalization of the complex. This effect has been described for the P2Y₂ R in the 1321N1 astrocytoma cell line, and UTP-induced activation and desensitization profiles have been previously defined. There is need to develop molecular vehicles for safe and …

Expression And Purification Of Cytochrome 2u1 And Canine 1a2 And Metabolism Of Caffeine By Cytochrome 1a2, Brenda M. Wekesa

MSU Graduate Theses

Cytochrome P450 enzymes are a large family of membrane heme proteins involved in eliminating drugs and the synthesis or elimination of steroids, eicosanoids, and vitamins. CYP2U1 is one of the poorly characterized P450 enzymes classified as orphan cytochrome P450 with unknown exact biological function. It is expressed in the brain and thymus as a catalyst in the hydroxylation of arachidonic acid. Unlike other cytochrome P450s, CYP2U1 is a bit unusual with forty extra amino acids. If a mutation occurs, it can lead to an early onset inherited disorder of the central nervous system called Hereditary Spastic Paraplegias (HSP). Most of …

Modulation Of Glucose Homeostasis By Nucleotide P2y2 Receptor And Biological Sex, Hailee Anne Marino

MSU Graduate Theses

Recent insights into the pathological role of Nucleotide P2Y₂ receptor suggest P2Y₂R involvement in high fat diet-induced obesity and potentiates insulin resistance. However, these recent insights do not demonstrate how P2Y₂R modulates glucose homeostasis under physiological conditions. Further, it remains unknown how sex biological factors influence P2Y₂R receptor signaling in the regulation of glucose homeostasis. The research objective for the present study is to elucidate the novel roles of P2Y₂ in fasting blood glucose and glucose tolerance (basal insulin sensitivity) under resting conditions in males and females. We expected that under physiological …

Stimuli Responsive Dye-Containing Peg-Pla Block Copolymer Micelles And Computationally Assisted Design Of A Stapled Peptide Bundle, Tyler L. Odom

MSU Graduate Theses

In this thesis, I report the preparation and characterization of dye-containing PEG-b-PLA block copolymer micelles and the computational design of a novel coiled-coil peptide bundle. The PEG-b-PLA micelles encapsulate hydrophobic molecules into their core and have strong potential as nanocontainers or delivery vesicles. In theory, these internalized molecules can be released upon exposure to mechanical forces that disrupt the micellar structures. This force-responsive nature is one of the inherent properties of micellar systems. However, there is a stark lack of research that utilize this property in applications. Along those lines, I have studied the behavior of …

Nucleotide P2y₂ Receptor-Dependent Leukocyte-Endothelial Interaction, Spencer E. Thomas

MSU Graduate Theses

Extracellular nucleotides (ATP, UTP) released from cells act on nucleotide receptors to promote vascular inflammation. Increased leukocyte-endothelial interaction is a hallmark of vascular inflammation. The nucleotide P2Y₂ receptor (P2Y₂R), activated by extracellular ATP≈UTP, plays a role in cardiovascular homeostasis and immune regulation. Moreover, accumulating evidence from studies in vitro and in vivo models have implicated the P2Y₂R in the inflammatory response significantly contributing to the progression and pathogenesis of asthma, atherosclerosis, sepsis, and ischemia. I hypothesized that P2Y₂R activation by UTP, an agonist of the receptor, increased leukocyte rolling and adhesion in the microvasculature from baseline. To test the hypothesis, …

Neuronal Migration In Developmental Hyperserotonmia: Assessment Of Vesicular Glutamate In The Raphe Nuclei, Trey M. Shupp

MSU Graduate Theses

The neurotransmitter serotonin is involved in the early development of the central nervous system and the organization of neurons throughout the cerebral cortex and cerebellum. It is proposed that serotonin indirectly interacts with cells in the marginal zone of the cerebral cortex known as Cajal-Retizus (CR) cells. These cells secrete the extracellular matrix protein reelin, which is known for its role in neuronal organization and migration during early neural development. It has been observed that low levels of serotonin are associated with similarly low levels of reelin during development and have been reported to result in disorganization of neurons in …

Predictors Of Student Success In An Upper Division Agriculture Chemistry Class, Maddison V. May

MSU Graduate Theses

To attempt to find what made students successful within a higher division class at Missouri State University’s (MSU) Darr College of Agriculture, de-identified academic records of 172 students enrolled in AGR 300 - Food and Agriculture Chemistry were studied over four semesters. Deidentified data were collected and compared to final letter grade received at the end of the class using correlation coefficients, analysis of variance, and stepwise regression. Analysis of variance was used to evaluate the influence of semester on letter grade. Correlation coefficients were used to determine and evaluate relationships between the variables and final grade. Stepwise regression was …

