ETHNOMEDICAL, ECOLOGICAL AND PHYTOCHEMICAL STUDIES OF THE PALAUAN FLORA
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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
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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)
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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
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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)
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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.
In Vitro Models to Study the Properties of the TCRα Locus Control Region
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Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene. LCR activities have been discovered in numerous T cell-lineage gene loci. These elements can be adapted to the design of gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein, we report two cell culture models to study the properties of the T cell receptor (TCR)-α LCR. The first is an in vitro embryonic stem cell differentiation model that has been optimized to manifest all key features of mouse TCRα LCR function. High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and are upregulated during the expected stage transition of T cell development. The ability of this LCR to overcome position effects may be due to barrier insulator-like activity within. To further explore this possibility, we established a second model that seems to support this notion. The characterization and identification of the sequences involved in this possible barrier insulator will provide an additional vertebrate model for the study of insulators. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.
Diet and feeding strategies of redbreast sunfish (Lepomis aurtitus) and bluegill sunfish (Lepomis macrochirus) in two suburban lakes.
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This is a study of the feeding habits of two species of sunfish, Lepomis auritus (Linnaeus, 1758), redbreast sunfish, and Lepomis macrochirus Rafinesque, 1819, bluegill sunfish, co-occurring in two suburban lakes with different degrees of shoreline development. Since it has been well-documented that other animal species change their dietary habits during or just prior to breeding season, it seemed sensible to determine if sunfish also exhibited this behavior. The two study lakes are close in proximity in Putnam County, New York. Lake Mahopac is a more urbanized setting, close to road traffic, surrounded by homes, and has little to no vegetation due to the introduction of grass carp. Long Pond is in a more pristine setting with one side being entirely wooded. It is not close to any main roads and there are few houses on the perimeter. The vegetation is for the most part undisturbed except for a small amount removed from its beach areas. Despite the lack of vegetation, Lake Mahopac still has as much species diversity as Long Pond. Unfortunately the bluegill population in Lake Mahopac has suffered from the lack of weed beds, which are necessary for successful breeding, which has resulted in a steady decline in numbers. The redbreast sunfish population in Long Pond is very small, most likely due to the fact that redbreast sunfish prefer moving water and Long Pond is relatively stagnant. Breeding season usually starts in May and ends in August; the exact time changes from year to year based upon weather conditions. Female bluegills from Lake Mahopac exhibited a dietary shift in which they fed opportunistically during the pre-breeding season (when water temperature is below 20° C), but shifted to that of a specialist during the breeding period (when water temperature is between 20° C to 28°C) and post-breeding (when water temperature once again begins to cool). When water temperature falls below 20° C in the fall, sunfish move to deeper waters until the following spring when the water once again warms up and they move to the shoreline to breed. There is some dietary overlap between the species, especially between females during pre-breeding, as well as between male and female redbreast sunfish from Lake Mahopac and male and female bluegills from Long Pond, indicating that if food sources become scarce they could develop both interspecific competition between females and intraspecific competition between the sexes in each lake.
IMPACT OF ODORS ON PATERNAL RESPONSIVENESS AND ASSOCIATED NEURONAL ACTIVITY IN "EXPECTANT" MALE PRAIRIE VOLES (Microtus ochrogaster),
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Dr. Maryam Bamshad-Alavi
Prairie voles (Microtus ochrogaster) are unusual mammals because they are socially monogamous. The breeding pairs form pair bonds and jointly care for offspring. Although the duration and intensity of parental behavior in male prairie voles is similar to that observed in females, there are sex differences in the onset and pattern of infant caring in this species. The factors that contribute to sex differences in parental behavior of prairie voles are unknown. As males and females show all components of active and inactive parental behaviors, it is possible that sensory inputs from infants arouse a different pattern of parental care in each sex. Males and females may also differ in the degree of attentiveness to infant sensory cues and to other environmental stimuli. Furthermore, they may focus their attention on different aspects of infant cues or perceive the same cues differently. I conducted three experiments to test these hypotheses. In the first experiment, I tested the attentiveness and sensitivity of male and female prairie voles towards infant-related odors across the reproductive period. Males and females showed increased attentiveness to infant-related odors at different times during the reproductive period. In the second experiment, I examined the impact of female sensory cues on male responsiveness towards infant odors. The data suggest that male's exposure to the female's tactile and distal cues during the gestation period elicited indirect paternal behavior in presence of infant odors. However, infant odors alone were insufficient to stimulate direct paternal responsiveness in these males. In the third experiment, I studied the neuronal activity of brain areas that could be involved in the enhanced indirect paternal behavior that was observed in males housed with their mate through mid gestation. In response to infant-related odors, males that had stayed with their mate had higher neuronal activity in the ventral tegmental area (VTA) compared to males that had stayed with their same-sex sibling. Together, these studies indicate that female sensory cues in prairie voles play an important role in enhancing indirect paternal care by increasing the male's attentiveness to odors and activating neurons in the VTA region of his brain.
