Cell Biology Commons^™

Partitioning The Fitness Components Of Rna Populations Evolving In Vitro, Carolina Diaz Arenas, Niles Lehman

Chemistry Faculty Publications and Presentations

All individuals in an evolving population compete for resources, and their performance is measured by a fitness metric. The performance of the individuals is relative to their abilities and to the biotic surroundings – the conditions under which they are competing – and involves many components. Molecules evolving in a test tube can also face complex environments and dynamics, and their fitnessmeasurements should reflect the complexity of various contributing factors as well. Here, the fitnesses of a set of ligase ribozymes evolved by the continuous in vitroevolution system were measured. During these evolution cycles there are three different catalytic …

The Osh Family Of Proteins And Their Role In Caloric Restriction-Mediated Lifespan Extension In The Budding Yeast Saccharomyces Cerevisiae, William Douglas Craft

Theses and Dissertations

Oxysterol-binding protein homolouges (OSH) have been implicated in mediating non-vesicular sterol transport. Caloric restriction (CR) down-regulates sterol synthesis in a variety of organisms, including the budding yeast Saccharomyces cerevisiae. OSH6, when up-regulated, extends replicative lifespan. In contrast, OSH5 up-regulation shortens replicative lifespan. In this thesis I obtained evidence to show that the deletion of OSH5 extends replicative lifespan in normal media. Deletion of OSH5 in the presence of caloric restriction blocks the lifespan-extending properties of the CR media. My data demonstrate that the total cellular sterol levels of the longevity mutants are indistinguishable from that of wild type. This suggests …

Secretion Of Interferon Gamma From Human Immune Cells Is Altered By Exposure To Tributyltin And Dibutyltin, Shanieek Lawrence, Jacqueline Reid, Margaret Whalen

Chemistry Faculty Research

Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion …

Purifying The Human Rna-Lariat Debranching Enzyme, Ammar Saigal

Honors Program Theses and Research Projects

This project is my first official research endeavor and it involved becoming acquainted with a large variety of biochemistry and molecular biology techniques. The goal is to elucidate the structure of a protein common to most if not all cells of organisms within the Domain Eukarya, which is one of the three taxonomic domains into which all life is categorized. This is the domain most relevant to human beings as it includes plants, fungi, and animals (from a strictly biologically-taxonomic perspective, the species Homo sapiens to which you and I belong is considered belonging to the Kingdom Animalia [Animal]).

Transformation Of Stimulus Correlations By The Retina, Kristina D. Simmons, Jason Prentice, Gašper Tkačik, Jan Homann, Heather K. Yee, Stephanie E. Palmer, Philip C. Nelson, Vijay Balasubramanian

Publications and Research

Redundancies and correlations in the responses of sensory neurons may seem to waste neural resources, but they can also carry cues about structured stimuli and may help the brain to correct for response errors. To investigate the effect of stimulus structure on redundancy in retina, we measured simultaneous responses from populations of retinal ganglion cells presented with natural and artificial stimuli that varied greatly in correlation structure; these stimuli and recordings are publicly available online. Responding to spatio-temporally structured stimuli such as natural movies, pairs of ganglion cells were modestly more correlated than in response to white noise checkerboards, but …

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 …

Developmental And Molecular Functions Of Plakophilin-3, William A. Munoz

Dissertations and Theses (Open Access)

Plakophilin-3, the less studied member of the plakophilin-catenin subfamily, and the larger catenin family, binds directly to desmosomal cadherin cytoplasmic domains and enhances desmosome formation and stability. In mammals, plakophilin-3 is expressed at the highest levels in desmosome-enriched tissues such as epithelia, with the knock-out in mice producing corresponding reductions in ectodermal integrity. In tissue, cellular and intracellular contexts where plakophilin-3 is not at the desmosomal plaque, little is known about its functions in the cytoplasm or nucleus, where it also localizes.

My work employed embryos of the amphibian, Xenopus laevis, to examine plakophilin-3’s developmental roles. I first evaluated …

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 …

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 …

Protective Effects Of Sphingomyelin Against Uv Photodamage In Human Keratinocytes, Kathleen De Guzman

Master's Theses

Ultraviolet (UV) radiation has been demonstrated in numerous studies to be a major risk factor for non-melanoma skin cancer development. Despite the emergence of current UV-preventative strategies, such as sunscreens and skin-protective clothing, the incidence of non-melanoma skin cancer has continued to rise. This has encouraged investigations on alternative methods for UV prevention. In particular, bovine milk sphingomyelin has been studied for its potential in protecting human skin against UV photodamage. While the previous studies have suggested that sphingomyelin exhibits UV-protective properties in a human skin equivalent model, the exact mechanisms behind sphingomyelin’s photoprotective effects are yet unknown.

