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- Title
- Genetic Interactions Between the Guanine Nucleotide Exchange Factor GEfmeso and GTPase signaling components in the drosophila wing reveal microenvironment dependent variation within gtpase signaling n.
- Creator
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Iketani, Ashley, Vonkalm, Laurence, Stern, Michael, Fernandez-Valle, Cristina, University of Central Florida
- Abstract / Description
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The Ras superfamily of GTPases are important regulators of morphogenesis involved in control of cytoskeletal dynamics, intracellular trafficking, apical-basal polarity and cell migration. Mis-regulation of GTPase signaling interferes with development and is linked to pathogenesis. Traditionally, GTPase signaling has been depicted as a series of independent linear pathways. However, recently it has become apparent that multiple GTPases can interact to regulate a single cellular process,...
Show moreThe Ras superfamily of GTPases are important regulators of morphogenesis involved in control of cytoskeletal dynamics, intracellular trafficking, apical-basal polarity and cell migration. Mis-regulation of GTPase signaling interferes with development and is linked to pathogenesis. Traditionally, GTPase signaling has been depicted as a series of independent linear pathways. However, recently it has become apparent that multiple GTPases can interact to regulate a single cellular process, functioning in poorly understood networks of cross talk between pathways during development. Jim Fristrom (unpublished data) identified a mutation (18-5) that interacts with components of the GTPases Rho1, Rala, and Cdc42 signaling in multiple developmental contexts. Genetic analysis, physical mapping studies, and sequencing of the mutant allele have indicated that the gene was an allele of GEFmeso (CG30115), which encodes guanine nucleotide exchange factor. To show that 18-5 is an allele of GEFmeso, I demonstrated that a GEFmeso transgene could functionally rescue developmental defects associated with the 18-5 mutation. I also investigated cross talk and network variation in signaling interactions between GEFmeso and other GTPase signaling components in the Drosophila wing. My data provide evidence for microenvironment-dependent variation in GTPase signaling networks in specific domains of the wing, and reveal intercellular variation in GTPase signaling within an otherwise uniform epithelium.
Show less - Date Issued
- 2012
- Identifier
- CFE0004252, ucf:49541
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004252
- Title
- The development of motuporamine derivatives and an investigation into their biological properties.
- Creator
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Skruber, Kristen, Phanstiel, Otto, Teter, Kenneth, Vonkalm, Laurence, University of Central Florida
- Abstract / Description
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This project investigates the synthesis of a class of compounds derived from a marine-based natural product and probes how iterative changes to its structure affect its derivatives' biological efficacy. The compound class of interest are the motuporamines which were isolated from the sea sponge Xestospongia exigua collected off the coast of Motupore island in Papua, New Guinea. The compounds for this project are predicated upon dihydromotuporamine C (Motu33), the compound that has been shown...
Show moreThis project investigates the synthesis of a class of compounds derived from a marine-based natural product and probes how iterative changes to its structure affect its derivatives' biological efficacy. The compound class of interest are the motuporamines which were isolated from the sea sponge Xestospongia exigua collected off the coast of Motupore island in Papua, New Guinea. The compounds for this project are predicated upon dihydromotuporamine C (Motu33), the compound that has been shown to be both cytotoxic to MDA-MB231 breast carcinoma cells and has antimetastatic efficacy. The motuporamine scaffold contains a large fifteen-membered saturated macrocycle and an appended polyamine component. A series of Motu33 derivatives were synthesized and evaluated for their ability to target the polyamine transport system as well as inhibit cell migration of human pancreatic cancer cells in vitro. By altering the polyamine component of the system we attempted to build smart antimetastatic compounds which target the upregulated polyamine transport system of human pancreatic cancers and block their migration.
Show less - Date Issued
- 2016
- Identifier
- CFE0006505, ucf:51390
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006505
- Title
- The Role of the Y-Chromosome in the Evolution of Autosomally Coded Traits.
- Creator
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Kutch, Ian, Fedorka, Kenneth, Vonkalm, Laurence, Hoffman, Eric, University of Central Florida
- Abstract / Description
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Recent work indicates that the Y-chromosome of the fruit fly Drosophila melanogaster can influence gene regulation on the autosomes and X chromosome. This newly discovered function of the Y has the potential to dramatically shape the regulatory evolution of numerous genes that reside throughout the genome; even for genes that code for both male and female traits. Given that the mechanism underlying the Y-linked influence on gene expression in D. melanogaster appears to exist in other...
