Proteome-Wide Analysis Of Single-Nucleotide Variations In The N-Glycosylation Sequon Of Human Genes, Raja Mazumder, Krishna Sudeep Morampudi, Mona Motwani, Sona Vasudevan, Radoslav Goldman

Biochemistry and Molecular Medicine Faculty Publications

N-linked glycosylation is one of the most frequent post-translational modifications of proteins with a profound impact on their biological function. Besides other functions, N-linked glycosylation assists in protein folding, determines protein orientation at the cell surface, or protects proteins from proteases. The N-linked glycans attach to asparagines in the sequence context Asn-X-Ser/Thr, where X is any amino acid except proline. Any variation (e.g. non-synonymous single nucleotide polymorphism or mutation) that abolishes the N-glycosylation sequence motif will lead to the loss of a glycosylation site. On the other hand, variations causing a substitution that creates a new N-glycosylation sequence motif can …

Elucidating The Mechanism Of Antimigratory Activity Of Cardiac Glycosides, Joshua H. Johnson

Honors Scholar Theses

The focus of this research is on cell migration and how it can be better understood through the use of small molecules that modulate cell migratory activity. The results have particular relevance in the realm of cancer pharmacology. Cardiac glycosides, which are known inhibitors of the eukaryotic Na⁺/K⁺-ATPase, have been determined to have antimigratory activities through the screening of several small molecule libraries. Here we investigate the antimigratory activities of the cardiac glycoside digitoxin as well as its analogs that we synthesized. Antimigratory activity was determined by conducting a wound closure assay with MDA-MB-231 human breast …

Improvements To The Forensic Analysis Of Mitochondrial Dna Typing, Elizabeth Montano

Honors Scholar Theses

Sequence analysis of human mitochondrial DNA (mtDNA) is an effective and reliable tool for the genetic characterization of forensic samples. The nature of the mitochondrial genome (mtgenome), its high copy number and small size (~17kb) makes it more resistant to degradation and more stable than nuclear DNA. For this reason mitochondrial DNA is often the only feasible option for the forensic analysis of environmentally compromised samples. Currently the forensic analysis of the mtgenome is restricted to the hypervariable regions, also known as the Displacement loop (d-loop). Previous studies, confirmed in the Strausbaugh lab, have demonstrated an increased variability in the …

Metallothionein Gene Dose And The Immune Response, Meaghan Roy-O-Reilly

Honors Scholar Theses

Metallothionein (MT) is a small, cysteine-rich protein with significant immunomodulatory activity. It has been shown to play a critical role in important cellular mechanisms including heavy metal detoxification, essential metal management and the inflammatory response. MT production can be induced by a number of cellular stressors and acts to lessen the harmful effects of oxidizing agents and heavy metal exposure. Previous studies have shown that the dose of the metallothionein gene present in an individual may have significant effects on the adaptive immune response, yet the mechanism behind this phenomenon remains unknown. We hypothesize that the gene dose of metallothionein …

Identifying Progenitor Cells Of Heterotopic Ossification, Eileen E. Semancik

Honors Scholar Theses

Heterotopic Ossification (HO) is the abnormal formation of bone within extraskeletal soft tissues. The condition can occur through both genetic and acquired means. Acquired cases of HO result from invasive surgery or traumatic injuries, with increasing prevalence of ectopic skeletogenesis as a result of combat-related blast injuries. HO has been characterized to some extent, including the histological features and the mutation underlying the genetic form, but the cells resident in skeletal muscle that represent the progenitors of heterotopic bone have yet to be determined. Only a few publications have attempted to definitively determine the progenitor cells in this disorder. Findings …

Fundamentals Of Protein Structure Alignment, Allen Holder, Mark Brandt, Yosi Shibberu

Mathematical Sciences Technical Reports (MSTR)

The central dogma of molecular biology asserts a one way transfer of information from a cell’s genetic code to the expression of proteins. Proteins are the functional workhorses of a cell, and studying these molecules is at the foundation of much of computational biology. Our goal here is to present a succinct introduction to the biological, mathematical, and computational aspects of making pairwise comparisons between protein structures. The presentation is intended to be useful for those who are entering this research area. The chapter begins with a brief introduction to the biology of protein comparison, which is followed by a …

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% …

Locking Down The N-Terminus Of Prmt1 In Order To Assess The Role Of Motion In Activity, Taylor James Rasmussen

