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The Sustainable Management and Conservation Santalum yasi (Sandalwood) in Fiji and Tonga: A Combined Ecological and Genetic Approach
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Valued internationally for the aromatic oil found within its heartwood, Sandalwood (Santalum, Santalaceae) is one of the most heavily exploited groups of plants across its range. While historically, most oil has been harvested from Santalum album in Southeast Asia and India, the decrease of S. album sources has caused a widening gap between supply and demand, which creates profitable market opportunities and increasing harvest pressure for alternative sandalwood oil sources. Santalum yasi, a quality alternative, has been harvested extensively in Fiji and Tonga, yet is vastly under-studied. The absence of basic data on population dynamics and genetic variation for remnant populations remains a major constraint to the sustainable management of this culturally and economically valued resource. This dissertation focuses on the ecological and genetic data and analyses that can aid in developing sustainable management strategies. Population size-class structure data was collected using transects in the three densest natural populations of Santalum yasi. Population dynamics, current species distribution, and ecological threats were investigated to find that the few remaining wild stands display discontinuous size class structures, are under regenerative stress and that the natural distribution has diminished significantly, even to local extinction in some areas. Using a nuclear microsatellite analysis, genetic variability within and between populations was investigated. Results suggest that there is no significant genetic variation between populations, but that most of the genetic variation lies within populations. This genetic distribution suggests that there is a significant level of gene flow between and among populations, most likely through human induced dispersal, showing a more panmictic trend than previously supposed. This may provide molecular evidence confirming the Western documentation and traditional oral history of extensive interaction between Fiji and Tonga and their trade of plants and culture. Based on these dwindling levels of yasi population size and unstructured genetic variation in Fiji and Tonga, further enumerations and resource surveys are not practical to conduct at this time. Rather, forestry and governmental efforts should be focused on promotion of local involvement in assisted natural regeneration of wild stands and preservation of genetic variation through in situ, community-mediated conservation.
The Ecology Of Winter Flounder From An Otolith Perspective
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THE CITY UNIVERSITY OF NEW YORK ABSTRACT THE ECOLOGY OF WINTER FLOUNDER FROM AN OTOLITH PERSPECTIVE By George W. Jackman Advisors: John Waldman (CUNY Queens College) and Karin E. Limburg (SUNY ESF) In this dissertation, sagittal otoliths were used as a lens to examine latent life history patterns in winter flounder (Pseudopleuronectes americanus) and also as a means of interpreting the species’ relationship to their abiotic and biotic environment. Otoliths provide a unique and powerful perspective into the lives of the telost fishes, because they permanently record the spatial and temporal histories through sequential growth patterns from conception to capture. The patterns of growth and dormancy in the otolith are regulated by endogenous and exogenous rhythms, and as the otolith grows, trace elements are absorbed from the ambient environment and incorporated into the calcium carbonate (CaCO3) matrix. Hence, concentric bands in the otolith reliably indicate age and growth conditions similar to the annuli in trees, whereas the chemical chronology contained within the CaCO3 crystal can function as a geochemical tag, to permit retrospective tracking of a fish’s movements and reconstruction of its environmental history. The first chapter in this study demonstrates that winter flounder sagittae are not morphologically, nor are they chemically identical, which is an essential distinction that sets them apart from those found in bilaterally symmetrical fishes. This finding has important implications when using otolith chemistry to investigate population structure of winter flounder and other flatfishes, because indiscriminate use of either otolith can bias statistical results. Consequently, method standardization is recommended when performing otolith chemistry in flatfishes. Furthermore, the results of this investigation provides evidence that the blind-side otolith in flatfishes may be absorbing chemicals differently from their eyed-side counterparts, though these results warrant further testing. In the second chapter, otolith microchemistry is utilized to examine the fine-scale stock structure of winter flounder that were sampled from the coastal margins of Long Island and surrounding areas. Using otolith microchemistry in this manner, group membership was recognizable an on a scale of tens of kilometers with a statistical accuracy that ranged from 83-87% depending upon spatial dimensions when re-classifying the specimens back to their location of capture. The second part of this chapter examines the feasibility of using otolith microchemistry with specific elemental markers to make qualitative assessments of inshore habitat of winter flounder. Through this investigation, it was revealed that some of the most chemically contaminated bodies of water still make important contributions to winter flounder recruitment, and juvenile growth in these systems can potentially exceed growth patterns in more pristine locations. The last chapter of this dissertation looks at the age and growth structure of winter flounder in the Hudson River Estuary (HRE) and western Long Island Sound (WLIS) and compares those results to several large-scale surveys that were performed last century and collectively form the historical record of winter flounder in New York waters. The ensuing results of this analysis show that winter flounder in the HRE and WLIS have incurred a faster growth rate, and larger, if not older fish comprise a greater percentage of the population than they did during the early and middle decades of the twentieth century. The increasing size-at-age shown by winter flounder in the HRE and WLIS correlates with a release from intra-specific competition and increasing pressure generated by size-selective mortality imposed by a resurgent and newly emerging suite of predators. Finally, the conclusions of this thesis summarize the results of these investigations and discuss potential directions for future research.
