View All Items

Title: DESIGN, CONSTRUCTION, AND CHARACTERIZATION OF THE YSGR MINIMAL CODON FAB LIBRARY FOR CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY.
Creator: Holmes, Sean, Ye, Jingdong, University of Central Florida
Abstract / Description: Of the entire human genome, 90% of all genetic information is transcribed but only a fraction of that subsequent RNA is translated into proteins. RNAs which are not translated into proteins are deemed non-coding RNAs. Little is known about this large category of noncoding RNAs, although they perform a variety of functions within the cell. RNA crystallography is used to study RNA tertiary structure, which gives insight to the function of these non-coding RNAs. However, complications associated...
Show moreOf the entire human genome, 90% of all genetic information is transcribed but only a fraction of that subsequent RNA is translated into proteins. RNAs which are not translated into proteins are deemed non-coding RNAs. Little is known about this large category of noncoding RNAs, although they perform a variety of functions within the cell. RNA crystallography is used to study RNA tertiary structure, which gives insight to the function of these non-coding RNAs. However, complications associated with RNA crystallography arise due to RNA's lack of surface functional group diversity, flexible tertiary structure, and conformational heterogeneity. A novel technique, Chaperone-assisted RNA crystallography (CARC), can greatly improve the success in crystallization of RNA. With this technique, synthetic antibodies called Antigen Binding Fragments (Fabs) are employed as crystallization chaperones to promote the structure elucidation of certain target RNAs. To identify Fabs that complex with RNA molecules of interest, we constructed a randomized library of synthetic antibodies enriched in ligand binding regions with tyrosine, serine, glycine, and arginine residues. This Fab protein library was constructed with a minimal codon design, and then screened against a variety of RNA targets. Several Fabs have been identified and isolated through phage display selection against three RNA targets. These Fabs were expressed and biochemically characterized for their binding affinities and specificities. To date, five Fabs have been identified and they have outstanding capabilities in binding their specific RNA antigen.
Show less
Date Issued: 2012
Identifier: CFH0004167, ucf:44856
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004167

Title: SURFACE ENTROPY REDUCTION TO INCREASE THE CRYSTALLIZABILITY OF THE FAB-RNA COMPLEX.
Creator: P.Ravindran, Priyadarshini, Ye, Jingdong, University of Central Florida
Abstract / Description: Crystallizing RNA has been an imperative facet and a challenging task in the world of RNA research. Assistive methods such as Chaperone Assisted RNA Crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by increasing the crystal contacts and providing initial phasing information. Using this technology the crystal structure of ΔC209 P4-P6 RNA (an independent folding domain of the self-splicing...
Show moreCrystallizing RNA has been an imperative facet and a challenging task in the world of RNA research. Assistive methods such as Chaperone Assisted RNA Crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by increasing the crystal contacts and providing initial phasing information. Using this technology the crystal structure of ΔC209 P4-P6 RNA (an independent folding domain of the self-splicing Tetrahymena group I intron) complexed to Fab2 (high affinity binding Fab) has been resolved to 1.95 Å. Although the complexed class I ligase ribozyme has also been crystallized using CARC, in practice, it has been found that the crystallization of, large RNA-Fab complex remains a confrontation. The possible reason for this difficulty is that Fabs have not been optimized for crystallization when complexed with RNA. Here we have used the Surface Entropy Reduction technique (SER) for the optimization process. Candidate residues for mutations were identified based on combining results from visual inspection of ΔC209 P4-P6/Fab2 crystal structure complex using pyMOL software and a web- based SER software. The protruding lysine and glutamate residues were mutated to a set of alanine (Super Mutant Alanine SMA) and serine (Super Mutant Serine SMS) mutant clones. Filter binding assay studies confirmed that the mutant clones bind to ΔC209 P4-P6 with similar binding affinities as that of the parent Fab2. Large scale expression of the mutants, parent clone and ΔC209 P4-P6 RNA were optimised. Crystal trays for ΔC209 P4-P6 complexed with Fab2, Fab2SMA and Fab2SMS were set-up side-by-side using Hampton crystal screen kits and ~600 conditions including temperature as a variable condition were screened. Crystal screening shows significantly higher crystal-forming ratios for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can be potentially applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of the Fab-RNA complex.
Show less
Date Issued: 2011
Identifier: CFE0003944, ucf:48711
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0003944

Title: Preparation, Characterization, and Delivery of Antibodies Binding to a Model Oncogenic RNA, Human Initiator tRNA.
Creator: Archer, Jennifer, Santra, Swadeshmukul, Ye, Jingdong, Ye, Jingdong, Self, William, Khaled, Annette, University of Central Florida
Abstract / Description: Non-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for...
Show moreNon-coding RNAs (ncRNAs) account for a higher percent of the genome than coding mRNAs, and are implicated in human disease such as cancer, neurological, cardiac and many others. While the majority of ncRNAs involved in disease were originally attributed to a class of RNAs called micro RNAs (miRNAs) with a small size of only about 19 -24 base pairs, emerging research has now demonstrated a class of long non-coding RNAs (lncRNAs) that have a size of over 200 base pairs to be responsible for gene regulation and other functional roles and have also found to contribute to pathogenesis in humans. The increased size and structural complexity require novel tools to study their interactions beyond RNA interference. Synthetic antibodies are classic tools and therapeutics utilized to study and treat proteins involved in human disease. Likewise we hypothesize that structured RNAs can also take advantage of synthetic antibodies to probe their functions and be utilized as therapeutics.Currently, antibodies have been raised against microbial riboswitches and other structured RNAs of single-celled organisms, and only one human structured RNA to the best of our knowledge. However, no one has yet to create a synthetic antibody capable of behaving as a therapeutic against a structured RNA. We therefore sought to raise an antibody Fab against a structured RNA, human initiator tRNA, a model oncogenic non-coding RNA and demonstrate its efficacy in vitro. We then characterized the antibody and explored delivery options in cancer cells including the use of nanoparticle delivery systems. With the emerging transcriptome revealing new ncRNAs implicated in human disease, our research has begun to address a new therapeutic strategy, laying down the foundation for the future of structured RNA-targeted therapies.
Show less
Date Issued: 2014
Identifier: CFE0005756, ucf:50072
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005756

