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Investigation Of Dna Interaction With Bis-Cationic Imidazolium Salts, Kyle Daily 6440928

Williams Honors College, Honors Research Projects

Imidazolium salt compounds have shown promise in vitro against non-small cell lung cancer (NSCLC) lines, but the exact mechanism of activity these compounds is still unknown. Due to their planar aromatic structure, we hypothesized that our bis-cation imidazolium salt series could act through DNA intercalation similar to known intercalators ethidium bromide, acridine orange, and echinomycin. Here, we tested bis-cation imidazolium salts with linkers of 2, 4, 10, and 12 carbons for DNA intercalation via viscosity and fluorescent intercalator displacement (FID) assays. Despite some solubility issues that may have decreased results, we found that our butyl and dodecyl linked compounds had …

Lipid-Protein Interactions Via Fluorescence Quenching In Small Unilamellar Vesicles, Talia Kordahi

Williams Honors College, Honors Research Projects

Studying peptide interactions with model membranes have aided in understanding protein-lipid interactions more clearly. In this report, fluorescent small unilamellar vesicles (SUVs) of a known composition were made. Two peptides, RND1 and MARCKS, were also fluorescently labeled and added to a solution of SUVs. The interactions between these were observed via fluorescence quenching, read out with a spectrofluorimeter. In addition, two environments of buffer were tested: 1X PBS and 0.1X PBS. After obtaining data, it was concluded that MARCKS quenched the SUVs more than RND1. This could be due to the presence of more lysine amino acids in MARCKS, which …

The Role Of Tgfβ1 And Ep Receptors In The Differentiation Of Normal And Diseased Lung Fibroblasts, Madison A. Hexter

Williams Honors College, Honors Research Projects

Fibroblasts, and their differentiation into myofibroblasts, are important in the pathology of several lung diseases including asthma and idiopathic pulmonary fibrosis (IPF). The differentiation process is dependent on several factors, such as soluble factors like transforming growth factor beta 1 (TGFβ1) and mechanical signals such as matrix stiffness. Additionally, the soluble arachidonic acid metabolite Prostaglandin E₂ (PGE₂) prevents TGFβ1 induced differentiation of fibroblasts to myofibroblasts, but the molecular mechanism is not completely understood¹⁵. Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel, has previously been implicated in the differentiation of cardiac fibroblasts^12,13. …

Improved Sugar Yields From Biomass Sorghum Feedstocks: Comparing Low‑Lignin Mutants And Pretreatment Chemistries, Bruno Godin, Nick Nagle, Scott Satler, Richard Agneessens, Jérôme Delcarte, Edward Wolfrum

United States Department of Agriculture-Agricultural Research Service / University of Nebraska-Lincoln: Faculty Publications

Background: For biofuel production processes to be economically efficient, it is essential to maximize the production of monomeric carbohydrates from the structural carbohydrates of feedstocks. One strategy for maximizing carbohydrate production is to identify less recalcitrant feedstock cultivars by performing some type of experimental screening on a large and diverse set of candidate materials, or by identifying genetic modifications (random or directed mutations or transgenic plants) that provide decreased recalcitrance. Economic efficiency can also be increased using additional pretreatment processes such as deacetylation, which uses dilute NaOH to remove the acetyl groups of hemicellulose prior to dilute acid pretreatment. In …

Effects Of Synthetic Ligands On Heterodimer Pairs Regarding Full-Length Human Pparα, Rxrα And Lxrα, Emily Delman

Browse all Theses and Dissertations

Nuclear receptor study is critically relevant in therapeutic medicine since the intricate details of disease states pertaining to atherosclerosis and diabetes are poorly understood. Three nuclear receptors of interest regulate target genes pertaining to cholesterol and fatty acid regulation, linking these receptors to therapeutic medicine. The first is the peroxisome proliferator-activated receptor alpha (PPARa), which resides in liver and muscle, coordinating lipoprotein and fatty acid homeostasis [1]. Cholesterol homeostasis is dictated by the liver X receptor alpha (LXRa), targeting genes pertaining to the kidney, intestine, liver and adipose tissues [2]. A common partner receptor to PPARa and LXRa is known …

