Biochemistry Commons^™

Liquid Chromatography Mass Spectrometry Method Development For The Determination Of Β–Defensins In Bovine Milk, Elisha Ogbebor

Masters Theses & Specialist Projects

Bovine mastitis is caused by a wide range of pathogens which results in a substantial economic loss for the dairy cattle livestock industry. β-defensins are a part of the innate immune system and act as the first line of defense against mastitis in bovine. β-defensins (~6- 10kDa) are antimicrobial peptides that contain about 28 to 67 amino acids, the presence of six cysteine residues results in their three disulfide bonds and a β-sheet structure. A few β- defensins have been identified in cows based on the site of occurrence and time of expression in tissue, but not in milk. A …

Visible Light Generation And Mechanistic Investigation Of High-Valent Metal-Oxo Species Supported By Different Ligands, Seth Ellis Klaine

Masters Theses & Specialist Projects

Numerous transition metal catalysts have been designed as biomimetic model compounds for the active site of metalloenzymes found throughout Nature, most notably cytochrome P450 monooxygenases that carry out the oxidative transformations of organic substrates with near-perfect chemo-, regio-, and stereo-selectivity. The primary active oxidants in catalytic and enzymatic cycles are fleeting high-valent metal-oxo intermediates where the oxo ligand can transfer to an organic substrate in a process known as oxygen atom transfer (OAT).

In the present work, porphyrin-manganese(III), salen-chromium(III), and salenmanganese( III) derivatives were successfully synthesized and spectroscopically characterized using 1H NMR and UV-Vis spectroscopies. A facile photochemical approach was …

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 …

Toxicity Of Novel Platinum Compounds In Mammalian Cancer Cells, Vanesa Veletanlic

Masters Theses & Specialist Projects

There are currently three FDA platinum compounds approved for use as chemotherapeutics, where each drug has variable efficacies for different cancer types depending on cancer’s tissue of origin. The approved compounds are platinum(II) complexes with four coordination sites on the platinum atom allowing two types of ligands to attach: leaving ligands, which are removed from the platinum atom in solution, and non-leaving ligands, which remain complexed to the platinum. Carboplatin, the preferred compound used to treat ovarian and small-cell lung cancers, has a characteristic cyclobutanedicarboxylic acid leaving ligand and two ammonia non-leaving ligands. A novel compound, 1,1-cyclobutanedicarboxylato(ethylenediamine)platinum (II), or Pt(en)CBDCA, …

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 …

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 …

Leaving Ligand Effects On Reactivity And Solubility Of Monofunctional Platinum(Ii) Anticancer Complexes, Heidi Linn Hruska Millay

Masters Theses & Specialist Projects

Monofunctional platinum(II) complexes, such as phenanthriplatin and pyriplatin, have notably different characteristics from the bifunctional anticancer complexes, such as cisplatin and oxaliplatin, which have detrimental toxicities and resistance associated with them. The unique properties of the monofunctional complexes may be exploited to target cancer cells without producing the toxic side effects associated with the current FDA-approved platinum-based anticancer drugs. To advance the understanding of these monofunctional platinum(II) complexes, this study replaced the chloride leaving ligand with an acetate group, which should increase solubility and alter the rate of reactivity with key amino acid and nucleotide targets. Phenanthriplatin and pyriplatin compounds …

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 …

Biomimetic Studies Of Oxidation Reactions By Metalloporphyrins Through Ligand Effect And Kinetic Studies Of Photo-Generated Porphyrin-Iron(Iv)- Oxocompound Ii Models, Dharmesh J. Patel

Masters Theses & Specialist Projects

High-valent iron(IV)-oxo porphyrins are the central oxidizing species in hemecontaining enzymes and synthetic oxidation catalysts. Many transition metal complexes have been extensively studied as models of the ubiquitous cytochrome P450 enzymes to probe the sophisticated oxygen atom transfer (OAT) mechanism as well as to invent enzyme-like oxidation catalysts. In this work, two metalloporphyrin complexes have been successfully synthesized, and spectroscopically characterized. A new photochemical entry to porphyrin-iron(IV)-oxo derivatives, commonly referred to as compound II models, was also investigated in two porphyrin ligands that differ in electronic and steric environments. As determined by their distinct UV-vis spectra and kinetic behaviors, iron(IV)-oxo …

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 …

Synthesis, Kinetic And Catalytic Studies Of Manganese Complexes With Corrole And Porphyrin Ligands, Haleh Jeddi

Masters Theses & Specialist Projects

High-valent transition metal-oxo intermediates play a significant role in the catalytic cycle of the ubiquitous cytochrome P450 enzymes and in biomimetic catalytic systems. In this work, manganese(III) porphyrin and corrole systems (2) were synthesized and characterized by UV-vis absorbance and 1H-NMR, matching literaturereported spectroscopic data. Manganese(V)-oxo corroles (3) and a manganese(IV)-oxo porphyrin (4) were successfully generated by chemical oxidation using mchloroperoxybenzoic acid (m-CPBA), and their oxidation reactions with organic reductants were comparatively investigated. Results from single-turnover kinetic studies indicate that in the tris(pentafluorophenyl)corrole system (3a), the active oxidizing intermediate differs in different solvents. The active oxidizing intermediate in acetonitrile is …

The Effects Of Carrier Ligands On Cisplatin Binding To Cysteine And Methionine, Adam C.R Smith

Masters Theses & Specialist Projects

We have reacted several derivatives of the anticancer drug cisplatin with N-acetyl-Lcysteine (N-AcCys) and N-acetyl-L-methionine (N-AcMet), which are two of the primary amino acid targets of platinum. NMR spectroscopy was used to monitor the reactions and determine the effect the different ligands would have on the platinum reactivity. Several of the platinum compounds were tested at pH of 4 and 7, and with platinum:amino acid ratios of 1:1, 2:1 and 1:2. Competition reactions between cysteine and methionine were done to confirm which would react with the platinum compound first. [Pt(dien)(NO3)]+ reacts faster with methionine than with cysteine at both pH …

Effects Of Binding Affinity Between Bovine Serum Albumin And Platinum Drugs, Nathan Puckett

Masters Theses & Specialist Projects

Platinum complex drugs such as cisplatin have been used as highly successful chemotherapy drugs since the 1970s. We are interested in how the ligands attached to cisplatin analogs influences their reactivity with biologically relevant targets along with time and amount. For this study, reactions were conducted to determine the reactivity between different platinum compounds and the protein bovine serum albumin. Various platinum compounds with different ligands were reacted in varying amounts with albumin in ammonium acetate buffer for either 1 hour, 4 hours, or 24 hours. Each reaction was quenched after the designated reaction time by dialysis and the platinum …

Effects Of Herbicides On Industrial Hemp (Cannabis Sativa) Phytotoxicity, Biomass, And Seed Yield, Brett A. Maxwell

Masters Theses & Specialist Projects

Field studies were established in 2015 at Bowling Green and Lexington, KY to evaluate industrial hemp (Cannabis sativa) tolerance to various herbicides. Hemp was planted into conventionally tilled soils in mid to late June at a seeding rate of 39 kg/ha in Bowling Green and 22 kg/ha in Lexington. Five herbicide active ingredients were applied preemergence (PRE) the day of planting and six postemergence (POST) treatments were applied to 30 cm hemp with a CO2-backpack sprayer delivering 140 L/ha. Plots were 3.1 m wide by 6.1 m long and were sprayed with a 2.1 m boom sprayer leaving a 0.46 …

Development Of Dihydrochalcone Functionalized Gold Nanoparticles For Augmented Antineoplastic Activity, Jason N. Payne

Masters Theses & Specialist Projects

Phloridzin, an antidiabetic and antineoplastic agent usually found in fruit trees, is a dihydrochalcone constituent that has a clinical/pharmaceutical significance as a sodiumglucose linked transport 2 (SGLT2) inhibitor. Phloridzin never experienced widespread clinical usage in the pharmaceutical market due to its side effects and poor bioavailability when compared to other antidiabetic therapeutics. The poor bioavailability is primarily attributed to the degradation of the glycosidic bond of the phloridzin, resulting in the formation of phloretin, the aglycone of phloridzin and glucose. While phloretin displays a reduced capacity of SGLT2 inhibition, this nutraceutical shows enhanced antineoplastic activity in comparison to phloridzin. Gold …

Visible-Light Generation Of High-Valent Metal-Oxo Intermediates And A Biomimetic Oxidation Catalyzed By Manganese Porphyrins With Iodobenzene Diacetate, Ka Wai Kwong

Masters Theses & Specialist Projects

High-valent iron-oxo intermediates play central roles as active oxidants in enzymatic and synthetic catalytic oxidations. Many transition metal catalysts are designed for biomimetic studies of the predominant oxidation catalysts in Nature, the cytochrome P450 enzymes.

In this work, a new photochemical method to generate high-valent iron-oxo porphyrin models was discovered. As controlled by the electronic nature of porphyrin ligands, iron(IV)-oxo porphyrin radical cations (Compound I model) and iron(IV)-oxo porphyrin derivatives (Compound II model) were produced. These observations indicate that the photochemical reactions involve a heterolytic cleavage of O-Br in precursors to give a putative iron(V)-oxo intermediate, which might relax to …

Identification Of Collagen Iv Associated Proteins In Drsophila Using Genetics And Mass Spectrometry, Mayank S. Kapadia

Masters Theses & Specialist Projects

Metastatic cancer cells invade and spread to other locations by disrupting the basement membrane (BM). The membrane plays a major role during the normal development of an organism as well. In order to understand the invasion mechanism it is important to know about the interactions occurring between the proteins of the BM during normal development. This study concentrates on isolating and identifying the major factors associated with collagen IV, a major component of BM, during the third instar larval development of Drosophila. Western blot and mass spectrometry analysis revealed that collagen IV associates with various growth factors, signaling molecules, and …

Synthetic Investigation On The Biomimetic Metal-Catalyzed Sulfoxidations And Photochemical Generation Of A Highly Reactive Ruthenium(V)-Oxo Porphyrin, Weilong Luo

Masters Theses & Specialist Projects

Catalytic oxidation plays a crucial role in current chemical and pharmaceutical industries which is also a leading technology for green chemical processes. In Nature, the ubiquitous cytochrome P450 enzymes can catalyze a wide variety of oxidation reactions with high efficiency and selectivity. Many transition metal catalysts are designed as the biomimetic model of cytochrome P450 enzymes. In this work, series of metalloporphyrins and metallocorroles have been successfully synthesized to investigate and develop catalytic selective oxidation of sulfides to sulfoxides.

Manganese(III) porphyrin complexes (2) and manganese(III) corrole complexes (6) with iodobenzene diacetate [PhI(OAc)2] as a mild oxygen source exhibited remarkable catalytic …

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 …

Effects Of Xanthan/Locust Bean Gum Mixtures On The Physicochemical Properties And Oxidative Stability Of Whey Protein Stabilized Oil-In-Water Emulsions, Goutham Puli

Masters Theses & Specialist Projects

Scientific evidence shows that dietary intake of the omega-3 polyunsaturated fatty acids is beneficial to human health. Fish oil is a rich source of omega-3 fatty acids. However, fish oil with high levels of omega-3 PUFA is very susceptible to oxidative deterioration during storage. The objective of this study was to investigate the effect of xanthan gum (XG)-locust bean gum (LBG) mixtures on the physicochemical properties of whey protein isolate (WPI) stabilized oil-in-water (O/W) emulsions containing 20% v/v menhaden oil. The O/W emulsions containing XG/LBG mixtures were compared to emulsions with either XG or LBG alone. The emulsions were prepared …

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 …

Exploring Zirconia As A Column Packing Material, Tushar Ghugare

Masters Theses & Specialist Projects

Zirconia is one of the most promising column packing materials for High Performance Liquid Chromatography (HPLC). The perfect HPLC support material should be energetically homogenous, have a high surface area on which different chemical species can reversibly attach and be physically and chemically stable over a wide range of pH, temperature and solvent conditions. Most existing supports do not have all of these properties. This project is also focused on a proteomics study. Zirconia, hafnium oxide and titanium oxide which are some of the more promising materials currently available, can be used for the separation and analysis of phosphorylated proteins. …

Cloning Of "Animal Cryptochrome" Cdna From The Model Organism Chlamydomonas Reinhardtii For Functional Analysis Of Its Protein Product, Shobha Lavanya Silparasetty

Masters Theses & Specialist Projects

reinhardtii, a unicellular green alga, is a model organism to study the circadian clock. Cryptochromes are the blue light photoreceptors that entrain the clock in some organisms. The CPH1 protein of C. reinhardtii resembles the cryptochromes of the plant model Arabidopsis, but whether CPH1 entrains the circadian clock in C. reinhardtii is not yet known. Recent reports have suggested the existence of one more cryptochrome in C. reinhardtii, which resembles the cryptochromes of animals. However, the amino acid sequence of this protein shows even higher sequence similarity with the 6-4 DNA photolyase of Arabidopsis. DNA photolyases …

The Study Of Variations In The Properties Of Biodiesel On Addition Of Antioxidants, Hiranmayee Kandala

Masters Theses & Specialist Projects

This research studies variations in biodiesel upon addition of different concentrations of antioxidant and petrodiesel additives. Oxidation onset temperature, oxidation induction time, oxidative stability, thermal stability, crystallization onset temperature and moisture retention properties of the biodiesel, with and without antioxidants have been studied. Antioxidants like BHT, BHA, PrG, Vit E and Vit C were added to the biodiesel during the study. These samples were analyzed using the PDSC, TGA, DSC and TGA-SA instrumentation.

The results of this research show an improvement in the OOT, OIT and Oxidative stability of biodiesel with the addition of antioxidants and by blending the biodiesel …

Geometric Build-Up Solutions For Protein Determination Via Distance Geometry, Robert Tucker Davis

Masters Theses & Specialist Projects

Proteins carry out an almost innumerable amount of biological processes that are absolutely necessary to life and as a result proteins and their structures are very often the objects of study in research. As such, this thesis will begin with a description of protein function and structure, followed by brief discussions of the two major experimental structure determination methods. Another problem that often arises in molecular modeling is referred to as the Molecular Distance Geometry Problem (MDGP). This problem seeks to find coordinates for the atoms of a protein or molecule when given only a set of pair-wise distances between …

Synthesis And Characterization Of Bis-Phosphine Complexes With Transition Metals, Alicia L. Mcdaniel

Masters Theses & Specialist Projects

Extractants and extraction methodologies play a vital role in many industrial processes, from the concentration of precious metals from ores to the separation of longlived nuclei from radioactive waste as well as the removal of heavy metals from soils and water for remediation. The vast majority of extractants rely on the use of nitrogen, oxygen, sulfur or selenium as Lewis base donor atoms to form coordination complexes with the metal ions of interest. These extractants often make use of the chelate effect and/or the macrocyclic effect in order to form stable complexes. Some of the best known types of chelate …

Development Of A Gene Transfer System In Clostridium Scindens Vpi 12708, Rashmi Ramasubbaiah

Masters Theses & Specialist Projects

Clostridium scindens VPI 12708 (previously known as Eubacterium sp. VPI 12708) is a bile-acid dehydroxylating bacterium originally isolated from the feces of a colon cancer patient. Many genes required for bile acid 7-a dehydroxylation are found on a large bile acid inducible operon (bai) that has been extensively studied. However the bai promoter, which directs expression of the bai operon, has yet to be characterized due, in part, to a lack of a functional genetic transfer system for this strain. A spontaneous rifampinresistant Clostridium scindens VPI 12708 mutant was used as a recipient to determine the efficacy of conjugation as …