Biochemistry, Biophysics, and Structural Biology Commons^™

Engineering And Characterizing Proteins: A Dual Study On Canine P53 Protein And Cyan Thermal Protein, Dhruv B. Sitapara

MSU Graduate Theses

This research aims to explore protein development and characterization, focusing on a dual study of canine p53 protein and cyan thermal protein. For the canine p53 protein, the goal is to comprehend its structure to better understand a key component of canine cancers. This multi-step process involved expressing the canine p53 DNA-binding domain in E. coli and purifying it through affinity and ion-exchange chromatography. The purified protein was studied to test the binding of canine p53 protein to human DNA sequences using Surface Plasmon Resonance (SPR) experiments and an Electrophoretic Mobility Shift Assay (EMSA), visualized with a SYBR-safe stain. The …

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-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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …