Biochemistry, Biophysics, and Structural Biology Commons^™

The Discovery Of Diverse Picophytoplankton Populations In The Columbia And Willamette Rivers Using Flow Cytometry, Kylee Brevick

Chemistry Undergraduate Departmental Honors Theses

As important primary producers, picophytoplankton determine the flow of carbon and energy in aquatic ecosystems. Picocyanobacteria are one picophytoplankton group known to be dominant in oceans and lakes, but they are still poorly understood in river systems. This project examined picophytoplankton communities in two distinct river systems: the Columbia and Willamette Rivers in Portland, Oregon. I aimed to characterize and quantify the picophytoplankton populations in the context of the environmental conditions of the two rivers. I used flow cytometry to detect cells based on their relative size and pigment fluorescence. I sampled nearly weekly for ten months to capture population …

Autofluorescence Microscopy: Identifying Bacterial Signals On Rock Samples, Noel Case

University Honors Theses

One common method for identifying microbes present in fluid and non-fluid samples is fluorescent dye staining, in which samples are stained with a fluorophore - a molecule excited by an external light source that re-emits energy as higher-wavelength light. The excitation wavelength used greatly impacts the observability of microbes in the resulting fluorescence images, and deep ultraviolet excitation (200-280 nm) seems to provide the highest signal-to-noise ratio of any excitation range. By inoculating rock samples (gypsum and marble) with B. subtilis, staining with SYTO 9, Acridine Orange and FM 1-43 dyes, and imaging with deep UV (275 nm), near …

Effects Of Pore-Forming Peptides (Melittin And Magainin 2) On The Phospholipid Bilayer Interior, Elmukhtar Ehmed Alhatmi

Dissertations and Theses

Antimicrobial peptides (AMPs) are one of the most promising solutions to drug-resistant bacteria. Melittin and magainin 2 are two of the most representative and extensively studied AMPs. In this research, I investigated the interaction of these two AMPs with three models of cell membranes: 80% POPC 20% POPG, 40%POPC 40% POPE and 20% POPG, and 80%POPC 20%POPG plus 30% mole fraction of cholesterol. Time-resolved fluorescence emission and fluorescence anisotropy decays of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were analyzed to determine the effects of AMPs on the bilayer headgroup packing and changes in the interior of the phospholipid bilayer during the …

Aerosolization Of Catalytic Rna For Prebiotic Transport And In Situ Reactivity, Brennan Roland Farrell

Dissertations and Theses

Recent theoretical and experimental work suggests that aqueous aerosols in the early Earth's atmosphere might have been an essential component to the development of life. These complex droplets would have served to compartmentalize emerging biomolecules, thereby concentrating them, increasing reactivity, and facilitating transport and exchange between ocean and atmosphere. This project tests an underexplored but potentially important environmental paradigm for the RNA world hypothesis of prebiotic evolution, probing the effects of aerosolization on catalytic RNA using a model ribozyme. Here we demonstrate the successful transport and in situ self-assembly of the Azoarcus ribozyme from multiple independent fragments via a laboratory-constructed …

A Tale Of Two-Hybrids: Investigating The Interactomes Of Cullin-Associated Proteins, Elyse Reitter

Dissertations and Theses

Cul3 is the major component of an E3 ligase in human cells. Cul3 was initially identified in the Singer lab as a protein that binds and degrades cyclin E, and subsequent studies have shown it to be part of a complex that is involved in a multitude of biological functions. The nature of this complex, its constituents, its regulation, and its dynamics is just beginning to be understood. The research presented here utilizes a series of two hybrid screens to identify families of interactomes with Cul3 at the center. This information will complement other work in the lab in which …

A Bioinformatic And Biochemical Analysis Of Cruciviruses, George William Kasun

Dissertations and Theses

Cruciviruses are novel ssDNA viruses discovered through metagenomics and direct environmental DNA amplification and cloning. The genomes of cruciviruses suggest that gene transfer between RNA and DNA viruses occurred due to the presence of putative protein-encoding genes that are homologous to both ssRNA and ssDNA viruses. In order to gain a better understanding of this group of viruses both bioinformatic analyses and in vitro biochemical experiments were employed. The results of the bioinformatic analyses show that cruciviruses are a highly diverse group of ssDNA viruses. Their placement within established ssDNA phylogenies is difficult due to heterogeneity in their putative replication-associated …