This thesis …

In-Situ Quantification Of The Interfacial Rheological Response Of Bacterial Biofilms To Environmental Stimuli, Patrick Rühs, Lukas Böni, Gerald Fuller, R. Inglis, Peter Fischer

Biology Department Faculty Works

Understanding the numerous factors that can affect biofilm formation and stability remain poorly understood. One of the major limitations is the accurate measurement of biofilm stability and cohesiveness in real-time when exposed to changing environmental conditions. Here we present a novel method to measure biofilm strength: interfacial rheology. By culturing a range of bacterial biofilms on an air-liquid interface we were able to measure their viscoelastic growth profile during and after biofilm formation and subsequently alter growth conditions by adding surfactants or changing the nutrient composition of the growth medium. We found that different bacterial species had unique viscoelastic growth …

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

Biology Faculty Research

Highlights

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

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

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

Faculty Publications - Department of Biological & Molecular Science

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

Novel Neuroprotective Function Of Apical-Basal Polarity Genecrumbs In Amyloid Beta 42 (Aβ42) Mediated Neurodegeneration, Andrew Steffensmeier, Meghana Tare, Oorvashi Roy Puli, Rohan Modi, Jaison Nainaparampil, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Alzheimer's disease (AD, OMIM: 104300), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). We have developed a transgenic Drosophilaeye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb) as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, …

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

Dartmouth Scholarship

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

Artificial Yeast Polarization Controlled By Chemical Gradient, James K. Nolan, Bernard Tao

The Summer Undergraduate Research Fellowship (SURF) Symposium

Engineering synthetic multicellular systems will lead to new synthetic biology technological platforms, inform developmental biology through recapitulation of natural systems and possibly unveil novel morphologies with practical applications not before reached throughout natural history (Maharbiz, 2012). Creating an exogenous molecular circuit that will polarize unicellular cells into “apical” and “basal” domains relative to a substrate plane would fulfill a missing component towards fully multicellular synthetic cellular communities (Maharbiz, 2012). To this end, a PIP₃ polarization network previously designed by Chau and associates (Chau, Walter, Gerardin, Tang, Lim 2012) was coupled to the specific activation by niacin of a recombinant …

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

Department of Biomedical Engineering Faculty Publications

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

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 …

Measuring And Modeling The Response Characteristics Of The Environmental Phosphate Transducer In Escherichia Coli, Chetan Sood

Open Access Dissertations

The PhoR/PhoB two-component system in Escherichia coli is a biological transducer that senses the limitation of environmental inorganic orthophosphate, the bacteria's preferred source of the essential nutrient phosphate, and transmits that information to the interior of the cell initiating a response that mitigates phosphate starvation. In the first part of this study, we present and apply a fluorescence microscopy technique to measure, in vivo, the dynamic response characteristics of the transducer with single-cell resolution. We report that the transience in the PhoR/PhoB TCS response is consistent with the transducer having a threshold sensitivity to the concentration of environmental phosphate, …

Establishing The Role Of The Pancreatic Transcription Factor Mist1 In Xbp1-Mediated Maintenance Of Pancreatic Acinar Cell Homeostasis, David Alan Hess

Open Access Dissertations

Pancreatic acinar cells (PACs) continuously produce more protein than any other cell type in the human body. As a result, PACs and other specialized secretory cells have a constant demand placed on their protein synthetic and packaging machinery. When demand for secreted products exceeds the capacity of the cell's basal protein production facilities, dangerous accumulations of misfolded proteins can build up, resulting in a condition known as ER stress. To ameliorate this stress, secretory cells activate a coordinated, three-part compensatory network collectively known as the unfolded protein response (UPR) to both expand the capacity of the ER and directly assist …

Protein Kinase Ck2 Phosphorylates And Activates P21-Activated Kinase 1, Yong Jae Shin, Yong-Bae Kim, Jeong-Ho Kim

Biochemistry and Molecular Medicine Faculty Publications

Activation of the p21-activated kinase 1 (PAK1) is achieved through a conformational change that converts an inactive PAK1 dimer to an active monomer. In this paper, we show that this change is necessary but not sufficient to activate PAK1 and that it is, rather, required for CK2-dependent PAK1S223 phosphorylation that converts a monomeric PAK1 into a catalytically active form. This phosphorylation appears to be essential for autophosphorylation at specific residues and overall activity of PAK1. A phosphomimetic mutation (S223E) bypasses the requirement for GTPases in PAK1 activation, whereas the constitutive activity of the PAK1 mutant (PAK1H83,86L), postulated to mimic GTPase-induced …

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 …

Effects Of Butyltins On Mitogen-Activated-Protein Kinase Kinase Kinase And Ras Activity In Human Natural Killer Cells, Lindsay J. Celada, Margaret M. Whalen

Chemistry Faculty Research

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT) diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The …

Pseudomonas Aeruginosa Exotoxin Y-Mediated Tau Hyperphosphorylation Impairs Microtubule Assembly In Pulmonary Microvascular Endothelial Cells, Ron Balczon, Nutan Prasain, Cristhiaan Ochoa, Jason Prater, Bing Zhu, Mikhail Alexeyev, Sarah Sayner, Dara W. Frank, Troy Stevens