Title: A Binary Approach for Selective Recognition of Nucleic Acids and Proteins.
Creator: Cornett, Evan, Kolpashchikov, Dmitry, Self, William, Ye, Jingdong, University of Central Florida
Abstract / Description: The design of probes for the selective recognition of biopolymers (nucleic acids and proteins) is a fundamental task for studying, diagnosing, and treating diseases. Traditional methods utilize a single component (small molecule or oligonucleotide) that binds directly to the target biopolymer. However, many biopolymers are unable to be targeted with this approach. The overarching goal of this dissertation is to explore a new, binary approach for designing probes. The binary approach requires...
Show moreThe design of probes for the selective recognition of biopolymers (nucleic acids and proteins) is a fundamental task for studying, diagnosing, and treating diseases. Traditional methods utilize a single component (small molecule or oligonucleotide) that binds directly to the target biopolymer. However, many biopolymers are unable to be targeted with this approach. The overarching goal of this dissertation is to explore a new, binary approach for designing probes. The binary approach requires two components that cooperatively bind to the target, triggering a recognition event. The requisite binding of two-components allows the probes to have excellent selectivity and modularity.The binary approach was applied to design a new sensor, called operating cooperatively (OC) sensor, for recognition of nucleic acids, including selectively differentiating between single nucleotide polymorphisms (SNPs). An OC sensor contains two oligonucleotide probe strands, called O and C, each with two domains. The first domain contains a target recognition sequence, whereas the second domain is complementary to a molecular beacon (MB) probe. Binding of both probe strands to the fully matched analyte generates a full MB probe recognition site, allowing a MB to bind and report the presence of the target analyte. Importantly, we show that the OC sensor selectively discriminates between single nucleotide polymorphisms (SNPs) in DNA and RNA targets at room temperature, including those with stable secondary structures. Furthermore, the combinatorial use of OC sensors to create a DNA logic gate capable of analyzing DNA sequences of Mycobacterium tuberculosis is described.The binary approach was also applied to design covalent inhibitors for HIV-1 reverse transcriptase (RT). In this application, two separate pre-reactive groups were attached to a natural RT ligand, deoxythymidine triphosphate (dTTP). Upon binding of both dTTP analogs in the RT active site, the pre-reactive groups are brought into the proper proximity and react with each other forming an intermediate that subsequently reacts with an amino acid side chain from the RT. This leads to covalent modification of RT, and inhibition of its DNA polymerase activity. This concept was tested in vitro using dTTP analogs containing pre-reactive groups derived from ?-lactamase inhibitors clavulanic acid (CA) and sulbactam (SB). Importantly, our in vitro assays show that CA based inhibitors are more potent than zidovudine (AZT), a representative of the dominant class of RT inhibitors currently used in anti-HIV therapy. Furthermore, molecular dynamics simulations predict that complexes of RT with these analogs are stable, and point to possible reaction mechanisms. The inhibitors described in this work may serve as the basis for the development of the first covalent inhibitors for RT. Moreover, the pre-reactive groups used in this study can be used to design covalent inhibitors for other targets by attaching them to different ligands. Overall, the work presented herein establishes the binary approach as a straightforward way to develop new probes to selectively recognize nucleic acids and proteins.
Show less
Date Issued: 2015
Identifier: CFE0006031, ucf:51010
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006031

Title: Development of Micro Volume DNA and RNA Profiling Assays to Identify the Donor and Tissue Source of Origin of Trace Forensic Biological Evidence.
Creator: Morgan, Brittany, Ballantyne, John, Kolpashchikov, Dmitry, Ye, Jingdong, University of Central Florida
Abstract / Description: In forensic casework analysis it is necessary to obtain genetic profiles from increasingly smaller amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is demonstrated with so-called 'touch DNA evidence' which is perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. However, the current method of recovery of trace DNA involves cotton...
Show moreIn forensic casework analysis it is necessary to obtain genetic profiles from increasingly smaller amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is demonstrated with so-called 'touch DNA evidence' which is perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. However, the current method of recovery of trace DNA involves cotton swabs or adhesive tape to sample an area of interest. This (")blind-swabbing(") approach may result in the recovery of biological material from different individuals resulting in admixed DNA profiles which are often difficult to interpret. Profiles recovered from these samples are reported to be from shed skin cells with no biological basis for that determination. A specialized approach for the isolation of single or few cells from 'touch DNA evidence' is necessary to improve the analysis and interpretation of recovered profiles. Here we describe the development of optimized and robust micro volume PCR reactions (1-5 uL) to improve the sensitivity and efficiency of 'touch DNA' analysis. These methods will permit not only the recovery of the genetic profile of the donor of the biological material, but permit an identification of the tissue source of origin using mRNA profiling. Results showed that the 3.5 uL amplification volume, a fraction of the standard 25 uL amplification volume, was the most ideal volume for the DNA assay, as it had very minimal evaporation with a 50% profile recovery rate at a single cell equivalent input (~5 pg) with reducing amplification volume alone. Findings for RNA showed that by reducing both amplification steps, reverse transcriptase PCR (20 uL) and body fluid multiplex PCR (25 uL), to 5 uL, ideal results were obtained with an increase in sensitivity and detection of six different body fluids down to 50 pg. Once optimized at the trace level, the assays were applied to the collection of single and few cells. DNA findings showed that about 40% of a full profile could be recovered from a single buccal cell, with nearly 80% of a full profile recovered from only two cells. RNA findings from collected skin particles of (")touched(") surfaces showed accurate skin detection down to 25 particles and detection in one clump of particles. The profiles recovered were of high quality and similar results were able to be replicated through subsequent experiments. More studies are currently underway to optimize these developed assays to increase profile recovery at the single cell level. Methods of doing so include comparing different locations on touched surfaces for highest bio-particle recovery and the development of physical characteristics of bio-particles that would provide the most ideal results.
Show less
Date Issued: 2013
Identifier: CFE0005385, ucf:50468
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005385

Title: Self-assembly of Amyloid Aggregates Simulated with Molecular Dynamics.
Creator: Berhanu, Workalemahu, Masunov, Artem, Kolpashchikov, Dmitry, Ye, Jingdong, Zou, Shengli, Schulte, Alfons, University of Central Florida
Abstract / Description: ABSTRACTAmyloids are highly ordered cross-? sheet aggregates that are associated with many diseases such as Alzheimer's, type II diabetes and prion diseases. Recently a progress has been made in structure elucidation, environmental effects and thermodynamic properties of amyloid aggregates. However, detailed understanding of how mutation, packing polymorphism and small organic molecules influence amyloid structure and dynamics is still lacking. Atomistic modeling of these phenomena with...
Show moreABSTRACTAmyloids are highly ordered cross-? sheet aggregates that are associated with many diseases such as Alzheimer's, type II diabetes and prion diseases. Recently a progress has been made in structure elucidation, environmental effects and thermodynamic properties of amyloid aggregates. However, detailed understanding of how mutation, packing polymorphism and small organic molecules influence amyloid structure and dynamics is still lacking. Atomistic modeling of these phenomena with molecular dynamics (MD) simulations holds a great promise to bridge this gap. This Thesis describes the results of MD simulations, which provide insight into the effects of mutation, packing polymorphism and molecular inhibitors on amyloid peptides aggregation. Chapter 1 discusses the structure of amyloid peptides, diseases associated with amyloid aggregation, mechanism of aggregation and strategies to treat amyloid diseases. Chapter 2 describes the basic principles of molecular dynamic simulation and methods of trajectory analysis used in the Thesis. Chapter 3 presents the results of the study of several all-atom molecular dynamics simulations with explicit solvent, starting from the crystalline fragments of two to ten monomers each. Three different hexapeptides and their analogs produced with single glycine replacement were investigated to study the structural stability, aggregation behavior and thermodynamics of the amyloid oligomers. Chapter 4 presents multiple molecular dynamics (MD) simulation of a pair polymorphic form of five short segments of amyloid peptide. Chapter 5 describes MD study of single-layer oligomers of the full-length insulin with a goal to identify the structural elements that are important for insulin amyloid stability, and to suggest single glycine mutants that may improve formulation. Chapter 6 presents the investigation of the mechanism of the interaction of polyphenols molecules with the protofibrils formed by an amyloidogenic hexapeptide fragment (VQIVYK) of Tau peptide by molecular dynamics simulations in explicit solvent. We analyzed the trajectories of the large (7(&)#215;4) aggregate with and without the polyphenols.Our MD simulations for both the short and full length amyloids revealed adding strands enhances the internal stability of wildtype aggregates. The degree of structural similarity between the oligomers in simulation and the fibril models constructed based on experimental data may explain why adding oligomers shortens the experimentally observed nucleation lag phase of amyloid aggregation. The MM-PBSA free energy calculation revealed nonpolar components of the free energy is more favorable while electrostatic solvation is unfavorable for the sheet to sheet interaction. This explains the acceleration of aggregation by adding nonpolar co-solvents (methanol, tri?uoroethanol, and hexa?uoroisopropanol). Free energy decomposition shows residues situated at the interface were found to make favorable contribution to the peptide -peptide association.The results from the simulations might provide both the valuable insight for amyloid aggregation as well as assist in inhibitor design efforts. First, the simulation of the single glycine mutants at the steric zipper of the short segments of various pathological peptides indicates the intersheet steric zipper is important for amyloid stability. Mutation of the side chains at the dry steric zipper disrupts the sheet to sheet packing, making the aggregation unstable. Thus, designing new peptidomimetic inhibitors able to prevent the fibril formation based on the steric zipper motif of the oligomers, similar to the ones examined in this study may become a viable therapeutic strategy. The various steric zipper microcrystal structures of short amyloid segments could be used as a template to design aggregation inhibitor that can block growth of the aggregates. Modification of the steric zipper structure (structure based design) with a single amino acid changes, shuffling the sequences, N- methylation of peptide amide bonds to suppress hydrogen bonding ability of NH groups or replacement with D amino acid sequence that interact with the parent steric zipper could be used in computational search for the new inhibitors. Second, the polyphenols were found to interact with performed oligomer through hydrogen bonding and induce conformational change creating an altered aggregate. The conformational change disrupts the intermolecular amyloid contact remodeling the amyloid aggregate. The recently reported microcrystal structure of short segments of amyloid peptides with small organic molecules could serve as a pharamcophore for virtual screening of aggregation inhibitor using combined docking and MD simulation with possible enhancement of lead enrichment. Finally, our MD simulation of short segments of amyloids with steric zipper polymorphism showed the stability depends on both sequence and packing arrangements. The hydrophilic polar GNNQQNY and NNQNTF with interface containing large polar and/or aromatic side chains (Q/N) are more stable than steric zipper interfaces made of small or hydrophobic residues (SSTNVG, VQIVYK, and MVGGVV). The larger sheet to sheet interface of the dry steric zipper through polar Q/N rich side chains was found to holds the sheets together better than non Q/N rich short amyloid segments. The packing polymorphism could influence the structure based design of aggregation inhibitor and a combination of different aggregation inhibitors might be required to bind to various morphologic forms of the amyloid peptides.
Show less
Date Issued: 2011
Identifier: CFE0004088, ucf:49131
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004088

