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The Diversity and Evolution of Transposable Elements in the Genome of The Lizard Anolis carolinensis
Year of Dissertation:
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
Year of Dissertation:
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.
Genetic, Morphological, and Ecological Relationships Among Populations of the Clam Shrimp, Caenestheriella gynecia.
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Little is known about the ecology of the clam shrimp, Caenestheriella gynecia. Caenestheriella gynecia was first discovered in 1939 in a single pool in Oxford, Ohio. Schmidt and Kiviat (2007) reported four new localities of C. gynecia in New York and New Jersey, three within the Hudson Valley of New York and one in northeastern New Jersey. Caenestheriella gynecia may have originated from a very small founder population due possibly to unusual dispersal vectors from its natural range to the west, in Ohio. Egg samples and hatched individuals were obtained from all study sites and specimens were raised in the lab to estimate several growth and survivorship traits. In the field, puddle habitats were observed between the months of May and August where water quality parameters (i.e., dissolved oxygen, temperature, conductivity and pH, and nutrient composition) were recorded. Genetic comparisons across the study sites were made using nuclear DNA sequencing and random amplified polymorphic DNA (RAPD) analysis. The results of this study presented a wide range in the hydro-chemical and physical characteristics of the ephemeral pools in which C. gynecia seem to tolerate. Morphologically, New Jersey and Massachusetts populations possessed meristics counts within the range of those discovered by Mattox in 1950. However, I recommend the placement of the New York population within the Cyzicus genus as their meristic measurements fell outside the range for Caenestheriella. RAPD results revealed the presence of more than one clone in puddles containing C. gynecia although mtDNA sequencing did not reveal any genetic variation within or among populations. The lack of males within C. gynecia's population and low levels of genetic variability support the clonal nature of a strictly parthenogenetic species. These investigations provide a substantial extension of fundamental knowledge of this poorly understood species.
FUNCTIONAL EVOLUTION OF THE APETALA1/FRUITFULL GENE LINEAGE
Natalia Pabon Mora
Year of Dissertation:
Several MADS-box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity, but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core-eudicot euAP1 and euFUL genes, but notably the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae-specific. Understanding how functional divergence of the core-eudicot duplicates occurred requires a careful examination of the function of pre-duplication (FUL-like) genes. Using Virus Induced Gene Silencing (VIGS), it is shown that FUL-like genes in Papaver somniferum (opium poppy) and Eschscholzia californica (California poppy) function in axillary meristem growth and in floral meristem and sepal identity, and play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal organ identity. In contrast, it is shown that the Aquilegia coerulea FUL-like homolog does not appear to play a role in flower or fruit development and instead has been recruited in leaf morphogenesis. In general the FUL-like gene functional repertoire encompasses all roles previously described for the core-eudicot euAP1 and euFUL genes, and subfunctionalization can be postulated as the functional outcome after the major AP1/FUL gene lineage duplication event. However, these results also point to significant functional variability of FUL-like genes within Ranunculales, most likely due to gene duplication and loss, as well as changes of FUL-like protein partners in different taxa.
Role of Toll-NF-kB Signaling in Inflammation and Immune Homeostasis in Drosophila melanogaster
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Inflammation is defined as a localized reaction in response to injury or tissue destruction. It serves to contain or sequester the causative agent. Fundamentally important to human health, inflammation and its aberrant regulation underlie many diseases including cancer, diabetes and heart disease. Like humans, fruit flies respond to infection by coordinating complex defense responses. Parasitic wasps are natural enemies of Drosophila and attack larvae or pupae. They inject one or more eggs, each approximately 100 mm in size, into the hemocoel of the fly larva. Oviposition results either in the development of the wasp larva that gradually eats the Drosophila host tissue, or the host's blood cells encapsulate the wasp egg. In the latter scenario, circulating host blood cells surround the wasp egg and inhibit its development. In addition to the activation and aggregation of the host blood cells in response to wasp attack, infected larvae also activate gene expression in the fat body. A number of antimicrobial peptides and other immune-related proteins are secreted into the hemolymph. The humoral arm is activated within the first couple of hours of infection, although the significance of this activation is not understood. It is also not clear how the encapsulation reaction