University Faculty and Staff Publications

Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that …

Early Innate Immunity Determines Outcome Of Mycobacterium Tuberculosis Pulmonary Infection In Rabbits, Selvakumar Subbian, Nirmalya Bandyopadhyay, Liana Tsenova, Paul O'Brien, Viraj Khetani, Nicole L. Kushner, Blas Peixoto, Patricia Soteropoulos, Joel S. Bader, Petros C. Karakousis, Dorothy Fallows, Gilla Kaplan

Publications and Research

Background: Pulmonary infection of humans by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), results in active disease in 5-10% of individuals, while asymptomatic latent Mtb infection (LTBI) is established in the remainder. The host immune responses that determine this differential outcome following Mtb infection are not fully understood. Using a rabbit model of pulmonary TB, we have shown that infection with the Mtb clinical isolate HN878 (a hyper-virulent W-Beijing lineage strain) leads to progressive cavitary disease similar to what is seen in humans with active TB. In contrast, infection with Mtb CDC1551 (a hyper-immunogenic clinical isolate) is efficiently …

P53'S Choice Of Myocardial Death Or Survival: Oxygen Protects Infarct Myocardium By Recruiting P53 On Nos3 Promoter Through Regulation Of P53-Lys118 Acetylation, Rajan Gogna, Esha Madan, Mahmood Khan, Uttam Pati, Periannan Kuppusamy

Dartmouth Scholarship

Myocardial infarction, an irreversible cardiac tissue damage, involves progressive loss of cardiomyocytes due to p53-mediated apoptosis. Oxygenation is known to promote cardiac survival through activation of NOS3 gene. We hypothesized a dual role for p53, which, depending on oxygenation, can elicit apoptotic death signals or NOS3-mediated survival signals in the infarct heart. p53 exhibited a differential DNA-binding, namely, BAX-p53RE in the infarct heart or NOS3-p53RE in the oxygenated heart, which was regulated by oxygen-induced, post- translational modification of p53. In the infarct heart, p53 was heavily acetylated at Lys118 residue, which was exclusively reversed in the oxygenated heart, apparently regulated …

Modulation Of Bax/Bak Dependent Apoptosis By Sirtuin 3 And Mitochondrial Permeability Transition By Sirtuin 4, Manish Verma

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondria are dynamic organelles that regulate a myriad of cellular functions, including energy production and metabolic regulation. Mitochondria are also a critical regulator of cell death signaling cascades modulating both apoptotic and necrotic cell death. However, what determines which cell death pathway is activated is still unclear. The mitochondrial/intrinsic pathway of apoptosis is dependent on the activation of pro-apoptotic proteins, Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). Once the integrity of outer mitochondrial membrane (OMM) is compromised, pro-apoptotic intermembrane space proteins like cytochrome c, Smac/Diablo, Omi/HtrA2 and AIF are released into the cytoplasm, which activates the post-mitochondrial …

Med13p Prevents Stress-Independent Mitochondrial Hyperfragmentation And Aberrant Apoptosis Activation In Saccharomyces Cerevisiae By Controlling Cyclin C Nuclear Localization, Svetlana Khakhina

Graduate School of Biomedical Sciences Theses and Dissertations

During aging, and as a result of environmental changes, cells are exposed to elevated levels of reactive oxygen species (ROS). High ROS levels induce lipid oxidation, protein aggregation, mitochondrial hyperfragmentation, DNA damage and programmed cell death (PCD), also called apoptosis. PCD is a highly regulated process and its misregulation has been linked to neurodegenerative diseases and cancer development.

Our hypothesis is that cyclin C plays a role in the initiation of apoptosis. During normal conditions, cyclin C represses the transcription of stress response genes (SRG). In response to stress, cyclin C translocates to the cytoplasm where it facilitates mitochondrial hyperfragmentation …

Recurrent Modification Of A Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity, William A. Rogers, Joseph R. Salomone, David J. Tacy, Eric M. Camino, Kristen A. Davis, Mark Rebeiz, Thomas M. Williams

Biology Faculty Publications

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. InDrosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development …

A Force Of Nature: Molecular Mechanisms Of Mechanoperception In Plants, Gabriele B. Monshausen, Elizabeth S. Haswell

Biology Faculty Research

The ability to sense and respond to a wide variety of mechanical stimuli-gravity, touch, osmotic pressure, or the resistance of the cell wall-is a critical feature of every plant cell, whether or not it is specialized for mechanotransduction. Mechanoperceptive events are an essential part of plant life, required for normal growth and development at the cell, tissue, and whole-plant level and for the proper response to an array of biotic and abiotic stresses. One current challenge for plant mechanobiologists is to link these physiological responses to specific mechanoreceptors and signal transduction pathways. Here, we describe recent progress in the identification …