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APTAMERIC SENSORS: IN VITRO SELECTION OF DNA THAT BINDS BROMOCRESOL PURPLE
Title
APTAMERIC SENSORS: IN VITRO SELECTION OF DNA THAT BINDS BROMOCRESOL PURPLE.
Creator
Miller, Derek B, Kolpshchikov, Dmitry, University of Central Florida
Abstract / Description
Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow's diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today's aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye...
Show moreAptamers being used as sensors is an emerging field that has capabilities of being tomorrow's diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today's aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to purple or vice versa. Previous studies have shown albumin contains the ability to facilitate this role and we now intend to use a DNA aptamer to achieve this as well. The BCP aptamer has the potential to serve as a signaling domain to any already selected aptamer thus making it a universal tool for both research and diagnostic measures. We have found that an alternative structure-switching systematic evolution of ligands by exponential enrichment (SELEX) method which left the dye unaltered was not sufficient for selecting an aptamer. We believe that a classical SELEX will enable us to select an aptamer that may be used to accomplish this role as a universal visual detector.
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Date Issued
2016
Identifier
CFH2000112, ucf:45951
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000112
FMF assay for assessing vaccine generated antibodies in a biomimetic manner
Title
FMF assay for assessing vaccine generated antibodies in a biomimetic manner.
Creator
Dhir, Vipra, Khaled, Annette, Self, William, Warren, William, University of Central Florida
Abstract / Description
Traditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to...
Show moreTraditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to study Influenza vaccine response in a more biomimetic manner and overcome the deficiencies of the traditional functional assays, we developed a fluorescent membrane fusion assay (fMF). The assay uses viruses labeled with Octadecyl Rhodmaine B Chloride (R18) to monitor two major neutralization pathways: blocking the attachment of virus to the target cells and blocking of virus membrane fusion in the endosomes. The latter was tested using endosomal acidification inhibitor Bafilomycin a1 which blocked membrane fusion by 85%. Specificity of the assay was tested using two different subtypes of viruses H1N1 (A/Puerto Rico/8/1934 and A/Brisbane/59/2007), and H3N2 virus (A/Aichi/68) with their respective subtype specific stem specific monoclonal antibodies: M145, Aca-1, Aca-2 (H1N1 specific) and Aca-3 (H3N2 specific). Subtype specific mAbs blocked membrane fusion, while a mismatch in virus subtype and the mAb resulted in lack of blocking. We also studied the effect of H1N1 head specific mAb Aca-4, which not only blocked attachment of the virus, but also demonstrated blocking of membrane fusion. Results were validated by testing pre- and post- sera from 2009 seasonal Influenza vaccination and to show that at higher Ab concentration the majority of virus (85%) was blocked from attaching cells, but at lower Ab concentration, where attachment could not be prevented, blocking of membrane fusion was still in effect - up to 50%. Sera screening experiments showed that sera antibodies work beyond just blocking attachment. They also may neutralize the already attached virus by blocking fusion of the viral membrane in the endosomes. The assay has the capacity to monitor blocking of attachment and fusion in a single run. Therefore, it is more representative regarding the natural process of infection and the corresponding neutralization pathways. The assay is unique in terms of assessing stem specific antibodies; stem specific response and its measurement are relevant for the advancement of a universal influenza vaccine.
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Date Issued
2015
Identifier
CFE0005604, ucf:50255
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005604
EXPRESSION OF CHOLERA TOXIN B SUBUNIT-ROTAVIRUS NSP4 ENTEROTOXIN FUSION PROTEIN IN TRANSGENIC CHLOROPLASTS
Title
EXPRESSION OF CHOLERA TOXIN B SUBUNIT-ROTAVIRUS NSP4 ENTEROTOXIN FUSION PROTEIN IN TRANSGENIC CHLOROPLASTS.
Creator
Kalluri, Anila, Daniell, Henry, University of Central Florida
Abstract / Description
Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae family and is considered to be the single most important cause of virus-based severe diarrheal illness in infants and young children particularly 6 months to 2 years of age in industrialized and developing countries. Infection in infants and young children is often accompanied by severe life threatening diarrhea, most commonly following primary infection. Diarrhea is the major cause of...
Show moreRotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae family and is considered to be the single most important cause of virus-based severe diarrheal illness in infants and young children particularly 6 months to 2 years of age in industrialized and developing countries. Infection in infants and young children is often accompanied by severe life threatening diarrhea, most commonly following primary infection. Diarrhea is the major cause of death among children around the world. Responsible for 4 to 6 million deaths per year according to the World Health Organization (WHO), diarrhea is especially dangerous for infants and young children. Globally, it is estimated that 1.4 billion episodes of diarrhea occur in children less than five years of age annually. In the United States alone, rotavirus causes more than 3 million cases of childhood diarrhea each year, leading to an estimated 55,000 to 100,000 hospitalizations and 20 to 100 deaths. And is a major cause of mortality for children in developing countries with approximately one million deaths annually. Rotaviruses belong to the family Reoviridae and are spherical 70-nm particles. The virus genome contains 11 segments of double-stranded RNA, each encoding a viral capsid or nonstructural protein. The identification of a rotavirus nonstructural protein gene (NSP4) encoding a peptide, which functions both as a viral enterotoxin and as a factor involved in the acquisition of host cell membrane during virus budding from cells, provides a new approach for mucosal immunization. Protein expression through chloroplast transformation system offers a number of advantages like high level of transgene expression, transgene containment via maternal inheritance, lack of gene silencing and position effect due to site specific gene integration and also the possibility of multi gene engineering in single transformation event. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen. To achieve an enhanced immune response to rotavirus infection, a fusion gene encoding the cholera toxin B subunit linked to rotavirus enterotoxin 90 aa protein (CTB-NSP490) was introduced into transgenic chloroplast and was transformed into chloroplast genome of Nicotiana tabacum by homologous recombination. The chloroplast integration of CTB-NSP4(90) fusion gene was confirmed in transgenic tobacco plants by PCR analysis. Southern blot analysis further confirmed site specific gene integration and homoplasmy. Immunoblot analysis of transformed chloroplast confirmed the expression of CTBNSP490 fusion protein both in monomeric and pentameric forms that retained the binding affinity to the enterocytes GM1 ganglioside receptor. Expression levels of CTB-NSP4 protein was quantified by GM1 ganglioside binding ELISA assay; mature leaves expressed CTB-NSP4 fusion protein to upto 2.45 % in total soluble protein, 100-400 fold higher than nuclear expression which was only 0.006%-0.026%. Antibody titration and virus challenge experiments will be performed in mice at Loma Linda University to evaluate the antigenic and protective properties of the chloroplast derived CTB-NSP4 fusion protein.
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Date Issued
2005
Identifier
CFE0000655, ucf:46540
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000655
IDENTIFICATION OF SMALL MOLECULES THAT INHIBIT PROSTATE CANCER CELL PROLIFERATION
Title
IDENTIFICATION OF SMALL MOLECULES THAT INHIBIT PROSTATE CANCER CELL PROLIFERATION.
Creator
Zelaya, Rainel, Chakrabarti, Ratna, University of Central Florida
Abstract / Description
Prostate cancer is the second most often diagnosed cancer and internationally the sixth foremost cause of cancer death in males, as of 2011. Within the United States it is the most common form of cancer in men with 186,000 new cases and with an overall 28,600 deaths in 2008, and it is the second leading kind of cancer-related death in men. The widespread threat that prostate cancer poses against men across the globe cannot be understated, and its initiation and progression must be understood...
Show moreProstate cancer is the second most often diagnosed cancer and internationally the sixth foremost cause of cancer death in males, as of 2011. Within the United States it is the most common form of cancer in men with 186,000 new cases and with an overall 28,600 deaths in 2008, and it is the second leading kind of cancer-related death in men. The widespread threat that prostate cancer poses against men across the globe cannot be understated, and its initiation and progression must be understood in order to truly comprehend its implicated risks and possible forms of treatment. As its name implies, prostate cancer is a form of cancer that develops in the prostate gland located in the male reproductive system. Its progress starts when standard semen-secreting prostate gland cells mutate into cancer cells. Although its developments may start at the prostate gland, cancer cells may metastasize to other parts of the body through circulation systems such as the lymph nodes. The main sites of metastasis for prostate cancer include the adrenal gland,the bones, the liver and the lungs. Although there are treatments available for prostate cancer, there is no definitive cure. The primary goal of this project was to find an alternative form of treatment, which is what will be necessary to combat this cancer.
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Date Issued
2014
Identifier
CFH0004595, ucf:45228
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004595
A Solid Phase Assay for Topoisomerase I interfacial Poisons and Catalytic Inhibitors
Title
A Solid Phase Assay for Topoisomerase I interfacial Poisons and Catalytic Inhibitors.
Creator
Cyril Sagayaraj, Vidusha, Muller, Mark, Zhao, Jihe, Chakrabarti, Debopam, University of Central Florida
Abstract / Description
We report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic...
Show moreWe report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a 'bimodal' readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. In addition to specific topoisomerase targeting drugs, the method also weakly detects other relevant anticancer agents, such as potent DNA alkylating and intercalating compounds; therefore, topoisomerase I HTS represents an excellent tool for searching and identifying novel genotoxic agents. This method is rapid, robust, economical and scalable for large library screens.
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Date Issued
2011
Identifier
CFE0004473, ucf:49304
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004473
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
A Preliminary Assessment of Steroid Reproductive Hormones in Archaeological Human Hair Utilizing a Modified Enzyme-Linked Immunosorbent Assay (ELISA) Technique
Title
A Preliminary Assessment of Steroid Reproductive Hormones in Archaeological Human Hair Utilizing a Modified Enzyme-Linked Immunosorbent Assay (ELISA) Technique.
