Biochemistry, Biophysics, and Structural Biology Commons^™

Conservation Of Lipid Storage Droplet Proteins In Evolution Of Animalia, Jacob D. Bowman

Undergraduate Honors Thesis Projects

Lipolysis is a conserved pathway used by organisms for the mobilization of fatty acid (FA) out of triacylglycerol (TAG). Neutral lipids are stored in lipid-storage droplets (LSD) which are in nearly every cell of an organism. LSDs are the location of lipolysis. Perilipin proteins are lipid-droplet associated proteins. Perilipins interact with other proteins such as ATGL that breaks down TAG into FA and DAG. Because of its conservation as a whole system, this suggests that proteins of the same pathway are evolving together. This present study traces the evolutionary history of lipolysis-related proteins amongst individual organisms and classes of organisms …

Transfer Rna Comes Of Age, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

"The year the journal RNA was founded was slated by some in scientific publishing to be the year that one particular type of RNA's run in the spotlight would end. In 1995 I had recently started as a post-doc with Dieter Söll at Yale when he came into the lab to solemnly inform us all that an editor at a certain (S)cience journal had just told him “we won't be publishing any more tRNA papers.” For a post-doc who had migrated across the Atlantic for the sole purpose of furthering his career by working on tRNA this was not great …

Introduction To Fifth Special Issue On Electroporation-Based Technologies And Treatments, Damijan Miklavčič, Lluis M. Mir, P. Thomas Vernier

Bioelectrics Publications

This special issue of the Journal of Membrane Biology contains reports on recent developments in the field of electroporation by participants in the International Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments held in November 2014 in Ljubljana. This was the eighth session of what is now an annual event, first organized in 2003.

Electroporation Of Mammalian Cells By Nanosecond Electric Field Oscillations And It's Inhibition By The Electric Field Reversal, Elena C. Gianulis, Jimo Lee, Chunqi Jiang, Shu Xiao, Bennet L. Ibey, Andrei G. Pakhomov

Bioelectrics Publications

The present study compared electroporation efficiency of bipolar and unipolar nanosecond electric field oscillations (NEFO). Bipolar NEFO was a damped sine wave with 140 ns first phase duration at 50% height; the peak amplitude of phases 2-4 decreased to 35%, 12%, and 7% of the first phase. This waveform was rectified to produce unipolar NEFO by cutting off phases 2 and 4. Membrane permeabilization was quantified in CHO and GH₃ cells by uptake of a membrane integrity marker dye YO-PRO-1 (YP) and by the membrane conductance increase measured by patch clamp. For treatments with 1-20 unipolar NEFO, at 9.6-24 …

In Situ Oh Generation From O2- And H2o2 Plays A Critical Role In Plasma Induced Cell Death, Dehui Xu, Dingxing Liu, Biqing Wang, Chen Chen, Zeyu Chen, Dong Li, Yanjie Yang, Hailan Chen, Michael G. Kong

Bioelectrics Publications

Reactive oxygen and nitrogen species produced by cold atmospheric plasma (CAP) are considered to be the most important species for biomedical applications, including cancer treatment. However, it is not known which species exert the greatest biological effects, and the nature of their interactions with tumor cells remains ill-defined. These questions were addressed in the present study by exposing human mesenchymal stromal and LP-1 cells to reactive oxygen and nitrogen species produced by CAP and evaluating cell viability. Superoxide anion (O₂-) and hydrogen peroxide (H₂O₂) were the two major species present in plasma, but their …

Thermodynamics Of Hhr23a Ubiquitin-Associated (Uba) Domains, Moses Joseph Leavens

Graduate Student Theses, Dissertations, & Professional Papers

Ubiquitin-associated (UBA) domains have been discovered in several proteins within the cell, and at least one domain plays an important functional role in cellular phenomena such as NF-κB signaling. Mutations within the UBA(2) domain of the ubiquitin-binding scaffold protein p62 are frequently observed in patients suffering from Paget’s disease of bone. The human DNA excision repair protein HHR23A contains the UBA(1) and UBA(2) domains, and nuclear magnetic resonance (NMR) structures depict their compact three-helix bundles. Moreover, the HHR23A UBA(1) and UBA(2) only share approximately 20% primary sequence identity. The p62 UBA(2) domain has been shown to contain quaternary structure in …

