Biochemistry Commons^™

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

University Scholar Projects

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

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

Renée Crown University Honors Thesis 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

Renée Crown University Honors Thesis 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 …

Determination Of Pore Size Distribution In Capillary-Channeled Polymer (C-Cp) Fiber Stationary Phases By Inverse Size-Exclusion Chromatography (Isec) And The Study Of The Role Of Interstitial Fraction On C-Cp Fibers On Protein Binding Capacity, Zhengxin Wang

All Theses

ABSTRACT High performance liquid chromatography (HPLC), first used in the 1960's, is a rapidly evolving analytical technique, widely employed for identification, separation, and purification in biotechnology and pharmaceutical industries. The development of the stationary phases has played an important role in improving this technique. Each stationary phase will have its own disadvantages. Polysaccharide-based stationary phases such as cross-linked dextran cannot tolerate high pressures and linear velocities; silica stationary phases are rigid enough but slow mass transfer in the pores on the surface causes another problem; with the introduction of non-porous and small bead packing materials, the low surface area and …

The Role Of Stress-Activated Protein Kinases In Parasites, Tamara Kreiss

Theses, Dissertations and Culminating Projects

Lymphatic filariasis and leishmaniasis are neglected tropical diseases that are caused by nematode and protozoal parasites. These diseases cause disfiguration, leaving their host socially marked, and in some cases cause more severe disease that can lead to death. These infections, which tend to persist for long periods of time, also lead to bacterial and fungal co-infections, which further exacerbate the disease. Currently there are insufficient treatment options. Current therapies are often too expensive, have toxicity associated with them and are subjected to growing resistance amongst parasite populations. In this thesis I investigated two potential drug targets. The first, is the …

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

Renée Crown University Honors Thesis 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 …

Microgenomic Approaches To Identify Clinically Relevant Gene Signatures That Discriminate Histologic Types Of Breast Carcinomas., Sean Thomas Butterbaugh

College of Arts & Sciences Senior Theses

Background: Breast cancer presents itself in a variety of histologic types, and the two most common types are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC). Based on comparative genomic hybridization (CGH) analyses, ILC is more closely related to low grade IDC than it is to intermediate and high grade IDC. Results from the BIG 1-98 trial demonstrate that post-menopausal women who are affected with estrogen receptor positive (ER+) ILC or luminal B (high grade) IDC experience a greater magnitude of benefit when they are treated with the aromatase inhibitor (AI) letrozole compared to treatment with the antiestrogen tamoxifen. …

Porphyrin Cross-Linkers For Generating Soluble Molecularly Imprinted Polymers From Polyethyleneimine, Mohammed R. Elshaer

Seton Hall University Dissertations and Theses (ETDs)

Molecular recognition is vital to many biochemical processes and is at the heart of promising bio-medically related technologies. Molecular imprinting has a long-standing history as a successful method for mimicking the molecular recognition phenomena exhibited by nature, whereby artificial receptors are prepared for a given target molecule based on synthetic polymers. The molecularly imprinted polymer (MIP) contains a three dimensional network with a memorized cavity specific to the shape and functionality of the templated target molecule. The utility of traditional MIPs has been limited due to an inherent lack of solubility. We have worked toward developing a system that allows …

Expression Of Human Neutrophil Cathepsin G In Pichia Pastoris, Evan T. Perry

Undergraduate Honors Theses

Cathepsin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG is a poorly understood enzyme, in part because it can only be obtained as mature enzyme purified from human blood, and because it seems to have dual specificity for chymotrypsin-like and trypsin-like substrates. Therefore, yeast Pichia pastoris was used to express immature recombinant human CatG to provide a source of the enzyme free of biohazards and to allow study of its dual specificity and C-terminal processing. To avoid potential cleavage by a yeast kexin protease, the amino sequence was modified without …

Propagation Of Oligomeric Α-Synuclein And Amyloid-Β: Implications For Parkinson's And Alzheimer's Diseases, Matthew Stephen Planchard

Master's Theses

The aggregation of amyloidogenic proteins is a critical event in the pathology of a variety of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). The proteins α-synuclein (αS) and amyloid-β (Aβ) are involved in the formation of amyloid lesions observed in PD and AD, respectively. Both PD and AD exhibit a significant amount of co-pathology in clinical settings, and the αS and Aβ proteins have been shown to interact in vitro. Recent experimental consensus has shown oligomeric species to be significant, if not primary, sources of toxicity in these diseases. …

Cloning, Expression And Interaction Studies Of The Potential Rubisco Activase Cbbq, Salma A. Dawoud

Honors Theses

Carboxysomes are polyhedral microcompartments found in all cyanobacteria and in many chemoautotrophs. Within their shell they contain the enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO), which fixes CO₂. Downstream of the carboxysome operon there is another gene cluster containing several genes that may enhance carboxysome function. Two of these genes, cbbQ and cbbO, encode potential RubisCO activases. Using recombinant CbbQ and CbbO protein, and RubisCO isolated from carboxysomes the interaction between these proteins was studied. The CbbO and CbbQ proteins were both His tagged, allowing them to be purified with Ni²⁺-NTA column chromatography. Each of these tagged …

Redox Active Motifs In Selenoproteins, Fei Li, Yuliya Pepelyayeva, Elias S. J. Arner, Craig A. Bayse, Sharon Rozovsky

