Investigations Of Protein-Lipid Interactions In Model Membranes: Influence Of Aromatic Anchoring Residues And Buried Polar Residues, Nick Gleason

Graduate Theses and Dissertations

To investigate in detail the interactions between transmembrane proteins and the lipid bilayers in which they are constituted, designed model peptides with selective isotopic labels were synthesized and analyzed by means of solid-state deuterium NMR spectroscopy. Starting from the well-characterized model peptide GWALP23, acetyl-GGALW(LA)6LWLAGA-amide, several Trp to Tyr mutations were compared to evaluate their respective interfacial anchoring abilities. It was found that Tyr, substituted on either or both termini, can effectively anchor the transmembrane alpha-helix, which then adopts a similar transmembrane topology in a range of bilayer thicknesses. Nevertheless, a consistent ~10° shift in tilt direction (helix rotation) is observed …

Lignin Modification In Arabidopsis And Populus For Studies Of Gene Function And Improving Lignin Degradation, Yi Xu

All Dissertations

Lignin is one of the most abundant biopolymers in plants and plays an important role in plant structure and stress defense. Lignin is also considered to be a hallmark of vascular plants because of its crucial role in plant terrestrialization. However, lignin is an undesired component in the pulp and paper industry, bioethanol production, and forage digestibility. Thus, understanding the functions and the evolution of lignin biosynthesis genes can not only advance our knowledge of the evolution of land-adaptation for vascular plants but also help guide the effort to exploit the potential for genetic manipulation of lignin for desirable traits …

An Analysis Of The Redox Properties And Stability Of Chlamydomonas Reinhardtii Cytochrome F, Cytochrome C6, And Mutants Thereof, Nicole Lynn Vanderbush

Graduate Theses and Dissertations

This body of work presents mutagenesis studies conducted on two c-type cytochromes from Chlamydomonas reinhardtii. Cytochrome f, a unique c-type cytochrome, is investigated in regards to its redox potential, the dependence of the redox potential, and the thermal stability of the protein. The mutations made were Y1F, Y9F, Y160F, Y160L, R156L, and R156K. The residues that were mutated surround the heme. It was found that, relative to the wild-type, only the Y160L and R156 mutants showed any difference in midpoint potential at pH 7. Wild-type and mutants both had a midpoint potential that was dependent upon pH indicating that none …

Plant Lectin Can Target Receptors Containing Sialic Acid, Exemplified By Podoplanin, To Inhibit Transformed Cell Growth And Migration, Jhon Ochoa-Alvarez, Harini Krishnan, Yongquan Shen, Nimish Acharya, Min Han, Dean Mcnulty, Hitoki Hasegawa

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors …

The Anti-Tumorigenic Mushroom Agaricus Blazei Murill Enhances Il-1Β Production And Activates The Nlrp3 Inflammasome In Human Macrophages, Tsung-Teng Huang, David M. Ojcius, John Ding-E. Young, Yi-Hui Wu, Yun-Fei Ko, Tsui-Yin Wong, Cheng-Yeu Wu, Chia-Chen Lu, Hsin-Chih Lai

All Dugoni School of Dentistry Faculty Articles

Agaricus blazei Murill (AbM) has been reported to possess immune activity against tumors and infections through stimulation of mononuclear phagocytes. Recently, AbM extract was shown to induce the production of the pro-inflammatory cytokine, interleukin-1β (IL-1β), in human monocytes. IL-1β is a key pro-inflammatory cytokine produced by activated macrophages and monocytes and its secretion is strictly controlled by the inflammasome. The purpose of this study is to investigate the effect of AbM water extracts on the regulation of IL-1β production and activation of the NLRP3 inflammasome in human THP-1 macrophages. The NLRP3 inflammasome consists of an NLRP3 receptor, an adaptor protein …

Discrimination Of Colon Cancer Stem Cells Using Noncanonical Amino Acid, Xinrui Duan, Honglin Li, Hexin Chen, Qian Wang

Faculty Publications

Cancer stem cells (CSCs) may be responsible for tumor recurrence. Metabolic labelling of newly synthesized proteins with non-canonical amino acids allows us to discriminate CSCs in mixed populations due to the quiescent nature of these cells.

A Pseudo-Trna Modulates Antibiotic Resistance In Bacillus Cereus, Theresa E. Rogers, Sandro F. Ataide, Kiley Dare, Assaf Katz, Stephanie Seveau, Hervé Roy, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNAOther, which does not function in translation. Deletion of tRNAOther led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNAOther. This suggested that tRNAOther might be expressed as part of a larger RNA, and RACE analysis …

Myxobacteria Versus Sponge-Derived Alkaloids: The Bengamide Family Identified As Potent Immune Modulating Agents By Scrutiny Of Lc-Ms/Elsd Libraries., Tyler A. Johnson, Johann Sohn, Yvette M. Vaske, Kimberly N. White, Tanya L. Cohen, Helene C. Vervoort, Karen Tenney, Frederick A. Valeriote, Leonard F. Bjeldanes, Phillip Crews

Natural Sciences and Mathematics | Faculty Scholarship

A nuclear factor-κB (NF-κB) luciferase assay has been employed to identify the bengamides, previously known for their anti-tumor activity, as a new class of immune modulators. A unique element of this study was that the bengamide analogs were isolated from two disparate sources, Myxococcus virescens (bacterium) and Jaspis coriacea (sponge). Comparative LC-MS/ELSD and NMR analysis facilitated the isolation of M. viriscens derived samples of bengamide E (8) and two congeners, bengamide E' (13) and F' (14) each isolated as an insperable mixture of diastereomers. Additional compounds drawn from the UC, Santa Cruz repository allowed expansion of the structure activity relationship …

Development Of A Chaperonin Activity Assay, Timothy P. Brown, Danielle Y. Martinez, Ricardo A. Bernal

COURI Symposium Abstracts, Summer 2012

Chaperonin-mediated protein folding by the Escherichia coli GroEL/GroES complex is pivotal in preventing premature aggregation and improper folding of proteins. In fact E. coli that have had the groE operon knocked out are not able to survive. The explicit details of facilitated folding in the interior of the chaperonin complex remain relatively unknown, although it is understood that ATP hydrolysis is essential to the chaperonin protein-folding cycle. The goal of this project is to develop an assay to detect the protein folding activity of chaperonins. ATP hydrolysis reactions allow chaperonins to undergo a series of conformational changes that alter the …

Purification And Characterization Of Mycobacterium Tuberculosis Esx-1 Secreted Protein Esat-6, Sebastian Montalvo, Gouzhong Jiang, Jianjun Sun

COURI Symposium Abstracts, Summer 2012

Mycobacterium tuberculosis ESX-1 secreted protein ESAT-6 has been shown to contribute to the pathogenesis and virulence of M. tuberculosis. Despite many studies, little is known about how this protein interacts with the host’s membrane and evades the host’s defense mechanism. In this project, we successfully purified and characterized of ESAT-6 wild type (E6-WT) and ESAT-6 mutant with either a single (E6-S) or double (E6-D) amino acid mutation that are supposed to diminish the membrane-interacting activity of ESAT-6. The methods used in our project include: cloning in plasmid vectors, cloning in PCR products with T-Vectors, chemical transformation of E. coli, affinity …

The Synthesis And Structural Characterization Of Metal Oxide Nanoparticles Having Catalytic Applications, Stacey Janel Smith

Theses and Dissertations

Nanotechnology is blossoming into one of the premiere technologies of this century, but the key to its progress lies in developing more efficient nanosynthesis methods. Variations in synthetic technique, however, can cause variations in size, structure, and surface characteristics, thereby altering the physical properties and functionality of the particles. Careful structural characterizations are thus essential for understanding the properties and appropriate applications for particles produced by new synthetic techniques.In this work, a new ‘solvent-deficient’ method is presented for the synthesis of an unprecedentedly wide range of metal oxide nanomaterials including at least one metal oxide from each group in Groups …

Inhibitory Effects Of Novel Immucillin Analogues On Borrelia Burgdorferi Bgp Nucleosidase, Christian Guerrero, Seth Eidemiller, Ken Cornell

IDeA Network for Biomedical Research Excellence (INBRE)

The pathogenic spirochaete Borrelia burgdorferi causes Lyme disease and is transmitted by deer ticks when they feed. Lyme disease is multisystemic—it adversely affects the heart, joints, and skin. Recent studies demonstrate that B. burgdorferi possesses three methylthioadenosine/Sadenosylhomocysteine (MTA/SAH) nucleosidases essential for the catabolic breakdown of both MTA and SAH. Both MTA and SAH are by-products of major pathways involving Sadenosylmethionine (SAM) and are kept at low micromolar concentrations due to their inhibitory activity.

This project examined the effect of transition state inhibitors on the surface binding Borrelia glycosaminoglycanbinding protein (Bgp) nucleosidase using recombinant Bgp and whole-cell B. burgdorferi activity assays. …

Centromeric Heterochromatin Assembly In Fission Yeast-Balancing Transcription, Rna Interference And Chromatin Modification, Benjamin Alper, Brandon R. Lowe, Janet F. Partridge

Chemistry & Physics Faculty Publications

Distinct regions of the eukaryotic genome are packaged into different types of chromatin, with euchromatin representing gene rich, transcriptionally active regions and heterochromatin more condensed and gene poor. The assembly and maintenance of heterochromatin is important for many aspects of genome control, including silencing of gene transcription, suppression of recombination, and to ensure proper chromosome segregation. The precise mechanisms underlying heterochromatin establishment and maintenance are still unclear, but much progress has been made towards understanding this process during the last few years, particularly from studies performed in fission yeast. In this review, we hope to provide a conceptual model of …

Identification Of Persistent Long Range Interactions In GA95 And GB95 Through Thermal Unfolding Simulations, Milen Redai Tesfamariam

Chemistry & Biochemistry Theses & Dissertations

For over five decades, different experiments have been performed to research how proteins attain their native three dimensional structures. However, the folding problem continues to be a puzzle in modern science. The design of two proteins that have maximal sequence identity but different folds and functions is one method that is being used to study the relationship between protein structure and amino acid sequence. In particular, mutant proteins of Streptococcus protein G, G_A and G_B, have 95% sequence identity and a 3a helix fold and β4/a fold, respectively. Molecular dynamics simulations of G_A95 …

Caehnorhabditis Elegans: A Low-Cost In Vivo Animal Model For Efficacy Studies Of Novel Antibiotics, Rylee Ann Gregory

Undergraduate Honors Capstone Projects

Since the 1940s, antibiotics have greatly reduced the adverse effects of infectious diseases caused by microbes. However, due to excessive, and often incorrect, use of known antibiotics, many organisms have adapted antibiotic resistance. Currently, over 70% of known infectious bacteria are resistant to at least one antibiotic. In the U.S. , 90,000 deaths occur each year due to infection by bacteria resistant to antibiotics. This number has increased by nearly 75,000 in the last 20 years. It is necessary, therefore, to continue developing new antibiotics in an effort to keep up with increasing antibiotic resistance. Traditional in vitro and whole …

Screening Synthetic Combinatorial Protein Libraries For Changes In Calmodulin Binding Specificity, Donald E. Dick

Kaleidoscope

No abstract provided.

The Role Of Argininosuccinate Synthase Serine 328 Phosphorylation In Nitric Oxide Production, Ricci Haines

USF Tampa Graduate Theses and Dissertations

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays …

(1)H, (15)N And (13)C Backbone Resonance Assignments Of The Kelch Domain Of Mouse Keap1., Elio Cino, Jingsong Fan, Daiwen Yang, Wing-Yiu Choy

Biochemistry Publications

Kelch-like ECH-associated Protein 1 (Keap1) is a multi-domain protein that functions as an inhibitor of the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cellular response to oxidative stress. Under normal conditions, Keap1 binds to Nrf2 via its C-terminal Kelch domain and the interaction ultimately leads to the ubiquitin-dependent degradation of Nrf2. It has been proposed that designing molecules to selectively disrupt the Keap1-Nrf2 interaction can be a potential therapeutic approach for enhancing the expression of cytoprotective genes. Here, we reported the (1)H, (13)C, and (15)N backbone chemical shift assignments of the Kelch domain of mouse Keap1. Further, …

Association Of A Multi-Synthetase Complex With Translating Ribosomes In The Archaeon Thermococcus Kodakarensis, Medha Raina, Sara Elgamal, Thomas J. Santangelo, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

In archaea and eukaryotes aminoacyl-tRNA synthetases (aaRSs) associate in multi-synthetase complexes (MSCs), however the role of such MSCs in translation is unknown. MSC function was investigated in vivo in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified together with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyltRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential …

Urinary Copper Elevation In A Mouse Model Of Wilson’S Disease Is A Regulated Process To Specifically Decrease The Hepatic Copper Load, Lawrence W. Gray, Fangyu Peng, Shannon A. Molloy, Venkata S. Pendyala, Abigael Muchenditsi, Otto Muzik, Jaekwon Lee, Jack H. Kaplan, Svetlana Lutsenko

Department of Biochemistry: Faculty Publications

Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson’s disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b^‒/^‒ mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated …

Host Pathogen Interactions: Is Arabidopsis Thaliana Remembered By Its Nemesis Pseudomonas Syringae?, Daniel Z. Kreiser

Lawrence University Honors Projects

Plants contain innate immune systems that deter pathogen infection. Pattern recognition receptors bind microbe-associated molecular patterns (MAMPs), triggering immunity. MAMPs are proteins exclusive to pathogens that are typically indispensable for their survival. For this reason, MAMPs cannot be mutated or removed without causing pathogen death. However, this does not necessitate constitutive expression of MAMPs. In this study, the MAMP response of Arabidopsis thaliana was utilized to determine differential detection of MAMPs expressed by Pseudomonas syringe pv. tomato DC3000 when pretreated with A. thaliana. Results demonstrated that more MAMPs are detected when P. syringae had previously encountered A. thaliana, …

Synthesis Of S-Ribosyl-L-Homocysteine And Analogs Modified At The Homocysteine-C3 Position, Ruoyi Liu

Master's Theses

Quorum sensing (QS) is a process of bacterial cell-to-cell communication that conveys population density information in order to coordinate gene expression to produce synchronized behaviors. QS regulates the expression of virulence genes in many species of bacteria; hence, the manipulation of QS pathways may lead to treatment options against many bacterial diseases. The LuxS enzyme converts S-ribosyl-L-homocysteine (SRH) into homocysteine (HCys) and 4(S),5-dihydroxypentane-2,3-dione (DPD), which is the precursor of autoinducer-2 (AI-2). Thus, inhibitors of LuxS could prevent QS by halting the conversion of SRH to AI-2 rendering the cell “uncommunicative”. This work shows the successful chemical synthesis …

Elevating Ascorbate Content In Tomato And Studying The Role Of Jasmonates In Modulating Ascorbate In Arabidopsis, Shashank S. Kulkarni

Student Theses and Dissertations

Vitamin C (ascorbate, AsA) is the most abundant water soluble antioxidant in plants and is essential for plant and human health. Ascorbate plays a key role in protecting plants from reactive oxygen species generated during normal metabolic processes and under stress conditions including wounding, pathogen attack, salt, heat, light, and ozone exposure. Jasmonates (JAs), a group of plants hormones involved in stress responses also protect plants from these stresses. There is evidence indicating that JAs modulate AsA content in plants. The recent discovery of the inositol pathway has opened possibilities for AsA enhancement via metabolic engineering in plants. Over-expression of …

Synthesis Of Fluorogenic Substrates For The Enzymatic Characterization Of Rv0045c From M. Tuberculosis, Kelly Jo Mckenna

Undergraduate Honors Thesis Collection

Mycobacterium tuberculosis is the pathogenic bacterial agent commonly responsible for tuberculosis, or TB. Although treatment exists for the active form of tuberculosis, no method has been developed for eliminating M tuberculosis in its dormant state. One hypothesized method for the elimination of dormant TB is to develop an inhibitor specific for M tuberculosis esterases and lipases, as these esterases and lipases are essential to the survival of dormant TB infection. In this research, the substrate specificity of the Rv0045c esterase from M tuberculosis was studied due to the essential role of Rv0045c in TB metabolism and its dissimilarity to other …

