Alumni Dissertations

 

Alumni Dissertations

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  • Effects of The Enzyme Inhibitor Prohexadione-calcium on Hops Determined by LC-TOF-MS

    Author:
    Adam Kavalier
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Edward Kennelly
    Abstract:

    Humulus lupulus L. (hops) is an agricultural crop valued for its inflorescences, commonly known as hop cones, which produce a diverse collection of secondary metabolites. Hop cones are most valued for their terpenophenolic contents, which are essential to beer production, and the subject of biomedical research. We studied two hop cultivars, Willamette and Zeus, over five stages of development, which were characterized by detailed flower morphology, gross cone measurements, and phytochemical quantitation. By combining morphological observations with phytochemical quantitation we produced an index to inform our developmentally dependent experiments. In order to understand these developmental processes and in an attempt to induce agronomically positive effects, we perturbed hop morphological and phytochemical development using enzyme inhibition. Prohexadione-calcium (Pro-Ca) is a known inhibitor of 2-oxoglutaric acid dependent dioxygenases present in the flavonoid, gibberellic acid, and ethylene biosynthetic pathways. We treated hops with Pro-Ca at each of the five time-points over two seasons; these time-points were later characterized as five developmental stages. Pro-Ca treatment induced significant increases in terpenophenolic content by 9.1-87.3%; however some treatments also induced significant decreases. Increases in cone biomass production by 1.5-19.6% were also measured in response to treatment in both seasons. Induced changes in cone biomass production and terpenophenolic accumulation were most dependent on cultivar and the developmental stage at which plants were treated. In a second series of experiments we conducted a targeted analysis of phenolic acids, flavonoids, and terpenophenolics over 22 days following a single Pro-Ca treatment conducted during early flowering. Terpenophenolics significantly increased following treatment, and coincided with changes in the flavonoid biosynthetic pathway including accumulation of metabolic precursors upstream from flavanone-3-hydroxylase, and decreases in flavonoid products downstream from flavanone-3-hydroxylase. In addition to changes in known compounds, marker analysis revealed the presence of two markers in treated samples not previously reported from hops. One of these markers has been tentatively identified as the antimicrobial compound luteoliflavan. This research provides insight into the relationship between secondary metabolic pathways in hops and indicates targets for future research into perturbation of metabolic pathways to increase medicinal and flavor compounds in hops.

  • The investigation of the antidiabetic Dominican traditional medicinal plants Costus spicatus Sw. and Momordica charantia L.

    Author:
    Amy Keller
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Edward Kennelly
    Abstract:

    Diabetes is a serious disease affecting many people throughout the world, and is expected to increase in the coming decades. Traditional medicine is used in many places around the globe, including the Dominican Republic, for the treatment of diabetes along with modern medicines. Fieldwork conducted in the Dominican community by the Institute of Economic Botany identified Costus species and Momordica charantia L. as being widely used for diabetes treatment, thus pointing to these plants for further investigation. In an in vivo study, Costus spicatus Sw. tea or water were fed ad libitum to a C57BLKS/J mice (KS) db/db mouse model of obesity and type 2 diabetes mellitus (T2DM). The C. spicatus tea did not improve glucose or insulin tolerance, or moderate hyperglycemia or insulin sensitivity. To analyze the hypoglycemic effect of Momordica charantia fruit, initial studies were conducted in vitro. Both an ethanol extract and saponin-rich fraction of fruit, along with the five isolated triterpene saponin compounds 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al, momordicine I, momordicine II, 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-β-glucopyranoside, and kuguaglycoside G, were tested to assess their potential stimulation of insulin secretion. The saponin-rich fraction, along with mormordicin II and kuguaglycoside G, were active in the assay, suggesting saponins as the active hypoglycemic compounds in M. charantia. To further characterize the antidiabetic activity of Momordica charantia, a saponin-rich fraction and ethanol extract of the fruit was gavaged daily to C57BL/6 mice fed a high-fat diet. Both treatments lowered fasting glucose and improved glucose tolerance after three weeks. Also, the ethanol-extract treated group had significantly less β-cell mass at the end of the study, pointing to improved β-cell function. The results of this study again suggest saponins in M. charantia as the therapeutic constituents. In conclusion, the studies described aimed to investigate the efficacy of traditional medicine in a rigorous scientific setting and found that although Costus spicatus was not active, Momordica charantia displayed significant antidiabetic activity. Information about safety and efficacy of herbal medicine will continue to be important as these traditional treatments increase in use around the world for health conditions, including diabetes.

  • Postnatal refinement of interareal circuits in ferret visual cortex

    Author:
    Reem Khalil
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Jonathan Levitt
    Abstract:

    Visual cortical areas are presumed to subserve different perceptual functions as a result of their rich network of interareal anatomical circuits. Interareal circuits have been shown to undergo extensive remodeling in the postnatal period. Revealing the timing of when brain circuits mature may help us assign particular neural substrates to particular visual functions. To illuminate perceptual development, we assessed the postnatal anatomical refinement of interareal feedforward and feedback projections in ferret visual cortex. We also described the developmental trajectory of zinc levels in ferret visual cortex, reflecting a subset of glutamatergic interareal feedforward and feedback processes. We find that the period of major reorganization in feedback circuits, feedforward circuits, as well as the dramatic decline in zinc levels in ferret visual cortex occurs in the month following eye opening. In chapter 1, we demonstrated that zinc histochemistry can be reliably used to distinguish visual cortical areas in juvenile visual cortex and further reveals circuit refinement. We show that the postnatal decline in levels of synaptic zinc follows a broadly similar timecourse in multiple areas of ferret visual cortex. In chapter 2, we assessed the developmental refinement of feedback projections between primary visual cortex and extrastriate areas in the juvenile ferret brain. We reveal substantial refinement in the spatial organization of feedback projections arising from multiple visual areas to primary visual cortex of the ferret during the period after eye opening. We find that while certain aspects of feedback circuitry refine with a similar timecourse in all areas, other aspects refine asynchronously. In chapter 3, we investigated the postnatal development of feedforward projections from V1 to different target areas. Before eye opening, at 4 weeks postnatal, synaptic bouton density is very high, and interbouton interval along individual axons is quite short. In all areas examined, both bouton density decreased, and interbouton interval increased substantially from 6 weeks to 8 weeks postnatal. Therefore, feedback and feedforward cortical circuits appear to share a broadly similar developmental trajectory. Our findings are consistent with the notion that visual experience is necessary and crucial in the refinement of these cortical circuits. Furthermore, our findings suggest that at least some aspects of cortical maturation occur largely synchronously in multiple visual areas.

  • The Interplay of Visual Attention and Saccade Planning in Active Vision

    Author:
    Afsheen Khan
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Jonathan Levitt
    Abstract:

    Vision is a highly active process. When we view the world, we do not hold our eyes still, but constantly move them around in order to view the object or area of interest with the fovea (the region of the retina with the highest acuity). Saccades are the step-like movements that we most often employ for this purpose. In addition, our attention is constantly being covertly attracted or directed to points of interest. Combining these different aspects of viewing: visual processing, the orienting of attention, and eye movements can be referred to as `active vision'. Most work on active vision or attention and saccades has concentrated on performance improvements preceding saccades, but relatively little is yet known about how attention affects later stages of saccade planning. That is the focus of this thesis. First we look at the temporal dynamics of the scaling of attention and what influence that attention scale exerts on the decision to make a saccade. We are able to infer the attention scale during individual trials from their saccade latencies. We find that the scale of attention changes very rapidly, and faster than previously reported. The remainder of the thesis concentrates on the effects of attention scale and locus on post-saccade adaptive processes: how the success of the current plan influences learning. Saccades maintain their accuracy through an adaptive process, slowly to compensate for muscle weakening, or rapidly in a lab setting using intra-saccadic steps. Little is known about how covert attention interacts with this process. The second study of this thesis looks at how the scale of attention can affect the magnitude of saccade adaptation. We use a novel paradigm in which the intrasaccadic steps change from trial-to-trial so that over many trials the displacement produces a sine wave pattern. We find that when attending to larger targets, there is proportionally greater adaptation than when attending to smaller targets. Finally, we demonstrate that the locus of attention at the end of a saccade contributes to the error signal for saccade adaptation. Instead of intra-saccadically moving the target in order to induce saccade adaptation, we present a distractor briefly after the saccade on the near side or far side of the target. By drawing attention away from the saccade target immediately after the saccade, the distractor is able to induce saccade adaptation. The magnitude of the saccade adaptation depends on the novelty of the distractor. These experiments highlight the interplay between attention and saccades. Using novel paradigms, we show that the locus of attention can induce saccade adaptation, and that the scale of attention influences both the magnitude of saccade adaptation as well as the decision to move. While conventionally, saccade experiments are performed using very small stimuli, we see that using larger stimuli can greatly change saccade performance. Use of larger, more complicated stimuli as compared to simple spot targets is a step closer to natural viewing and very important to our understanding of active vision.