Picosecond To Terahertz Perturbation Of Interfacial Water And Electropermeabilization Of Biological Membranes, P. Thomas Vernier, Zachary A. Levine, Ming-Chak Ho, Shu Xiao, Iurii Semenov, Andrei G. Pakhomov

Bioelectrics Publications

Non-thermal probing and stimulation with subnanosecond electric pulses and terahertz electromagnetic radiation may lead to new, minimally invasive diagnostic and therapeutic procedures and to methods for remote monitoring and analysis of biological systems, including plants, animals, and humans. To effectively engineer these still-emerging tools, we need an understanding of the biophysical mechanisms underlying the responses that have been reported to these novel stimuli. We show here that subnanosecond (≤500 ps) electric pulses induce action potentials in neurons and cause calcium transients in neuroblastoma-glioma hybrid cells, and we report complementary molecular dynamics simulations of phospholipid bilayers in electric fields in which …

Diffuse, Non-Polar Electropermeabilization And Reduced Propidium Uptake Distinguish The Effect Of Nanosecond Electric Pulses, Iurii Semenov, Christian W. Zemlin, Olga N. Pakhomova, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Ca²⁺ activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca²⁺ transients was expectedly higher for nsEP (36 kV/cm)than forms EP (0.09 kV/cm) but the respective doses were similar (190 and460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca²⁺ level by N10 nM in cells with blocked voltage-gated Ca²⁺ channels and depleted Ca²⁺ depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These …

The Role Of Artificial Structures In Facilitating Range Expansion Of The Introduced Barnacle Megabalanus Coccopoma In The Southeastern U.S.A., Alicia M. Reigel

College of Graduate Studies: Theses & Dissertations

The barnacle Megabalanus coccopoma is a recent invader of the southeastern U.S.A. from the tropical eastern Pacific. In Georgia, M. coccopoma populations along the immediate coastline often suffer extensive mortality during the winter, but population rebuilding is common after these events suggesting that there may be nearby larval sources. I investigated the hypothesis that artificial structures (i.e., buoys, towers), occurring far enough offshore of Georgia for water temperatures to be moderated by the Gulf Stream, provide refuges for breeding adults of M. coccopoma and can serve as the larval source. I investigated this hypothesis by first developing thirteen microsatellite primer …

Connecting Proline Metabolism And Signaling Pathways In Plant Senescence, Lu Zhang, Donald F. Becker

Department of Biochemistry: Faculty Publications

The amino acid proline has a unique biological role in stress adaptation. Proline metabolism is manipulated under stress by multiple and complex regulatory pathways and can profoundly influence cell death and survival in microorganisms, plants, and animals. Though the effects of proline are mediated by diverse signaling pathways, a common theme appears to be the generation of reactive oxygen species (ROS) due to proline oxidation being coupled to the respiratory electron transport chain. Considerable research has been devoted to understand how plants exploit proline metabolism in response to abiotic and biotic stress. Here, we review potential mechanisms by which proline …

Chloroplast Lipid Transfer Processes In Chlamydomonas Reinhardtii Involving A Trigalactosyldiacylglycerol 2 (Tgd2) Ortholog, Jaruswan Warakanont, Chia-Hong Tsai, Elena J.S. Michel, George R. Murphy Iii, Peter Y. Hsueh, Rebecca L. Roston, Barbara B. Sears, Christoph Benning

Department of Biochemistry: Faculty Publications

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl-constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologs of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER-tochloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized …

Phenotypic Screening Identifies Brefeldin A/Ascotoxin As An Inducer Of Lipid Storage In The Algae Chlamydomonas Reinhardtii, Nishikant Wase, Boqiang Tu, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

The use of microalgae as a biofuel feedstock is highly desired, but current methods to induce lipid accumulation cause severe stress responses that limit biomass and, thus oil yield. To address these issues, a high throughput screening (HTS) method was devised to identify chemical inducers of growth and lipid accumulation. Optimization was performed to determine the most effective cell density, DMSO and Nile Red (NR) concentrations to monitor growth and lipid accumulation. The method was tested using 1717 compounds from National Cancer Institute (NCI) Diversity Set III and Natural Products Set II in Chlamydomonas reinhardtii. Cells were inoculated at …