Determining A Method For Expressing And Purifying Cytochrome P450 4v2: A Protein Involved In Bietti's Crystalline Dystrophy, Cody Lane Turner

MSU Graduate Theses

Within the Cytochrome P450 class of enzymes, there are a group known as the “orphan” cytochromes. The “orphan” classification comes from the poorly understood in vivo functionality and substrate specificity. Cytochrome P450 4V2 (CYP4V2) is one of these “orphans” and belongs to the CYP4 family. The CYP4 family is known for the omega oxidation of endogenous fatty acids. This family is most commonly found on chromosome 1 (CYP4ABXZ). CYP4V2 is unique in that its location is bound to chromosome 4 as discovered by Jiao in 2004. Mutations within the CYP4V2 gene have been associated with the …

The Role Of Rna Editing Within Calcium-Dependent Activator For Secretion 1 In Large Dense Core Vesicle Fusion, Kaylee M. Mathiason

MSU Graduate Theses

Calcium-dependent activator protein for secretion 1 (CAPS1) is involved in synaptic vesicle and large dense core vesicle fusion by bridging vesicles and the plasma membrane prior to vesicle fusion and cargo release. CAPS1 is subject to a post-transcriptional modification called RNA editing. RNA editing recodes a single amino acid in the region of the protein that interacts with the vesicle. This recoding event within CAPS1 has cellular consequences in neurons where edited CAPS1 overexpression increases basal secretion from neural synaptic vesicles. Our goal was to investigate how RNA editing within CAPS1 affects large dense core vesicle secretion in an insulinoma …

Multifaceted Mechanism Of Vps1 Mediated Endosome-To-Golgi Fusion In Vitro, Ehsan Suez

MSU Graduate Theses

To maintain cell homeostasis, protein recycling through intracellular membrane fusion is an important cellular process. Both Vps1 and Ypt6 have been implicated in protein recycling from the endosome to the trans-Golgi Network (TGN). SNARE proteins are thought to be the key regulator in this membrane fusion mediated protein recycling mechanism. I studied membrane fusion events by incorporating purified proteins into liposomes. A series of data suggest that high concentration of SNARE proteins inhibits fusion unlike the opposite popular notion. Also, the data suggests that Vps1 acts on membrane fusion dynamics in a manner that lower concentrations of Vps1 enhance …

Application Of Carbon Nanoparticles As Dna Detection Probe And Fluorescent Ink, Luckio Frank Owuocha

MSU Graduate Theses

There is a significant interest in developing a sensitive, selective, efficient, and inexpensive method for rapid molecular diagnostic tests. This research aims to develop an inexpensive nucleic acid detection method by using DNA-conjugated carbon nanoparticles that exhibit fluorescence in the visible region. Carbon nanoparticles of this class can be detected without specialized equipment and have great promise toward the development of analytical methods that can be used in resource-limited environments with a lack of access to proper diagnostic and healthcare. We employed EDC-NHS (two-step) and EDC (one-step) coupling techniques to prepare DNA-conjugated carbon nanoparticles. The dot blotting method was adapted …

Investigating Chitosan Modified With Triethylammonium Butanamide And Triethylphosphonium Butanamide As Non-Viral Gene Delivery Vectors By Examining Cytotoxicity And Transfection Efficiency, Deborah C. Ehie

MSU Graduate Theses

Gene therapy is a very challenging field, especially with new emerging genetic disorders. Chitosan (CS), due to chitosan’s flexibility, biocompatibility, and biodegradability, has been of interest in the world of gene therapy especially as researchers are gravitating towards non-viral vectors due to the problems caused by viral vectors. Nevertheless, there are still issues regarding solubility, cellular uptake of cargos being transported in vitro or in vivo, increased cytotoxicity levels, as well as many other things that prevent chitosan from being an efficient gene delivery agent. Here I present five derivatives of chitosan, which were all modified with either triethylphosphonium …

Novel Cyanoximates As An Alternative In Cancer Chemotherapy, Kafayat Aderonke Yusuf

MSU Graduate Theses

Chemotherapy is one of the most effective treatment plans for several cancer types. The recurrent side effects derived from chemotherapy agents have warranted the search for novel chemical compounds with better efficacy and minimal side effects. In line with this idea, I investigated effects of a group of newly synthesized metal based chemical compounds called cyanoximates on HeLa human cancer cells. Cyanoximates used were Pt(DECO)₂, Pt(MCO)₂, and Pd(DECO)₂ along with the chemotherapy drug cisplatin as a positive control. I found that the metal cyanoximates reduced cell viability via apoptosis, and that Pt(DECO)₂ was most …

