Biochemistry, Biophysics, and Structural Biology Commons^™

A Novel In Vivo Protein Refolding Technique, Yuefei Huang

Wayne State University Dissertations

Proteins perform their functions in their native folded states and misfolding of proteins may cause severe diseases, including Alzheimer's disease, Parkinson's disease, prion disease and diabetes. Understanding protein folding is important for us to engineer proteins to treat these diseases. For protein therapeutics, large quantities of properly folded and functional proteins are required. The current technology produces recombinant proteins using either eukaryotic or prokaryotic expression system, both of them have major problems that prevent production of large quantities of properly folded and functional human proteins for protein therapeutics.

Although the eukaryotic cells have comprehensive folding machinery that contains chaperones and …

Human Trophoblast Survival And Invasion In The Developing Placenta: Autocrine Regulation By Hbegf, Philip Jessmon

Wayne State University Dissertations

HBEGF is a multifunctional protein in early pregnancy that induces cytotrophoblast (CTB) cell differentiation to an invasive phenotype, protects against apoptosis, and is involved in an autocrine signaling mechanism that leads to its own protein synthesis. CTBs exist in a low O₂ environment during the first 10 weeks of implantation, during which they invade the decidualized uterine stroma. Inhibitors of intracellular signaling pathways demonstrated that at 20% O₂ HBEGF induces an increase in cell migration through the ERK, MAPK14, JNK, or PIK3 pathways downstream of signaling through its ERBB receptors. Also downstream of these four pathways, HBEGF induces …

Destruction Of Biological Tetrapyrrole Macrocycles By Hypochlorous Acid And Its Scavenging By Lycopene, Dhiman Maitra

Wayne State University Dissertations

Hypochlorous acid (HOCl) is a potent oxidant generated by the hemoprotein myeloperoxidase. Although HOCl plays an important role in the innate immune response,sustained high levels of HOCl has been implicated to play a harmful role. In several pathological conditions such as atherosclerosis, endometriosis and sickle cell disease where HOCl is elevated there are reports of significant free iron accumulation. Free iron is toxic since it can lead to the generation of other secondary free radicals such as hydroxyl radical by Fenton reaction. The exact source and mechanism by which the free iron is generated is not clearly understood. This work …

Mechanical Forces And Tumor Cells: Insight Into The Biophysical Aspects Of Cancer Progression, Indrajyoti Indra

Wayne State University Dissertations

Mechanical forces play an important role in the regulation of cellular behavior and physiological processes including adhesion, migration, proliferation, tissue repair, embryogenesis and development. In addition, a number of diseases including cancer, have been linked to changes in cellular and extracellular mechanical properties. However, whether a correlation exists between the progression of cancer towards metastasis and mechanical factors has not been clearly defined. Additionally, how a cell responds to changes in extracellular mechanical cues as it gains metastatic abilities is poorly understood. To address these questions, we have utilized a panel of murine breast cancer cell lines with progressive metastatic. …

Rox1 Function In Dosage Compensation: Structural / Functional Analysis Of A Non-Coding Rna, Ying Kong

Wayne State University Dissertations

roX1 is a long non-coding RNA involved in the chromosome-wide gene regulation that occurs during dosage compensation in Drosophila. Dosage compensation in Drosophila melanogaster occurs by a global two-fold increase of transcription from the single male X chromosome. This essential process compensates for X chromosome monosomy. The male-specific lethal (MSL) complex, containing five proteins, localizes to the male X chromosome and alters chromatin to modify gene expression. roX1 and roX2 RNAs are redundant components of MSL complex that are required for its exclusive X-localization. Recent studies in our lab have revealed a second role of roX RNAs in heterochromatic gene …

Epigenetic Effects Of Benzo(A)Pyrene In Fundulus Heteroclitus And Danio Rerio, Xiefan Fang

Electronic Theses and Dissertations

DNA methylation is one of the epigenetic mechanisms that controls gene expression and is vulnerable to early life environmental toxicant exposures. Our goal was to use two fish models, Fundulus heteroclitus and Danio rerio (zebrafish), to study the benzo(a)pyrene (BaP) effects on DNA methylation status and whether the alterations could contribute to BaP-mediated reproductive and developmental toxicities. Initially, we used Fundulus to study BaP effects on glycine N-methyltransferase (GNMT) expression throughout development. Fundulus embryos were exposed to waterborne BaP at nominally 10 or 100 μg/L and both GNMT mRNA expression and enzyme activity were measured. Quantitative PCR and whole mount …