Instability At Trinucleotide Repeat Dnas, Rujuta Yashodhan Gadgil

Browse all Theses and Dissertations

Trinucleotide repeats (TNRs) are sequences prone to formation of non-B DNA structures and mutations; undergo expansions in vivo to cause various inherited neurodegenerative diseases. Hairpin structures formed during DNA replication or repair can cause replication fork stalling and if left unrepaired could cause single or double strand DNA breaks. To test and study this hypothesis we have devised a novel two color marker gene assay to detect DNA breaks at TNRs. By inducing replication stress our results show that TNRs are prone to DNA strand breaks and it is dependent on the repeat tract length. Double strand breaks at structured …

Investigation Of Mutations In Nuclear Genes That Affect The Atp Synthase, Russell Dsouza

Wayne State University Dissertations

The F1 domain is the catalytic subunit of the mitochondrial ATP synthase. Studies with respiratory-deficient yeast identified ATP1 and ATP2 as nuclear genes encoding the alpha and beta subunits, respectively, of the mitochondrial F1-ATPase. The mutations in the atp1 and atp2 genes were cloned and sequenced, and they appear to affect the ATP synthase. Most yeast strains with mutations in the or the subunit primarily show an F1 assembly defective phenotype. This feature is similar to the assembly-defective mutants missing the chaperones required for assembly of the F1 oligomer or either the alpha/beta subunits.

Some of the atp2 and atp1 …

Insights Into De Novo Fes-Cluster Biogenesis Via The Eukaryotic Fes-Cluster (Isc) Pathway In Vitro, Stephen Paul Dzul

Wayne State University Dissertations

Fe-S clusters are iron-containing cofactors utilized by numerous proteins within several biological pathways essential to life. In eukaryotes, the primary pathway for Fe-S cluster production is the iron-sulfur cluster (ISC) pathway. The eukaryotic ISC pathway, localized primarily within the mitochondria, has been best characterized within Saccharomyces cerevisiae. In yeast, de novo Fe-S cluster formation is accomplished through coordinated assembly of the substrates iron and sulfur on the primary scaffold assembly protein “Isu1”. The sulfur used for cluster assembly is provided by the cysteine desulfurase “Nfs1”, a protein that works in union with its accessory protein “Isd11”. Frataxin “Yfh1” helps direct …

Effective Drug Treatment Induces Drug Resistance Through Rapid Genome Alteration-Mediated Cancer Evolution, Steven Horne

Wayne State University Dissertations

The central paradox associated with current cancer therapeutic strategies is initially effective treatment, which eliminates a high tumor cell count, consistently results in successful drug resistance. Mathematical and evolutionary modeling have previously suggested that therapeutic intervention could provide selective pressure for the expansion of resistant variants. Drug-related stress has been associated with genome chaos, a common phenomenon in cancer characterized as rapid, stochastic genomic fragmentation and reorganization. Since cancer represents an evolutionary process, analysis within the context of genome-mediated cancer evolution can shed light on this key problem of therapeutics. We propose that genomic change is a general response to …

Genome Wide Analysis Identifies Sphingolipid Metabolism As A New Target Of Valproic Acid, Shyamalagauri Jadhav Jadhav

Wayne State University Dissertations

Bipolar disorder (BD), which is characterized by depression and mania, affects about 1% of the total world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproic acid (VPA), a branched short-chain fatty acid, is one of the most widely used drugs for the treatment of BD. Although many hypotheses have been postulated to explain the molecular mechanism of action of this drug in BD, the therapeutic mechanism is not understood. This knowledge gap has hampered the development of new drugs to treat this disorder. To identify candidate pathways affected by VPA, I performed …

A Novel Role For Repetitive Sequences In Recognition Of The Drosophila Melanogaster X Chromosome, Sonal Suresh Joshi

Wayne State University Dissertations

In humans and fruit flies, males have one X chromosome while females have two. This imbalance in gene dosage is potentially lethal, and the process of dosage compensation corrects it. The MSL (Male Specific Lethal) complex, which is composed of five proteins and one of two functionally redundant long non-coding roX (RNA on the X) RNAs, brings about dosage compensation in Drosophila melanogaster. In fruit fly dosage compensation, all the genes on the single male X chromosome are upregulated approximately twofold, via chromatin modifications, to equalize gene dosage with the two X chromosomes of females. This process calls for highly …

An Analysis Of The Interaction Between Sin3 And Methionine Metabolism In Drosophila, Mengying Liu

Wayne State University Dissertations

Chromatin modification and cellular metabolism are tightly connected. The mechanism for this cross-talk, however, remains incompletely understood. SIN3 controls histone acetylation through association with the histone deacetylase RPD3. In this study, my major goal is to explore the mechanism of how SIN3 regulates cellular metabolism.

