Biochemistry, Biophysics, and Structural Biology Commons^™

Role Of Cdx4 And Sp5l In Zebrafish Development, Wesley Tsai

Honors Theses

The Caudal Type Homeobox transcription factors cdx are a family of genes found in vertebrates that regulates body regionalization and anterior-posterior patterning. They are also responsible for regulating axial elongation, but the mechanisms behind this behavior are not known. Previous studies in mouse embryonic stem cells have shown that the cdx genes are necessary for upregulating the gene sp5 which may be linked to axial elongation. Sp5 is a zinc-finger transcription factor belonging to the specificity protein (sp) family. Our group has used in-situ hybridization experiments on zebrafish embryos to show that sp5-like (sp5l) is transcribed within tailbud tissues that …

Probing Amyloid-Beta Protein Structure And Dynamics With A Selective Antibody, Shikha Grover

Dissertations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The AD brain is characterized by significant neuronal loss and accumulation of insoluble fibrillar amyloid-β protein (Aβ) plaques and tau protein neurofibrillary tangles in the brain. However, over the last decade, many studies have shown that the neurodegenerative effect of Aβ may in fact be caused by various soluble oligomeric forms as opposed to the insoluble fibrils. Furthermore, the data suggest that a pre-fibrillar aggregated form, termed protofibrils, mediates direct neurotoxicity, and triggers a robust neuroinflammatory response.

Antibodies targeting the various conformation of Aβ are important therapeutic agents to prevent the progression …

Impact Of Sample Conditions On Dna Phosphodiester Backbone Bi/Bii Conformational Equilibrium Dynamics, Autumn C. Pilarski

MSU Graduate Theses

DNA damage, such as single base lesions and mismatches, is highly prevalent within cells. If these DNA damage events are not repaired, they could lead to mutations and thus disease and cancer. Intricate repair mechanisms are in place to fix these damage events, one such being Base Excision Repair (BER) and associated enzyme: Thymine DNA Glycosylase (TDG). The first step of this repair process, recognition of the lesion by TDG, is not well understood. The following thesis presents results to better understand the fundamental biophysical question of how a DNA lesion within a mismatch context is recognized in a million …

Functional Characterization Of The Newly Discovered Type V Crispr-Cas Protein Cas12a2, Dylan J. Keiser

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Similarly to people, bacteria are under the treat of infection by viruses. To circumvent these threats, bacteria evolve complex immune systems. Our understanding of some of these immune systems has led to many advancements in the field of Biotechnology including tools that made expressing proteins for study in a lab easier, tools that revolutionized the feasibility of gene editing, and tools that could change the way we think about viral diagnostics and cancer therapeutics. A certain type of immune system that bacteria use to fight virus is called a CRISPR system. Presented here is work to understand the function of …

The Effects Of Crowding Agents On The Pka Of Physiologically Stable I-Motif Dna, Courtney Turner

Honors Theses

The overall goal of this thesis was to investigate the environmental conditions that induce i-Motif folding of the DNA strand DUX4L22, a cytosine-rich segment of DNA found in the human genome. Cellular conditions were simulated using sodium cacodylate buffer and multiple weights of polyethylene glycol as a crowding agent. The presence of i-motifs were confirmed by Circular Dichroism spectroscopy. I found that DUX4L22 does form i-motifs under these physiological simulations at both acidic and neutral pHs. DUX4L22 therefore shows potential for use in studies of a wide variety of biotechnological advances, such as regulatory switches in nanomachines or drug-delivery systems.

Bci Validation: Yfiler Plus Kit Validation, Chloe Koon

Honors Projects

In order to have access and use the Combined DNA Index System (CODIS), it is required by the Federal Bureau of Investigation's Quality Assurance Standards (FBI-QAS) for Forensic DNA Units to complete comprehensive validation studies on all equipment, materials, and methods used in the process of DNA analysis. This study continues validation previous done within the Ohio Bureau of Criminal Investigation (BCI). YFiler Plus is a DNA amplification kit that is used in cases where it is most useful to examine only male DNA profile(s) within a sample by amplifying loci only on the Y-Chromosome. Components of this study include …

Reproducibility Of Individual Dna Deposits Detected Through Cellular Fluorescence, Natalee Small-Davidson

