Alumni Dissertations

 

Alumni Dissertations

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  • Genomic analyses reveal putatively pathogenic genes and functional elements in Borrelia burgdorferi, the Lyme disease bacterium

    Author:
    Che Martin
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Weigang Qiu
    Abstract:

    B. burgdorferi s.l. (B. burgdorferi sensu lato) represents a Gram-negative bacterial species complex that includes causative agents of Lyme disease. As an obligate parasite, B. burgdorferi`s persistence in nature depends on its innate ability to exist and survive in two distinct biological environments, the hard-bodied tick (vector) and small vertebrates (hosts), as it progresses through different stages of its enzootic cycle. To accomplish this feat, B. burgdorferi heavily depends on its ability to tightly regulate differentially expressed host- and vector-specific genes. However, very little is known about the genes and regulatory elements contributing to the pathogenicity of this increasingly prevalent pathogen. This is primarily due to the fact that B. burgdorferi is not a model organism and is difficult to culture and transform. Additionally, we were previously limited in our ability to do comparative genomic studies in this organism due to the unavailability of whole-genome sequences. The recent release of whole-genome sequences from 22 strains spanning 8 different genospecies of B. burgdorferi has since made it possible to conduct a comprehensive genome study in this organism. Here, we employ phylogenomics analyses (analysis of the genomes of a group of closely related species) on the core genome of B. burgdorferi in order to identify putative genes and functional elements that may contribute to the pathogenicity of this organism. We conducted three main analyses: i) test of positive natural selection within the coding regions of the core genome replicons, ii) test of evolutionary constraints within the non-coding regions of cp26 and lp54, and iii) structural and phylogenetic comparison of OspA and OspB (Outer Surface Protein A and B), two paralogous virulence-associated lipoproteins. Consequently, we identified three genes putatively involved in adaptive host immunity, fifteen genes putatively involved in adaptive divergence, two new genes putatively under direct transcription RpoS (an alternative regulatory subunit of RNA polymerase), a number of putative cis and trans-acting regulatory elements, and fourteen fixed differences between OspA and OspB concentrated in the region proximal to the C-terminus barrel domain and the N-terminus globular domain. These findings highlight a number of coding and non-coding sequences that may contribute to the virulence of Borrelia burgdorferi in humans. These findings provide a basis for future experimental studies towards the discovery of therapeutic, diagnostic, and preventive approaches to Lyme disease.

  • The Role of Soluble Adenylyl Cyclase in the BDNF-Dependent Block of MAG/Myelin-Mediated Inhibition

    Author:
    Jennifer Martinez
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Marie Filbin
    Abstract:

    In the adult mammalian central nervous system axons do not spontaneously regenerate following injury. This lack of axonal regeneration is partly due to the presence of inhibitory proteins, such as myelin-associated glycoprotein (MAG). Previously, we showed that elevating cyclic AMP (cAMP) by pretreating (priming) neurons with neurotrophins, such as brain-derived neurotrophic factor (BDNF), is sufficient to overcome the block of axonal outgrowth by MAG. Additionally, we demonstrated this BDNF-mediated effect to be PKA-, ERK-, calcium- and CREB-dependent. However, increasing cAMP levels in response to BDNF could be dependent on several factors. A balance between the production of cAMP by adenylyl cyclases and its degradation by PDEs will determine intracellular cAMP levels. Given that the source of the cAMP produced in response to BDNF is unknown, we sought to investigate which adenylyl cyclase is activated, transmembrane adenylyl cyclase (tmAC) or soluble adenylyl cyclase (sAC). tmACs and sAC differ in their spatial localization within the cell, structure and regulation. Our hypothesis is that the rise in cAMP in response to BDNF priming is partially dependent on sAC activation. In this study, we have detected an isoform of sAC, somatic sAC, expressed in various postnatal rat primary neurons and demonstrated that specifically blocking sAC with the pharmacological inhibitors, KH7 and OH-E or by knocking down sAC expression with siRNA, abolishes the ability of BDNF to overcome inhibition by MAG. Additionally, infection of primary neurons with a lentivirus that expresses sAC is sufficient to overcome the block of axonal growth by MAG and myelin in vitro and promotes optic nerve regeneration in vivo. As previously mentioned, priming with BDNF leads to ERK activation, which results in overcoming MAG-induced inhibition of neurite growth. We found that blocking sAC with pharmacological inhibitors blocked the BDNF-dependent phosphorylation of ERK whereas blocking tmAC had no effect on ERK activation by BDNF. Lastly, we sought to determine if alternative modes of sAC regulation exist, such as interactions with TrkB. Our data demonstrated that sAC does not associate with inactive or active TrkB receptors, yet does not rule out that other potential modes of regulation may exist. Taken together, our data suggest that sAC plays an integral role in BDNF signaling to overcome inhibition of axonal growth by MAG.