Show moreRecent work indicates that the Y-chromosome of the fruit fly Drosophila melanogaster can influence gene regulation on the autosomes and X chromosome. This newly discovered function of the Y has the potential to dramatically shape the regulatory evolution of numerous genes that reside throughout the genome; even for genes that code for both male and female traits. Given that the mechanism underlying the Y-linked influence on gene expression in D. melanogaster appears to exist in other independently evolved heterogametic sex chromosomes, the evolutionary implications of Y-linked regulatory variation (YRV) deserves to be explored. These implications include the potential for Y-chromosomes to facilitate the adaptive evolution of sexually dimorphic gene expression, and the potential for the Y to constrain evolutionary rates in both males and females (depending on the nature of the YRV effect). Unfortunately, the evolutionary implications of this potentially widespread and significant phenomenon have yet to be explored. My dissertation addresses this knowledge gap by determining the influence YRV has on the evolution of autosomally coded traits in D. melanogaster. First, we address the potential for selection to shape YRV by determining if YRV (i) exists within natural populations (i.e. where natural selection operates), and (ii) has any influence on male fitness-related autosomal traits. Second, we address if YRV can facilitate the adaptive evolution of sexually dimorphic gene expression by testing for the presence of Y-linked additive genetic variation. To this end, we investigate the physiological properties of select Y-chromosomes across multiple genetic backgrounds. Third, we address if YRV can constrain adaptive evolution for autosomally coded traits by employing artificial selection on replicate populations that contain either multiple Y-chromosomes (i.e. contain YRV) or only a single Y-chromosome (no YRV). The following studies present evidence that YRV does exist within populations where natural selection operates. We show significant levels of YRV on X-linked and autosomal immune gene expression in wild caught D. melanogaster from a single natural population. Furthermore, YRV effects on immune related genes show a significantly positive correlation to a male fly's ability to fight an immune challenge (an important aspect of organismal fitness). Estimated physiological properties of YRV support previous interpopulation studies showing strong non-additive effect dependent on the autosomal genetic background with which Y-chromosome's are paired with. Physiological epistasis can manifest as additive genetic variation on a population level, but our experimental evolution study suggest that YRV constrains rather than facilitates the evolution of the autosomal coded geotaxis behavior. Ultimately, this dissertation provides evidence that YRV has the potential to influence how autosomal traits evolve and that population level studies of YRV indicate a potential constraint to the adaptive evolution of autosomal traits. If these trends are common and YRV is a wide spread phenomenon, Y-chromosomes have the potential to influence how autosomal traits evolve.
Show less - Date Issued
- 2017
- Identifier
- CFE0006756, ucf:51873
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006756
- Title
- The Role of LIM Kinase 1 and its Substrates in Cell Cycle Progression.
- Creator
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Ritchey, Lisa, Chakrabarti, Ratna, Zervos, Antonis, Zhao, Jihe, Vonkalm, Laurence, University of Central Florida
- Abstract / Description
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LIM Kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, has been shown to be involved in cell cycle progression. In this study we examine the role of LIMK1 in G1 phase and mitosis. We found ectopic expression of LIMK1 resulted in altered expression of p27Kip1, the G1 phase Cyclin D1/Cdk4 inhibitor. Overexpression of LIMK1 resulted in lower levels of p27Kip1 and p27Kip1-pY88 (inactive p27Kip1). Knockdown of LIMK1 resulted in elevated levels of p27Kip1 and p27Kip1-pY88. Together,...
Show moreLIM Kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, has been shown to be involved in cell cycle progression. In this study we examine the role of LIMK1 in G1 phase and mitosis. We found ectopic expression of LIMK1 resulted in altered expression of p27Kip1, the G1 phase Cyclin D1/Cdk4 inhibitor. Overexpression of LIMK1 resulted in lower levels of p27Kip1 and p27Kip1-pY88 (inactive p27Kip1). Knockdown of LIMK1 resulted in elevated levels of p27Kip1 and p27Kip1-pY88. Together, these results suggest LIMK1 regulates progression of G1 phase through modulation of p27Kip1 expression.LIMK1 is involved in the mitotic process through inactivating phosphorylation of Cofilin. Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with ?-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one other's function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation.The substrates of LIMK1, Aur-A and Cofilin, are also involved in the mitotic process. Aur-A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through regulation of the actin cytoskeleton. Aur-A phosphorylates Cofilin at multiple sites including S3 resulting in inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during progression of prophase to metaphase. Inhibition of Aur-A activity altered subcellular localization of Cofilin and induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the early mitotic stage through regulation of actin cytoskeleton reorganization.?