Undergraduate Honors Capstone Projects

Protein arginine methyltransferases (PRMTs) are involved In many major biological pathways in the human body. Processes that demonstrate arginine methylation by PRMTs include, but are not limited to, histone modification, DNA transcription, and post-translational protein modifications. Although recent research has allowed the identification of several PRMT isoforms and exposed their involvement in these processes, relatively little is known about the details of how these enzymes perform their biochemical duties. It is currently hypothesized that the N-terminus of PRMT variant I is involved in recognizing substrates and aiding in catalysis by virtue of a change in its conformation. To understand how …

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 …

Transport And Spectroscopic Studies Of The Effects Of Fullerene Structure On The Efficiency And Lifetime Of Polythiophene-Based Solar Cells, Emilee L. Sena, Justin H. Peel, Devin Wesenberg, Shreya Nathan, Marianne Wallis, Maxwell J. Giammona, Thorsteinn Adalsteinsson, Brian J. Mcnelis, Richard P. Barber Jr.

Physics

Time-dependent measurements of both power conversion efficiency and ultraviolet-visible absorption spectroscopy have been observed for solar cell blends containing the polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) with two different functionalized C60 electron acceptor molecules: commercially available [6,6]-phenyl C61 butyric acid methyl ester (PCBM) or [6,6]-phenyl C61 butyric acid octadecyl ester (PCBOD) produced in this laboratory. Efficiency was found to decay with an exponential time dependence, while spectroscopic features show saturating exponential behavior. Time constants extracted from both types of measurements showed reasonable agreement for samples produced from the same blend. In comparison to the PCBM samples, the stability of the PCBOD blends was …

The Time-Course Of The Effects Of Growth Hormone During Zebrafish (Danio Rerio) Auditory Hair Cell Regeneration, Yajie Wang

Masters Theses & Specialist Projects

Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the …

Determination Of Selectivity And Potential For Drug Resistance Of Novel Antimalarial Compounds From Nature-Inspired Synthetic Libraries, Eric Keasler

HIM 1990-2015

As malaria, caused by Plasmodium spp., continues to afflict millions of people worldwide, there is a dire need for the discovery of novel, inexpensive antimalarial drugs. Although there are effective drugs on the market, the consistent development of drug resistant species has decreased their efficacy, further emphasizing that novel therapeutic measures are urgently needed. Natural products provide the most diverse reservoir for the discovery of unique chemical scaffolds with the potential to effectively combat malarial infections, but, due to their complex structures, they often pose extreme challenges to medicinal chemists during pharmacokinetic optimization. In our laboratory we have performed unbiased, …

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 …

Interaction Of Environmental B. Cenocepacia Strains With Cystic Fibrosis And Non-Cystic Fibrosis Bronchial Epithelial Cells In Vitro., Annamaria Bevivino, Luisa Pirone, Ruth Pilkington, Noemi Cifani, Claudia Dalmastri, Máire Callaghan, Fiorentina Ascenzioni, Siobhan Mcclean

Articles

Burkholderia cenocepacia is an important human pathogen in patients with cystic fibrosis (CF). Non-clinical reservoirs may play a role in the acquisition of infections, so it is important to evaluate the pathogenic potential of environmental B. cenocepacia isolates. In this study, we investigated the interactions of two environmental B. cenocepacia strains (Mex1 and MCII-168) with two bronchial epithelial cell lines,16HBE14o- and CFBE41o-, which have a non-CF and a CF phenotype, respectively.

The environmental strains showed a significantly lower level of invasion into both CF- and non-CF cells in comparison with the clinical B. cenocepacia LMG16656^T strain. Exposure of polarized …

Designing An "Information-Experience" Using Creativity Science Theory And Tools, Stephanie Belhomme

Creativity and Change Leadership Graduate Student Master's Projects

An “information-experience” encapsulated by a technological/digital audio-visual tool presents data and potentially meaningful information to prompt actionable knowledge concerning: “unspoken creative process elements;” their profound impacts on both how well our “physiology of creativity” functions; but also on how well foundational creative thinking and behavioral prerequisites (energy, motivation, imagination, and ownership) are leveraged.

The product: 1) introduces the user to one component of the CPS (Creative Problem Solving) Facilitation Process - Exploring the Challenge; 2) features a content specific component which prompts exploration of the many correlations between societal, organizational / community, human physiological / behavioral data, and the direct …

Conformational Motions Associated With Ligand Binding In Dihydrofolate Reductase From Bacillus Stearothermophilus, Mayam Tobilola Alapa

Theses, Dissertations and Culminating Projects

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently attached fluorophore, N-[2-(lmaleimidyl) ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR …

A Leishmania Mitogen Activated Protein Kinase As A Potential Anti-Parasitic Drug Target : Purification, Characterization And Inhibitor Interactions, Aysenur Sayakci

Theses, Dissertations and Culminating Projects

The disease, Leishmaniasis, is caused by the protozoal parasite Leishmania, which is transmitted by the bite of an infected Phlebotomine female sandfly. It is a significant health problem in tropical regions of the world and new therapeutic approaches for treating this disease are urgently needed.