Stable Expression Of Tuberculosis Vaccine Antigen In Lettuce Chloroplasts, Priya Saikumar Lakshmi

Electronic Theses and Dissertations

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific transgene integration into the chloroplast genome was confirmed by polymerase chain reaction and Southern blot analysis. In transplastomic leaves, expression levels of fusion protein (CTB-ESAT6) varied depending upon …

Evolutionary Relationships Among Staphylococci And The Prevention Of Staphylococcus Aureus Nasal Colonization, Ryan Paul Lamers

Electronic Theses and Dissertations

Staphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of this bacterium is a known risk factor for autoinfection. The predisposition to infection by nasal carriers of S. aureus, and the ease with which strains are transferred between individuals, suggests that …

Synthesis, Characterization And Antibacterial Activity Of Silver Embedded Silica Nanoparticle/Nanogel Formulation, Roseline Menezes

Electronic Theses and Dissertations

The antibacterial property of silver (Ag) has been known since ancient time. It is reported in the literature that silver nanoparticles (AgNPs) exhibit improved antibacterial properties in comparison to silver ions of equivalent metallic Ag concentration. Such improvement in antibacterial activities is due to the high surface area to volume ratio of AgNPs (which facilitates interaction with the bacterial cells), increased release of silver ions and direct intra-cellular uptake of AgNPs leading to localized release of Ag ions. To date, over 300 consumer products containing AgNPs are available in the market and the inventory is rapidly expanding. The antibacterial efficacy …

Expression And Functional Evaluation Of Exendin 4 Fused To Cholera Toxin B Subunit In Tobacco Chloroplast To Treat Type 2 Diabetes, Ramya Nityanandam

Electronic Theses and Dissertations

The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the …

Expression Of Trichoderma Reesei Beta]-Mannanase In Tobacco Chloroplasts And Its Utilization In Lignocellulosic Woody Biomass Hydrolysis, Pankaj M. Agrawal

Electronic Theses and Dissertations

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plantbased production of these enzymes on large scale offers a cost effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β- mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed the site-specific …

Identification And Characterization Of Equine Rotaviruses In Southern Ireland, Crystal Cronin

Theses

Group A Rotaviruses(GARVs) are enteric pathogens which induce acute gastroenteritis in infants, neonates and the young of many animal species. During a 4 year study (2005-2009), 64 fecal samples from symptomatic foals, from several counties, in the south of Ireland, were molecularly characterized to determine the VP& (G type), VP4 (P type), and NSP4 antigenic specificities. Globally, G3 [P12] and G14[P12] are recognized as the predominant equine GARV strains. Twenty-two samples (34.38%) were found to contain G3 viruses, while 21 samples (32.81%) contained G14 viruses. In this study, there was a distinct increase in the incidence of mixed infections circulating …

Characterization Of Pregnancy Induced Cardiac Hypertrophy In Rats, Patricia Holden

Online Theses and Dissertations

During pregnancy, an increase in blood volume occurs to compensate for fetal development, resulting in cardiac hypertrophy. For the majority of women, this physiological cardiac hypertrophy resolves following pregnancy. Prolonged cardiac hypertrophy can lead to heart failure. We propose that by studying the biochemical mechanisms that mediate healthy cardiac remodeling associated with pregnancy, we will gain a better understanding of mechanisms involved in pathological cardiac hypertrophy. To determine the biochemical changes that occur during pregnancy induced cardiac remodeling, we subjected rats to timed matings and collected morphological and biochemical data from not pregnant, 19 days pregnant, and 24 hours postpartum. …

Quantitative Proteomics Analysis Of Differentially Expressed Proteins In Aβ(17-42) Treated Synaptosomes, Jaffer Mohammed

Online Theses and Dissertations

Oxidative stress has been associated in the pathogenesis of numerous diseases such as neurodegenerative disorders, ischemia, and cancer. The brain is susceptible to oxidative stress due to its high content of peroxidizable unsaturated fatty acids, high consumption of oxygen per unit weight, high levels of free radicals, and comparatively low levels of antioxidant defense systems. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can react with biomolecules such as proteins, lipids, carbohydrates, DNA, and RNA, which can lead to oxidative damage, cellular dysfunction, and can ultimately cause cell death. Down syndrome (DS) is the most common form of chromosomal …

The Incidence Of Type-1 Diabetes In Nod Mice Is Modulated By Restricted Flora Not Germ-Free Conditions., Cecile King, Nora Sarvetnick