Cytotoxic Activity Of Triazole-Containing Alkyl Β-D-Glucopyranosides On A Human T-Cell Leukemia Cell Line, E. Davis Oldham, Larissa M. Nunes, Armando Varela-Ramirez, Stephen E. Rankin, Barbara L. Knutson, Renato J. Aguilera, Hans-Joachim Lehmler

Chemistry and Physics

Simple glycoside surfactants represent a class of chemicals that are produced from renewable raw materials. They are considered to be environmentally safe and, therefore, are increasingly used as pharmaceuticals, detergents, and personal care products. Although they display low to moderate toxicity in cells in culture, the underlying mechanisms of surfactant-mediated cytotoxicity are poorly investigated.

An Investigation Of Bag3 Knockdown And Its Effects On The Cytotoxicity Of Laromustine, Colin Sheehan

Honors Theses

Laromustine is a sulfonylhydrazine anticancer prodrug whose main cytotoxic arises from its ability to interfere with DNA replication of dividing cells. Multiple studies have suggested that Laromustine induces a form of cell death known as apoptosis. In a previous study investigating the mechanism of apoptosis, bcl2-associated athanogene 3 (BAG3) demonstrated significant upregulation in the presence of Laromustine. Given its anticancer ability, we selected BAG3 as a target for further investigation. BAG3 knockdown through transient shRNA transfections was performed in U138 glioblastoma multiforme cells and verified using qRT-PCR analysis. Finally, cell death assays were used to assess Laromustine’s cytotoxic effect on …

Divergent Responses Of Atlantic Coastal And Oceanic Synechococcus To Iron Limitation, Katherine R. M. Mackey, Anton F. Post, Matthew R. Mcilvin, Gregory A. Cutter, Seth G. John, Mak A. Saito

OES Faculty Publications

Marine Synechococcus are some of the most diverse and ubiquitous phytoplankton, and iron (Fe) is an essential micronutrient that limits productivity in many parts of the ocean. To investigate how coastal and oceanic Atlantic Synechococcus strains acclimate to Fe availability, we compared the growth, photophysiology, and quantitative proteomics of two Synechococcus strains from different Fe regimes. Synechococcus strain WH8102, from a region in the southern Sargasso Sea that receives substantial dust deposition, showed impaired growth and photophysiology as Fe declined, yet used few acclimation responses. Coastal WH8020, from the dynamic, seasonally variable New England shelf, displayed a multitiered, hierarchical cascade …

The Relative Importance Of Methanogenesis In The Decomposition Of Organic Matter In Northern Peatlands, J. Elizabeth Corbett, Malak M. Tfaily, David J. Burdige, Paul H. Glaser, Jeffrey P. Chanton

OES Faculty Publications

Using an isotope-mass balance approach and assuming the equimolar production of CO₂ and CH₄ from methanogenesis (e.g., anaerobic decomposition of cellulose), we calculate that the proportion of total CO₂ production from methanogenesis varies from 37 to 83% across a variety of northern peatlands. In a relative sense, methanogenesis was a more important pathway for decomposition in bogs (80 ± 13% of CO₂ production) than in fens (64 ± 5.7% of CO₂ production), but because fens contain more labile substrates they may support higher CH4 production overall. The concentration of CO₂ produced from methanogenesis (CO …

The Geotraces Intermediate Data Product 2014, Edward Mawji, Reiner Schlitzer, Elena M. Dodas, Cyril Abadie, Wafa Abouchami, Robert F. Anderson, Gregory A. Cutter

OES Faculty Publications

The GEOTRACES Intermediate Data Product 2014 (IDP2014) is the first publicly available data product of the international GEOTRACES programme, and contains data measured and quality controlled before the end of 2013. It consists of two parts: (1) a compilation of digital data for more than 200 trace elements and isotopes (TEls) as well as classical hydrographic parameters, and (2) the eGEOTRACES Electronic Atlas providing a strongly inter-linked on-line atlas including more than 300 section plots and 90 animated 3D scenes. The IDP2014 covers the Atlantic, Arctic, and Indian oceans, exhibiting highest data density in the Atlantic. The TEI data in …

