Biotechnology Commons^™

Dna Purification Process Optimization At Life Technologies Corporation, Trevor J. Shepherd

Master's Theses

This project focused on optimizing the plasmid DNA purification process at Life Technologies. These plasmids are designed to code for specialized proteins used by research universities, national laboratories, or research companies. Once cultivated and harvested, the plasmids must be analyzed for quality and quantity. The project is divided into improving three aspects of the process: 1) plasmid identification, 2) plasmid purity evaluation, and 3) process yield. Plasmid identification is now simpler, more robust and has zero ambiguity. Plasmid purity evaluation is now measured with computer software, which reduces user error and eliminates subjectivity. Using the nascent metrics provided by the …

Validation Of Antibodies Used To Study Hypoxia Inducible Factors In Two Species Of Fundulus, Jenna D. Hill

LSU New Orleans Theses and Dissertations

Hypoxia inducible factors (HIFs) are transcription factors and the master regulators of oxygen-dependent gene expression in animals. The focus of this thesis is the distribution of HIF protein in tissues of the fish Fundulus heteroclitus and F. grandis, two widespread species that occur in naturally hypoxic waters. Polyclonal antibodies against HIF-1α, HIF-2α, and HIF-3α were tested on proteins made in vitro and on extracts made from several tissues of normoxic and hypoxic fish. Antibodies against HIF-1α and 3α bound specifically to full length protein made in vitro, and produced bands on western blots of nuclear extracts of near …

Regulation Of The Target Of Rapamycin Signaling Pathway In Saccharomyces Cerevisiae, Tammy Pracheil

LSU New Orleans Theses and Dissertations

An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and in vivo protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, Saccharomyces cerevisiae. Mutations in SAC7 and FAR11 suppressed lethality of both lst8 and tor2-21 mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2.

Far11, a component of a six-member complex, was found to interact …

Aerobic Degradation Of Α-, Β-, Γ-Hexachlorocyclohexane By Narragansett Bay Bacterioplankton, Ian M. Rambo

Senior Honors Projects

Hexachlorocyclohexanes (HCHs) are a family of chlorinated organic compounds that were previously used as agricultural insecticides. HCHs are recognized as persistent organic pollutants due to their toxicity, recalcitrant properties, and tendency to bioaccumulate in food webs. Although HCH was first synthesized in 1825, its use was not widespread until the discovery of the insecticidal activity of the γ-HCH isomer in 1942. γ-HCH and its toxic waste isomers α-HCH and β-HCH were banned from production and use by the United Nations in 2009, yet these chemicals still present environmental problems due to their persistence in soils and surface waters. HCHs continue …

Expression, Production, And Purification Of Novel Therapeutic Proteins, Mckinzie Shea Fruchtl

Graduate Theses and Dissertations

Interest in the production of recombinant proteins consisting of collagen binding domain (CBD) fused to a bioactive material has increased due to the targeting/attachment capabilities of CBD. For example, CBD fusions can be applied to the reversing of bone density loss and the repair of the eardrum, specifically, by choosing an appropriate fusion partner (parathyroid hormone or epidermal growth factor). The production of CBD fusions was examined using batch and fed-batch culturing of Escherichia coli to express the fusion proteins, and affinity chromatography to isolate the final product.

Different medium formulations, feeding strategies, and induction methods were tested in order …

Biomass Gasification: An Alternative Solution To Animal Waste Management, Hanjing Wu

Department of Agricultural and Biological Systems Engineering: Dissertations, Theses, and Student Research

The overall goal of this research was to evaluate gasification of animal waste as an alternative manure management strategy, from the standpoints of syngas production and biochar application.

To meet the overall objective, the thermogravimetric characteristics of dairy manure, as a thermochemical conversion feedstock, were studied firstly. Then, gasification technology was applied to dairy manure and feedlot manure using a fluidized-bed laboratory-scale gasifier. In addition, biochar derived from the feedlot manure was examined for its effects on nutrient leaching as a soil amendment. Finally, a life cycle assessment was conducted to evaluate greenhouse gas emissions of two feedlot manure management …

A Cell Simulator Platform: The Cell Collective, Tomáš Helikar, B. Kowal, J. A. Rogers

Department of Biochemistry: Faculty Publications

Diseases are often a result of multiple malfunctions in complex, nonlinear biological/biochemical networks. As such, these processes are far more complicated to understand because they tend to give rise to functions that are emergent in nature, i.e., higher-level (mal)functions that are more than the sum of their parts. Systems biology provides a new approach to understanding biological systems and diseases from a holistic perspective.