Creator
Tisdale, Elisha, Schultz, John, Williams, Lana, Wheeler, Sandra, University of Central Florida
Abstract / Description
Hair has become an invaluable resource in forensic, clinical, and bioarchaeological research. The unique interaction between the growing hair fiber, the hair follicle, and the endocrine system inundates the growing hair fiber with an incremental record of many of the discreet physiological processes of the body. Recently, a novel study by Webb et al. (2010) demonstrated that endogenous records of cortisol, the (")stress hormone("), are capable of being extracted from archaeological human hair...
Show moreHair has become an invaluable resource in forensic, clinical, and bioarchaeological research. The unique interaction between the growing hair fiber, the hair follicle, and the endocrine system inundates the growing hair fiber with an incremental record of many of the discreet physiological processes of the body. Recently, a novel study by Webb et al. (2010) demonstrated that endogenous records of cortisol, the (")stress hormone("), are capable of being extracted from archaeological human hair through a modified enzyme-linked immunosorbent assay (ELISA) technique, thus providing insight into the (")invisible(") stress experiences of an individual that would otherwise not be detectable through skeletal analysis. The present study seeks to apply this novel ELISA technique to archaeological hair to determine whether endogenous patterns of secretion are detectable for the steroid reproductive hormones estradiol and testosterone. Here, hair from 10 individuals from the Kellis 2 cemetery in the Dakhleh Oasis, Egypt is analyzed for endogenous concentrations of the steroid hormones cortisol, estradiol, and testosterone. A control sample consisting of hair from 10 modern cadavers is also assessed for each hormone to ensure method efficacy. Cortisol, estradiol, and testosterone were successfully identified in all 10 archaeological individuals and in each of the 10 individuals in the modern control group. Results revealed that archaeological preservation of each hormone was favorable, and incremental patterning of each hormone seem to reflect endogenous hormone secretion in life. Values for cortisol, estradiol in pre-menopausal females, and testosterone extracted from the archaeological and modern control samples fall within reference values taken from archaeological and clinical research; however, estradiol values for males and postmenopausal females exceeded projected reference values. Explorations for variables which could contribute to discrepancies between reported and observed estradiol values are provided, along with two case studies on female individuals from the archaeological sample. The results of this study demonstrate that steroid reproductive hormones can be preserved in archaeological human hair, and that these hormones can be analyzed to create additional lines of inquiry into bioarcheological studies of ancient health and fertility.
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Date Issued
2017
Identifier
CFE0006921, ucf:51700
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006921
Chlamydia trachomatis Transformants Show a Significant Reduction in Rates of Invasion upon Removal of Key Tarp Domains
Title
Chlamydia trachomatis Transformants Show a Significant Reduction in Rates of Invasion upon Removal of Key Tarp Domains.
Creator
Parrett, Christopher, Jewett, Travis, Roy, Herve, Moore, Sean, University of Central Florida
Abstract / Description
Chlamydia trachomatis is an obligate, intracellular bacterium which is known to cause multiple human infections including nongonococcal urethritis (serovars D-K), lymphogranuloma venereum (serovars L1, L2, L3) and trachoma (serovars A-C). The infectious form of the bacterium, called the elementary body (EB), harbors a type III secreted effector known as Tarp (translocated actin recruiting phosphoprotein) which is a candidate virulence factor and is hypothesized to play a role in C....
Show moreChlamydia trachomatis is an obligate, intracellular bacterium which is known to cause multiple human infections including nongonococcal urethritis (serovars D-K), lymphogranuloma venereum (serovars L1, L2, L3) and trachoma (serovars A-C). The infectious form of the bacterium, called the elementary body (EB), harbors a type III secreted effector known as Tarp (translocated actin recruiting phosphoprotein) which is a candidate virulence factor and is hypothesized to play a role in C. trachomatis' ability to invade and grow within epithelial cells in a human host. C. trachomatis L2 Tarp harbors five unique protein domains which include the Phosphorylation Domain, the Proline Rich Domain, the Actin Binding Domain, and two F-Actin Binding Domains. Tarp has been biochemically characterized in vitro, but it has yet to be characterized in vivo due to a lack of genetic tools in C. trachomatis. Through the recent generation of a chlamydial transformation system, we have created transformants which express epitope tagged wild type or mutant Tarp effectors. In this thesis, C. trachomatis transformants expressing Tarp lacking one of the five biochemically defined protein domains were used to examine both bacterial invasion and bacterial development within mammalian host cells. Our results demonstrate that those EBs which harbor mutant Tarp missing either its Phosphorylation Domain or its Actin Binding Domain were less capable of host cell invasion. However, these transformants, once internalized, were capable of normal development when compared to wild type C. trachomatis or C. trachomatis harboring an epitope tagged wild type Tarp effector. These results suggest that transformant expressed Tarp lacking the Phosphorylation Domain or Actin Binding Domain may be acting as a dominant-negative effector protein. Ultimately, these results support the hypothesis that Tarp is a virulence factor for Chlamydia trachomatis. Furthermore, this data indicates that through the manipulation of the Tarp effector, C. trachomatis pathogenesis may be attenuated.
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Date Issued
2016
Identifier
CFE0006159, ucf:51142
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006159