  • Systematics and Taxonomy of Solanum sections Dunaliana and Irenosolanum (Solanaceae)

    Author:
    Donald McClelland
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Michael Nee
    Abstract:

    The monophyly and phylogenetic relationships of the Solanum dunalianum group have been the subject of controversy. The S. dunalianum group was investigated using fifty-eight morphological characters and DNA sequence data: molecular markers ITS, trnT-trnF, and waxy. The individual datasets, combined molecular dataset, and total evidence dataset were analyzed under the parsimony criterion. In all analyses, the S. dunalianum group was resolved as not monophyletic, necessitating taxonomic realignment. In the total evidence strict consensus, all species of the Solanum dunalianum group, except S. tetrandrum, fell into either of two clades, compatible with the previously proposed Solanum sections Dunaliana and Irenosolanum. Solanum tetrandrum was resolved with S. melongena indicating an affinity with species from tropical Asia. Solanum section Dunaliana was sister to New Guinean species of the S. ferocissimum group. Solanum section Irenosolanum was sister to a clade of Australian and New Guinean species of various groups. No unique synapomorphies support either section, but morphological trends exist. Taxonomic treatments for each section are presented. Both include a key to species, distribution maps, and images of type specimens. Solanum section Dunaliana, centered on New Guinea, is restricted to a morphologically coherent clade of six species including S. labyrinthinum named herein. It is characterized by a large shrub or small tree habit,

  • Influenza A and Flavivirus Manipulation of Cell Death

    Author:
    Jeffrey McLean
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Zahra Zakeri
    Abstract:

    Viruses employ a variety of strategies to manipulate cell fate and maximize their replication potential during infection. Interacting specifically with cell death pathways allows viruses direct control over the cellular decision to survive or die during infection. Influenza A virus, of the family Orthomyxoviridae, encodes proteins that interact with the cell at several points along the apoptosis pathway to induce apoptosis. We demonstrate that caspase activation through pro-apoptotic Bax, a downstream target of Bcl-2, is a critical determinant of the nature of cell death induced by Influenza A infection. Influenza A virus cannot establish an apoptotic response without functional Bax, and instead elicits autophagy after infection. Efficient virus replication is dependent upon Bax-mediated apoptosis as Bax KO also causes a 99% reduction in Influenza A viral titer. In stark contrast, cells infected with Dengue-2 or Modoc virus of the family Flaviviridae do not die, even at high MOI. Instead, infection with either of these flaviviruses generates an autophagy-dependent protection against several toxins. Autophagy upregulation following infection is also critical to maximum flavivirus replication. Expression of NS4A from either virus is uniquely sufficient to elicit both autophagy and protection against death similar to whole virus infection. As autophagy upregulation is essential for maximum flavivirus replication, flavivirus NS4A is therefore identified as a potential target for the development of specific anti-viral therapy for flavivirus infection.

