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POPULATION DYNAMICS OF THE AMERICAN OYSTERCATCHER (HAEMATOPUS PALLIATUS) NEAR THE NORTHERN LIMIT OF ITS RANGE
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American Oystercatchers (Haematopus palliatus) expanded their range north along the Atlantic Coast, reaching Massachusetts 40 years ago. Concurrent to northward range expansion, oystercatchers have declined in the core part of their range, and have been designated a "Species of High Concern" by the U.S. Shorebird Plan. I examined the demographics of a migratory population of American Oystercatchers in Nantucket, Massachusetts to explore factors influencing local populations. I estimated fecundity (0.20 females fledged per female), annual survival, and dispersal using a variety of field and statistical techniques. I analyzed data from four nesting seasons for oystercatchers in Nantucket and examined temporal patterns in reproductive success. All my measures of reproduction were higher than those from other Atlantic Coast populations. I hypothesized that island populations experience higher reproductive success due to the absence of mammalian predators, and these populations may be sustaining mainland populations. Furthermore, I found that egg survival was exceptionally high but chick survival was low. Therefore, identification of chick loss should be a priority in conservation efforts. I used advanced mark-recapture techniques to estimate annual adult survival and breeding-site fidelity for American Oystercatchers. My results revealed a high rate of true annual survival (0.94) and a strong, but variable, degree of breeding-site fidelity (0.93). Additionally, I used mark-recapture data to estimate juvenile annual survival (0.51) and subadult transition probability (0.18). Using perturbation analyses, I found adult survival and breeding-site fidelity had the greatest impact on local population growth. My modeling also indicated that current reproductive success is sufficient to maintain the population (λ=0.970, 95% CI: 0.90 - 1.02), but not sufficient to predict the recent increase. Using reverse encounter histories, I estimated movement parameters and found approximately 7% of the population permanently emigrates while 16% of the population is likely comprised of adults immigrating into Nantucket. Demographic analyses confirmed that high immigration rates are responsible for the observed growth (λ = 1.08, 95% CI: 0.99 - 1.16). My dissertation provides a foundation for continued examination of shorebird population demographics, a model for demographic analyses of a species of conservation concern, and a preliminary work illuminating the advantages of dispersal and the relationship between breeding-site fidelity and reproductive success.
UBIQUITIN PROTEASOME PATHWAY, AUTOPHAGY AND A NOVEL THERAPEUTIC APPROACH TO PREVENT NEURODEGENERATION LINKED TO INFLAMMATION IN ALS
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Neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases as well as amyotrophic lateral sclerosis (ALS), are a heterogeneous group of clinical disorders characterized by the selective loss of neurons in specific regions of the CNS. Despite their variability they have similar features including the accumulation of proteins that develop into inclusion bodies. Ubiquitinated proteins are major components of these inclusions suggesting that impaired proteasome activity and/or dysfunction of the ubiquitination pathway may be main players in this process. Emerging data reveal that autophagosomes are also components of inclusion bodies, implicating autophagy in neurodegenerative disorders as well. The research described in this thesis led to two discoveries: 1) The UPP, not autophagy, is the main pathway implicated in the degradation of soluble ubiquitinated proteins. Furthermore, proteasome impairment, and not autophagy dysfunction, causes the intracellular accumulation/aggregation of ubiquitinated proteins. We demonstrate that proteasome impairment leads to accumulation of ubiquitinated proteins. Most importantly, inhibition of autophagy failed to cause ubiquitin protein accumulation. We also established that the soluble p62/sqstm1, a polyubiquitin shuttling factor, is associated with the proteasomes and not with autophagosomes. It is thought that p62/sqstm1 delivers polyubiquitinated proteins to proteasomes and/or autophagosomes for degradation. From a therapeutic point of view our data support the notion that pharmacological means to sustain or enhance proteasome activity and p62/sqstm1 levels could be an efficacious strategy to remove ubiquitinated proteins and prevent or delay the onset of neurodegeneration associated with protein aggregation. 2) The activity of the proteasome and the levels of p62/sqstm1 are significantly enhanced by dibutyryl-cAMP in spinal cord cultures. We demonstrate that db-cAMP enhances the activity of the proteasome and p62/sqstm1 levels. Furthermore, pre-treatment of the neuronal cultures with db-cAMP mitigate cell toxicity induced by prostaglandin J2 (PGJ2). PGJ2 is an inflammatory mediator found to be elevated in post mortem slices of spinal cord motor neurons in ALS patients. Our data support the notion that enhancing of proteasome activity by the cAMP/PKA pathway provides an effective neuroprotective strategy against ALS and other neurodegenerative diseases associated with proteinaceous aggregates and signs of neuroinflammation. In conclusion, our data support that impaired proteasome activity occupies the center stage in the development of neurodegenerative diseases. Enhancing proteasome activity can have therapeutic implications in drug development aiming at prevention and treatment of neurodegenerative disorders.
