Biotechnology Commons^™

The Development Of Motuporamine Derivatives And An Investigation Into Their Biological Properties, Kristen Skruber

Electronic Theses and Dissertations

This project investigates the synthesis of a class of compounds derived from a marine-based natural product and probes how iterative changes to its structure affect its derivatives' biological efficacy. The compound class of interest are the motuporamines which were isolated from the sea sponge Xestospongia exigua collected off the coast of Motupore island in Papua, New Guinea. The compounds for this project are predicated upon dihydromotuporamine C (Motu33), the compound that has been shown to be both cytotoxic to MDA-MB231 breast carcinoma cells and has antimetastatic efficacy. The motuporamine scaffold contains a large fifteen-membered saturated macrocycle and an appended polyamine …

Chlamydia Trachomatis Transformants Show A Significant Reduction In Rates Of Invasion Upon Removal Of Key Tarp Domains, Christopher Parrett

Electronic Theses and Dissertations

Chlamydia trachomatis is an obligate, intracellular bacterium which is known to cause multiple human infections including nongonococcal urethritis (serovars D-K), lymphogranuloma venereum (serovars L1, L2, L3) and trachoma (serovars A-C). The infectious form of the bacterium, called the elementary body (EB), harbors a type III secreted effector known as Tarp (translocated actin recruiting phosphoprotein) which is a candidate virulence factor and is hypothesized to play a role in C. trachomatis' ability to invade and grow within epithelial cells in a human host. C. trachomatis L2 Tarp harbors five unique protein domains which include the Phosphorylation Domain, the Proline Rich Domain, …

Neurological Profile Of Older Apoe-Pon1 Double Knockout Mice, Connie Mitra

Electronic Theses and Dissertations

Atherosclerosis is a cardiovascular disease where plaques made up of lipids in the form of cholesterol ester build up in the carotid and innominate arteries that supply blood to the brain. Accumulation of the plaques limit the flow of blood and nutrients to the brain, leading to diminished oxygen supply, increased oxidative stress and cell death. All these have been implicated in Alzheimer's disease (AD). Alzheimer's disease, a chronic, progressive, age related neurodegenerative disorder is the most common form of dementia in the elderly accounting for 60-80% of the cases. Clinically, Alzheimer's disease is characterized by loss of memory, damage …

Apolipoprotein-Ai Regulates Hepatic Vldl Secretion By Controlling Intracellular Vldl-Trafficking, Bhavesh Gurwani

Electronic Theses and Dissertations

Cardiovascular diseases cause 17 million deaths annually, which is estimated to increase to 23 million deaths by the year 2030. One of the major risk factors for the pathogenesis of cardiovascular diseases is increased secretion of very-low density lipoproteins (VLDL) by the liver; however, reduced VLDL-secretion causes fatty liver disease. Synthesis and secretion of VLDL by the liver plays an important role in maintaining overall lipoprotein homeostasis. Assembly of VLDL occurs along with the expression of apolipoproteinB-100 (apoB100) and its lipidation at the endoplasmic reticulum (ER) level. Once formed in the ER lumen, the nascent VLDL is transported to the …

Vo-Ohpic Treatment Reduces Cardiac Remodeling In Doxorubicin-Induced Cardiomyopathy, Taylor Johnson

Electronic Theses and Dissertations

Doxorubicin (Doxo) is one of multiple anthracycline drugs used to effectively treat various forms of cancer. Unfortunately, Doxo treatment, as a side effect, induces cardiomyopathy and subsequent heart failure. We have previously demonstrated that transplanted embryonic stem (ES) cells and their conditioned medium (CM) modulate the PTEN pathway and reduce apoptosis, fibrosis and hypertrophy in a Doxo induced cardiomyopathy (DIC) model. However, mechanisms of inhibited apoptosis mediated through PTEN pathway are completely unknown. Therefore, we used VO-OHpic (VO), a potent PTEN inhibitor to understand the mechanism of apoptosis as well as its effect on cardiac remodeling in DIC. Animals were …

Malondialdehyde (Mda) And Glutathione Peroxidase (Gpx) Are Elevated In Crohns Disease-Associated With Mycobacterium Avium Subspecies Paratuberculosis (Map), Ahmad Qasem

