Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • 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.

  • Lizards and LINEs: Phylogeography and Genome Evolution of the Green Anole (Anolis carolinensis)

    Author:
    Marc Tollis
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Stephane Boissinot
    Abstract:

    The sequencing of the green anole lizard (Anolis carolinensis) genome has already provided insights into how vertebrate genomes have evolved since the phylogenetic split between reptiles and mammals ~300 million years ago. For instance, the diversity and abundance transposable elements (TEs) in the Anolis genome, particularly the non-LTR retrotransposons (nLTR-LTs), shows more similarity to fish than to mammals, which suggests that mammals have significantly diverged from the amniote ancestor in terms of genome structure. The fate of TEs in a genome relies on the relative strengths of purifying selection against deleterious elements and genetic drift in host populations. Surprisingly, the geographic distribution and demographic history of populations within A. carolinensis has largely escaped scrutiny. We studied the patterns of mitochondrial and nuclear DNA sequences and found that there are five evolutionary lineages of green anoles, which diverged ~2 million years ago. Climatic shifts during the early Pleistocene may have driven their early diversification, particularly on ancient island refugia in what is now Florida, where a remarkable phylogeographic diversity of green anoles is found. More recently, the dispersal of green anoles onto the continental mainland led to a dramatic westward range expansion across the Gulf Coastal Plain. These insights into the evolutionary history of A. carolinensis allowed us to infer the population dynamics of nLTR-RTs in the Anolis genome. While nLTR-RTs are rare in Anolis, we find that they reach fixation in populations quite readily. We also find that full-length (FL) nLTR-RT insertions may be subjected to purifying selection as they are found at lower population frequencies than truncated (TR) insertions. This suggests deleterious effects of ectopic recombination or the process of retrotransposition are limiting the copy number of FL elements in Anolis. Finally, we find that FL elements are much more likely to be fixed in populations of small effective size, where purifying selection may not be acting as efficiently due to strong genetic drift. While FL elements are subjected to purifying selection, the fixation of TR elements suggests that another mechanism, such as DNA loss, may account for the relative paucity of nLTR-RTs in the Anolis genome.

  • SURVIVAL SIGNALS IN HUMAN CANCER CELLS MEDIATED BY PHOSPHOLIPASE D AND mTOR

    Author:
    Alfredo Toschi
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    David Foster
    Abstract:

    Phospholipase D (PLD), which is commonly elevated in renal and other cancers, provides a survival signal that suppresses apoptosis induced by serum deprivation in renal cancer cells. Hypoxia-Inducible Factors α (HIFα), important effectors of hypoxic response, have been shown to play a pivotal role in the tumorigenesis of renal cancer cells that lack the von Hipple Lindau tumor suppressor gene (VHL), a critical mediator of HIFα proteolytic degradation. We report here a role for PLD as another regulatory component of HIFα expression in renal cancer cells where accumulation of both HIF1α and HIF2α in require functional PLD for efficient translation, independently from pVHL expression. The expression of HIF1α has been widely shown to be dependent on mTOR, the mammalian target of rapamycin and its sensitivity to rapamycin has been established. In contrast, HIF2α has been reported to be insensitive to rapamycin. mTOR, a critical node for control of cell growth and survival, exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that while HIF2α is insensitive to rapamycin in renal cancer cells, HIF2α expression is still dependent on mTOR expression and we are able to show in the fourth chapter of this work that while HIF1α is dependent on both mTORC1 and mTORC2, HIF2α depends solely on the rapamycin resistant mTORC2. However, while much is known about the regulation of mTORC1, little is known about the regulation of mTORC2. PLD and its metabolite phosphatidic acid (PA) have been implicated in the regulation mTOR but its role has been controversial. In light of the concomitant regulation by PLD and mTOR of HIFα expression, we investigated the role of PLD in the regulation mTOR activity. We report in the fifth chapter of this work that PA, in competition with rapamycin, is required for functional mTORC1 and mTORC2 complex formation. Suppression of PLD prevented phosphorylation of the mTORC1 substrate S6 kinase at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated phosphorylation of Akt and the mTORC2- and Akt-dependent phosphorylation of PRAS40 indicating that PA is required for the association of mTOR with Raptor to form mTORC1 and mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with the PA-mTORC1 interaction. However, suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. The data provided here reveal a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes. The competition between PA and rapamycin for mTOR suggests a mechanism for the suppression of mTOR by rapamycin and explains the rapamycin resistance of mTORC2 and HIF2α. The last part of this work implicates PLD in the regulation of another hallmark of cancer cells: aerobic glycolisis. The metabolic shift from oxidative phosphorylation to aerobic glycolysis, also known as the "Warburg effect", is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. A shift to aerobic glycolysis is also a response to hypoxia, which stimulates the accumulation of HIFα necessary for the expression of proteins involved in glucose uptake and glycolysis. We are able to show here that the metabolic shift from oxidative phosphorylation to aerobic glycolysis in human cancer cells is dependent on the elevated PLD activity in breast and renal cancer cells. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on mTORC1 in breast cancer cells and on mTORC2 in renal cancer cells, consistently with a role for PLD in activating mTOR. We are able to conclude that elevated PLD signaling, which is common in human cancer cells, is critical for the activation of mTOR complexes and accounts for mTORC2 insensitivity to Rapamycin. Moreover, elevated PLD activity is required for the expression of HIFα and the consequent transcriptional activation of many genes involved in tumorigenesis including genes involved in the metabolic shift to aerobic glycolysis. Taken together, this data provides evidence that targeting PLD could prove therapeutically significant in cancers with elevated PLD activity such as renal and breast cancer.

  • Using Tissue Culture as an Alternative Source of Polyphenols Produced by

    Author:
    George Tsalokostas
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Dominick Basile
    Abstract:

    Plants have long been used as sources of pharmaceuticals or other commercial products. Many of these products are difficult to synthesize at affordable prices. Furthermore, the extensive testing that is required of synthetic products in order to meet safety standards, compared to requirements for natural products, has stimulated interest in replacing many synthetic chemicals, especially food additives, by natural plant extracts. "Plantations" of medicinal plants, as with any cropped plants, are vulnerable to diminished yields due to outbreaks of disease or unfavorable changes in growing conditions, resulting in considerable economic loss to growers. Furthermore, some plants can not be grown as crops in the geographical areas where there is the most need for their products. In such cases, an alternative means of obtaining natural products from plants is through plant tissue culture. The fruit and leaves of Ficus carica (the edible fig) produce many polyphenolic antioxidants of potential therapeutic value. However, the plant is vulnerable to fig mosaic virus disease, and does not grow well out of semitropical and Mediterranean climates. In this work, Ficus carica tissue cultures were investigated as an alternate source of antioxidant polyphenols. It is well know that ntioxidants have anticarcinogenic, antibacterial, and antiviral properties and can be used as food supplements. In an effort to determine culture conditions that resulted in the production of polyphenols comparable to those produced by intact plants, chemical and physical factors that affect yield, such as basal media composition, light intensity, temperature, growth hormones, and elicitors were tested. As a result of this research, callus cultures were developed that contained an average of 4.26% polyphenols of their dry weight. Analysis based on TLC and HPLC, showed that the main antioxidants found in callus tissue are apigenin, isoquercitrin, astragalin (kaempferol glycoside), rutin, emodin, cyanidin, caffeic acid, tannic acid, chlorogenic acid, quercitin, kaempferol, taxifolin, catechin and epichatechin. These results show that tissue cultures of Ficus carica can be used successfully for production of antioxidant compounds used as food supplements. It is also shown that tissue cultures initiated from vegetative tissue can produce polyphenols usually found in fig fruit as well as in leaves and other parts of the plant.

  • Systems biology-based study of provitamin A carotenoid biosynthesis in Arabidopsis thaliana

    Author:
    Oren Tzfadia
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Eleanore Wurtzel
    Abstract:

    Due to their great nutritional and health value, understanding the regulatory mechanisms and recognizing new points of control in the carotenoid pathway can be the goal of breeding plans for increasing carotenoids accumulation in crop plants. Systems biology is an inter-disciplinary field, which integrates computational models and tools with molecular biology and different types of data including in silico transcriptomics, co-expression correlation, metabolomics, proteomics and phylogenetic information in order to develop hypotheses with statistically sound robustness. In the first steps of my work I describes the sequential use of freely available databases to explore the regulation of carotenoid biosynthesis in Arabidopsis during chloroplast development. The findings suggested that coordinated transcriptional regulation of genes along the isoprenoid-related biosynthesis pathways, play a major role in coordinating the synthesis of functionally related, chloroplast-localized isoprenoid-derived compounds. Next I aspired to find candidate genes that are participating in or regulating the carotenoid pathway. A model was developed to integrate several types of high-throughput data, in order to optimize candidate gene ranking in an effort to best define associated genes for a specific studied pathway. The candidate ranking was achieved by using an iterative algorithm (called MORPH), which is built on implementation of machine learning techniques. Application of the method on several biological pathways in Arabidopsis proved the ability of the algorithm to capture experimentally proven gene candidates related to known biological pathways. The robustness of the predictions provided by MORPH creates an exciting research methodology to explore regulation of biological pathways in plants. Although the development of the computational algorithm was initially triggered by the specific needs of our laboratory, namely, for close analysis of the carotenoid pathway, the algorithm is suitable for almost any biological pathway in plants. Moreover the method could be applied to any other model system that has enough available high-throughput data.