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- Title
- EXPRESSION OF AN EPITOPE TAGGED TARP EFFECTOR IN CHLAMYDIA TRACHOMATIS.
- Creator
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Nguyen, Brenda, Jewett, Travis, University of Central Florida
- Abstract / Description
-
Previous studies performed on Chlamydia trachomatis have demonstrated how these obligate intracellular microbes invade host cells through the utilization of secreted effector proteins. One secreted effector called Tarp (translocated actin recruiting protein) is implicated in cytoskeleton rearrangements that promote bacterial entry into the host cell. The focus of our study is to create a plasmid that carries the tarP gene that when transcribed and translated from within Chlamydia trachomatis...
Show morePrevious studies performed on Chlamydia trachomatis have demonstrated how these obligate intracellular microbes invade host cells through the utilization of secreted effector proteins. One secreted effector called Tarp (translocated actin recruiting protein) is implicated in cytoskeleton rearrangements that promote bacterial entry into the host cell. The focus of our study is to create a plasmid that carries the tarP gene that when transcribed and translated from within Chlamydia trachomatis will generate a c-Myc epitope tagged Tarp. The tag will be used in future studies to track the progression of the protein through the infectious process and will allow us to distinguish this protein from the Tarp effector expressed from the endogenous wild type gene. The epitope-tagged Tarp expression plasmid will be used as a template to construct Tarp deletion mutants. The mutant forms will be created in regions that have been biochemically characterized and predicted to be important to the invasion process of the pathogen. Observations on the potential phenotypes of these mutants and the possibility of allelic exchange will also be pursued in the future.
Show less - Date Issued
- 2013
- Identifier
- CFH0004385, ucf:45022
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004385
- Title
- PG-VTV Biogenesis Requires ATP to Facilitate Phosphorylation of Syntaxin 17.
- Creator
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Saxena, Anika, Siddiqi, Shadab, Jewett, Travis, Tigno-Aranjuez, Justine, University of Central Florida
- Abstract / Description
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The uptake of cytotoxic free fatty acids (FFA) and their conversion to physiologically expedient triglycerides (TAG) which are later on assimilated to very low density lipoproteins (VLDL) is of utmost value among the multifarious tasks performed by the liver. Inflated concentration of VLDL in the blood stream directly correlates with the reinforcement of atherosclerosis. VLDL is synthesized in the hepatic endoplasmic reticulum (ER) and transported to the Golgi where it encounters several...
Show moreThe uptake of cytotoxic free fatty acids (FFA) and their conversion to physiologically expedient triglycerides (TAG) which are later on assimilated to very low density lipoproteins (VLDL) is of utmost value among the multifarious tasks performed by the liver. Inflated concentration of VLDL in the blood stream directly correlates with the reinforcement of atherosclerosis. VLDL is synthesized in the hepatic endoplasmic reticulum (ER) and transported to the Golgi where it encounters several alterations. It is then enclosed in distinct post-Golgi VLDL transport vesicles (PG-VTVs) and released into the blood. The data generated in our lab has proved the requirement of ATP for PG-VTV biogenesis however, ATP substitution with non-hydrolyzable ATP analogue (ATP?S) had no effect on this process. Therefore, the present study is based on the hypothesis that ATP mediated protein phosphorylation regulates PG-VTV biogenesis. First, hepatic subcellular organelles were isolated and their purity was determined by performing Western blot. A cell-free in vitro budding assay was performed in presence or absence of ATP, GTP and cytosol using 3[H]-TAG labelled hepatic Golgi to generate PG-VTVs. We performed Western blotting to confirm distinct protein phosphorylation at tyrosine residue during PG-VTV formation however, protein phosphorylation event did not occur when PG-VTV budding was blocked. Two-dimensional gel electrophoresis identified Syntaxin 17 (STX17) as the phosphorylated protein required for PG-VTV formation. ATP mediated phosphorylation of STX17 during biogenesis of PG-VTVs was confirmed by its presence on PG-VTVs. PG-VTV budding was found to be significantly reduced on performing budding assay using STX17 immunodepleted cytosol compared to positive control. RNAi mediated knockdown of STX17 in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3[H]-TAG liquid scintillation counter. Based on these results, it can be justified that STX17 plays a vital role in regulating PG-VTV budding and overall VLDL secretion from hepatocytes.
