Molecular Biology Commons^™

Enzymatic Post-Translational Halogenation For Adding Functionality To Biomaterials, Alexander L. Compean

Browse all Theses and Dissertations

Silk fibroin from the silkworm, Bombyx mori, is a unique biomaterial that has been extensively studied for a variety of applications due to its promising properties such as controllable self-assembly, robust mechanical properties, and biological compatibility. Previously, there have been numerous methods describing the chemical modification of silk fibroin that utilize synthetic or enzymatic means that do not use halogens as a means of functionalization. Herein, a halogenation strategy is presented to modify silk fibroin with the aim of developing a novel functional material through the carbon-halogen (C-X) bond. Modification with NaX (X = Cl, Br, and I) salts, hydrogen …

Microsatellites And Their Association With Break Induced Replication, French J. Damewood Iv

Browse all Theses and Dissertations

To study microsatellites instability and their repair pathways a dual fluorescent (DF2) and selectable (ganciclovir sensitive/ thymidine kinase (TK) expressing) cell system was assayed using replication fork stalling agents hydroxyurea and telomestatin. These cell lines carried ectopically integrated microsatellites derived from the Dystrophia Myotonica Protein Kinase (DMPK) gene ((CTG)102 microsatellite), or an 88 bp polypurine/ polypyrimidine (Pu/Py) repeat from the PKD-1 locus, inserted into a FLP recombinase target site. These microsatellites form non-B DNA structures in -vivo and in-vitro causing replication fork stalling and double strand breaks. DF2 myc (CTG)102 -TK cells treated with hydroxyurea were assayed for mutagenesis of …

The Effect Of Cxcl12 Ligand On Internalization And Dimerization Of Cxcr4 Receptors In Live Cells, Loga Iyer

Williams Honors College, Honors Research Projects

The primary objective of this project was to determine the effect of CXCL12 ligand binding on the CXCR4 receptor, specifically, how it would impact receptor internalization and dimerization. The CXCL12 ligand derives from the stromal cell-derived alpha family [8]. The CXCR4 receptors, known as C-X-C chemokine receptor type 4 play an essential role in controlling cell proliferation. When misregulated, these receptors can drive tumorigenesis and are thus important targets of cancer therapy. These G protein-coupled receptors stimulate a cascade of signaling pathways in specific tissues [1]. These pathways include the positive transcriptional control of CXCR4 via the Nuclear Respiratory Factor-1 …

Targeted Combination Therapy Of Triple-Negative Breast Cancer, Ketki Bhise

Wayne State University Dissertations

TARGETED COMBINATION THERAPY OF TRIPLE-NEGATIVE BREAST CANCERby KETKI BHISE May 2021 Advisor: Dr. Arun Iyer Major: Pharmaceutical Sciences Degree: Doctor of Philosophy Triple Negative Breast Cancer (TNBC) accounts for 10-20% of the total breast carcinoma cases. There is no FDA-approved targeted therapy for TNBC due to lack estrogen, progesterone and HER-2. We have identified the role of a hypoxia biomarker, carbonic anhydrase IX (CAIX) in proliferation and metastasis of TNBC. Based on this observation, we have developed CAIX-targeted Doxorubicin (Dox) prodrug, abbreviated as DoxAtz. To improve the delivery of DoxAtz to the tumor, we developed long circulating liposomes (LipoDoxAtz) and …

Mnrr1: Understanding The Role Of A Novel Mitochondrial-Nuclear Regulator, Stephanie L. Gladyck

Wayne State University Dissertations

Mitochondria are complex organelles that generate most of the energy required to sustain life and function in metabolic and signaling pathways required to maintain cellular homeostasis. MNRR1 (mitochondrial nuclear retrograde regulator 1 or CHCHD2) is a small, bi-organellar twin CX9C protein that is emerging as an important regulator of mitochondrial function, apoptosis, and cellular stress by participating in mitochondrial-nuclear crosstalk. Our lab has previously shown that in the mitochondria, MNRR1 regulates complex IV (Cytochrome c oxidase or COX) and is able to finetune the oxidase function through phosphorylation status. We have also shown that during stress, mitochondrial MNRR1 levels deplete, …

Entry And Replication Of Negative-Strand Rna Viruses, Kerri Boggs

Theses and Dissertations--Molecular and Cellular Biochemistry

Hendra virus (HeV) and human metapneumovirus (HMPV) are negative-sense, singled-stranded RNA viruses. The paramyxovirus HeV is classified as a biosafety level 4 pathogen due to its high fatality rate and the lack of a human vaccine or antiviral treatment. HMPV is a widespread pneumovirus that causes respiratory tract infections which are particularly dangerous for young children, immunocompromised individuals, and the elderly. Like HeV, no vaccines or therapies are available to combat HMPV infections. These viruses fuse their lipid envelopes with a cell to initiate infection. Blocking cell entry is a promising approach for antiviral development, and many vaccines are designed …

Glycosaminoglycan Modulation Of Key Proteins In Alzheimer's Disease, Tamim Chiba

Theses and Dissertations

Glycosaminoglycans (GAGs) are linear polysaccharides whose disaccharide building blocks consist of an amino sugar (D-glucosamine) and either uronic acid (D-glucuronic acid or L-iduronic acid) or galactose. Nearly all GAGs (with the exception of hyaluronic acid) are covalently attached to proteins, forming proteoglycans, and are ubiquitous in nature. The monomeric units, chain length, degree and pattern of sulfation, and epimerization are important properties that determine the influence of these molecules. Through Coulombic forces, hydrogen bonds and hydrophobic interactions, they regulate many physiological and pathophysiological processes such as blood coagulation, cell growth and differentiation, inflammatory processes, host defense and viral infection mechanisms. …

Microsatellites And Their Association With Break Induced Replication, French J. Damewood Iv

Browse all Theses and Dissertations

To study microsatellites instability and their repair pathways a dual fluorescent (DF2) and selectable (ganciclovir sensitive/ thymidine kinase (TK) expressing) cell system was assayed using replication fork stalling agents hydroxyurea and telomestatin. These cell lines carried ectopically integrated microsatellites derived from the Dystrophia Myotonica Protein Kinase (DMPK) gene ((CTG)102 microsatellite), or an 88 bp polypurine/ polypyrimidine (Pu/Py) repeat from the PKD-1 locus, inserted into a FLP recombinase target site. These microsatellites form non-B DNA structures in -vivo and in-vitro causing replication fork stalling and double strand breaks. DF2 myc (CTG)102 -TK cells treated with hydroxyurea were assayed for mutagenesis of …

Quantitative Pcr And Sanger Sequencing Of Mitochondrial Dna Recovered From Waterlogged Bone, Kailey Babcock

Master of Science in Forensic Science Directed Research Projects

In forensic contexts, samples containing heavily fragmented DNA are commonly encountered. Compromised biological samples are especially prevalent in instances where human remains have been submerged in an aqueous environment for extended periods of time. Nuclear DNA is particularly vulnerable to the prolonged exposure to heat, moisture, and bacterial degradation that are prevalent in aquatic settings. Paired with the difficulty of recovering DNA from skeletal remains, which are often the only remaining component after the soft tissues have been stripped away, mitochondrial DNA (mtDNA) analysis serves as an invaluable alternative. In this multifaceted study, mtDNA analysis was performed on waterlogged bone …

Molecular And Genetic Studies On A Sipd/Ipad-Related Virulence Spike Protein From Immune-Suppressive Particles Of A Drosophila Parasitoid, Carlo Miguel A. Sevilla

Dissertations and Theses

Parasitoid wasps are effective biological control agents that have evolved intricate host immune suppression strategies to overcome host immune defenses. The most important anti-parasite defense mechanism is encapsulation, where the host’s blood cells surround the wasp egg. In the Drosophila/Leptopilina host-parasitoid system, extracellular vesicles (EVs) are oviposited along with the wasp egg. The EVs possess spikes and contain hundreds of proteins, some of which enable wasp eggs to escape encapsulation by changing the morphology and function of hemocytes, or killing them. Previous studies have identified an abundant surface/spike protein (SSp40) from Leptopilina heterotoma EVs. While p40, a predicted …

Using Nmr Spectroscopy And Computational Chemistry To Confirm The Structure Of Novel Antibiotic Nocamycin O, Stephanie Lewis

CMC Senior Theses

In recent years, many medically promising antibiotics have been discovered in nature, especially in insect-microbe symbioses. One of the better-studied examples of this kind of defensive relationship is that of fungus-growing ants and the antibiotic-producing Actinobacteria. These bacteria produce several defensive chemicals with myriad uses, including one antibiotic that inhibits the growth of several bacterial strains, including other Actinobacteria. This antibiotic (known as nocamycin O) is a promising candidate for medicinal use due to its similarities to bacterial RNA polymerase inhibitors tirandamycin and streptolydigin, which inhibit several human pathogens. The determination of the structure of nocamycin O will be an …

Understanding The Epigenetic Role Of 8-Oxoguanine And Ogg1 In Non-Small Cell Lung Cancer, Kyrellos Ibrahim

CMC Senior Theses

Oxidative damage to the genome can form 8-oxoguanine (oxoG), a premutagenic lesion suggested to play an epigenetic role in the regulation of various cellular pathways. Alongside oxoG in this regulation is the 8-oxoguanine DNA glycosylase (OGG1), which primarily functions to repair oxoG damage via base excision repair, but is also implicated in recruiting NFκB and impacting gene expression associated with cancer growth. This proposal aims to build genome-wide maps of oxoG occupancy, and indirectly OGG1 localization, in healthy lung cells and in non-small cell lung cancer adenocarcinoma cells in order to identify regulatory regions in the genome at which oxoG …

The Tumor Suppressor Par-4 Regulates Hypertrophic Obesity, Nathalia Araujo

Theses and Dissertations--Toxicology and Cancer Biology

Prostate Apoptosis Response-4 (Par-4) is a tumor suppressor ubiquitously expressed in all tissues and able to selectively induce apoptosis in cancer cells. Although well established in the context of cancer, relatively little is known about the function of Par-4 in the healthy and non-tumorigenic context. Observations from our lab showed that Par-4 knockout mouse lines were obese and displayed adipocyte hypertrophy under a normal chow diet when compared to Par-4 wild-type mice. These Par-4 knockout mice exhibited hepatic steatosis and hyperinsulinemia as secondary consequences of obesity. Par-4 knockout mice displayed increased intestinal dietary fat absorption and its subsequent storage in …

Initial And Advanced Stages Of Microbiota Establishment Within The Tsetse Fly, Miguel Eduardo Medina Munoz

Graduate Theses, Dissertations, and Problem Reports (ETD)

Symbiosis is a long-term physical association between two or more species, although little is known regarding its evolutionary origins, particularly at the genetic level. Tsetse flies are the vector of African trypanosomes, causative agents of Human and Animal African Trypanosomiases. Tsetse provide an ideal model for studying initial and advanced stages of symbiosis. Tsetse have a simple digestive tract microbiota primarily consisting of two bacteria; the ancient mutualist Wigglesworthia glossinidia and the recently acquired Sodalis glossinidius. This work presents a chronological study in evolutionary terms of the history of a microbial-insect association. First, I present concepts on symbiosis and …

From Inner Segment To Outer Segment: Palmitoylation Of Photoreceptor Na+, K+-Atpase And The Importance Of Prcd In Photoreceptor Outer Segment Morphogenesis, Emily R. Sechrest

Graduate Theses, Dissertations, and Problem Reports (ETD)

Photoreceptors are specialized neuroepithelial cells which are optimized for efficient capture of light and initiation of visual transduction. These cells have several compartments which are very important for proper visual function and segregation of cellular processes, including the outer segment (OS), inner segment (IS), nucleus, and synapse. The IS houses all of the cellular organelles and biosynthetic molecular machinery the cell requires and is the site of protein synthesis. The light-sensing OS is a highly modified, primary cilium, which contains many stacks of double membranous discs which house proteins required for formation and maintenance of OS structure, as well as …

Fxr Agonists Induce Distinct H-12 Structural States, Vikash Kumar

Graduate Student Theses, Dissertations, & Professional Papers

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. The FXR LBD shows multiple low-energy conformational states of the activation function-2 (AF-2) coregulator binding surface upon ligand binding, indicating the complexity of FXR activation. However, it is unknown how ligand binding leads to different conformational states within the AF-2 region centered on helix 12 (H-12) of the LBD. Here we observe the conformation of the coregulator binding surface (H-12 specifically) of FXR upon ligand binding in solution using fluorine-19 (19F) nuclear magnetic resonance (NMR) and simulations of this surface using molecular dynamics. Fluorescence anisotropy of fluorescein-labeled coregulator peptides …

Photoreceptor Phosphodiesterase (Pde6): Activation And Inactivation Mechanisms During Visual Transduction In Rods And Cones, Rick H. Cote

Faculty Publications

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary

intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes …

The Receptor Basis Of Serotonergic Modulation In An Olfactory Network, Tyler Ryan Sizemore

Graduate Theses, Dissertations, and Problem Reports (ETD)

Neuromodulation is a nearly ubiquitous process that endows the nervous system with the capacity to alter neural function at every level (synaptic, circuit, network, etc.) without necessarily adding new neurons. Through the actions of neuromodulators, the existing neural circuitry can be adaptively tuned to achieve flexible network output and similarly dynamic behavioral output. However, despite their near ubiquity in all sensory modalities, the mechanisms underlying neuromodulation of sensory processing remain poorly understood. In this dissertation, I address three main questions regarding the mechanisms of one modulator (serotonin) within one sensory modality (olfaction). I begin by establishing a "functional atlas" of …

A Mechanism Behind The Mechanotransduction Of Surface Characteristics In Osteoblasts, Otto J. Juhl Iv

Theses and Dissertations

Biomaterials for use in bone regeneration and healing range from metal and metal alloy implants to hydrogel-based solutions. These materials can be optimized to increase bone healing and integration by improving the mechanical and biological properties. Regardless of the material itself, the cell-substrate interaction is key to the success of the biomaterial once implanted. Substrate surface characteristics such as roughness, wettability, and particle density are well-known contributors to a substrate’s overall osteogenic potential, and therefore the substrate's overall success. Unfortunately, it is still unknown how these substrate surface characteristics are transduced into intracellular signals by cells, preventing specific tailoring of …

Clpxp Degradation System In Escherichia Coli, A Study Of Its Energy Sources And Its Applications In Managing The Expression Levels Of Targeted Membrane And Soluble Proteins, Thilini Abeywansha

Theses and Dissertations--Chemistry

ClpXP is an Escherichia coli protease that carries out energy-dependent intracellular proteolysis. In recent years, this system has been widely studied due to its importance as protein regulatory machinery and a virulence factor. Protein substrates of ClpXP contain degrons with a specific protein sequence. SsrA tag is one of the five degrons known to designate proteins for ClpXP degradation. SsrA is an 11 amino acid peptide added to the C-terminus of nascent polypeptide chains translated from aberrant messenger RNAs lacking stop codons via a process called trans-translation.

ClpXP was known to targets only cytosolic proteins with degrons until recently, …

Investigation Of Multidrug Efflux Transporter Acrb In Escherichia Coli: Assembly, Degradation And Dynamics, Prasangi Irosha Rajapaksha

Theses and Dissertations--Chemistry

The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.

In the …

Development Of Fluorescence Microscopy Approaches To Study Subcellular Protein Transport And Enzymatic Activity, Anchal Singh

Masters Theses

Understanding the subcellular localization of proteins and their activity is important in understanding their normal function in eukaryotic cells. Fluorescence cellular imaging techniques can selectively and sensitively visualize subcellular biochemistry. Using this approach, two different methods were employed in this thesis. The first focused on studying protein import into peroxisome and the other on monitoring the activity of an endoplasmic reticulum (ER)-localized enzyme, human carboxylesterase 1 (CES1).

Peroxisomes are mainly known as the center for long chain fatty acid b-oxidation as well as the production and detoxification of hydrogen peroxide. Proteins which are needed in the peroxisomes are encoded in …

Flavin Modification And Redox Tuning In The Bifurcating Electron Transfer Flavoprotein From Rhodopseudomonas Palustris: Two Arginines With Different Roles, Nishya Mohamed-Raseek

Theses and Dissertations--Chemistry

Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism in addition to two well-known mechanisms, substrate level phosphorylation and Oxidative phosphorylation, in electron bifurcation endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens.

Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: …

Development Of Linked-Domain Protein Inhibitors Of The E2-Conjugating Enzyme Ube2d, Anneroos E. Nederstigt

University of the Pacific Theses and Dissertations

In most eukaryotic organisms, the ubiquitination pathway is one of the most important and versatile signaling systems in use. It is integral to processes such as protein degradation and homeostasis, DNA repair cell cycle regulation, signaling and regulation, epigenetics, and many more. Ubiquitin (Ub) is a short polypeptide of 8.6 kDa, 76 residues that functions as a reversible post-translation modification (PTM). It furthermore contains 7 different lysine residues (K6, K11, K27, K29, K33, K48, K63), all of which can form isopeptide linkages with one another to link individual Ub moieties to form unique polyUb chains onto substrates. The type of …

Function Of Mllt3 In Liver Metabolism, Grace Ann Hammack

Master's Theses

The chromatin regulator MLLT3 recognizes target genes through the YEATS domain that identifies post-translationally modified histones, with preference for crotonyl and acetyl marks, and recruits different multiprotein effector complexes through its C-terminal domain to target genes. To study the role of MLLT3 in gene regulation, the Zeleznik-Le and Hemenway labs developed Mllt3 whole-body knockout (Mllt3;Rosa26-CreERT2) mice. These mice have a hematopoietic stem cell phenotype and an unexpected obesity and hepatic steatosis phenotype. It was unknown whether these phenotypes were from liver intrinsic effects or influenced by other parts of the body. To study this fatty liver phenotype further, Mllt3;Alb-Cre were …

Beta-Adrenergic Receptor Mediated Transcriptional Dysregulation In Hematopoeitc Stem And Progenitor Cells Leads To Bone Marrow Erythroid Suppresion In Multiple Myeloma Patients - Ex Vivo Investigations, Vimal Ravi Subramaniam

Master's Theses

The sympathetic nervous system (SNS) regulates our body’s involuntary response to stress or harm. Once activated, it releases hormones and neurotransmitters known as catecholamines throughout the body to mediate its recovery. The catecholamines bind to adrenergic receptors located on various cells in a process designated as adrenergic signaling to elicit this response. This process is not always to our benefit. Adverse symptoms such as anemia can result due to adrenergic signaling. Our laboratory previously showed that burn injury triggers adrenergic receptor stimulation resulting in anemia induced by diminished erythropoiesis. The development of anemia in these studies was found to be …

Mechanistic Insights Of Mitochondrial Reactive Oxygen Species In Alzheimer's Disease Models, Jakob C. Green

Theses and Dissertations

Alzheimer’s disease (AD) is a neurodegenerative disease that results in impaired cognition, disorientation, confusion, poor judgement, and behavioral changes. The mitochondrial cascade hypothesis (MCH), which postulates that accumulating oxidative stress and mitochondrial dysfunction plays an integral role in the development of the disease, potentially upstream, in conjunction with or independent of Aβ and tau. The literature supports the critical role of mitochondrial dysfunction in the progression and development of AD, but is inconclusive about what precise role, as initiator or delegate, it performs. Regardless, the impact that oxidative stress and mitochondrial dysfunction have on AD deserves further study and investigation. …

Development And Evaluation Of A Combinatorial Rt-Qpcr Multiplex For Forensic Body Fluid Identification, Carolyn A. Lewis

Theses and Dissertations

Body fluid identification is essential in the forensic biology workflow that assists DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the sensitivity and specificity molecular techniques can achieve; therefore, molecular methods, such as microRNA and microbial signatures, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance. A disadvantage of RNA analysis in forensic …

Detrimental Role Of Irf1 In Myeloid Cells During Influenza Infection, Bianca L. Baum

Theses and Dissertations

The influenza virus is constantly evolving new ways to overcome both innate and vaccine-induced host defenses, making the innate immune response a critical therapeutic target for minimizing morbidity and mortality caused by infection. Here we examined the role that interferon regulatory factor 1 (IRF1) plays in regulating host innate immune responses in myeloid cells. We found that knocking out IRF1 from myeloid cells in mice (IRF1^DMy) led to a partial protection during IAV infection. Interestingly, recruitment of bone marrow derived myeloid cells as well as NLRP3 inflammasome expression were decreased in the lungs of IRF1^DMy mice, whereas …

On The Structure And Function Of Mitochondrial Uncoupling Proteins: The Case Of Ucp2, Afshan Ardalan

Theses and Dissertations (Comprehensive)

Uncoupling proteins (UCPs) are regulated proton transporters of the mitochondrial inner membrane. UCP-mediated proton leak negatively impacts the rate of ATP synthesis. Despite the importance of their physiological role(s) in certain tissues, molecular aspects of UCPs’ structure-function relationships are not fully understood. The current study explores the tertiary and quaternary structure of UCP2, as well as its proton transport mechanism in lipid membranes. The proteins were expressed in the E. coli inner membrane, purified and reconstituted into liposomes. Proteins were characterized by semi-native SDS-PAGE. Circular dichroism spectroscopy (CD) and fluorescence quenching assays were utilized to study the conformation of proteins …