is so tightly controlled or how it is terminated. Previous studies from our laboratory have focused on the role of Toll-NF-kB signaling in hematopoiesis. Larvae deficient in either IkB/cactus or the SUMO-conjugating enzyme, Ubc9, exhibit hematopoietic defects (overproliferation and abnormal differentiation of the blood cells) accompanied with aggregation and microtumor formation. These mutants also express antimicrobial peptides such as Drosomycin from the fat body even in the absence of infection. Given that both the humoral and cellular immune systems of these mutants are hyperactive, we hypothesized that changes induced after wasp infection represent acute inflammation, and these effects become chronic in cactus or Ubc9 mutants. Studies in the first chapter explore the cellular and molecular parallels between wasp-induced gene expression and encapsulation versus constitutive gene expression and encapsulation (in microtumors) of Ubc9 mutants. We show that several core components (including SPE, Toll, and cactus) of the Toll-Dorsal pathway are activated in each case. However, while gene expression after wasp infection shows acute phase profile (activation followed by downregulation), expression in mutants remains high. We show that the cytokine Spätzle (the Toll ligand) is expressed in the immune cells, hemocytes and the fat body. Spätzle protein levels are higher after wasp infection and in Ubc9 mutants. Misexpression of either SPE or Spz protein in immune tissues of wild type larvae leads to blood cell proliferation, differentiation, infiltration of the fat body tissue and melanized microtumors. We propose a pro-inflammaotry role for SPE/Spätzle whose downregulation is essential for limiting acute inflammation. Accordingly, loss of spz suppresses Ubc9- defects and loss of SPE in the immune cells fails to encapsulate the parasitic egg. In contrast to the pro-inflammatory role of SPE/Spz, SUMO conjugating enzyme, Ubc9, and IkB homologue, Cactus, are anti-inflammatory agents. RNAi knockdown of Ubc9, or even Uba2/Aos1 subunits of SUMO activating enzymes leads to systemic chronic inflammation. Affected animals activate Drosomycin in the fat body in the absence of infection. Blood cells infiltrate the fat body and these changes are accompanied with microtumor development. Extensive staining of fat body and blood cells reveals that while cactus transcription and protein levels increase after infection, Ubc9 mutants cannot sustain normal cytoplasmic levels of Cactus protein. These studies demonstrate the existence of a sumoylation dependent Ubc9/IkB modulated immune homeostasis mechanism that balances the pro- and anti-inflammatory factors in the hosts. This study on Drosophila inflammatory responses complements the existing mammalian models for cancer inflammation. Knowledge gained from our system should be highly relevant in developing novel strategies for medical and agricultural pest control.
The Sex of the Cell Dictates its Response
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Male and female differences in frequency of occurrence in disease have perplexed scientists for some time. This in part derives from limitations in the systems in which one can evaluate sex differences. At the organismal level, differences can be hidden by a myriad of extensive and complex factors. Additional limitations exist since most biomedical studies are performed almost exclusively on male subjects, as the female hormonal milieu is intrinsically more variable and too troublesome for routine inclusion in research protocols. Research documenting sex differences continues to grow, and while most researchers suggests that sex hormones are at the core of these differences, more evidence is suggesting other innate biological factors are at play. To address these limitations, we asked whether differences could be induced and assessed in a controlled cellular system. We then asked whether any aspect of sex dimorphism could be attributed to chromosomal rather than hormonal differences; and finally established a role for differential DNA methylation in sex differences. We found that male and female cells responded differently to cell death inducers as measured by cell viability. We then evaluated gene expression between the sexes, and found that many genes were differentially expressed in vivo. These differences persisted in our cultures, affording us the ability to further characterize these sex differences. Furthermore, we found that cell death induction led to dimorphic gene expression; at instances having opposite effects on cells, where one sex is repressed, and the other is induced. We then evaluated DNA methylation to characterize the differential gene regulation. Blocking DNA methylation globally, using 5-Aza-2-deoxycytidine, led to a loss of gene expression differences between the sexes and found that differences in methylation patterns correlated directly with differences in gene expression. Here we provided a model system, whereby we can test for individual differences resulting in sex dimorphisms at the cellular level. It is critical to continue to expand our knowledge in this area, as this work can be extrapolated and applied to other instances where differences are being measured between the sexes; providing more tools to better characterize those conditions.
