Biochemistry, Biophysics, and Structural Biology Commons^™

Transcriptomic Analyses Of The Co2-Concentrating Mechanisms And Development Of Molecular Tools For Chlamydomonas Reinhardtii, Andrew J. Brueggeman

Department of Biochemistry: Dissertations, Theses, and Student Research

Microalgae, such as Chlamydomonas reinhardtii, account for a large percentage of photosynthesis that occurs on the planet. Many algae possess a Carbon-Concentrating Mechanism, or CCM, that actively transports inorganic carbon (C_i) into the cell to create artificially high internal levels of CO₂, enhancing their rate of carbon fixation. The production of biofuels from algal sources can serve as both a renewable and carbon-neutral energy source. This thesis details research in Chlamydomonas, in the effort to both better understand the CCM in algae and improve laboratory and industrial manipulations with algae.

In the first chapter of this …

The Development And Applications Of Nmr Metabolomics Analysis Of Bacterial Metabolomes, Steven M. Halouska

Department of Chemistry: Dissertations, Theses, and Student Research

Metabolomics is a relatively new field that involves the study of metabolic responses that are occurring within a biological system. Metabolite profiles of an organism, tissue extract, and biofluids are important indicators to determine the physiological state of a biological profile. Comparison of such profiles from different phenotypes can be used to identify specific metabolic changes leading to the understanding of metabolic pathways, disease progression, drug toxicity and efficacy, and cellular responses to different intracellular and extracellular conditions. Metabolomics investigations often use sophisticated analytical techniques such as NMR spectroscopy to provide an unbiased and comprehensive approach to evaluate metabolic perturbation …

Obstacles And Solutions To Studying Functional Adhesives Using Vibrational Sum-Frequency Generation Spectroscopy, Angela Renee Andersen

Theses and Dissertations

Important aspects of adhesion occur at interfaces, including structures that may be different from those in the bulk materials. However, probing the orientation of molecules in functional adhesives poses a significant challenge because adhesive molecules are always located at a buried interface. The limited penetration depth of surface-specific analysis prohibits the study of buried interfaces using those techniques. The large quantity of bulk molecules relative to the adhesive molecules interacting at the interface results in the bulk signal swamping out adhesive signal in bulk analysis techniques. An interface-specific technique is required to study functional adhesives. One such technique that has …

Investigation Into The Effects Of Pegylation On The Thermodynamic Stability Of The Ww Domain, Sam S. Matthews

Theses and Dissertations

The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins.This thesis details the results of two studies aimed at …

An Examination Of The Inhibitory Effects Of Antibiotic Combinations On Ribosome Biosynthesis In Staphylococcus Aureus, Justin Beach

Electronic Theses and Dissertations

Bacteremia initiated by Staphylococcus aureus infections can be a serious medical problem. Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. Combination therapy for certain infections has been used to reduce the emergence of resistance when a single agent has become ineffective. We hypothesize that the use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects resulting from indirect effects on ribosomal subunit synthesis.

To determine this we measured the effects of single and multiple antibiotics on cell growth …

The Staphylococcus Pseudintermedius Adhesin Spsd Contains A Central Fibronectin-Binding Domain, Andrea S. Bordt

Dissertations and Theses (Open Access)

Staphylococcus pseudintermedius is a Gram-positive bacterium significant because of its ability to cause costly and difficult to treat veterinary infections worldwide. It exhibits several similarities to Staphylococcus aureus, however, very little is known about its surface adhesins. Surface adhesins in S. aureus are significant contributors to pathogenesis. S. pseudintermedius encodes the surface protein SpsD, which contains characteristics of the microbial surface components recognizing adhesive matrix molecules family and confers attachment of the heterologous host Lactococcus lactis to fibronectin. This work has identified a centrally-located fibronectin binding domain in SpsD which binds the 30 kDa N-terminal domain of fibronectin with …