Protozoal MAPKs (Mitogen Activated Protein Kinases) play important roles in parasite viability and infectivity and as such, represent viable drug targets. It has been demonstrated that LmxMPKl is an essential MAPK required for the parasite to establish infection and for proliferation of the amastigote stage (the mammalian stage of the parasite) of the parasite. In …

Characterization Of Novel Histone H2b Mutants Associated With Chromosome Segregation Defects In Saccharomyces Cerevisiae, Thiruchelvam Rajagopal

Graduate Theses and Dissertations

Histones are small basic proteins that associate with DNA to form the basic unit of chromatin, the nucleosome. Histones H3 and H4 form a tetramer that is bound by two H2A-H2B dimers to form the histone octamer, to which approximately 146 bp of DNA wrap around to form the nucleosome. High resolution structural information and recent advances in the understanding of histone post-translational modifications have illuminated the many regulatory functions chromatin exerts in the cell, from the transcriptional control of gene expression to chromosome segregation. However, the specific role that histones play in these processes is not well understood. Previous …

Structural Analysis Of The Drosophila Innexin Shakb: Role Of The N-Terminus In Rectifying Electrical Synapses, William Marks

Biology Theses

Gap junction channels mediate direct intercellular communication in all multicellular animals. They are comprised of the connexin family of proteins in vertebrates and the innexin family in prechordates. Connexins and innexins share many functional and structural similarities as orthologous proteins. Both types are capable of forming electrical synapses. Rectifying junctions are specialized electrical synapses found in neural systems that control escape responses. It has been shown that heterotypic gap junction channels mediate asymmetric properties of rectifying junctions. Shaking B N+16 and Shaking B lethal are variants of the ShakB locus in Drosophila and the organization of these innexins into heterotypic …

Does Vibrational Loading Modulate The Effects Of Radiotherapy On Growing Bone?, Samantha Hay

Renée Crown University Honors Thesis Projects - All

Radiotherapy is an important part of cancer therapy, used in addition to surgery for treatment of patients with soft tissue sarcomas, and alternatively for treatment of patients with Ewing sarcoma of bone. Treating pediatric extremity tumors with radiotherapy has been shown to have harmful effects on the epiphyseal plate, resulting in permanent limb shortening and deformity when bone growth centers are exposed to radiation. Mechanical signals, specifically low-magnitude high-frequency vibrations (LMHFV), have been shown to be non-invasive and non-pharmacological growth factors in bone that have the potential to serve as a safe treatment for a number of clinical conditions. Thus, …

Expression And Purification Of Recombinant Saposin B For Coenzyme Q10 Purification, Vivian Yaci Yu

Renée Crown University Honors Thesis Projects - All

Coenzyme Q10 (CoQ10) plays a critical role in energy production in humans. Low levels of CoQ10 have been linked to diseases such as Parkinson's and Huntingtons's disease. CoQ10 levels decrease as we age, and use of statin drugs also lowers CoQ10 levels. Oral supplementation however increases CoQ10 levels. However, the lack of industrial sources of CoQ10 and the difficulty in CoQ10 purification has resulted the need to address these problems. Sporidiobolus johnsonii (S. johnsonii) has been reported as a natural producer of CoQ10. This work is aim to increase the production of CoQ10 in S. johnsonii through genetic …

Gold (Iii) Macrocycles Are Dna Intercalators That Inhibit Topoisomerase I And Ii, Alexander Fagenson

HIM 1990-2015

Human Topoisomerase IB (TOP1) and Topoisomerase II? (TOP2?) are essential nuclear enzymes that control DNA topology during DNA replication, gene transcription and cell division. These enzymes carry out their catalytic function by making transient single-strand (type I) or double-strand (type II) breaks in the DNA. In vivo, these complexes are short-lived but can be exploited by anti-cancer drugs to mechanistically kill cancer cells. Two general classes of compounds can kill cancer cells through a topo-targeted mechanism. Interfacial Poisons (IFPs) act at the enzyme-DNA interface to inhibit the religation reaction, resulting in the accumulation of DNA double-stand breaks (DSBs) in the …

Design, Construction, And Characterization Of The Ysgr Minimal Codon Fab Library For Chaperone-Assisted Rna Crystallography, Sean Holmes