Methionine metabolism generates the major methyl donor S-adenosylmethionine (SAM) for histone methylation. In collaboration with others, I report that reduced levels of some enzymes involved in methionine metabolism and histone demethylases lead to lethality, as well as wing development and cell proliferation defects in Drosophila melanogaster. Additionally, disruption of methionine metabolism can directly affect histone …

Modeling The Mechanism Underlying Environmental And Genetic Determinants Of Gene Expression And Complex Traits, Gregory Alan Moyerbrailean

Wayne State University Dissertations

Advances in next-generation sequencing technologies and functional genomics strategies have allowed researchers to identify both common and rare genetic variation, to deeply profile gene expression, and even to determine regions of active gene transcription.

While these technologies and strategies have contributed greatly to our understanding of complex traits and diseases, there are many biological questions and analytical issues to be addressed.

Genome-wide association studies (GWAS) have successfully identified large numbers of genetic variants associated with complex traits and diseases. However, in many cases the mechanistic link between the phenotype and associated variant remains unclear. This may be because most variants …

Molecular Basis Of Sex Differences In Cardiac Hypertrophy And Heart Failure, Maya James

Online Theses and Dissertations

Transforming growth factor beta ligands and receptors are known to be pro-hypertrophic and pro-fibrotic factors in the heart, and are known to contribute to the development of cardiac hypertrophy and heart failure. It is well established that premenopausal females possess a lower incidence of these pathologies. We and others have observed a greater level of fibrosis in male hearts compared to female hearts in rodent models of cardiac hypertrophy. It is well established that estrogen is cardioprotective in that it can prevent the development of cardiac hypertrophy, as well as abrogate the development of heart failure following sustained cardiac hypertrophy. …

Enzyme Analysis Of Oxidatively Modified Proteins Post-Tbi, Brittany Rice

Online Theses and Dissertations

The brain is one of the most important organs in the body. It functions as a control center by regulating and coordinating actions and reactions, which is facilitated via signal transduction pathways. Its function is primarily dependent upon sufficient supply of glucose for energy metabolism. The dysfunction of the brain resulting from an external force is known as traumatic brain injury (TBI). Symptoms range from physical to psychological and effects can be mild, moderate, or severe depending on the extent of injury. TBI is associated with oxidative damage, the overproduction of reactive oxygen/nitrogen species. Reduced energy metabolism is a consequence …

A Novel Forensic Dna Profiling Method Based On Molecular Beacons Without Dna Purification, Benjamin C. Shaw

Online Theses and Dissertations

Analysis of polymorphisms in nucleic acid sequences provides the basis for identification of individuals and their genetic deficiencies. Currently, the accepted method of analysis for profiling is Short Tandem Repeat (STR) profiling. This is a lengthy process, typically taking up to 3 days. The time necessary to generate an STR profile, along with the ever-increasing reliance on DNA to solve crimes, has led to a large DNA sample backlog, with violent crime turnaround taking an average of 103 days. The time and resource investment required for STR analysis is significant, and not all samples generate useful profiles.

The current methods …

Recruitment Of Monocytes To The Pre-Ovulatory Ovary, Alex Paige Whitaker

Online Theses and Dissertations

Ovulation is a recurring biological process that involves inflammatory reactions that degrade and restructure tissue. It requires the chemotaxis of leukocytes to the ovary to help regulate and promote these events. Hormonal signals and chemokines that are released during the peri-ovulatory period initiate the release of specific leukocyte populations, possibly from the spleen, that infiltrate the ovary and facilitate the release of oocytes from ovarian follicles. These studies utilized a super-ovulation protocol to initiate ovulation in immature balb/c and cd1 mice. Ovaries were collected at several times post human chorionic gonadotropin (hCG) injection and analyzed by multi-color flow cytometry. Oviducts …

Efficiency Of The Targetron Gene Knockout System As A Transformative Protocol For The Mutagenesis Of Listeria Monocytogenes, Hilaree Noele Frazier

