Category Archives: Defenses

ON THE PROTECTIVE PROPERTIES OF GLYCINE BASED OSMOLYTES IN A THIOL REDUCING ENVIRONMENT

Michigan Technological University
Department of Chemistry
John Hausman
Advisor: Dr. Ashutosh Tiwari
Master’s Defense
Monday August 10, 2015
9:30 am, Chem-Sci 101

ON THE PROTECTIVE PROPERTIES OF GLYCINE BASED OSMOLYTES IN A THIOL REDUCING ENVIRONMENT

The protective properties of osmolytes have been studied intently for decades. Originally used to aid in the crystallization of proteins in x-ray diffraction studies, these cosolvents have been shown to reverse protein denaturation and aggregation. Osmolytes aid extremophiles in surviving harsh environments by preferentially excluding themselves from the surface of the protein, thus directing water molecules to the protein’s surface. Their use is increasing and affects the everyday consumer as these osmolytes are naturally found in many health foods and also in many daily use products such as shampoo. Consumers also supplement their diets with thiol-based antioxidants such as glutathione as part of healthier life style. However, thiol-based antioxidants in high concentration have been known to cause un-intended health issues that relate to its disulfide reducing property. The disulfide-reducing agent can cleave the proteins’ disulfide-bonds and promote misfolding and aggregation. In this thesis, we investigated the influence of commonly consumed glycine-based osmolytes on providing stability to proteins against a disulfide-reducing agent. We chose glycine, sarcosine (N-methyl glycine), di-methyl glycine (DMG), and betaine (N,N,N-trimethylglycine) with increasing number of methyl groups. An added benefit to studying this class of osmolytes is to also investigate the impact N-methyl substitution has on the osmolytes’ protective properties. We studied the effect of these osmolytes on protein aggregation using spectroscopic techniques such as UV-visible absorbance, intrinsic fluorescence and extrinsic fluorescence measurements. In addition, we carried out non-reducing SDS-PAGE to check for higher order aggregates and characterized morphology of these aggregates using scanning electron microscopy. Overall, our results show that of all the osmolytes used, glycine was the best stabilizer followed by sarcosine. Betaine and dimethylglycine did not provide effective protection against disulfide-reducing influence.


Gold Nanorod-based Theranostics for Specific Cancer Imaging and Therapy

Jianheng Bi
Original Research Proposal
Michigan Technological University
Department of Chemistry
Advisor- Dr. Haiying Liu
Tuesday August 4, 2015
9:00 am
Chem-Sci 101

ABSTRACT
This proposal describes the preparation, characterization and application of gold nanorod-based theranostics for specific cancer imaging and therapy. Cancer theranostics will be constructed by modifying the gold nanorods with near-infrared dyes and cancer-imaging peptides via silica layers and poly(ethylene glycol) tethered spacers. The thickness of the silica layers will be carefully controlled to significantly enhance the fluorescence intensity of near-infrared dyes via a surface plasma of gold nanrods while specific targeting and imaging will be achieved through strong specific multivalent interactions of cancer-homing peptide residues with cancer cells and tissues. Once specific targeting to cancer cells or tissues are achieved, thermotherapy and photodynamic therapy will be conducted to kill cancer cells and tissues via thermal heat from gold nanorods and singlet oxygen generated by photodynamic agent under near-infrared pulse laser.


Xu Xiang
PhD Defense
Doctoral Candidate, Department of Chemistry
Advisor: Dr. Patrica Heiden

Friday July 24, 2015
Memorial Union Ballroom A-1
9:00 a.m.

Novel Drug Delivery Systems: Thermo-responsive drug delivery designs and Multi-drug delivery scaffold designs

Abstract:
This research has three parts: two deal with novel medical devices for drug delivery, and one looks at properties of fish scales as an abundant waste resource that can be modified to have value in medical and other areas. This presentation discusses two novel drug delivery designs, including their synthesis and characterization. These nanoparticle devices address two current challenges in drug delivery. The first is designing a drug-carrier to more efficiently deliver toxic (e.g. chemotherapy) drugs, which harm all cells. So non-specific delivery of the drug is wasteful and harmful to the patient. Knowledge gained from a fundamental study of the effects of polymer architecture (linear amphiphilic diblock and triblock copolymers and star copolymers) and end group polarity, on thermally triggered drug delivery led to selecting two diblock copolymers to assemble into novel gold-core hybrid nanoparticles. These nanoparticles were tested for their ability to reduce low temperature release and efficiently release the drug at a triggered temperature. The second drug delivery system extended the capabilities of a new type of tissue support device (a scaffold) by showing that the system can independently control the simultaneous release of different drugs, and that the spatial distribution of these drugs within the device, called a “nanoparticle fiber”, can be controlled. Three different model drugs were assembled and their release proven, within the nanoparticle fiber. This work succeeded in yielding a novel, flexible, and robust system of assembled fibers of nanoparticles that could independently control the release of multiple drugs.


Total Synthesis of Belizentrin

Total Synthesis of Belizentrin

Shahien Shahsavari
Original Research Proposal
Michigan Technological University
Department of Chemistry

Advisor- Dr. Shiyue Fang
Monday July 6, 2015
10:00 am
Chem-Sci 101

Abstract

Marine organisms are known to produce some of the most bioactive secondary metabolites found in nature. Currently, many of these marine-derived molecules are used as “first-in-class” drugs in the market. This proposal describes the total synthesis of belizentrin, a novel polyketide-derived macrocycle isolated from the marine dinoflagellate Prorocentrum belizeanum. Belizentrin has already demonstrated effective changes in neuronal survival assays in vitro at nanomolar concentrations. Additionally, its highly bioactive properties and structural similarities with other known immunosuppressant compounds make it a valuable target for medical research. However, since the extraction yield is minimal, it is essential to produce this compound in larger quantities from readily available starting materials for a complete investigation of its biological activities. The overall synthetic strategy involves a convergent late-stage coupling of three major fragments. The key features of the synthesis include iterative asymmetric allylic substitutions, titanium-catalyzed asymmetric epoxidation of terminal olefins, and the Yamaguchi macrolactonization to form the macrocyclic core of the molecule.


T Synthesis of Isobractatin and Neobractatin

Shanshan Hou
Original Research Proposal
Michigan Technological University
Department of Chemistry
Advisor- Dr. Lanrong Bi
Tuesday April 21, 2015
9:00 am
Chem-Sci 106

T Synthesis of Isobractatin and Neobractatin

Abstract
The new Garcinia derivatives, neobractatin and isobractatin, will be synthesized. These compounds are caged-prenylxanthones which are isolated from Chinese native Garcinia bracteata. They displayed strong inhibition of cancer cells, which could significantly induce cell apoptosis and inhibit autophagy. However, the compounds which are extracted from the Chinese native Garcinia species have very low yield. Total synthesis of these derivatives will provide adequate quantities for biological studies. In addition, the new synthetic methodologies could not only produce the target compounds but also give rise to their isomers which could be potential anti-tumor candidates and used for future research.


Synthesis of Lipodiscamide A

Ashok Khanal
Original Research Proposal
Michigan Technological University
Department of Chemistry
Advisor: Dr. Shiyue Fang
Thursday, March 5, 2015
10:00 am Chem Sci ~ Room 211

Synthesis of Lipodiscamide A

Abstract:
Lipodiscamide A is a cytotoxic lipopeptide extracted from the marine sponge Discodermia kiiensis. Research on lipodiscamide A has been fueled by its properties such as cytotoxicity, and antimicrobial and immunosuppressive activities. In this proposal, the total synthesis of lipodiscamide A will be described. Structurally, lipodiscamide A contains an unsaturated fatty acid moiety and a hexapeptide moiety. The two moieties are linked together with two ester bonds to form a macrocycle. The yield of this compound from natural sources is very low. Therefore, its synthesis from readily available starting materials resulting in large amounts of lipodiscamide A is important for full investigation of its biological activities.


Synthesis of Taiwaniadducts I and J

Xin Yan
Original Research Proposal
Michigan Technological University
Department of Chemistry
Advisor-Dr. Lanrong Bi
Thursday, February 26, 2015
9:00 am Admin Building Room 404
Abstract
The Taiwaniadducts I and J, as cycloadducts of terpene quinones from taiwania cryptomerioides, will be synthesized. These two compounds are isolated from Taiwania cryptomerioides hayata, a large coniferous evergreen species common to Taiwan. They have potential in medical applications due to the breadth of their biological activities. However, low yields of these naturally extracted compounds limits further research on their biological activities and no synthetic approaches to these two compounds have been reported before. Herein, we design a synthetic route which can provide adequate quantities of target compounds for further biological studies. Moreover, the design methodology involved in this proposal may show a reasonable approach to the synthesis of this kind of cycloadducts.


Assessing the Binding Capabilities of Bromodomain-Containing Protein 9

Sarah Hopson (Advisor- Dr. Martin Thompson)

Doctoral Student, Department of Chemistry,Michigan Technological University

Monday, March 2, 2015-9:00 am- Admin 404

Abstract

Post-translational modifications of histones, such as the acetylation of lysines, play an importantrole in regulating transcription. Histone tails have a large proportion of positively-charged residues, which create electrostatic interactions with the negatively-charged DNA backbone. Lysine acetylation is thought to weaken these interactions, because it neutralizes lysine’s positively-charged side chain.

Proteins recognize the acetylated lysines using bromodomains; bromodomains are acetylated lysine “readers” and play a critical role in modulation of gene expression. Of the 46 bromodomain-containing proteins in the human proteome, 15 function as transcriptional regulators and 8 function as chromatin remodelers. Nearly all of the other bromodomain proteins influence transcription in some manner (histone acetyltransferase, transcription repressor, transcription initiation, etc.). Due to their significant influence on transcription, mutations of bromodomains are often linked with cancers.

Bromodomain-containing protein 9 (BRD9) has not yet been studied. The aim of this proposed research is to determine the specificity and affinity of BRD9 toward acetyl-lysine sites on the tails of the four core histone proteins.

A high-throughput examination of possible histone interactions with the bromodomain of BRD9 will be conducted using a modified SPOT array. The peptides demonstrating the strongest interactions with the bromodomain will be synthesized using standard Fmoc peptide synthesis. A quantitative examination of the binding affinities of these peptides to the bromodomain, the bromodomain and DUF3512 (domain of unknown function), and the full length BRD9 will be conducted using isothermal titration calorimetry. The results will be compared to determine how the surrounding amino acid sequences affect the bromodomain’s binding capabilities.


NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIOOLIGOMERS

Durga Pokharel

Advisor: Dr. Shiyue Fang

Doctoral candidate, Department of Chemistry

PhD Defense

Friday December 12, 2014   9:30am     Fisher 130

 

NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIOOLIGOMERS

Abstract

 

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications.

To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The savings with the new techniques compared with HPLC are estimated to be 70% to 90% depending on purification scale and throughput. We expect these new oligonucleotide and peptide purification technologies to be widely used in academic research labs, biotechnology companies, and pharmaceutical companies in the near future.


Purification and Carbohydrate Binding Properties of Two New Plant Proteins

Mr. Robert K Brown

Advisor: Dr. Tarun K Dam

Master’s Candidate Department of Chemistry

Michigan Technological University

“Purification and Carbohydrate Binding Properties of Two New Plant Proteins”

 

Friday, December 12, 2014

10:00 – 11:00 AM 

Room 404 ~ Administration Building
Purification and Carbohydrate Binding Properties of Two New Plant Proteins

Abstract:

 

Protein glycosylation is an important post-translational modification for many biological processes such as cell recognition, intercellular communication and cell death. Proteins that are able to bind to glycosylated proteins via carbohydrates are called lectins. Hemolytic lectins are proteins or glycoproteins that undergo specific interactions with cell surface carbohydrates and subsequently induce cellular lysis. They are termed “hemo” lytic because of their ability to lyse erythrocytes. We have isolated a novel hemolytic lectin named HelyX from the bulbs of a monocot plant, as well as a mannose-binding lectin named DIL from a separate monocot species. HelyX is a uniquely robust hemolytic lectin. It shows concentration dependent reversible hemolytic/agglutinating properties against both human and rabbit erythrocytes. The activity was found to be carbohydrate dependent. HelyX was isolated using ammonium sulfate precipitation, size exclusion chromatography, and analyzed by gel electrophoresis. DIL was purified using a modified version of a newly developed protocol. DIL interacts with the plant enzyme invertase with high affinity. This high affinity interaction suggests that the binding site of DIL is complimentary to glycoproteins containing larger high mannose glycans. Invertase is central to plant metabolism and defense. Therefore DIL might play a modulatory role in plant metabolism and defense through its interaction with invertase. HelyX and DIL did not show lytic activity on free living amoeba, Acanthamoebae. Instead the lectins promoted cyst formation of amoeabae trophozoites indicating a lectin-mediated rearrangement of membrane architecture. This result indicates that the lytic activity of HelyX or DIL depends on the macromolecular landscape of the cell membrane.