HIM 1990-2015

Of the entire human genome, 90% of all genetic information is transcribed but only a fraction of that subsequent RNA is translated into proteins. RNAs which are not translated into proteins are deemed non-coding RNAs. Little is known about this large category of noncoding RNAs, although they perform a variety of functions within the cell. RNA crystallography is used to study RNA tertiary structure, which gives insight to the function of these non-coding RNAs. However, complications associated with RNA crystallography arise due to RNA's lack of surface functional group diversity, flexible tertiary structure, and conformational heterogeneity. A novel technique, Chaperone-assisted …

Determination Of The Hydrogen Peroxide Concentration In Rotenone Induced Dopaminergic Cells Using Cyclic Voltammetry And Amplex Red, Kishan Patel

HIM 1990-2015

Parkinson's disease (PD) is a neurodegenerative condition that affects millions of people worldwide. The exact etiology of PD is unknown. However, it is well established that environmental factors contribute to the onset of PD. In particular, chemicals such as the insecticide Rotenone have been shown to increase the death of dopaminergic (DA) neurons by increasing levels of reactive oxygen species (ROS). ROS such as hydrogen peroxide (H2O2) have been shown to be elevated above basal levels in PD patients. Currently, to measure H2O2 concentrations, a commercially available (Amplex® Red) fluorescent assay is used. However, the assay has limitations: it is …

Identification Of Plasmodium Falciparum Protein Kinase Substrates And Interacting Proteins, Jessica Yap

HIM 1990-2015

Characterization of PfPKA and PfPK5 substrates, as well as the proteins they interact with, will help us to develop innovative therapies targeting binding sites.; Malaria is a devastating disease that results in almost one million deaths annually. Most of the victims are children under the age of five in Sub-Saharan Africa. Malaria parasite strains throughout developing countries are continually building resistance to available drugs. Current therapies such as mefloquine, chloroquine, as well as artemisinin are becoming less effective, and this underscores the urgency for therapeutics directed against novel drug targets. In order to identify new drug targets, the molecular biology …

Biochemical Characterization Of Sac9, A Putative Phosphoinositide Phosphatase In Arabidopsis Thaliana, And Its Role In Cellular Abnormalities, Almut H. Vollmer

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Since the early colonization of land by fungi and plants some 700 million years ago, plants have been continuously faced with changes in their environment. Unlike animals, plants are not free to move about, and can therefore not evade many stress factors. How plants sense and respond to their environment has been of interest not only to scientific research but also in more practical applications such as agriculture.

Signals (such as light or salinity) from the outside of plant cells trigger a flow of information to the inside of the cell. The final target for most of the information is …

Structural And Mechanistic Investigations Of Phosphothreonine Lyase Class Of Enzymes, Alok Gopalkrishna Shenoy

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Phosphorylation and dephosphorylation are a highly pervasive mechanism in biology that is used by the cell to modulate enzymes and proteins. The presence of a phosphate group can activate or deactivate an enzyme. The phosphate group is linked to a protein by a phosphoester bond that is known to be highly stable in cytoplasmic pH range. Thus the breaking and formation of these bonds need to be effected by enzymes.

Recent discovery of the activity carried out by certain virulence related proteins (OspF released by Shigella and SpvC released by Salmonella) have resulted in a necessity to create a new …

1. Improving The Yield Of Biodiesel From Microalgae And Other Lipids. 2. Studies Of The Wax Ester Biosynthetic Pathway And Potential Biotechnological Application, Bradley D. Wahlen

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels.

Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain high amounts of free fatty acids, which are unsuitable for base-catalyzed transesterification. Herein an approach is described that efficiently accomplishes the simultaneous esterification and transesterification of both free fatty …

Structure Of The Catalytic Chain Of Methanococcus Jannaschii Aspartate Transcarbamoylase In A Hexagonal Crystal Form: Insights Into The Path Of Carbamoyl Phosphate To The Active Site Of The Enzyme, Jacqueline Vitali, Aditya K. Singh, Alexei S. Soaresb, Michael J. Colaneri

Physics Faculty Publications

Crystals of the catalytic chain of Methanococcus jannaschii aspartate transcarbamoylase (ATCase) grew in the presence of the regulatory chain in the hexagonal space group P6₃22, with one monomer per asymmetric unit. This is the first time that crystals with only one monomer in the asymmetric unit have been obtained; all known structures of the catalytic subunit contain several crystallographically independent monomers. The symmetry-related chains form the staggered dimer of trimers observed in the other known structures of the catalytic subunit. The central channel of the catalytic subunit contains a sulfate ion and a K⁺ ion as …