  • ETHNOMEDICAL, ECOLOGICAL AND PHYTOCHEMICAL STUDIES OF THE PALAUAN FLORA

    Author:
    Christopher Kitalong
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Michael Balick
    Abstract:

    ABSTRACT ETHNOMEDICAL, ECOLOGICAL AND PHYTOCHEMICAL STUDIES OF THE PALAUAN FLORA By Christopher U. Kitalong Adviser: Michael J. Balick There has been a serious deterioration of Palauan culture, language and traditional practices over the last century. To promote health and avoid this deterioration of tradition, ecological, ethnobotanical and phytochemical studies have been carried out on the plant Phaleria nisidai Kaneh. (Thymelaeaceae), "Delal a Kar", Palauan for "Mother of Medicine". This study is the first study that sets the foundations for the development of natural Palauan therapeutics, through validation of ethnomedically significant plants. Validations of these plants is done through documenting Palauan plant ethnomedical data; mapping the distribution of Palauan plants on limestone Rock Islands; and analyzing ethnopharmacological and phytochemical properties of a Palauan panacea, Phaleria nisidai. Ethnographical data was collected through interviews with Palauan traditional leaders and elders concerning the general uses of Palauan plant flora. From this pool of interviews, the two most frequently described medicinal plants were Premna obtusfolia L. (Verbenaceae) and Phaleria nisidai (Thymelaeaceae). Then ecological studies on plant diversity and distribution were done on the limestone islands in the southern lagoon of Palau, which have the most intact anthropogenic sites in Palau. The Rock Islands of Palau had a relatively homogenous plant distribution with high percent of indigenous plant species. Ethnomedically significant plant species, Phaleria nisidai and Premna serratifolia, were only found on islands with human activity. Furthermore, Phaleria nisidai was only found in close proximity to former habitation/anthropogenic sites. Phaleria nisidai was important and useful to past inhabitants and may have even been brought by original immigrants to Palau. Obesity and ensuing diabetes mellitus type II are among the most prevalent NCDs in Palau and throughout the Pacific. In order to validate an ethnomedically significant plant species, Phaleria nisidai, and its major phytochemical component, mangiferin, a blood glucose lowering compound, were analyzed. Mangiferin from different populations of Phaleria nisidai used in Palau was quantified from different extraction methods. Aqueous extraction proved to be the most effective method for extracting the highest percentage of the mangiferin, from Phaleria nisidai leaves. These studies on Phaleria nisidai, have set the foundation for future clinical testing for treatment of diabetes mellitus type II.