Show less - Date Issued
- 2018
- Identifier
- CFE0007094, ucf:51931
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007094
- Title
- AB Toxins: Recovery from Intoxication and Relative Potencies.
- Creator
-
Cherubin, Patrick, Teter, Kenneth, Naser, Saleh, Jewett, Travis, Zervos, Antonis, University of Central Florida
- Abstract / Description
-
AB-type protein toxins have a catalytic A subunit attached to a cell-binding B subunit. Ricin, Shiga toxin (Stx), exotoxin A, and diphtheria toxin are AB toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. Our overall goal is to help elucidate the cellular basis of intoxication for therapeutic development. According to the current model of intoxication, the effect of AB toxins is irreversible. To test this model, we...
Show moreAB-type protein toxins have a catalytic A subunit attached to a cell-binding B subunit. Ricin, Shiga toxin (Stx), exotoxin A, and diphtheria toxin are AB toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. Our overall goal is to help elucidate the cellular basis of intoxication for therapeutic development. According to the current model of intoxication, the effect of AB toxins is irreversible. To test this model, we developed a system that uses flow cytometry and a fluorescent reporter to examine the cellular potency of toxins that inhibit protein synthesis. Our data show that cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin exposure. This work challenges the prevailing model of intoxication by suggesting ongoing toxin delivery to the cytosol is required to maintain the inhibition of protein synthesis and ultimately cause apoptosis. We also used our system to examine the basis for the greater cellular potency of Stx1 in comparison to Stx2. We found that cells intoxicated with Stx1a behave differently than those intoxicated with Stx2: cells exposed to Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Additional experiments indicated the identity of the Stx B subunit is a major factor in determining the uniform vs. bimodal response to Stx subtypes. This work provides evidence explaining, in part, the differential toxicity between Stx1 and Stx2. Overall, our collective observations provide experimental support for the development of inhibitors and post-exposure therapeutics that restrict, but not necessarily block, toxin delivery to the host cell.
Show less - Date Issued
- 2019
- Identifier
- CFE0007613, ucf:52523
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007613
- Title
- Unraveling PDI and its Interaction with AB Toxins.
- Creator
-
Guyette, Jessica, Teter, Kenneth, Self, William, Jewett, Travis, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
Protein disulfide isomerase (PDI) is an essential endoplasmic reticulum (ER) protein that acts as both an oxidoreductase and chaperone. It exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin (Ctx), which is a unique property of PDI. This unfolding allows PDI to disassemble the Ctx holotoxin, which is required for Ctx activity. Here, we investigated the unfolding and refolding property of PDI and how this affects its...
Show moreProtein disulfide isomerase (PDI) is an essential endoplasmic reticulum (ER) protein that acts as both an oxidoreductase and chaperone. It exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin (Ctx), which is a unique property of PDI. This unfolding allows PDI to disassemble the Ctx holotoxin, which is required for Ctx activity. Here, we investigated the unfolding and refolding property of PDI and how this affects its interaction with bacterial toxins. PDI showed remarkable redox-linked conformational resilience that allows it to refold after being thermally stressed. Deletion constructs of PDI showed that both active domains play opposing roles in stability, and can both refold from an unfolded state, indicating that either domain could unfold during its interaction with Ctx. Its ability to refold suggests that the cycle of unfolding and refolding with Ctx is a normal mechanism that prevents protein aggregation. Disruption of this cycle with the polyphenol, quercetin-3-rutinoside, prevented the disassembly of Ctx, which blocked Ctx intoxication of cultured cells. Loss of PDI function was also found to inhibit intoxication with Escherichia coli heat-labile toxin but not with ricin and Shiga toxins. Toxin structure also contributes to efficiency of PDI binding and disassembly, which may explain the differential potencies between toxins. While Ctx and Ltx share similar structures, Ctx is more potent and efficiently disassembled compared to Ltx. We believe that PDI-mediated disassembly is the rate-limiting step in intoxication, thus dictating toxin potency. Overall, PDI can be targeted for a potential therapeutic for many bacterial toxins because of its unique unfolding properties and its key role in cell intoxication.