Lichen Taxonomy for the 21st Century: A Revision of the Genus Lepraria s.l. in North America north of Mexico
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The results of a series of studies of lichens are presented. These studies are hoped to codify a forward for taxonomic progress in the group. Chapter one presents a standardized morphological terminology and descriptive scheme for the sterile crustose genus Lepraria. Chapters two through seven present a series of studies where molecular phylogenetic analyses of nrITS sequence were used to resolve species boundaries in Cladonia (III), Lepraria (IV-VII), and Punctelia (II). Chapter eight reveals not all sterile crustose lichens resemble their congeners. Chapter nine presents a study in the use of molecular data to place an unknown sterile crustose lichen in higher level classification of fungi. Chapter ten uses that method to revise the circumscriptions of Lepraria and the sterile fruticose genus Leprocaulon. Finally, chapter eleven comprises a taxonomic revision of the crustose species of Lepraria s. str. that occur in North America north of Mexico.
Regulation of anti-dsDNA B-cells in mice transgenic for the heavy and light chains of an Anti-dsDNA antibody
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Regulation of anti-dsDNA B-cells in mice transgenic for the heavy and light chains of an Anti-dsDNA antibody By Rita H. Lewis Advisor: Professor Linda A. Spatz The diversity of the B-cell repertoire is important for the development of antibodies to a multitude of pathogens. However, in the process of generating antibody diversity, B-cells arise that produce antibodies to self-antigens. These autoreactive B-cells must be kept in check lest they secrete autoantibodies that can induce autoimmune disease. There are several mechanisms inherent in the immune system for regulating autoreactive B-cells. I have been using a transgenic mouse model, in which mice were made transgenic for the R4A IgM heavy and Vê1 light chain genes of an anti-double stranded DNA (anti-dsDNA) antibody, to study the regulation of anti-dsDNA B cells. Anti-dsDNA antibodies are the hallmark of the autoimmune disease Systemic Lupus Erythematosus (SLE). I observed that the transgenic anti-dsDNA B cells are targeted to anergy in the R4A-Cì/Vê1 mice as evidenced by arrested development, receptor down modulation, and functional unresponsiveness and reduced calcium flux in response to B-cell receptor stimulation. I also observed a relatively high frequency of transgenic B cells in the T1 but not the T2 or mature stages of development. In addition, transgenic T1 B cells were observed to display features of anergy suggesting that the T1 stage may be a regulatory checkpoint where anti-dsDNA B cells are anergized and subsequently eliminated. Interestingly, transgenic B-cells that were able to transition to the T2 and T3 stages of development tended to co-express an endogenous heavy chain while the majority of mature B cells in these mice expressed the endogenous heavy chain only. Surprisingly, however, B-cells expressing only the endogenous heavy chain on their membrane were also observed to express transgenic heavy chain transcripts. These results have led me to propose a model whereby autoreactive B cells can escape a regulatory checkpoint if they express more than one heavy chain. These dual receptor expressing B-cells may have diminished autoreactivity if the level of membrane expression of the transgenic heavy chain is low relative to the level of expression of the endogenous heavy chain. Subsequent down-regulation of the transgenic heavy chain as a B-cell matures in the periphery may be a novel mechanism for averting autoreactivity.