The Geometry And Sensitivity Of Ion-Beam Sculpted Nanopores For Single Molecule Dna Analysis, Ryan Connor Rollings

Graduate Theses and Dissertations

In this dissertation, the relationship between the geometry of ion-beam sculpted solid-state nanopores and their ability to analyze single DNA molecules using resistive pulse sensing is investigated. To accomplish this, the three dimensional shape of the nanopore is determined using energy filtered and tomographic transmission electron microscopy. It is shown that this information enables the prediction of the ionic current passing through a voltage biased nanopore and improves the prediction of the magnitude of current drop signals when the nanopore interacts with single DNA molecules. The dimensional stability of nanopores in solution is monitored using this information and is improved …

Steered Molecular Dynamics Simulations Of A Type Iv Pilus Probe Initial Stages Of A Force-Induced Conformational Transition, Joseph L. Baker, Nicolas Biais, Florence Tama

Publications and Research

Type IV pili are long, protein filaments built from a repeating subunit that protrudes from the surface of a wide variety of infectious bacteria. They are implicated in a vast array of functions, ranging from bacterial motility to microcolony formation to infection. One of the most well-studied type IV filaments is the gonococcal type IV pilus (GC-T4P) from Neisseria gonorrhoeae, the causative agent of gonorrhea. Cryo-electron microscopy has been used to construct a model of this filament, offering insights into the structure of type IV pili. In addition, experiments have demonstrated that GC-T4P can withstand very large tension forces, and …

An Integrated Research Approach To The Development Of Fungal Isolates As Bioherbicides For The Control Of Woody Weeds, Patrick Mershon

Independent Study Project (ISP) Collection

Prickly acacia (Acacia nilotica) is an invasive weed tree in Australia, ranked seventh on the list of Weeds of National Significance because of its aggressiveness and its economic and environmental impacts. The significant downsides to manual and chemical control of this species drove the search for a biological control mechanism. Surveying and sampling stands of A. nilotica naturally exhibiting disease symptoms yielded several native Australian fungi capable of pathogenesis in this species, which are currently under assessment as potential bioherbicides for this weed. Three field trials were established at the same site in order to further advance current …

The Unstable Ground Of Low Hierarchies, Joshua Dinsmore

The Transdisciplinary STEAM+ Journal

Broad Vision is a collaborative project between the Sciences and Arts. It involves students and lecturers from six different departments, across three schools at the University of Westminster, London, UK. In the first year of the project we worked with the microscope as the locus for our interconnections.

A Single Point Mutation In The Listerial Betl Sigma(A)-Dependent Promoter Leads To Improved Osmo- And Chill-Tolerance And A Morphological Shift At Elevated Osmolarity, Ronald F. Hoffman, Susan Mclernon, Audrey Feeney, Colin Hill, Roy D. Sleator

Department of Biological Sciences Publications

Betaine uptake in Listeria monocytogenes is mediated by three independent transport systems, the simplest of which in genetic terms is the secondary transporter BetL. Using a random mutagenesis approach, based on the E. coli XL1 Red mutator strain, we identified a single point mutation in a putative promoter region upstream of the BetL coding region which leads to a significant increase in betL transcript levels under osmo- and chill-stress conditions and a concomitant increase in stress tolerance. Furthermore, the mutation appears to counter the heretofore unreported “twisted” cell morphology observed for L. monocytogenes grown at elevated osmolarities in tryptone soy …

Detection And Quantification Of Methylation In Dna Using Solid-State Nanopores., Jiwook Shim, Gwendolyn I Humphreys, Bala Murali Venkatesan, Jan Marie Munz, Xueqing Zou, Chaitanya Sathe, Klaus Schulten, Farhad Kosari, Ann M Nardulli, George Vasmatzis, Rashid Bashir

College of Science & Mathematics Departmental Research

Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single …

Functional Heterologous Expression Of An Engineered Full Length Cipa From Clostridium Thermocellum In Thermoanaerobacterium Saccharolyticum, Devin H. Currie, Christopher D. Herring, Adam M. Guss, Daniel G. Olson, David A. Hogsett, Lee R. Rynd

Dartmouth Scholarship

Background: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. Results:We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to …

Colloidal Nano-Apatite Particles With Active Luminescent And Magentic Properties For Biotechnology Applications, Rajendra Kasinath, Kumar Ganesan