POPULATION DYNAMICS AND OCCUPANCY PATTERNS OF EASTERN SCREECH OWLS (MEGASCOPS ASIO) IN NEW YORK CITY PARKS AND ADJACENT SUBURBS
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Eastern screech owls (Megascops asio) are one of the few raptor species that permanently reside in New York City (NYC). To better inform management of this charismatic species in urban parks, I sought to determine the present status and future viability of existing screech owl populations in NYC and identify potential landscape characteristics that affect park occupancy by screech owls. Using captive, non-releasable owls and isolated free-living owls, I developed a method of identifying individual screech owls via vocalization analysis. Using call-broadcast surveys and subsequent recording of owl responses, I gathered capture-recapture histories of urban owls in three NYC parks and one comparison nature preserve, the Mianus River Gorge Preserve (MRGP), a rural/suburban nature preserve in Bedford, NY. These histories were used to estimate abundance and survival rate in each of the parks. I then projected simulated populations using my estimated adult survival and previously published yearling survival and reproductive rates. I built my projection model to include density dependence based on park area, realistic sex ratio fluctuations, and periodic drops in survival rate due to hypothetical environmental events. Survival in urban parks was 0.98 - 1.0, much greater than in the MRGP, 0.57 ± 0.15. Despite the high adult survival, populations in parks could be extirpated within 10 - 20 years by severe drops in survival rate (e.g., extreme winters, storms, or secondary poisoning) if such conditions occurred more often than once every ten years or if parks were smaller than 1.0km2. In 2008 and 2009, my colleagues and I conducted a citizen science-based study on screech owl occupancy patterns across three counties adjacent to NYC: Westchester and Putnam, NY, and Fairfield, CT. Volunteers conducted call-broadcast surveys on their own properties and sent my colleagues and me the results in 2008 and 2009. Occupancy and detection were modeled as functions of the amount of forest cover and impervious surface cover at each survey point. These models were validated against an independent dataset collected by myself and other trained scientists. Validated models indicated a negative association between occupancy and percent forest cover or, similarly, a positive association with percent impervious cover. Both the citizen science and the systematic datasets supported similar owl-habitat patterns of higher occupancy probabilities in developed areas compared to rural. The above patterns described eastern screech owl habitat use in rural and suburban areas outside of NYC. I hypothesized that at some point urbanization would become too intense for owls to tolerate, and they would be primarily relegated to protected greenspaces in extremely urbanized cities, as I observed in NYC. In 2010, I surveyed 13 additional parks in NYC and the more urban southern sections of Westchester to characterize occupancy patterns in highly urban areas. I used similar landscape measurements as in the citizen science study, only these measurements were taken across the entire park. Occupancy appeared to decline sharply if the percent impervious cover surrounding a park rose above 50 - 60%. I interpreted this pattern as evidence that high urbanization around a park acts primarily as a barrier to immigration. It is also possible that high urbanization around a park leads to higher mortality from vehicles. In terms of management, my work has indicated that in large parks (e.g., >1km2), extinction probability is relatively low, but parks larger than 3km2 may be less suitable if big parks allow the establishment of larger raptors such as barred (Strix varia) or great horned owls (Bubo virginianus). Managers can enhance population persistence by increasing the amount of available habitat via habitat restoration and reforesting or re-meadowing developed but unused parks (e.g., large lawns and paved areas) and increasing over-winter survival and reproductive rates by installing nest boxes. However, frequent chance events can extirpate any park-bound population, and thus managers and city planners should also look to enhance the probability of dispersal and recolonization via corridors or reducing the general level of urbanization around protected parks.
