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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.
Postnatal refinement of interareal circuits in ferret visual cortex
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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
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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
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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.