Mechanisms Of Substrate Recognition By The Cul3-Based E3 Ligase, Katia Graziella De Oliveira Rebola

Dissertations and Theses

Cul3-based E3 ligase is responsible for regulating a variety of cellular pathways, many of which are known to have profound effects on the proper function of multicellular organisms. Although progress over the past years has been truly impressive, our understanding of the mechanisms of E2 recruitment and selection by the BCR complex and all the roles that Cul3 plays on kidneys remains in its infancy. To explore these aspects, this dissertation aims to analyze the Cul3 complex using two different approaches: (1) We used the powerful tool of chimeric analysis to map the essential domain binding characteristics of Cul3 taking …

Complete Mitochondrial Genome Sequence Of The Gulf Coast Tick (Amblyomma Maculatum), Amanda E. Brenner, Rahul Raghavan

Biology Faculty Publications and Presentations

The complete circularized mitochondrial genome sequence of Amblyomma maculatum is 14,803 bp long. It encodes 13 protein coding genes, 2 rRNA genes, 22 tRNA genes, 2 tick box motifs, and 2 control regions. The gene arrangement and content are consistent with those of previously reported Metastriata tick mitochondrial genomes.

Mechanisms Of Connexin-46 And -50 Intercellular Channel Function And Stability By Molecular Dynamics Simulations, Bassam George Haddad

Dissertations and Theses

Gap junctions make up a class of intercellular channels that characteristically connect the cytoplasm of directly apposed cells through large assemblies, or plaques, constituted by a multitude of intercellular channels. Gap junction mediated intercellular communication is critical for a variety of physiological functions, from coordinating electrical impulses in the heart and brain to maintaining homeostasis in most tissues. There are 21 isoforms of connexins, the constituent subunit of the gap junction, expressed in a tissue dependent manner. Gap junctions formed from different isoforms exhibit distinct biophysical properties, such as gating kinetics and sensitivity, as well as unique permeability and selectivity …

Functional Role Of The N-Terminal Domain In Connexin 46/50 By In Silico Mutagenesis And Molecular Dynamics Simulation, Umair Khan

University Honors Theses

Connexins form intercellular channels known as gap junctions that facilitate diverse physiological roles, from long-range electrical and chemical coupling to nutrient exchange. Recent structural studies on Cx46 and Cx50 have defined a novel and stable open state and implicated the amino-terminal (NT) domain as a major contributor to functional differences between connexin isoforms. This thesis presents two studies which use molecular dynamics simulations with these new structures to provide mechanistic insight into the function and behavior of the NTH in Cx46 and Cx50. In the first, residues in the NTH that differ between Cx46 and Cx50 are swapped between the …

The Role Of Native Lens Α-Crystallin In Amyloid Suppression Using Β-Amyloid As A Model Amyloid Client, Leilani Lopes

University Honors Theses

Cataracts are a result of age-related protein aggregate formation in the eye lens, and the leading cause of blindness worldwide. α-crystallin, a small heat shock protein, acts a molecular chaperone that serves as the primary defense mechanism against protein aggregate formation in the lens. Recent evidence suggest amyloid formation in the lens may contribute to cataract formation, and that β-amyloid is present in lens epithelia of age-related cataracts. Previous studies have shown that αB-crystallin in other parts of the body, such as the brain, is protective against neurodegenerative diseases such as Alzheimer’s disease that are associated with plaque deposits containing …

Steady-State Transmembrane Water Exchange In Proliferating Cultures Of Saccharomyces Cerevisiae, Joseph O'Malley Armstrong

Dissertations and Theses

Cellular water exchange is often considered in terms of a change in volume, where a net flux of water moves across the cell membrane due to a change in osmotic pressure. Osmotic pressure can cause a cell to shrink or swell, however, rapid water exchange persists across the membrane even when the volume of the cell is constant. Steady-state transmembrane water exchange describes the exchange of water across the cell membranes which results in no net change in cell volume. This exchange is astonishingly rapid; the entire pool of intracellular water of a Saccharomyces cerevisiae cell may exchange 2-5 times …

Thermodynamics Of Ligand Binding And Global Structural Stability Of Human Serum Albumin, Matthew Walter Eskew

Dissertations and Theses

Protein structure is integral to its function. For the past 70 years differential scanning calorimetry has been used to measure protein structural stability. More recently it has been used to study macromolecular interactions. Interactions between proteins and ligands can manifest on differential scanning calorimetry melting curves or thermograms. Utilizing differential scanning calorimetry thermograms to detect or diagnose diseases has been a major goal in disease diagnostics. However, correlating specific ligand-protein interactions, as manifested in a thermogram, with a disease-specific plasma thermogram, has proven elusive.

Modified human serum albumin was utilized to develop a process to capture and retrieve ligands from …

Urban Air Quality Modeling: Evaluating Impacts Of Ecoroofs On Urban Air Quality In Portland, Oregon, Danlyn L. Brennan, Selina Teng

altREU Projects

In this work, the authors present a mathematical model simulating the impact of ecoroof vegetation on air quality in an urban microclimate. An ecoroof is a layer of vegetation established on rooftops to reduce stormwater runoff, mitigate heat, and enhance air quality. Certain plant species in ecoroofs may however have a detrimental effect on air quality, due to their emission of biogenic volatile organic compounds (BVOCs), which include potentially reactive species such as isoprene. These chemical species can interact with urban air pollution, particularly NOx produced by car traffic, to create other harmful pollutants such as ozone and particulate matter …

Connexin-46/50 In A Dynamic Lipid Environment Resolved By Cryoem At 1.9 Å, Jonathan A. Flores, Bassam G. Haddad, Kimberly A. Dolan, Janette Bernadette Myers, Craig C. Yoshioka, Jeremy Yoshioka, Daniel M. Zuckerman, Steve L. Reichow

Chemistry Faculty Publications and Presentations

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid …

No Oxygen, No Water: The Unique Physiology Of Annual Killifish Embryos Across Development, Daniel Erik Zajic

Dissertations and Theses

For most vertebrates, an abundance of oxygen is necessary for the production of ATP and the maintenance of cellular homeostasis. The absence of oxygen, even for brief periods, quickly leads to ATP depletion which can lead to irreparable damages to sensitive organs, such as the brain and heart. However, certain vertebrates demonstrate an extraordinary ability to thrive and recover fully from periods of no oxygen (anoxia). The annual killifish (Austrofundulus limnaeus) lives in ephemeral ponds in the Maracaibo basin of Venezuela and their embryos have the remarkable ability to not only survive anoxic periods for months, but also …

The Role Of Redox Chemistry Of Disulfide Bonds In Cysteine Residues Of Membrane Proteins By Cuprous And Cupric Ions In Cell Death Of E. Coli, Morgan R. Stewart

University Honors Theses

The antimicrobial properties of copper have been thoroughly researched, but is still unclear what the actual mechanism of cell death is. This study explores the theory that copper ions and other copper sources act as an antibiotic for E. coli by cleaving the disulfide bonds of membrane proteins through redox chemistry, disrupting the cell membrane and causing cell death. The focus of this study is Cu(I) and Cu(II) interactions with the thiol containing amino acid, cysteine, and how these interactions may be responsible for copper’s toxicity. Cuprous ions have been found to be more toxic to E.coli than cupric …

Archaeosine Modification Of Archaeal Trna - A Role In Structural Stabilization, Ben Turner, Brett W. Burkhart, Katrin Weidenbach, Robert Ross, Patrick A. Limbach, Ruth A. Schmitz, Valérie De Crécy-Lagard, Kenneth M. Stedman, Thomas J. Santangelo, Dirk Iwata-Reuyl

Biology Faculty Publications and Presentations

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside of the Archaea. G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted …

Size-Dependent Interactions Of Lipid-Coated Gold Nanoparticles: Developing A Better Mechanistic Understanding Through Model Cell Membranes And In Vivo Toxicity, Arek M. Engstrom, Ryan A. Faase, Joe E. Baio, Marilyn R. Mackiewicz, Stacey L. Harper