THE ROLE OF Na+/H+ EXCHANGER-1 (NHE1) IN MAMMARY BRANCHING MORPHOGENESIS AND MAINTENANCE OF TISSUE ARCHITECTURE
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Branching morphogenesis in vivo is a highly ordered process that necessitates spatially and temporally choreographed cues by growth factors and hormones, as well as mechanical and signal feedback from the extracellular matrix. Successful completion of this developmental process results in the architectural, and thereby the functional, basis for the lung, collecting ducts of the kidney, salivary, and mammary glands. The quest to understand the basic biological mechanisms underlying this developmental morphogenesis has lead to many seminal findings in the field of epithelial tube generation, as well as provided valuable insight into the pathogenesis of cancer. The primary focus of this thesis was elucidating the role of the Na+/H+ exchanger type 1 (NHE1) in branching morphogenesis of the mouse mammary gland. To accomplish this goal, we used three-dimensional (3D) primary tissue culture of mammary gland pieces (organoids) in a four day organotypic assay of growth factor induced branching morphogenesis. NHE1 is a ubiquitously expressed master regulator of intracellular pH (pHi). We found that blocking the function of this exchanger in the presence of growth factor stimulation led to altered kinase signaling, inhibition of growth factor induced alkalization, sustained proliferation after four days, ectopic expression of keratin 6 (K6), and a dramatic failure to undergo branching morphogenesis. These findings led us to question the role of NHE1 in the maintenance of branched mammary tissue architecture. We, therefore, inhibited NHE1 function on fully branched structures in our assay and found that NHE1 inhibition led to rapid loss (within 24 hours) of branched architecture by a process of branch fusion, with complete loss of the branched morphology after four days. This was not accompanied by cell death or altered proliferation, however, we did record altered intracellular pH (pHi) in the end buds of branched structures that had NHE1 inhibited. NHE1 localization, F-actin organization, and myoepthelial cell location were altered in structures that had undergone a loss of architecture, indicating a loss of tissue organization. Finally, we found that NHE1 inhibition resulted in a decrease in mammary Ecadherin. Having found that NHE1 function is vital for both branching morphogenesis and the maintenance of branched architecture, we considered the role that NHE1 could be playing in the pathology of breast cancer. Both intracellular and extracellular pH is deregulated in cancer. This could be attributed to over activity of NHE1. Additionally, NHE1 is over expressed in many cancers. We used the ER+ breast cancer cell line MCF7 to investigate the therapeutic potential of chemotherapy augmentation by NHE1 inhibition. We found that Cycylophosphamide, a DNA alkylating chemotherapeutic agent known to be more effective in an acidic environment, was roughly 5 times more effective when used along with NHE1 inhibition. These findings indicate that NHE1 is a critical regulator of branching morphogenesis and tissue stability, as well as suggests a potential therapeutic target for the treatment of breast cancer.