The Role Of Heparin In Acidic Fibroblast Growth Factor Signaling, Rebecca Kerr

Graduate Theses and Dissertations

Fibroblast growth factors (FGFs) are a superfamily of 23 heparin-binding proteins. Acidic fibroblast growth factor (FGF1) is a 16 kDa protein that is involved in many important biological processes such as cell differentiation, angiogenesis, and wound healing. FGF1 exhibits its activity by binding to cell-surface tyrosine kinase receptors (FGFRs). In addition to binding to FGFRs, FGF1 binds to heparin/HLGAGs. Heparin is proposed to have two functions: it protects FGFs from degradation by proteases and from thermal denaturation; and upon binding it induces FGF1 oligomerization, which is mandatory for binding to FGFR. However, it remains controversial whether or not heparin is …

Investigating The Importance Of The N-Terminal Negative Residues In Human Prmt1, Brooke Siler

Undergraduate Honors Capstone Projects

Many essential physiological pathways, such as cell proliferation, gene expression, and cardiovascular health are regulated by Protein Arginine Methyltransferases (PRMTs) through methylation of arginine residues in protein substrates. Understanding how PRMTs interact with their substrates is pivotal to understanding the biological role of these enzymes, and fundamental to the goal of identifying possible sites to be inhibited through drug therapy. Natural variations in the N-terminus of the PRMTl enzymes and data collected in our lab suggest that the N-terminus is important for activity and/or the binding of protein substrates. Preliminary data collected had led us to hypothesize that the negatively …

Mechanisms Of The Anti-Pneumococcal Function Of C-Reactive Protein, Toh B. Gang

Electronic Theses and Dissertations

Human C-reactive protein (CRP) increases survival of and decreases bacteremia in mice infected with Streptococcus pneumoniae. Such protection of mice against pneumococcal infection is seen only when CRP is administered into mice 6 hours before to 2 hours after the injection of pneumococci, but not when CRP is given to mice at a later time. Our first aim was to define the mechanism of CRP-mediated initial protection of mice against infection. It was proposed that CRP binds to phosphocholine (PCh) moieties present in the cell wall and activates the complement system on the pneumococcal surface that kills the pathogen. …

Biochemical And Biophysical Studies Of Novel Features Of Ras-Related Protein Interactions, Kyla Marie Morinini Morris

Graduate Theses and Dissertations

The Ras superfamily of G-proteins are of great research interest for structure-function relationships among proteins as they act as molecular switches in the regulation of various biochemical reactions in the cell. They are regulated by protein-protein interactions targeted to the highly flexible switch regions. Mutations in G-proteins or their effectors may cause alterations in structure and/or function that can lead to overactivity.

The Ras-related protein Cell division cycle 42 (Cdc42) is important in regulating cell-signaling processes. The T35A mutation in Cdc42 leads to a decrease in flexibility of the Switch I region responsible for effector binding. The kinetics of the …

Identification Of Disufide Bond Formation Between Mitoneet And Glutamate Dehydrogenase 1., Morgan Roberts, Jacquelyn Crail, Megan Laffoon, William Fernandez, Michael Menze, Mary Konkle

Faculty and Staff Scholarship

MitoNEET is a protein that was identified as a drug target for diabetes, but its cellular function as well as its role in diabetes remains elusive. Protein pull-down experiments identified glutamate dehydrogenase 1 (GDH1) as a potential binding partner. GDH1 is a key metabolic enzyme with emerging roles in insulin regulation. MitoNEET forms a covalent complex with GDH1 through disulfide bond formation and acts as an activator. Proteomic analysis identified the specific cysteine residues that participate in the disulfide bond. This is the first report that effectively links mitoNEET to activation of the insulin regulator GDH1.

