Life Sciences Commons^™

Lipid Genetics In Pregnant Women: Maneuvering The Challenges Of Using Degraded Samples, Kylee Meece

Mahurin Honors College Capstone Experience/Thesis Projects

The results of this project will explore a novel approach to lipid metabolism in pregnant women by exploring the relationship between maternal gene expression of genes critical for fat metabolism, protein expression, and maternal and neonatal outcomes. The study will also examine gene expression in lean pregnant women compared to obese pregnant women, shedding light into the adverse pathology of obesity during pregnancy. These findings may uncover a mechanism related to gene-expression that connects unfavorable maternal fat metabolism to inflammation and/or insulin resistance, which may help to uncover future targets for interventions.

The analysis of lipid gene expression requires the …

Synthetic Pathways For Potential Platinum 1,10-Phenanthroline Compounds, Jacob Young

Mahurin Honors College Capstone Experience/Thesis Projects

There are extremely important uses for platinum when it comes to the treatment of cancers and dementias. One potential therapeutic combines platinum with a structure known as phenanthroline. This platinum phenanthroline structure is normally extremely water-insoluble, rendering it difficult to study and use in biological systems. The Williams’ Biochemistry Laboratory endeavored to find a simpler synthetic pathway for a water-soluble phenanthroline product. While unsuccessful in identifying a product as of yet, many synthetic pathways have been ruled out, and more hypothetical pathways are being formulated for testing.

Among the strategies analyzed are varying solvent, reagents, apparatus, methodology, and temperature in …

Development Of A Dna Biosensor Using Crispr/Dcas9 On A Graphene Oxide Surface For Detecting Antibiotic Resistance Genes, Wendy Cecil

Mahurin Honors College Capstone Experience/Thesis Projects

Antibiotic resistance is a rapidly spreading global threat to human health. Previously in our lab, we developed a simple method for facile detection of antibiotic resistance genes (ARGs) in bacteria. This DNA detection system takes advantage of the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeat and CRISPRAssociated Protein). We designed CRISPR in complex with nuclease deactivated Cas9 (dCas9) on graphene oxide (GO) sheets conjugated with Cas9 antibodies. Single guide RNA (sgRNA) was customized to program dCas9 for target DNA binding. sgRNA was covalently labeled with fluorescein to provide fluorescence signal. Graphene oxide (GO) is utilized for its quenching properties …

Investigation Of The Phenotypic Effect Of Mutating A Highly-Conserved Cysteine Residue In The Rna Polymerase Beta Prime Subunit Of E. Coli Rna Polymerase, Meg Dillingham

Mahurin Honors College Capstone Experience/Thesis Projects

All bacteria contain a multi-subunit RNA polymerase (RNAPs) that is essential for gene expression. Because of the centrality of these enzymes in cellular life, the structure and function of the various subunits is intensely studied. The primary sequence of the RNAP β’ subunit contains five cysteine residues that are highly conserved. Four of the cysteines coordinate a zinc atom and form the beta prime subunit zinc binding domain (ZBD). Mutation of any one of the ZBD cysteines is lethal to the cell. However, the role of the fifth residue (C58), which is located upstream of the ZBD cysteines, has not …

A Look At Gene Control: Tracking The Ccnd1 Gene, Bryan Anders

Mahurin Honors College Capstone Experience/Thesis Projects

Cancer occurs when the cell does not properly control its own cell cycle. It then replicates in an out of control fashion leading to the death of various organs and then the demise of the organism as a whole. As it seems to have always been a problem for cell-based life, certain safeguards against cancer have been evolved over time. One such method comes in the form of prevention via cyclin proteins, which are encoded from cyclin genes. The gene that is the focus of this research is the CCND1, or cyclin D1, gene that controls the progression through various …

Ototoxicity Of Cisplatin, Pyriplatin, And Phenathriplatin In The Auditory Hybridoma Cell Line, Hei-Oc1, Alexandra Johnston

Mahurin Honors College Capstone Experience/Thesis Projects

Cisplatin is an anti-cancer drug which is effective against several cancers, but also causes harmful side-effects, including ototoxicity and hearing loss. While cisplatin is a bifunctional compound that forms coordinate covalent bonds with both strands of DNA, recently investigated monofunctional platinum(II) compounds bind to only one DNA strand, and may activate different cell-death mechanisms. As several monofunctional platinum(II) compounds have anti-cancer properties, but could target different cell-death pathways, they could potentially have different and reduced side-effects. In this study, the HEI-OC1 auditory hybridoma cell line was used to investigate the ototoxicity of cisplatin and two monofunctional platinum(II) compounds, phenanthriplatin and …

Developing Tale Proteins As A Biosensor For Detecting Pathogen Specific Double-Stranded Dna, Kathrine Gaiko

Mahurin Honors College Capstone Experience/Thesis Projects

Transcription activator-like effector (TALE) proteins are important for DNA binding. They bind to specific nucleotide sequences by the use of two residues in each repeat allowing them to target specific DNA sequences. Their modular structure makes TALEs advantageous over other DNA binding proteins such as zinc finger proteins. Zinc finger proteins (ZFP) use a finger like projection to bind 3-4 subsequence base pairs while TALE proteins use two amino acid residues in each repeat to bind one nucleotide. ZFP can use SEER-Lac system for colorimetric detection, while TALE proteins can use fluorescence labeling with Alexa for detection of binding to …

Investigation Of Transient Expression System In Nicotiana Bethamiana To Produce Novel Tales And The Development Of (Ds)Dna Detection With Quantum Dot-Labeled Proteins In Graphene Oxide Quenching Arrays, Colleen Jackson

Mahurin Honors College Capstone Experience/Thesis Projects

Our objective was to develop a faster method of quantitively detecting double stranded (ds)DNA of pathogenic bacteria such as the Shiga Toxin 2 gene present in E. coli O157. Transcription Activator-Like Effectors (TALEs) are a new class of DNA-binding proteins which selectively bind to dsDNA with the 12th and 13th amino acids of each repeat, called repeat variable diresidues (RVDs). Novel TALE proteins were designed to target the stx2 gene and were cloned into existing AvrBs3 TALE protein in the pMAL c2x vector system for bacterial BL-21 E. Coli expression. The protein’s DNA-binding region was then subcloned pEAQ vectors for …

An Approach To Identify Mycobacteriophage Diversity Prior To Dna Sequencing, Charles Gregory

Mahurin Honors College Capstone Experience/Thesis Projects

Over 6,869 Mycobacteriophages have been isolated and purified. Of these, 1,367 genomes have been sequenced at the DNA level and more are added each year through the SEA-PHAGES program. Sequenced mycobacteriophages are grouped into clusters based on a 50% or greater nucleotide identity. The number and breadth of these clusters represents the diversity present in the environment. Each year, as new phages are discovered by students in the SEA-PHAGES program, the question arises, “Which isolates should we sequence?” In order to sequence phages that represent the greatest possible diversity, and thus broaden under-represented clusters and identify new singletons, we need …

Investigating The Synergistic Effects Of Cisplatin And Two Curcuminoid Compounds On Cancer, Denis Hodzic

Mahurin Honors College Capstone Experience/Thesis Projects

Cisplatin is an anti-cancer drug effective against several cancers which can produce the serious side-effect of hearing loss. Curcumin, a natural plant compound, can increase the activity of cisplatin against cancer and counteract cisplatin’s effect against hearing. Because curcumin exhibits poor bioavailability, there is considerable interest in developing synthetic curcumin analogs (curcuminoids) that are more soluble and which retain anti-cancer activity and otoprotective function. This study investigated whether two curcuminoids, EF-24 and CLEFMA, increase the cytotoxic and ototoxic effects of cisplatin against the lung cancer cell line, A549, and the colorectal cancer cell line, Caco2. Cytotoxicity was measured by using …

The Creation Of Α2nc1 Transgenic Drosophila Melanogaster, Jordan Olberding

Mahurin Honors College Capstone Experience/Thesis Projects

The α2 isoform of collagen IV’s noncollagenous 1 domain (α2NC1 or canstatin) has shown promise as an anti-cancer molecule, possessing both angiostatic and pro-apoptotic properties. This work aims to further the knowledge of α2NC1 by conducting a truly in vivo study in which the Gal4-induced endogenous overexpression of α2NC1 in Drosophila melanogaster will be used to assess the effects of α2NC1 upon normal development as well as tumorigenesis. To this end, transgenic fly lines capable of overexpressing α2NC1 were created. Initial overexpression studies indicated no developmental phenotype was caused by abundance of α2NC1. However, future overexpression studies in which α2NC1 …

Reactions With Platinum (Ll) Complexes And Selenium-Containing Amino Acids, Stephanie Robey

Mahurin Honors College Capstone Experience/Thesis Projects

We have reacted [Pt(Me₄en)(D₂O)₂]²⁺ [Me₄En=N,N,N’N’-tetramethylethylenediamine] with Selenomethionine (SeMet), Methionine (Met), and Methylselenocysteine (MeSeCys). When MeSeCys was reacted with [Pt(Me₄en)(D₂O)₂]²⁺, we observed both stereoisomers of Se,N chelates, as well as [Pt(Me₄en)(MeSeCys)Cl]⁺ from ­¹­H NMR Spectroscopy; the latter formed due to the presence of Cl^- in the solution. Both isomers of the chelate seemed to form proportionally to one another, not favoring a specific stereoisomer. Eventually the [Pt(Me₄en)(MeSeCys)Cl]⁺ products became Se,N chelates. We incubated SeMet with …

Pcr Amplification Of Trypanosoma Cruzi - Specific Dna From Raccoon Blood Samples, Colin Moss

Mahurin Honors College Capstone Experience/Thesis Projects

The parasitic protist Trypanosoma cruzi is the causative agent of Chagas Disease. Chagas Disease causes greater than 15,000 deaths each year, and nearly 28 million people are believed to be at risk of infection in Central and South America. This parasite has been described in many mammalian host species and has also been described in the United States. The purpose of this study was to attempt to use PCR to amplify T. cruzi-specific DNA directly from blood samples obtained from raccoons (Procyon lotor) trapped in Warren and Barren Counties of Kentucky in 2007 and 2008. DNA was …

Biomechanical Modeling Of Forelimb Adduction In The Eastern Mole, Scalopusaquaticus, Mark Sandefur

Mahurin Honors College Capstone Experience/Thesis Projects

The hypertrophied musculoskeletal system driving adduction of Sealopus aquatieus's forelimb supports the locomotion of these burrowing subterranean mammals. Due to the excessive bulk of the major muscles contributing to this action, it was thought that this particular species may represent a rare case of a mammalian species with a greatly enhanced force contribution from each muscle, correlating with a reduction in the potential for the forelimb to adduct at a high velocity. By assessing the lever ratios of five significant muscles contributing to forelimb adduction, combined with vector analyses for the angles of these muscles' insertions, a model was established …

The Establishment Of Standard Methods And Experimental Procedures For The Prototypical Icp/Tof-Ms Using An Icp-Aes, Joseph Riley

Mahurin Honors College Capstone Experience/Thesis Projects

The ICP-AES was used to develop a standard test method for the preparation of samples for analysis purposes, a method that could also be transposed onto the new prototypical ICP/TOF-MS. Whereas both of these instruments are analytical spectroscopic instruments and use the ICP's plasma as an energy source, the ICP/TOF-MS also has a mass spectrometer attached to it. This addition gives the instrument better reliability, accuracy, and precision in analysis as well as faster analysis time. Since the ICP-AES is a precursor of the ICP/TOF-MS, it can be used to develop a standard development method. The experiment involved using a …