Alumni Dissertations

 

Alumni Dissertations

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  • Regulation of Microtubule Stability in Saccharomyces Cerevisiae

    Author:
    Aya Shohat
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Dan Eshel
    Abstract:

    The integrity of chromosome segregation during mitosis is essential for the propagation of genetic information to daughter cells during cell division. In yeast, it is achieved in four microtubule-dependent steps: first is spindle assembly, which involves the migration of duplicated microtubule organizing centers to form a bipolar spindle by prophase; second is orientation of the mitotic spindle at the site of cytokinesis; third- chromosome movement along kinetochore microtubules (anaphase A); and fourth, complete chromosome segregation through spindle elongation through interpolar microtubules (anaphase B). Signaling pathways have been implicated in the regulation of microtubule dynamics and stability, which is required for these processes. This work identifies additional protein regulators of microtubule stability using mutants of key mitotic motor proteins, specifically, Cin8p, Kip1p, and Dyn1p. Loss of Cin8p function in the absence of either Kip1p or Dyn1p is lethal. Haploid cells that carry the cin8-3 temperature sensitive allele in a deletion background of either KIP1 or DYN1 cannot grow above 35◦C. Our studies suggest that suppressors of these mutant genotypes act by stabilizing microtubules. We propose that the mechanism of suppression involves enhancing signal-transduction cascades that regulate microtubule stability and dynamics. We found that FCP1 overexpression supressess the microtubule defect in our background and that this suppression requires the following genes to manifest: SWI6, SWI4, CLB2, ELM1, HSL1 and MRS6. These proteins, with a previously uncharacterized role in microtubule stability, may be candidate microtubule-associated proteins (MAPs) or novel regulators of MAPs (direct or indirect). In addition, a putative pathway to MT stability was drawn based on genetic interactions we established, epistatic experiments that were done and physical data we produced, combined with existing knowledge.

  • The Systematics and Evolution of the Nightjars and their allies (Aves: Caprimulgiformes)

    Author:
    Snorri Sigurdsson
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Joel Cracraft
    Abstract:

    Recent studies have shown that the avian order Caprimulgiformes includes eight families: the owlet-nightjars (Aegothelidae), the nightjars (Caprimulgidae), the potoos (Nyctibiidae), the frogmouths (Podargidae), the monotypic oilbird (Steatornithidae) and the three families traditionally placed in the order Apodiformes: the swifts (Apodidae), the tree-swifts (Hemiprocnidae) and the hummingbirds (Trochilidae). In this study, a total-evidence approach was utilized to address the relationships of these families. A phylogenetic analysis of a combined dataset of 134 skeletal morphological characters, 14 nuclear loci (exons and introns) and a presence/absence indel matrix, with a taxon sampling of all eight families as well as 16 outgroup taxa provided a better resolved phylogeny for the group than obtained by previous studies. New relationships include the placement of the frogmouths (Podargidae) as the sistergroup to a clade containing the owlet-nightjars (Aegothelidae) and the three "apodiform" families. Both morphological and molecular data supported the sister-relationship of the nightjars (Caprimulgidae) and the potoos (Nyctibiidae). A phylogeny for the three New World radiations of nightjars (Caprimulgidae) was produced from a four-loci molecular dataset. The taxon sampling was the densest of any phylogenetic study of the group, not only were all but three New World nightjar species sampled, but also 78 of 101 recognized subspecies. This provided an opportunity to address species- and intraspecific-level relationships. The taxonomic modifications resulting from the phylogeny included a reduction in the number of genera for the three radiations, from 14 to 10, and nine subspecies were elevated to full phylogenetic species status resulting in an increase in total species numbers from 89 to 98 for the family. The modified phylogeny of the New World nightjars was utilized to investigate temporal patterns of diversification, historical biogeography and evolution of habitat choice and migratory behavior. The three New World radiations are for the most part temporally congruent but they show highly independent histories of spatial and ecological diversification that have resulted in divergent patterns of extant species distributions as well as ecology, impacted by multiple independent vicariant events, long-distance dispersal and habitat shifts.