Monodentate, Bidentate And Photocrosslinkable Thiol Ligands For Improving Aqueous Biocompatible Quantum Dots, Hiroko Takeuchi

Graduate Theses and Dissertations

Water-soluble Quantum Dots (QDs) are highly sensitive fluorescent probes that are often used to study biological species. One of the most common ways to render QDs water-soluble for such applications is to apply hydrophilic thiolated ligands to the QD surface. However, these ligands are labile and can be easily exchanged on the QD surface, which can severely limit their application. As one way to overcome this limitation while maintaining a small colloidal size of QDs, we developed a method to stabilize hydrophilic thiolated ligands on the surface of QDs through the formation of a crosslinked shell using a photocrosslinking approach. …

Quantitative Analysis Of Neurotransmitter Pathways Under Steady State Conditions - A Perspective, Arthur J L Cooper

NYMC Faculty Publications

In a contribution to this Research Topic Erkki Somersalo and Daniela Calvetti carried out a mathematical analysis of neurotransmitter pathways in brain, modeling compartmental nitrogen flux among several major participants - ammonia, glutamine, glutamate, GABA, and selected amino acids. This analysis is important because cerebral nitrogen metabolism is perturbed in many diseases, including liver disease and inborn errors of the urea cycle. These diseases result in an elevation of blood ammonia, which is neurotoxic. Here, a brief description is provided of the discovery of cerebral metabolic compartmentation of nitrogen metabolism - a key feature of cerebral glutamate-glutamine and GABA-glutamine cycles. …

Mutational Analysis Of The Rotavirus Nsp4 Enterotoxic Domain That Binds To Caveolin-1, Judith M. Ball, Megan E. Schroeder, Cecelia V. Williams, Friedhelm Schroeder, Rebecca D. Parr

Faculty Publications

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction.

Methods: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed …

Microtubule Severing At Crossover Sites By Katanin Generates Ordered Cortical Microtubule Arrays In Arabidopsis, Quan Zhang, Erica Fishel, Tyler Bertroche, Ram Dixit

Biology Faculty Research

Highlights

• Severing primarily depolymerizes the overlying CMT at crossover sites
• Severing probability increases nonlinearly with crossover time
• Katanin localizes to crossover sites and is required for severing
• Loss of katanin activity prevents the formation of coaligned CMT arrays

Summary
The noncentrosomal cortical microtubules (CMTs) of land plants form highly ordered parallel arrays that mediate cell morphogenesis by orienting cellulose deposition [1, 2 and 3]. Since new CMTs initiate from dispersed cortical sites at random orientations [4], parallel array organization is hypothesized to require selective pruning of CMTs that are not in the dominant orientation. Severing of CMTs at crossover sites …

Immunoproteomic Analysis Of Proteins Expressed By Two Related Pathogens, Burkholderia Multivorans And Burkholderia Cenocepacia, During Human Infection., Minu Shinoy, Ruth Dennehy, Lorraine Coleman, Stephen Carberru, Kirsten Schaffer, Máire Callaghan, Sean Doyle, Siobhan Mcclean

Articles

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We …

Identification And Rna Binding Characterization Of Plant Virus Rna Silencing Suppressor Proteins, Jeff Vargason, Carissa J. Burch, Jesse W. Wilson

Faculty Publications - Department of Biological & Molecular Science

Suppression is a common mechanism employed by viruses to evade the antiviral effects of the host’s RNA silencing pathway. The activity of suppression has commonly been localized to gene products in the virus, but the variety of mechanisms used in suppression by these viral proteins spans nearly the complete biochemical pathway of RNA silencing in the host. This review describes the agrofiltration assay and a slightly modified version of the agro-infiltration assay called co-infiltration, which are common methods used to observe RNA silencing and identify viral silencing suppressor proteins in plants, respectively. In addition, this review will provide an overview …

Investigation Of Proline Utilization A: Kinetic Analysis Of Substrate Channel-Blocking Mutants And Creation Of A Trifunctional Chimera Enzyme, Benjamin W. Arentson

Department of Biochemistry: Dissertations, Theses, and Student Research

Proline metabolism is known to be involved in many cellular processes such as cell signaling, cellular redox balance, and cell survival. One of the enzymes involved in proline catabolism, proline utilization A, plays a role in oxidizing proline to glutamate in a two-step oxidation pathway involving enzymes proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH).