Environmental Engineering

Colloidal Nano-apatite Particles with Active Luminescent and Magentic Properties for Biotechnology Applications. The synthesis of functional nano-materials is a burgeoning field that has produced remarkable and consistent breakthroughs over the last two decades. Individual particles have become smaller and shown potential for well defined functionality. However, there are still unresolved problems, a primary one being the loss of functionality and novelty due to uncontrolled aggregation driven by surface energy considerations. As such the first design criteria to harness the true potential of nanoparticles is to prevent unwanted agglomeration by: (1) improving, and, if possible, (2) controlling aggregation behavior. This requires …

Disocvering Ionic Liquid Resistant Genes, Bree Person, Douglass Higgins, Michael Thelen

STAR Program Research Presentations

: Plant biomass is a rich source of sugars that can be converted to biofuels by engineered microbes. However, because the lignocellulose in biomass is insoluble in aqueous conditions and recalcitrant to enzymatic degradation, thermochemical treatment is required to break apart the lignin and cellulose polymers before sugars can be released. The most effective chemicals for doing this are known as ionic liquids, which are salts that are molten at temperatures below 100° C. Although these solvents have many unique properties that are ideal for solubilizing lignocellulose, they have been found to inhibit the growth of bacterial strains used to …

Intellectual Property And Opportunities For Food Security In The Philippines, Jane Payumo, Howard Grimes, Antonio Alfonso, Stanley P. Kowalski, Keith Jones, Karim Maredia, Rodolfo Estigoy

Law Faculty Scholarship

By 2050, the Philippine population is projected to increase by as much as 41 percent, from 99.9 million to nearly 153 million people. Producing enough food for such an expanding population and achieving food security remain a challenge for the Philippine government. This paper argued that intellectual property rights (IPR) can play a key role in achieving the nation’s current goal to be food-secure and provided examples to illustrate that the presence of sound intellectual property (IP) helps foster research, development, and deployment of agricultural innovations. This paper also offered key recommendations about how the IP system can be further …

Mitochondrial Glutathione Transport: Implications For Bcl-2 And Neuronal Survival, Heather Marie Wilkins

Electronic Theses and Dissertations

Oxidative stress is a contributing factor to many neurodegenerative diseases. In particular, mitochondria are a key source of oxidative stress due to electron leakage at the level of the electron transport chain. To combat the endogenous production of reactive oxygen and reactive nitrogen species mitochondria are equipped with several redox-cycling systems, such as glutathione (GSH). Mitochondrial GSH has been shown to be a critical reservoir of this antioxidant, where selective depletion of mitochondrial GSH can induce apoptosis in several systems. Many studies have intricately linked Bcl-2 to cellular GSH status and it has been previously shown that Bcl-2 is a …

The National Institutes Of Health, Patents, And The Public Interest: An Expanded Rationale Of Justice Breyer’S Dissent In Stanford V. Roche, Nida Shakir

Marquette Intellectual Property Law Review

In February 2010, the Alzheimer’s Institute of America (AIA) filed a patent infringement lawsuit against Jackson Laboratory, the largest repository of research mice in the world. AIA sued Jackson Laboratory for infringing on AIA’s patent covering a DNA mutation linked to Alzheimer’s disease. Jackson Lab allegedly violated that patent by distributing mice especially bred for Alzheimer’s research. READ MORE, download the article.

Proline Mechanisms Of Stress Survival, Xinwen Liang, Lu Zhang, Sathish Kumar Natarajan, Donald F. Becker

Department of Biochemistry: Faculty Publications

Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria …

Comparative Metabolic Profiling Of Haberlea Rhodopensis, Thellungiella Halophyla, And Arabidopsis Thaliana Exposed To Low Temperature, Maria Benina, Toshihiro Obata, Nikolay Mehterov, Ivan Ivanov, Veselin Petrov, Valentina Toneva, Alisdair R. Fernie, Tsanko S. Gechev

Department of Biochemistry: Faculty Publications

Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea's remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4^◦C) and subsequent return to optimal temperatures (21^◦C) was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive Arabidopsis thaliana. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21^◦C. H. rhodopensis …

Is Breakdown Of Fatty Acid Peroxides Involved In The Induction Of Apolipoprotein A1?, Rajat Gupta

Electronic Theses and Dissertations

Over the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the susceptibility of serum lipoproteins to undergo oxidation, thereby contributing to the progression of atherosclerosis. The intestinal cells are responsible for the absorption of dietary fatty acid …

Next-Peak: A Normal-Exponential Two-Peak Model For Peak-Calling In Chip-Seq Data, Nak-Kyeong Kim, Rasika V. Jayatillake, John L. Spouge

Mathematics & Statistics Faculty Publications

Background: Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) can locate transcription factor binding sites on genomic scale. Although many models and programs are available to call peaks, none has dominated its competition in comparison studies.

