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Novel copper loaded core-shell silica nanoparticles with improved copper bio-availability : Synthesis, characterization and study of antibacterial properties
Title
Novel copper loaded core-shell silica nanoparticles with improved copper bio-availability : Synthesis, characterization and study of antibacterial properties.
Creator
Maniprasad, Pavithra, Santra, Swadeshmukul, Self, William, Naser, Saleh, University of Central Florida
Abstract / Description
A novel core-shell silica based antimicrobial nanoparticle was synthesized. The St(&)#246;ber silica shell has been engineered to accommodate copper. Synthesis of the core-shell Cu-silica nanoparticle (C-S CuSiNP) involves preparation of base-hydrolyzed St(&)#246;ber silica (")seed(") particles first, followed by the acid-catalyzed seeded growth of the Cu-silica shell layer around the core. Scanning electron microscopy and transmission electron microscopy showed monodispersed, spherical...
Show moreA novel core-shell silica based antimicrobial nanoparticle was synthesized. The St(&)#246;ber silica shell has been engineered to accommodate copper. Synthesis of the core-shell Cu-silica nanoparticle (C-S CuSiNP) involves preparation of base-hydrolyzed St(&)#246;ber silica (")seed(") particles first, followed by the acid-catalyzed seeded growth of the Cu-silica shell layer around the core. Scanning electron microscopy and transmission electron microscopy showed monodispersed, spherical shaped nanoparticles with smooth surface morphology. Characterization of particle size distribution in solution by the Dynamic Light Scattering (DLS) technique was fairly consistent with the electron microscopy results. Loading of Cu to nanoparticles was confirmed by the SEM-Energy Dispersive X-Ray Spectroscopy (EDS) and Atomic Absorption Spectroscopy (AAS). Antibacterial efficacy of C-S CuSiNP was evaluated against E.coli and B.subtilis using Cu hydroxide ((")Insoluble(") Cu compound) and copper sulfate as positive control and silica (")seed(") particles (without Cu loading) as negative control. Minimum Inhibitory Concentration (MIC) of C-S CuSiNP was evaluated by measuring the fluorescent intensity of resorufin to determine the decrease in viable cells with increase in copper concentration in C-S CuSiNP. The MIC value of C-S CuSiNP against both E.coli and B.subtilis was estimated to be 4.9 ppm. Bac-light fluorescence microscopy based assay was used to count relative population of the live and dead bacteria cells. Antibacterial study clearly shows that C-S CuSiNP is more effective than insoluble Cu hydroxide particles and copper sulfate at equivalent metallic Cu concentration, suggesting more soluble Cu in C-S CuSiNP material due to its core-shell design.
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Date Issued
2011
Identifier
CFE0004479, ucf:49300
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004479
Signals Delivered By Interleukin-7 Regulate The Activities Of Bim And JunD In T Lymphocytes
Title
Signals Delivered By Interleukin-7 Regulate The Activities Of Bim And JunD In T Lymphocytes.
Creator
Ruppert, Shannon, Khaled, Annette, Self, William, Zervos, Antonis, Teter, Kenneth, University of Central Florida
Abstract / Description
Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal...
Show moreInterleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal transducers of the IL-7 growth signal: BimL and JunD. IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. After a bioinformatics survey to reveal possible JunD-regulated genes activated early in the IL-7 signaling cascade, our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism, including Pim-1. Pim-1, an IL-7 induced protein, was inhibited upon JNK or JunD inhibition. Our hypothesis that JunD positively regulated proliferation was confirmed when the proliferation of primary CD8+ T-cells cultured with IL-7 was impaired upon treatment with JunD siRNA. These results show that the IL-7 signal is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth through the expression of metabolic factors like HXKII and Pim-1. When metabolic activities are inhibited, cells undergo autophagy, or cell scavenging, to provide essential nutrients. Pro-apoptotic Bim was evaluated for its involvement in autophagy. Bim is a BH3-only member of the Bcl-2 family that contributes to T-cell death. Partial rescue of T-cells occurs when Bim and the interleukin-7 receptor are deleted, implicating Bim in IL-7-deprived T-cell apoptosis. Alternative splicing results in three different isoforms: BimEL, BimL, and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on IL-7 for growth, were used. Loss of Bim in IL-7-deprived T-cells delayed apoptosis, but blocked the degradative phase of autophagy. The conversion of LC3-I to LC3-II was observed in Bim-deficient T-cells, but p62, which is degraded in autolysosomes, accumulated. To explain this, BimL, was found to support acidification of lysosomes associated with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon IL-7 withdrawal only in Bim-containing T-cells, indicating that in these cells autophagy was protective. IL-7 dependent T-cells lacking Bim were insensitive to inhibition of autophagy or lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, associated with intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of functional autolysosomes during autophagy.
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Date Issued
2012
Identifier
CFE0004435, ucf:49331
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004435
A Binary Approach for Selective Recognition of Nucleic Acids and Proteins
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.
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Date Issued
2015
Identifier
CFE0006031, ucf:51010
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006031
Cerium Oxide Nanoparticles Sensitize Pancreatic Cancer Cells to Radiation by Promoting Acidic pH, ROS, and JNK Dependent Apoptosis
Title
Cerium Oxide Nanoparticles Sensitize Pancreatic Cancer Cells to Radiation by Promoting Acidic pH, ROS, and JNK Dependent Apoptosis.
Creator
Wason, Melissa, Zhao, Jihe, Self, William, Altomare, Deborah, Baker, Cheryl, University of Central Florida
Abstract / Description
Side effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl...