Genetic Indicators Of Iron Limitation In Wild Populations Of Thalassiosira Oceanica From The Northeast Pacific Ocean, P. Dreux Chappell, Leann P. Whitney, Joselynn R. Wallace, Adam I. Darer, Samua Jean-Charles, Bethany D. Jenkins

OES Faculty Publications

Assessing the iron (Fe) nutritional status of natural diatom populations has proven challenging as physiological and molecular responses can differ in diatoms of the same genus. We evaluated expression of genes encoding flavodoxin (FLDA1) and an Fe-starvation induced protein (ISIP3) as indicators of Fe limitation in the marine diatom Thalassiosira oceanica. The specificity of the response to Fe limitation was tested in cultures grown under Fe-and macronutrient-deficient conditions, as well as throughout the diurnal light cycle. Both genes showed a robust and specific response to Fe limitation in laboratory cultures and were detected in small volume samples collected from …

Elucidation Of A Novel Pathway In Staphylococcus Aureus: The Essential Site-Specific Processing Of Ribosomal Protein L27, Erin A. Wall

Theses and Dissertations

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are …

Regulation Of Tie2 Extracellular Complex Formation In Angiogenesis, Annamarie Dalton

Theses and Dissertations

Pathological angiogenesis is an essential component of tumor growth, development, and metastasis for which few effective therapeutic options exist. Though many cancer therapies target the function of cell surface receptors, mechanisms regulating membrane receptor crosstalk remain unclear. Two important families of receptors in angiogenesis, the Ties and Integrins, respond to the extracellular environment via outside-in and, in the case of Integrins, also inside- out signaling. Recent reports showed that the endothelial specific tyrosine kinase receptor, Tie2, forms complexes with two of the endothelial Integrin heterodimers, α₅β₁ and α_Vβ₃, providing a convenient mechanism for …

Mysteries Of The Trypanosomatid Maxicircles: Characterization Of The Maxicircle Genomes And The Evolution Of Rna Editing In The Order Kinetoplastida, Preethi Ranganathan Iyengar

Theses and Dissertations

The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates.

This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 …

Characterization Of A Novel Protease In Staphylococcus Aureus, Adam L. Johnson

Theses and Dissertations

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms …

Numerical Geometry Of Map And Model Assessment, Willy Wriggers, Jing He

Mechanical & Aerospace Engineering Faculty Publications

We are describing best practices and assessment strategies for the atomic interpretation of cryo-electron microscopy (cryo-EM) maps. Multiscale numerical geometry strategies in the Situs package and in secondary structure detection software are currently evolving due to the recent increases in cryo-EM resolution. Criteria that aim to predict the accuracy of fitted atomic models at low (worse than 8 angstrom) and medium (4-8 angstrom) resolutions remain challenging. However, a high level of confidence in atomic models can be achieved by combining such criteria. The observed errors are due to map-model discrepancies and due to the effect of imperfect global docking strategies. …

The Role Of Rnai In Downregulation Of Physcomitrella Patens Defense Genes And The Preliminary Steps Of Peg Mediated Transformation Of Mosses, Adam Beswick

Undergraduate Honors Thesis Collection

When attacked by a pathogen, the moss Physcomitrella patens will undergo both a hypersensitive response (HR) as well as a systematic acquired resistance (SAR) response. The SAR response turns on genes that ready the plant for future pathogen attacks. The hormone jasmonic acid (JA) is one of the products of SAR response in P. patens. In this study interference RNA (RNAi) was used to decrease gene expression of the allene oxide cyclase (ACe) gene, a key gene in the production pathway of JA. Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), RNAi was found to significantly lower ACC gene levels …

Annotation And Transcription Start Site Analysis Of Contig70 In Drosophila Biarmipes, Robin Wolschendorf

Honors Projects

The following is my annotation and transcription start site analysis report for contig70 in D. biarmipes to be submitted to the Genomics Education Partnership (GEP) at Washington University in St. Louis. The report was completed in accordance with the guidelines and template set forth by the GEP. There are eight complete genes found in contig70, for which I found the protein coding exon boundaries as well as the transcription start sites for all isoforms. All conclusions and reasoning for them are outlined in the report.