Student Theses

Contact traces are an important part of DNA casework, but the probative value of any identified associations depends on the possibility of passive transfer. There is known individual variation in DNA left behind during contact, this DNA shedding propensity has an effect on whose DNA is detected. This study evaluated this variability using a cell staining approach. Volunteers were asked to deposit a fingerprint on a clean glass slide, then wash their hands and deposit a second fingerprint after a 30-minute wait without touching anything. Three sets of samples were collected over three consecutive weeks. Fingerprints were stained with a …

Analysis Of Foki Cleavage Resistance Observed In Dna Sequences Generated Via The Combinatorial Selection Method, Repsa, Andre Berry

Master of Science in Chemical Sciences Theses

FokI is a thoroughly investigated and highly utilized restriction endonuclease that recognizes the DNA sequence, 5’-GGATG-3’, and cleaves outside of this site 9 and 13 base-pairs downstream. The shifted cleavage function possessed by this kind of endonuclease is utilized in many applications including the combinatorial selection method, REPSA. FokI employment in the REPSA procedure has demonstrated the tendency to select for an unmodified sequence that possesses the recognition site yet is refractory to cleavage by the enzyme. Sequencing of the cleavage resistant DNA has revealed the inhibitory event to be induced by the presence of an additional inversely oriented recognition …

Chemical And Co-Solute Effects Of Polyethylene Glycol On I-Motif Formation, Lindsey Rutherford

Honors Theses

DNA typically forms Watson and Crick double helix structures in which adenine, thymine, guanine, and cytosine pair with their complimentary DNA base. However, DNA i-motif structures can form in cytosine rich DNA, typically under slightly acidic conditions (~pH 6). DNA i-motifs are four stranded secondary structures in which cytosine pairs with cytosine to form a quadruplex. The i-motifs are typically formed in acidic conditions because of the protonation in the C•C base pair between one of the three hydrogen bases. Recent studies have suggested i-motifs can also form under neutral conditions, which is more realistic for a cell. It is …

Effects Of Crowding Agents On I-Motif Dna, Hayden Brines

Honors Theses

Deoxyribonucleic acid (DNA) is a well-known double stranded, helical, biological molecule. In addition to its more commonly known structure, DNA can also form more complicated structures like G-quadruplexes and i-motifs (iM). The iMs are formed by cytosine rich DNA and are a four stranded structure that is typically looped around itself. The iM formation is typically pH-dependent and is favored in more acidic conditions; the pKa value is approximately 6.5. This pKa value allows for potential in vivo formation, since the cells have a pH of approximately 7.3. Due to this, iMs are thought to be powerful, innovative molecules for …

Nucleic Acids Promote Oligomerization Of Immunoglobulin G, Alexa Gomez

Electronic Theses and Dissertations

Nucleic acids have been found to prevent aggregation as chaperones, as well as act as co-factors and promote aggregation of amyloidogenic proteins leading to various diseases. Immunoglobulin G, IgG, are prone to aggregate as therapeutic proteins, and light chains of IgG can form amyloid fibrils, causing a disease known as light chain amyloidosis. Here we discuss the effect nucleic acids have on full-length immunoglobulin G aggregation. Our results show G-quadruplex DNA, and bulk DNA lead to oligomerization of full-length IgG, and induce increases in secondary structure. Tryptophan fluorescence indicates structural changes are occurring in the presence of DNA. Additionally, IgG …

Structural Characterization And Selective Drug Targeting Of Higher-Order Dna G-Quadruplex Systems., Robert Chandos Monsen

Electronic Theses and Dissertations

There is now substantial evidence that guanine-rich regions of DNA form non-B DNA structures known as G-quadruplexes in cells. G-quadruplexes (G4s) are tetraplex DNA structures that form amid four runs of guanines which are stabilized via Hoogsteen hydrogen bonding to form stacked tetrads. DNA G4s have roles in key genomic functions such as regulating gene expression, replication, and telomere homeostasis. Because of their apparent role in disease, G4s are now viewed as important molecular targets for anticancer therapeutics. To date, the structures of many important G4 systems have been solved by NMR or X-ray crystallographic techniques. Small molecules developed to …

Diversity Of The Major Histocompatibility Complex In African Penguins (Spheniscus Demersus) In Situ, Athena Schalk

Undergraduate Honors Thesis Projects

The Major Histocompatibility Complex (MHC) is responsible for the immune response in all jawed vertebrates and protects individuals against a variety of pathogens and diseases. Maintaining genetic diversity within the MHC exons is critical to protecting endangered species. African penguins (Spheniscus demersus) are in danger of losing their MHC diversity in isolated populations due to reductions in population size associated with environmental changes and human activity. This study analyzes the diversity within the exons in the DNA encoding the MHC by amplifying the exons through polymerase chain reaction and identifying alleles through denaturing gradient gel electrophoresis. Wild populations of …

Development Of Dual Functional Dna/Rna Nanostructures For Drug Delivery, Vibhav Amit Valsangkar

Legacy Theses & Dissertations (2009 - 2024)

In addition to the traditional biochemical functions, DNA and RNA have been increasingly studied as building blocks for the formation of various 2D and 3D nanostructures. DNA has emerged as a versatile building block for programmable self-assembly. DNA-based nanostructures have been widely applied in biosensing, bioimaging, drug delivery, molecular computation and macromolecular scaffolding. A variety of strategies have been developed to functionalize these nanostructures. The major advantage is that DNA is a very stable molecule and its base-pairing properties can be easily utilized to control and program the formation of desired nanostructures. In addition, some of these DNA/RNA nanostructures have …

Effects Of The Dihydrouracil Lesion On Dna Using 1h/31p 1d And 2d Solution Nmr, Benjamin M. Boyd

MSU Graduate Theses

The effects of the dihydrouracil lesion in DNA were studied using two dimensional NMR spectroscopy. The sequence used was based off of the Drew-Dickerson Dodecamer, with the cytosine in the three position replaced by a dihydrouracil. All of the nonexchangeable proton chemical shifts, with the exception of the H2, H5’, and H5’’, of the lesioned DNA were identified using NOESY spectra and then compared to the chemical shift values of the Drew Dickerson Dodecamer. The largest differences in chemical shifts were observed in the nucleotides neighboring the lesion, both within the strand and on the opposite strand. The imino exchangeable …

Insights Into Nucleic Acid-Platinum(Ii) Compound Interactions And Structural Impacts, Supuni Duneeshya Kamal Thalalla Gamage

Wayne State University Dissertations

With the discovery of cisplatin in the 1960s, it has been widely studied as a precursor for anticancer drug development. Despite its effectiveness against certain cancers, clinical usage of cisplatin is restricted by a number of side effects and resistance. In the past decade, scientists have been exploring biologically important ligands such as sugar derivatives in the hope of overcoming such challenges. Attachment of a sugar moiety could facilitate lower accumulation of platinum drugs in the body as well as enhance cellular uptake. In this study, a carbohydrate-linked cisplatin analog, cis-dichlorido[(2-β-D-glucopyranosidyl)propane-1,3-diammine]platinum (5) has been studied. The aim was to evaluate …

Unintended Consequences Of Dna Analysis Delays In North Carolina, Pamela Cook Woodard

Walden Dissertations and Doctoral Studies

The processing of DNA recovered from felony crime scenes often causes delays in trials of up to 3 years, calling into question defendants' rights to a speedy trial. Using Lewin's force field analysis as the theoretical framework, the purpose of this quantitative, comparative study was to compare the processing and reporting of results related to DNA testing in 4 states. Survey data were collected from state bar members (n=137), members of a professional law organization (n=149), and members of a state DNA laboratory (n=20). The purpose of this quantitative, comparative study was to determine whether these variables (interagency communications, staff …

The Evaluation Of The Rapidhittm 200 On Degraded Biological Samples, Alice Kim

Graduate Theses, Dissertations, and Problem Reports

DNA (deoxyribonucleic acid) has become an integral part of forensic science in the last couple of decades since its discovery to this application by Alec Jeffreys. Although there have been many advances throughout the years, the time it takes to obtain a DNA profile using conventional methods in a laboratory setting is approximately 24 to 72 hours. Due to this length of time and the increase in demand for DNA testing, it has caused a tremendous amount of backlog throughout the country. In 2009, the FBI (Federal Bureau of Investigation) in collaboration with the US Department of Defense of Homeland …