Show less - Date Issued
- 2014
- Identifier
- CFE0005701, ucf:50156
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005701
- Title
- A Time-Course Analysis of Behavioral Plasticity and Differential Gene Expression Patterns in Response to Density in Schistocerca americana (Orthoptera: Acrididae).
- Creator
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Gotham, Steven, Song, Hojun, Vonkalm, Laurence, Fedorka, Kenneth, University of Central Florida
- Abstract / Description
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Phenotypic plasticity is the ability of the genotype to express alternative phenotypes in response to different environmental conditions and this is considered to be an adaptation in which a species can survive and persist in a rapidly changing environment. Some grasshoppers and locusts are capable of expressing an extreme form of density-dependent phenotypic plasticity, known as locust phase polyphenism. At low population density, the individuals typically have a cryptic coloration as nymphs...
Show morePhenotypic plasticity is the ability of the genotype to express alternative phenotypes in response to different environmental conditions and this is considered to be an adaptation in which a species can survive and persist in a rapidly changing environment. Some grasshoppers and locusts are capable of expressing an extreme form of density-dependent phenotypic plasticity, known as locust phase polyphenism. At low population density, the individuals typically have a cryptic coloration as nymphs, are less active, and only seek out conspecifics for reproductive purposes. At high density, however, they develop a drastically different phenotype in which they have a conspicuous coloration, are much more active, and tend to stay together in large groups. The American Birdwing grasshopper, Schistocerca americana, is a non-swarming species related to the desert locust, S. gregaria, which shows density-dependent phenotypic plasticity in behavior, color, and morphology. In this thesis, I have identified the duration of crowding necessary for a 6th instar S. americana reared in the isolated condition to express the typical crowded behavior. The behavior changed after just one hour of crowding and the effect of crowding diminished after 48 hours to near-complete isolated behavior. In reverse, the crowded condition was isolated, but behavior did not significantly change over time. Gene expression of the following three genes suspected of having a role in behavior change were investigated based on studies of S. gregaria: protein kinase A (PKA), L-Tryptophan-5-monooxygenase (T-5), and Aromatic L-amino acid decarboxylase (Decarb). T-5 was up-regulated in the long-term isolated condition compared to the long-term crowded condition. T-5 and Decarb were up-regulated in isolated individuals that were crowded for 10 hours compared to the long-term isolated condition. This study represents a novel contribution in the study of phenotypic plasticity as it establishes the time course of behavioral and molecular plasticity in a non-swarming grasshopper for the first time.
Show less - Date Issued
- 2014
- Identifier
- CFE0005799, ucf:50049
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005799
- Title
- Genetic and biochemical characterization of the roles of two putative purine transporters in the infectious cycle of Borrelia burgdorferi.
- Creator
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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.
Show less - Date Issued
- 2014
- Identifier
- CFE0005511, ucf:50303
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005511
- Title
- A chemical and genetic approach to study the polyamine transport system in Drosophila.
- Creator
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Wang, Minpei, Vonkalm, Laurence, Phanstiel, Otto, Teter, Kenneth, Ballantyne, John, University of Central Florida
- Abstract / Description
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Polyamines are small cationic molecules that play important roles in most vital cellular processes including cell growth and proliferation, regulation of chromatin structure, translation and programmed cell death. Cellular polyamine pools are maintained by a balance between biosynthesis and transport (export and import). Increased polyamine biosynthesis activity and an active transport system are characteristics of many cancer cell lines, and polyamine depletion has been shown to be a viable...