Title: Photoactivatable Organic and Inorganic Nanoparticles in Cancer Therapeutics and Biosensing.
Creator: Mathew, Mona, Gesquiere, Andre, Hickman, James, Ye, Jingdong, Campiglia, Andres, Schoenfeld, Winston, University of Central Florida
Abstract / Description: In photodynamic therapy a photosensitizer drug is administered and is irradiated with light. Upon absorption of light the photosensitizer goes into its triplet state and transfers energy or an electron to oxygen to form reactive oxygen species (ROS). These ROS react with biomolecules in cells leading to cell damage and cell death. PDT has interested many researchers because of its non-invasiveness as compared to surgery, it leaves little to no scars, it is time and cost effective, it has...
Show moreIn photodynamic therapy a photosensitizer drug is administered and is irradiated with light. Upon absorption of light the photosensitizer goes into its triplet state and transfers energy or an electron to oxygen to form reactive oxygen species (ROS). These ROS react with biomolecules in cells leading to cell damage and cell death. PDT has interested many researchers because of its non-invasiveness as compared to surgery, it leaves little to no scars, it is time and cost effective, it has potential for targeted treatment, and can be repeated as needed. Different photosensitizers such as porphyrines, chlorophylls, and dyes have been used in PDT to treat various cancers, skin diseases, aging and sun-damaged skin. These second generation sensitizers have yielded reduced skin sensitivity and improved extinction coefficients (up to ~ 105 L mol-1 cm-1). While PDT based on small molecule photosensitizers has shown great promise, several problems remain unsolved. The main issues with current sensitizers are (i) hydrophobicity leading to aggregation in aqueous media resulting in reduced efficacy and potential toxicity, (ii) dark toxicity of photosensitizers, (iii) non-selectivity towards malignant tissue resulting in prolonged cutaneous photosensitivity and damage to healthy tissue, (iv) limited light absorption efficiency, and (v) a lack of understanding of where the photosensitizer ends up in the tissue. In this dissertation research program, these issues were addressed by the development of conducting polymer nanoparticles as a next generation of photosensitizers. This choice was motivated by the fact that conducting polymers have large extinction coefficients ((>) 107 L mol-1 cm-1), are able to undergo intersystem crossing to the triplet state, and have triplet energies that are close to that of oxygen. It was therefore hypothesized that such polymers could be effective at generating ROS due to the large excitation rate that can be generated. Conducting polymer nanoparticles (CPNPs) composed of the conducting polymer poly[2-methoxy-5-(2-ethylhexyl-oxy)-p-phenylenevinylene] (MEH-PPV) were fabricated and studied in-vitro for their potential in PDT application. Although not fully selective, the nanoparticles exhibited a strong bias to the cancer cells. The formation of ROS was proven in-vitro by staining of the cells with CellROX Green Reagent, after which PDT results were quantified by MTT assays. Cell mortality was observed to scale with nanoparticle dosage and light dosage. Based on these promising results the MEH-PPV nanoparticles were developed further to allow for surface functionalization, with the aim of targeting these NPs to cancer cell lines. For this work targeting of cancers that overexpress folate receptors (FR) were considered. The functionalized nanoparticles (FNPs) were studied in OVCAR3 (ovarian cancer cell line) as FR+, MIA PaCa2 (pancreatic cell line) as FR-, and A549 (lung cancer cell line) having marginal FR expression. Complete selectivity of the FNPs towards the FR+ cell line was found. Quantification of PDT results by MTS assays and flow cytometry show that PDT treatment was fully selective to the FR+ cell line (OVCAR3). No cell mortality was observed for the other cell lines studied here within experimental error. Finally, the issue of confirming and quantifying small molecule drug delivery to diseased tissue was tackled by developing quantum dot (Qdot) biosensors with the aim of achieving fluorescence reporting of intracellular small molecule/drug delivery. For fluorescence reporting prior expertise in control of the fluorescence state of Qdots was employed, where redox active ligands can place the Qdot in a quenched OFF state. Ligand attachment was accomplished by disulfide linker chemistry. This chemistry is reversible in the presence of sulfur reducing biomolecules, resulting in Qdots in a brightly fluorescent ON state. Glutathione (GSH) is such a biomolecule that is present in the intracellular environment. Experimental in-vitro data shows that this design was successfully implemented.
Show less
Date Issued: 2014
Identifier: CFE0005839, ucf:50923
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005839