  • Phylogeny and Population Genetics of the Endangered Dwarf Bear-poppy, Arctomecon humilis Coville (Papaveraceae) Using Microsatellite Markers

    Author:
    Joshua Simpson
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Dwight Kincaid
    Abstract:

    The genus Arctomecon (Papaveraceae) is comprised of three narrowly endemic rare species that are largely restricted to gypsum soils of the eastern Mojave Desert. The small, remaining populations of these species have become increasingly isolated by urban development and habitat fragmentation. Arctomecon humilis is federally listed as endangered due to its limited distribution within a ~15 km radius of an actively expanding city. Organizations involved with land management and conservation have called for greater insight into the genetic variation and population structure of the remaining subpopulations as they make important decisions regarding where to focus their efforts and resources. The goal of this study was to provide answers to some of the remaining research gaps involving Arcotmecon species particularly conservation genetics by developing microsatellite markers and comparing community dynamics. First, a phylogenetic study using six gene regions (nrITS, cpDNA (matK, rbcL, trnH-psbA, rpl32-trnL, ndh-rpl32)) was conducted for members of the genus and three outgroup species. Thorough sampling throughout the geographic range of Arctomecon was conducted in order to obtain a complete representation of the genetic variability present across multiple populations. A total of 1176 plants were sampled from 35 locations with DNA extraction being performed on 949 of those, to be included in different stages of research. Outgroup taxa included a member of the sister genus Argemone, a Meconopsis species from within the subfamily, and an Eschscholzia from a separate subfamily. It was hypothesized that this increased sampling and number of gene regions would provide a more robust species tree, as compared to previous studies. Additionally, I hypothesized that new genetic markers could identify isolated populations that would be more informative to conservation management. The phylogenetic analysis did result in a well-supported species tree in addition to exhibiting broad structure among populations within each species. Notably, the population sampled in the Grand Canyon is genetically and morphologically divergent from all the other populations of A. californica that were sampled. Polymorphic microsatellite markers revealed the micro-evolutionary structure from within and between populations of A. humilis. This was the first time that genetic markers of this type have been developed for any Arctomecon species. Sixteen markers with 2 to 31 alleles (mean=12) per marker were used to determine the level of variation and admixture among 341 individual plants from thirteen sampling localities. The number of individuals per locality ranged from 26 at Price Hills to 49 at Boomer Hill. Each marker was tested for amplification and variability within the sister species A. californica and A. merriamii where cross-amplification occurred with less success and fewer alleles than in A. humilis. Population genetic analyses identified localities with greater amounts of admixture, as well as those more isolated and at risk of inbreeding depression. Through Bayesian analysis and genetic cluster assignment the overall trend suggests that populations are becoming more isolated. Analysis of Molecular Variance found 30% of the genetic variability between populations, and the FST analogues indicated substantial genetic differentiation (G'ST=0.427). A concern among land managers and conservation organizations concerned the effectiveness of the reserve system. An analysis of the allele frequencies located within the protected areas does indicate that the reserve system is effectively capturing genetic diversity. However, allele frequency data also suggest that a small number of new annual recruits represent only a subset of potential alleles. Due to the small effective population sizes and the already rare habitat supporting Arctomecon humilis the conservation efforts should continue to monitor and protect this unique species in all locations.

  • Functions of Notch and Neuralized in Drosophila hematopoiesis

    Author:
    Chiyedza Small
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Shubha Govind
    Abstract:

    In vertebrates, hematopoiesis is commonly divided into two temporal phases, primitive (embryonic) and definitive (adult). Genetic studies in zebrafish and mice have implicated signaling pathways and molecular networks of transcription factors in the control of primitive and definitive hematopoietic programs. Notch signaling is essential for the proper execution of a wide array of cell fate decisions and developmental processes, including hematopoiesis. Many of these same signaling and transcriptional mechanisms also control hematopoiesis in simpler animals, such as the fruit fly Drosophila melanogaster. Because of its simple organization and genetic accessibility, Drosophila hematopoiesis has recently gained attention. Drosophila larvae produce three cell types: plasmatocytes, crystal cells and lamellocytes. While plasmatocytes and crystal cells arise in embryonic stages, lamellocytes do not. In fact very few lamellocytes are typically found in healthy third instar larvae. However, their differentiation is induced in large numbers upon oviposition by parasitic wasps. Circulating blood cells divide continuously as the animal grows in size. In addition, a small hematopoietic organ flanking the dorsal vessel supports the growth and development of blood cells. In larval stages, the Notch pathway regulates the differentiation of crystal cells: loss or reduction of Notch signaling results in the reduction of crystal cells while an increase in Notch signaling leads to the expansion of the crystal cells population in circulation and in the lymph gland (Duvic et al., 2002; Lebestky et al., 2003). Notch encodes the receptor/transcription factor that mediates short range cell-cell signaling. Notch ligand Serrate is expressed in the niche of the lymph gland. Serrate activates Notch in the pro-crystal cells and promotes commitment of the crystal cell fate. This step requires the functions of transcription factor Suppressor of Hairless (Lebestky et al., 2003). Duvic et al. (2002) also reported that Notch function is essential for lamellocyte differentiation, although how this occurs was not explored in the Duvic study. The goal of this work was to analyze the contributions of Notch and Neuralized in lamellocyte differentiation. Neuralized encodes an E3 ligase for ubiquitination of Notch ligands, and its role in hematopoiesis remains unexplored. The thesis contains three chapters. In Chapter 1, I report the expression of Notch in the lymph gland and circulating blood cells. Using RNA interference and clonal analysis, I show that Notch maintains lamellocytes in their progenitor state. This requirement is non cell-autonomous. Lamellocytes induced by loss of Notch appear mostly in the peripheral cortical zone of the anterior lobes, that houses both mature cells and progenitors. Notch target genes are expressed in most anterior lobe cells. In Chapters 2 and 3, the functions of Neuralized are explored in three ways: RNA interference, clonal analysis (Chapter 2), and using putative alleles of neuralized, l(3)hem1 and l(3)hem2 (Chapter 3). These studies show that Neuralized function is essential in maintaining hematopoietic stem-like progenitors in their undifferentiated state. In the medullary zone, where these undifferentiated progenitors reside, Neuralized plays an essential role in cell division and differentiation. Further, like Notch, it provides an inhibitory non cell-autonomous influence on pro-lamellocytes in the cortical zone, and keeps them from differentiating in the absence of infection. In chapter 3, I characterize a classical hematopoietic mutation, l(3)hem1, a putative weak allele of neur. The lymph glands and blood cells of this homozygous mutant are severely affected, with multiple defects in cell division and differentiation. These studies support the three functions of Neuralized uncovered in Chapter 2. Our studies provide novel insights into hematopoieitic stem/progenitor division and differentiation. Notch signaling plays an essential role in mammalian hematopoiesis. Misregulation of the Notch pathway leads to hematopoietic malignancies in humans. Because of the high molecular conservation between flies and mammals, understanding the regulation of Notch signaling in Drosophila hematopoiesis will yield insights into its role in mammalian hematopoiesis and potentially in developing therapies for treatment of human malignancies.

  • prenatal cocaine dysregulates BDNF-TrkB and P75 Signaling in The Hippocampus and Prefrontal Cortex of Adolescent Rats

    Author:
    Andres Stucky
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Andres Stucky
    Abstract:

    Brain-derived neurotrophic factor (BDNF) upregulates glutamatergic transmission and N-Methyl-D-Aspartate receptor (NMDAR) function through the activation of tropomyosin-related kinase receptor type B (TrkB). Conversely, NMDAR activation influences BDNF release. Because prenatal cocaine exposure can markedly alter glutamatergic transmission and NMDAR activation, we hypothesized that a dysregulation of the glutamatergic system following prenatal cocaine exposure could result in long-lasting alteration of TrkB signaling, thereby influencing the interaction between TrkB and glutamatergic NMDARs. In agreement with this hypothesis, we found that activated (i.e. tyrosine-phosphorylated) TrkB (pY-TrkB) levels in response to exogenous BDNF were increased in both the prefrontal cortex and hippocampus of 21-day-old rats that were exposed prenatally to cocaine. This cocaine-induced effect was corroborated by an elevated pY-TrkB-associated phospholipase, C-1, and adapter protein, Shc, as well as increases in downstream extracellular signal-regulated kinase 2 (ERK2) and PI3K signaling. We report a significant decrease in the levels of BDNF released at the synapse of prenatal cocaine- exposed rats compared to control after NMDA and K+ stimulation and a marked increased affinity of receptor TrkB to its ligand BDNF. This suggests that increased activation and signaling of TrkB in prenatal cocaine- exposed rats is the result of increased affinity of TrkB to BDNF, possibly as a functional compensation for decreased levels of activity-dependent BDNF released at the synapse. Moreover, we found a decreased activity of the p75 neurotrophin receptor(p75NTR) death-inducing pathways, as assessed by p75NTR recruitment of adaptor proteins TRADD, FADD, and TRAF2/6, and corroborated by decreased downstream Janus kinase 1 (JNK1) activation, as indicated by lower JNK1 phosphorylation (p-JNK1) levels. Our data suggest that BDNF-TrkB and BDNF-/proBDNF-p75NTR activities are reduced following prenatal cocaine exposure due to a marked reduction in BDNF/Thrown Away proBDNF release. Given that neurotrophins and glutamate receptors interact to modulate the health and excitability of glutamatergic synapses, upregulation of BDNF-TrkB signaling and downregulation of BDNF-/proBDNF p75NTR pathways suggests a more efficient neurotrophin signaling in an attempt to reestablish synaptic homeostasis when supplies of BDNF are restored.

  • Molecular and Evolutionary Properties of Non-Gene-Coding Regions in Bacteria Using Comparative Genome Bioinformatics

    Author:
    Tika Sukarna
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Weigang Qiu
    Abstract:

    Although most non-gene-coding regions of the genome have long been thought of as nonfunctional, a growing body of literature now show that many sites act as important sources of phenotypic variation and complexity. In eukaryotes, this has been attributed to the sophisticated gene regulatory apparatus that includes cis-acting regulatory elements acting on multiple levels. In bacteria, this level of regulatory multiplicity is reduced, as is reflected by the lower percentage of intergenic segments in their genomes and the lower capacity for metabolic and catabolic activities. Most non-gene-coding intergenic portion of the genome of bacteria is thought of as functionally compact, mostly transcriptional and translational regulatory in nature, containing only limited number of infrastructural or regulatory RNAs. This study addresses the extent of this cis-regulatory organization on noncoding genomic regions in the Lyme bacteria Borrelia burgdorferi as apparent in their molecular and evolutionary properties and how they can influence and be applied to the bioinformatic predictions of cis-regulatory function. Several general molecular and evolutionary properties of a bacterial non-coding genome were identified. Overall, most non-gene-coding intergenic portion of Borrelia are constrained, functionally compact and degenerate. A phylogenetic footprinting approach for very closely related species (> 90% nucleotide sequence identity) was developed to test for specific sites of transcriptional regulation, which was additionally tested using the Escherichia coli genome dataset. The method finds most constrained regions to coincide with several general properties of promoter binding, suggesting that constraint levels are differentiable even amongst these very closely related bacterial species, providing a way to measure for molecular function at a fine phylogenetic level through the understanding of the patterns of DNA sequence evolution.

  • Systematics of Grammitid ferns (Polypodiaceae): using a combined approach to resolve the circumscription of Melopmene, and portions of the polyphyletic genera Lellingeria and Terpsichore

    Author:
    Michael Sundue
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Robbin Moran
    Abstract:

    Recent phyogenetic analyses of grammitid ferns (Polypodiaceae) demonstrated that many genera recognized within this clade are not monophyletic. Focus here is upon circumscription of genera within one clade identified in previous analyses that includes the monophyletic Melpomene, plus portions of two polyphyletic genera, Lellingeria and Terpsichore. Morphology of grammitid ferns is reviewed and used to compile a matrix of 111 qualitative characters for 150 terminals. Phylogenetic analysis of the morphological matrix offer no support for a monophyletic Terpsichore as orginally circumscribed, but otherwise have limited value due to the lack of resolution in the consensus tree. Phylogenetic analyses using chloroplast markers atpB, rbcL, and trnLF, along with 111 qualitative morphological characters resolve this ingroup as monopyletic and sister to a clade that includes CeradeniaEnterosora, and Zygophlebia. Melpomene is monophyletic, but is nested within Lellingeria in most trees. Ingroup species of Terpsichore form three well supported monophyletic groups that together are paraphyletic with regards to Melpomene plus Lellingeria. Two clades of species currently combined in Terpsichore are recognized as new genera. One of these clades, sometimes referred to as the Terpsichore anfractuosa clade, is described as the new genus Ascogrammitis. Sixteen species of Ascogrammitis are recognized, including five new ones, and new combinations are made for the previously recognized species. A key is provided to distinguish the species, and illustrations are provided for 12 species. The genus occurs primarily in neotropical cloud forests, with the greatest diversity in the Andes.

  • ANGIOGENESIS OF HUMAN RETINAL MICROVASCULAR ENDOTHELIAL CELLS: ROLE OF INSULIN-LIKE GROWTH FACTOR-1 AND HYPOXIA-INDUCIBLE FACTOR-1 ALPHA IN THE PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY

    Author:
    Janto Tachjadi
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    William L'Amoreaux
    Abstract:

    Retinal vascular formation is a complex process that requires a precise temporospatial regulation of various elements, including the growth factors. Insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) are ligands of specific receptor tyrosine kinases (RTKs), which, if activated, will initiate downstream pathways that ultimately promote cell survival, cell proliferation, vascular permeability and cell migration; all of which permit blood vessel development. Formation of blood vessels from pre-existing vessels may be normal (angiogenesis) or pathological (neovascularization) processes. Examples of required angiogenesis include wound healing and repair of the endometrium. In proliferative retinopathies, such as retinopathy of prematurity or diabetic retinopathy, there is a dysregulation of IGF-1 that results in the neovascular formation of abnormal retinal blood vessels from pre-existing vessels. In these proliferative retinopathies, neovascularization is intended to bring nutrients to tissues, with unfortunate risk of loss of vision. To add to the chemical mixture that promotes neovascularization, the transcription factor hypoxia-inducible factor (HIF) is important in inducing VEGF secretion in cells stimulated by either hypoxia or IGF-1. Therefore it is a convergence of several signaling pathways that ultimately leads to neovascularization. The goals of this thesis are to demonstrate the role of IGF-1 in the formation of retinal vasculature using human retinal microvascular endothelial (HRMVE) primary cell line; and to study the in vitro effect of IGF-1 stimulation on HIF-1 alpha; in human retinal angiogenesis through the phosphatidylinositol 3-kinase (PI3K) pathway.

  • The role of BAFF overexpression in the loss of anti-dsDNA B cell tolerance

    Author:
    Mitchell Thorn
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Linda Spatz
    Abstract:

    Overexpression of BAFF is believed to play an important role in Systemic Lupus Erythematosus and elevated levels of serum BAFF have been found in lupus patients. Excess BAFF also leads to overproduction of anti-dsDNA antibodies and a lupus-like syndrome in mice. In the present study, we use mice transgenic for the R4A-Cμ (IgMa) heavy chain of an anti-dsDNA antibody, to study the effects of BAFF overexpression on anti-dsDNA B-cell regulation. We observe that overexpression of BAFF promotes anti-dsDNA B cell maturation and secretion of antibody and enriches for transgenic anti-dsDNA B cells in the marginal zone and follicular splenic compartments. In addition, our data suggests that BAFF rescues a subset of anti-dsDNA B cells from a regulatory checkpoint in the transitional stage of development. The subset of B cells identified as transitional type 3 (T3) subset present in R4A-Cμ and R4A-Cμ/BAFF mice, exhibits an anergic phenotype and contains a high frequency of anti-dsDNA B cells. Our findings suggest that BAFF may enhance survival of this subset and promote anti-dsDNA Ab secretion in synergy with environmental stimuli such as CpG.

  • Ecological Effects of Road De-icing Salt on Adirondack Forests and Headwater Streams

    Author:
    Athena Tiwari
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Joseph Rachlin
    Abstract:

    Water samples from upstream and downstream sites on eighteen study streams in the Adirondacks, New York State, were collected over three years and analyzed for the presence of road salt runoff as measured by chloride ion content. Streams crossed by state roads receive more road salt runoff than streams crossed by county roads. High levels of road salt runoff were not associated with lower levels of Plecoptera or Trichoptera in headwater streams in the Adirondacks. However, Ephemeroptera were affected by high levels of road salt runoff. Forest composition in ten transects above and below state roads was analyzed by point-centered quarter method. Trees in the lowest quartile of circumference in each transect, representing recruitment, were further analyzed by point-centered quarter method. Transects were centered on study streams. Mean chloride ion content of study streams, indicating forest exposure to road salt runoff, was seen to favor recruitment of balsam, Abies balsamea.