Show morePolyamines are small cationic molecules that play important roles in most vital cellular processes including cell growth and proliferation, regulation of chromatin structure, translation and programmed cell death. Cellular polyamine pools are maintained by a balance between biosynthesis and transport (export and import). Increased polyamine biosynthesis activity and an active transport system are characteristics of many cancer cell lines, and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by ?-difluoromethylornithine (DFMO), an inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase. However, malignant cells often circumvent DFMO therapy by up-regulating polyamine import; therefore, there is a need to develop compounds that inhibit polyamine transport. Collectively, DFMO and polyamine transport inhibitors provide the basis for a combination therapy leading to effective intracellular polyamine depletion. Using a Drosophila leg imaginal disc model for polyamine transport, I studied three candidate transport inhibitors (Ant444, Trimer44 and Triamide44) for their ability to inhibit transport in the Drosophila model. Ant444 and Trimer44 effectively inhibited the uptake of the toxic polyamine analog Ant44 that gains entry to cells via the polyamine transport system. Ant444 and Trimer44 were also able to inhibit the import of exogenous polyamines into DFMO-treated imaginal discs. Triamide44 was an ineffective inhibitor, however a structurally redesigned compound, Triamide444, showed a 50-fold increase in transport inhibition and was comparable to Ant444 and Trimer44. Ant444 and Trimer44 showed differences in their relative abilities to block import of specific polyamines, and I therefore asked if a cocktail of these inhibitors would be more effective than either alone. My data show that a cocktail of polyamine transport inhibitors is more effective than single inhibitors when used in combination with DFMO, and suggests the existence of multiple polyamine transport systems. To further the development of effective transport inhibitors it is important to identify components of the transport system. The mechanism of polyamine transport in multicellular organisms including mammals is still unknown. Our laboratory has developed a simple assay to detect components of the transport system using RNAi knockdown and over-expression of candidate genes. However, the assay requires that animals live until the pupal stage of development. Pleiotropic effects of individual gene products following over-expression or knockdown may result in early developmental lethality for reasons unrelated to polyamine transport. Our assay is based on the GAL4/UAS system and involves the use of enhancers driving GAL4 expression (GAL4 driver). GAL4 in turn determines the expression level of UAS-candidate gene constructs (UAS responder). I reasoned that in some cases it might be possible to bypass early lethality by judicious choice of drivers that reduce responder expression, thus permitting survival to the pupal phase. To this end, I used five imaginal disc drivers (30A, 71B, 32B, 69B, and T80) as well as a ubiquitously expressed control driver to over-express and knockdown EGFR and components of the Rho signaling pathway. The relative strength of each driver was ranked, and I was able to demonstrate in principle that animals could survive to later stages of development in a manner that correlated with the relative strength of the driver. The approach I developed is broadly applicable to other studies of Drosophila development.To identify new components of the polyamine transport system I studied the role of proteoglycans in this process. The proteoglycan glypican-1 has been previously implicated in mammalian polyamine transport. In particular, the heparin sulfate side chains of glypican-1 appear to play an important role. In order to extend our knowledge of the role of proteoglycans in polyamine transport, I examined the role of the core proteoglycans perlecan and syndecan as well as genes encoding enzymes in the heparin sulfate and chondroitin sulfate biosynthetic pathways. I was able to confirm a role for glypican-1 in polyamine transport in imaginal discs but not in whole animals. This may indicate that glypican-1 is not required for polyamine uptake through the gut. Studies of genes encoding perlecan, syndecan and enzymes in the heparin sulfate and chondroitin sulfate biosynthetic pathways did not reveal a role for these genes in polyamine transport. These studies were conducted in whole animals and my data may reflect tissue-specific differences between the imaginal disc and gut transport systems where transport in imaginal discs is proteoglycan dependent and transport in the gut is not.
Show less - Date Issued
- 2017
- Identifier
- CFE0007297, ucf:52162
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007297
- Title
- Behavioral and disease ecology of Gopher Tortoises (Gopherus polyphemus) post exclusion and relocation with a novel approach to homing determination.
- Creator
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Napier, Johnathan, Savage, Anna, Moore, Sean, Vonkalm, Laurence, Fedorka, Kenneth, University of Central Florida
- Abstract / Description
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In the wake of human expansion, relocations and the loss of habitat can be stressful to an organism, plausibly leading to population declines. The gopher tortoise (Gopherus polyphemus) is a keystone species that constructs burrows it shares with 362 commensal species. Frequent exclusions and relocations and long generation times have contributed to G. polyphemus being State-designated as Threatened in Florida. Prior studies have indicated that G. polyphemus may possess homing behavior and...
Show moreIn the wake of human expansion, relocations and the loss of habitat can be stressful to an organism, plausibly leading to population declines. The gopher tortoise (Gopherus polyphemus) is a keystone species that constructs burrows it shares with 362 commensal species. Frequent exclusions and relocations and long generation times have contributed to G. polyphemus being State-designated as Threatened in Florida. Prior studies have indicated that G. polyphemus may possess homing behavior and thus be able to counteract stressors due to relocation and exclusion. I radiotracked a cohort of G. polyphemus for 11 months following excavation, relocation, and exclusion due to a pipeline construction project. In conjunction with analyzing G. polyphemus movement patterns post-release, I developed novel statistical methodologies with broad application for movement analysis and compared them to traditional analyses. I evaluated habitat usage, burrowing behavior, movements, growth, and disease signs among control versus relocated and excluded individuals and among sexes and size classes, forming predictors for behavior and disease risk. I found statistical support that my new methodology is superior to previous statistical tests for movement analyses. I also found that G. polyphemus engages in homing behavior, but only in males. Behavioral differences were also found between the sexes with respect to burrowing behavior. Overall health, disease prevalence, and immune response were unaffected by relocation and exclusion, nor were they statistically correlated. Signs were unreliable as etiological agents, outperformed by serological detection. I determined that the Sabal Trail pipeline as a potential stressor did not affect movement behavior, homing, nor the disease/immune profile of G. polyphemus in this study.
Show less - Date Issued
- 2018
- Identifier
- CFE0007581, ucf:52581
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007581
- Title
- Design, Synthesis, and Biological Evaluation of Novel Polyamine Transport System Probes and their Application to Human Cancers.
- Creator
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Muth, Aaron, Phanstiel, Otto, Ye, Jingdong, Elsheimer, Seth, Miles, Delbert, Vonkalm, Laurence, University of Central Florida
- Abstract / Description
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The mammalian polyamine transport system (PTS) has been of interest due to its roles in cancer and maintaining cellular homeostasis. Polyamines are essential growth factors which are tightly controlled via a balance of biosynthesis, metabolism, import, and export. This work focused on the development and biological testing of polyamine transport probes to help understand the molecular requirements of the PTS. This was mediated through the use of a CHO (PTS active) and CHO-MG* (PTS deficient)...
Show moreThe mammalian polyamine transport system (PTS) has been of interest due to its roles in cancer and maintaining cellular homeostasis. Polyamines are essential growth factors which are tightly controlled via a balance of biosynthesis, metabolism, import, and export. This work focused on the development and biological testing of polyamine transport probes to help understand the molecular requirements of the PTS. This was mediated through the use of a CHO (PTS active) and CHO-MG* (PTS deficient) screen, where compounds demonstrating high toxicity in CHO and low toxicity in CHO-MG* were considered PTS selective. The first chapter focused on the development of polyamine-based drugs which are both metabolically stable to polyamine oxidase (PAO) activity and are hyperselective for targeting the PTS. This approach was optimized by combining a di-substituted aryl design with terminal N-methylation of the appended polyamine chains to generate a new class of superior PTS agonists. The metabolic stability of these compounds was demonstrated in CHO and CHO-MG* in the presence and absence of a known PAO inhibitor, aminoguanidine (AG). Highly PTS selective compounds were then tested in the NCI-60 cell line screen to demonstrate the effectiveness of polyamine-based drugs in cancer therapy. During this screen, the MALME-3M (human melanoma) cell line was identified as being very sensitive to these PTS targeting drugs. Further studies using MALME-3M and its normal counterpart, MALME-3, showed excellent targeting of the cancer line over MALME-3. For example, The MeN44Nap44NMe compound showed 59-fold higher toxicity in MALME-3M over MALME-3.The second chapter focused on the development of potential polyamine transport inhibitors (PTIs) for use in combination therapy with ?