Journal Articles: Regenerative Medicine

In the NOD mouse, the incidence of type-1 diabetes is thought to be influenced by the degree of cleanliness of the mouse colony. Studies collectively demonstrate that exposure to bacterial antigen or infection in the neonatal period prevents diabetes [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], supporting the notion that immunostimulation can benefit the maturation of the postnatal immune system [11]. A widely accepted extrapolation from this data has been the notion that NOD mice maintained under germ-free conditions have an increased incidence of diabetes. However, evidence supporting this influential concept is surprisingly limited [12]. In this …

Short Peptides As Inhibitors Of Amyloid Aggregation, Bradley Neddenriep, Anastasia Calciano, Daniel Conti, Erin Sauve, Marissa Paterson, Edward Bruno, David A. Moffet

Chemistry and Biochemistry Faculty Works

The misfolding and aggregation of proteins into amyloid has been linked to a variety of age-related diseases. Aggregation of proteins, such as Aβ in Alzheimer's disease and Islet Amyloid Polypeptide (IAPP, amylin) in type 2 diabetes, appears to lead to the formation of toxic assemblies. These assemblies range in size from small oligomers (2-8 proteins) to large fibrils (thousands of proteins). It remains unclear how these amyloidogenic proteins misfold and form toxic species, but growing evidence suggests that inhibiting the aggregation of these proteins could slow, if not prevent altogether, the progression of these diseases. We describe the use of …

Bio-Functionalized Clay Nanoparticles For Wound Healing Applications, Christopher Anthony Vaiana

Browse all Theses and Dissertations

Wound healing is a complex, multi-step process that can be summarized into three stages, namely hemostasis and inflammation, proliferation, and finally tissue remodeling. Battlefield wound healing demands rapid hemostasis using clotting or cauterizing agents to immediately limit blood loss, but this occurs at the expense of proper tissue repair beyond hemostasis. Layered silicate clays such as kaolin and montmorillonite (MMT) have been previously shown to induce blood clotting due to their ability to form charged interactions with clotting factors. The charge characteristics of sodium MMT (Na-MMT) also enable functionalization with active biomolecules. Herein we first functionalize three types of alumoinosilicate …

Interrogation Of The Distal Gut Microbiota Of Healthy Adolescents And Those With Irritable Bowel Syndrome, Laura J. Rigsbee

Browse all Theses and Dissertations

The human-associated microbiota has been the focus of much current research, with the microbiota inhabiting the gastrointestinal tract of particular interest. These organisms play many roles in human health and well-being. However, shifts in the composition of the intestinal microbiota have been associated with diseases such as irritable bowel syndrome, inflammatory bowel disease, and colon cancer. Several recent studies have reported on the distal gut microbiota composition of healthy adults and those with IBS, while there is a lack of studies devoted to adolescents. This study utilized a custom-designed Affymetrix Microbiota Array capable of detecting 775 phylo-species of intestinal bacteria …

Functional Metabolomics' Enhances Assessment Of Tissue Dysfunction As Demonstrated In A Rat Model Of Sub-Acute D-Serine Exposure, Isaie Sibomana

Browse all Theses and Dissertations

We describe a methodology that combines urinary metabolomics with a tissue-specific stressor administration to enhance assessment of tissue function. Kidney function in rats was mildly compromised with a sub-acute dose of D-serine and stressed with furosemide. NMR-based metabolomics analyses showed no detectable effects due to D-serine alone; but furosemide or D-serine + furosemide groups, classified separately from each other, and from control. Furosemide alone caused a ca. 2-fold increase in glucose, lactate, choline, and a 30% decrease in TCA intermediates (p≤0.05). D-serine suppressed these effects and produced a 1.7-fold increase in a p-phenolic acid-derivative of tyrosine (PAdY) relative to control …

Novel Role Of Mecp2 In Developing Oligodendrocytes And Myelination, Daniel Moore

Theses and Dissertations

Methyl-CpG-binding protein 2 (MeCP2 is) is an epigenetic regulator that binds to methylated DNA. Initially identified as transcriptional repressor, MeCP2 also binds to different proteins functioning as gene activator. Importantly, MecCP2 gene mutations and changes in MeCP2 levels are associated to several forms of mental retardation and autism-related disorders; including Rett, a neurodevelopmental disorder affecting primarily girls. While brain MeCP2 was considered to be exclusively neuronal, this regulator is also present in glia. We found that oligodendrocytes, the myelinating cells of the central nervous system (CNS), express particularly high MeCP2 levels at a developmental stage that precedes their final maturation. …