Intermediate P5C/GSA has been shown to use an intramolecular channel to move from the PRODH active site to the P5CDH active site in a phenomenon called substrate channeling. In this work, one of the main objectives was to learn more about the channel usage. …

Intrinsic Disorder Within An Akap-Protein Kinase A Complex Guides Local Substrate Phosphorylation, F. Donelson Smith, Steve L. Reichow, Jessica L. Esseltine, Dan Shi, Lorene K. Langeberg, John D. Scott, Tamir Gonen

Chemistry Faculty Publications and Presentations

Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme …

Killerflip: A Novel Lytic Peptide Specifically Inducing Cancer Cell Death, B Pennarun, G. Gaidos, O Bucur, A Tinari

Dartmouth Scholarship

One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killer FLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using …

Cellular Uptake Mechanism Of Paclitaxel Nanocrystals, Iris K. Archer, Zhaohui Wang, Tonglei Li

The Summer Undergraduate Research Fellowship (SURF) Symposium

Therapeutic options for metastasized human cancer in current practice remain limited and, sadly, there is no cure for metastatic cancer. The typical approach, chemotherapy, has both low efficacy due to poor drug solubility, and cytotoxic side effects to healthy tissue when delivered indiscriminately. To address both of these issues, we are pursuing the use of nanocrystal formulations of current chemotherapeutic agents as delivery platforms. Herein, we have studied cellular uptake mechanisms in cancer cells of nanocrystals of a chemotherapeutic agent, paclitaxel. Our goal in this study is to determine whether the nanocrystals can be taken up via endocytosis, especially when …

Identification Of Set1 Target Genes, William Beyer, Scott D. Briggs

The Summer Undergraduate Research Fellowship (SURF) Symposium

The Set1 complex, a histone methyltransferase complex found in S. cerevisiae (budding yeast), is the only histone methyltransferase responsible for catalyzing methylation of histone H3 at Lysine 4. It possesses homologues in other species, humans included. While yeast only have the Set1 complex, the human homologues of the yeast Set1 complex include mixed-lineage leukemia family (MLL1-4), Set1 A, Set1 B, among others. MLL1-4 has been shown to play a role in transcription, cell type specification, and the development of leukemia. One application of characterizing the role of a protein is that the information gained can provide insight into the function …

Involvement Of Post-Transcriptional Histone Modifications In Chromosome Missegregation, Jessica N. Gabbard, Ann Kirchmaier

The Summer Undergraduate Research Fellowship (SURF) Symposium

Proper chromosome segregation during mitosis ensures the equal inheritance of parental DNA by two daughter cells. Errors in chromosome segregation result in aneuploidy, the inheritance of abnormal chromosome numbers. Aneuploidy is a characteristic of tumors cells; therefore, understanding the factors that cause chromosome missegregation will provide insight into carcinogenesis. Certain post-transcriptional histone modifications in centromeric and pericentromeric regions are associated with maintaining kinetochore integrity and ensuring proper chromosome segregation. However, whether loss or improper distribution of modifications directly or indirectly causes chromosome missegregation is yet to be determined. To compare the DNA content of mutated yeast strains relative to WT, …

Saccharomyces Cerevisiae Cdc7 Homology In Drosophila Melanogaster, Marcus R. Hosler, Robert E. Stephenson, Vikki M. Weake

The Summer Undergraduate Research Fellowship (SURF) Symposium

Saccharomyces cerevisiae Dbf4(Dumbbell former 4) and Cdc7(Cell Division Cycle 7) form a complex that phosphorylates Mcm2 (Minichromosome maintenance 2) to initiate DNA replication. Cdc7 is a target for cancer research because there is a Cdc7 ortholog in humans that is necessary for DNA replication and cell survival. Our goal is to characterise a putative Cdc7 homolog in Drosophila melanogaster (dCdc7). We have previously shown that expression of the known Drosophila Dbf4 ortholog, Chiffon, and dCdc7 can rescue yeast cells deficient in active Cdc7. Our hypothesis is that the dCdc7 is activated by Chiffon to phosphorylate MCM2. To test this hypothesis, …

Development Of A Metabolomic Method To Define The Phenylalanome In Arabidopsis Thaliana, Cole G. Wunderlich, Clint Chapple, Xu Li

The Summer Undergraduate Research Fellowship (SURF) Symposium

In the study of metabolomics, one of the greatest challenges can be accurately identifying compounds detected in biological extracts, especially when standards are not readily available. Current metabolomic methods are also limited in that they provide little to no information about a compound’s metabolic origin. In this study, we sought to address these issues by developing a novel metabolomic method that employs stable isotope feeding, LC-MS, Xcms, and an analytical software algorithm to study the ‘phenylalanome’ of Arabidopsis thaliana. Using this approach we were able to develop a method that, based on current results, is capable of detecting over …

The Effects Of Exogenous Extracellular Matrix And Substrate Stiffness On Mouse Tendon Cells In Vitro, Caleb J. Mcdaniel, Sarah Calve

The Summer Undergraduate Research Fellowship (SURF) Symposium

To improve the treatment of musculoskeletal injuries, a better understanding of the transitional environment in which progenitor cells form mature musculoskeletal constructs is necessary. This need arises because injury repair requires restructuring of tissue, similar to the initial tissue construction that occurs during embryonic development by progenitor cells. Differences in both the biochemical and mechanical environments between a transitional and a differentiated state are known to take place, but how these differences affect cell behavior had not yet been characterized in mammalian tendon cells. In order to investigate this, we have determined the effects of exogenous extracellular matrix and the …

Membrane Vesicles Nucleate Mineralo-Organic Nanoparticles And Induce Carbonate Apatite Precipitation In Human Body Fluids, Cheng-Yeu Wu, Jan Martel, Wei-Yun Cheng, Chao-Chih He, David M. Ojcius, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that …

Development Of A Conditional Mesd (Mesoderm Development) Allele For Functional Analysis Of The Low-Density Lipoprotein Receptor-Related Family In Defined Tissues, Andrew V. Taibi, Janet K. Lighthouse, Richard C. Grady, Kenneth R. Shroyer, Bernadette Holdener

Department of Biomedical Engineering Faculty Publications

The Low-density lipoprotein receptor-Related Protein (LRP) family members are essential for diverse processes ranging from the regulation of gastrulation to the modulation of lipid homeostasis. Receptors in this family bind and internalize a diverse array of ligands in the extracellular matrix (ECM). As a consequence, LRPs regulate a wide variety of cellular functions including, but not limited to lipid metabolism, membrane composition, cell motility, and cell signaling. Not surprisingly, mutations in single human LRPs are associated with defects in cholesterol metabolism and development of atherosclerosis, abnormalities in bone density, or aberrant eye vasculature, and may be a contributing factor in …

Identification Of Leptospira Interrogans Phospholipase C As A Novel Virulence Factor Responsible For Intracellular Free Calcium Ion Elevation During Macrophage Death, Jing-Fang Zhao, Hong-Hu Chen, David M. Ojcius, Xin Zhao, Dexter Sun, Yu-Mei Ge, Lin-Li Zheng, Xu'ai Lin, Lan-Juan Li, Jie Yan

All Dugoni School of Dentistry Faculty Articles

Background: Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca²⁺ concentration ([Ca²⁺]i) elevation induced by infection can cause cell death, but [Ca²⁺]i changes and high [Ca²⁺]i-induced death of macrophages due to infection of Leptospira have not been previously reported.

Methodology/Principal Findings: We first used a Ca²⁺-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca²⁺]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination …

An Analysis Of The Impact Of Storage Temperature, Moisture Content & Duration Upon The Chemical Components & Bioprocessing Of Lignocellulosic Biomass, Arun Athmanathan

Open Access Dissertations

The successful utilization of lignocellulosic biomass as a feedstock for fuels and chemicals necessitates storage for 2-6 months. It is correspondingly important to understand the impact of storage parameters - moisture concentration, temperature and duration - on biomass quality.

As aerobic storage is the most viable large-scale solution, aerobic storage experiments were carried out with three projected bioenergy feedstocks - sweet sorghum (Sorghum bicolor) bagasse, corn (Zea mays) stover and switchgrass (Panicum virgatum). Stored samples of each were examined for dry matter loss and composition change to develop a material balance around carbohydrates and lignin.

A mean dry matter loss …