Show less - Date Issued
- 2019
- Identifier
- CFE0007646, ucf:52511
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007646
- Title
- Chlamydia trachomatis Transformants Show a Significant Reduction in Rates of Invasion upon Removal of Key Tarp Domains.
- Creator
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Parrett, Christopher, Jewett, Travis, Roy, Herve, Moore, Sean, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2016
- Identifier
- CFE0006159, ucf:51142
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006159
- Title
- Synthesis and Characterization of Core-Shell Zinc Silica Nanoparticles and Zinc Silica Nanogels for Agricultural Applications.
- Creator
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Berroth, Megan, Santra, Swadeshmukul, Moore, Sean, Jewett, Travis, University of Central Florida
- Abstract / Description
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Plant pathogens are a serious problem facing the agricultural industry today. Current methodologies use copper based biocides as the main form of defense. Unfortunately this can lead to damaging environmental effects and increased rates of antimicrobial resistance. In this study, antimicrobial activity of multiple alternative zinc-based nanoformulations were tested against three important plant pathogens: Xanthomonas alfalfae, Pseudomonas syringae, and Clavobacter michiganensis. Xanthomonas...
Show morePlant pathogens are a serious problem facing the agricultural industry today. Current methodologies use copper based biocides as the main form of defense. Unfortunately this can lead to damaging environmental effects and increased rates of antimicrobial resistance. In this study, antimicrobial activity of multiple alternative zinc-based nanoformulations were tested against three important plant pathogens: Xanthomonas alfalfae, Pseudomonas syringae, and Clavobacter michiganensis. Xanthomonas sub species cause Citrus canker, a devastating disease that affects millions of citrus trees worldwide while the latter two affect tomato crops. Materials synthesis was completed and the resulting nanoformulations were characterized by Atomic Absorption Spectroscopy, Scanning Electron Microscopy, High Resolution Transmission Electron Microscopy, and X-Ray Photoelectron Spectroscopy. The antimicrobial efficacy of the newly synthesized formulas and two commercially available products, Kocide 3000 (DuPont) and Nordox (Brandt), were determined by Minimum Inhibitory Concentration Assays followed by Bacterial Viability Assays. The subsequent data demonstrated a marketed difference in the way the antimicrobial agents acted upon the bacterial species. The core-shell zinc silica nanoparticles (C-SZnSiNP) proved to be ineffective, while the zinc silica nanogel (ZnSiNG) was as successful at killing the bacteria as the commercial products. This shows promise for a new alternative material with zinc at the forefront of the fight against plant pathogens.
Show less - Date Issued
- 2015
- Identifier
- CFE0006209, ucf:51099
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006209
- Title
- Synthesis and Characterization of Antimicrobial Non-Color Forming Silica-Silver Nanocomposite.
- Creator
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Bazata, Joshua, Santra, Swadeshmukul, Moore, Sean, Jewett, Travis, University of Central Florida
- Abstract / Description
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Silver has been utilized for its antimicrobial properties for thousands of years in a variety of fields, extending the shelf life of food and water, rendering eating utensils sanitary, and more recently in biomedical applications such as silver based antiseptic creams. While effective as an antimicrobial agent at very low concentrations ((&)#181;g/mL), silver imparts a strong color to objects it is incorporated into, due to its high plasmonic efficiency. The goal of this study was to...
Show moreSilver has been utilized for its antimicrobial properties for thousands of years in a variety of fields, extending the shelf life of food and water, rendering eating utensils sanitary, and more recently in biomedical applications such as silver based antiseptic creams. While effective as an antimicrobial agent at very low concentrations ((&)#181;g/mL), silver imparts a strong color to objects it is incorporated into, due to its high plasmonic efficiency. The goal of this study was to determine if incorporating silver nanoparticles into a silica matrix could reduce or eliminate the plasmonic signal, while retaining the antimicrobial effects of the silver nanoparticles.Citrate capped silver nanoparticles (AgNP) were synthesized using a borohydride reduction method as outlined by Zheng et. al., and incorporated into silica nanoparticles using a method adapted from Fleger et. al. To test the antimicrobial efficacy of these synthesized silica coated silver nanoparticles (SiAgNP), minimum inhibitory concentration testing at three time points, 1, 4, and 8 hours, was carried out against E. coli and S. aureus using broth microdilution and Alamar Blue as an indicator of microbial growth. Efficacy was judged against uncoated AgNP and aqueous silver nitrate (AgNO3) solutions at equivalent Ag concentrations. Silica nanoparticles (SiNP) were utilized as a negative control. Further antimicrobial characterization using a bacterial viability assay revealed a time dependent killing trend in the SiAgNP, suggesting a controlled release of Ag+ from within the silica matrix. Efficacy of the SiAgNP was determined to fall between the most effective antimicrobial form of silver tested, AgNO3, and least effective, AgNP. However, the SiAgNP material exhibited no visible plasmon peak when UV-Visible spectrophotometric readings were taken, as well as remaining colorless when coated onto a ceramic substrate. Zeta potential revealed a high degree of colloidal stability of the SiAgNP. TEM imaging studies were carried out, verifying the presence of Ag within and on the silica nanoparticles, as well as the crystalline structure of the uncoated AgNP. It was determined that coating AgNP synthesized through borohydride reduction with silica through a St(&)#246;ber synthesis mechanism yields a material with enhanced antimicrobial effects compared to AgNP, but with no detectable plasmon signal, effectively producing a non-color forming silver based antimicrobial.
Show less - Date Issued
- 2015
- Identifier
- CFE0006208, ucf:51097
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006208
- 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.
Show less - Date Issued
- 2017
- Identifier
- CFE0006785, ucf:51815
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006785
- 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.
Show less - Date Issued
- 2012
- Identifier
- CFE0004792, ucf:49748
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004792
- Title
- Characterization of Innate Immunity in the Female Reproductive Tract for the Prevention of HIV Acquisition.
- Creator
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Eade, Colleen, Cole, Alexander, Jewett, Travis, Naser, Saleh, Khaled, Annette, University of Central Florida
- Abstract / Description
-
Human immunodeficiency virus (HIV) infects 30 million people worldwide. In sub-Saharan Africa, the region most affected by HIV, women comprise 60% of the infected population. Heterosexual transmission is a major mode of viral acquisition, mandating further research of the process and prevention of HIV acquisition via the female reproductive tract (FRT). The FRT is a dynamic environment, protected by host immune mechanisms and commensal microbes. The disruption of either of these elements can...
Show moreHuman immunodeficiency virus (HIV) infects 30 million people worldwide. In sub-Saharan Africa, the region most affected by HIV, women comprise 60% of the infected population. Heterosexual transmission is a major mode of viral acquisition, mandating further research of the process and prevention of HIV acquisition via the female reproductive tract (FRT). The FRT is a dynamic environment, protected by host immune mechanisms and commensal microbes. The disruption of either of these elements can increase susceptibility to HIV. Accordingly, one common risk factor for HIV acquisition is the microbial shift condition known as bacterial vaginosis (BV), which is characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. Our model showed good agreement with clinical BV trends; we observed a distinct cytokine and human ?-defensin-2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. We next evaluated the effect of this interaction on HIV infection by investigating the soluble effectors secreted when FRT epithelial cells were cocultured with A. vaginae. We observed increased proviral activity mediated by secreted low molecular weight effectors, and determined that this activity was not likely mediated by cytokine responses. Instead, we identified a complex mixture containing several upregulated host proteins. Selected individual proteins from the mixture exhibited HIV-enhancing activity only when applied with the complex mixture of proviral factors, suggesting that HIV enhancement might be mediated by synergistic effects.In addition to characterizing the immune interactions that mediate the enhanced HIV acquisition associated with BV, we also evaluated the safety and efficacy of RC-101, a candidate vaginal microbicide being developed for the prevention of HIV transmission. RC-101 has been effective and well tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation. Here, we treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate compatibility of this microbicide with FRT tissue and microflora. RC-101 was well tolerated by host tissues and commensal vaginal bacteria, while BV-associated bacteria were inhibited by RC-101. By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection.