A Systematic Revision of North American Tolypella A. Braun (Charophyceae, Charophyta)
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Abstract A SYSTEMATIC REVISION OF NORTH AMERICAN TOLYPELLA A. BR. (CHAROPHYCEAE, CHAROPHYTA) by William Pérez Advisor: Dr. Kenneth G. Karol Charophyta comprises the algal classes Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Coleochaetophyceae and Zygnematophyceae and the land plants. However, the precise phylogenetic position of these algal classes with respect to land plants is unresolved as are the phylogenetic relationships among genera in Charophyceae (Characeae). Characeae contains two tribes with six genera: tribe Chareae (Chara, Lamprothamnium, Lychnothamnus and Nitellopsis) and tribe Nitelleae (Nitella and Tolypella). Tolypella was considered the third most species-rich genus but, in the most comprehensive taxonomic treatment of Characeae, 16 Tolypella species were consolidated into two species, T. nidifica and T. intricata in sections Rothia and Tolypella, respectively. It was further suggested that Tolypella might be a derived group within the closely related genus Nitella. Currently, there are no comprehensive molecular phylogenetic studies of Tolypella. In this investigation into North American Tolypella, sequence data from the plastid genes atpB, psbC and rbcL and the nuclear ribosomal operon 18S-ITS1-5.8S-ITS2-28S were assembled for a broad range of charophytic algae and land plants in order to address Tolypella species diversity and resolve their relationship to other genera in Characeae. In addition, mitochondrial and plastid genomes were completely sequenced for three Tolypella species for a comparative analysis of genome architecture and gene content and for inclusion into a multi-gene phylogeny of the Charophyta. Phylogenetic analyses of the plastid and nuclear gene data showed that Nitelleae is paraphyletic with Nitella sister to Chareae. Genus Tolypella and Tolypella section Tolypella were recovered as monophyletic. Tolypella section Rothia was weakly to moderately supported as monophyletic or weakly supported as paraphyletic in several analyses. Several lineages of the traditionally recognized Tolypella species in addition to a new species, T. ramosissima sp. nov., were identified, which suggests greater species diversity in Tolypella than currently recognized. Comparative analyses of mitochondrial and plastid genomes of three Tolypella species, T. canadensis, T. glomerata and T. intricata, showed that gene content, gene order and genome architecture are relatively conserved, however, T. glomerata showed a complete loss of the Ndh 1-complex genes. Phylogenetic analyses also recovered a monophyletic Tolypella and sections Rothia and Tolypella but showed conflicting results regarding the sister taxon to land plants with either Charophyceae or Zygnematophyceae as their closest living relatives.
Search for host factors involved in attachment of Agrobacterium tumefaciens to plants.
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Agrobacterium tumefaciens is able to infect a diverse array of plants and causes crown gall disease. Typically these bacteria attach to plant roots and transform the plant cells to induce tumors. The mechanism of this attachment in the infection process is not yet fully understood. Using wild type Arabidopsis thaliana, Columbia-0, and several Arabidopsis mutant lines as a binding target, we screened for A. thaliana mutants with altered adhesion. The A. thaliana mutant lines were selected in The Arabidopsis Information Resource (TAIR) according to possible location of the resulting protein and similarity to known transformation mutants. Of these mutants nine showed a variation in attachment from the wild type, of which two were known transformation mutants rat1 and rat3. Of these, the two were higher and seven were lower. Two mutants showed a growth phenotype with one having more roots and the other having wavy root hair growth, but both had wildtype attachment. I also attempted to quantify the adhesion in these mutants using several approaches. However, I was not able to find a quantitative method that correlated well with microscopic observations of adhesion. Real-time PCR (qPCR) assay showed measurable differences between the mutants lines and the wildtype, suggesting some effect of the mutation on the interaction of A. thaliana and A. tumefaciens. Using this assay the level of bacterial attachment to the root surface can be indirectly measured. In the process of selecting this method several other approaches were attempted. These included flow cytometry of bacterial cells and of cells bound to beads, 96-well plate binding assay and the previously used plate colony counting. Mutants used in this study were also evaluated for transformation efficiency. Most of the mutants had not been previously tested for attachment or transformation. The attachment and transformation phenotypes provide a better understanding of the gene that has been affected by these mutant Arabidopsis lines. The affected gene sequence and the data available on that gene were used to analyze the functional domains of the proteins showing an altered phenotype. There should be specific results here, rather than generalizations. These showed that kinase, extensin and heat shock protein domains were present in low attachment mutants and fasciclin, CDC48 and VirB2 domains were in high attachment mutants. The leucine-rich repeat (LRR) domains were strongly represented in all of the attachment mutants.. The SALK_ 040891C and SALK_085076C mutants that had high clumping but low attachment had heat shock, extensin and LRR domains. The putative protein functional domains may give insight to the possible function of the gene in both Arabidopsis and in possible interaction with A. tumefaciens. From these phenotypes, along with bioinformatic analysis, we can analyze mutant plant lines that exhibit enhanced or inhibited attachment. The combination of these methods may yield insight on the attachment mechanism as well as the infection process as a whole.