Electronic Theses and Dissertations

Inflamed tissue in Crohn’s disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The level of MAP antibodies in bovine sera was determined by IDEXX kit whereas detection of MAP DNA was performed by IS900-based nPCR. A total of 42 cattle (21 infected with MAP and 21 …

Chloroplast Membrane Remodeling During Freezing Stress Is Accompanied By Cytoplasmic Acidification Activating Sensitive To Freezing 2, Allison C. Barnes, Christoph Benning, Rebecca Roston

Department of Biochemistry: Faculty Publications

Low temperature is a seasonal abiotic stress which restricts native plant ranges and crop distributions. Two types of low temperature stress can be distinguished: chilling and freezing. Much work has been done on the mechanisms by which chilling is sensed, but relatively little is known about how plants sense freezing. Recently, SENSITIVE TO FREEZING 2 (SFR2) was identified as a protein which responds in a non-transcriptional manner to freezing. Here, we investigate the cellular conditions which allow SFR2 activation. Using a combination of isolated organelle, whole tissue and whole plant assays, we provide evidence that SFR2 is activated by changes …

Caloric Restriction Of Db/Db Mice Reverts Hepatic Steatosis And Body Weight With Divergent Hepatic Metabolism, Kyung Eun Kim, Youngae Jung, Soonki Min, Miso Nam, Rok Won Heo, Byeong Tak Jeon, Dae Hyun Song, Chin-Ok Yi, Eun Ae Jeong, Hwajin Kim, Jeonghyun Kim, Seon-Yong Jeong, Woori Kwak, Do Hyun Ryu, Tamas L. Horvath, Gu Seob Roh, Geum-Sook Hwang

Department of Biochemistry: Faculty Publications

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of liver disease and its prevalence is a serious and growing clinical problem. Caloric restriction (CR) is commonly recommended for improvement of obesity-related diseases such as NAFLD. However, the effects of CR on hepatic metabolism remain unknown. We investigated the effects of CR on metabolic dysfunction in the liver of obese diabetic db/db mice. We found that CR of db/db mice reverted insulin resistance, hepatic steatosis, body weight and adiposity to those of db/m mice. H-NMR- and UPLC-QTOF-MS-based metabolite profiling data showed significant metabolic alterations related to lipogenesis, …

Inhibiting Hexamer Disassembly Of Human Udp-Glucose Dehydrogenase By Photoactivated Amino Acid Crosslinking, George Grady, Ashley Thelen, Jaleen Albers, Tong Ju, Jiantao Guo, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD⁺-dependent oxidation steps. Human UGDH apoprotein purifies as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking was achieved by encoding p-benzoyl-L-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing to long wavelength …

Synthesis And Transfer Of Galactolipids In The Chloroplast Envelope Membranes Of Arabidopsis Thaliana, Amélie Kelly, Barbara Kalisch, Georg Hölzl, Sandra Schulze, Juliane Thiele, Michael Melzer, Rebecca L. Roston, Christoph Benning, Peter Dörmann

Department of Biochemistry: Faculty Publications

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol

(DGDG)] are the hallmark lipids of photosynthetic

membranes. The galactolipid synthases MGD1 and DGD1

catalyze consecutive galactosyltransfer reactions but localize to the

inner and outer chloroplast envelopes, respectively, necessitating

intermembrane lipid transfer. Here we show that the N-terminal

sequence of DGD1 (NDGD1) is required for galactolipid transfer

between the envelopes. Different diglycosyllipid synthases (DGD1,

DGD2, and Chloroflexus glucosyltransferase) were introduced into

the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions

(NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction

of DGDG synthesis in the outer envelope membrane was

observed only with diglycosyllipid synthase fusion …

Role Of Apoptotic Hepatocytes In Hcv Dissemination: Regulation By Acetaldehyde, Murali Ganesan, Sathish Kumar Natarajan, Jinjin Zhang, Justin L. Mott, Larisa I. Poluektova, Benita L. Mcvicker, Kusum K. Kharbanda, Dean J. Tuma, Natalie A. Osna

Department of Biochemistry: Faculty Publications

Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, …

Cadmium And Secondary Structure-Dependent Function Of A Degron In The Pca1p Cadmium Exporter, Nathan Smith, Wenzhong Wel, Miaoyun Zhao, Xiaojuan Quin, Javier Seravalli, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting degron that exists at the 250–350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the degron. This suggested that cadmium-mediated masking of the degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the …

Atomic Resolution Experimental Phase Information Reveals Extensive Disorder And Bound 2-Methyl-2,4-Pentanediol In Ca2+-Calmodulin, Jiusheng Lin, Henry Van Den Bedem, Axel T. Brunger, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca²⁺-CaM, but no assignment of these features has been made. In addition, Ca²⁺-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 A resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca²⁺-CaM. …

Mutual Information Upper Bound Of Molecular Communication Based On Cell Metabolism, Massimiliano Pierobon, Zahmeeth Sakkaff, Jennie L. Catlett, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Synthetic biology is providing novel tools to engineer cells and access the basis of their molecular information processing, including their communication channels based on chemical reactions and molecule exchange. Molecular communication is a discipline in communication engineering that studies these types of communications and ways to exploit them for novel purposes, such as the development of ubiquitous and heterogeneous communication networks to interconnect biological cells with nano and biotechnology-enabled devices, i.e., the Internet of Bio-Nano Things. One major problem in synthetic biology stands in the development of reliable techniques to control the engineered cells from the external environment. In this …

Novel Acyltranserases And Methods Of Using, Edgar B. Cahoon, Umidjon Iskandarov, Hae Jin Kim, Jillian Collins-Silva

Department of Biochemistry: Faculty Publications

Provided herein are novel acyltransferases and methods of using Such novel acyltransferases in making medium-chain fatty acids.

An Evaluation Of New And Established Methods To Determine T-Dna Copy Number And Homozygosity In Transgenic Plants, Katarzyna Glowacka, Johannes Kromdijk, Lauriebeth Leonelli, Krishna K. Niyogi, Tom E. Clemente, Stephen P. Long

Department of Biochemistry: Faculty Publications

Stable transformation of plants is a powerful tool for hypothesis testing.Arapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations.Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher …

High-Throughput Mutation, Selection, And Phenotype Screening Of Mutant Methanogenic Archaea, Mary E. Walter, Alicia Ortiz, Casey Sondgeroth, Nathan M. Sindt, Nikolas Duszenko, Jennie L. Catlett, You Zhou, Shah R. Valloppilly, Christopher Anderson, Samodha Fernando, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or …

Nitrogenase Femoco Investigated By Spatially Resolved Anomalous Dispersion Refinement, Thomas Spatzal, Julia Schlesier, Eva-Maria Burger, Daniel Sippel, Limei Zhang, Susana L.A. Andrade, Douglas C. Rees, Oliver Einsle

Department of Biochemistry: Faculty Publications

The [Mo:7Fe:9S:C] iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation. Only recently its atomic structure was clarified, while its reactivity and electronic structure remain under debate. Here we show that for its resting S 1⁄4 3/2 state the common iron oxidation state assignments must be reconsidered. By a spatially resolved refinement of the anomalous scattering contributions of the 7 Fe atoms of FeMoco, we conclude that three irons (Fe1/3/7) are more reduced than the other four (Fe2/4/5/6). Our data are in agreement with the …

Pluralistic And Stochastic Gene Regulation: Examples, Models And Consistent Theory, Elisa N. Salas, Jiang Shu, Matyas F. Cserhati, Donald P. Weeks, Istvan Ladunga

Department of Biochemistry: Faculty Publications

We present a theory of pluralistic and stochastic gene regulation. To bridge the gap between empirical studies and mathematical models, we integrate pre-existing observations with our meta-analyses of the ENCODE ChIP-Seq experiments. Earlier evidence includes fluctuations in levels, location, activity, and binding of transcription factors, variable DNA motifs, and bursts in gene expression. Stochastic regulation is also indicated by frequently subdued effects of knockout mutants of regulators, their evolutionary losses/gains and massive rewiring of regulatory sites. We report wide-spread pluralistic regulation in ≈800 000 tightly co-expressed pairs of diverse human genes. Typically, half of ≈50 observed regulators bind to both …

Tumor Suppressive Role Of Sestrin2 During Colitis And Colon Carcinogenesis, Seung-Hyun Ro, Xiang Xue, Sadeesh K. Ramakrishnan, Chun-Seok Cho, Sim Namkoong, Insook Jang, Ian A. Semple, Allison Ho, Hwan-Woo Park, Yatrik M. Shah, Jun Hee Lee

Department of Biochemistry: Faculty Publications

The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery …