Biochemistry Commons^™

Chemical Control And Understanding Of Horizontal Gene Transfers, Drug-Resistance Development, And Filament And Biofilm Formation, Yuchen Jin

Dissertations - ALL

Biofilms formed by microbes on surfaces are the sources for persistent infectious diseases and environmental problems. The mechanism and details of how antibiotics promote biofilm formation is largely unknown. For instance, it not clear what stages of biofilm growth are promoted to proceed faster than without antibiotics, what phenotypes of bacteria form in an antibiotic-promoted biofilm, and what different biofilm compositions and structures are caused by the presence of antibiotics. Among other effects, antibiotics can cause bacteria to form filaments of living bacteria. Here, we conduct a real-time study of the adherence of bacteria and antibiotic-induced filamentous bacteria on surfaces …

The Phase Behavior Of Ubqln Proteins And Implications For Protein Quality Control, Yiran Yang

Dissertations - ALL

The ubiquitin-proteasome system (UPS) and autophagy are essential pathways for maintaining protein quality control (PQC) in cells. Misfolded proteins and large aggregates are cleared by UPS and autophagy signaled by ubiquitin (Ub) or polyubiquitin (polyUb) chains. Shuttle proteins facilitate cargo transporting by interacting with both ubiquitin and degradation machineries. Previously, our lab discovered that the shuttle protein Ubiquilin-2 (UBQLN2) is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. LLPS is a biophysical process by which proteins separate themselves from the surrounding aqueous solution by forming protein-rich droplets. The overarching goals of this work are …

Direct Single Molecule Imaging Of Enhanced Diffusion For Enzymes And Enzyme-Conjugated Dna Origami, Mengqi Xu

Dissertations - ALL

Enzymes have been shown to diffuse faster in the presence of their substrates. Recently, we revealed new insights into this process of enhanced diffusion using single-particle tracking (SPT) with total internal reflection fluorescence (TIRF) microscopy. We found that the mobility of individual enzymes was enhanced three-fold in the presence of the substrate, and the motion remained Brownian. We showed that the relative increase in diffusion is independent of the total enzyme concentrations; and the oligomerization state of enzymes did not change during the catalytic turnover. These experiments ruled out the possibility that the enhanced enzyme diffusion was caused by the …

Characterizing Multivalent Interactions Between Folded Protein Domains And Intrinsically Disordered Regions Or Peptide Substrates, Tongyin Zheng

Dissertations - ALL

Protein-protein interactions (PPIs) play central roles in most biological processes. Studying PPIs is a fundamental step in understanding the molecular basis of cellular processes such as cell-cell contact, enzyme activity, and transient assembly of signaling complexes or cellular structures. In this work, we employed a combination of biophysical and biochemical methods to characterize PPIs, with a focus on interactions between structured domains and intrinsically disordered regions or peptide substrates.The subject of Chapter two is prolyl isomerase Ess1, which is an essential enzyme found in Saccharomyces cerevisiae. Ess1 regulates the transcription and co-transcriptional RNA processing by catalyzing the isomerization of serine-proline …

Coordination Geometries In Metallobundles Enforcing Oxidative And Hydrolytic Catalysis And Designing Biomaterials For Use As Antimicrobials, Jennifer Yoon

Dissertations - ALL

De novo protein design permits discovery of intricate folds and functions emulating natural enzymes in simpler, yet robust model constructs. Helical bundles serve as premier scaffolds to incorporate diverse reactivities from oxidative, reductive, to hydrolytic transformations observed in much grander O2-utilizing metalloproteins such as radical-generating ribonucleotide reductases and catalases. One notable family of de novo proteins is the Due Ferri or DF series of four-helix bundles providing a dinuclear site for metal incorporation and are amenable for tailoring active site reactivity. As reactive oxygen species and radical-based intermediates are prevalent and necessary for steering life-essential processes through reactive radicals stabilized …

An Exploration Of Ruthenium Catalyzed Alder-Ene Reactions, Emily Bonner

Honors Capstone Projects - All

This project focuses on expanding the reaction scope of the ruthenium (Ru) catalyzed Alder-ene reaction with both exocyclic enol ethers and simple alkenes. Knowledge gained from these reactions may be applied to the development of efficient methods for the synthesis of both oxygen heterocycles and acyclic compounds that incorporate a 1,4-diene unit. Each of the two double bonds in the diene possesses different reactivity which increases the utility of these products as synthetic intermediates by allowing for greater versatility in synthesizing natural product targets. A breadth of alkyne and alkene couplings were attempted in this project via the Ru catalyzed …

Ubr-5, A Conserved Hect-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling In Caenorhabditis Elegans, Anniya Gu

Honors Capstone Projects - All

Germline stem cell proliferation in C. elegans is regulated by the distal tip cell (DTC) located at the distal end of each gonad arm. The DTC signals germ cells, through a Notch-type signaling pathway, to proliferate. As germ cells move farther away from the DTC, they transition into meiosis and produce gametes. There are two Notch-type receptors in C. elegans, GLP-1 and LIN-12, which play a role in regulating cell fate decisions. Whenever GLP-1 function is lost, all the germ cells exit mitosis and enter meiosis. If this happens early in development, then the animal is sterile because it does …

The Influence Of The Estrous Cycle On Acute Seizure Activity, Alexis Yagielski

Honors Capstone Projects - All

Epilepsy is a neurological disease that is characterized by spontaneous seizure activity. Seizures are excitatory events that result from the abnormal excessive and hypersynchronous firing of populations of neurons in the brain. Catamenial epilepsy is a condition in which seizure threshold fluctuates during the female menstrual cycle. It is commonly hypothesized that this fluctuation is due to the changes in estradiol:progesterone ratio that occur over the course of the menstrual cycle (Herzog, 2008). It has been shown in many studies that estradiol has proconvulsant properties whereas progesterone possesses anticonvulsant properties. Considering the pattern of hormonal variation and the effects of …

Using Simple Self-Assembling Peptides To Attain Novel Protein-Like Functions, Tyler Smith

Honors Capstone Projects - All

Proteins carry out many extremely efficient functions, including catalysis and biomolecule recognition. Underlying this efficiency is their extraordinary complexity and ability to fold into unique three-dimensional structures. Attempts to replicate this efficiency through de novo design have only shown moderate success, and it is unclear how modern-day proteins may have evolved. However, short peptides that alternate hydrophobic and hydrophilic residues can self-assemble into amyloid fibrils to achieve well-defined secondary structure. These aggregates may have served as a template from which the first proteins were derived. We designed self-assembling seven-residue peptides that are able to act as Zn²⁺-dependent esterases. …

The Use Of Shape Memory Polymers As A Tool To Study Human Fibrosarcoma And Murine Mesenchymal Stem Cell Migration, Justin N. Elkhechen

Honors Capstone Projects - All

Shape memory polymers (SMPs) are a class of “smart” materials that can transform between two distinct conformations through external stimuli, such as heat or electricity. Their usage in bioengineering has led to a promising field of research that lies at the interface of cell and mechanobiology, potentially providing insight into cancer therapies and tissue development—two processes that exist in dynamic environments in vivo. The present work involves creating new, shape changing, scaffolds for studies to analyze cell migration upon changes to the environmental topography. Specifically, this Capstone has been primarily focused on the development of a “half and half” fibrous …

Overexpression And Gene Profiling Of Asparagine Synthetase In Hybrid Poplar, Kristopher Murray

Honors Capstone Projects - All

Plants with polysaccharide-rich secondary cell walls have great potential as a source of carbohydrates for bioethanol production. However, the presence of phenolic lignin inhibits the isolation of bioethanol precursors cellulose and hemicellulose from the secondary cell wall. Recent studies have linked nitrogen availability to secondary cell wall development and composition, making nitrogen metabolism genes an interesting target in the improvement of plant cell walls for biofuels production. Plants use a nitrogen assimilation pathway to convert inorganic nitrogen sources into organic sources, included amino acids, which play key roles in metabolism and cell wall development. Asparagine synthetase (AS), a key enzyme …

The Effect Of High Terperature On Mating: Developmental Buffering Of S. Cerevisiae To The Environment, Sarah Hosie

Honors Capstone Projects - All

In Saccharomyces cerevisiae, cellular polarization is an essential structural and functional aspect of growth and development. It is responsible for yielding and maintaining cellular asymmetry, and allows for cells to function. Mating in S. cerevisiae is a process that incorporates cell-to-cell signaling, signal transduction, cellular polarization, plasmogamy, karyogamy, and many other cellular processes. Each of these steps is mediated by a myriad of signaling proteins that are involved in a signaling cascade that is regulated by both extracellular and intracellular signals. Much is known about the mating process and pathway in S. cerevisiae. However, this project aimed to …

Characterization Of Ghrelin O-Acyltransferase Active Site, Leslie Patton

Honors Capstone Projects - All

Ghrelin, first discovered in 1999, is a 28-amino acid peptide hormone involved in the regulation of appetite, insulin secretion and sensitivity, and many neurological effects such as learning, memory, and depression.^1-6 Ghrelin has been identified to have a unique posttranslational octanoylation carried out by the enzyme ghrelin O-acyltransferase (GOAT). This distinctive modification is a point of interest in studying GOAT whereby blocking the acylation of the ghrelin could potentially halt the activity of the peptide hormone and provide a means of treating obesity, diabetes, and other diseases affected by ghrelin levels. The duration of my project involved working …

Synthesis And Characterization Of Co(Ii) And Cu(Ii) Pyrophosphate Coordination Complexes For Treatment Of Pathogenic Agents., Leann Jade Miles

Honors Capstone Projects - All

Candidiasis, the yeast infection of the mouth, genitals, and blood can be fatal in individuals who are immunocompromised. In humans, Candidiasis is most commonly caused by the yeast, Candida albicans (C. albicans), but can also be caused by other Candida species .^[1]

C. albicans demonstrates marked resistance towards Cu(II) but more sensitivity towards Co(II) compounds, especially in mutants that lack the CaCRP1 and CaCUP1 gene that encodes for the copper-transporting P-type ATPase and Metallothionein proteins, respectively.^[2] Here, I will describe the effects of Co(II) and Cu(II) metal based complexes that incorporate a bridging pyrophosphate …

Molecular Mechanisms For Regulating The Assembly Of The Mixed Lineage Leukemia-1 (Mll1) Core Complex, Michael Hu

Honors Capstone Projects - All

Epigenetics is the study of heritable traits that are not caused by alterations to DNA. An example of such a mechanism is histone methylation, specifically histone H3 lysine 4 (H3K4). Histones are proteins that wrap and package DNA into nucleosomes. Modifications to a histone’s chemistry can up or down regulate specific gene expression programs through changes in nucleosome positioning. Human mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases. Previous studies in the Cosgrove lab have shown that an interaction between two protein sub-complexes, MLL1 and WRAD (WDR5, RbBP5, Ash2L, DPY30), leads to dimethylation. …

The Purification And Characterization Of The Drosophila Melanogaster Trithorax Protein And Its Implications In The Studies Of The Set Domain Family Of Proteins, Kelsey Monteith

Honors Capstone Projects - All

Methylation at histone H3 lysine 4 (H3K4) is a post-translational modification often associated with transcriptional regulation through altering the structural state of chromatin. The human mixed lineage leukemia protein-1 protein (MLL1) is a known histone methyltransferase that catalyzes the transfer of methyl groups to H3K4. MLL1 works in a core complex with other essential components, proteins WDR5, RbBP5, Ash2L, DPY-30 (WRAD), which is required for H3K4 dimethylation. Trithorax (TRX) protein is the Drosophila melanogaster ortholog to human MLL1, and although structurally similar is unable to perform dimethylation when in complex with the human components. The goal of this study is …

The Use Of P53-Derived Stapled Peptides As Affinity Isolation Reagents, Kathryn Margaret Headley

Honors Capstone Projects - All

The transcription factor p53 can induce apoptosis and cell cycle arrest in response to cellular distress. Cancer cells often display increased cell survival. In most cases, this is due to a p53-related defect, such as mutation, deletion, degradation, or sequestration. HDM2 and HDMX are homologous proteins that regulate the function of p53, and their over-expression can lead to an ineffective p53 response. Various inhibitors, including hydrocarbon stabilized alpha-helices of p53 (SAH-p53s), have been developed to target HDM2 and HDMX and restore functionality to the p53 pathway. It has been recently found that SAH-p53 factors also elicit cell death responses in …

Vitamin B12-Based Bioconjugate Probes For In Vitro And In Vivo Imaging, Anna Kahkoska

Honors Capstone Projects - All

Cancer is the second leading cause of death in the United States. This year, an estimated 577,190 Americans will die as a result of this family of diseases. Finding cancer at its most treatable stage gives patients the greatest chance of recovery; novel imaging agents that target primary and metastasized tumors offer hope for improved prognoses in the future. Based on the hypothesis that vitamin B12 (B12) and its association with specific transport proteins could offer selective access to cancer cell lines, a series of B12-based imaging agents were synthesized, characterized, and assayed for both in vitro and in vivo …

