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(1 - 7 of 7)
- Title
- Manipulation of host signal transduction pathways and cytoskeleton functions by invasive bacterium Listeria monocytogenes and Chlamydia trachomatis.
- Creator
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Jiwani, Shahanawaz, Jewett, Travis, Zervos, Antonis, Khaled, Annette, Teter, Kenneth, University of Central Florida
- Abstract / Description
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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
- 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
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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
- The Actin-Severing Protein Cofilin Is Downstream Of Neuregulin Signaling, Is Regulated By The Tumor Suppressor Merlin, And Is Essential For Schwann Cell Myelination.
- Creator
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Sparrow, Nicklaus, Fernandez-Valle, Cristina, Lambert, Stephen, Ebert, Steven, Altomare, Deborah, University of Central Florida
- Abstract / Description
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Myelination is a complex process requiring coordination of directional motility and an increase in Schwann cell (SC) size to generate a multi-lamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin1-type III (NRG) expressed on the axon surface. NRG binding to ErbB2/3 receptors on the Schwann cell surface initiates signaling cascades necessary for myelination. We identify...
Show moreMyelination is a complex process requiring coordination of directional motility and an increase in Schwann cell (SC) size to generate a multi-lamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin1-type III (NRG) expressed on the axon surface. NRG binding to ErbB2/3 receptors on the Schwann cell surface initiates signaling cascades necessary for myelination. We identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG-ErbB2/3 signaling in rat SC. A five minute exposure of SCs to NRG triggers phosphorylation of ErbB2 with concomitant dephosphorylation, and activation, of cofilin, and its upstream regulators, LIM domain kinase (LIMK) and Slingshot-1 phosphatase (SSH). This leads to cofilin activation and recruitment to the leading edge of the SC plasma membrane. These changes are associated with rapid plasma membrane expansion yielding a 35(-)50% increase in SC size within 30 minutes of NRG1 exposure. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs co-cultured with sensory neurons fail to elaborate myelin. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 hours of co-culturing with neurons, cofilin-deficient SCs fail to align and elongate on axons and often adhere to the underlying substrate rather than to axons. We show that the Neurofibromatosis Type II (NF2) tumor suppressor, merlin, is an upstream regulator of cofilin1, and that merlin knockdown in Schwann cells inhibits their elaboration of normal myelin sheaths in vitro. Merlin-deficient SCs form shorter myelin segments in DRG neuron/SC co-cultures. Merlin-deficient Schwann cells have increased levels of both active Rac (Rac-GTP) and F-actin indicative of a stable actin cytoskeleton. Surprisingly merlin-deficient Schwann cells fail to dephosphorylate and activate cofilin1 in response to NRG stimulation. Inhibition of LIMK restores the ability of merlin-deficient SCs to activate cofilin in response to NRG. In developing rat sciatic nerve, merlin becomes hyper-phosphorylated at S518 during the time of peak myelin formation. During this time, cofilin is localized to the inner mesaxon, and subsequently to Schmidt-Lanterman incisures in mature myelin. This study: 1) identifies cofilin and its upstream regulators, LIMK and SSH, as end targets of a NRG-ErbB2/3 signaling pathway in Schwann cells, 2) demonstrates that cofilin modulates actin dynamics in Schwann cells allowing for motility needed to effectively engage and myelinate axons, 3) shows that merlin regulates NRG-ErbB2/3-cofilin-actin signaling during SC myelination to determine the myelin segment length.
Show less - Date Issued
- 2017
- Identifier
- CFE0006664, ucf:51217
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006664
- Title
- Mechanism of Actin Bundle Assembly, Mechanics and Structure by Ion Interaction.
- Creator
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Castaneda, Nicholas, Kang, Hyeran, Huo, Qun, Gesquiere, Andre, University of Central Florida
- Abstract / Description
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The assembly of actin filaments into bundles plays an essential role in mechanical strength and dynamic reorganization of cytoskeleton. Divalent counterions at high concentrations promote bundle formation through electrostatic attraction between charged filaments. Although it has been hypothesized that specific cation interactions may contribute to salt-induced bundling, molecular mechanisms of how salt modulates bundle assembly and mechanics are not well established. Here we determine the...
Show moreThe assembly of actin filaments into bundles plays an essential role in mechanical strength and dynamic reorganization of cytoskeleton. Divalent counterions at high concentrations promote bundle formation through electrostatic attraction between charged filaments. Although it has been hypothesized that specific cation interactions may contribute to salt-induced bundling, molecular mechanisms of how salt modulates bundle assembly and mechanics are not well established. Here we determine the mechanical and dynamic properties of actin bundles with physiologically relevant cations. Using total internal reflection fluorescence (TIRF) microscopy, we measure the bending stiffness of actin bundles determined by persistence length analysis. We characterize real-time formation of bundles by dynamic light scattering intensity and direct visualization using TIRF microscopy. Our results show that divalent cations modulate bundle stiffness as well as time-dependent average bundle size. Furthermore, molecular dynamic simulations propose specificity for cation binding on actin filaments to form bundles. The work suggests that cation interactions serve a regulatory function in bundle assembly dynamics, mechanics, and structure.