Show less - Date Issued
- 2013
- Identifier
- CFE0004677, ucf:49867
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004677
- Title
- Molecular Regulators of Post-Golgi VLDL Transport Vesicle (PG-VTV) Biogenesis.
- Creator
-
Riad, Aladdin, Siddiqi, Shadab, Jewett, Travis, Naser, Saleh, University of Central Florida
- Abstract / Description
-
Amongst its numerous functions, the liver is responsible for the synthesis and secretion of very low-density lipoprotein (VLDL). VLDL particles play the important role of facilitating the transport of lipids within the aqueous environment of the plasma; yet high plasma concentrations of these particles result in the pathogenesis of atherosclerosis, while low VLDL secretion from the liver results in hepatic steatosis. VLDL synthesis in the hepatocyte is completed in the Golgi apparatus, which...
Show moreAmongst its numerous functions, the liver is responsible for the synthesis and secretion of very low-density lipoprotein (VLDL). VLDL particles play the important role of facilitating the transport of lipids within the aqueous environment of the plasma; yet high plasma concentrations of these particles result in the pathogenesis of atherosclerosis, while low VLDL secretion from the liver results in hepatic steatosis. VLDL synthesis in the hepatocyte is completed in the Golgi apparatus, which serves as the final site of VLDL maturation prior to its secretion to the bloodstream. The mechanism by which VLDL's targeted transport to the plasma membrane is facilitated has yet to be identified. Our lab has identified this entity. Our findings suggest that upon maturation, VLDL is directed to the plasma membrane through a novel trafficking vesicle, the Post-Golgi VLDL Transport Vesicle (PG-VTV). PG-VTVs containing [3H] radiolabeled VLDL were generated in a cell-free in vitro budding assay for study. First, the fusogenic capabilities of PG-VTVs were established. Vesicles were capable of fusing with the plasma membrane and delivering the VLDL cargo for secretion in a vectorial manner. The next goal of our study is to characterize key regulatory molecular entities necessary for PG-VTV biosynthesis. A detailed analysis was undertaken to determine the PG-VTV proteome via western blot and two-dimensional difference in gel electrophoresis. The identification of key molecular regulators will potentially offer therapeutic targets to control VLDL secretion to the bloodstream.
Show less - Date Issued
- 2013
- Identifier
- CFE0005236, ucf:50602
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005236
- Title
- Manipulation of host signal transduction pathways and cytoskeleton functions by invasive bacterium Listeria monocytogenes and Chlamydia trachomatis.
- Creator
-
Jiwani, Shahanawaz, Jewett, Travis, Zervos, Antonis, Khaled, Annette, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Infectious disease remains one of the leading causes of morbidity and mortality worldwide. Many bacteria that cause disease have the capacity to enter into eukaryotic cells such as epithelial cells and tissue macrophages. Gaining access into the intracellular environment is one of the most critical steps in their survival and/or in pathogenesis. The entry mechanisms employed by these organisms vary considerably, but most mechanisms involve sabotaging and manipulating host cell functions....
Show moreInfectious disease remains one of the leading causes of morbidity and mortality worldwide. Many bacteria that cause disease have the capacity to enter into eukaryotic cells such as epithelial cells and tissue macrophages. Gaining access into the intracellular environment is one of the most critical steps in their survival and/or in pathogenesis. The entry mechanisms employed by these organisms vary considerably, but most mechanisms involve sabotaging and manipulating host cell functions. Invasion of epithelial cells involves triggering host signal transduction mechanisms to induce cytoskeleton rearrangement, thereby facilitating bacterial uptake. My work focuses on understanding the molecular mechanisms employed by bacterial pathogen Listeria monocytogenes and Chlamydia trachomatis to gain access into the host cells in order to cause the disease.In first part of my thesis I investigated the mechanism of Listeria monocytogenes entry. Listeria, a facultative intracellular organism, is responsible for causing meningitis, septicemia, gastroenteritis and abortions. Critical for Listeria virulence is its ability to get internalized, replicates and spread into adjacent host cells. One of the pathways of Listeria internalization into mammalian cells is promoted by binding of its surface protein Internalin B (InlB) to host receptor MET. Studies done in the past demonstrated a critical role of host type IA Phosphoinositide (PI) 3-kinase in controlling cytoskeleton rearrangement and entry of Listeria downstream of MET. An important unresolved question was how activation of PI3K results in cytoskeleton rearrangements that promote Listeria entry. In this work, we identified 9 host signaling molecules, that includes Rab 5c, SWAP 70, GIT1, PDK1, mTor, ARAP2, ARNO, DAPP1 (&) PKC-?, acting downstream of type IA Phosphoinositide (PI) 3-kinase to regulate changes in host cytoskeleton to cause Listeria entry.Second part of my thesis involved studying the functions of chlamydial effector protein Tarp in its invasion. Infection caused by Chlamydia Trachomatis is the most common sexually transmitted disease resulting in uro-genital diseases, LGV, ectopic pregnancy and infertility. It is also responsible for causing trachoma, the leading cause of preventable blindness in third world countries. Being an obligate intracellular pathogen, gaining access into intracellular environment is the most critical step in lifecycle and pathogenesis of Chlamydia. Previous studies demonstrate the role of both chlamydial and host actin nucleators, Tarp and Arp2/3 complex respectively, in mediating Chlamydial entry into non-phagocytic cells. But the molecular details of these processes were not well understood. In this study, we demonstrate novel function of Tarp protein to form actin bundles by its ability to bind filamentous actin through newly identified FAB domains. And we also provide bio-chemical evidence that Tarp and Arp2/3 complex works in conjunction to cause changes in host cytoskeleton that effectively culminate into bacterial uptake by host cells.Overall, this research was a significant step in enhancing our understanding, at a molecular level, to pathogenesis of infections caused by Listeria monocytogenes and Chlamydia trachomatis.
Show less - Date Issued
- 2012
- Identifier
- CFE0004555, ucf:49225
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004555
- Title
- Development of Molecular Diagnostic Tools for Mycobacterium Species.
- Creator
-
Bengtson, Hillary, Kolpashchikov, Dmitry, Rohde, Kyle, Self, William, Jewett, Travis, Masternak, Michal, University of Central Florida
- Abstract / Description
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This dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and...
Show moreThis dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and the leading cause of infectious disease related deaths worldwide. The control of TB is limited by the lack of sensitive and specific diagnostic tools available at the point of care (POC). The studies presented here illustrate the advances in our technology for the detection and differentiation of M.tb and NTM. The use of BiDz sensors enables the selective recognition of DNA/RNA analytes containing single nucleotide polymorphisms associated with species-specific identification, drug susceptibility testing (DST) and strain typing. First, we developed a platform for the detection of M.tb and drug susceptibility using multiplex PCR and BiDz sensors. However, this method relies on the use of expensive instrumentation which is often not available in high TB burden countries. Therefore, additional studies focused on the development of tools for the detection of isothermal amplification products and the direct detection of rRNA. Based on these findings, we also developed an NTM species typing tool using BiDz sensors for species identification in ~1 hour. Despite the advantages of BiDz sensor technology, their use is limited to the detection of a few selected mutations. To address this limitation, we developed a 15-loci multiplex PCR followed by analysis with a custom microarray for high-throughput identification of SNPs. The work presented in this dissertation has the potential to enable the rapid, specific and sensitive identification of mycobacterial species necessary to reduce the diagnostic delay, ensure initiation of effective therapy, and prevent further transmission.
