Life Sciences 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 …

Functional Analysis Of Legionella Pneumophila Effector Protein, Shreya Neupane

Masters Theses & Specialist Projects

Legionella pneumophila is a gram-negative bacterium that causes Legionnaire’s disease (a severe form of pneumonia) in humans. L. pneumophila can cause infection by utilizing its Type IV secretion system, a protein secretion system that transports proteins from the bacterial cytosol into the infected macrophage. Effectors released from the Type Iv secretion system allow L. pneumophila to create a safe environment to survive, replicate and cause infection. One such effector, RavQ, inhibits cell proliferation of mammalian HEK 293T cells and localizes to the cell’s nucleus, leading us to hypothesize that RavQ interferes with cellular activity in the nucleus. To detect its …

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 …

Developing Novel Tale-Based Rapid Detection Of Pathogenic Dna Utilizing 2d Graphene Oxide Nanosheets And Quantum Dots, Narayan Neupane

Masters Theses & Specialist Projects

A faster method of quantitative detection of specific dsDNA of pathogenic bacteria such as the Shiga toxin 2 gene (stx2) present in E. coli O157:H7 is of great importance. There is a need for a simple and facile method for sensitive detection of pathogenic gene which is crucial for the prevention and earlier treatment of any infectious diseases. A Transcriptional Activator-Like Effector (TALE) is a novel class of DNA-binding proteins with the unique modularity, flexibility and easy programmability compared to previously discovered DNA- binding proteins. TALEs can bind to any DNA sequences through its unique variable di- residues …

Synthesis And Molecular Mechanics Study Of Platinum Triamine Complex With N-Acetyl-L-Methionine And Guanosine 5’Monophosphate, Nitin Tanajirao Katare

Masters Theses & Specialist Projects

Cisplatin and its analogues have been used and tested over decades for cancer treatment, but because of the serious side effects, it is important to find ways to overcome those side effect by synthesizing a novel platinum molecule. We have synthesized triamine complex and conducted its reaction with biomolecules such as 5’GMP and Methionine in the lab as well as conducted molecular mechanics (MM). MM methods are used for predicting and designing new structures and properties, and to model geometries of small molecules. MM and dynamics calculations have been used to study the 9 Ethylguanine and N-Ac-Lmethionine complexes with platinum …

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, …

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 …

Effects Of Ef-24 And Cisplatin On Cancer, Renal, And Auditory Cells, Denis Hodzic

Masters Theses & Specialist Projects

Cisplatin is a chemotherapy drug effective against several forms of cancer, but can also cause serious side-effects, including nephrotoxicity and ototoxicity. Curcumin, a natural plant compound, can increase cisplatin’s anti-cancer activity and counteract cisplatin’s deleterious effect on the auditory and renal systems. Unfortunately, curcumin exhibits poor bioavailability, which has promoted interest in the development of synthetic curcumin analogs (curcuminoids) that are soluble, target cancer, and do not cause side effects. This study investigated whether the curcuminoid (3E,5E)-3,5-bis[(2-fluorophenyl) methylene]-4-piperidinone (EF-24) increases the anti-cancer effects of cisplatin against a human ovarian cancer cell line (A2780) and its cisplatin-resistant counterpart (A2780cis), while preventing …

Developing A New Sensing Technology For Double-Stranded Dna Detection Utilizing Engineered Zinc Finger Proteins And Nanomaterials, Dat Thinh Ha

Masters Theses & Specialist Projects

A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of TEM-1 β- lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogenspecific DNA sequences present in E. coli O157:H7 on the lab-on-a chip. ZFPs customdesigned to detect shiga toxin …

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 …

Monitoring And Quantifying Tetracycline Resistance Genes In A Swine Waste Anaerobic Digester Over A 100-Day Period, Melanie Couch

Masters Theses & Specialist Projects

Unregulated use of growth promoting antibiotics like Tetracyclines in agricultural feeds is becoming an increasing problem in antibiotic resistance. Undigested antibiotics leads to significant concentrations in livestock waste. These concentrations provide continuous selection pressure for the development of antibiotic resistance genes in the environment. Antibiotic resistance related deaths are projected to surpass cancer related deaths by 2050 making antibiotic resistance a pressing public health issue. The purpose of this study is to determine the abundance and persistence of tetracycline (tet) resistance genes in swine waste over a period of 100 days in an anaerobic digester system. Tet(A), tet(B), tet(G), tet(M), …

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 …

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 …

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 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 …

Trimethylated Lysine 4 At Histone 3 Shows The Same Circadian Rhythm At Promoters Of Diversely-Expressed Genes In Chlamydomonas Reinhardtii, Robyn M. Wilson

Masters Theses & Specialist Projects

Circadian clocks are biochemical mechanisms that allow eukaryotic and some prokaryotic organisms to coordinate their physiology with daily environmental changes. It enables organisms to increase their fitness by taking advantage of beneficial environmental conditions while also avoiding or restricting certain sensitive processes during harsh conditions. Similarly, post-translational histone modifications allow eukaryotic organisms to regulate gene expression in response to environmental or developmental factors. Some post-translational modifications of histones are associated with active transcription while others are associated with repressed transcription depending upon the location, type and degree of modification. Trimethylation of lysine 4 on the N-terminal tail of histone H3 …

Multiplexed Detection Of Double-Stranded Pathogenic Dna With Engineered Zinc Finger Proteins, Juhwa Kim

Masters Theses & Specialist Projects

The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated 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 …

Functional And Expression Analysis Of A Novel Basement Membrane Degrader In Drosophila Melanogaster, Christopher J. Fields

Masters Theses & Specialist Projects

The Srivastava Lab is focused on the identification and characterization of genes that play a role in basement membrane remodeling. Previously, we identified putative basement membrane degraders through a genetic screen. One such gene has been suggested to play a role in the maintenance of the stem cell niche in Drosophila melanogaster, but no other information about the role this gene plays in development or disease has been published. Here, data are presented from experiments utilizing Drosophila genetics and immunohistochemistry that provide important insights on the biological role of this gene.

Collagenase activity was up-regulated upon overexpression of this gene, …

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 …

Using Zinc Finger Proteins As A Diagnostic Tool For The Detection Of A Cancer Biomarker, Anu Kini

Masters Theses & Specialist Projects

RASSF1A is a tumor suppressor gene which loses its function due to methylation of CpG islands on its promoter region. Detection of methylation leads to early diagnosis of cancer.

Zinc finger proteins are capable of detecting a specific DNA sequence and Methyl binding domain can bind to the methyl group on the CpG, using this idea mCpG SEER- Lac system makes use of a split protein, β-lactamase. Lac A attached to the ZFP and Lac B attached to the MBD protein. On binding to the DNA, the Lac A and Lac B come in close proximity with each other causing …

Binding Of Oxaliplatin And Its Analogs With Dna Nucleotides At Variable Ph And Concentration Levels, Rippa Sehgal

Masters Theses & Specialist Projects

Oxaliplatin is one of the three FDA-approved platinum anticancer drugs and considered a third generation drug, discovered after the first generation drug cisplatin and second generation drug carboplatin. It is known to react with proteins and DNA nucleotides in the body. Reaction with DNA occurs primarily at guanosine residues and secondarily at adenine residues for oxaliplatin and other platinum drugs. We have previously studied oxaliplatin and an analog with additional steric hindrance in the amine ligand and found that the analog had different reactivity with methionine. Now, we have prepared oxaliplatin and its three analogs Pt(Me2dach)(ox), Pt(en)(ox) and Pt(Me4en)(ox) and …

Expression And Purification Of Unlabelled And Isotopically Labelled Human Fibroblast Growth Factor-1 And Its Receptor Relevance In Cancer Research, Oluwadamilola Filani

Masters Theses & Specialist Projects

Studies show that fibroblast growth factors (FGFs) control variety of cellular activities such as mitosis, cell differentiation, survival and angiogenesis. The FGF family consists of 23 different heparin-binding proteins. One of the most intensively studied members is human FGF-1 (hFGF-1) because of its critical role in the formation of blood vessels and cell proliferation in some types of cancer. The biological activities of FGFs are primarily mediated via interactions with fibroblast growth factor receptors (FGFR) and are a potent target in cancer. In this study, we report an efficient affinity column purification of hFGF-1 and the D2 domain of FGFR-2 …

Functional Characterization Of A Cathepsin L In Drosophila Melanogaster, Qian Dong

Masters Theses & Specialist Projects

The Drosophila dorsal Air Sac Primordium (ASP) is a tracheal tube that invasively grows toward and into the wing imaginal disc. The unfolding of Drosophila wing is a process following eclosion with a cuticular bilayer replacing epithelial cells originally packing the wing. We reasoned that protease functions might be needed for the invasion of ASP into the wing imaginal disc as well as the rearrangement of epithelia cells during wing unfolding. Our study is particularly focused on understanding the role of a Cathepsin L like cysteine protease (CP1) in the development of dorsal ASP and wing development of Drosophila melanogaster. …

An Apache Hadoop Framework For Large-Scale Peptide Identification, Harinivesh Donepudi

Masters Theses & Specialist Projects

Peptide identification is an essential step in protein identification, and Peptide Spectrum Match (PSM) data set is huge, which is a time consuming process to work on a single machine. In a typical run of the peptide identification method, PSMs are positioned by a cross correlation, a statistical score, or a likelihood that the match between the trial and hypothetical is correct and unique. This process takes a long time to execute, and there is a demand for an increase in performance to handle large peptide data sets. Development of distributed frameworks are needed to reduce the processing time, but …

Understanding The Molecular Level Interactions Of Cancer Inhibitor Imatinib With Human Fibroblast Growth Factor-1, Tulsi Modi

Masters Theses & Specialist Projects

Fibroblast growth factors (FGFs) lack signal sequences, and are exported through endoplasmic reticulum (ER)-Golgi-independent non-classical routes. FGFs work as modulators of various cellular activities like mitosis, differentiation, survival etc. Among the FGF family, which comprises of 23 different heparin proteins, human FGF-1 (hFGF-1), a potent angiogenic factors are one of the targets in cancer inhibition, as they are involved in blood vessel formation in tissues. There has been intensive research directed at the development of drugs that could effectively inhibit angiogenesis. In this context, the purpose of this study is to fully understand the molecular principles essential to determine probability …

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 …

The Time-Course Of The Effects Of Growth Hormone During Zebrafish (Danio Rerio) Auditory Hair Cell Regeneration, Yajie Wang

Masters Theses & Specialist Projects

Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the …