Results: We propose a rigorous statistical model, the normal-exponential two-peak (NEXT-peak) model, which parallels the physical processes generating the empirical data, and which can naturally incorporate mappability information. The model therefore estimates total strength of binding (even if some binding locations do not map uniquely into a reference genome, effectively censoring them); it also assigns an error to an estimated binding location. The comparison study …

The Contested Vision For Agriculture's Future: Sustainable Intensive Agriculture And Agroecology, Drew L. Kershen

Faculty Articles

No abstract provided.

Response To "Sodium Current Inhibition By Nanosecond Pulsed Electric Field (Nspef) - Fact Or Artifact?" By Verkerk Et Al, Andrei G. Pakhomov

Bioelectrics Publications

It was nice to learn that our studies of nanosecond pulsed electric field (nsPEF) effects on membrane currents [Nesin et al., 2012; Nesin and Pakhomov, 2012] gained the attention of scientists outside the immediate field of bioelectromagnetics.

The Hyaluronan Receptor For Endocytosis (Hare) Activates Nf-Κb-Mediated Gene Expression In Response To 40–400-Kda, But Not Smaller Or Larger, Hyaluronans, Madhu S. Pandey, Bruce A. Baggenstoss, Jennifer Washburn, Edward N. Harris, Paul H. Weigel

Department of Biochemistry: Faculty Publications

Background: HARE mediates systemic clearance of hyaluronan (HA), which turns over continuously in tissues.

Results: HARE uptake of 40–400-kDa, but not larger or smaller, HA stimulated NF-κB activation.

Conclusion: HA-HARE signal complexes activate NF-κB and gene transcription only with optimally sized HA.

Significance: HARE responsiveness to a narrow size range ofHAdegradation products may be a sensing system to detect tissue ECM stress.

Electric Field Standing Wave Effects In Ft-Ir Transflection Spectra Of Biological Tissue Sections: Simulated Models Of Experimental Variability, Tomasz P. Wrobel, Barbara Wajnchold, Hugh Byrne, Malgorzata Baranska

Articles

The so-called electric field standing wave effect (EFSW) has recently been demonstrated to significantly distort FT-IR spectra acquired in a transflection mode, both experimentally and in simulated models, bringing into question the appropriateness of the technique for sample characterization, particularly in the field of spectroscopy of biological materials. The predicted effects are most notable in the regime where the sample thickness is comparable to the source wavelength. In this work, the model is extended to sample thicknesses more representative of biological tissue sections and to include typical experimental factors which are demonstrated to reduce the predicted effects. These include integration …

Using Printed Anhydrous Carbohydrate Glass To Create Water Soluble Scaffolding For Use In Tissue Engineering, Tanner Carden, Devon Bane, Courtney Bane, Gavon Carden

Summer Community of Scholars Posters (RCEU and HCR Combined Programs)

No abstract provided.

The Effects Of Hydrostatic Pressure On Early Endothelial Tubulogenic Processes, Ryan M. Underwood

Theses and Dissertations--Biomedical Engineering

The effects of mechanical forces on endothelial cell function and behavior are well documented, but have not been fully characterized. Specifically, fluid pressure has been shown to elicit physical and chemical responses known to be involved in the initiation and progression of endothelial cell-mediated vascularization. Central to the process of vascularization is the formation of tube-like structures. This process—tubulogenesis—is essential to both the physiological and pathological growth of tissues. Given the known effects of pressure on endothelial cells and its ubiquitous presence in the vasculature, we investigated pressure as a magnitude-dependent parameter for the regulation of endothelial tubulogenic activity. To …

Role Of Host Immune Response And Bacterial Autolysin Atl In Human Nasal Colonization By Staphylococcus Aureus, Vanathy Paramanandam

Electronic Theses and Dissertations

Staphylococcus aureus (SA) is a major human pathogen that colonizes the anterior nares in 30% of the human population. Though nasal carriage of SA is a known risk factor for the subsequent spread of SA infections, the dynamics of SA nasal colonization is poorly understood. Our research focuses on understanding the host and bacterial factors that might contribute to the human nasal colonization by SA. In an attempt to elucidate the host response to SA, we performed an autologous human in vivo nasal colonization study, which showed decreased survival rates of SA in hosts who elicited a robust immune response. …