Molecular 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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

Ex Vivo Dna Cloning, Adam B. Fisher

Theses and Dissertations

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are …

Biophysical And Computational Investigations Into G-Quadruplex Structural Polymorphism And Interaction With Small Molecules., Huy Tuan Le

Electronic Theses and Dissertations

In the cell, guanine-rich nucleic acids can self-assemble into unique four stranded tertiary structures known as G-quadruplexes. G-quadruplex formation in the telomere leads inhibits telomerase, an enzyme activated in cancer cells to maintain the telomere and allowing for cancer cells to achieve immortality. G-quadruplex formation in the promoters and 5’-untranslated regions regulates the expression of many oncogenes. Furthermore, G-quadruplex formation during cellular replication promotes genomic instability, a characteristic which enables tumor development. Because of their implication in cancer, G-quadruplex structures have emerged as attractive drug targets for anti-tumor therapeutics. In the current dissertation work, we present three experimental approaches to …

Helicase-Ssb Interactions In Recombination-Dependent Dna Repair And Replication, Christian Jordan

Graduate College Dissertations and Theses

Dda, one of three helicases encoded by bacteriophage T4, has been well- characterized biochemically but its biological role remains unclear. It is thought to be involved in origin-dependent replication, recombination-dependent replication, anti- recombination, recombination repair, as well as in replication fork progression past template-bound nucleosomes and RNA polymerase. One of the proteins that most strongly interacts with Dda, Gp32, is the only single-stranded DNA binding protein (SSB) encoded by T4, is essential for DNA replication, recombination, and repair.

Previous studies have shown that Gp32 is essential for Dda stimulation of replication fork progression. Our studies show that interactions between Dda …

Meiotic Dna Re-Replication And The Recombination Checkpoint, Nicole Ann Najor

Wayne State University Dissertations

Progression through meiosis occurs through a strict sequence of events, so that one round of DNA replication precedes programmed recombination and two nuclear divisions. Cyclin dependent kinase 1 (Cdk1) is required for meiosis, and any disruption in its activity leads to meiotic defects. The Cdk1 inhibitor, Sic1, regulates the G1-S transition in the mitotic cell cycle and the analogous transition in meiosis. We have employed a form of Sic1, Sic1deltaP^HA, that is mutated at multiple phosphorylation sites and resistant to degradation. Meiosis specific expression of Sic1deltaP^HA disrupts Cdk1 activity and leads to significant accumulation of over replicated …

Due-B, A New Human Dna Replication Protein, Is The Functional Homolog Of S. Cerevisiae Sld3, Jianhong Yao

Browse all Theses and Dissertations

DNA unwinding elements (DUEs) are commonly found at DNA replication origins. The DUE binding protein (DUE-B) is crucial for the initiation of DNA replication in eukaryotes. The unique 59 amino acid C-terminal part of DUE-B shares nearly 50% similarity with yeast the C-terminus of Sld3. DUE-B plays a key role in eukaryotic DNA replication because it is required for the loading of Cdc45, the MCM helicase activator, on chromatin. Here we show that DUE-B, just like yeast Sld3, binds to Cdc45 and TopBP1 through its C-terminus in Sf9 cells and in vitro. We also show that DUE-B, Cdc45 and TopBP1 …

An Analysis Of Mitochondrial Dna In Rett Syndrome And Other Neurodegenerative Disorders, Catherine Erickson Burgess

Theses and Dissertations in Biomedical Sciences

Mitochondrial dysfunction resulting from mutations on mitochondrial DNA (mtDNA) is being recognized in a growing spectrum of diseases. These diseases, resulting from single base mutations, large deletions, or insertions, have been largely neuromuscular in origin. However, as an understanding of the effects of mtDNA mutations progresses, attention is now focusing on neurodegenerative diseases. Rett Syndrome (RS), a progressive neurodegenerative disease with predominantly female cases, demonstrates morphologic mitochondrial changes, mitochondrial enzyme deficiencies and maternal inheritance (characteristic of mtDNA diseases). No investigation of mtDNA involvement has been previously conducted and, to date, no biological marker exists for this disorder.

Our preliminary studies …

Unusual Structure Of A Human Middle Repetitive Dna, Duminda D. Ratnasinghe

Electronic Theses and Dissertations

The L2Hs sequences are a polymorphic, interspersed, middle repetitive DNA family unique to human genomes. Genomic fingerprinting indicates that these DNAs vary from one individual to another and between tissues of the same individual. Sequence analysis reveals that they are AT-rich (76%) and contain many unusual sequence arrangements (palindromes, inverted and direct repeats). These sequence properties confer on the L2Hs elements the potential to fold into non-B-form structures, a characteristic of recombination hot spots. To test this hypothesis carbodiimide, osmium tetroxide and S$\sb1$ nuclease were used as single-strand specific probes to study a recombinant plasmid, pN6.4.39, containing a single L2Hs …

The Role Of The Voltage Gradient In The Agarose Gel Electrophoresis Of Dna, David Wheeler

Theses and Dissertations in Biomedical Sciences

In Part I of this dissertation, empirical equations for predicting DNA mobility during agarose gel electrophoresis (AGE) from voltage gradient are developed from the data of McDonnel (36) for electrophoresis in a 1.6% agarose gel. These equations represented the data well for DNA between 2 and 10 kilobase pairs (KBp) in length. A computer program, called GELSIM, which incorporates these equations is described in Part II. GELSIM was designed to allow researchers to analyze electrophoresis data by predicting the effect on DNA migration of altering the voltage of electrophoresis. In this way, electrophoretic banding patterns produced using different voltages could …

Molecular Study Of The B19 (Human) Pathogenic Parvovirus, Jamshed Ayub

Theses and Dissertations in Biomedical Sciences

The B19 (human) parvovirus is a small single stranded DNA virus of 5.4 kilobases. B19 is specific for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 differs from other parvoviruses in some important aspects, which include the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter. B19 has an unusual transcription map which is described in this study.

The transcription map of …