EXPLORATIONS IN ADVANCING MICROALGAE PRODUCTIVITY
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This thesis is part of an effort to advance microalgae cultivation for biofuel and high value products. The first chapters describe a large scale screening effort in which novel strains that hold great promise in large outdoor cultivation systems were isolated. Among these are strains of Scenedesmus obliquus, Borodinellopsis texensis, Chlorella sorokiniana, Ankistrodesmus, Coelastrella, and a previously uncharacterized species in the Chlamydomonadales. These species are characterized in terms of their phylogenetic relationship, biomass productivity, and, for some, lipid and carotenoid profiles under growth and stress conditions. Also discussed in regards to growth metabolism and oil productivity is a unique enzymatic characteristic found in S. obliquus, the presences of a chloroplast localized enolase. Lastly, we demonstrate a novel bacteria-algae mutualistic relationship that may be of particular interest in cultivating microalgae in wastewater. Overall, the work advances algae productivity with algal species and bacteria that may be extended further upon to push forward the field of algae biofuel production.
THE CHARACTERIZATION OF Pb2+ TOXICITY IN RAT NEURAL DEVELOPMENT: AN ASSESSMENT OF Pb2+ EFFECTS ON THE GABA SHIFT IN NEURAL NETWORKS AND IMPLICATIONS FOR LEARNING AND MEMORY DISRUPTION
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Abdeslem El Idrissi
The toxic effects of Pb2+ on the developing rat nervous system has been investigated to assess early developmental GABAergic disruption and its implications with altering inhibitory learning and memory. This goal was achieved using a multi-systems approach: blood lead levels (clinical physiology), qRT-PCR (molecular genetics), brain and primary neuronal culture immunology (immunohistochemical and cellular approaches), physiological cellular components (synaptosomes and protein expression) and finally through learning and memory assessment with GABA mimetic drug manipulations in the intact animal (behavioral pharmacology). The influence of a 956ppm Pb2+ gestational diet (i.e. from birth to sacrifice) resulted in pup mean blood lead levels (BLLs measured in ìg/dL) (Range 28-47) and Dams (Range 33-51) respectively. In contrast, control pups and dams were Pb2+ negative. These ages were selected to determine neurodevelopmental trajectories of the GABA-shift from excitation-to-inhibition postnatally in our model. qRT-PCR studies evinced a delay in mRNA expression regulating GAD 80, 65, CACAN â3, GABAAR and were differentially regulated cortex and hippocampus as a function of age in response to Pb2+. Brain slice immunohistochemistry revealed an early shift of KCC2 expression in both cortex and hippocampus. Notably, these alterations were differentially regulated by age, brain region and subcellular circuitry within structures (i.e. DG vs. CA3). Neuronal cultures revealed that in response to Pb2+ at low micro molar concentrations induced VSCC-â3 nuclear translocation and GABAAR upregulation. KCC2 expression was inhibiting in cultures by Pb2+. Synaptosomal effects of Pb2+ revealed altered glutamate accumulation and handling with increased spontaneous and decreased evoked release in significantly modulated by Pb exposure suggesting altered brain synaptogenesis. Pb2+ exposure resulted in increased binding suggesting post synaptic modification in cortex and hippocampus increasing brain excitability. Behaviorally, Pb2+ exposure resulted in increased anxiety, impulsivity, stress, and disrupted learning and memory regulated by inhibitory circuits that were recovered with taurine, a GABAAR agonist, administration. Specifically, Pb2+ disrupted contextual and auditory associative learning. Taken together, these results suggest that Pb2+ interferes with early VSCCs and GABAAR synergistic action that establishes GABAergic neural networks and in turn produces increased brain excitability and over reactivity as a consequence of reduced inhibition.