  • Down Regulation of Neuronatin by MicroRNA-151 Overcomes Inhibition of Axonal Growth by Myelin-based Inhibitors

    Author:
    Dawn Kochanek
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Marie Filbin
    Abstract:

    Down Regulation of Neuronatin by microRNA-151 Overcomes Inhibition of Axonal Growth by Myelin-based Inhibitors by Dawn Marie Kochanek Thesis Advisor: Dr. Marie T. Filbin After injury, the axons of the adult central nervous system (CNS) fail to regenerate. This failure is due to the cellular environment and the neuronal response to that environment. One factor for environmentally-mediated axonal inhibition are the proteins that are present in myelin, such as myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp). Previously, our lab has shown that elevating the ubiquitous second messenger cyclic-adenosine monophosphate (cAMP) overcomes MAG/myelin inhibition. MicroRNAs (miRNA or miR), are small fragments of RNA that have been shown to bind to target mRNAs and regulate their translation. We hypothesized that miRNAs might be playing a role in the ability of cAMP to overcome MAG/myelin-based axonal growth inhibition. To investigate if miRNAs have a role in the cAMP effect we performed a miRNA microarray with cAMP treated vs. control dorsal root ganglion (DRG) neurons. One miRNA that increased more than two fold with cAMP treatment was miRNA-151. As previously stated, the axons of the mammalian CNS do not regenerate after injury. However, there is one situation in which they have been shown to spontaneously regenerate. DRG axons are bifurcated with one branch extending into the CNS and the other into the peripheral nervous system (PNS). Studies have shown that if a lesion is made to the PNS branch and then subsequently to the CNS branch, the CNS branch will regenerate. This conditioning lesion-induced regeneration has been found to be dependent upon increased levels of cAMP. We next investigated whether like after treatment with cAMP, miR-151 was also increased after a peripheral conditioning lesion. We found similar significant increases in miR-151 levels in DRG neurons following a peripheral conditioning lesion. To determine a functional role for miR-151 in overcoming MAG/myelin-mediated neurite outgrowth inhibition we next performed overexpression and knockdown analyses of miR-151 and then subsequently subjected the neurons to a neurite outgrowth assay. Overexpression of pre-miR-151 in DRG neurons overcame MAG/myelin-mediated neurite outgrowth inhibition and conversely, knockdown of miR-151 with anti-miR-151 in DRG neurons attenuated the ability of db-cAMP to overcome MAG/myelin-mediated inhibition. To investigate the mechanism by which overexpression of miR-151 overcomes MAG/myelin-mediated inhibition we sought to identify miR-151 target mRNAs, using target prediction algorithms. One putative target was Neuronatin, a 9 kD transmembrane proteolipid protein with unknown neuronal function. We next wanted to assess if miR-151 could bind to the 3'UTR of Neuronatin and inhibit its translation. To test this, 293-T cells were co-transfected with miR-151 and a luciferase reporter gene fused to a wildtype or mutated Neuronatin 3'UTR. MiR-151 overexpression decreased the luciferase activity of the wildtype, but not the activity of the mutated Neuronatin 3'UTR, thus validating that Neuronatin is a miR-151 target. Likewise, we found that both treatment of DRG neurons with db-cAMP or overexpressing miR-151 led to a significant decrease in Neuronatin protein levels, while Neuronatin mRNA levels were unaffected. Finally, using siRNA we knocked-down Neuronatin in DRG neurons and then subjected the neurons to a neurite outgrowth assay. Knockdown of Neuronatin led to a significant increase in total neurite length on both MAG-expressing CHO cells and purified myelin. Our findings suggest that the cAMP-induced miR-151 plays an important role in overcoming MAG/myelin-mediated axonal growth inhibition.

  • Down Regulation of Neuronatin by MicroRNA-151 Overcomes Inhibition of Axonal Growth by Myelin-based Inhibitors

    Author:
    Dawn Kochanek
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Marie Filbin
    Abstract:

    Down Regulation of Neuronatin by microRNA-151 Overcomes Inhibition of Axonal Growth by Myelin-based Inhibitors by Dawn Marie Kochanek Thesis Advisor: Dr. Marie T. Filbin After injury, the axons of the adult central nervous system (CNS) fail to regenerate. This failure is due to the cellular environment and the neuronal response to that environment. One factor for environmentally-mediated axonal inhibition are the proteins that are present in myelin, such as myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp). Previously, our lab has shown that elevating the ubiquitous second messenger cyclic-adenosine monophosphate (cAMP) overcomes MAG/myelin inhibition. MicroRNAs (miRNA or miR), are small fragments of RNA that have been shown to bind to target mRNAs and regulate their translation. We hypothesized that miRNAs might be playing a role in the ability of cAMP to overcome MAG/myelin-based axonal growth inhibition. To investigate if miRNAs have a role in the cAMP effect we performed a miRNA microarray with cAMP treated vs. control dorsal root ganglion (DRG) neurons. One miRNA that increased more than two fold with cAMP treatment was miRNA-151. As previously stated, the axons of the mammalian CNS do not regenerate after injury. However, there is one situation in which they have been shown to spontaneously regenerate. DRG axons are bifurcated with one branch extending into the CNS and the other into the peripheral nervous system (PNS). Studies have shown that if a lesion is made to the PNS branch and then subsequently to the CNS branch, the CNS branch will regenerate. This conditioning lesion-induced regeneration has been found to be dependent upon increased levels of cAMP. We next investigated whether like after treatment with cAMP, miR-151 was also increased after a peripheral conditioning lesion. We found similar significant increases in miR-151 levels in DRG neurons following a peripheral conditioning lesion. To determine a functional role for miR-151 in overcoming MAG/myelin-mediated neurite outgrowth inhibition we next performed overexpression and knockdown analyses of miR-151 and then subsequently subjected the neurons to a neurite outgrowth assay. Overexpression of pre-miR-151 in DRG neurons overcame MAG/myelin-mediated neurite outgrowth inhibition and conversely, knockdown of miR-151 with anti-miR-151 in DRG neurons attenuated the ability of db-cAMP to overcome MAG/myelin-mediated inhibition. To investigate the mechanism by which overexpression of miR-151 overcomes MAG/myelin-mediated inhibition we sought to identify miR-151 target mRNAs, using target prediction algorithms. One putative target was Neuronatin, a 9 kD transmembrane proteolipid protein with unknown neuronal function. We next wanted to assess if miR-151 could bind to the 3'UTR of Neuronatin and inhibit its translation. To test this, 293-T cells were co-transfected with miR-151 and a luciferase reporter gene fused to a wildtype or mutated Neuronatin 3'UTR. MiR-151 overexpression decreased the luciferase activity of the wildtype, but not the activity of the mutated Neuronatin 3'UTR, thus validating that Neuronatin is a miR-151 target. Likewise, we found that both treatment of DRG neurons with db-cAMP or overexpressing miR-151 led to a significant decrease in Neuronatin protein levels, while Neuronatin mRNA levels were unaffected. Finally, using siRNA we knocked-down Neuronatin in DRG neurons and then subjected the neurons to a neurite outgrowth assay. Knockdown of Neuronatin led to a significant increase in total neurite length on both MAG-expressing CHO cells and purified myelin. Our findings suggest that the cAMP-induced miR-151 plays an important role in overcoming MAG/myelin-mediated axonal growth inhibition.

  • Phylogeny, taxonomy and morphological evolution in Conostegia (Melastomataceae: Miconieae)

    Author:
    Ricardo Kriebel
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Fabian Michelangeli
    Abstract:

    The genus Conostegia comprises 77 species of shrubs and trees ranging from Central America to northern South America and the Caribbean. They are ecologically important as they provide pollen for native bees and fruits for birds. One of the main questions of this study is if the genus Conostegia is actually monophyletic. I address this question for the first time by gathering genetic data from four chloroplast regions and two nuclear ribosomal regions of DNA. Phylogenetic analyses of these data revealed that Conostegia is not monophyletic and that a group of species in the genera Clidemia and Miconia fall within it. It is noteworthy that species of these genera that fall within Conostegia are for the most part restricted to southern Central America. Morphological studies that include the use of anatomy, morphometrics, as well as different types of microscopy were conducted to attempt and identify potential characters that support a more broadly circumscribed Conostegia. These studies revealed several characters such as structured variation in herkogamy, a stele within the style and presence of mucilage inside the ovary in many species as potential synapomorphies for clades within the Conostegia clade. Not only are some of these characters useful but also novel in the systematics of the family Melastomataceae. Having identified a broader Conostegia clade, a taxonomic revision was conducted, including ample documentation of the morphology of all the species in the clade, descriptions and maps for 77 species. Lastly, taking advantage of the results of the molecular phylogeny as guidance, three main hypotheses were tested using morphometric approaches. The first hypothesis stated that diversification in floral morphology had indeed occurred within the Conostegia clade, all species of which are buzz pollinated. It has been said in the past that groups in which buzz pollination has evolved, tend to conserve their floral morphology and pollinators. That being said, no study has addressed how conserved flowers in these lineages actually are. This question was addressed by quantifying floral morphology over a broad sample of species within the Conostegia clade and found four major floral types. Transitions between floral types and their possible biological significance are discussed. The second hypothesis that was tested was that leaf venation in Conostegia is in fact quantifiable using geometric morphometrics and that such variation has a phylogenetic component. Variation in leaf venation, in particular the position of origin of the main parallel veins that characterize the family, has been historically used to distinguish groups of species. These different types of leaf veins have also been proposed as potential synapomorphies for major clades within the family. Possibly because variation in leaf venation is a continuous character, its use in Melastomataceae systematics has been difficult. A general framework is here proposed and it is shown that geometric morphometrics is an efficient tool for grasping leaf venation. In addition, it is shown that one of the three major clades in Conostegia is in fact different in its leaf venation from the other two clades and that the other two clades are similar to each other. The third and last hypothesis tested was that seeds in Conostegia can be quantified using elliptic Fourier analysis (EFA). The latter is a powerful geometric morphometric technique based on outlines. Traditionally, seeds in the Melastomataceae have been used in the systematics of many groups in a qualitative framework by coding binary or multistate discrete variables. For the first time a continuous framework using EFA was attempted. The results show that EFA can efficiently quantify many seeds in a small amount of time and summarize their variation using multivariate statistics in few axes. Furthermore, the EFA analyses revealed that one of the clades within Conostegia is significantly different than the other two but that the latter two are not different between each other. These results corroborate, in a different structure, the results obtained in the leaf venation analyses. In conclusion, identifying a natural or monophyletic group allowed for a revision of the taxonomy of a noteworthy component of Neotropical forests providing a tool to the general public for their identification. Subsequently, having clarity with respect to the relationships and identity of the species within the clade, biological questions were addressed experimenting with new available tools. This process yielded several discoveries.

  • An ethnobotanical, ecological and LC-MS-based chemometric investigation of Phaleria nisidai, a traditional adaptogen containing diterpene esters from Palau, Micronesia

    Author:
    Daniel Kulakowski
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Edward Kennelly
    Abstract:

    An ethnobotanical, ecological and LC-MS-based chemometric investigation of Phaleria nisidai, a traditional adaptogen containing diterpene esters from Palau, Micronesia Palau is a country with a rich heritage of traditional medicine still being practiced. One of the most popular and respected remedies in Palau is a tea made from fresh leaves of Phaleria nisidai Kaneh. (Thymelaeaceae). Interviews conducted to determine the use of this plant revealed that it is employed non-specifically to treat a variety of general health concerns. Its use as a prophylactic to keep away sickness, as a 'system cleaner', as well as for strength and energy indicate that it is being used as an adaptogen, a medicine taken routinely to help adapt to external pathogenic, mental or physical stress. A series of in vitro immunological assays were conducted to determine the effect of crude extracts and guide phytochemical fractionation of this plant to identify immunostimulant compounds. Phaleria nisidai is in the family Thymelaeaceae, one of two plant families known to produce diterpene ester compounds. Multivariate analysis to compare mass spectral data of active and inactive fractions allowed for the identification of several daphnane diterpene esters as tentative active marker compounds. Simplexin, the marker contributing most to differentiation of active and inactive fractions, was active when tested alone for stimulation of cytokine output by peripheral blood mononuclear cells (PBMCs). This demonstrated that a chemometric approach to active compound determination as opposed to traditional time- and resource-intensive bioactivity-guided purification methods to determine active compounds in a plant matrix was successful. This work also confirms a methanol extract of this plant and one of its constituents has activity as an immunostimulant, verifying its traditional use. This is also the first report of diterpene esters in the genus Phaleria. Simplexin and other diterpene esters have shown cocarcinogenic and irritant activity in cell and mouse models and have caused gastric and pulmonary problems in animals grazing on plants containing these compounds. However, these compounds have also shown anti-HIV and anticancer activity. To determine if Palauans are ingesting diterpene esters in traditional preparations of Phaleria nisidai, simplexin, acetoxyhuratoxin and huratoxin were quantified from methanol and aqueous extracts prepared in the lab and in aqueous infusions prepared by six traditional healers in Palau. Diterpenes were not detected in traditional aqueous extracts prepared by healers in Palau, or in aqueous extracts prepared in our lab, but were detected in the methanol extract. PBMC proliferation as well as their production of IFNγ was measured and it was found that aqueous extracts induce both PBMC proliferation as well as an increase in IFNγ production, although these effects were milder and significantly less than the activity demonstrated by the methanol extract. This further validated the traditional use of P. nisidai as an immunestimulating adaptogen and allayed concerns about public health issues with chronic ingestion of aqueous P. nisidai infusions. Levels of simplexin, acetoxyhuratoxin, huratoxin and a bioactive xanthone, mangiferin, were analyzed in leaf samples from 227 trees collected from 92 populations of Phaleria nisidai. All Rock Island populations contained minute daphnane concentrations and lower amounts of mangiferin than populations on the largest Palauan island, Babeldaob. Savannah, scrub savanna and mature forest habitats all contained populations with exceptionally high levels of daphnanes, while the mangiferin content across habitats was more homogenous. Analysis of these compounds by geographic variables can be useful to identify high-yielding chemotypes for biomedical or toxicological studies. These studies demonstrate that Palauan traditional medicine can offer a source of plant metabolites with potent biological activity that corresponds to their traditional use. The discovery of simplexin and other diterpene esters in a plant consumed daily indicates that Palauans have found a way to selectively extract beneficial compounds while minimizing exposure to potentially harmful metabolites. The possibility also exists that Palauans have uncovered a low-dose therapeutic window at which diterpene esters can provide immunostimulant benefits while avoiding toxicological risks. The pharmacological potential of simplexin and related diterpenes in Phaleria nisidai should be reevaluated in light of these findings.

  • The Behavioral Effects of Exposure to the Endocrine Disrupting Chemical, Ethinyl Estradiol, on Fourspine Stickleback (Apeltes quadracus)

    Author:
    Kristine Kuroiwa
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Jennifer Basil
    Abstract:

    There is growing evidence that endocrine disrupting chemicals polluting the environment have the potential to affect animal health and behavior. Ethinyl estradiol (EE2), the active ingredient in contraceptive pills and hormone-replacement therapies, is a particularly potent endocrine disrupting chemical found in our waterways. In this study, adult male and female fish were used to investigate the effects of a 60 day EE2 exposure on 1) ecologically relevant behaviors, 2) gonadal state, and 3) male reproductive coloration. Exposure to levels of EE2 documented in the environment (10, 70 and 100 ng/L) detrimentally altered fish behavior. Fish exposed to EE2 were less active, more aggressive, and avoided an artificial predator less often. 100 days in clean water reversed some but not all of these behavioral changes. In both males and females, gonads in exposed groups differed from controls, and included the presence of ovotestes found only in males exposed to EE2. Reproductive coloration in males was not affected by exposure. This is the first report of long-lasting behavioral aberrations caused by EE2 exposure in adult fish.