The GEP does genomics research, specifically comparing the genomes of all species of Drosophila. …

Studying Cellular And Molecular Interaction With Two-Photon Fluorescence Microscopy, Arifur Rahaman

Open Access Theses & Dissertations

Advances in microscopy and fluorescent probes provide new insight into studying cellular and molecular interactions. Two-photon fluorescence microscopy is one of the most important recent inventions in cellular and molecular study. This technology enables noninvasive study at cellular and molecular levels in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. In this study, we used our two-photon microscope to observe predatorial behavior of fast moving bacterivorous marine organism Cafeteria roenbergensis and quantify Mycobacterium …

Self-Cleaning Applications Of Tio2 By Photo-Induced Hydrophilicity And Photocatalysis, Swagata Banerjee, Dionysios Dionysiou, Suresh Pillai

Articles

Self-cleaning materials have gained considerable attention for both their unique properties and practical applications in energy and environmental areas. Recent examples of many TiO2-derived materials have been illustrated to understand the fundamental principles of self-cleaning hydrophilic and hydrophobic surfaces. Various models including those proposed by Wenzel, Cassie-Baxter and Miwa-Hashimoto are discussed to explain the mechanism of self-cleaning. Examples of semiconductor surfaces exhibiting the simultaneous occurrence of superhydrophilic and superhydrophobic domains on the same surface are illustrated, which can have various advanced applications in microfluidics, printing, photovoltaic, biomedical devices, anti-bacterial surfaces and water purification.

Several strategies to improve the efficiency of …

Raman Micro-Spectroscopy For Rapid Screening Of Oral Squamous Cell Carcinoma, Luis Felipe Carvalho, Franck Bonnier, Kate O'Callaghan, Jeff O'Sullivan, Stephen Flint, Hugh Byrne, Fiona Lyng

Articles

Raman spectroscopy can provide a molecular-level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution and could be useful to monitor changes in composition for dysplasia and early, non-invasive cancer diagnosis (carcinoma in situ), both ex-vivo and in vivo. In this study, we demonstrate this potential by collecting Raman spectra of nucleoli, nuclei and cytoplasm from oral epithelial cancer (SCC- 4) and dysplastic (pre-cancerous, DOK) cell lines and from normal oral epithelial primary cell cultures, in vitro, which were then analysed by principal component analysis (PCA) as a multivariate statistical method to discriminate the spectra. Results …

Identification, Description, And Activity Of Proteins In The Tergal Glands Of The German Cockroach, Blattella Germanica (L.), Aaron Myers

Open Access Theses

German cockroaches are important urban pests that have been linked to asthma and serious allergic reactions in sensitized individuals. In this research project I, (i) identified different proteins expressed in the tergal glands of male German cockroaches, (ii) determined the expression levels of these proteins in different cockroach life stages and tissues, and (iii) investigated the role of the tergal gland alpha-amylase (BGTG-1) protein. ^ Four major tergal gland proteins were separated on denaturing polyacrylamide gels. With peptide sequencing two of these proteins were identified as alpha-amylase (BGTG-1) and Blattella germanica allergen 2 (Bla g 2). Both of these proteins …

Transcriptional Stress Response In Foodborne Pathogens, Aaron M. Pleitner

Open Access Dissertations

Novel molecular method use is leading to better identification and understanding of microbial presence in food and food processing environments. The application of molecular methods targeting foodborne pathogens provides genetic profiling and targeted intervention methods. Whole-genome sequencing (WGS) provides information on the genomic uniqueness of specific pathogens and is being implemented in epidemiological investigations. Transcriptomic profiling allows for tracking the differential expression of genes and elucidating stress responses. Effective methods and treatments require an understanding of intervention (e.g. sanitizers) and treatment (e.g. antibiotics) mechanisms and efficacies. Accurate novel molecular method use requires proper learning of theory and data management. ^ …