FORAGING ECOLOGY OF SHOREBIRDS AT A STOPOVER SITE: NICHE DYNAMICS, AGGRESSION AND RESOURCE USE IN DELAWARE BAY
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Classical ecological theory predicts that generally similar species ought to partition resources in order to minimize competition amongst themselves. This basic idea becomes complex when one is dealing with species that migrate over thousands of miles and forage in a broad diversity of habitats and geographical locations. I studied a suite of migratory sandpipers, and asked whether they partitioned niches at a major migratory stopover in Delaware Bay. During migration, shorebirds form large, usually mixed-species flocks, which forage on marshes, mudflats, beaches or similar two-dimensional habitats where all individuals are distributed on the same horizontal plane. These habitats are often affected by the tidal cycle forcing birds to feed at the same time, which leads to intensified competition through both depletion and interference. Using multidimensional niche approach, I explore whether coexisting shorebirds separate by time of passage, habitat use and foraging behavior, during northbound migration, at the time shorebirds gather in large numbers to capitalize on eggs of spawning horse shoe crabs (Limulus polyphemus) (Chapter 1). I hypothesize that differential migration timing is the most important dimension for separation of species. Also, I investigate aggressive interactions of shorebirds (Chapter 2), hypothesizing that birds will exhibit more aggression toward conspecifics than to heterospecifics, as individuals of the same species, due to morphological similarity, more often compete for resources. Finally, I examine the importance of horseshoe crab eggs for study species through PCR of prey DNA from birds' fecal samples with horseshoe crab specific primers that I designed for this study (Chapter 3). My research demonstrates that shorebird species mostly separate by differential timing of spring migration during stopover in Delaware Bay. Also, the study confirms higher measures of aggression between conspecifics than between heterospecifics, although the incidence of interspecific interactions was higher than previously reported for shorebirds, most likely due to interspecific dominance relationships. The presence of interspecific aggression in mixed-species foraging flocks emphasizes the importance of temporal segregation between migratory species, as such ecological segregation may reduce the opportunity for interspecific aggressive encounters, which in turn can have positive effects on birds' time and energy budget during stopover period. In addition, this study corroborates the importance of horse shoe crab eggs for migrating shorebirds in this crucial stopover area.
The Diversity and Evolution of Transposable Elements in the Genome of The Lizard Anolis carolinensis
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Eukaryotic genomes are littered with repetitive DNA sequences called transposable elements (TEs). Though once considered "junk DNA," these elements can greatly impact their host genomes by influencing genome size, providing novel proteins and promoter sequences, as well as disrupting gene function or causing chromosomal rearrangements. However, the impact TEs have on their host's genome depends on their abundance and diversity, which differ greatly among vertebrate genomes. The genome of teleostean fish contain a very diverse community of elements that are represented only by recent inserts found in very low copy number (<100). On the other hand, most mammalian genomes have a very low diversity of TEs dominated by L1 retrotransposons, yet elements in mammals accumulate to reach extraordinary copy numbers (>100,000). This difference accounts, for the most part, for the difference in genome size between these two groups. Until recently, we did not have a good model to study the transition from the small repeat-poor genome of fish to the larger repeat-rich genome of mammals. The first non-avian reptile genome sequence, the lizard Anolis carolinensis (the North American green anole), bridges the large phylogenetic gap between fish and mammals and provides a better understanding of early amniotes genomic evolution. We performed the first comprehensive analysis of TEs in the anole genome. We found that the anole genome contains an extraordinary diversity of active TEs. This genome contains several concurrently active clades of non-LTR retrotransposons (CR1, L1, L2, RTE, and R4) each represented by multiple families. The vast majority of insertions are very young, suggesting that most elements do not reach fixation and when they do, they decay rapidly. In addition the anole genome is inhabited by multiple superfamilies of DNA transposons (hAT, Helitron, Maverick and Chapaev), some of which were laterally transferred to the anole. We conclude that the genomic landscape of the lizard is strikingly similar to the one of fish and shows little resemblance to mammalian genomes.