Show moreSide effects of radiation therapy (RT) remain the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl peroxide resulting in accumulation of the latter whereas in neutral pH CONPs scavenge both. CONP treatment prior to RT markedly potentiated the cancer cell apoptosis both in culture and in tumors and the inhibition of the pancreatic tumor growth without harming the normal tissues or host mice. Mechanistically, CONPs were not able to significantly impact RT-induced DNA damage in cancer cells, thereby ruling out sensitization through increased mitotic catastrophe. However, JNK activation, which is known to be a key driver of RT-induced apoptosis, was significantly upregulated by co-treatment with CONPs and RT in pancreatic cancer cells in vitro and human pancreatic tumors in nude mice in vivo compared to CONPs or RT treatment alone. Further, CONP-driven increase in RT-induced JNK activation was associated with marked increases in Caspase 3/7 activation, indicative of apoptosis. We have shown CONPs increase ROS production in cancer cells; ROS has been shown to drive the oxidation of thioredoxin (TRX) 1 which results in the activation of Apoptosis Signaling Kinase (ASK) 1. The dramatic increase in ASK1 activation following the co-treatment of pancreatic cancer cells with CONPs followed by RT in vitro suggests that increased the c-Jun terminal kinase (JNK) activation is the result of increased TRX1 oxidation. The ability of CONPs to sensitize pancreatic cancer cells to RT was mitigated when the TRX1 oxidation was prevented by mutagenesis of a cysteine residue, or the JNK activation was blocked by an inhibitor,. Additionally, angiogenesis in pancreatic tumors treated with CONPs and RT was significantly reduced compared to other treatment options. Taken together, these data demonstrate an important role and mechanisms for CONPs in specifically killing cancer cells and provide novel insight into the utilization of CONPs as a radiosensitizer and therapeutic agent for pancreatic cancer.
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Date Issued
2013
Identifier
CFE0005116, ucf:50725
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005116
Structural and functional characterization of enzymes of a novel group of tryptophylquinone cofactor containing oxidases
Title
Structural and functional characterization of enzymes of a novel group of tryptophylquinone cofactor containing oxidases.
Creator
Sehanobish, Esha, Davidson, Victor, Khaled, Annette, Self, William, Tatulian, Suren, University of Central Florida
Abstract / Description
Protein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas...
Show moreProtein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas mediterranea, have been shown to possess the latter. This study involves the characterization of two such proteins, lysine-?-oxidase (LodA) and glycine oxidase (GoxA). They have been reported to contain the same cysteine tryptophylquinone (CTQ) cofactor. Both require the co-expression of a second protein, LodB and GoxB respectively to generate matured CTQ containing active protein. Kinetic analysis of the reaction catalyzed by LodA showed that it followed the usual Michaelis-menten mode of interaction with its substrates. GoxA on the other hand exhibited allosteric cooperativity for its substrate glycine. This was attributed to the dimeric conformation of the wildtype GoxA based on size exclusion chromatographic studies. Mutagenesis study of amino acid residues based on the crystal structure of LodA and a homology model of GoxA, have given a detailed idea about their structure-function relationship. Kinetic studies on mutants of Tyr211 of LodA along with Lys530 present at the substrate channel, showed effects on both Km for the substrates and kcat for the reaction. As a result these residues have shown their involvement in forming a gate-like structure to control the to and fro movement of the substrate and products. Corresponding to this residue, the Phe237 of GoxA has proved to be important in maintaining the allostericity, by mediating the stable dimer formation. From the kinetic parameters, Cys448 of LodA was found to be responsible for substrate specificity and affinity. Whereas, mutants of His466 of GoxA that correspond to the Cys448 residue, were unable to yield CTQ containing active GoxA. On the other hand, Asp512 of LodA and Asp547 of GoxA that correspond to each other, have been implicated for their involvement in CTQ biogenesis. This study therefore highlights how even though this new pool of enzymes have great degrees of similarity in terms of the cofactors and conserved active site residues, there are major differences in the mechanism of the reaction that they catalyze which on a broader sense could influence the overall physiological importance of the enzyme in the biological system.
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Date Issued
2016
Identifier
CFE0006390, ucf:51494
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006390
Discovery and characterization of antimalarial compounds with novel cellular mechanisms of action
Title
Discovery and characterization of antimalarial compounds with novel cellular mechanisms of action.
Creator
Roberts, Bracken, Chakrabarti, Debopam, Jewett, Travis, Self, William, University of Central Florida
Abstract / Description
Malaria kills over 500,000 people each year and over a third of the global population is at risk of infection. Though the human race has been fighting the malaria war for over 4,000 years and we have made great strides in eliminating malaria from many countries, we are treading on the edge of what could be another malaria epidemic primarily due to widespread drug resistance. There are documented cases of resistance for every known antimalarial in use today, including Artemisinins. It is...
Show moreMalaria kills over 500,000 people each year and over a third of the global population is at risk of infection. Though the human race has been fighting the malaria war for over 4,000 years and we have made great strides in eliminating malaria from many countries, we are treading on the edge of what could be another malaria epidemic primarily due to widespread drug resistance. There are documented cases of resistance for every known antimalarial in use today, including Artemisinins. It is critical that we open a new window of discovery in development of next generation antimalarials that circumvent current resistance paradigms. These compounds must attack new targets, have different speeds of action, and ideally possess powerful transmission blocking potential if they are to be successful antimalarial candidates. Screening endeavors historically focused on either synthetic or natural product libraries. Recent efforts have focused on combining privilege elements of natural products into synthetically tractable compounds to create hybrid libraries. To discover novel antimalarial pharmacophores, we have screened natural products derived from marine biodiversity as well as natural product-inspired synthetic libraries. Our phenotypic screening of 3,164 marine natural products from the Harbor Branch Oceanographic Institute, 56 high density combinatorial natural product based libraries from the Torrey Pines Institute for Molecular Studies, alkaloid, terpene, and macrocyclic libraries from Memorial Sloan Kettering Cancer Center, and 594 natural productinspired compounds from Asinex have identified several new selective antiplasmodial hit chemotypes. iv In this study, we have focused on compounds that exhibit cellular actions differing from current antimalarials. Two of the scaffolds, UCF 201 and 501, a spirocyclic chromane and a nitroquinoline, respectively, act early in the development cycle and block invasion. The alkaloid derived compound M03 blocks egress. UCF 501 cures malaria in the rodent model and significantly inhibits stage V gametocytogenesis. Given that discovery of transmission blocking agents are a priority in the malaria elimination strategies, this result is significant. This work is of high impact as it addresses a critical need in the field- next generation antimalarial scaffolds for malaria therapy and elimination campaign.