Chemistry Faculty Publications and Presentations

Introduction: Humans are intentionally exposed to gold nanoparticles (AuNPs) where they are used in variety of biomedical applications as imaging and drug delivery agents as well as diagnostic and therapeutic agents currently in clinic and in a variety of upcoming clinical trials. Consequently, it is critical that we gain a better understanding of how physiochemical properties such as size, shape, and surface chemistry drive cellular uptake and AuNP toxicity in vivo. Understanding and being able to manipulate these physiochemical properties will allow for the production of safer and more efficacious use of AuNPs in biomedical applications.
Methods and Materials: Here, …

Application Of Single Particle Electron Microscopy To Native Lens Gap Junctions And Intrinsically Disordered Signaling Complexes, Janette Bernadette Myers

Dissertations and Theses

Gap junctions are a class of membrane proteins that facilitate cell-to-cell communication by forming channels that directly couple the cytoplasm of neighboring cells. The channels are composed of monomers called connexins. Humans express 21 connexin isoforms in a cell-type specific fashion, and each isoform has distinct mechanisms of permeation and regulation. Co-assembly of multiple isoforms into a single intercellular channel can change channel properties, such as conductance and selectivity to substrates (e.g., ions, metabolites and signaling molecules). However, the mechanistic basis for this functional diversity has remained poorly understood. This lack of mechanistic insight has been due in large part …

The Effect Of Dynamic Kinetic Selection On An Evolving Ribozyme Population, Patrick David Poletti

Dissertations and Theses

Dynamic Kinetic Selection (DKS) suggests that kinetic, rather than thermodynamic, stability will dictate the composition of a replicating population of biomolecules. Here, the results obtained from a series of five related reactions involving gradually increasing percentages of randomly-mutated substrate fragments to generate variants of full-length Azoarcus group I intron through an autocatalytic self-assembly reaction involving a series of recombination events, showed DKS as a driving factor in dictating the population composition of full-length product assembled from substrates that had fewer positions available to randomization.

In trying to elucidate a plausible scheme for the origins of complex biomolecules on the prebiotic …

Cullin-3 Dependent Deregulation Of Actn1 Represents A New Pathogenic Mechanism In Nemaline Myopathy, Jordan Blondelle, Kavya Tallapaka, Jane T. Seto, Majid Ghassemian, Madison Clark, Jenni M. Laitila, Adam Bournazos, Jeffrey Singer, Stephan Lange

Biology Faculty Publications and Presentations

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy, and presence of nemaline bodies within myofibers. However, understanding of the underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40, and KLHL41, three substrate adaptors for the E3 ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for disease development. Using Cullin-3–knockout mice, we identified accumulation of non-muscle α-actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in patients with mutations in KBTBD13. Our …

Limited Sequence Diversity Within A Population Supports Prebiotic Rna Reproduction, Ryo Mizuuchi, Niles Lehman

Chemistry Faculty Publications and Presentations

The origins of life require the emergence of informational polymers capable of reproduction. In the RNA world on the primordial Earth, reproducible RNA molecules would have arisen from a mixture of compositionally biased, poorly available, short RNA sequences in prebiotic environments. However, it remains unclear what level of sequence diversity within a small subset of population is required to initiate RNA reproduction by prebiotic mechanisms. Here, using a simulation for template-directed recombination and ligation, we explore the effect of sequence diversity in a given population for the onset of RNA reproduction. We show that RNA reproduction is improbable in low …

The Effect Of Styrene-Maleic Acid (Sma) Copolymers On Solubilizing Lipid Bilayers And Forming Nanodiscs, Ghada Alramadan

Dissertations and Theses

Cell membranes, or plasma membranes, play an essential role in the structure and the function of living cells. In 1972, the fluid mosaic membrane model was the first unifying paradigm of membrane structure. It is no longer considered adequate because evidence of many non-homogeneous lipid structures in both natural and model membranes have been discovered over the past thirty years. The field of membrane biophysics now uses updated versions of the mosaic model, which consists of the complex mixture of different lipid species. The lipid species found in natural membranes produce a range of dynamic, laterally segregated, non-homogeneous domains, which …

Small Noncoding Rna Profiles Along Alternative Developmental Trajectories In An Annual Killifish, Amie L. Romney, Jason E. Podrabsky