BIODIVERSITY, TAXONOMY AND SYSTEMATICS OF NEW WORLD FRESHWATER LEECHES (ANNELIDA: HIRUDINEA) WITH PARTICULAR EMPHASIS ON GLOSSIPHONIID LEECHES AND THEIR BACTERIAL ENDOSYMBIONTS
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The phylum Annelida Lamarck, 1809 includes segmented worms such as leeches, earthworms, lugworms, sandworms and clamworms that inhabit almost all possible environments and places of the world. Leeches (Class Hirudinea) represents only one group of around 680 species out of the approximately 16,500 described species of Annelida. The class Hirudinea has been divided in two groups based on their mouthparts. The order Rhynchobdellida, a paraphyletic assemblage, includes species with a large and eversible proboscis and the order Arhynchobdellida that includes species with a muscular pharynx with or without jaws. Both orders include organisms specialized to feed on vertebrate blood. This study includes the description of eight species of leeches new to science that belong to three families (Glossiphoniidae, Macrobdellidae and Praobdellidae). The phylogenetic relationships of species of three families (Glossiphoniidae, Macrobdellidae and Praobdellidae) and one suborder (Erpobdelliformes) were investigated using molecular and morphological data and a suite of phylogenetic methods (Parsimony, Maximum Likelihood and Bayesain Inference). The description of the new species Tyrannobdella rex (Praobdellidae) and Oxyptychus bora (Macrobdellidae) are discussed in the context of their placement in phylogenies. The phylogenetic study of Erpobdelliformes includes the comparison of alternative classification schemes. Based on the results, the phylogenetic position of the terrestrial and macrophagous Orobdella octonaria (Gastrostomobdellidae) within the Erpobdelliformes is established for the first time. The phylogenetic relationships of the proboscis-bearing species of the genera Haementeria, Helobdella and Placobdella were investigated using a combination of nuclear and mitochondrial markers and Parsimony and Bayesian Inference methods. In addition to the monophyly of Haementeria, Helobdella and Placobdella, the 3 genera formed a monophyletic group notwithstanding their different feeding preferences. The correlation with phylogeny and some morphological traits is shown. These include, eyespot morphology, annulation patterns, shape of the ovisacs, sensory organs on the dorsal surface and presence of bacteriomes. Species of Haementeria and Placobdella have specialized organs called bacteriomes associated with their salivary complex that harbor symbiotic proteobacteria. Using DNA bacterial sequences (16S rRNA), the exclusive association of Haementeria spp. with gammaproteobacteria and Placobdella spp. with alphaproteobacteria is shown. Using pyrosequencing technology, the nucleotide sequences of a DNA sample extracted from the bacteriomes of Placobdella parasitica were analyzed. A total of 1,053,345 DNA fragments were obtained and assembled. Leech and symbiont DNA fragments were separated using Blast tools and 50 bacterial and Helobdella robusta genomes for reference. Finally, the so-called DNA barcoding protocol is discussed and some recommendations were given to increase the information content of the database (Bold system). In addition, DNA barcoding protocol was used to estimate the diversity of species of Helobdella from Mexico.