Online Theses and Dissertations

The process of using electroporation to introduce plasmid DNA into host cells is a valuable molecular technique that is increasingly employed in labs worldwide. Electroporators are generally small and relatively inexpensive, making them attractive systems to use for a variety of purposes. Electroporation protocols are numerous in the published literature and encompass all cell types, from prokaryotic bacterial cells to eukaryotic human cells. The TargeTron Gene Knockout System by Sigma-Aldrich is an affordable option for the electroporation of numerous bacterial species. However, its use in Listeria monocytogenes has not been extensively characterized. Here we sought to discuss the effectiveness of …

Characterization Of A Phosphonate-Specific Cytidylyltransferase, Kissa Batul

Theses and Dissertations (Comprehensive)

Antibiotic resistance is a major global health concern that requires new therapeutic approaches. Furthermore, a lack of narrow spectrum antibiotics on the market produces unintended consequences with respect to changes in our microbial make up. Phosphonates are reduced versions of phosphates that possess a C-P bond which is more resistant to enzymatic and chemical degradation. The role of phosphonate containing macromolecules (e.g. cell surface polysaccharides) remains enigmatic, however their presence suggests that they may confer an advantage. The biosynthesis of phosphonate-containing macromolecules is unknown, but a pathway is proposed involving aLicC type cytidylyltransferase-catalyzed conjugation to a phosphonate followed by a …

The Role Of Foxo Transcription Factors In Alcohol-Induced Deficient Fracture Repair, Philip M. Roper

Dissertations

Proper and complete repair of a bone fracture is essential in quality of life maintenance, but poor healing and fracture malunion are still medically and socially relevant problems. Alcohol abuse impairs normal fracture healing, leading to delayed or incomplete union. This dissertation aims to clarify mechanisms behind this alcohol-induced impaired healing, thereby elucidating potential methods of intervention.

Alcohol-induced oxidative stress has been linked to many morbidities associated with alcohol abuse. This dissertation elucidates a potential mechanism through which alcohol inhibits fracture healing by increasing oxidative stress. Using a rodent model, I found that alcohol exposure decreases fracture callus formation and …

Integrin Α6Β4 Promotes Pancreatic Cancer Invasion By Altering Dna Repair-Mediated Epigenetics, Brittany L. Carpenter

Theses and Dissertations--Molecular and Cellular Biochemistry

Integrin α6β4 is upregulated in pancreatic carcinoma, where signaling promotes metastatic properties, in part by altering the transcriptome. Such alterations can be accomplished through DNA demethylation of specific promoters, as seen with the pro-metastatic gene S100A4. I found that signaling from integrin α6β4 dramatically upregulates expression of amphiregulin (AREG) and epiregulin (EREG), ligands for the epidermal growth factor receptor (EGFR), and that these ligands promote pancreatic carcinoma invasion. To determine if AREG and EREG are regulated by DNA methylation, pancreatic cancer cells with low AREG and EREG expression were treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR), resulting in stable …

Calcineurin: From Activation To Inhibition, Erik C. Cook

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin is a Ser/Thr phosphatase whose function is implicated in critical physiological processes such as immune system activation, fetal heart development, and long-term depression in neurons. Calcineurin has been implicated in the progression of Alzheimer’s disease and cardiac hypertrophy. It is not well understood how calcineurin is activated on a molecular level by Ca²⁺ and its activating protein calmodulin. Previous data from our lab show that calmodulin interaction induces the folding of the intrinsically disordered regulatory domain of calcineurin in two discrete and distant regions into α-helical conformations and that this folding is critical for complete activation of calcineurin. …

A Pipeline For Creation Of Genome-Scale Metabolic Reconstructions, Shaun W. Norris

Theses and Dissertations

The decreasing costs of next generation sequencing technologies and the increasing speeds at which they work have lead to an abundance of 'omic datasets. The need for tools and methods to analyze, annotate, and model these datasets to better understand biological systems is growing. Here we present a novel software pipeline to reconstruct the metabolic model of an organism in silico starting from its genome sequence and a novel compilation of biological databases to better serve the generation of metabolic models. We validate these methods using five Gardnerella vaginalis strains and compare the gene annotation results to NCBI and the …

Sensing And Mapping Of Surface Hydrophobicity Of Proteins By Fluorescent Probes, Nethaniah Dorh