Chemistry & Biochemistry Faculty Publications

Selenoproteins use the rare amino acid selenocysteine (Sec) to act as the first line of defense against oxidants, which are linked to aging, cancer, and neurodegenerative diseases. Many selenoproteins are oxidoreductases in which the reactive Sec is connected to a neighboring Cys and able to form a ring. These Sec-containing redox motifs govern much of the reactivity of selenoproteins. To study their fundamental properties, we have used Se-77 NMR spectroscopy in concert with theoretical calculations to determine the conformational preferences and mobility of representative motifs. This use of Se-77 as a probe enables the direct recording of the properties of …

Tht And Capillary Electrophoresis To Monitor The Effects Of Solutions Conditions On Amylin Aggregation, Michael May

Graduate Theses and Dissertations

Amylin (hIAPP) aggregates have been found in 90% of patients with type II diabetes at autopsy, and are suspected to play a role in the death of islet &beta-cells¹. However, this aggregation process is not well understood. Here, we explore methods that utilize capillary electrophoresis (CE) as a means to better understand amylin's aggregation process.

We examined the effects of solutions conditions: agitation, pH, salt, and temperature on amylin aggregation using Thioflavin T, dot blots, and capillary electrophoresis. Thiofalvin T was used to predict the lag time to &beta-sheet formation. Our results indicated all variables with the exception …

Metal Stopping Reagents Facilitate Discontinuous Activity Assays Of The De Novo Purine Biosynthesis Enzyme Pure, Kelly L. Sullivan, Loredana C. Huma, Elwood Mullins, Michael E. Johnson, T. Joseph Kappock

Department of Biochemistry Faculty Publications

The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24 h, as assessed by the Bratton-Marshall assay (BMA) for diazotizable amines. The ZnSO₄ stopping reagent is proposed to chelate CAIR, enabling delayed analysis of this acid-labile product by BMA or other HTS methods

Characterization Of Udp-Arabinopyranose Mutase Genes In The Arabidopsis Cell Wall Mutant Mur5, Christopher A. Hart

Honors Scholar Theses

The genome of Arabidopsis thaliana contains several coding regions for UDP-arabinopyranose mutases (UAMs) that are also known as reversibly glycosylated polypeptides (RGPs). The mur5 cell wall mutant of Arabidopsis shows a 30% decrease in cell wall arabinose content, and a missense mutation in the Reversibly Glycosylated Polypeptide 2 gene was recently proposed to cause this mutant phenotype. Through a traditional complementation analysis, mur5 and a T-DNA insertion mutant in the RGP2 gene were shown not to complement each other, indicating that the two genes are mutant alleles of the same locus. The mur5 SNP located in RGP2 caused a more …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Functional Analysis Of Cytosolic Hsp70 Nucleotide Exchange Factor Networks In Yeast, Jennifer Lynn Abrams

Dissertations and Theses (Open Access)

The Hsp70 class of molecular chaperones play critical roles in protein homeostasis via an ATP-dependent folding cycle. Cytosolic Hsp70s in the budding yeast Saccharomyces cerevisiae, Ssa and Ssb, interact with up to three distinct nucleotide exchange factors (NEFs) homologous to human counterparts; Sse1/Sse2/HSP110, Fes1/HspBP1, and Snl1/Bag1. In an effort to understand the differential functional contributions of the cytosolic NEFs to protein homeostasis (“proteostasis”), I carried out comparative genetic, biochemical and cell biological analyses. For these studies, I developed protocols to monitor protein disaggregation and reactivation in a near real-time coupled assay that revealed the importance of aggregate dynamics in the …

Pop2: A Potential Regulator Of Hmt1-Catalyzed Arginine Methylation In Yeast, Celeste Excell

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methylation is an important post-translational modification that is vital in regulating various cellular processes such as gene transcription, cell signaling, and RNA processing. Protein arginine methyltransferases (PRMTs) are responsible for performing this important modification. PRMT1 (protein arginine methyltransferase 1) and Hmt1 (hnRNP methyltransferase 1) are the predominant PRMTs in humans and yeast, respectively. Despite growing momentum in this field, relatively little is understood about PRMT regulation. Further work discovering how PRMTs are regulated will greatly advance our understanding of diseases where PRMTs have been implicated, such as heart disease, viral pathogenesis, and cancer.

It has been discovered that …

Electron Transfer And Substrate Reduction In Nitrogenase, Karamatullah Danyal

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The reduction of nitrogen to ammonia by the industrial Haber-Bosch process is considered one of the major scientific breakthroughs of the last century. It is considered to be responsible for approximately one third of the world's current population. This growth over the past 50 or so years accompanied by the changes in dietary habits due to economic growth have markedly increased the demand for fixed nitrogen in the form of fertilizer. The Haber-Bosch process and biological nitrogen fixation has been able to fulfil this demand. However, this industrial process is costly due to its high temperature and pressure requirements. Every …

Mechanisms Of Action And Co-Optive Evolution For Hypervariable Courtship Pheromones In Plethodontid Salamanders., Wilburn Beau Wilburn

Electronic Theses and Dissertations

Pheromones are an important type of chemical cue used by most animals to convey information between individuals. For more than 100 million years, male plethodontid salamanders have utilized a system of non-volatile, proteinaceous pheromones to regulate female mating behavior and receptivity. One of these pheromone components, Plethodontid Modulating Factor (PMF), is a hypervariable protein related to the three-finger protein (TFP) superfamily. Previous studies revealed that PMF persists as a rapidly evolving multi-isoform mixture. However, many characteristics of PMF as a pheromone remained undetermined, including gene structure and transcriptional regulation, translational regulation, protein structure, evolutionary mechanisms, and the isoform effects on …