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 …

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 …

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 …

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 …

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 …

Synthesis And Structural Studies Of Calcium And Magnesium Diphosphonate Compounds, Seungmo Suh

Renée Crown University Honors Thesis Projects - All

The purpose of this project is to study synthetic methodologies leading to the designing of various calcium and magnesium based diphosphonates with possible applications as bone scaffolding materials. In tissue engineering scaffolds are frequently employed to enhance the growth of new cells, providing the motivation to study alkaline earth metal based diphosphonates as a possible source for scaffold for bone growth.

While the transition metal phosphonates based frameworks are well studied, alkaline earth metal based phosphonates and diphosphonates remain at its infancy. The challenge to the chemistry of the alkaline earth metal phosphonates and diphosphonates based frameworks includes poor solubility …

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 …

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 …

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 …