Medicinal Plants of Northern Thailand for the Treatment of Cognitive Impairment in the Elderly
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Dementia is a progressive neurological disease affecting memory and behavior. The diagnosis of dementia is increasing exponentially worldwide and with it the potential risk for a severe social and economic burden of caring for an increasing debilitated elderly population. Cognitive impairment, and especially memory loss, can be the first indication of dementia. This study documents the treatment of cognitive impairment in the elderly by Thai traditional healers in northern Thailand using medicinal plants. Interviews were conducted from 2008-2012 to investigate the etiology of dementia in Thai Traditional Medicine and identify plants used to treat memory loss in the pharmacopeia of northern Thailand. Multi-plant herbal formulas from ancient manuscripts of the Lanna Kingdom were obtained from Thai traditional healers. These formulas were analyzed through ethnobotanical inquiry for plant species with potential bioactivity against memory loss in the elderly. Crude extracts of eleven selected plant species were screened through four in vitro bioassays to measure their general antioxidant activity, total phenolic content and acetylcholinesterase inhibition activity. Of these eleven species, five plants exhibited high levels of acetylcholinesterase inhibition activity: Cinnamomum bejolghota (Buch-Ham.) Sweet, Dracaena loureiroi Gagnep., Diospyros decandra Lour., Jasminum sambac (L.) Aiton., and Eurycoma longifolia Jack. One plant, Cinnamomum bejolghota demonstrated high activity in all four in vitro bioassays. Three different doses of an ethanol extract of Cinnamomum bejolghota were evaluated for their memory enhancing ability on in vivo rat behavioral models and enzymatic marker tests on their brain tissue. Results from the Morris Water Maze navigation task showed that the two highest dosages of the extract produced significant memory improvement after two weeks of treatment. Enzymatic marker analyses in three portions of the rats' brains associated with memory formation, the hippocampus, striatum and cortex, showed significant acetylcholinesterase inhibition activity thereby increasing acetylcholine levels in these parts of the brain. This study identified a plant with memory enhancing activity that, with further study, could help to alleviate the suffering of those afflicted with age-related memory decline. Ethnopharmacological studies support the viability of traditional medicine to treat diseases that are relevant in modern society.
The Non-canonical Growth Activating Functions of HIghly Expressed Mdm2-C
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Mdm2 is an oncoprotein that regulates the tumor suppressor protein, p53 via the Mdm2 canonical pathway. The pathway involves p53 protein degradation and transcriptional repression. Mdm2 is often found over-expressed in cancers. In the presence of Mdm2 over-expression, the activity of p53 is frequently attenuated and the protein levels remain paradoxically high. Cancers with Mdm2 over-expression also over-express mdm2 splice variant transcripts. There are over forty identified spliced variants of mdm2. Therefore, we hypothesized that in the presence of Mdm2 over-expression, a different form of Mdm2 protein exists that does not function in the Mdm2 canonical pathway. In this study, the functions of an Mdm2 isoform, Mdm2-C, were investigated. We observed that Mdm2 over-expressing cells have high basal levels of mdm2-C transcript. We have cloned and expressed mdm2-C in vitro. We created an Mdm2-C specific antibody, Mdm2 C410, to the splice junction of exons four and ten (Mdm2 C410) and validated the C410 antibody using in vitro translated full-length Mdm2 compared to Mdm2-C. The Mdm2 C410 antibody did not detect Mdm2-FL. We saw that different human cancer cell lines, liposarcoma and breast cancer tissues, over-expressed endogenous Mdm2-C protein. We also observed that there was an estrogen-dependent increase in endogenous Mdm2-C protein in ER+ mdm2 SNP309 breast cancer cells that was p53-independent. In addition, the exogenous expression of Mdm2-C in human p53-null cancer cells showed that Mdm2-C does not function in the Mdm2 canonical pathway. Immunofluorescence utilizing the Mdm2 C410 antibody displayed that Mdm2-C was localized to the cytoplasm and nucleolus in a speckled pattern that might be integral to its cellular functions. We observed that the over-expression of Mdm2-C in the presence or absence of p53 in human and mouse cell lines promoted cell growth. Furthermore, the partial down regulation of mdm2-C via siRNA in mutant p53 G/G mdm2SNP309 breast cancer cells, T47D resulted in increased cell death. Thus suggesting that unlike other Mdm2 isoforms and full-length Mdm2, Mdm2-C has distinct roles in cell survival and p53-independent Mdm2 molecular pathways. Here we report the first identification of an endogenous tumor-associated splice variant Mdm2 protein, and document that Mdm2-C functions through a non-canonical growth activation pathway that is p53-independent.