The yeast 14-3-3 proteins Bmh1 and Bmh2 alter the transcription of genes under rapamycin treatment, an environment that mimics nutrient-limited conditions in yeast. Nutrient-limited genes are regulated by the TOR pathway in response to metabolic demands, nutrient availability, and stress. Under nutrient-limited environments and rapamycin treatment, TOR proteins are inhibited and transcription factors are localized to the nucleus, which then up regulate nutrient-limited genes. Even though both of these 14-3-3 proteins overlap in function, a deletion of one but not necessarily both, effects the growth and cell division of the yeast. A double deletion of both of these proteins has been shown to be lethal in most but not all strains. Further investigation reveals that both single deletions of Bmh1p and Bmh2p alter the transcription of over 286 genes after treatment with rapamycin. A deletion of one of these proteins will show the effect of that protein in absence of the other but deleting both of these proteins will show their overall role during nutrient-limited conditions. Since these proteins are shown to regulate nutrient-limited genes in a single knockout, the principle behind this experiment is to determine whether the double deletion of the 14-3-3 proteins alters the transcription of the genes any differently then the single deletions. Through a microarray analysis it has been determined that the strain with the double knockout of both 14-3-3 proteins, BMH1 and BMH2, affects the transcription of numerous genes throughout the entire genome, some different and some the same as the single deletion strains.
Chemical Models of Anti-Cancer Pharmaceuticals Based on Ruthenium Compounds
with Organic Side Chains.
Molecules that bind to DNA inhibit replication, which is important for anti-cancer pharmaceuticals. This work focuses on DNA binding analysis of ruthenium compounds that have a modifiable chain attached to one of three ligands surrounding the metal center. Modifying the shape, chirality and hydrophobicity of the binding molecule affects where binding occurs as well as the strength of the interaction. Generally molecules that bind strongly and at specific locations are better candidates for drug-therapy. The syntheses of the modifiable ligand begins with 4-formyl-4’-methyl-bipyridine, which is an aldehyde, produced using a selenium oxidation reaction with yields up to 78%. The aldehyde is then reduced to an alcohol (95 % yield). The alcohol undergoes a Williamson Ether synthesis to produce a bipyridine ligand with a modifiable chain. This ligand is bound to [Ru(phenanthroline)2Cl2] to produce the final product shown in Figure 1. All products were analyzed by 1HNMR, 13CNMR, and infrared spectroscopy. The [Ru(phen)2bpy-naphthalene](PF6)2 was bound to DNA in fluorescence titration studies. The DNA binding strengths were calculated and compared to a control ruthenium complex, [Ru(phenanthroline)3](PF6)2. The mode of binding is unknown and expected to depend on the nature of the modified ligand. We expect that binding strength will increase with the addition of the naphthalene group compared to control, because naphthalene is planar and aromatic that favors intercalation between the base pairs of DNA.
Figure 1. Final ruthenium compound, [Ru(phen)2(bpy-R)]2+. This Ru compound has a charge of =2, which will attract the negatively charged DNA and a modifiable bipyridine ligand expected to affect DNA binding mode and strength.
In upstate New York, the burning of wood fuels for heating purposes is very common in the winter months. Woodstoves and pellet stoves are conventional and practical options for residents seeking to heat their homes. Wood combustion may also pose a potential threat to indoor air quality, and in effect, public health. The purposes of this research are threefold. First, emissions of carbon monoxide, carbon dioxide, and particulate matter (10 µm and 2.5 µm) are monitored at homes before (for background) and during wood burning. Second, the concentrations of toxic species (such as formaldehyde and nitrogen oxides) released from each appliance as a result from the combustion of wood are measured. Polyaromatic compounds in the emissions will be collected and analyzed through gas chromatography-mass spectrometry. Last, several wood and pellet samples have been collected from the homes of volunteers and analyzed using adiabatic oxygen bomb calorimetry to determine the gross heat of combustion for different wood fuel types. Analysis of all results will provide information on the effects that wood burning has on air within residential buildings and the heating efficiency of wood fuels.