Origin and development of hematopoietic tumors in sumoylation mutants of Drosophila melanogaster
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The larval hematopoietic system of Drosophila melanogaster consists of freely circulating cells, the sessile population, and the hematopoietic organ called the lymph gland. Most hemocytes function as macrophages and play a significant role in innate immunity. Hemocytes also remodel tissues, aiding in development of the organism. Constitutive activation of immune signaling pathways, as well as various mutations in genes which are not linked to immune pathways, result in the overabundance of circulating hemocytes and tumor formation. Many aspects of tumor development, such as the identity of the affected hematopoietic population and mechanisms of tumor growth are either not characterized or not well understood. The overall goal of this project was to understand the origin and development of hematopoietic tumor formation in Ubc9 mutants. Ubc9 is an E2 enzyme, which conjugates SUMO (small ubiquitin-like modifier) to a range of target proteins. Sumoylation targets vary in function from structural components to enzymes and transcription factors. Thus, sumoylation affects multiple cellular functions via modification of protein localization, stability or activity. Loss-of-function Ubc9 mutants of Drosophila exhibit severe defects in hematopoietic and immune tissues, including hemocyte overproliferation and tumor formation during larval stages. In this dissertation, we report that the hemolymph of Ubc9 mutants contains hematopoietic cells and structures that range from aggregates (composed of only few cells) to small and large tumors. The largest tumors are less than 1 mm3 in volume. Most cells and aggregates exhibit high ration of mitotic cells, but only a few of the largest tumors in the hemolymph contain actively dividing cells. Based on staining patterns and genetic rescue experiments, we propose that the large tumors are derived specifically from overgrown posterior lobes of the hematopoietic organ. The origin of smaller tumors and aggregates is less clear. Our data suggest that these structures likely derive from circulating hemocytes and fragments of dispersed anterior lobes. Microtumors in Ubc9 mutants arise from the highly-mitotic mutant stem/progenitor cells of the lymph gland. Loss of sumoylation cascade enzymes E1 (Aos1/Uba2), E2 (Ubc9), or E3 (PIAS) leads to loss of proliferative quiescence in hematopoietic precursors localized within the lymph gland. Proliferative quiescence of these precursors is at least in part mediated by the activity of the cyclin-dependent kinase inhibitor Dacapo/p21. Expression of Dacapo homolog, human p21, in the Ubc9 progenitor cells relieves tumor formation. These studies suggest that sumoylation provides a cell-intrinsic mechanism to preserve stem/progenitor cell states for stress response, immunity and development of the fly.
Effects of endophyte infection, environmental stress and competition on Lolium perenne populations from the Mediterranean region
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The role of ubiquitin-mediated proteolysis in Drosophila glia development
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Biological processes are dynamic, requiring both simple and complex mechanisms that enable cells to adapt with the ever-changing environment. Ubiquitination is one of many posttranslational modifications that result in a change in cellular activity. Mono-ubiquitination, the addition of a single ubiquitin moeity leads to endocytosis and membrane trafficking, while the addition of a multiple ubiquitin chains primarily results in protein degradation. Rap/Fzr acts as an activator of the E3 ubiquitin ligase, Anaphase Promoting Complex (APC) which has been studied for its role in the timely degradation of cell cycle regulators. My thesis work focuses on novel roles Rap/Fzr during nervous system development and specifically investigates its role during glia development. My results show that Rap/Fzr regulates glia development through its interaction with Loco, an RGS protein and Nonstop, a ubiquitin specific protease. Bioinformatic analysis revealed that both Loco and Nonstop contain Destruction box (D-box) motifs and KEN box motifs, which are amino acid sequences used by Rap/Fzr for substrate recognition. My thesis work shows that Rap/Fzr targets Loco for ubiquitination, and subsequent degradation and thus, inhibits the formation of glia from dividing neuroblasts. Furthermore, Rap/Fzr together with Nonstop, regulates the migration and the endoreplication of glia cells.
Effects of bisphenol-A on oxidative stress, mitochondrial dysfunction and behavior: lymphoblasts and Drosophila melanogaster studies - Potential implications in autism
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Autism is a behaviorally defined neurodevelopmental disorder characterized by impairments in three main areas of social interaction, communication, and repetitive, restricted interests and behaviors. There has been an increase in the prevalence of autism with recent estimation of 1 in every 50 children diagnosed with autism. Though there is no single identifiable cause for autism, several studies have shown an increase in oxidative stress and decrease in antioxidants in autism. The role of environmental factors has also been implicated in autism. Bisphenol A (BPA) is a widely used chemical in the manufacturing of plastics, and its exposure has raised concerns in a variety of conditions. The present study with lymphoblastoid cells and Drosophila melanogaster identifies BPA as an environmental risk factor for the increased oxidative stress, mitochondrial dysfunction and behavioral impairments in lymphoblasts and Drosophila melanogaster. When lymphoblastoid cells were exposed to BPA, there was an increase in lipid peroxidation and free radicals (reactive oxygen species) and decrease in mitochondrial membrane potential generation suggesting BPA induced oxidative stress and mitochondrial dysfunction. The study also illustrates an increase in the mitochondrial DNA (mtDNA) copy number in the lymphoblasts in response to the BPA exposure. In neurodevelopmental disorders such as autism, behavior is an important component of the condition. We therefore attempted to detect behavioral modifications in Drosophila melanogaster following exposure to BPA. In this study, we used an open field assay to help identify disturbances in locomotion along with repetitive behavior in BPA-exposed flies. We also observed an abnormal social interaction between the BPA-exposed flies in a social setting. Along with the behavioral modifications, there was also an increase in the lipid peroxidation in the brains of the BPA-exposed flies. Furthermore there was also a delay in the development of the Drosophila embryos, although we did not detect any gross morphological changes in the peripheral nervous system of the embryos following BPA exposure. We have therefore demonstrated that Drosophila may be used as an animal model for complex neurodevelopmetal disorders, which have a poorly understood etiology.