Characterization Of Dap2 (Defense-­‐Associated Aspartic Protease-­‐2) In Arabidopsis Thaliana And Determining Its Role In Plant Defense Responses, Kemardo Kitaro Henry

Honors Capstone Projects - All

Plants defend themselves from invading pathogens by coordinating activity of several plant defense-­‐associated genes. Defense-­‐Associated Aspartic Protease-­‐1 (DAP1) gene of Arabidopsis encodes for an atypical aspartic protease with a putative proteolytic activity. The DAP1 is a negative regulator of plant immunity and the transgenic plants ectopically expressing this genes support pathogen growth and pathogenicity. The Arabidopsis genome has another putative protese that has ~75% homology to DAP1 present in close proximity and therefore was named DAP2. Here, I describe the role of the DAP2 gene in plant defense responses. Two independent knockout dap2 mutants are found to be more susceptible …

Development Of Protein Farnesyltransferase Variants With Altered Substrate Selectivity, Susan Zhang

Honors Capstone Projects - All

Post-translational modifications play an essential role in regulating protein structure and function. Enzymes catalyzing these modifications must often recognize and modify multiple substrate proteins from among a plethora of non-substrates with similar structures and amino acid sequences. For example, protein farnesyltransferase (FTase) catalyzes the addition of an isoprenoid group to a cysteine near the C-terminus of a substrate protein and is proposed to modify a pool of substrates numbering more than one hundred. We seek to understand the interactions in the FTase active site that engender substrate selectivity. By mutating two residues within FTase, we have developed FTase variants with …

Investigation Of Ghrelin Acylation By Ghrelin O-Acyltransferase, Edward P. Prybolsky

Honors Capstone Projects - All

Ghrelin is a peptide hormone involved in hunger signaling and other physiological processes. To become active, ghrelin first must be acylated by ghrelin O-acyltransferase (GOAT). This enzyme carries out a post-translational modification that attaches an octanoyl group to the side chain of the serine 3 (S3) residue of ghrelin. With ghrelin linked to hunger signaling, memory, and other physiological processes, GOAT has been identified as a potential drug target. However, inhibitor design is difficult because not much is known regarding the active site structure and catalytic mechanism of GOAT. This study reports the design of a novel fluorescence-based assay that …

Design Of Metalloproteins For Catalysis And Bioimaging, Krystyna Zhezherya

Honors Capstone Projects - All

Abstract

Metalloenzymes constitute a large subclass of proteins with diverse functions including metal-sensing, signal transduction pathways, and metal transport. In this report, I aim to study the intricate relationship between structure and function as it relates to metalloproteins in the fields of catalysis and bioimaging. For catalysis, I used existing platforms in addition to de novo design to study small scaffolds with varying secondary structure, and their function in catalyzing the hydrolysis of a p-nitrophenol ester. For bioimaging, I used an existing enzyme known as human carbonic anhydrase I, hCA, to develop a bioimaging agent that can be fused to …

Spatial Patterns Of Mercury In Atmospheric Deposition, Soils And Lake Biota In The Adirondack Park, New York, Xue Yu

Civil and Environmental Engineering - Dissertations

Mercury (Hg), as a trace element cycling in the environment, poses a serious health threat to both humans and wildlife due to its toxicity. Atmospheric deposition is the main source of Hg to most remote environments. The Adirondack Park in New York State of the United States receives moderate Hg deposition, and is a region characterized by relatively high concentrations of Hg in the terrestrial and especially aquatic biota. It is important to understand the mechanisms that contribute to the sensitivity of this region to Hg inputs. In my research, studies of the spatial patterns of Hg in atmospheric deposition, …

Recombinant Expression And Purification Of Human Intrinsic Factor (If) And Mutants K159d And Q201a Designed To Interfere With Cubilin Receptor Binding, Soreen Cyphers

Honors Capstone Projects - All

Although both the α- and β-domains of intrinsic factor (IF) have been previously expressed, the full crystal structure of the protein has yet to be reported. The purpose of this research is to (1) express IF in order to obtain a complete crystal structure and (2) utilize a mutant form of IF in order to orally deliver rotavirus to the ileum.The first goal of this research is to express IF in the yeast Pichia pastoris. The second goal is to express a K159D IF mutant protein. K159 of IF plays a role in salt bridge formation between IF and …

Synthesis And Structural Studies Of Calcium And Magnesium Diphosphonate Compounds, Seungmo Suh

Honors Capstone Projects - All

The purpose of this project is to study synthetic methodologies leading to the designing of various calcium and magnesium based diphosphonates with possible applications as bone scaffolding materials. In tissue engineering scaffolds are frequently employed to enhance the growth of new cells, providing the motivation to study alkaline earth metal based diphosphonates as a possible source for scaffold for bone growth.

While the transition metal phosphonates based frameworks are well studied, alkaline earth metal based phosphonates and diphosphonates remain at its infancy. The challenge to the chemistry of the alkaline earth metal phosphonates and diphosphonates based frameworks includes poor solubility …

Expression And Purification Of Recombinant Saposin B For Coenzyme Q10 Purification, Vivian Yaci Yu

Honors Capstone Projects - All

Coenzyme Q10 (CoQ10) plays a critical role in energy production in humans. Low levels of CoQ10 have been linked to diseases such as Parkinson's and Huntingtons's disease. CoQ10 levels decrease as we age, and use of statin drugs also lowers CoQ10 levels. Oral supplementation however increases CoQ10 levels. However, the lack of industrial sources of CoQ10 and the difficulty in CoQ10 purification has resulted the need to address these problems. Sporidiobolus johnsonii (S. johnsonii) has been reported as a natural producer of CoQ10. This work is aim to increase the production of CoQ10 in S. johnsonii through genetic …

Effects Of Ethanol On Social Behavior & Brain Structure, Lua Jafari

Honors Capstone Projects - All

Autism spectrum disorders are a developmental disorder characterized by abnormal social behavior. Those with autism have problems with social interactions, repetitive behaviors, and both verbal and non-verbal communication. While the cause of autism is not yet know, studies have shown that there are both genetic and environmental factors linked with autism. The amygdala is considered to be part of the “social brain”, and atypical activation and morphology of the amygdala has been seen in those with autism.

For this research project, I utilized rats subjected to acute ethanol exposure during gestation and examined exposure effects on the anatomy and biochemistry …

Determination Of The Amino Acids Involved In The Interactions Between Dpy-30 And Ash2l, Key Components Of The Mll1 Core Complex, Carey Stuart

Honors Capstone Projects - All

The human version of the DPY-30 protein is homologous to the DPY-30 protein in Caenorhabditis elegans (nematode), along with other DPY-30 homologous proteins in other organisms. This protein is involved in dosage compensation of X-linked genes, balancing the levels of expression of these genes between the sexes. The mechanism by which the balancing is carried out varies from organism to organism. For example, in C. elegans hermaphrodites (XX), transcript levels of the X-linked genes are cut in half. In Drosophila, the genes on the male’s (XY) X chromosome are transcribed at twice the rate of the female’s (XX) genes. …

Structural And Functional Characterization Of A Putative Citmhs Protein From Bacillus Anthracis, Qi Wen Li

Honors Capstone Projects - All

The growing number of strains of antibiotic-resistant bacteria poses a new threat to human health. The development of innovative strategies to treat bacterial infections is essential. The recent discovery of a new class of membrane transporters, CitMHS, unique to bacteria, presents a potential new target for antibacterial therapeutics. CitMHS is a class of secondary metal citrate membrane transporters, with only five members characterized to date (2,3,5,6,7). No crystal structures have been reported. Detailed structural and functional studies of CitMHS are essential to the understanding of infection pathways and, consequently, the ability to block such pathways. Cit_Ba is a CitMHS …

Germline Cyst Formation And Development In Zebrafish, Tess Cherlin

Honors Capstone Projects - All

In developing pre-natal mice, germline cysts are clusters or packages of cells consisting of individual oocytes. They are formed after the primordial germ cells move to the ovary during embryogenesis. The primordial germ cells divide, but do not completely separate. The cells within the cysts are linked by intercellular bridges, which end up breaking down leading to the formation of primordial follicles. The primordial follicles are oocytes surrounded by somatic cells called granulosa cells. Only one third of the original oocytes survive cyst breakdown and become the eggs that will be used by the mouse for her reproduction. This developmental …