-difluoromethylornithine (DFMO). This therapy is predicated upon reducing sustained polyamine depletion within cells by inhibiting both polyamine biosynthesis with DFMO and polyamine transport with the PTI ligand. Potential PTIs were identified by blocking the uptake of spermidine in DFMO-treated CHO and L3.6pl cells. Previous work has identified a tri-substituted polyamine-based design as an effective PTI. Low toxicity and a low Ki value in a L1210 screen were good predictors for PTI efficacy. The structural requirements for a potent PTI were explored by modulating the toxicity through the introduction of amide bonds, and also by determining the number and orientation of the polyamine messages (appended to an aryl core) required for efficient inhibition of polyamine uptake. These experiments showed that a tri-substituted design and a triamine message (homospermidine) appended was optimal for PTI potency. The final chapter focused on the development of Dihydromotuporamine C derivatives as non-toxic anti-metastatic agents. Dihydromotuporamine C demonstrated good anti-invasive properties with tumor cells. Derivatives were made in an effort to reduce the cytotoxicity of the parent and improve the anti-migration potency. The motuporamine derivatives all have a polyamine message (norspermidine or homospermidine) appended to make a macrocycle core, making them prime targets to evaluate as potential PTS ligands in the CHO and CHO-MG* screen. Each compound was also tested in the highly metastatic pancreatic cancer cell line L3.6pl to determine both its IC50 value and maximum tolerated dose (MTD). The anti-migration assay was performed at the lowest MTD obtained (0.6 (&)#181;M) in order to compare the series at the same non-toxic dose. The results suggested that as the N1-amine center was moved further from the macrocyclic ring, an increased ability to inhibit cell migration and reduced toxicity was observed. These collective findings provide new tools for cell biologists to modulate and target polyamine transport in mammalian cells. Future applications of these technologies include new cancer therapies which are cell-selective and inhibit the spread of tumors.
Show less - Date Issued
- 2012
- Identifier
- CFE0004636, ucf:49895
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004636
- Title
- Biochemical Characterization of the NifB Enzyme and NifB-cofactor.
- Creator
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Gevorkyan, Jirair, Igarashi, Robert, Belfield, Kevin, Hernandez, Florencio, Kuebler, Stephen, Vonkalm, Laurence, University of Central Florida
- Abstract / Description
-
The Mo-nitrogenase complex is composed of two components, Fe-protein and MoFe-protein. This complex is able to catalyze the reduction of N2 through the MgATP dependent transfer of electrons from the Fe-protein Fe4S4 cluster to the MoFe-protein P-cluster and, subsequently, to the iron-molybdenum cofactor (FeMo-co). FeMo-co is a Fe7S9MoC-(R)-homocitrate cluster and has two biosynthetic precursors, NifB-co and L-cluster, of unknown structure and composition. The biosynthesis of FeMo-co is an...
Show moreThe Mo-nitrogenase complex is composed of two components, Fe-protein and MoFe-protein. This complex is able to catalyze the reduction of N2 through the MgATP dependent transfer of electrons from the Fe-protein Fe4S4 cluster to the MoFe-protein P-cluster and, subsequently, to the iron-molybdenum cofactor (FeMo-co). FeMo-co is a Fe7S9MoC-(R)-homocitrate cluster and has two biosynthetic precursors, NifB-co and L-cluster, of unknown structure and composition. The biosynthesis of FeMo-co is an enigmatic process that minimally requires NifB, NifEN, Fe-protein, MoO42-, (R)-homocitrate and S-adenolsylmethionine.A means to isolate the NifB enzyme for characterization has been developed through use of a GST-fusion tag. Double recombination of A. vinelandii strains with a constructed vector has yielded strains capable of nif promoter regulated expression of GST-NifB. Extracts of strains containing GST-NifB were shown to activate the Mo-nitrogenase complex in biochemical complementation assays. Mass spectroscopy was then used to verify successful isolation of GST-NifB by GSH-Sepharose affinity purification.The number of NifB-co ligand binding sites and ligand types were examined by EXAFS analysis of samples containing selenol and thiol ligands. A Fe6S9C model for NifB-co was optimized to best fit the EXAFS data, where a 2-fold discrepancy in binding sites implied by thiol or selenol only ligand samples suggests Fe-(?2S)-Fe binding in the absence of Se. Samples containing heterogeneous ligand types indicated that NifX bound NifB-co ligates to four cysteine residues and one molecule of DTT.
Show less - Date Issued
- 2013
- Identifier
- CFE0004682, ucf:49865
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004682