Show less - Date Issued
- 2017
- Identifier
- CFE0006572, ucf:51307
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006572
- Title
- GENETIC ANALYSIS OF RHOA SIGNALING DURING EPITHELIAL MORPHOGENESIS IN DROSOPHILA.
- Creator
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Leppert, Amanda Fitch, von Kalm, Laurence, University of Central Florida
- Abstract / Description
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Epithelial morphogenesis is contingent upon cell shape changes. Cell shape changes are the driving force for the metamorphosis of the adult Drosophila leg from the leg imaginal disc precursor. Genetic analysis has identified several Drosophila genes involved in regulating cell shape changes during leg disc morphogenesis. These include members of the RhoA signaling pathway and the product of the Stubble-stubbloid (Sb-sbd) locus, a transmembrane serine protease. Mutations in the Sb-sbd gene...
Show moreEpithelial morphogenesis is contingent upon cell shape changes. Cell shape changes are the driving force for the metamorphosis of the adult Drosophila leg from the leg imaginal disc precursor. Genetic analysis has identified several Drosophila genes involved in regulating cell shape changes during leg disc morphogenesis. These include members of the RhoA signaling pathway and the product of the Stubble-stubbloid (Sb-sbd) locus, a transmembrane serine protease. Mutations in the Sb-sbd gene interact genetically with the members of the RhoA signaling pathway, however the nature of the relationship between Sb-sbd serine protease activity and RhoA signaling is not understood.To identify additional components of the RhoA signaling pathway that may help us to understand the role of the Sb-sbd protease in RhoA signaling the Drosophila genome was systematically scanned for genes that interact with Sb-sbd and RhoA mutations using deletions/deficiencies of specified regions of each chromosome. A total of 201 deficiencies uncovering approximately 84.9-91% of the euchromatic genome and spanning the X, second, and third chromosoms were tested. Of the 201 deficiencies tested, five putative interacting genetic regions and one gene within these deficiencies were identified. The candidate gene Eip78C encodes a nuclear steroid hormone receptor previously identified as having an important role in metamorphosis.
Show less - Date Issued
- 2004
- Identifier
- CFE0000046, ucf:46104
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000046
- Title
- Nano-Biophysical Approaches for Assessing Nanoparticle Interactions with Biological Systems.
- Creator
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Untracht, Zachary, Kang, Ellen, Gesquiere, Andre, Santra, Swadeshmukul, University of Central Florida
- Abstract / Description
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Understanding interactions between nanoparticles and biological systems is fundamental for the development of emerging nano-biotechnology applications. In this thesis, I present an investigation of zinc oxide (ZnO) nanoparticles interactions with biomolecules in two separate studies. The first section of my thesis covers tracking and detection of ZnO nanoparticles using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). ZinkicideTM is a bactericidal ZnO nanoparticle which...
Show moreUnderstanding interactions between nanoparticles and biological systems is fundamental for the development of emerging nano-biotechnology applications. In this thesis, I present an investigation of zinc oxide (ZnO) nanoparticles interactions with biomolecules in two separate studies. The first section of my thesis covers tracking and detection of ZnO nanoparticles using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). ZinkicideTM is a bactericidal ZnO nanoparticle which has been developed for agriculture. The characterization of Zinkicide in biological media and in solution has been difficult due to its high dispersibility and ultra-small size. SDS-PAGE is considered a golden standard for protein qualitative interpretations. In this study, we have modified this typical protein assay and developed protocols for quantifying Zinkicide concentration, fluorescence intensity, and relative molecular weight changes in aqueous solutions. We found that SDS-PAGE is a novel and fundamental approach for assessing ZnO nanoparticles.The second part of my thesis is focused on investigating biological toxicity induced by nanoparticles. Recent studies have shown that nanoparticles have the capabilities to induce abnormalities on cellular networks including actin cytoskeleton. We have studied the effects of ZnO nanoparticles on filamentous actin assembly dynamics utilizing total internal reflection fluorescence (TIRF) microscopy imaging and biophysical analysis. The combination of these studies has provided pertinent information for the future development of nanoparticles designed for biological applications.
Show less - Date Issued
- 2019
- Identifier
- CFE0007738, ucf:52423
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007738
- 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