  • Wild Oysters, Crassostrea virginica, in the Hudson River Estuary: Growth, Health and Population Structure

    Author:
    Tiffany Medley
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    John Waldman
    Abstract:

    It has been estimated that the Hudson River Estuary (HRE) once had 350 square miles of oyster beds. Overharvesting and pollution during the Industrial Age ultimately led to the near eradication of the species from the estuary. Oysters are known for their filtering effects in minimizing eutrophication and oyster reefs provide habitat to many species of fish, invertebrates and algae. Today, there are no known functional oyster reefs in the HRE, but individual oysters can be found attached to rock and other hard substrate along shorelines. Their distribution, abundance, growth, and health were unknown. This study identifies locations of where wild oysters, Crassostrea virginica, can be found living in the HRE. They were found to exist in geographically separate areas of the estuary identified as Hudson River (HDS), East River, Queens (ERQ), East River, Bronx (ERB), Hackensack River (HKS) and western Long Island Sound (LIS). Two known oyster diseases, MSX and Dermo, were found to be present in the HRE oysters, with the highest prevalence at the HKS location where 100% of oysters sampled tested positive for MSX. Annual shell growth did not differ among the HRE populations and oysters were found to have the highest condition index at the LIS location. It was discovered that HRE oysters have significantly lighter shells than that of oysters sampled from Delaware Bay. An analysis of metals resulted in highest overall metal concentrations at HKS and significantly different chromium and nickel concentrations at the LIS location between two sampling years. A genetic analysis using mitochondrial DNA and microsatellite markers indicated that HRE oysters show some genetic differentiation from one another, in addition to Delaware Bay and Rhode Island oysters, and that the populations do not exhibit low genetic diversity. Though there is a long history of pollution in the HRE, existing wild oysters in the East River and western Long Island Sound appear to be tolerating their environmental conditions and provide assurance that oyster restoration efforts in these areas of the estuary could be successful.

  • cAMP AND CHAPERONES: POTENTIAL THERAPEUTIC STRATEGIES TO PREVENT INFLAMMATION-LINKED TAU PATHOLOGY IN ALZHEIMER'S DISEASE

    Author:
    Maria Jose Metcalfe
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Maria Figueiredo-Pereira
    Abstract:

    Senile plaques and neurofibrillary tangles are hallmarks of Alzheimer's disease (AD). The main component of neurofibrillary tangles (NFTs) is Tau, a highly soluble microtubule-associated protein whose major function is to stabilize microtubules, specifically in axons, in a phosphorylation-dependent manner. Neurodegenerative diseases collectively designated "Tauopathies" are linked to Tau mutations and/or Tau post-translational modifications. Accordingly, Tau hyperphosphorylation and cleavage are important events leading to Tau intracellular accumulation, aggregation and neuronal cell death. Caspase-cleaved Tau is detected in NFTs supporting the view that the apoptosis cascade is involved in the formation of NFTs. It is thought that Tau cleavage at its C-terminus by caspases renders Tau prone to aggregation and formation of NFTs. At the sites of damage, AD brains also exhibit signs of chronic inflammation manifested by reactive astrocytes and microglia, which produce cytotoxic agents among them prostaglandins. There is a profound gap in our understanding of how cyclooxygenases and their prostaglandin products redirect cellular events to promote neurodegeneration. In our studies we treated E18 cortical neurons with prostaglandin J2 (PGJ2), because it is potently neurotoxic. We show that PGJ2, a neurotoxic product of inflammation, induces the formation of the aggregate-prone form of Tau (Tau cleaved at Asp421, Delta-Tau) in a time- and dose-dependent manner. Furthermore, PGJ2 activates caspase 8 (extrinsic apoptotic pathway) and the effector caspase 3, thus inducing apoptosis in the cortical neuronal cultures. In addition, we addressed the potential of increasing cAMP levels to prevent the toxic effects of neuroinflammation. Our studies focused on increasing cAMP because PGJ2 signals through a Gi protein-coupled receptor that reduces cAMP levels, promoting neuronal loss. Notably, increasing intracellular cAMP levels with dibutyryl-cAMP (db-cAMP) or PACAP27 prior to PGJ2 treatment decreased the levels of Delta-Tau and caspase activation, mitigating the loss of cell viability. These protective results of cAMP were only observed at early time points (4h and 8h) upon treatment with PGJ2, indicating that they are only effective when applied before the neurons reach a point of no return. We confirmed that PGJ2 treatment for 24h inhibits the proteasome and induces the accumulation and aggregation of ubiquitinated proteins. Elevating cAMP moderately increased the activity of the 26S proteasome. Surprisingly, db-cAMP or PACAP27 pre-treatment failed to prevent the accumulation/aggregation of ubiquitinated proteins induced by PGJ2. We also addressed the potential of targeting molecular chaperones, such as Hsp90 and Hsp105 to prevent the toxic effects of neuroinflammation. Our studies focused on Hsp105 because it is a newly characterized chaperone that is highly abundant in the brain, and it is active under low ATP conditions. Nevertheless its role in neurodegeneration has yet to be determined. Promoting Hsp105 overexpression by its transient transfection in human neuroblastoma SK-N-SH cells improved cell survival upon treatment with PGJ2 for 24h. Furthermore, we investigated the protective effect of EC102, a small molecule Hsp90 inhibitor, in PGJ2-induced cell toxicity. EC102 blocks the Hsp90 ATPase activity, inhibiting its foldase capacity, instead targeting substrates for degradation by the proteasome. Rat E18 primary cortical neurons showed an increase in cell viability when cells were pre-treated with EC102 prior to PGJ2 treatment. These beneficial effects of molecular chaperones support targeting them as a potential therapeutic target for AD. Based on our studies, we propose a model in which any stimulus (physical, chemical or infectious) capable of inducing inflammation in a particular brain area activates microglia and astrocytes. The toxic products released by glia, including PGJ2, act on the neighboring neurons causing among other effects, intracellular protein misfolding. If these proteins fail to be cleared by the ubiquitin/proteasome pathway (UPP) or fail to be refolded by chaperones, apoptosis is triggered launching caspase-mediated proteolysis. Caspase activation is responsible for generating protein fragments, including truncated Tau, which serve as seeds for cytotoxic protein aggregation. This sequence of events could explain many pathological features of the AD neurodegenerative process. In conclusion, these studies showed that products of inflammation affect proteasome activity and alter protein turnover, which leads to protein aggregation, and neuronal injury. All of these processes are relevant to the pathology in AD. Moreover, our data indicate that the accumulation/aggregation of ubiquitinated proteins is a very stable phenomenon, and that once formed, the cell has difficulty in removing these aggregates. Overall, our studies suggest a new potential therapeutic approach for AD that involves maintenance of intracellular levels of cAMP and, in a separate approach, enhancing the activity of heat shock proteins. Elucidation of neurotoxic mechanisms linked to products of inflammation is highly significant, as it will offer new targets for anti-inflammatory drugs that more effectively prevent AD neurodegeneration linked to chronic inflammation and protein aggregation.

  • Chemical, genetic, and ethnobotanical diversity in Asian eggplant

    Author:
    Rachel Meyer
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Amy Litt
    Abstract:

    Eggplants (Solanum melongena L.) were domesticated in tropical Asia where they are used abundantly as both food and medicine. Human selection has produced hundreds of landraces that differ in morphology and chemistry in ways that may be related to local ethnobotanical preferences. Here I present a study of the genetic, phytochemical, and ethnobotanical diversity of Asian eggplant landraces and wild relatives, which together aimed to identify the molecular and chemical differences among lineages, correlate these differences with domestication and possible selection pressures, and generate hypotheses about shifts in gene regulation that could have caused these differences. Phylogeographic analyses revealed that eggplants were domesticated at least three times, in India, southern China, and the Indo-Malayan islands (Malesia). Interviews and literature review of contemporary and historic ethnobotanical data on eggplant from India, China, and Malesia, revealed largely different medicinal uses of eggplant among these regions, suggesting separate domestications could have produced fruit with different phytochemistry. Analyses of eggplant chemical profiles spanning 43 phenolic compounds, many of which have therapeutic and flavorful qualities, were compared to genetic and geographic data. Results showed that these putatively independently domesticated lineages have significantly different levels of hydroxycinnamic acid polyamine amides, which actually may contribute to fruit texture and size, in addition to health beneficial qualities of the fruit. Analyses also revealed all three domestication events produced eggplants with lower total phenolic abundance, suggesting milder flavor was preferred that lowered health beneficial compounds. Study of genes expression levels of 13 enzymes in the phenolic pathway revealed that differential expression of six genes may underlie the different phytochemical profiles; these genes encode spermine and spermidine hydroxycinnamoyl transferases, hydroxycinnamoyl transferase, and cinnamoyl 3- hydrolase. These studies allowed me to propose detailed routes of phenolic biosynthesis in eggplant, and suggest that expression levels of these potential key regulatory genes of hydroxycinnamic acid synthesis were altered during the domestication process in multiple centers of domestication.

  • The Role of Serotonin 1A Receptor-Mediated Signaling in PSD95 Induction and Synaptogenesis During Neonatal Hippocampal Development

    Author:
    Amit Mogha
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Probal Banerjee
    Abstract:

    Previous studies have shown that the 5-HT1A-R is essential for neonatal front brain development. A polymorphism in 5-HT1A-R gene leading to its aberrant expression has been reported to result in severe depression in humans, while its absence up to postnatal day 15 (P15) results in anxiety like behavior in mice. How this receptor functions to regulate the behavior of an animal is still poorly understood. Our prior studies have shown that 5-HT1A-R mediated signaling pathway leads to stimulation of a neuro-protective MAPKèPKCα pathway in differentiated hippocampal neuron-derived HN2-5 cells. Further studies showed that this pathway is also functional in cultured P15 hippocampal slices and that it causes an induction in field EPSP (fEPSP) in the P15 slices. To further understand the mechanistic role of 5-HT1A-R in synaptogenesis we used organotypic hippocampal slice cultures from C57BL6 mice at P15 to show that the activation of 5-HT1A-R mediated signaling pathway in the hippocampus leads to a massive increase in PSD95 expression and dendritic spine as well as synapse number. The results presented in our study demonstrate that 5-HT1A-R mediated signaling pathway acts via sequential activation of ERK and PKC. In vivo studies by intrahippocampally infusing 5-HT1A-R agonist and different signaling inhibitors revealed that the same pathway is involved in the induction of PSD95 and synaptogenesis in vivo via activation of PKCα in C57 as well as Swiss Webster mice. We further demonstrated that intra-hippocampal infusion of antidepressant drug like Fluoxetine, which is Selective Serotonin Reuptake Inhibitor (SSRI), leads to the induction of PSD95 and synaptogenesis through the same pathway. Equipped with this knowledge, we activated the 5-HT1A-R mediated signaling pathway in 5-HT1A-R (-/-) mice by activating the downstream effector PKCα by intrahippocampally infusing its activator Bryostatin, which is also an Alzheimer's drug, to boost PSD95 expression and synaptogenesis. We also observed that the 5-HT1A-R (-/-) mice have less number of synapses in the hippocampus. We, therefore for the first time elucidate the signaling pathway which explains how 5-HT1A-R regulates hippocampal sculpting and function, which may determine the affective normalcy of an adult.

  • Production of Phenolic Diterpenes in Tissue Culture of Rosmarinus officinalis L.