Refining Vibrationally-Resonant Sum Frequency Generation Spectroscopy For Studies Of Interfacial Interactions, Alexander D. Curtis

Theses and Dissertations

Many phenomena of interest to science and engineering occur at interfaces; however, access to, or discrimination of, interfacial interactions has been challenging, especially at buried interfaces. Vibrationally resonant sum-frequency generation (VR-SFG) spectroscopy is a powerful tool for investigating the molecular structure of free or buried interfaces, but spectral analysis has relied on many assumptions. To claim accurate new insights, practitioners must be able to make unique determinations from the data without experimental artifacts affecting the final results. For example, two independent and overlapping studies for the polystyrene/air interface were carried out, but reported different surface structures. Initially, this difference was …

Conservation Of Oxidative Protein Stabilization In An Insect Homologue Of The Parkinsonism-Associated Protein Dj-1, Jiusheng Lin, Janani Prahlad, Mark A. Wilson

Department of Biochemistry: Faculty Publications

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well conserved in other DJ-1 homologues, including two (DJ-1α and DJ-1β) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1β. The structure of D. melanogaster DJ-1β is similar to that of human DJ-1, although two important residues in …

Cholesterol Lowering Effects Of Milk With Added Phytosterols, Laura Kells Cusack

Master's Theses

Foods incorporating plant sterols (PS) reduce Low-density lipoprotein (LDL) on average approximately 10%. PS with a higher lipid solubility may promote greater reductions. We examined the cholesterol lowering effect of a novel triglyceride recrystallized phystosterol (TRP). Twenty subjects (mean ± SD; age, 56 ± 10 years; BMI, 27 ± 5) with elevated LDL (>100 mg/dL) participated in three 4-week phases; Phase I, 2% milk; Phase II, milk with 2.0 grams (g) free PS; Phase III, milk with 2.0 g TRP. Before and after each phase two fasting blood draws were obtained for determination of serum cholesterol. Between the 2% …

Mutation And Complementation Of A Cellulose Synthase (Cesa) Gene, Ahmed Y. El-Araby

Senior Honors Projects

Cellulose is a carbohydrate polymer that is composed of repeating glucose subunits. Being the most abundant organic compound in the biosphere and comprising a large percentage of all plant biomass, cellulose is extremely plentiful and has a significant role in nature. Cellulose is present in plant cell walls, in commercial products such as those made from wood or cotton, and is of interest to the biofuel industry as a potential alternative fuel source. Although indigestible by humans, cellulose is nutritionally valuable, serving as a dietary fiber. Because of its ubiquity and importance in many areas, studying cellulose will prove to …

Time-Dependent Density-Functional-Theory Calculation Of High-Order-Harmonic Generation Of H-2, Xi Chu, Gerrit C. Groenenboom

Chemistry and Biochemistry Faculty Publications

The observation of the isotope effect in the high-order-harmonic generation (HHG) of H-2 presents a challenge for time-dependent density-functional-theory (TDDFT) methods, since this effect is related to the dynamics of the ion created in the tunneling ionization step of HHG and it depends on the harmonic order. As an initial step toward describing this effect within current computational capacity, we benchmark a method in which the nuclear and electronic degrees of freedom are separated and both treated quantum mechanically. For the electrons two TDDFT formalisms are adopted. Although the ion-dynamics effect is not described in our method, it reproduces the …

Recombinant Expression And Purification Of Human Intrinsic Factor (If) And Mutants K159d And Q201a Designed To Interfere With Cubilin Receptor Binding, Soreen Cyphers

Renée Crown University Honors Thesis Projects - All

Although both the α- and β-domains of intrinsic factor (IF) have been previously expressed, the full crystal structure of the protein has yet to be reported. The purpose of this research is to (1) express IF in order to obtain a complete crystal structure and (2) utilize a mutant form of IF in order to orally deliver rotavirus to the ileum.The first goal of this research is to express IF in the yeast Pichia pastoris. The second goal is to express a K159D IF mutant protein. K159 of IF plays a role in salt bridge formation between IF and …