Myosin V-Mediated Cargo Traffic Toward The Trans-Golgi Network And Myosin V Implications In Snc1 Exocytosis, Vy Ngoc Khanh Nguyen

MSU Graduate Theses

Retrieval of cargo proteins from the endosome towards the trans-Golgi network (TGN) is a crucial intracellular process for cellular homeostasis. Its dysfunction is associated with pathogenesis of Alzheimer and Parkinson's diseases. Myosin family proteins are cellular motors walking along actin filaments by utilizing the chemical energy from ATP hydrolysis, known to involve in pleiotropic cellular trafficking pathways. However, the question of whether myosins play a role in the trafficking of Snc1 and Vps10 has not been addressed yet. The present study assesses the potential roles of all five yeast myosins in the recycling of two membrane cargo, Snc1 and …

Effects Of The Dihydrouracil Lesion On Dna Using 1h/31p 1d And 2d Solution Nmr, Benjamin M. Boyd

MSU Graduate Theses

The effects of the dihydrouracil lesion in DNA were studied using two dimensional NMR spectroscopy. The sequence used was based off of the Drew-Dickerson Dodecamer, with the cytosine in the three position replaced by a dihydrouracil. All of the nonexchangeable proton chemical shifts, with the exception of the H2, H5’, and H5’’, of the lesioned DNA were identified using NOESY spectra and then compared to the chemical shift values of the Drew Dickerson Dodecamer. The largest differences in chemical shifts were observed in the nucleotides neighboring the lesion, both within the strand and on the opposite strand. The imino exchangeable …

The Dynamin-Like Protein Vps1 Stimulates Endosome-To-Golgi Fusion In Vitro, Jared Christopher Smothers

MSU Graduate Theses

Intracellular membrane fusion events can be reconstituted by exploiting isolated organelles from cellular hosts or artificial membranes made of purified phospholipid components. Artificial construction of membranes provides two significant advantages. First, cellular isolation of the endosome-derived vesicles and TGN (trans-Golgi Network) compartments needed for the fusion assay would be extremely challenging. Second, reconstituting the membranes provides the added benefit of controlling size and lipid compositions to functionally mimic the individual membrane architectures and introduce only the purified proteins that are under investigation. For these reasons, I have developed the first simultaneous lipid and content mixing fusion assays that measures the …

Synthesis, Stabilization, And Modification Of Zinc Oxide Nanoparticles For Biological Applications, Allison Kimberly Freese

MSU Graduate Theses

Nanoparticles have become very useful as delivery systems in biomedicine. The nanoparticles can be layered with different compounds to produce a vessel for transport of biological materials. Specifically, gold nanoparticles layered with a reducing agent, lysozyme, and polyelectrolytes can be synthesized to transport lysozyme into a cell. However, zinc oxide nanoparticles are cheaper, biocompatible nanoparticles that can be used for the same process. Here in, zinc oxide nanoparticle conjugates were synthesized, modified, and analyzed to be used as a biological material delivery system. The zinc oxide nanoparticles were synthesized using zinc chloride and sodium hydroxide. The particles were then layered …

Further Characterization Of The Skeletal Phenotype In A Hurler Syndrome Mouse Model And The Ethical Treatment Of Children In Medicine, Anna Marie Mcwoods

MSU Graduate Theses

Mucopolysaccharidosis type I (MPS I) is a rare, autosomal recessive disorder caused by the deficiency of the lysosomal enzyme α-L-iduronidase (IDUA). Absence of IDUA results in the accumulation of dermatan and heparin sulfate and ultimately causes multi-system dysfunction. The most severe form of MPS I is Hurlers syndrome, a rapidly progressive disorder that, if left untreated, is fatal. Current treatment options for diagnosed individuals includes hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). These treatments are able to ameliorate the majority of symptoms with the exception of the bone phenotype. This investigation aimed to further characterize the bone …

Kinetics Of Hiv-1 Uncoating In C20 Microglial Cells, Melanie Anne Taylor

MSU Graduate Theses

Uncoating is a poorly understood yet required step of HIV-1 replication that is defined as the disassembly of the viral capsid structure. The goal of this project is to characterize uncoating in C20 microglial cells. These cells are a natural target of HIV-1 that are infected to establish latent viral reservoirs and HIV-associated neurological disorders. A stable C20 cell line that expresses TRIM-CypA was established to study the kinetics of uncoating with the CsA washout assay. The expression of TRIM-CypA was confirmed by western blot and the functionality of the protein was confirmed by a viral infectivity assay. Using this …

Development Of Rapid, Homogeneous Assay For Investigating Isopeptide Bond Formation Using Fluorescence Polarization/Depolarization Measurements, Samuel Patricc Kasson