Biology Faculty Publications and Presentations

Embryonic development of Austrofundulus limnaeus can occur along two phenotypic trajectories that are physiologically and biochemically distinct. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately non-diapausing embryos and older females produce mostly diapausing embryos. Embryonic incubation temperature can override this pattern and alter trajectory. We hypothesized that temperature-induced phenotypic plasticity may be regulated by post-transcriptional modification via noncoding RNAs. As a first step to exploring this possibility, RNA-seq was used to generate transcriptomic profiles of small noncoding RNAs in embryos developing along the two alternative trajectories. We find distinct profiles of …

Prebiotic Rna Network Formation: A Taxonomy Of Molecular Cooperation, Cole Mathis, Sanjay N. Ramprasad, Sara Imari Walker, Niles Lehman

Chemistry Faculty Publications and Presentations

Cooperation is essential for evolution of biological complexity. Recent work has shown game theoretic arguments, commonly used to model biological cooperation, can also illuminate the dynamics of chemical systems. Here we investigate the types of cooperation possible in a real RNA system based on the Azoarcusribozyme, by constructing a taxonomy of possible cooperative groups. We construct a computational model of this system to investigate the features of the real system promoting cooperation. We find triplet interactions among genotypes are intrinsically biased towards cooperation due to the particular distribution of catalytic rate constants measured empirically in the real system. For other …

The T6a Modification Acts As A Positive Determinant For The Anticodon Nuclease Prrc, And Is Distinctively Nonessential In Streptococcus Mutans, Jo Marie Bacusmo, Silvia S. Orsini, Jennifer Hu, Michael Demott, Patrick C. Thiaville, Ameer Elfarash, Mellie June Paulines, Diego Rojas-Benítez, Birthe Meineke, Christopher Deutsch, Dirk Iwata-Reuyl, Patrick A. Limbach, Peter C. Dedon, Kelly C. Rice, Stewart Shuman, Valérie De Crécy-Lagard

Chemistry Faculty Publications and Presentations

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALys UUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t⁶A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t⁶A-deficient yeast derivatives, it is shown that t⁶A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t …

Group I Intron Internal Guide Sequence Binding Strength As A Component Of Ribozyme Network Formation, Elizabeth Satterwhite, Jessica Anne Mellor Yeates, Niles Lehman

Chemistry Faculty Publications and Presentations

Origins-of-life research requires searching for a plausible transition from simple chemicals to larger macromolecules that can both hold information and catalyze their own production. We have previously shown that some group I intron ribozymes possess the ability to help synthesize other ribozyme genotypes by recombination reactions in small networks in an autocatalytic fashion. By simplifying these recombination reactions, using fluorescent anisotropy, we quantified the thermodynamic binding strength between two nucleotides of two group I intron RNA fragments for all 16 possible genotype combinations. We provide evidence that the binding strength (K_D) between the 3-nucleotide internal guide sequence …

The Foundations Of Network Dynamics In An Rna Recombinase System, Jessica Anne Mellor Yeates

Dissertations and Theses

How life originated from physical and chemical processes is one of the great questions still unanswered today. Studies towards this effort have transitioned from the notion of a single self-replicating entity to the idea that a network of interacting molecules made this initial biological leap. In order to understand the chemical kinetic and thermodynamic mechanisms that could engender pre-life type networks we present an empirical characterization of a network of RNA recombinase molecules. We begin with 1-, 2-, and 3-molecular ensembles and provide a game theoretic analysis to describe the frequency dependent dynamics of competing and cooperating RNA genotypes. This …

Quantitative Comparison Of A Nanoengineered Alumina Coated Cnt Arrays To Sio2 Coated Cnts And Solution Based Delivery System, Shree Aier

Undergraduate Research & Mentoring Program

To meet the growing need for nanoengineered biocompatible materials to serve as drug delivery platforms, in this research, carbon nanotube arrays were fabricated by chemical vapor deposition, followed by an alumina coating by the high yielding, tightly controlled atomic layer deposition. This nanoengineered vertically aligned alumina nanowire array serves as a platform for delivering antigens, which act as cancer adjuvants. The physicochemical characteristics of the nanowires (NWs) can significantly influence the delivery of a biomolecule to immune cells. To investigate the material characteristics, the delivery efficiency of the antigen using NWs was quantitatively assessed by flow cytometry. Further, the mechanism …