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Date Issued
2017
Identifier
CFE0006785, ucf:51815
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006785
The mauC gene encodes a versatile signal sequence and redox protein that can be utilized in native and non-native protein expression and electron trnasfer systems
Title
The mauC gene encodes a versatile signal sequence and redox protein that can be utilized in native and non-native protein expression and electron trnasfer systems.
Creator
Dow, Brian, Davidson, Victor, Self, William, Rohde, Kyle, Tatulian, Suren, University of Central Florida
Abstract / Description
The redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding...
Show moreThe redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding network of amicyanin from the aforementioned study were attempted to be used as a model to increase the stability of another beta barrel protein, the immunoglobulin light chain variable domain (VL). In addition, amicyanin was used as an alternative redox partner with MauG. MauG is a diheme protein from the mau gene cluster that catalyzes the biogenesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase (MADH). The amicyanin-MauG complex was used to study the free energy dependence and impact of reorganization energy in biological electron transfer reactions.The sole tryptophan of amicyanin was converted to a tyrosine via site-directed mutagenesis. This mutation had no effect on the electron transfer parameters with its redox partners, methylamine dehydrogenase and cytochrome c-551i. However, the pKa of the pH-dependence of the redox potential of the copper site was shifted +0.5 pH units. This was a result of an additional hydrogen bond between Met51 and the copper coordinating residue His95 in the reduced form of amicyanin. This additional hydrogen bond stabilizes the reduced form. Also, the stability of the copper site and the protein overall was significantly decreased, as seen by the temperature dependence of the visible spectrum of the copper site and the circular dichroism spectrum of the protein. This destabilization is attributed to the loss of an interior, cross-barrel hydrogen bond.The VL is structurally similar to amicyanin, but it does not contain any cross-barrel hydrogen bonds. The importance of the cross-barrel hydrogen bond in stabilizing amicyanin is evident. A homologous bond in VL was attempted to be engineered by using site-directed mutagenesis to insert neutral residues with protonatable groups into the core of the protein. Wild-type (WT) VL was purified from the periplasm and found to be properly folded as determined by circular dichroism and size exclusion chromatography. Mutants were expressed in E. coli using the amicyanin signal sequence for periplasmic expression. Folded mutant protein could not be purified from the periplasm.When amicyanin is used in complex with MauG, it retains the pH-dependence of the redox potential of its copper site due to the looseness of the interprotein interface. The free energy of the reaction was manipulated by variation in pH from pH 5.8 to 8.0. The ET parameters are reorganization energy of 2.34 eV and an electronic coupling constant of 0.6 cm-1. P94A amicyanin has a potential that is 120 mV higher than WT amicyanin and was used to extend the range of the free energy dependence studied. The ET parameters of the reaction of WT and P94A amicyanin with MauG were within error of each other. This is significant because the ET reaction of P94A amicyanin with its natural electron acceptor was not able to be studied due to a kinetic coupling of the ET step with a non-ET step. This kinetic coupling obscured the parameters of the ET step because it is not kinetically distinguishable from the ET step.A Y294H MauG mutant was also studied. This mutation replaced the axial tyrosine ligand of the six-coordinate heme of MauG with a histidine. No reaction is observed with Y294H MauG in its native reaction. However, the high valent oxidation state of the five-coordinate heme of Y294H MauG reacts with reduced amicyanin. The ET rate was analyzed by ET theory to study the high valent heme in Y294H MauG. The reorganization energy of Y294H MauG was calculated to be nearly 20% lower as compared to the same reaction with WT MauG. These results provide insight into the obscured nature of reorganization energy of large redox cofactors in proteins, particularly heme cofactors, as well as to how the active sites of enzymes are optimized to perform long range ET vs catalysis with regard to balancing redox potential and reorganization energy.
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Date Issued
2016
Identifier
CFE0006100, ucf:51192
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006100
Identification of Novel Antimalarial Scaffolds From Marine Natural Products
Title
Identification of Novel Antimalarial Scaffolds From Marine Natural Products.
Creator
Roberts, Bracken, Chakrabarti, Debopam, Jewett, Travis, Self, William, University of Central Florida
Abstract / Description
Malaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater.Screening of natural product libraries is known to have higher hit rates than synthetic...
Show moreMalaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater.Screening of natural product libraries is known to have higher hit rates than synthetic chemical libraries. This elevated hit rate is somewhat attributed to the greater biodiversity available in natural products. Marine life is the most biodiverse system on the planet, containing 34 of the 36 known phyla of life, and is expected to be a rich source of novel chemotypes. In collaboration with the Harbor Branch Oceanographic Institute in Ft. Pierce we have screened a library of over 2,800 marine macroorganism peak fractions against Plasmodium falciparum using the SYBR green I fluorescence-based assay. In this screening process we have identified six compounds from five novel chemical scaffolds all of which have low micromolar to submicromolar IC50 values and excellent selectivity indices. Additionally, one of these chemical scaffolds, the bis(indolyl)imidazole, was selected for further in vitro pharmacological and structure-activity relationship (SAR) profiling, key steps in the challenging process of identifying a new antimalarial drug lead compound.
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Date Issued
2012
Identifier
CFE0004792, ucf:49748
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004792
Molecular Mechanisms involved in inflammatory angiogenesis induced by monocyte chemotactic protein induced protein-1 (MCPIP1)
Title
Molecular Mechanisms involved in inflammatory angiogenesis induced by monocyte chemotactic protein induced protein-1 (MCPIP1).
Creator
Roy, Arpita, Kolattukudy, Pappachan, Ebert, Steven, Parthasarathy, Sampath, Self, William, University of Central Florida
Abstract / Description
Major diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes...
Show moreMajor diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes involved in angiogenesis mediated via MCPIP. To determine if angiogenesis induced by inflammatory cytokines, TNF-?, IL-1? and IL-8 is mediated via induction of MCPIP, knockdown of MCPIP by its specific siRNA, in human umbilical vein endothelial cells was performed. Oxidative stress, ER stress and autophagy are known to be involved in mediating inflammation. We hypothesized that MCPIP-induced angiogenic differentiation is mediated via induction of oxidative stress, ER stress and autophagy. Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3?methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as an in vitro tool to assay angiogenic differentiation. Inhibition of each step inhibited the subsequent steps postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via sequential induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. MCPIP has deubiquitinase and anti-dicer RNase activities. If and how the dual enzymatic activities of MCPIP mediate angiogenesis was unknown. Our results showed that hypoxia-induced angiogenesis is mediated via MCPIP. MCPIP deubiquitinated ubiquitinated hypoxia-inducible factor (HIF-1?) and the stabilized HIF-1? entered the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and vascular endothelial growth factor causing the activation of p38 MAP kinase involved in angiogenesis. MCPIP expression promoted angiogenesis by inhibition of thrombospondin-1 synthesis via induction of silent information regulator (SIRT)-1 and/or via suppression of VEG-inhibitor levels caused by inhibition of NF-?B activation. MCPIP inhibited the production of the anti-angiogenic microRNAs (miR)-20b and miR-34a that repress the translation of HIF-1? and SIRT-1, respectively. Cells expressing the RNase-dead mutant of MCPIP, D141N, that had lost the ability to induce angiogenesis had deubiquitinase activity but did not inhibit the production of miR-20b and miR-34a. Mimetics of miR-20b and miR-34a inhibited MCPIP-induced angiogenesis. These results show for the first time that both deubiquitinase and anti-dicer RNase activities of MCPIP are involved in inflammatory angiogenesis. Results from our study delineate key processes that could be potential targets for therapeutic intervention against inflammatory angiogenesis.
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Date Issued
2012
Identifier
CFE0004793, ucf:49760
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004793
Cerium oxide Nanoparticles: Their Phosphatase Activity and its Control
Title
Cerium oxide Nanoparticles: Their Phosphatase Activity and its Control.
Creator
Dhall, Atul, Self, William, Seal, Sudipta, Zervos, Antonis, University of Central Florida
Abstract / Description
Cerium oxide nanoparticles are established scavengers of reactive oxygen and nitrogen species. They have many potential biomedical applications that depend on their physicochemical properties and mode of preparation. Recent studies have found these nanoparticles possess phosphatase mimetic activity. Studying such catalytic activities will qualify their biomedical applications and render information on their bioavailability and potential toxicity.Two oxidation states of cerium exist in these...
Show moreCerium oxide nanoparticles are established scavengers of reactive oxygen and nitrogen species. They have many potential biomedical applications that depend on their physicochemical properties and mode of preparation. Recent studies have found these nanoparticles possess phosphatase mimetic activity. Studying such catalytic activities will qualify their biomedical applications and render information on their bioavailability and potential toxicity.Two oxidation states of cerium exist in these nanoparticles (3+ or 4+). It is hypothesized that the oxidation state of cerium in the nanoparticles determines the amount of adsorbed water on the crystal lattices. This in turn governs their activity as phosphatases. Nanoparticles with higher levels of cerium in the 4+ state exhibit phosphatase activity while those with higher levels of cerium in the 3+ state do not. This phosphatase activity may be controlled with the addition of inhibitory anions. It is hypothesized that anions with structures similar to phosphate can inhibit phosphatase activity by leading to the production of complexes on the surface of cerium oxide nanoparticles.Substrates that were used to test this activity include para-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (MUP) and adenosine triphosphate (ATP). To highlight the role of adsorbed water, we also performed experiments on pNPP with methanol as a solvent. The activity was measured by absorbance (pNPP and ATP) or fluorescence (MUP) and reported as nmol of phosphate/min. In some cases this rate was calculated through coupled reactions or by measuring the rate of formation of other colored products formed along with the release of phosphate such as pNP (para-nitrophenol).The phosphatase activity increased as the amount of adsorbed water increased implying that the abundance of adsorbed water makes the surface of 4+ ceria nanoparticles more active. Phosphatase activity for all the substrates exhibited Michaelis-Menten kinetics. Although the phosphatase activity of these nanoparticles is slow (turnover rate) as compared to real biological phosphatases, it can be used as a model catalytic activity to follow other catalytic activities that are associated with nanoparticles that have an abundance of cerium in the 4+ state, such as catalase activity. These results also provide information on the nature of the active sites involved in the catalytic activities associated with these nanoparticles.We identified three inhibitors, tungstate, molybdate and arsenate, which decreased the phosphatase activity of these nanoparticles in a dose dependent manner. Vmax, Km and Ki values were determined by varying substrate concentrations in the presence and absence of inhibitors. A partial mixed inhibition model was fit for each of these inhibitors.Summary: Phosphatase activity of cerium oxide nanoparticles with higher levels of cerium in the 4+ oxidation state was used as a model catalytic activity to study the nature of the active sites involved in catalysis. The study of inhibitors can reveal more information as to the surface binding of substrates in catalysis.
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Date Issued
2014
Identifier
CFE0005603, ucf:50261
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005603
Host and Bacterial Determinants of Staphylococcus aureus Nasal Colonization in Humans
Title
Host and Bacterial Determinants of Staphylococcus aureus Nasal Colonization in Humans.
Creator
Muthukrishnan, Gowrishankar, Cole, Alexander, Moore, Sean, Self, William, Parkinson, Christopher, University of Central Florida
Abstract / Description
Staphylococcus aureus (SA), an opportunistic pathogen colonizing the anterior nares in approximately 30% of the human population, causes severe hospital-associated and community-acquired infections. SA nasal carriage plays a critical role in the pathogenesis of staphylococcal infections and SA eradication from the nares has proven to be effective in reducing endogenous infections. To understand SA nasal colonization and its relation with consequent disease, assessment of nasal carriage...
Show moreStaphylococcus aureus (SA), an opportunistic pathogen colonizing the anterior nares in approximately 30% of the human population, causes severe hospital-associated and community-acquired infections. SA nasal carriage plays a critical role in the pathogenesis of staphylococcal infections and SA eradication from the nares has proven to be effective in reducing endogenous infections. To understand SA nasal colonization and its relation with consequent disease, assessment of nasal carriage dynamics among a diverse population and determining factors responsible for SA nasal carriage have become major imperatives.Here, we report on an extensive longitudinal monitoring of SA nasal carriage in 109 healthy individuals over a period of up to three years to assess nasal carriage dynamics. Phylogenetic analyses of SA housekeeping genes and hypervariable virulence genes revealed that not only were SA strains colonizing intermittent and persistent nasal carriers genetically similar, but no preferential colonization of specific SA strains in these carriers was observed over time. These results indicated that other non-SA factors could be involved in determining specific carriage states. Therefore, to elucidate host responses during SA nasal carriage, we performed human SA nasal recolonization in a subset of SA nasal carriers within our cohort. In these studies, SA colonization levels were determined, and nasal secretions were collected and analyzed for host immune factors responsible for SA nasal carriage. Interestingly, we observed that stimulation of host immune responses lead to clearance of SA while sustained SA colonization was observed in hosts that did not mount a response during carriage. Further, analysis of nasal secretions from hosts revealed that proinflammatory cytokines and chemokines were significantly induced during SA nasal clearance suggesting that innate immune effectors influence carriage.SA utilizes a repertoire of surface and secreted proteins to evade host immune response and successfully colonize the nose. Analysis of the most abundant immunoevasive proteins in the exoproteome of SA nasal carrier strains revealed that expression levels of Staphylococcal protein A (SPA) produced by SA nasal carrier strains in vitro corresponded to the level of persistence of SA in the human nose. To determine if SPA is involved in modulating the host's response to SA colonization, a subset of participants in our cohort was nasally recolonized with equal concentrations of both wild-type (WT) and spa-disrupted (?spa) autologous strains of SA. Interestingly, ?spa strains were eliminated from the nares significantly faster than WT when the host mounted an immune response, suggesting that the immunoevasive role of SPA is a determinant of carriage persistence. Collectively, this report augments our understanding of SA nasal carriage dynamics, in addition to identifying important host and microbial determinants that influence SA nasal colonization in humans. Better understanding of this phenomenon can lead to improved preventative strategies to thwart carriage-associated SA infections.
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Date Issued
2014
Identifier
CFE0005673, ucf:50173
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005673
Screening of Quantum Dots for Toxicity on the Growth and Viability of Escherichia coli
Title
Screening of Quantum Dots for Toxicity on the Growth and Viability of Escherichia coli.
Creator
Tharkur, Jeremy, Santra, Swadeshmukul, Self, William, Moore, Sean, University of Central Florida
Abstract / Description
Heavy metal (HM) containing quantum dots (Qdots) are increasingly used in commercial products due to their unique electronic, optoelectronic, optical and magnetic properties. Once disposed to the landfill, environmental weathering is likely to compromise HM Qdot integrity, leading to release of heavy metal ions. To minimize any negative environmental impact of HM Qdots, there is an increasing demand for developing HM free or environmentally-friendly surface modified HM Qdot alternatives. In...
Show moreHeavy metal (HM) containing quantum dots (Qdots) are increasingly used in commercial products due to their unique electronic, optoelectronic, optical and magnetic properties. Once disposed to the landfill, environmental weathering is likely to compromise HM Qdot integrity, leading to release of heavy metal ions. To minimize any negative environmental impact of HM Qdots, there is an increasing demand for developing HM free or environmentally-friendly surface modified HM Qdot alternatives. In this study, synthesis of HM free ZnS:Mn/ZnS and surface modified HM CdS:Mn/ZnS Qdots (using N-acetylcysteine, NAC, and Dihydrolipoic acid, DHLA) and their potential toxicity assessment using E. coli as a model system is reported. NAC and DHLA are known antioxidants and therefore expected to reduce HM induced toxicity and improve colloidal stability of Qdots. All Qdots were synthesized at room temperature using a reverse micelle microemulsion method. Qdots were fully characterized using UV-visible absorption spectroscopy, fluorescence emission spectroscopy, zeta potential, Nuclear Magnetic Resonance spectroscopy (NMR) and High Resolution Transmission Electron Microscopy (HRTEM). Qdot environmental weathering was simulated by treating Qdots with concentrated acid (6N HCl). Qdot toxicity was evaluated on E. coli growth and viability using growth curves, turbidity and bactericidal assays (CFU). Results show that Zn based Qdots exhibit reduced toxicity on E.coli growth and viability when compared to Cd based Qdots. In addition, surface modification with NAC and DHLA minimized toxicity of Cd based Qdots. In summary, Zn based Qdots appear to be more environmental-friendly than Cd based Qdots.
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Date Issued
2013
Identifier
CFE0005426, ucf:50416
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005426
Modulation of cholera toxin structure and function by host proteins
Title
Modulation of cholera toxin structure and function by host proteins.
Creator
Burress, Helen, Teter, Kenneth, Self, William, Zervos, Antonis, Tatulian, Suren, University of Central Florida
Abstract / Description
Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the...
Show moreCholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.
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Date Issued
2014
Identifier
CFE0005310, ucf:50511
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005310
Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis
Title
Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis.
Creator
Nelson, Samantha, Self, William, Moore, Sean, Rohde, Kyle, University of Central Florida
Abstract / Description
A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH),...
Show moreA previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.
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Date Issued
2014
Identifier
CFE0005388, ucf:50455
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005388
Role of host immune response and bacterial autolysin Atl in human nasal colonization by Staphylococcus aureus
Title
Role of host immune response and bacterial autolysin Atl in human nasal colonization by Staphylococcus aureus.
Creator
Paramanandam, Vanathy, Cole, Alexander, Naser, Saleh, Self, William, University of Central Florida
Abstract / Description
Staphylococcus aureus (SA) is a major human pathogen that colonizes the anterior nares in 30% of the human population. Though nasal carriage of SA is a known risk factor for the subsequent spread of SA infections, the dynamics of SA nasal colonization is poorly understood. Our research focuses on understanding the host and bacterial factors that might contribute to the human nasal colonization by SA. In an attempt to elucidate the host response to SA, we performed an autologous human in vivo...
Show moreStaphylococcus aureus (SA) is a major human pathogen that colonizes the anterior nares in 30% of the human population. Though nasal carriage of SA is a known risk factor for the subsequent spread of SA infections, the dynamics of SA nasal colonization is poorly understood. Our research focuses on understanding the host and bacterial factors that might contribute to the human nasal colonization by SA. In an attempt to elucidate the host response to SA, we performed an autologous human in vivo nasal colonization study, which showed decreased survival rates of SA in hosts who elicited a robust immune response. We also identified a significant correlation between SA nasal colonization and the expression of host proinflammatory, chemotactic and growth factors.Additionally, we functionally disrupted a major autolysin, atl a surface expressed bacterial protein that plays multiple roles in cell separation, adhesion and biofilm formation of SA. Microscopic analysis of the ?atl strains showed phenotypic differences, including cell clumping and cluster formation due to defective cell separation, which confirmed the functional loss of atl. Subsequent analysis of the ?atl and wild-type strains revealed that there was no significant difference in their ability to adhere to human nasal epithelial cells (hNEC) in an ex vivo hNEC model. Similarly, our competitive in vivo human nasal colonization study, in which equal colony-forming units of each wild-type and ?atl SA strain were inoculated in the anterior nares of donors, showed similar survival rates between wild-type and ?atl. These results suggest that Atl might not be directly involved in the adherence and colonization of SA to the anterior nares. Furthermore, our study suggests that host factors might play a predominant role in determining SA colonization to human anterior nares.
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Date Issued
2013
Identifier
CFE0005393, ucf:50463
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005393
Genetic and biochemical characterization of the roles of two putative purine transporters in the infectious cycle of Borrelia burgdorferi
Title
Genetic and biochemical characterization of the roles of two putative purine transporters in the infectious cycle of Borrelia burgdorferi.
Creator
Jain, Sunny, Jewett, Mollie, Naser, Saleh, Self, William, Vonkalm, Laurence, University of Central Florida
Abstract / Description
Lyme disease, the most common tick borne disease in United States, is caused by the bacterial pathogen Borrelia burgdorferi. In nature, B. burgdorferi exists in an enzootic infectious cycle between an arthropod vector and mammalian hosts. Identification and characterization of the genes essential for B. burgdorferi survival throughout its infectious cycle is an important step toward understanding the molecular mechanisms involved in B. burgdorferi pathogenesis. B. burgdorferi contains a small...
Show moreLyme disease, the most common tick borne disease in United States, is caused by the bacterial pathogen Borrelia burgdorferi. In nature, B. burgdorferi exists in an enzootic infectious cycle between an arthropod vector and mammalian hosts. Identification and characterization of the genes essential for B. burgdorferi survival throughout its infectious cycle is an important step toward understanding the molecular mechanisms involved in B. burgdorferi pathogenesis. B. burgdorferi contains a small genome, which lacks the genes encoding for the enzymes required for de novo synthesis of amino acids, fatty acids and nucleic acid precursors. Therefore, the spirochete is dependent upon the host environment for the uptake of these essential nutrients. Purines are required for the synthesis of nucleotides for the biosynthesis of DNA and RNA. Due to the lack of de novo purine synthesis, the ability of B. burgdorferi to salvage purines from its host environments is essential to its survival. While the enzymes critical for the B. burgdorferi purine salvage pathway are known, the transporters involved in the uptake of purines from the host environments are not. The work in this thesis is focused on identification of the genes encoding purine permeases in B. burgdorferi and genetic and biochemical characterization of their functions in the infectious cycle of B. burgdorferi. Here, we demonstrate that homologous genes bbb22 and bbb23 present on circular plasmid 26 encode for purine permeases, which are important for transport of hypoxanthine, adenine and guanine. Furthermore, genes bbb22-23 together were essential for B. burgdorferi infection in mice. BBB22 and BBB23 share 78% amino acid identify. And although, individually both BBB22 and BBB23 were found to be capable of purine transport, BBB22 has higher affinity for hypoxanthine and adenine compared to BBB23. Moreover, the bbb22 gene alone was sufficient to restore mouse infectivity to spirochetes lacking both bbb22 and bbb23, whereas, bbb23 was not. Nonetheless, the spirochete loads in the tissues of mice infected with B. burgdorferi carrying bbb22 alone were significantly reduced compared to B. burgdorferi carrying both bbb22 and bbb23, demonstrating the importance of the two genes together for the spirochetes to achieve wild type levels of infection. In ticks, genes bbb22 and bbb23 were dispensable for spirochete survival but contributed to spirochete replication in fed larvae. The replication of spirochetes lacking bbb22-23 in larval ticks was restored to wild type levels by the reintroduction of the low affinity purine transporter encoded by bbb23 alone. Overall, we have identified a purine transport system in B. burgdorferi, which is essential for spirochete survival in the mammalian host and contributes to spirochete replication in the tick vector. As B. burgdorferi lacks typical virulence factors and toxins, these studies highlight the critical role of physiological functions in the virulence of this pathogen. Moreover, the BBB22-23 in vivo essential transport system may represent a novel therapeutic target to deliver antimicrobial drugs to treat Lyme disease.
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Date Issued
2014
Identifier
CFE0005511, ucf:50303
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005511
Mixed Valence Copper(Cu)/Silica Nanocomposite: Synthesis, Characterization and Systematic Antimicrobial Studies
Title
Mixed Valence Copper(Cu)/Silica Nanocomposite: Synthesis, Characterization and Systematic Antimicrobial Studies.
Creator
Young, Mikaeel, Santra, Swadeshmukul, Self, William, Moore, Sean, University of Central Florida
Abstract / Description
Copper (Cu) compounds are widely used as effective agricultural bactericides. Continuous use of these materials has led to Cu accumulation in soil over time. The United States Environmental Protection Agency (US EPA) is concerned about potential Cu contamination in the environment. Improving biocidal efficacy of Cu is an attractive alternative, allowing reduction of Cu amount per application. In this research, we focused on making water-soluble mixed-valence Copper/Silica composite nanogel ...
Show moreCopper (Cu) compounds are widely used as effective agricultural bactericides. Continuous use of these materials has led to Cu accumulation in soil over time. The United States Environmental Protection Agency (US EPA) is concerned about potential Cu contamination in the environment. Improving biocidal efficacy of Cu is an attractive alternative, allowing reduction of Cu amount per application. In this research, we focused on making water-soluble mixed-valence Copper/Silica composite nanogel (CuSiNG) material. The objective is to improve the efficacy of Cu by manipulating Cu valence states. It has been shown in the literature that Cu (0) and Cu (I) states are more potent that Cu (II) states in terms of their antimicrobial efficacy. It is hypothesized that mixed valence Cu will exhibit improved efficacy over Cu (II). A water-soluble mixed valence Cu/silica nanogel (MV-CuSiNG) composite has been synthesized and characterized. Structure, morphology, crystallinity and composition of the MV-CuSiNG material was characterized using High-Resolution Transmission Electron Microscopy (HRTEM), HRTEM Selected Area Electron Diffraction (SAED) and X-ray Photoelectron Spectroscopy (XPS). Amount of Cu loading in MV-CuSiNG composite material was estimated by Atomic Absorption Spectroscopy (AAS). To confirm presence of Cu (I) in the MV-CuSiNG material, Neocuproine (Nc, a Cu (I) specific chelator) assay was used. Antimicrobial efficacy of MV-CuSiNG and CuSiNG was evaluated against X.alfalfae, B.subtilis and E.coli using Kocide(&)#174; 3000 ((")Insoluble Cu (II)(") compound), Copper sulfate ((")Soluble Cu (II)(") compound) and Cuprous chloride (Copper (I) compound) as positive controls and silica (")seed(") particles (without Cu loading) as negative control. Antimicrobial studies included observing bacterial growth inhibition and determining the Minimum Inhibitory Concentration (MIC). Improved antimicrobial efficacy was observed in MV-CuSiNG when compared to CuSiNG and other controls. For the assessment of plant safety of MV-CuSiNG and CuSiNG materials, phytotoxicity studies were conducted using Vinca sp and Hamlin orange under environmental conditions. It was observed that MV-CuSiNG material was safe to plants at commercially used (standard) spray application rate.
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Date Issued
2013
Identifier
CFE0005282, ucf:50550
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005282
Novel Immunogens of Cellular Immunity Revealed using in vitro Human Cell-Based Approach
Title
Novel Immunogens of Cellular Immunity Revealed using in vitro Human Cell-Based Approach.
Creator
Schanen, Brian, Self, William, Warren, William, Khaled, Annette, Seal, Sudipta, Zervos, Antonis, University of Central Florida
Abstract / Description
Nanotechnology has undergone rapid expansion largely as a result of its enormous potential for applications as biomaterials, drug delivery vehicles, cancer therapeutics, and immunopotentiators. Despite this wave of interest and broad appeal for nanoparticles, evidence of their effect to the human immune system remains scarce. Concerns rise as studies on nanoparticle toxicology continue to emerge indicating that nanomaterials can be acutely toxic and can have long term inflammatory effects as...
Show moreNanotechnology has undergone rapid expansion largely as a result of its enormous potential for applications as biomaterials, drug delivery vehicles, cancer therapeutics, and immunopotentiators. Despite this wave of interest and broad appeal for nanoparticles, evidence of their effect to the human immune system remains scarce. Concerns rise as studies on nanoparticle toxicology continue to emerge indicating that nanomaterials can be acutely toxic and can have long term inflammatory effects as seen in animal models. Based on these findings and the rise in the development of nanoparticle technologies targeting in vivo applications, the urgency to characterize nanomaterial immunogenicity is paramount.Nanoparticles harbor great potential because they possess unique physicochemical properties compared to their larger counter parts as a result of quantum-size effects and their inherent large surface area to volume ratio. These physicochemical properties govern how a nanoparticle will behave in its environment. However, researchers have only just begun to catalogue the biological effect these properties illicit. We took it upon ourselves to investigate nanoparticle size-induced effects using TiO2, one of the most widely manufactured nanoparticles, as a model. We studied these effects in dendritic cells across a human donor pool. We examined dendritic cells because they have an inimitable functional role bridging the innate and adaptive arms of immunity. From this work we found that TiO2 nanoparticles can activate human dendritic cells to become pro-inflammatory in a size-dependent manner as compared to its micron-sized counterpart, revealing novel immune cell recognition and activation by a crystalline nanomaterial.Having identified nanomaterial size as a contributing feature of nanoparticle induced immunopotentiation, we became interested if additional physicochemical properties such as surface reactivity or catalytic behavior could also be immunostimulatory. Moreover, because we witnessed a stimulatory effect to dendritic cells following nanoparticle treatment, we were curious how these nanoparticle-touched dendritic cells would impact adaptive immunity. Since TiO2 acts as an oxidant we chose an antioxidant nanoparticle, CeO2, as a counterpart to explore how divergent nanoparticle surface reactivity impacts innate and adaptive immunity. We focused on the effect these nanoparticles had on human dendritic cells and TH cells as a strategy towards defining their impact to cellular immunity. Combined, we report that TiO2 nanoparticles potentiate DC maturation inducing the secretion of IL-12p70 and IL-1?, while treatment with CeO2 nanoparticles induced IL-10, a hallmark of suppression. When delivered to T cells alone TiO2 nanoparticles induced stronger proliferation in comparison to CeO2 which stimulated TReg differentiation. When co-cultured in allogeneic T cell assays, the materials directed alternate TH polarization whereby TiO2 drives largely a TH1 dominate response, whereas CeO2 was largely TH2 bias. Combined, we report a novel immunomodulatory capacity of nanomaterials with catalytic activity. While unintentional exposure to these nanomaterials could pose a serious health risk, development and targeted use of such immunomodulatory nanoparticles could provide researchers with new tools for novel adjuvant strategies or therapeutics.
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Date Issued
2012
Identifier
CFE0004629, ucf:49927
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004629
Discovery and characterization of novel antimicrobials against Mycobacterium tuberculosis
Title
Discovery and characterization of novel antimicrobials against Mycobacterium tuberculosis.
Creator
Rodrigues Felix, Carolina, Rohde, Kyle, Jewett, Mollie, Self, William, Phanstiel, Otto, University of Central Florida
Abstract / Description
Tuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M....
Show moreTuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M. tuberculosis, therefore a long treatment time is required for stable cure without relapse. Poor patient compliance increases the emergence of drug resistant strains, posing yet another challenge for the eradication of TB. There is dire need for novel compounds targeting not only drug-resistant, but also dormant bacteria so as to effectively eliminate drug-resistant strains and also shorten treatment time. This requires compounds with novel modes of action and novel drug screening approaches which focus on dormant M. tuberculosis. In the current work a method was optimized which induces the dormant phenotype of M. tuberculosis in vitro allowing large scale screening of compounds against these tolerant bacteria. The high chemical diversity of marine natural products was explored to increase the chances of finding novel compounds with novel mechanisms of action. Additionally, gold-complexed scaffolds were examined for their putative ability to inhibit topoisomerase 1, which is a highly conserved and essential protein of mycobacteria, not currently targeted in classical treatment regimens. Several marine natural products were identified with selective bactericidal activity against dormant bacteria, emphasizing the powerful tool that was developed for drug screening. Moreover, the gold-complexes were also bactericidal against not only replicating and dormant bacilli, but also mycobacteria resistant to front-line TB drugs. Compounds characterized in this study represent a promising starting point for the development of novel TB therapeutics and discovery of new conditionally essential pathways of dormant bacteria.
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Date Issued
2017
Identifier
CFE0007294, ucf:52172
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007294
Development of in vitro point of care diagnostics (IVPCD) based on Aptamers integrated Biosensors.(&)nbsp;
Title
Development of in vitro point of care diagnostics (IVPCD) based on Aptamers integrated Biosensors.(&)nbsp;.
Creator
Saraf, Nileshi, Seal, Sudipta, Fang, Jiyu, Florczyk, Stephen, Dong, Yajie, Self, William, University of Central Florida
Abstract / Description
The global market for the medical diagnostic industry is worth 25 billion dollars in the United States and is expected to grow exponentially each year. Presently available methods for biodetection, such as immunoassays, chemiluminescence and fluorescent based assays are expensive, time consuming and require skilled labor with high-end instruments. Therefore, development of novel, passive colorimetric sensors and diagnostic technologies for detection and surveillance is of utmost importance...
Show moreThe global market for the medical diagnostic industry is worth 25 billion dollars in the United States and is expected to grow exponentially each year. Presently available methods for biodetection, such as immunoassays, chemiluminescence and fluorescent based assays are expensive, time consuming and require skilled labor with high-end instruments. Therefore, development of novel, passive colorimetric sensors and diagnostic technologies for detection and surveillance is of utmost importance especially in resource constrained communities. The present work focusses on developing novel and advanced in vitro biodiagnostic tools based on aptamer integrated biosensors for an early detection of specific viral proteins or small biomolecules used as potential markers for deadly diseases. Aptamers are short single stranded deoxyribonucleic acid (DNA) which are designed to bind to a specific target biomolecule. These are readily synthesized in laboratory and offers several advantages over antibodies/enzymes such as stable in harsh environment, easily functionalized for immobilization, reproducibility etc. These undergo conformational changes upon target binding and produces physical or chemical changes in the system which are measured as colorimetric or electrochemical signals. Here, we have explored the aptamer-analyte interaction on different platforms such as microfluidic channel, paper based substrate as well as organic electrochemical transistor to develop multiple compact, robust and self-contained diagnostic tools. These testing tools exhibit high sensitivity (detection limit in picomolar) and selectivity against the target molecule, require no sophisticated instruments or skilled labor to implement and execute, leading a way to cheaper and more consumer driver health care. These innovative platforms provide flexibility to incorporate additional or alternative targets by simply designing aptamers to bind to the specific biomolecule.
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Date Issued
2018
Identifier
CFE0007766, ucf:52388
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007766

Pages