    Author:
    Kiyo Mondo
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Dominick Basile
    Abstract:

    Rosemary (Rosmarinus officinalis L.) is an herb native to the Mediterranean that has been used to prevent the oxidation of fats and oils in foods and cosmetics. The leaves of rosemary are a powerful source of natural antioxidants; the potent antioxidant properties of its extracts are mainly due to the phenolic diterpenes, carnosol and carnosic acid. At present, field-grown plants are the sole source of carnosic acid and carnosol. Rosemary is relatively easy to grow in many parts of world; however the variation of seasonal stresses such as temperature, solar irradiation, and rainfall can significantly alter the level and quality of phenolic diterpenes. An alternative method with more consistent results to obtain phenolic diterpenes from rosemary is a biotechnological approach, specifically, plant tissue culture. In this research, different culture types (callus cultures, suspension cultures, and shoots from in vitro propagated plants) of rosemary were established, and the effects of temperature, light intensity, and UV radiation for enhancing the synthesis of carnosic acid, carnosol, and related phenolic diterpenes in these cultures were evaluated. MS medium supplemented with either 10 mg/L indole-3-acetic acid (IAA), 4 mg/L benzylaminopurine (BAP) and 1 mg/L 6-furfurylaminopurine (kinetin) or 0.05 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg/L BAP at 18 and 25°C with 67.5 to 81.0 µmol m-2s-1 proved to be the best medium and conditions for developing and sustaining rosemary callus cultures. Shoots from in vitro regenerated plants of rosemary accumulated various amounts of carnosic acid and carnosol, which were also present in callus culture, but the yields of these compounds were considerably lower than in regenerated shoots or in dried field grown leaves. Only carnosol and rosmanol, not carnosic acid, were detected in cell aggregates in suspension cultures, whereas spent medium contained only rosmanol. Exposure of the callus cultures to several stress conditions by increasing temperatures and light intensities, and UV radiation treatments enhanced the level of the phenolic diterpenes, carnosic acid and carnosol.

  • The Liochelidae Fet & Bechly, 2001 (Scorpiones) of the Indo-Pacific Region: systematics and biogeography

    Author:
    Lionel Monod
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Lorenzo Prendini
    Abstract:

    The aim of the present project was to reassess the systematics, phylogeny and biogeography of the Indo-Pacific taxa of the scorpion family Liochelidae Fet & Bechly, 2001 based on morphology and molecules. The morphological matrix comprised of 85 terminal taxa and 263 characters was compiled based on the examination of more than 4000 specimens including most of the types and fresh material collected during several field surveys in Australia, Malaysia, New Caledonia, the Solomon Islands and Thailand. 101 DNA samples representing 42 taxa, half of the known diversity, were obtained for the molecular analysis. Three mitochondrial and two nuclear genes (approximately 4180 base-pairs) were sequenced from each sample. Cladistic analyses were then performed on the morphological, the molecular and the combined datasets respectively. Finally, dates of cladogenesis events estimated using molecular clocks were used to infer a plausible biogeographical hypothesis. Results of the taxonomic revision lead to the following emendations: (1) the elevation of the subfamily Hormurinae Laurie, 1896 to family level, (2) the revalidation of Hormiops Fage, 1933 and Hormurus Thorell, 1876 that were placed into synonymy with Liocheles Sundevall, 1883, (3) the revalidation of 10 species placed into synonymy (eight Hormurus spp., two Liocheles spp.), (4) the discovery of 51 new species (1 Hormiops, 48 Hormurus, 1 Liocheles, 1 Opisthacanthus Peters, 1861 (Monodopisthacanthus Lourenço, 2001)), (6) the elevation of two subspecies to species rank, and (5) the synonymy of one genus (Tibetiomachus Lourenço & Qi, 2006), three species and one subspecies. The biogeographical analysis indicates that vicariance, dispersal and vicariance are not mutually exclusive and that all three mechanisms played a significant role in the development of the Indo-Pacific liochelids. The phylogeny and dating do not corroborate the "out-of- India" origin for the Southeast Asian taxa, but rather suggest that they may be part of an ancient Laurasian lineage. On the other hand, the colonisation of the Australo-Papuan Archipelago from Sundaland by liochelids is confirmed. However, the age estimate of divergence between Melanesian Hormurus and Asian Liocheles in the Paleocene contradicts the current geological model and suggests the existence of land connections between the two provinces well before the Eocene.