Effects of Liposomes Composed of Phosphatidylcholine and Phosphatidyl-serine on the Structures of Bovine Serum Albumin and Cytochrome C
Advisor: Dr. Nadia Marano
Abstract : Protein aggregation contributes to neurodegenerative diseases such as Parkinson's and Alzheimer's disease. The earliest stages of prefibrillar aggregation are believed to have a significant role in disease pathogenesis by increasing cytotoxicity within the cell and disrupting physiological ion concentrations leading to eventual cell death. Phospholipids that make up cellular membranes are capable of making proteins adopt non-native confirmations by causing the proteins to initially unfold, and then allowing them to take up non-native conformations. Inferences can be made regarding the unfolding and folding processes of proteins in the presence of phospholipids, specifically phosphatidylcholine (PC) and phosphatidylserine (PS). In order to study the effect of these lipids on the structure of model proteins intrinsic fluorescence was used to study changes in the environment surrounding the tryptophan residues within the protein. Fluorescence was measured when samples of bovine serum albumin and cytochrome c were incubated in vitro with or without small-unilamellar vesicles containing PS and PC for different times. From initial studies, PS had a larger affect on the structure of BSA and cytochrome c than PC and mixed liposomes of PS/PC. Next we will compare this observation with changes in fluorescence brought about by urea, which denatures the protein.
Cancer is the uncontrolled proliferation of abnormal cells. Highly conserved DNA motifs are recognized by replication machinery in the promoter region of a gene such as the stem cell leukemia gene (SCL) which act as regulatory elements (Göttgens, 2002). Through targeting these motifs and inhibiting DNA synthesis of these regions with antibiotic drugs, cancer therapy could become more selective. Anthracycline drugs have been shown to treat various cancer lines through intercalation. Nogalamycin is an anthracycline antitumor natural antibiotic that uses threading to inhibit DNA-directed RNA synthesis which prevents a cell from dividing and proliferating. The binding mechanism of nogalamycin has been studied extensively, including SLU graduate Matt Millard, but its specificity for various DNA structures has not been determined. Though the cardiotoxicity of nogalamycin will prevent its commercial use, a similar compound could be synthesized with less adverse effects. Common techniques for studying DNA binding require large DNA sample volumes. Footprinting with topoisomerase and agarose gel electrophoresis require much smaller samples but have only been used to study intercalating molecules, never threading molecules (Webb, 2003). Agarose gel electrophoresis reveals the degree of intercalation post relaxation with topoisomerase by the migration distance of the plasmid (Webb, 2003). The main focus of this research is to discover if this technique works with threading molecules like nogalamycin and implementing it using a variety of DNA motifs to determine the structural specificity of the antibiotic.
Göttgens, B., Barton, L.M., Chapman, M.A., Sinclair, A.M., Knudsen, B., Grafham, D., Gilbert, J.G.R., Rogers, J., Bentley, D., and Green, A. (2002). Transcriptional Regulation of the Stem Cell Leukemia Gene (SCL): Comparative Analysis of Five Vertebrate SCL Loci. Genome Res.12: 749-759.
Webb, M.R. and Ebeler, S.E. (2003) A gel electrophoresis assay for the simultaneous determination of topoisomerase I inhibition and DNA intercalation. Analy. Biochem. 321: 22-30.
Lead and cadmium, known as heavy metals, have been shown to have detrimental health effects in humans, including bioaccumulation in many organs causing brain damage, kidney damage, and gastrointestinal distress, as well as negative reproductive and developmental effects. The goal of this project is to examine lead and cadmium levels in household items, primarily leaded crystal glassware, glazed ceramics, and children’s toys, and how they might be ingested through contact. The project was created due to many product recalls in recent years due to lead and cadmium levels. The dinnerware items were soaked in water, wine, brandy, tea and lemonade to imitate consumption and use of the pieces. The children’s toys were soaked in solutions of artificial sweat and saliva to mimic toy-human contact by hand or by mouth. Analysis was performed using the flame atomic absorption spectroscopy technique and the results were compared to accepted values from the United States Consumer Product Safety Commission and Food and Drug Administration.
Metallacrowns are a unique family of inorganic molecules with metal-containing ring structures at the center. Oximes such as are one type of organic molecule that can form metallacrowns, and this project explores the use of the oxime phenyl 2- pyridyl ketoxime (pko). This project began by investigating the use of copper 9-metallacrown-3 (9-MC-3, 9 indicating the total number of ring members with 3 metal atoms) complexes as dye sensitizers for solar cells. A researcher from another university suggested that modifying the ligand by adding a carboxylic acid functional group on the phenyl ring might improve the complex’s utility as a dye sensitizer. These molecules, however, did not show positive results, and modifying the ligand would not have helped. However, no carboxylate derivatives of pko have been utilized to form metallacrowns. This project’s overall goal is now the synthesis of modified pko ligands for use in the synthesis of an inverse copper 9-MC-3 complex. Another interesting feature of this project would be that the modified pko ligand could allow the metallacrowns to be linked together by coordinating the carboxylic acids to other metal ions. This could then produce extended solids or molecular containers.
At least three synthetic routes are possible for the new ligand. Two of the more common have been tried. The first multistep synthetic route was adapted from a procedure reported in 1998 by Takeuchi , et al., that couples 3-bromopyridine with methyl 4-formylbenzoate. Product was not achieved in sighnificant amounts when 3-bromopyridine was replaced with 2-bromopyridine to create the modified phpyko. When tested, the 3-bromopyridine synthesis worked as well as had been reported. The current approach was outlined by Villani and Papa. This route uses a Friedel-Crafts acylation followed by oxidation with potassium permanganate. Results from these synthetic approaches and any new metallacrowns will be presented.
Hydroxamic acids coordinated to first row transition metals is well documented in the chemical literature, specifically in relation to copper(II), iron(III), and nickel(II), but there is very little concerning hydroxamic coordination with soft less labile second and third row transition metals such as platinum and palladium. The first goal of this study explores the synthesis and characterization of five previously cited platinum hydroxamic acid complexes using 195Pt-NMR; an aptitude of nuclear magnetic resonance spectroscopy that has never been collected on this class of molecules. The prior compounds show (O,O), (O,N), and (N,N) hydroxamate coordination modes about the platinum and were discovered to coordinate to platinum via deprotonated hydroxyl and carbonyl oxygen atoms giving rise to stable five member chelate rings. The second goal of this study explores numerous coordination chemistry investigations of platinum in an attempt to synthesize the first platinum metallacrown; a molecule that has not been previously reported in the chemical literature. Metallacrowns are unique symmetric inorganic compounds with ring structures at their core. Metallacrowns based on hydroxamic acids form a cyclic ring structure consisting of an alternating metal ion (e.g. Pt), oxygen atom (O), and nitrogen atom (N) respectively (Pt-O-N), with a metal ion or lanthanide ion (La) encapsulated at its core. Metallacrowns are presently being investigated for use as chiral recognition agents, catalysts, antibiotics, single molecular magnets, and MRI contrasting agents. Water molecules in the human body bind to the lanthanide ion in commercially used contrasting agents such as [Gd(DOTA)]2-, which enhances the relaxation of water protons and is measured by the MRI machine and used to create an image. In lanthanide-metallacrowns, up to four water molecules can bind to the central lanthanide ion compared to one water molecule in commercially used contrasting agents. When more water molecules are able to bind to the lanthanide, a clearer and more defined MRI image is possible. Currently no metallacrowns are used as MRI contrasting agents because metallacrowns tend to degrade and become labile inside the pH of the human body. Platinum is a harder less labile metal compared to first row transition metals currently used to synthesize metallacrowns. Platinum has been shown to remain intact and withstand the thermodynamic requirements of human tissue in that it positively intercalates DNA and is currently used in 50% of all anti-cancer treatments.
The purpose of this research is to synthesize several new metallacrown ligands: N,N-dimethylglycine hydroxamic acid, N, N-dimethylβ-alanine hydroxamic acid, N, N-bis(2-pyridylmethyl)glycine hydroxamic acid and N, N-bis(2-pyridylmethyl)β-alanine hydroxamic acid. Metallacrown are a relatively new class of molecule that have various potential applications. These molecules have shown potential in bioactivity, molecular recognition, organic catalysis, liquid crystals, building one, two and three dimensional solids, and as single molecular magnets. The dimethyl ligands are being synthesized so that thermodynamic studies can be conducted in order to further understand the self-assembly of the more complicated pyridylmethyl ligands. Synthesis of these molecules have been attempted following four different synthetic pathways with no successful synthesis of the final ligands, but all precursor material has been synthesized and characterized.
Rapamycin treatment of Saccharomyces cerevisiae (yeast) cells has been shown to regulate genes involved in nutrition pathways. Such regulation often involves activation or repression of certain proteins, and it is barely understood. Herein, we investigated the recruitment of the Histone Deacetylase (HDAC) enzyme RPD3 onto the promoter sequences of nutrition associated genes, hypothesizing that certain transcription factors linked to RPD3 recruit it to these specific promoter sequences. Computational analysis of these promoter sequences revealed a common CTCATC motif (the Polymerase A and C element- PAC element) shown to bind PBF1 and PBF2 transcription factors. Reverse Transcription Quantitative PCR of GAP1, RPA43 and RPC25 genes on doubly deleted PBF1 and PBF2 cells compared to Wild Type cells showed transcription profiles slightly similar to those of RPD3.
To demonstrate sufficiency of the PAC element in recruiting, a strain of cells without GAP1 gene was unsuccessfully developed via transformation using DNA pringle cassette. Mutagenesis of the same GAP1 gene on a plasmid was inconclusive too. However, microarray experiment on the doubly deleted PBF1 and PBF2 transcription factors revealed the regulation of ribosomal protein genes. This analysis in essence increases understanding of a mechanism that could be mimicked in cancerous growth.
The goal of this research is to synthesize a [Ru(phen)2dppz]2+ amide linked dimer, which can bis-intercalate with DNA. By intercalating with DNA, this compound has the potential to stop the DNA replication process and in turn stop cell division. This may be a harmful effect, but the molecule may prove useful as a chemotherapeutic agent. Ruthenium polypyridyl complexes are composed of ruthenium atoms surrounded by aromatic pyridyl groups. Based on research by Battaglia and Thomas, it was determined that compounds not unlike the [Ru(phen)2dppz]2+ amide linked dimer are actively transported into cells. With a possible active transport pathway into the cell, the compounds mode of action is legitimated by having access to the nucleus. The [Ru(phen)2dppz]2+ amide linked dimer is expected to use its positive charge to attract the negative phosphate backbone of DNA, then use the hydrophobic interaction and pi-pi stacking between base pairs to intercalate its head groups into the DNA strand. The mechanism is not totally understood, but bis-intercalators have larger binding constants and larger dissociation times than mono-intercalators of the same type.
The mechanism and method of binding cannot be studied further until the compound's synthesis is completed; however, a selenium deprotection step has proven difficult. My focus has been increasing the yield of this selenium removal step, since Jen Achtyl has optimized the other steps. Following the Zn and HCl deprotection method outlined in the literature, only 0.1% yield of product has been achieved. Subsequent reactions from the literature like a deprotection with HCl and HI, and a deprotection with Mg and methanol have been attempted. Both of these reactions show relevant product peaks in the NMR, but they display just as much unreacted material. A final reaction with triphenylphosphine will be attempted, and the previous reactions may be researched further for proper separation methods. The final goal of this research will be the creation of a complete [Ru(phen)2dppz]2+ amide linked dimer, which after the removal of selenium should not prove difficult. This compound has already been synthesized by other research groups, but we are hoping to perfect the synthesis ourselves and then alter the linker molecule for DNA binding studies.
Boron Containing Heterocycles: Suitability of Dioxaboroles and Boronate Esters
in Diels-Alder Reactions.
Boron containing compounds are useful in synthesis reactions because of their versatility and affinity for Lewis acid base chemistry and are found in natural as well as in complex synthetic products. Usually found in a trigonal planar bonding geometry, boron has an empty p-orbital perpendicular to the bonding plane. In the presence of a Lewis base, boron will adopt a tetrahedral geometry and a negative charge and can for instance form a boronate ester. The p-orbital can also promote aromaticity by allowing circulation of electron density around the ring, as is the case for a dioxaborole. Both boronate esters and dioxaboroles are heterocyclic ring systems, with boron attached to two alkyl-bridged oxygen atoms. The dioxaborole system is aromatic in nature, with electron pair donation from the two oxygen atoms and an alkene pi bond, using the boron to share electron density. Boronate esters have alkane chains connecting the two oxygen atoms, however, and are therefore non-aromatic. Instead, the boronate esters are useful in coordination chemistry, allowing reagents to tether to one another through boron, causing it to switch bonding geometry. Both of these heterocycles types are useful in Diels-Alder reactions, though they perform opposite roles. Dioxaboroles are used as dienophiles while boronate esters act as dienes, the function dependent on what the boron is attached to. In boronate ester chemistry, because a dienophile such as methyl vinyl ketone coordinates with boron through a lone pair on the ketone, an intramolecular Diels-Alder reaction may then happen between the alkene bond and the furan. Attempts at synthesis of various boroles and boronate esters have so far been unsuccessful.
Abstract: The androgen receptor is a transcription factor with three domains. The carboxy-terminal domain is the androgen-binding domain, which is activated by the binding of one of several select steroid hormones, such as testosterone. The activation of the androgen receptor by the hormone ligand interaction causes the protein to induce the transcription of androgen mediated genes. My project focuses on the conformational changes of the androgen-binding domain upon ligand binding. We have a construct consisting of the androgen-binding domain of the androgen receptor linked with fluorophores on both the N-terminus and C-terminus of the domain. The goal of the project is to obtain the construct in high yield and subsequently use FRET (fluorescence resonance energy transfer) techniques to investigate the conformational changes of the protein in the presence of various synthetic steroid hormone-like and natural steroid hormone ligands.
Abstract: This project consists of the synthesis and analysis of novel molecules that feature a newly proposed binding site for interaction with the Transient Receptor Potential Vanillin Type-1 (TRPV1) Receptor. The human TRPV1 receptor is responsible for transmitting the sensation of pain when triggered by noxious stimuli. These stimuli include capsaicin (the active ingredient in chili peppers), endovanilloids (chemicals released during tissue inflammation and some diseases that cause pain signals to be sent to the brain), low pH and heat (ii). The proposed molecules have been designed to effectively bind to this receptor, blocking the interaction between the noxious stimuli and the TRPV1 receptor without causing a response from the receptor. Molecules that behave in this manner are referred to as antagonists (iii). Through this mechanism, the novel molecules work as analgesics to relieve pain in people who suffer from chronic pain conditions such as osteoarthritis, multiple sclerosis, diabetes, and complications due to cancer. A number of such TRPV1 antagonists have progressed into human clinical studies. A synthetic route, based on the utilization of dimethyldithiocyano-iminocarbonate to facilitate linkage of two large aminosubstituted precursors via a cyanoguanidine group will be used to create these molecules. These antagonists feature the proposed D-region binding site (iv) that is expected to increase affinity to the TRPV1 receptor. After the molecules have been synthesized and purified, their activities will be measured in a nematode bioassay. Results will be compared to previously synthesized antagonists from Dr. French's lab to determine the effectiveness of the D-region binding site. This research will help to build upon recent discoveries in the field of analgesic research and serve as a template for further exploration of TRPV1 antagonists containing the D-region binding site.
i. Szallasi, Arpad. (2003) Journal of Medicinal Chemistry, (47) 11, 2717-2723
ii. Szallasi, Arpad, et al. (2007) Nature, (6) 5, 357-372
iii. Tamayo, Nurai, et al. (2008) Journal of Medicinal Chemistry (51), 2744-2757
iv. Lee, Jeewoo. (2001) Bioorganic and Medicinal Chemistry (9) 19-32
Methods for the Analysis of Isothermal Titration Calorimetry Data
in Drug/DNA Complexes
Isothermal titration calorimetry (ITC) has rapidly become an extremely important tool in studying biomolecular interactions such as ligand/DNA complexes. By directly measuring the heat released or absorbed in a biomolecular binding event, ITC can simultaneously determine binding parameters such as n, K, ΔH and ΔS in a single experiment, effectively obtaining an entire thermodynamic profile of the interaction. As the methods and technology behind ITC have advanced over the last 20 years since the availability of the first commercial instruments, researchers have begun to study ever increasingly complex binding interactions that stretch the method’s capabilities. Some complex systems currently being studied can lead to a myriad of separate interactions with heat effects that obscure binding effects. Interactions such as the heat of a ligand mixing with buffer, buffer with itself, or potential ligand self-aggregation before injection are just a few of the important interactions that can alter the raw data for the experiment; especially in systems with inherently small heat values. Given data for these complex ITC systems, how can we correct for these issues and how can we determine the error within the experiment as a whole? Research by Niklaas J. Buurma and Ihtshamul Haq have shown that it is possible to account for ligand self-aggregation and other equilibria through simulated annealing of the data through novel software, IC-ITC. One possible approach to improved analysis of complex ITC data that will be studied is the use of statistical regression techniques to examine the extra heat effects.
Analysis of the Components of "5 Hour Energy" in a High School Lab
Abstract: Caffeine is a very common and well known compound. It is the perfect compound for high school students to study because of the prior knowledge they already possess. In the experiment that is being developed, students will be able to extract caffeine from 5 Hour Energy (a popular energy drink) using liquid extraction. They will then recrystallize the caffeine and verify the identity using TLC. TLC will also be used to verify the presence of the other ingredients of 5 Hour Energy including amino acids, vitamins, and sugars. Lessons will be developed that allow the students to study the biochemical aspects of each of the ingredients. Students will be able to decide if energy drinks are good for them, based on the information they gain.