Dissertations, Master's Theses and Master's Reports

Surface hydrophobic interactions in proteins play a critical role in molecular recognition, influence biological functions, and play a central role in many protein misfolding diseases. As significance of surface hydrophobic interactions in age-related proteinopathies is becoming clear; it has led to an increased demand for better probes and tools to sense and characterize protein surface hydrophobicity. Current commercially available fluorescent probes such as 8-anilino-1-naphthalene sulfonic acid (ANS), 4,4′ -dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS), 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN), tetraphenylethene derivative, and Nile Red can sense proteins average hydrophobicity. However, probe limitations prevents their application for measuring the protein surface hydrophobicity. Some of the major deficiencies …

Functional Characterization Of Cancer-Related Mutations Of Erk3, Hadel Mohammed Alsaran

Browse all Theses and Dissertations

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer. Importantly, ERK3 promotes cancer cell migration and invasion by phosphorylating steroid receptor activator 3 (SRC-3), hence upregulating pro-invasive matrix metalloproteinase genes. While the link between ERK3 and cancers has been recognized, little is known about ERK3 mutations in cancer progression. In this study, we have investigated ERK3 mutations on arginine 64 (arginine 64 mutated to cysteine or histidine, R64C or R64H) and leucine 290 (leucine …

Biomimetic Production Techniques For Mechanical And Chemical Characterization Of Sucker Ring Teeth Isoform-12 From The Dosidicus Gigas Squid, Marcus T. Grant

Browse all Theses and Dissertations

The unique protein-based structure of Sucker Ring Teeth (SRT) of cephalopods have spurned research into the molecular design, physical characteristics, functionality and mechanical properties to explore biomimetic engineering and biochemical potential for eventual industrial production. Previous research has elucidated the potential for scientific and industrial exploitation. However, much of the previous research focused on the most abundant protein isoform of the sucker ring teeth, suckerin-19 (also known as suckerin-39) from the Jumbo or Humboldt Squid (Dosidicus Gigas). There is little known about the characteristics of the other 37 protein isoforms of Sucker Ring Teeth. Although the other isoforms have similar …

Autoimmunity In Picornavirus Infections, Chandirasegara Massilamany, Andreas Koenig, Jay Reddy, Sally Huber, Iwona Buskiewicz

School of Veterinary and Biomedical Sciences: Faculty Publications

Enteroviruses are small, non-enveloped, positive-sense singlestrand RNA viruses, and are ubiquitously found throughout the world. These viruses usually cause asymptomatic or mild febrile illnesses, but have a propensity to induce severe diseases including type 1 diabetes and pancreatitis, paralysis and neuroinflammatory disease, myocarditis, or hepatitis. This pathogenicity may result from induction of autoimmunity to organ-specific antigens. While enterovirus-triggered autoimmunity can arise from multiple mechanisms including antigenic mimicry and release of sequestered antigens, the recent demonstration of T cells expressing dual T cell receptors arising as a natural consequence of Theiler’s virus infection is the first demonstration of this autoimmune mechanism.

Genome-Wide Search For Host Association Factors During Ovine Progressive Pneumonia Virus Infection, Jesse Thompson, Fangrui Ma, Meghan Quinn, Shi-Hua Xiang

School of Veterinary and Biomedical Sciences: Faculty Publications

Ovine progressive pneumonia virus (OPPV) is an important virus that causes serious diseases in sheep and goats with a prevalence of 36% in the USA. Although OPPV was discovered more than half of a century ago, little is known about the infection and pathogenesis of this virus. In this report, we used RNA-seq technology to conduct a genome-wide probe for cellular factors that are associated with OPPV infection. A total of approximately 22,000 goat host genes were detected of which 657 were found to have been significantly up-regulated and 889 down-regulated at 12 hours post-infection. In addition to previously known …

Guidelines For The Use And Interpretation Of Assays For Monitoring Autophagy (3rd Edition), Daniel J. Klionsky, Rodrigo Franco, More Than 2,000 Co-Authors

School of Veterinary and Biomedical Sciences: Faculty Publications

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that …

From Crystal Structure Of Α-Conotoxin Gic In Complex With Ac-Achbp To Molecular Determinants Of Its High Selectivity For Α3Β2 Nachr, Bo Lin, Manyu Xu, Xiaopeng Zhu, Yong Wu, Xi Liu, Dongting Zhangsun, Yuanyan Hu, Shi-Hua Xiang, Igor E. Kasheverov, Victor I. Tsetlin, Xinquan Wang, Sulan Luo

School of Veterinary and Biomedical Sciences: Faculty Publications

Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking …