Matching Items (18)

Filtering by

Clear all filters

134935-Thumbnail Image.png

Aptamer-functionalized Hydrogel for the Capture and Release of CCRF-CEM Leukemia Cancer Cells

Description

The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a

The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.

Contributors

Agent

Created

Date Created
2016-12

149699-Thumbnail Image.png

Synthesis and evaluation of a new class of cancer chemotherapeutics based on purine-like extended amidines

Description

A potential new class of cancer chemotherapeutic agents has been synthesized by varying the 2 position of a benzimidazole based extended amidine. Compounds 6-amino-2-chloromethyl-4-imino-1-(2-methansulfonoxyethyl)-5-methyl-1H-benzimidazole-7-one (1A) and 6-amino-2-hydroxypropyl-4-imino-1-(2-methansulfonoxyethyl)-5-methyl-1H-benzimidazole-7-one (1B) were assayed at the National Cancer Institute's (NCI) Developmental Therapeutic Program (DTP)

A potential new class of cancer chemotherapeutic agents has been synthesized by varying the 2 position of a benzimidazole based extended amidine. Compounds 6-amino-2-chloromethyl-4-imino-1-(2-methansulfonoxyethyl)-5-methyl-1H-benzimidazole-7-one (1A) and 6-amino-2-hydroxypropyl-4-imino-1-(2-methansulfonoxyethyl)-5-methyl-1H-benzimidazole-7-one (1B) were assayed at the National Cancer Institute's (NCI) Developmental Therapeutic Program (DTP) and found to be cytotoxic at sub-micromolar concentrations, and have shown between a 100 and a 1000-fold increase in specificity towards lung, colon, CNS, and melanoma cell lines. These ATP mimics have been found to correlate with sequestosome 1 (SQSTM1), a protein implicated in drug resistance and cell survival in various cancer cell lines. Using the DTP COMPARE algorithm, compounds 1A and 1B were shown to correlate to each other at 77%, but failed to correlate with other benzimidazole based extended amidines previously synthesized in this laboratory suggesting they operate through a different biological mechanism.

Contributors

Agent

Created

Date Created
2011

152245-Thumbnail Image.png

Novel strategies for producing proteins with non-proteinogenic amino acids

Description

The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using

The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.

Contributors

Agent

Created

Date Created
2013

152086-Thumbnail Image.png

Study of ribosomes having modifications in the peptidyltransferase center using non-alpha-L-amino acids and synthesis and biological evaluation of topopyrones

Description

The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino

The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino acid in response to one codon. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome enabled the incorporation of both alpha-D-amino acids and beta-amino acids into full length protein. Described in Chapter 2 are five modified ribosomes having modifications in the peptidyltrasnferase center in the 23S rRNA. These modified ribosomes successfully incorporated five different beta-amino acids (2.1 - 2.5) into E. coli dihydrofolate reductase (DHFR). The second project (Chapter 3) focused on the study of the modified ribosomes facilitating the incorporation of the dipeptide glycylphenylalanine (3.25) and fluorescent dipeptidomimetic 3.26 into DHFR. These ribosomes also had modifications in the peptidyltransferase center in the 23S rRNA of the 50S ribosomal subunit. The modified DHFRs having beta-amino acids 2.3 and 2.5, dipeptide glycylphenylalanine (3.25) and dipeptidomimetic 3.26 were successfully characterized by the MALDI-MS analysis of the peptide fragments produced by "in-gel" trypsin digestion of the modified proteins. The fluorescent spectra of the dipeptidomimetic 3.26 and modified DHFR having fluorescent dipeptidomimetic 3.26 were also measured. The type I and II DNA topoisomerases have been firmly established as effective molecular targets for many antitumor drugs. A "classical" topoisomerase I or II poison acts by misaligning the free hydroxyl group of the sugar moiety of DNA and preventing the reverse transesterfication reaction to religate DNA. There have been only two classes of compounds, saintopin and topopyrones, reported as dual topoisomerase I and II poisons. Chapter 4 describes the synthesis and biological evaluation of topopyrones. Compound 4.10, employed at 20 µM, was as efficient as 0.5 uM camptothecin, a potent topoisomerase I poison, in stabilizing the covalent binary complex (~30%). When compared with a known topoisomerase II poison, etoposide (at 0.5 uM), topopyorone 4.10 produced similar levels of stabilized DNA-enzyme binary complex (~34%) at 5 uM concentration.

Contributors

Agent

Created

Date Created
2013

152375-Thumbnail Image.png

Bleomycin, from start to finish: total synthesis of novel analogues to in vitro fluorescence microscopy imaging

Description

The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of

The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. Chapter 1 describes bleomycin and related DNA targeting antitumor agents as well as the specific structural domains of bleomycin. Various structural analogues of pyrimidoblamic acid were synthesized and subsequently incorporated into their corresponding full deglycoBLM A6 derivatives by utilizing a solid support. Their activity was measured using a pSP64 DNA plasmid relaxation assay and is summarized in Chapter 2. The specifics of bleomycin—DNA interaction and kinetics were studied via surface plasmon resonance and are presented in Chapter 3. By utilizing carefully selected 64-nucleotide DNA hairpins with variable 16-mer regions whose sequences showed strong binding in past selection studies, a kinetic profile was obtained for several BLMs for the first time since bleomycin was discovered in 1966. The disaccharide moiety of bleomycin has been previously shown to be a specific tumor cell targeting element comprised of L-gulose-D-mannose, especially between MCF-7 (breast cancer cells) and MCF-10A ("normal" breast cells). This phenomenon was further investigated via fluorescence microscopy using multiple cancerous cell lines with matched "normal" counterparts and is fully described in Chapter 4.

Contributors

Agent

Created

Date Created
2013

137623-Thumbnail Image.png

Intelligent Input Parser for Organic Chemistry Reagent Questions

Description

Due to its difficult nature, organic chemistry is receiving much research attention across the nation to develop more efficient and effective means to teach it. As part of that, Dr. Ian Gould at ASU is developing an online organic chemistry

Due to its difficult nature, organic chemistry is receiving much research attention across the nation to develop more efficient and effective means to teach it. As part of that, Dr. Ian Gould at ASU is developing an online organic chemistry educational website that provides help to students, adapts to their responses, and collects data about their performance. This thesis creative project addresses the design and implementation of an input parser for organic chemistry reagent questions, to appear on his website. After students used the form to submit questions throughout the Spring 2013 semester in Dr. Gould's organic chemistry class, the data gathered from their usage was analyzed, and feedback was collected. The feedback obtained from students was positive, and suggested that the input parser accomplished the educational goals that it sought to meet.

Contributors

Agent

Created

Date Created
2013-05

153728-Thumbnail Image.png

Isolation and functional studies of the F-type ATP synthase from spinach chloroplasts and Heliobacterium modesticaldum

Description

Adenosine triphosphate (ATP) is the universal chemical energy currency in most living cells, used to power many cellular reactions and generated by an enzyme supercomplex known as the ATP synthase, consisting of a hydrophilic F1 subcomplex and a membrane-bound FO

Adenosine triphosphate (ATP) is the universal chemical energy currency in most living cells, used to power many cellular reactions and generated by an enzyme supercomplex known as the ATP synthase, consisting of a hydrophilic F1 subcomplex and a membrane-bound FO subcomplex. Driven by the electrochemical gradient generated by the respiratory or photosynthetic electron transport chain, the rotation of the FO domain drives movements of the central stalk in response to conformational changes in the F1 domain, in which the physical energy is converted into chemical energy through the condensation of ADP and Pi to ATP. The exact mechanism how ATP synthesis is coupled to proton translocation is not known as no structure of the intact ATP-synthase nor the intact FO subcomplex has been determined to date. Structural information may shed light on these mechanisms and aid in understanding how structural changed relate to its coupling to ATP synthesis. The work in this thesis has successful established a defined large-scale CF1FO isolation procedure resulting in high purity and high yield of this complex from spinach thylakoid membranes by incorporating a unique combination of biochemical methods will form the basis for the subsequent structural determination of this complex. Isolation began from the isolation of intact chloroplasts and the separation of intact thylakoid membranes. Both native and denaturing electrophoresis analyses clearly demonstrated that the purified CF1FO retains its quaternary structure consisting of the CF1 and CFO subcomplexes and nine subunits (five F1 subunits: α, β, γ, δ and ε, and four FO subunits: a, b, b' and c). Moreover, both ATP synthesis and hydrolysis activities were successfully detected using protein reconstitution in combination with acid-base incubation and in-gel ATPase assays, respectively. Furthermore, the ATP-synthase of H. modesticaldum, an anaerobic photosynthetic bacterium, was also isolated and characterized at the biochemical level. These biochemical characterizations directly influenced recent studies on the high-resolution structure determination of intact CF1FO using electron crystallography on two-dimensional crystals. The availability of the functionally intact CF1FO purified at a large scale will lead to studies that investigate the possible crystallization conditions to ultimately determine its three-dimensional structure at atomic resolution.

Contributors

Agent

Created

Date Created
2015

153337-Thumbnail Image.png

Studying biomolecular structures and their interaction using atomic force microscopy

Description

Atomic force microscopy (AFM) has become an important tool to characterize and image surfaces with nanoscale resolution. AFM imaging technique has been utilized to study a wide range of substances such as DNA, proteins, cells, silicon surfaces, nanowires etc. Hence

Atomic force microscopy (AFM) has become an important tool to characterize and image surfaces with nanoscale resolution. AFM imaging technique has been utilized to study a wide range of substances such as DNA, proteins, cells, silicon surfaces, nanowires etc. Hence AFM has become extremely important in the field of biochemistry, cell biology and material science. Functionalizing the AFM tip made it possible to detect molecules and their interaction using recognition imaging at single molecule level. Also the unbinding force of two molecules can be investigated based on AFM based single molecule force spectroscopy.

In the first study, a new chemical approach to functionalize the AFM tip in a simple and user-friendly way has been described. Copper-free click chemistry and a vinyl sulfone PEG linker have been utilized during the process. Using this technique, human thrombin and integrin were detected in separate experiments. Then a novel tri-arm linker with two recognition molecules on it was designed and two proteins (human thrombin and integrin) were detected simultaneously in the same experiment using recognition imaging. This technique can be applied to understand many multivalent interactions taking place in nature. Using the same tri-arm linker functionalized with two biotin molecules, the interaction of streptavidin with mono-biotin and bis-biotin ligands were investigated. The thermal stability of streptavidin-biotin complex was also studied using SDS-PAGE analysis.

In the final study, structure of native chromatin extracted from normal and cancer cell lines were analyzed using AFM imaging and agarose gel electrophoresis. Different salt fractions were used to extract chromatin region depending on their solubility. Mnase sensitivity of the chromatin sample was used to understand the open and closed structures of chromatin from different sources. The amount of chromatin in different salt fractions could act as an indicator of amount of open and condensed chromatin in normal and cancer cells. Eventually this ratio of closed and open structure of chromatin could be an indicator of tumorigenic nature of particular cell lines.

Contributors

Agent

Created

Date Created
2015

153396-Thumbnail Image.png

Self-assembly of complex DNA nanostructures and reconfigurable DNA devices

Description

Deoxyribonucleic acid (DNA) has emerged as an excellent molecular building block for nanoconstruction in addition to its biological role of preserving genetic information. Its unique features such as predictable conformation and programmable intra- and inter-molecular Watson-Crick base pairing interactions make

Deoxyribonucleic acid (DNA) has emerged as an excellent molecular building block for nanoconstruction in addition to its biological role of preserving genetic information. Its unique features such as predictable conformation and programmable intra- and inter-molecular Watson-Crick base pairing interactions make it a remarkable engineering material. A variety of convenient design rules and reliable assembly methods have been developed to engineer DNA nanostructures. The ability to create designer DNA architectures with accurate spatial control has allowed researchers to explore novel applications in directed material assembly, structural biology, biocatalysis, DNA

computing, nano-robotics, disease diagnosis, and drug delivery.

This dissertation focuses on developing the structural design rules for "static" DNA nano-architectures with increasing complexity. By using a modular self-assembly method, Archimedean tilings were achieved by association of different DNA motifs with designed arm lengths and inter-tile sticky end interactions. By employing DNA origami method, a new set of design rules was created to allow the scaffolds to travel in arbitrary directions in a designed geometry without local symmetry restrictions. Sophisticated wireframe structures of higher-order complexity were designed and constructed successfully. This dissertation also presents the use of "dynamic" DNA nanotechnology to construct DNA origami nanostructures with programmed reconfigurations.

Contributors

Agent

Created

Date Created
2015

151401-Thumbnail Image.png

DNA directed self-assembly of plasmonic nanoparticles

Description

Deoxyribonucleic acid (DNA), a biopolymer well known for its role in preserving genetic information in biology, is now drawing great deal of interest from material scientists. Ease of synthesis, predictable molecular recognition via Watson-Crick base pairing, vast numbers of available

Deoxyribonucleic acid (DNA), a biopolymer well known for its role in preserving genetic information in biology, is now drawing great deal of interest from material scientists. Ease of synthesis, predictable molecular recognition via Watson-Crick base pairing, vast numbers of available chemical modifications, and intrinsic nanoscale size makes DNA a suitable material for the construction of a plethora of nanostructures that can be used as scaffold to organize functional molecules with nanometer precision. This dissertation focuses on DNA-directed organization of metallic nanoparticles into well-defined, discrete structures and using them to study photonic interaction between fluorophore and metal particle. Presented here are a series of studies toward this goal. First, a novel and robust strategy of DNA functionalized silver nanoparticles (AgNPs) was developed and DNA functionalized AgNPs were employed for the organization of discrete well-defined dimeric and trimeric structures using a DNA triangular origami scaffold. Assembly of 1:1 silver nanoparticle and gold nanoparticle heterodimer has also been demonstrated using the same approach. Next, the triangular origami structures were used to co-assemble gold nanoparticles (AuNPs) and fluorophores to study the distance dependent and nanogap dependencies of the photonic interactions between them. These interactions were found to be consistent with the full electrodynamic simulations. Further, a gold nanorod (AuNR), an anisotropic nanoparticle was assembled into well-defined dimeric structures with predefined inter-rod angles. These dimeric structures exhibited unique optical properties compared to single AuNR that was consistent with the theoretical calculations. Fabrication of otherwise difficult to achieve 1:1 AuNP- AuNR hetero dimer, where the AuNP can be selectively placed at the end-on or side-on positions of anisotropic AuNR has also been shown. Finally, a click chemistry based approach was developed to organize sugar modified DNA on a particular arm of a DNA origami triangle and used them for site-selective immobilization of small AgNPs.

Contributors

Agent

Created

Date Created
2012