MSU Graduate Theses

Autocatalytic intramolecular isopeptide bonds have been found in nature in certain gram-positive bacterial pilus structures. Recently, splitting of these domains that are capable of autocatalytic intramolecular isopeptide bond formation have been applied to create stable, selective, bio-orthogonal Catcher/Tag systems. The CnaB2 domain found in the FbaB pilus structure of Streptococcus pyogenes, has yielded the Catcher/Tag, Protein/Peptide systems termed SpyCatchter and SpyTag. Recent study has focused on tag optimization, stability and bio-orthogonality evaluation, along with applications in bioconjugation. I have recombinantly expressed SpyCatcher and SpyTag-fused proteins in E.coli, and conjugated them to fluorescent probes in order for use in fluorescence polarization/depolarization …

The Role Of Rad4 In Dna Repair And Its Interplay With Telomeres In Tetrahymena Thermophila, Emily Nischwitz

MSU Graduate Theses

Telomeres are repetitive parts of the genome that act as a protective end cap to the chromosomes. Telomeres are critical to the integrity and stability of the genome, therefore, ensuring that their sequence is maintained, even after damage, is crucial. Much of the pioneering work responsible for explaining telomeres has been conducted in ciliates, specifically in Tetrahymena thermophila. Telomeres in T. thermophila have a high amount of tandem thymine repeats (GGGGTT) and, thus, are susceptible to ultraviolet light (UV) induced lesions called pyrimidine dimers, which must be repaired by nucleotide excision repair (NER). In humans, Xeroderma Pigmentosum C (XPC) …

Sodium Pyruvate Alters The Immune Response To Influenza A Virus Infection In Macrophages, Hazzar Abysalamah

MSU Graduate Theses

ABSTRACT

Pyruvate is the end product of glycolysis. It can either be transported into the mitochondria for use in the TCA cycle or be used to regenerate NAD⁺ during fermentation or aerobic glycolysis (also called the Warburg Effect). I recently discovered that addition of sodium pyruvate to the culture medium during infection of macrophages with influenza A virus affects the production of cytokines involved in immune signaling. While infection of macrophages with influenza A virus resulted in high levels of cytokines (IL-6, IL-1β, and TNF-α) in the absence of sodium pyruvate, the addition of sodium pyruvate significantly impaired cytokine …

Investigation Of The Homologs Rad51 And Dmc1 Role In Cell Division And Homologous Recombination, Amaal Abulibdeh

MSU Graduate Theses

RecA-like proteins homologs Rad51 and Dmc1 (disruption of meiotic control) promote recombination between homologous chromosomes by repairing programmed DNA Double-Strand Breaks (DSBs). Dmc1 is a Recombinase involved in meiosis-specific repair of DSBs, whereas Rad51 has been found to be involved in meiotic and non-meiotic DSBs repair. Previous studies showed that when RAD51 is overexpressed, interhomologous recombination still occurs even when DMC1 is knocked out. Dmc1 and Rad51 have not been fully characterized in the ciliate Tetrahymena thermophila. In order to more fully investigate the role of Rad51 and Dmc1 in Homologous Recombination Repair (HHR), this work focuses on using …

Development Of Endogenous Tagging Plasmids For Characterization Of Protein Interactions, Localization, And Post-Translational Modifications Of Tetrahymena Thermophila Rad23, Evan Andrew Wilson

MSU Graduate Theses

Rad23 is a protein involved in both nucleotide excision repair (NER) and proteasome-mediated degradation, and has been suggested to facilitate interactions between these two pathways. The model organism Tetrahymena thermophila, which has a transcriptionally silent micronucleus, provides a useful platform for studying the role of Rad23 in global genome NER (GG-NER). However, the ectopic expression systems used thus far in T. thermophila to study Rad23 are repressed by UV light and do not account for the background expression of endogenous RAD23; these phenomena prevent insightful gains to the true dynamics of Rad23. In this thesis, endogenous tagging …

Functionalization Of Indium-Based Quantum Dots For Use As A Non-Viral Gene Therapy Vector, Nicholas A. Mundt

MSU Graduate Theses

This work aims to develop functionalized, water-soluble indium-based quantum dots (QDs) as a non-viral gene therapy vector. The QDs were solubilized in water by exchanging native hydrophobic surface ligands with 11-mercaptoundecanioc acid (MUA); an amphiphilic ligand providing terminal carboxylate groups that impart water solubility to the QDs. The aqueous QDs were then functionalized with a terminal tertiary amine to impart a positive surface charge, allowing negatively-charged DNA to complex with the nanoparticles. The QDs were characterized via electrophoresis to determine their ability to bind DNA. Results show that further work is needed to optimize DNA binding. In addition, this work …