Effects of The Enzyme Inhibitor Prohexadione-calcium on Hops Determined by LC-TOF-MS
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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.
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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.
The function of the phospholipid flippase Atp8a1 in neurotransmission, brain development and autistic behavior
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Abstract The function of the phospholipid flippase Atp8a1 in neurotransmission, brain development, and autistic behavior By Daniel J. Kerr The plasma membrane consists of lipids and proteins. Among the integral membrane proteins are P-type ATPases, widely expressed in both prokaryotes and eukaryotes, which use ATP to translocate ions across a plasma membrane. Type IV enzymes are putative aminophospholipid translocases (APLTs) and catalyze phosphatidylserine (PS) transfer into the cytosolic leaflet of a lipid bilayer. Previously, our group showed pronounced PS externalization in Atp8a1 (-/-) mice but not wild type (WT). Subsequent behavioral testing demonstrated that these Atp8a1 (-/-) mice display significant deficiency in spatial learning (p = 0.0001), increased hyperactivity, and decreased anxiety, all indicating aberrant hippocampus-dependent behavior. The putative flippase ATP10C gene, located within chromosome 15q11-q13 has been identified as an autism susceptibility locus and that the Atp8a1 gene is located in the middle of an autism-associated 4p12-15.3 inversion domain also containing a chromosome 4p GABAA receptor gene cluster. Based on such information, I asked whether there is a link between Atp8a1 and autism. Using Western blotting analysis of human brain homogenates (tissue specimens from brain bank), I demonstrated a pronounced induction of Atp8a1 in the hippocampus of juvenile autistic subjects compared to control. The difference was more evident when only the juvenile males were examined. This increase in the putative flippase was also observed in the temporal lobe of autistic children. I saw no significant differences in the level of post-synaptic density 95 (PSD95), a well-known marker for synaptic connectivity, in neither the hippocampus nor temporal lobe of autistic juvenile individuals. The role of Atp8a1 was further investigated by using mouse models. Using electron microscopy (EM), Atp8a1 (-/-) mice were shown to have fewer and weaker glutamatergic excitatory synapses in the CA1 hippocampus region compared to wild type controls. Paired-pulse analysis of the Schaffer collateral pathway demonstrates inhibition at 20-ms inter-stimulus interval of the Atp8a1 (-/-) mice but not controls. No differences in social interaction were observed between the two groups. In separate experiments the level of Atp8a1 was boosted by injecting a lenti virus-Atp8a1 construct into the hippocampi of C57/BL6 mouse pups at the early developmental stage of postnatal day 6 (P6). EM analysis revealed that the mice with elevated Atp8a1 had fewer and weaker glutamatergic excitatory synapses in the hippocampal CA1 region compared to mice injected with the empty construct. Although not significant, a trend toward inhibition of the Schaffer collateral pathway at 20-ms inter-stimulus was observed in the mice receiving the Atp8a1 construct. Social interaction tests indicated possible autistic-like behavior in the mice with increased Atp8a1 in the hippocampus. These findings suggest that either enhanced or diminished levels of the flippase Atp8a1 may be detrimental to brain connectivity. Furthermore, increased levels of Atp8a1 in the mouse hippocampus may be associated with deficits in social behavior. It is therefore possible that mice with enhanced hippocampus Atp8a1 may serve as a future model in autism research.