Gq Noncanonical Roles In Translational Regulation, Brett Demarco

Electronic Theses and Dissertations

This study investigates protein nucleic acid interactions, focusing on G-quadruplex (GQ) forming DNA/RNA in human disease. GQ structures are formed in DNA/RNA, when four guanine residues form planar tetrads stabilized by Hoogsteen base pairing, that stack forming a GQ structure stabilized by potassium ions. These GQ structures are targeted by the arginine glycine-glycine (RGG) RNA-binding domain. Fragile X mental retardation protein (FMRP), a translation regulator protein implicated in the fragile X syndrome, has an RGG domain and has been previously shown to interact with neuronal GQ forming messenger RNA (mRNA). We have investigated three neuronal FMRP mRNA targets that we …

Development Of Lc-Ms For The Identification And Characterization Of Non-Adjacent Dna Photoproduct Formation In G-Quadruplex Forming Sequences, Claudia Posadas

Arts & Sciences Electronic Theses and Dissertations

Ultraviolet light is well known to induce cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts in duplex DNA, which interfere with DNA replication and transcription. Recently, a new class of DNA photoproducts known as anti cyclobutanepyrimidine dimers have been discovered, which form in G-quadruplex forming sequences in solution. G-quadruplex structures have been proposed to form in human DNA telomeres and certain promoters in vivo but evidence for their existence has been lacking. Since anti-cyclobutante pyrimidine dimers have been shown to form in G-quadruplex forming sequences, their formation in irradiated human cells could be used to confirm the existence …

Thermodynamics In Large Hairpin Polyamide-Dna Interactions, Yang Song

Dissertations

Human papillomavirus (HPV) is a common sexually transmitted virus responsible for cervical cancers, and its infection is currently incurable. Only a few vaccines against high-risk HPV strains are available. Hairpin polyamides (PAs) in different sizes (8-20 units long) bind DNA in different lengths. They have been shown to have different anti-HPV activities in cell culture.

The interaction between PA and DNA is stabilized by two types of molecular forces: attractive and repulsive forces. Attractive forces include hydrogen bonds, van der Waals contacts and electrostatic forces between PA and DNA. Repulsive forces include the hydrophobic effect, which forces the PA out …

Dna Functionalized Nanoparticles In Nanobiosensor And Sensor Array Development For Molecular Diagnostics And In Vitro Identification Of Biomolecules, Mustafa Salih Hizir

Legacy Theses & Dissertations (2009 - 2024)

Nucleic acid technology along with vast variety of nanomaterials has demonstrated a great potential in many applications from biosensing studies to molecular diagnostics, from biomedical and bioanalytical research to environmental analysis. Especially short single stranded (ss) DNA molecules, called oligonucleotides, are extraordinary biopolymers featuring diverse functionality on the nanoparticles thanks to their high degree of programmability, target-specific binding or cleavage, molecular recognition ability, structure-switching capability, and unique interactions at the bio-nano interfaces. Among those, there have been many biosensing applications utilizing ss DNAs and numerous nanomaterials through various detection techniques such as fluorometric, colorimetric or electrochemical methods. Although many groundbreaking …

A Novel Method To Analyze Dna Breaks And Repair In Human Cells, Caitlin Elizabeth Goodman

Browse all Theses and Dissertations

Microsatellites repeat sequences are prone to forming non-canonical DNA structures and mutations. These areas of the genome can undergo expansions and contractions and are responsible for a variety of inherited neurological and neuromuscular disorders. Hairpin structures formed by trinucleotide repeats can lead to replication fork stalling, and fork collapse causing DNA double strand breaks. Various mechanisms are involved in processing microsatellites including mismatch repair, base excision repair, and crossover junction endonuclease cleavage. These processes, which are supposed to protect the genome, could also be the culprits which are causing mutations. In order to test and study this hypothesis, the use …

Mutagenic And Spectroscopic Investigation Of Ph Dependent Cooa Dna Binding, Brian R. Weaver

Chemistry Honors Papers

The carbon monoxide (CO) sensing heme protein, CooA, is a transcription factor which exists in several bacteria that utilize CO as an energy source. CooA positively regulates the expression of coo genes in the presence of CO such that the corresponding proteins may metabolize CO. The present studies have yielded the unexpected result that Fe(III) CooA binds DNA tightly at pH < 7, deviating from all previously reported work which indicate that CooA DNA binding is initiated only when the exogenous CO effector reacts with the Fe(II) CooA heme. This observation suggests that the disruption of one or more salt bridges upon effector binding may be a critical feature of the normal CooA activation mechanism. To test this possibility, several protein variants that eliminated a selected salt bridge for the CooA homolog from Rhodospirillum rubrum were prepared via site-directed mutagenesis. Samples of these variant proteins, which were overexpressed in Escherichia coli, were then characterized by spectroscopic methods and functional assays to investigate the impact these mutations had on CooA heme coordination …

Silica Nanoparticles For The Delivery Of Dna And Rnai In Cancer Treatment, Michael Aaron Vrolijk

Graduate College Dissertations and Theses

DNA and interfering RNA (RNAi) – short interfering RNA (siRNA) and micro RNA (miRNA) – are promising new cancer therapies, especially for drug resistant lines. However, they require a delivery system in vivo to prevent degradation and off target effects. Silica based nanoparticles, both solid and mesoporous, are a promising option due to their biocompatibility, ease of preparation and morphology control, reproducibility, and facile addition of functional groups including targeting ligands.

After a brief introduction to cancer treatment and review of the current nanoparticle treatments undergoing clinical trials, this thesis details the many methods explored over the past ten years …

The Functions Of The Cid And Lrg Operons In S. Aureus Programmed Cell Death, Xinyan Zhang

Theses & Dissertations

Staphylococcus aureus cid/lrg operons regulate the formation of S.aureus biofilm formation and programmed cell death based on previous in vivo work done in Dr. Bayles's lab. cid operon, which encodes CidA/CidB/CidC proteins, has been shown to be an effector in leading to the lysis and death of the S.aureus; While lrg operon, encoding LrgA and LrgB proteins, is an inhibitor of the lysis and death. Recent studies suggest that CidA behaves like holin proteins from bacterial phage, by increasing the murein hydrolysis activity under aerobic culturing conditions. LrgA, together with LrgB, appears to inhibit this function. …

Coarse-Grained Simulations Of The Self-Assembly Of Dna-Linked Gold Nanoparticle Building Blocks, Charles Wrightsman Armistead

Graduate Theses and Dissertations

The self-assembly of nanoparticles (NPs) of varying shape, size, and composition for the purpose of constructing useful nanoassemblies with tailored properties remains challenging. Although progress has been made to design anisotropic building blocks that exhibit the required control for the precise placement of various NPs within a defined arrangement, there still exists obstacles in the technology to maximize the programmability in the self-assembly of NP building blocks. Currently, the self-assembly of nanostructures involves much experimental trial and error. Computational modeling is a possible approach that could be utilized to facilitate the purposeful design of the self-assembly of NP building blocks …

X-Ray Characterization Of Mesophases And Phase Transitions Of Dna Analogues In Solutions, Mustafa Selcuk Yasar

Doctoral Dissertations

We think of DNA as double-stranded helices (duplex), but the polymer exists in many conformations. Several triplex and quadruplex DNA structures can be formed in laboratory settings and exist in nature. This thesis first provides a brief description of the nature of the order in arrays of duplex DNA under biologically relevant molecular crowding conditions. Then we compare the duplex DNA mesophases with the corresponding liquid crystalline phase behavior of the triplex and quadruplex DNA analogues. In particular, we focus on G-quadruplexes. Observed in the folds of guanine-rich oligonucleotides, G-quadruplex structures are based on G-quartets formed by hydrogen bonding and …

Specific Binding Affinity Of The Non-Catalytic Domain Of Eukaryotic Like Type Ib Topoisomerase Of Vaccinia Virus, Benjamin R. Reed

Dissertations, Theses, and Capstone Projects

Topoisomerases are ubiquitous proteins that alter supercoiling in double stranded DNA (dsDNA) during transcription and replication and. vaccinia and the closely related poxvirus variola virus, at 314 amino acids in length, encode the smallest of the type I topoisomerases(TopIB). TopIB is a two domain protein that recognizes the sequence 5’-T/CCCTT, cleaves at the 3’-end and relaxes supercoiling through rotation. The C-terminal domain (CTD) alone contains the catalytic activity and specificity. Deletion of the N-terminal domain results in a greatly reduced rate of relaxation and rapid dissociation. Biochemical data suggests that the N-terminal domain (NTD) is important for pre-cleavage binding and …

Aptameric Sensors: In Vitro Selection Of Dna That Binds Bromocresol Purple, Derek B. Miller

Honors Undergraduate Theses

Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow’s diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today’s aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to …