Title: Electrical, Optical and Chemical Properties of Organic Photo Sensitve Materials.
Creator: Shi, Zheng, Liao, Yi, Kolpashchikov, Dmitry, Ye, Jingdong, Zou, Shengli, Su, Ming, University of Central Florida
Abstract / Description: Light as a (")green(") source of energy has become increasingly attractive throughout the past century and has shown versatility for the application of activating chemical reactions. Compared with traditional energy sources, it provides a more direct, selective and controllable method. My PhD study was focused on the study of photochemistry of organic materials in two different systems. The first system is regarding reversible photoacids which generate protons on irradiation. With the aim of...
Show moreLight as a (")green(") source of energy has become increasingly attractive throughout the past century and has shown versatility for the application of activating chemical reactions. Compared with traditional energy sources, it provides a more direct, selective and controllable method. My PhD study was focused on the study of photochemistry of organic materials in two different systems. The first system is regarding reversible photoacids which generate protons on irradiation. With the aim of systematically studying these novel types of long lived photoacids, a series of photoacids was designed, synthesized and whose chemical mechanism was thoroughly investigated. This type of photoacid changes from a weak acid to a strong acid with a pH change of several units, which achieves nearly complete proton dissociation upon visible light irradiation. The whole process is reversible and the half-life of the proton-dissociation state is long enough to be used in many applications. Besides fundamental studies, different applications based on this type of photoacids were also completed. An esterification reaction was catalyzed and the volume of a pH-sensitive polymer was altered due to the large amount of photo generated protons from this photoacid. A reversible electrical conductivity change of polyaniline (PANI) was also achieved by doping with this reversible photoacid. In order to induce a large conductivity increase, an irreversible photoacid generator (PAG) was embedded in a novel PANI/PAG/PVA novel composition. In this system, Poly (vinyl alcohol) (PVA) forms a hydrogen-bonding network to facilitate proton transfer between the PAG and PANI. A final electrical conductivity of 10-1 S cm-1 was successfully achieved after irradiation. The second system in which I explored photochemistry of organic molecules concerns Photo-retro-Diels-Alder (PrDA) reactions and a variety of Diels-Alder (DA) adducts were designed for these studies. UV light was used to trigger the retro-Diels-Alder reactions. Quantum yield of each DA adducts was investigated. This revealed that the photo-reactivity of this process depends on the electron-donating ability of the diene and the electron-withdrawing ability of the dienophile component. Mechanistic studies of this PrDA reaction reveal that a charge-separated intermediate is generated from a singlet excited state. This was applied to an unsaturated cyclic ?-diketones (DKs), which underwent PrDA reactions and generated anthracene derivatives and carbon monoxide (CO), which itself plays profound and important roles in biological systems. These unsaturated cyclic ?-diketones (DKs) encapsulated in micelles are effective CO-releasing molecules (CORMs) and are capable of carrying and releasing CO in cellular systems. This novel type of organic CORMs has potentially low toxicity and generates fluorescence, which provides a useful tool for the study of the biological functions of CO.
Show less
Date Issued: 2013
Identifier: CFE0005114, ucf:50748
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005114

Title: Design, Synthesis, and Biological Evaluation of Novel Polyamine Transport System Probes and their Application to Human Cancers.
Creator: Muth, Aaron, Phanstiel, Otto, Ye, Jingdong, Elsheimer, Seth, Miles, Delbert, Vonkalm, Laurence, University of Central Florida
Abstract / Description: The mammalian polyamine transport system (PTS) has been of interest due to its roles in cancer and maintaining cellular homeostasis. Polyamines are essential growth factors which are tightly controlled via a balance of biosynthesis, metabolism, import, and export. This work focused on the development and biological testing of polyamine transport probes to help understand the molecular requirements of the PTS. This was mediated through the use of a CHO (PTS active) and CHO-MG* (PTS deficient)...
Show moreThe mammalian polyamine transport system (PTS) has been of interest due to its roles in cancer and maintaining cellular homeostasis. Polyamines are essential growth factors which are tightly controlled via a balance of biosynthesis, metabolism, import, and export. This work focused on the development and biological testing of polyamine transport probes to help understand the molecular requirements of the PTS. This was mediated through the use of a CHO (PTS active) and CHO-MG* (PTS deficient) screen, where compounds demonstrating high toxicity in CHO and low toxicity in CHO-MG* were considered PTS selective. The first chapter focused on the development of polyamine-based drugs which are both metabolically stable to polyamine oxidase (PAO) activity and are hyperselective for targeting the PTS. This approach was optimized by combining a di-substituted aryl design with terminal N-methylation of the appended polyamine chains to generate a new class of superior PTS agonists. The metabolic stability of these compounds was demonstrated in CHO and CHO-MG* in the presence and absence of a known PAO inhibitor, aminoguanidine (AG). Highly PTS selective compounds were then tested in the NCI-60 cell line screen to demonstrate the effectiveness of polyamine-based drugs in cancer therapy. During this screen, the MALME-3M (human melanoma) cell line was identified as being very sensitive to these PTS targeting drugs. Further studies using MALME-3M and its normal counterpart, MALME-3, showed excellent targeting of the cancer line over MALME-3. For example, The MeN44Nap44NMe compound showed 59-fold higher toxicity in MALME-3M over MALME-3.The second chapter focused on the development of potential polyamine transport inhibitors (PTIs) for use in combination therapy with ?-difluoromethylornithine (DFMO). This therapy is predicated upon reducing sustained polyamine depletion within cells by inhibiting both polyamine biosynthesis with DFMO and polyamine transport with the PTI ligand. Potential PTIs were identified by blocking the uptake of spermidine in DFMO-treated CHO and L3.6pl cells. Previous work has identified a tri-substituted polyamine-based design as an effective PTI. Low toxicity and a low Ki value in a L1210 screen were good predictors for PTI efficacy. The structural requirements for a potent PTI were explored by modulating the toxicity through the introduction of amide bonds, and also by determining the number and orientation of the polyamine messages (appended to an aryl core) required for efficient inhibition of polyamine uptake. These experiments showed that a tri-substituted design and a triamine message (homospermidine) appended was optimal for PTI potency. The final chapter focused on the development of Dihydromotuporamine C derivatives as non-toxic anti-metastatic agents. Dihydromotuporamine C demonstrated good anti-invasive properties with tumor cells. Derivatives were made in an effort to reduce the cytotoxicity of the parent and improve the anti-migration potency. The motuporamine derivatives all have a polyamine message (norspermidine or homospermidine) appended to make a macrocycle core, making them prime targets to evaluate as potential PTS ligands in the CHO and CHO-MG* screen. Each compound was also tested in the highly metastatic pancreatic cancer cell line L3.6pl to determine both its IC50 value and maximum tolerated dose (MTD). The anti-migration assay was performed at the lowest MTD obtained (0.6 (&)#181;M) in order to compare the series at the same non-toxic dose. The results suggested that as the N1-amine center was moved further from the macrocyclic ring, an increased ability to inhibit cell migration and reduced toxicity was observed. These collective findings provide new tools for cell biologists to modulate and target polyamine transport in mammalian cells. Future applications of these technologies include new cancer therapies which are cell-selective and inhibit the spread of tumors.
Show less
Date Issued: 2012
Identifier: CFE0004636, ucf:49895
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004636

Title: Structural and Functional Studies of Glycine Riboswitches and Development of Fab Chaperone Assisted RNA Crystallography.
Creator: Sherman, Eileen, Ye, Jingdong, Kolpashchikov, Dmitry, Koculi, Eda, Harper, James, Self, William, University of Central Florida
Abstract / Description: The glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few...
Show moreThe glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few nucleotides upstream of the established glycine riboswitch sequence which changed its ligand binding characteristics (Chapter 1). Previously, the two aptamers of the riboswitch were thought to cooperatively bind glycine, but with the inclusion of this leader sequence which forms a kink turn motif with the linker between the two aptamers, glycine binding in one aptamer no longer requires glycine binding in the other. Furthermore, the Kd from three species tested are now a similar, lower value of about 5 (&)#181;M, indicating authenticity of this new consensus sequence. Glycine binding and interaptamer interaction both enhanced one another in trans aptamer assays. Another discovery from this was a shortened construct including all of aptamer II but only part of aptamer I in which a few specific nucleotides prevented glycine binding in aptamer II (Chapter 2). This may provide insight into the nature of interaptamer interactions in the full switch; addition of an oligonucleotide complimentary to these nucleotides restored glycine binding ability to aptamer II. With future development, this could also be a useful molecular biology tool, using two signals, glycine and an oligonucleotide, to allow gene expression.To precisely understand how any macromolecule functions, a 3D structure, obtainable by x-ray crystallography, is vital. A new technique to accomplish that for RNA, precedented in the protein world, is Fab chaperoned crystallography, which has advantages compared to RNA alone. A phage displayed library of Fabs with reduced codon diversity designed for RNA was created, the YSGR Min library (Chapter 3). Its Fabs had specificities and affinities equal to or greater than previous libraries which were originally created for phage displayed selection against proteins. Fab chaperoned RNA crystallography is currently in progress for the glycine riboswitch; the best resolution thus far is 5.3 (&)#197; (Chapter 4). In addition to providing molecular insight into its gene regulation mechanism, a structure of the glycine riboswitch could be applied for use in structure based drug design of novel antibiotics targeting the riboswitch to disrupt important downstream carbon cycle genes in pathogenic bacteria.
Show less
Date Issued: 2014
Identifier: CFE0005549, ucf:50285
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005549

Title: Synthesis and Study of Chemo-Hydrothermally Derived Water-Soluble Chitosan and Chiosan-Metal Oxide Composites.
Creator: Basumallick, Srijita, Santra, Swadeshmukul, Kolpashchikov, Dmitry, Zou, Shengli, Ye, Jingdong, Seal, Sudipta, University of Central Florida
Abstract / Description: Chitosan (CS) is a man-made sugar based biopolymer derived from chitin, the second most abundant natural polymer after cellulose. Chitin is sourced from crustacean species such as shrimps and crabs. The chemical structure of chitin contains N-Acetyl D-glucosamine monomer units which forms CS upon deacetylation. In CS, ?-(1-4) linked D-glucosamine units are randomly distributed. Approximately 75% - 80% sugar units contains primary amine groups in commercially available low molecular weight CS....
Show moreChitosan (CS) is a man-made sugar based biopolymer derived from chitin, the second most abundant natural polymer after cellulose. Chitin is sourced from crustacean species such as shrimps and crabs. The chemical structure of chitin contains N-Acetyl D-glucosamine monomer units which forms CS upon deacetylation. In CS, ?-(1-4) linked D-glucosamine units are randomly distributed. Approximately 75% - 80% sugar units contains primary amine groups in commercially available low molecular weight CS. Biodegradability, low toxicity, mucoadhesive and transfecting properties of CS polymer are attractive for applications as oral and nasal drug delivery systems. Chitosan polymer is water insoluble at neutral pH. To solubilize CS, dilute mineral acid (such as hydrochloric acid and nitric acid) or organic acid (such as acetic acid) is often used. CS contains both hydroxyl and primary amine groups in its structure. In acidic solution, the amine functional groups become protonated (positively charged). Positively charged CS remains stable only in low pH condition due to electrostatic repulsion of charged polymer segments. Therefore, by using a suitable anionic (negatively charged) cross-linker, stable CS particles (such as nanoparticles and microspheres) can be prepared. This is popularly known as ionic gelation method. Extensive studies have been done on the synthesis of drug loaded CS particles where particle integrity is maintained by ionic gelation using tripolyphosphate (TPP, an anionic cross-linker). Drug encapsulated CS-TPP composite particles are shown to maintain biodegradability and biocompatibility. The CS-TPP composite particles exhibits very limited dispersibility at neutral pH conditions specifically in neutral buffered conditions. A number of biomedical applications (including systemic drug formulations) however demands buffer-stable CS composite particles for achieving optimal therapeutic outcome.To overcome the above dispersibility issues, CS polymer and CS particles units have been chemically modified using water soluble motifs (such as water soluble polymer or ligands). This approach is very cumbersome and usually involves multiple purification steps. Chemical modification of natural CS chain introduces risks of compromising biodegradability and biocompatibility. Therefore, there is a strong need for developing a straightforward method of making water soluble CS and CS particles.Chapter 1 of this dissertation presents an overview of the CS polymer, various applications of CS polymers, methods of making CS polymers and CS particles, current limitations of synthesis methods for preparing stable chitosan particles at neutral pH conditions and finally delineates the scope of the proposed research work.Chapter 2 describes development of chemo-hydrothermal synthesis method for producing water soluble CS polymer and water dispersible CS composite particles. In this method, a chemical (depolymerizing agent) is used to treat CS polymer in a hydrothermal (high temperature and high pressure) condition. Two types of depolymerizing agents have been used, an inorganic acid (e.g. hydrochloric acid, HCl) and a bicarboxylic organic acid (e.g. tartaric acid, TA). In both cases, 100% depolymerized CS polymer was obtained. Chemical characteristics of the depolymerized CS were comparable to acid solubilized CS. CS polymer exhibits weak fluorescence. Interestingly, hydrothermally depolymerized CS shows strong fluorescence properties irrespective of the nature of depolymerizing agent used. TA not only depolymerized CS but also formed CS-TA composite particulate structures in solution via self-assembly. The CS-TA composite particles are stable in a wide pH range from 5 to 11. Detailed spectroscopic and microscopic studies have been done to understand the basic mechanism of particle formation and increase in fluorescence properties (i.e. structure-property relationship). Usefulness of CS-TA in solubilizing water-insoluble cargos (such as fluorescein isothiocyanate, FITC) has been demonstrated.Chapter 3 is focused on hydrothermal synthesis of mixed-valence copper (Cu) oxide loaded CS-TA composite particles and their characterization. Crystalline Cu oxide nanoparticles were coated with the CS-TA layer. Water dispersibility of Cu oxide greatly improved upon coating with CS-TA material. To demonstrate catalytic activity of Cu-oxide loaded CS-TA film in sequestering carbon dioxide (CO2), an electrochemical setup was used. Electrochemical reduction of CO2 was successfully demonstrated. It was observed that CS-TA environment not only maintained catalytic properties of Cu oxide but also allowed solution processing of Cu-oxide film onto the electrode surface.Chapter 4 discusses a convenient method of making monodispersed water dispersible Cu loaded chitosan nanoparticles (Cu-CS) using HCl depolymerized CS polymer. The purpose of this study was to investigate if there was any improvement in antibacterial properties of Cu-CS nanoparticles prepared using hydrothermally treated CS polymer. Interestingly, it was observed that the antibacterial efficacy of Cu was not compromised in Cu-CS nanoparticles. Moreover, the materials exhibited improvement in antibacterial efficacy against both Gram-negative and Gram-positive bacteria species. A plausible mechanism has been proposed to explain antibacterial results.Chapter 5 summarizes major findings of this dissertation research and presents future research directions.
Show less
Date Issued: 2014
Identifier: CFE0005461, ucf:50395
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005461

Title: Characterization of composite broadband absorbing conjugated polymer nanoparticles by steady-state, time-resolved and single particle spectroscopy.
Creator: Bonner, Maxwell, Gesquiere, Andre, Campiglia, Andres, Santra, Swadeshmukul, Hernandez, Florencio, Perez Figueroa, Jesus, Ye, Jingdong, Fernandez-Valle, Cristina, University of...
Show moreBonner, Maxwell, Gesquiere, Andre, Campiglia, Andres, Santra, Swadeshmukul, Hernandez, Florencio, Perez Figueroa, Jesus, Ye, Jingdong, Fernandez-Valle, Cristina, University of Central Florida
Show less
Abstract / Description: As the global economy searches for reliable, inexpensive and environmentally friendly renewable energy resources, energy conservation by means of photovoltaics has seen near exponential growth in the last decade. Compared to state-of-the-art inorganic solar cells, organic photovoltaics (OPVs) composed of conjugated polymers are particularly interesting because of their processability, flexibility and the potential for large area devices at a reduced fabrication cost. It has been extensively...
Show moreAs the global economy searches for reliable, inexpensive and environmentally friendly renewable energy resources, energy conservation by means of photovoltaics has seen near exponential growth in the last decade. Compared to state-of-the-art inorganic solar cells, organic photovoltaics (OPVs) composed of conjugated polymers are particularly interesting because of their processability, flexibility and the potential for large area devices at a reduced fabrication cost. It has been extensively documented that the interchain and intrachain interactions of conjugated polymers complicate the fundamental understanding of the optical and electronic properties in the solid-state (i.e. thin film active layer). These interactions are highly dependent on the nanoscale morphology of the solid-state material, leading to a heterogeneous morphology where individual conjugated polymer molecules obtain a variety of different optoelectronic properties. Therefore, it is of the utmost importance to fundamentally study conjugated polymer systems at the single molecule or nanoparticle level instead of the complex macroscopic bulk level.This dissertation research aims to develop simplified nanoparticle models that are representation of the nanodomains found in the solid-state material, while fundamentally addressing light harvesting, energy transfer and interfacial charge transfer mechanisms and their relationship to the electronic structure, material composition and morphology of the nanoparticle system. In preceding work, monofunctional doped nanoparticles (polymer-polymer) were fabricated with enhanced light harvesting and F?rster energy transfer properties by blending Poly[(o-phenylenevinylene)-alt-(2-methoxy-5-(2-ethylhexyloxy)-p-phenylenevinylene)] (BPPV) and Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) at various MEH-PPV doping ratios. While single particle spectroscopy (SPS) reveals a broad distribution of optoelectronic and photophysical properties, time-correlated single photon counting (TC-SPC) spectroscopy displays multiple fluorescence lifetime components for each nanoparticle composition, resulting from changing polymer chain morphologies and polymer-polymer aggregation. In addition, difunctional doped nanoparticles were fabricated by doping the monofunctional doped nanoparticles with PC60BM ([6,6]-phenyl-C61-butyric acid methyl ester) to investigate competition between intermolecular energy transfer and interfacial charge transfer. Specifically, the difunctional SPS data illustrated enhanced and reduced energy transfer mechanisms that are dependent on the material composition of MEH-PPV and PC60BM. These data are indicative of changes in inter- and intrachain interactions of BPPV and MEH-PPV and their respective nanoscale morphologies. Together, these fundamental studies provide a thorough understanding of monofunctional and difunctional doped nanoparticle photophysics, necessary for understanding the morphological, optoelectronic and photophysical processes that can limit the efficiency of OPVs and provide insight for strategies aimed at improving device efficiencies.
Show less
Date Issued: 2011
Identifier: CFE0004089, ucf:49143
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004089

Title: Multifunctional and Responsive Polyelectrolyte Nanostructures.
Creator: Malhotra, Astha, Zhai, Lei, Kolpashchikov, Dmitry, Ye, Jingdong, Chumbimuni Torres, Karin, Santra, Swadeshmukul, Fang, Jiyu, University of Central Florida
Abstract / Description: A polyelectrolyte complex is formed by mixing two oppositely charged polyelectrolytes in a solution. The electrostatic interactions between partially charged polymeric chains lead to the formation of a stable complex while avoiding the use of covalent cross linkers. Since complex formation can improve the stability of polyelectrolyte and metal ions in polyelectrolyte can provide various functionalities, PECs incorporated with metal ions are promising candidates for manufacturing stable and...
Show moreA polyelectrolyte complex is formed by mixing two oppositely charged polyelectrolytes in a solution. The electrostatic interactions between partially charged polymeric chains lead to the formation of a stable complex while avoiding the use of covalent cross linkers. Since complex formation can improve the stability of polyelectrolyte and metal ions in polyelectrolyte can provide various functionalities, PECs incorporated with metal ions are promising candidates for manufacturing stable and multifunctional structures. While the coordination of metal ions and polyelectrolytes has been extensively investigated in solutions and multilayer films, to our knowledge, no research has been performed to study the effect of metal ion/polyelectrolyte interactions on PECs structures and properties. The following research demonstrates the impact of different metal ions in controlling PEC structure morphology and applications. These discoveries indicate great potential of metal ions in PECs to fabricate functional PEC nanostructures.The research investigates the effect of the interactions between different metal ions and polyelectrolytes on the morphology and properties of PECs, explore the fabrication of different structures using embedded metal ions and understand the impact of metal ion/polyelectrolyte interactions on the nanoparticle structures. The research concludes: 1) incorporating metal ions of different valence into PECs introduces metal ion/polyelectrolyte interactions that can tune the morphology of PECs; 2) metal ion/polyelectrolyte interactions can be used to control the PECs swelling properties and stability in aqueous solutions; 3) the release of embedded metal ions from PECs to aqueous solutions is affected by metal ion/polyelectrolyte interactions; and 4) the embedded metal ions function as a reagent reservoir for various applications to produce functional structures. ?
Show less
Date Issued: 2014
Identifier: CFE0005833, ucf:50918
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005833

Fab (3)	+ -
RNA (3)	+ -
A549 (1)	+ -
Amyloid fibril (1)	+ -
Antibody (1)	+ -

Beta sheet inhibitor (1)	+ -
Biological (1)	+ -
Body Fluid (1)	+ -
Cancer (1)	+ -
Chaperone Assisted RNA Crystallography (1)	+ -
Chemo Hydrothermally Treated (1)	+ -
Chitin (1)	+ -
Chitosan (1)	+ -
Clumps (1)	+ -
Copper-Chitosan (1)	+ -
Covalent Inhibitors (1)	+ -
DNA (1)	+ -
Donor (1)	+ -
Evidence (1)	+ -
Fab chaperone assisted RNA crystallography (1)	+ -
Few Cells (1)	+ -
Forensic Biology (1)	+ -
Forensics (1)	+ -
H-bond occupancy (1)	+ -
Identity (1)	+ -
Irreversible inhibitors (1)	+ -
MD simulation (1)	+ -
MDA-MB-231 (1)	+ -
MIA PaCa-2 (1)	+ -
MM-PBSA binding free energy (1)	+ -

ETD Collection (12)	+ -
Honors Collection (1)	+ -

University of Central Florida (13)	+ -
Ye, Jingdong (13)	+ -
Kolpashchikov, Dmitry (7)	+ -
Santra, Swadeshmukul (4)	+ -
Self, William (3)	+ -

Zou, Shengli (3)	+ -
Campiglia, Andres (2)	+ -
Gesquiere, Andre (2)	+ -
Archer, Jennifer (1)	+ -
Ballantyne, John (1)	+ -
Basumallick, Srijita (1)	+ -
Berhanu, Workalemahu (1)	+ -
Bonner, Maxwell (1)	+ -
Chumbimuni Torres, Karin (1)	+ -
Cornett, Evan (1)	+ -
Elsheimer, Seth (1)	+ -
Fang, Jiyu (1)	+ -
Fernandez-Valle, Cristina (1)	+ -
Harper, James (1)	+ -
Hernandez, Florencio (1)	+ -
Hickman, James (1)	+ -
Holmes, Sean (1)	+ -
Khaled, Annette (1)	+ -
Koculi, Eda (1)	+ -
Liao, Yi (1)	+ -
Malhotra, Astha (1)	+ -
Masunov, Artem (1)	+ -
Mathew, Mona (1)	+ -
Miles, Delbert (1)	+ -
Morgan, Brittany (1)	+ -

Search In

Sort Results By:

Narrow results by:

Topical Subject

Collection

Type of Resource

Language

Creator

Format

Owner