Molecular Biology Commons^™

The Function Of Carmil1 In Migrating Cells, Marc Edwards

All Theses and Dissertations (ETDs)

This dissertation describes the physiological role of the Capping Protein- CARMIL interaction in migrating cells. I establish the CARMIL-CP complex as a key regulator of lamellipodial actin assembly and of lamellipodial dynamics. Membrane ruffling at the leading edge of motile cells and macropinocytosis were also found to be dependent on the CARMIL1-CP interaction. This is consistent with macropinocytosis and ruffling being dependent on a functional and dynamic lamellipodium.: Kerr and Teasdale, 2009).

In chapter two I demonstrate that the CBR of CARMIL1 is competent to inhibit CP in cells. I show that overexpression of the CBR in cells leads to …

The Effects Of Salinity, Ph, Temperature, And Dissolved Oxygen On Sensitivity Of Pcr Identification Of T4 Bacteriophage, Joesph F. Cannon, Nicholas A. Thurn, Paul E. Richardson

Journal of the South Carolina Academy of Science

Bacteriophages are used as indicators of pathogenic bacteria in drinking, and wastewaters. They also show potential in limiting aquatic bacterial populations through their lytic properties. The effect of different water characteristics (salinity, pH, dissolved oxygen, and temperature) on the sensitivity of the PCR identification of virus particles were analyzed to determine at what levels bacteriophage can be detected in environmental samples. Results from this preliminary study indicate that a PCR bacteriophage detection technique has potential as a relatively efficient and economical indicator of coliform contamination in multiple aquatic environments. While further evaluation is needed, the protocol appears to function in …

Valproic Acid Causes Proteasomal Degradation Of Dicer And Influences Mirna Expression, Zhaiyi Zhang, Paolo Convertini, Manli Shen, Xiu Xu, Frédéric Lemoine, Pierre De La Grange, Douglas A. Andres, Stefan Stamm

Molecular and Cellular Biochemistry Faculty Publications

Valproic acid (VPA) is a commonly used drug to treat epilepsy and bipolar disorders. Known properties of VPA are inhibitions of histone deacetylases and activation of extracellular signal regulated kinases (ERK), which cannot fully explain VPA's clinical features. We found that VPA induces the proteasomal degradation of DICER, a key protein in the generation of micro RNAs. Unexpectedly, the concentration of several micro RNAs increases after VPA treatment, which is caused by the upregulation of their hosting genes prior to DICER degradation. The data suggest that a loss of DICER protein and changes in micro RNA concentration contributes to the …

Molecular Evolution Of Protein-Rna Mimicry As A Mechanism For Translational Control, Assaf Katz, Lindsey Solden, S. Betty Zou, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-β-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the …

The Glycine And Proline Reductase Systems: An Evolutionary Perspective And Presence In Enterobacteriaceae, Joshua Witt

HIM 1990-2015

The Glycine and Proline Reduction systems are two of the best characterized selenoenzymes in bacteria and have been found to occur in a wide variety of clostridia [1-5]. These enzymes are utilized to reduce glycine or D-proline to obtain energy via substrate level phosporylation or membrane gradients, respectively [6, 7]. This includes the pathogens C. difficile and C. botulinum [5, 8]. Strains of C. difficile are activate toxigenic pathways whenever either of these pathways is active within the cell [5, 8]. Though evolutionary studies have been conducted on ammonia producing bacteria [9] none has been done to directly characterize these …

Development Of Novel Subunit Vaccine Against H5n1 Influenza, Lu Zhang

Graduate Theses and Dissertations

Influenza is a common infectious disease resulting from a frequently mutated RNA virus. Vaccination is currently the most effective method to prevent people from seasonal or pandemic influenza. The production of traditional egg-based influenza vaccine is time-consuming and provides limited effect against new strains. Therefore, it is necessary to develop a rapid method to produce influenza vaccines. We proposed a novel influenza vaccine based on the E.coli expression system. Hemagglutinin (HA) is the major target surface protein of influenza virus for vaccine development. In this study, we sub-cloned the HAs encoding gene into an E. coli expression vector; the signal …

Prolactin And Isplatin Combination Treatment Inhibit Tumorspheres Formation And Tumor Growth In Mice, Eric Hingleung Lee

All Dissertations

Cancer stem cells (CSCs) are defined as a small population of tumor initiating cells that are responsible for the initiation, development, progression, and recurrence of cancer. The chemo and radiation resistance of CSCs remains one of the major obstacles in conventional anti-cancer therapies. One of the reasons that conventional chemotherapeutics are not effective in targeting CSCs is that CSCs are usually in a non-proliferative or dormant state. In this perspective, targeting CSCs by inducing its proliferation and differentiation and simultaneously applying chemotherapeutics may be an alternative approach. The current study investigates the effect of prolactin (PRL), a hormone intimately involved …

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 …

Characterization Of The Rna Binding And Rna Degrading Subunits Of The Eukaryotic Exosome, Borislava Tsanova

Dissertations and Theses (Open Access)

The exosome is an essential complex of ten proteins involved in the processing and degradation of many RNAs in the cell. These include various stable RNAs, mRNAs, and aberrant transcripts both in the nucleus and in the cytoplasm.

In this work I characterize the three members of the exosome “cap”, the RNA binding proteins Rrp4, Rrp40, and Csl4. I determine that in spite of their structural similarity, they each have a unique essential role. Second, I determine that two of the cap proteins Rrp4 and Rrp40 have a role in bridging subunits of the PH ring of the exosome. The …

New Insights Into The Roles Of Human Dna Damage Checkpoint Protein Atr In The Regulation Of Nucleotide Excision Repair And Dna Damage-Induced Cell Death, Zhengke Li

Electronic Theses and Dissertations

Integrity of the human genome is frequently threatened by endogenous and exogenous DNA damaging reagents that may lead to genome instability and cancer. Cells have evolved multiple mechanisms to repair DNA damage or to eliminate the damaged cells beyond repair and to prevent diverse diseases. Among these are ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage checkpoint and nucleotide excision repair (NER) that are the major pathways by which cells handle ultraviolet C (UV-C)- or other exogenous genotoxin-induced bulky DNA damage. However, it is unclear how these 2 pathways may be coordinated. In this study we show that ATR physically interacts …

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. …

The Development Of An In Vivo Microdialysis Collection Method Of Cytokines From Brain Tissue, Anthony W. Herbaugh

Graduate Theses and Dissertations

In this thesis, different methods to improve the microdialysis collection procedure for cytokines from brain tissue are presented. The first method was based on stopped flow and results indicating that no significant difference in relative recovery between stopped flow and continuous flow are shown. The second method is an antibody bead-based enhancement method. With the antibody bead-based method, a 3.5 fold increase in the collected concentrations of Chemokine (C-C motif) Ligand 2 (CCL2) were observed. However, there was no significant increase in the in vivo collection efficiency of Interleukin-6 (IL-6) using the antibody enhancement. Finally the development of an in-house …

The Role Of Histone H3 And H4 In Centromere Function And Genome Integrity, Payel Chaudhuri

Graduate Theses and Dissertations

Histone H2A plays an important role in chromosomal segregation among parent and daughter cells during mitosis. While it is established that this histone is important in maintaining chromosome number in cell, further work is carried out to explore the role of other histones like H3 and H4 for similar effects. A systematic study is initiated by screening a library based on mutation of different amino acid residues in these histones. This detailed screening identified specific regions within H3 and H4, which are critically important for centromeric function. These histones residing near the DNA entry/exit region of nucleosome effects the functionality …

Molecular And Biochemical Mechanisms Of Pathogenesis In The Maize Foliar Pathogen Cercospora Zeae-Maydis, Winfred-Peck Dorleku

Graduate Theses and Dissertations

GLS is a serious foliar disease of maize, a major staple crop grown commercially in the USA for both human and animal feed production, and increasingly, for ethanol production. The disease is caused by two Cercospora species, C. zeae-maydis and C. zeina, both of which infect maize in the USA and in other parts of the world, with yield losses potentially greater than 50%, depending on local conditions. In culture, C. zeae-maydis produces a phytotoxic, host non-specific perylenequinone, cercosporin, and abscisic acid (ABA), for which there is no known pathological or physiological function in the fungus. Experimental evidence indicates …

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. …

Cooperative Tumor Suppression By Arf And P53, Jason Thomas Forys

All Theses and Dissertations (ETDs)

Cancer is a complex genetic disease characterized by the inactivation of tumor suppressor genes and enhanced activity of oncogenes leading to deregulated cellular proliferation. Two tumor suppressor genes, p53 and Arf, play important roles in protecting cells against numerous biological stresses. In response to oncogenic signals, increased ARF expression leads to the activation of p53, which in turn leads to the cessation of cell division or induction of an apoptotic response. Interestingly, p53 coordinates repression of Arf transcription, setting up a negative feedback loop with currently unknown physiological significance. Cells that lack p53 express elevated levels of ARF, but it …

Homeotic Gene Teashirt (Tsh) Has A Neuroprotective Function In Amyloid-Beta 42 Mediated Neurodegeneration, Michael T. Moran, Meghana Tare, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Background: Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Aβ42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive.

Methodology/Principal Findings: We have generated a transgenic Drosophila eye model where …

Gene Expression Studies For The Analysis Of Domoic Acid Production In The Marine Diatom Pseudo-Nitzschia Multiseries, Katie Boissonneault, Brooks M. Henningsen, Stephen S. Bates, Deborah L. Robertson, Sean Milton, Jerry Pelletier, Deborah A. Hogan, David E. Housman

Dartmouth Scholarship

Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions.

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 …

Bacterial Transcription Factors Grea, Greb And Dksa: Characterization Of Their Interaction With Rna Polymerase And Molecular Mechanism Of Action, Andrey Parshin

Graduate School of Biomedical Sciences Theses and Dissertations

The activity of RNA polymerase (RNAP), the key enzyme of transcription process, is regulated by a large number of transcription factors acting at each step of the transcription cycle. Our research project is focused on studying the structure and function of bacterial transcript cleavage factors GreA, GreB and the stringent response regulator DksA. Gre factors bind in the secondary channel of RNAP in backtracked (inactive) ternary elongation complexes and stimulate the enzyme's intrinsic RNase activity, which is required for suppression of transcription pause and arrest, enhancement of transcription fidelity, and efficient promoter escape. DksA also acts through the RNAP secondary …

Genetic Engineering Of Black Cherry (Prunus Serotina) For Reproductive Sterility And Insect Pest Resistance, Ying Wang

Open Access Dissertations

Black cherry (Prunus serotina Ehrh.) is one of the most valuable hardwoods for high- end cabinetry, furniture, architectural millwork, paneling, and veneer. However, the damage caused by cambial-mining insect pests triggers gummosis in black cherry, a non-specific defense response in which resinous gum is deposited at the site of injury. The gum defects dramatically decrease the yield of high-quality black cherry lumber, and the value can be reduced by as much as 90%. The goal of this project was to optimize the transformation and in vitro rooting system, and to develop transgenic black cherry for reproductive sterility and insect pest …

Characterization Of Caxck31, A Bacterial Calcium/Proton Antiporter, Marc Robert Ridilla

Open Access Dissertations

To better understand a class of transporters known as Calcium/Cation Antiporters (CaCAs), the bacterial calcium/proton antiporter CAXCK31 was purified and characterized. New methods were developed for its heterologous overexpression and purification. These methods help to define stress responses to toxic membrane overproduction in E. coli and may be broadly applicable to studies of membrane proteins. The results from a variety of biochemical and biophysical experiments demonstrated that CAXCK31 exists as a dimer in the membrane and can be purified in the dimeric state. The methods used include chemical cross-linking, FRET, and SEC-MALS. In addition, various transport properties of CAXCK31, including …

Structural Studies On The Rubella Virus Capsid Protein And Its Organization In The Virion, Vidya Mangala Prasad

Open Access Dissertations

Rubella virus is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, rubella infection leads to severe damage in fetuses. Despite its medical importance, very little is known about the structure of the pleomorphic rubella virus as compared to its alphavirus relatives. The rubella capsid protein is a critical structural component of virions as well as a key factor in virus-host interactions. Three crystal structures of the structural domain of the rubella capsid protein have been described here. The polypeptide fold of the capsid protomer has not been observed previously. The capsid protein structure, along …

The Termite Digestome: Understanding The Digestive Physiology Involved In Lignocellulosic Biomass Degradation, Zachary John Karl

Open Access Dissertations

The purpose of this research was to advance the understanding of lower termite digestive physiology and discover potential biocatalysts that can aid in the degradation of lignocellulosic biomass. Various protein characterization and gene expression methods were used throughout this research in order to accomplish these objectives. The results of this dissertation indicate that: 1) termites and their symbionts act in a synergistic manner to degrade biomass in vitro, 2) the host fraction of the gut (i.e., foregut and midgut) is the likely site of glucose absorption, 3) the termite and its symbionts contribute specific enzymes to the digestive process, 4) …

Generation And Statistical Modeling Of Active Protein Chimeras: A Sequence Based Approach, Nicholas Fico

Open Access Dissertations

Generation of active protein chimeras is a valuable tool to probe the functional space of proteins. Statistical modeling is the next logical step, allowing us to build a model of gene fragment replaceability between species. In this thesis I begin to develop the statistical tools that are needed to systematically describe combinatorial protein libraries. I present three sets of diverse chimeric protein libraries developed using sequence information. The statistical model of the human N-Ras and human K-Ras-4B genes reveal a set previously unidetifed surface residues on the N-Ras G-Domain that may be involved in cellular localization. Statistical modeling of a …

Identification Of Cell Cycle–Regulated Genes Periodically Expressed In U2os Cells And Their Regulation By Foxm1 And E2f Transcription Factors, Gavin D. Grant, Lionel Brooks Iii, Xiaoyang Zhang, J. Matthew Mahoney, Viktor Martyanov, Tammara A. Wood, Gavin Sherlock, Chao Cheng, Michael L. Whitfield

Dartmouth Scholarship

We identify the cell cycle–regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle–regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle–regulated genes. FOXM1 is bound to cell cycle–regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly …

The Tethering Complex Hops Catalyzes Assembly Of The Soluble Snare Vam7 Into Fusogenic Trans-Snare Complexes, Michael Zick, William Wickner

Dartmouth Scholarship

The fusion of yeast vacuolar membranes depends on the disassembly of cis–soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The …

Differential Biosynthesis Of Antioxidant Stilbenoids In Hairy Root Cultures Of Muscadine Grape And Peanut Treated With Methyl Jasmonate And Hydrogen Peroxide, Cesar Augusto Nopo-Olazabal

Student Theses and Dissertations

Stilbenoids are polyphenolic compounds that are believed to protect plants against abiotic and biotic stresses. Importantly, an increasing number of these specialized plant metabolites have been associated with potential health benefits in humans. Therefore, understanding the molecular mechanisms involved in their biosynthesis is of importance to enhance the levels of these beneficial compounds in plants. In this research we studied the biosynthesis of stilbenoids in two economically important food crops: muscadine grape and peanut. Hairy root cultures were used as biological systems because their genetic and biochemical stability facilitates the study of specialized metabolism. Selected hairy root cultures from muscadine …

Identification Of Proteins Potentially Involved In The Formation Of Lafora Bodies, A Hallmark Of Lafora Disease, Elham Schokraie, Oliver Kötting, Matthew S. Gentry

Molecular and Cellular Biochemistry Presentations

Lafora Disease (LD) is a fatal teenage-onset progressive myoclonus epilepsy. It is characterized by the formation of Lafora bodies (LBs), deposits of abnormally branched, insoluble, hyperphosphorylated glycogen-like polymers that are generally believed to trigger the development of the clinical symptoms of LD. 58% and 35% of the LD cases are caused by mutations in EPM2A (laforin) and EPM2B (malin), respectively. However, little is known about their function in LB formation. Two different mechanisms have been proposed to explain the accumulation of insoluble LBs: first, excessive glycogen phosphorylation and, second, an imbalance between glycogen synthesizing enzymes. The present study aims at …

Serdemetan Antagonizes The Mdm2-Hif1Α Axis Leading To Decreased Levels Of Glycolytic Enzymes, Jason Alexander Lehman, Paula M. Hauck, Jaimie M. Gendron, Christopher N. Batuello, Jacob A. Eitel, Allan Albig, Madhavi P. Kadakia, Lindsey D. Mayo

Biochemistry and Molecular Biology Faculty Publications

Serdemetan (JNJ-26854165), an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1), were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises …