Molecular Biology Commons^™

Dna Repair Fidelity And Cancer : Structural And Kinetic Insights From Dna Polymerase Beta Mutator Variants, Chelsea Lynne Gridley

Legacy Theses & Dissertations (2009 - 2024)

DNA polymerases are essential for genome replication and DNA repair in all living organisms. Precise DNA replication is critical for the preservation of genomic stability. Any insult, endogenous/exogenous, to cellular DNA requires properly functioning repair polymerases. In eukaryotes, DNA polymerase beta, a small enzyme (39 kDa), plays an important role in DNA repair during the base excision repair pathway. Pol beta catalyzes the incorporation of nucleotides in small stretches (1-6 nucleotides) of damaged double-stranded DNA. Should gap-filling synthesis by pol beta be compromised, mutations in genomic DNA accumulate, which are frequently linked to human diseases, including cancers. For this reason, …

Gld-1 Represses Its Puf Mrna Targets Prior To/At Initiation Of Translation In The C.Elegans Germline, Gautham Sarathy

Legacy Theses & Dissertations (2009 - 2024)

The C.elegans germline offers an ideal system to study posttranscriptional regulation of gene expression as it is a major mechanism through which the control over gene expression is achieved. GLD-1 (defective in GermLine Development) is a maxi-KH motif containing RNA binding protein that controls various aspects of germline development from decision over germcell proliferation vs. meiotic entry to the production of mature gametes suggesting that GLD-1 likely controls many mRNA targets.

Investigation Of A 16s Rna Central Domain Pseudoknot, Jenna Marie Jasinski-Bolak

Wayne State University Theses

X-ray crystallography of the prokaryotic 30S ribosomal subunit revealed a myriad of complex RNA-RNA, RNA-protein, and protein-protein interactions. Among these are several phylogenetically conserved RNA pseudoknots. Pseudoknots are structurally and functionally diverse RNA secondary structures. They are generally formed by two short complimentary sequences separated by many bases of single stranded regions or loops. These relatively simple folds are often yield complex structures that are key components of functionally important conformational changes in RNA structure. One such pseudoknot is located in the central domain of the 16S rRNA.

The central domain pseudoknot is formed by Watson-Crick base pairing between G570-C866 …

Mechanistic Studies Of A Novel Ppar-Gamma Mutant That Causes Lipodystrophy And Diabetes, Olga Astapova

Wayne State University Dissertations

PPAR-gamma is a nuclear receptor that plays a central role in metabolic regulation by regulating extensive gene expression networks in adipose, liver, skeletal muscle and many other tissues. Human PPAR-gamma mutations are rare and cause a monogenetic form of severe type II diabetes with metabolic syndrome, known as familiar partial lypodystrophy. The E157D PPAR-gamma mutant causes atypical lipodystrophy in a large Canadian kindred, presenting with multiple musculoskeletal, neurological and hematological abnormalities in addition to the classic lipodystrophy features of insulin-resistant diabetes, hypertension and dyslipidemia. This mutation is localized to the p-box of PPAR-gamma, a small region that interacts directly with …

Protection Of The Female Reproductive Tract In The Prevention Of Hiv, Camila Diaz

HIM 1990-2015

Worldwide, more than half of all HIV-infected individuals are women. Since mucosal surfaces are the primary gateway for HIV entry, maintaining the integrity of the female reproductive tract (FRT) is essential for preventing infection. The FRT employs many immune mechanisms that serve as the first line of defense against HIV transmission. Among these are vaginal fluid secretions rich in antimicrobial peptides, and commensal bacteria that colonize the vagina and prevent infections. We sought to study vaginal fluid as an innate immune component of the FRT in the prevention of HIV infection. Additionally, we investigated the anti-HIV microbicide candidate RC-101 as …

Physiological Relevance Of A Trna-Dependent Mechanism For Membrane Modification In Enterococcus Faecium, Jesse Harrison

HIM 1990-2015

Enterococci were once thought to be harmless, commensal organisms that colonize the gastrointestinal tract of humans and other mammals. In the last 30 years, however, concern has grown in the clinical setting over two particular species, Enterococcus faecalis and Enterococcus faecium, which are frequently found to be the etiologic agents of nosocomial infections. Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that add amino acids to phosphatidylglycerol (PG) in the cellular envelope of bacteria. Addition of amino acids to PG confers resistance to various therapeutic antimicrobial agents, and contributes to evasion of the host immune response in a number of clinically …

Amelioration Of Amyloid Burden In Advanced Human And Mouse Alzheimer's Disease Brains By Oral Delivery Of Myelin Basic Protein Bioencapsulated In Plant Cells, Neha Kohli

Electronic Theses and Dissertations

One of the pathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque deposition in aging brains by aggregation of amyloid-β (Aβ) peptides. In this study, the effect of chloroplast derived myelin basic protein (MBP) fused with cholera toxin subunit B (CTB) was investigated in advanced diseased stage of human and mouse AD brains. The CTB-fusion protein in chloroplasts facilitates transmucosal delivery in the gut by the natural binding ability of CTB pentameric form with GM1 receptors on the intestinal epithelium. Further, bioencapsulation of the MBP within plant cells confers protection from enzymes and acids in the digestive system. Here, …

Identification Of Novel Antimalarial Scaffolds From Marine Natural Products, Bracken Roberts

Electronic Theses and Dissertations

Malaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater. Screening of natural product libraries is known to have higher hit rates than synthetic chemical libraries. This elevated hit rate is somewhat attributed to the greater biodiversity available in natural products. Marine life is the most biodiverse system on the …

Light-Activated Binary Nucleotide Reagent For Inactivation Of Dna Polymerase, Evan M. Cornett

Electronic Theses and Dissertations

This work explores a binary reagent approach to increase the specificity of covalent inhibitors. In this approach, two ligand analogs equipped with inert pre-reactive groups specifically bind a target biopolymer. The binding event brings the pre-reactive groups in proximity with each other. The two groups react, generating active chemical intermediates that covalently modify and inactivate the target. In the present study we compare the new approach with the traditional single-component reagent strategy using DNA polymerase from bacteriophage T4 as a model target biopolymer. We report the design and synthesis of two analogs of deoxythymidine triphosphate, a natural DNA polymerase substrate. …

Insights Into Hepatic Alpha-Fetoprotein Gene Regulation During Liver Development And Disease, Erica Leigh Clinkenbeard

Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics

The liver is an essential organ for cholesterol homeostasis. If this process becomes dysregulated, cardiovascular disease (CVD) develops. Zinc-fingers and homeoboxes 2 (Zhx2) as an important hepatic gene regulator and contributes to CVD. BALB/cJ mice, with mutant Zhx2 allele, have fewer atherosclerotic plaques compared to other strains on a high fat diet. In my dissertation, I focused on the liver phenotype in BALB/cJ mice on a high-fat diet and found increased liver damage compared to wild-type Zhx2 mice. These data indicates that reduced Zhx2 in the liver leads to CVD resistance, but increases liver damage. Therefore, Zhx2 has an important …

Role Of Cyclooxygenase-2 In Abdominal Aortic Aneurysms In Mice, Kamalika Mukherjee

Theses and Dissertations--Pharmacy

Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease with no available pharmacological treatment. AAA formation reduces the structural integrity of the vessel and increases the susceptibility to rupture. The inflammatory response within human aneurysmal tissue is characterized by increased expression of cyclooxygenase-2 (COX-2). Similarly, in a mouse model of the disease induced by chronic Angiotensin II (AngII) infusion, we have shown that COX-2 expression in the abdominal aortic smooth muscle layer increases early in the development of the disease. Furthermore, genetic or pharmacological inactivation of COX-2 prior to disease initiation reduces AAA incidence.

The current study utilized nonhyperlipidemic mice …

Microfluidic Impedance Spectroscopy As A Tool For Quantitative Biology And Biotechnology, Ahmet C. Sabuncu, Jie Zhuang, Juergen F. Kolb, Ali Beskok

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to …

Expansion Dating: Calibrating Molecular Clocks In Marine Species From Expansions Onto The Sunda Shelf Following The Last Glacial Maximum, Eric D. Crandall, Elizabeth J. Sbrocco, Timery S. Deboer, Paul H. Barber, Kent E. Carpenter

Biological Sciences Faculty Publications

The rate of change in DNA is an important parameter for understanding molecular evolution and hence for inferences drawn from studies of phylogeography and phylogenetics. Most rate calibrations for mitochondrial coding regions in marine species have been made from divergence dating for fossils and vicariant events older than 1-2 My and are typically 0.5-2% per lineage per million years. Recently, calibrations made with ancient DNA (aDNA) from younger dates have yielded faster rates, suggesting that estimates of the molecular rate of change depend on the time of calibration, decaying from the instantaneous mutation rate to the phylogenetic substitution rate. aDNA …

Protein Folding By 'Levels Of Separation': A Hypothesis, Lesley H. Greene, Terri M. Grant

Chemistry & Biochemistry Faculty Publications

The protein folding process has been studied both computationally and experimentally for over 30 years. To date there is no detailed mechanism to explain the formation of long-range interactions between the transition and native states. Long-range interactions are the principle determinants of the tertiary structure. We present a theoretical model which proposes a mechanism for the acquisition of these interactions as they form in a modified version of ‘degrees of separation’, that we term ‘levels of separation’. It is based on the integration of network science and biochemistry. (C) 2012 Federation of European Biochemical Societies.

Further Characterization Of The Nodamura Virus Rna2 3'-Terminal Stem Loop Structure And Its Role In Viral Rna Replication, Joshua Frederick

Open Access Theses & Dissertations

Nodamura virus (NoV) is an excellent model system for studying many aspects of the life cycle of positive-strand RNA viruses. NoV's simple genome organization and ability to replicate its genome to high levels in a wide variety of host cells, including plants, yeast, insects and mammals (Ball et al., 1992; Garzon et al., 1978, 1990; Murphy et al., 1970; Price et al., 2005; Scherer & Hurlbut, 1967; Selling et al., 1990; reviewed by Ball & Johnson, 1998), make it a prime model system for studying the basic mechanisms of viral RNA replication. Other positive-strand RNA viruses, including those that are …

The Effects Of Extended Access To Methamphetamine Self-Administration On Dopaminergic Markers In The Striatum, Joe Luevano

Open Access Theses & Dissertations

Methamphetamine (METH) abuse is a persistent problem in the U.S. and abroad. Escalation of METH use among independent users occurs for a variety of physiological and psychological reasons. Methamphetamine dependence may be attributed to the rewarding effect of this drug via the dopaminergic systems of the central nervous system (CNS). The presence of METH in the CNS increases synaptic release of dopamine. This increase in dopaminergic neurotransmission is thought to be directly attributed to the rewarding effects of METH. Following METH use, compensatory changes have been found to occur in the dopaminergic system during various periods of abstinence. It is …

Oxidative Effects Of Nanosecond Pulsed Electric Field Exposure In Cells And Cell-Free Media, Olga N. Pakhomova, Vera A. Khorokhorina, Angela M. Bowman, Raminta Rodaitė-Riševičienė, Gintautas Saulis, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′, 7′-dichlorodihydrofluoresein (H₂DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H₂DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat …

Signals From Intraventricular Depth Electrodes Can Control A Brain-Computer Interface, Jerry J. Shih, Dean J. Krusienski

Electrical & Computer Engineering Faculty Publications

A Brain-Computer Interface (BCI) is a device that enables severely disabled people to communicate and interact with their environments using their brain waves. Most research investigating BCI in humans have used scalp-recorded electroencephalography (EEG). We have recently demonstrated that signals from intracranial electrocorticography (ECoG) and stereotactic depth electrodes (SDE) in the hippocampus can be used to control a BCI P300 Speller paradigm. We report a case in which stereotactic depth electrodes positioned in the ventricle were able to obtain viable signals for a BCI. Our results demonstrate that event-related potentials from intraventricular electrodes can be used to reliably control the …

Identifying Sm22 As A Key Player In Arterial Diseases, Jianbin Shen

Wayne State University Dissertations

Background : Expression of vascular smooth muscle cell (VSMC) cytoskeleton markers including SM22 is down-regulated in arterial diseases including atherosclerosis where inflammation and osteochondrogenesis are present. However, the role of this downregulation in arterial pathogenesis is unknown. Hypothesis : Downregulation of SM22 may actively contribute to arterial pathogenesis. Methods : Five Sm22 knockout (Sm22^-/-) mice and their wild type littermates were subjected to carotid artery denudation, an artery injury model. Analyses were conducted on carotid arteries 2 weeks after injury. Primary VSMCs were isolated from mouse aortas and investigated individually at passage 2 to 4. Sm22 knockdown was …

Identification Of Transcriptional Mechanisms Downstream Of Nf1 Gene Defeciency In Malignant Peripheral Nerve Sheath Tumors, Daochun Sun

Wayne State University Dissertations

Malignant peripheral nerve sheath tumor (MPNST) is a type of soft tissue sarcoma that occurs in carriers of mutations in the neurofibromatosis type I gene (Nf1) as well as sporadically. Plexiform neurofibromas in NF1 patients have a significant risk of developing into MPNSTs leading to increased morbidity and mortality from this syndrome. Surgery is the primary intervention but it is not always effective due to the tendency of MPNSTs to infiltrate the surrounding tissue or grow in an inoperable location. Neurofibromin, the protein coded by the Nf1 gene, functions as a GTPase activating protein (GAP) whose mutation leads to constitutive …