Show less - Date Issued
- 2017
- Identifier
- CFE0006856, ucf:51735
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006856
- Title
- The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin.
- Creator
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Bader, Carly, Teter, Kenneth, Zervos, Antonis, Jewett, Travis, Tatulian, Suren, University of Central Florida
- Abstract / Description
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Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much...
Show moreCholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gs? target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals.The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs?, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of post-intoxication treatment options.
Show less - Date Issued
- 2013
- Identifier
- CFE0004810, ucf:49759
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004810
- Title
- Dubious role of Mycobacterium paratuberculosis in pathogenesis of Type I diabetes.
- Creator
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Thanigachalam, Saisathya, Naser, Saleh, Singla, Dinender, Siddiqi, Shadab, Jewett, Travis, University of Central Florida
- Abstract / Description
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INTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto...
Show moreINTRODUCTION: Type 1 Diabetes Mellitus (T1DM) is a chronic disorder with unknown etiology and associated with insulin deficiency. Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis in cattle, has been implicated in many autoimmune diseases including Crohn's disease, TIDM and others. We hypothesize that the molecular mimicry including epitope homology between MAP-Hsp65 and pancreatic Glutamic Acid Decarboxylase65 (GAD65) may play a role in the auto destruction of pancreatic beta cells leading to insufficient insulin production and the development of TIDM, following exposure to MAP. METHODOLOGY: Peptide sequences of MAP-Hsp65 and GAD65 were analyzed using BLAST and PyMOL bioinformatics tools. Cross reactivity between the two proteins were evaluated using immunoblot and ELISA. Furthermore, coded EDTA blood samples were collected from 18 subjects (12 DM and 6 controls) and investigated for the presence or exposure to MAP. Peripheral leukocytes were investigated for harboring viable MAP using long-term culture followed by nested PCR. Clinical plasma samples were used for measurement of anti-MAP IgG titer as well as glucose and Insulin concentrations. Moreover, coded bovine sera from 100 cattle (50 MAP infected and 50 healthy) were investigated for possible correlation between MAP infection and plasma levels of glucose and insulin. RESULT: Peptide BLAST analysis revealed a 44% identity between MAP Hsp65 and GAD65 proteins with 75% positive identities in a 16 amino acid region. PyMOL 3-D structural analyses identified a shared epitope region within the 16 amino acid motif which is known to be an antigenic site on GAD65 antigen. MAP DNA and anti-MAP IgG were detected in the blood of TD8, a TIDM subject. Strong cross reactivity was observed between plasma from TD8 and MAP Hsp65 in proteins samples from M. tuberculosis, and E. coli recombinant clone expressing MAP Hsp65. A weak cross reactivity was also observed between rat pancreatic tissue homogenate and rabbit anti-MAP IgG. Long term culture of leukocytes from 18 blood samples resulted in the detection of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects whereas anti-MAP IgG were detected in 5/10 (50%) TIDM samples compared to 3/8 (37.5 %) controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle (P(<)0.0001). CONCLUSION: We identified and confirmed a shared epitope region between MAP Hsp65 and human pancreatic GAD65. The shared epitope is a known antigenic binding site. Although MAP DNA was detected in both TIDM and control subjects, a strong correlation was found between anti-MAP IgG titer and MAP-positive culture in clinical samples, regardless of diagnosis. The correlation between MAP infection and insulin level in cattle is significant. Overall the result is intriguing and requires further investigation of MAP in well-characterized clinical samples.
Show less - Date Issued
- 2012
- Identifier
- CFE0004608, ucf:49924
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004608