Alumni Dissertations

 

Alumni Dissertations

Filter Dissertations By:

 
 
  • Characterization of the Sinorhizobium meliloti ExoR protein

    Author:
    Haiyang Lu
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Hai-Ping Cheng
    Abstract:

    The soil bacterium Sinorhizobium meliloti is capable of establishing a symbiotic relationship with its leguminous plant host alfalfa by forming nitrogen-fixing root nodules through a series of signal exchanges and structural changes. The presence of a potential bacterial signal molecule, succinoglycan, is required for the invasion step of this symbiosis. The production of succinoglycan, which is inversely coupled with the production of flagella, is tightly regulated by the S. meliloti ExoR protein and the ExoS/ChvI two-component regulatory system. To better understand the regulatory function of ExoR and its relationship with the ExoS/ChvI system, I have carried out extensive genetic and biochemical analyses of ExoR and ExoS proteins. I found that ExoR is a periplasmic protein and it functions only in the periplasm. Interestingly, the C-terminal 20 amino acids appear not to be essential for the regulatory function of ExoR. Most importantly, the ExoR protein is digested in the periplasm, which appears to be the molecular mechanism regulating the amount of functional ExoR protein in the periplasm. The genetic analysis of my collection of exoR, exoS, and chvI mutants suggests that ExoR functions upstream of the ExoS/ChvI two-component signal transduction pathway. This conclusion was further supported by the analysis of the exoR expression in different genetic backgrounds, which suggests the ExoR-ExoS-ChvI pathway is feedback regulated by ExoR. The combination of biochemical and genetic analyses suggest that ExoR functions in the periplasm through interaction with the ExoS sensing domain to keep ExoS in the off state. The proteolysis of ExoR would reduce the amount of functional ExoR and lead to the activation of ExoS and the expression of the genes regulated by the pathway. With the continuous discovery of ExoR/ExoS/ChvI homologous systems in a wide range of bacteria, my findings will contribute to a better understanding of the S. meliloti-alfalfa symbiosis and the pathogenicity of some bacterial plant and animal pathogens.

  • Antimalarial Benzophenones and Xanthones from Garcinia species

    Author:
    James Lyles
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Edward Kennelly
    Abstract:

    Garicina, a genus in the Clusiaceae, is a source of antiparasitic and antimalarial phenolic secondary metabolites, including benzophenones and xanthones. The methanolic extracts of G. livingstonei, G. mangostana, G. spicata, and G. xanthochymus were tested in vitro for antiplasmodial activity against the P. falciparum clone D6. The crude methanolic G. mangostana extract inhibited the clone by 89%, while the G. xanthochymus extract inhibited it by 24%. Neither of the extracts showed any cytotoxicity toward Vero cells. Hexanes, EtOAc, and n-BuOH partitions of a G. xanthochymus seed extract were also screened against the P. falciparum clone D6, the hexanes partition inhibited the clone by 58%, the EtOAc and n-BuOH partitions showed no inhibitory activity. Additionally, compounds identified from Garcinia species were screened in the plasmodial lactase dehydrogenase (pLDH) activity assay. The compounds tested were: aristophenone A (1), cycloxanthochymol (2), gambogenone (3), guttiferone A (4), guttiferone E (5), guttiferone H (6), isoxanthochymol (7), xantochymol (8), xanthone (9), mangiferin (10), alloathyriol (11), alpha-mangostin (12), beta-mangostin (13), 3-isomangostin (14), 8-desoxygartanin (15), 4-methoxyxanthone (16), 1,5,6-trihydroxyxanthone (17), and 32-hydroxy-ent-guttiferone M (18). Only compounds 5, 6, 7, 12, 13, and 14 showed antiplasmodial activity. The antiplasmodial activities of compounds 5, 6, and 14 have not been previously reported. This is the first report of 7, 12, and 13 having antiplasmodial activity against the chloroquine-sensitive P. falciparum clone D6 and chloroquine-resistant clone W2. For the benzophenones 8 and 2 the absence of a terminal methylene on the C-8 side chain (cf. 5 and 7) correlates with antiplasmodial activity. Compound 6 with a terminal methylene on C-30 and a cyclohexane from C-7 to C-8 is antiplasmodial. It was concluded that, for benzophenones neither the C-5 nor the C-14 side groups affect antiplasmodial activity. The substitution pattern of both the A and B rings of the xanthones was shown to be important in determining the extent of antiplasmodial activity. An isoprenyl chain was necessary on C-8 for antiplasmodial activity (cf. 12 and 14). The results indicate that the antiplasmodial activity is determined by factors other than the hydroxylation of C-4 and C-5 alone as the current structure activity studies indicate.

  • Habitat association and spatial distribution of Procellariiform seabirds along the continental shelf of the northeast of the United States and southeastern Canada: Relationships to prey and other top predators

    Author:
    Marie Martin
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Richard Veit
    Abstract:

    In the first chapter, I compare habitat association among seven species within the order of Procellariiform seabirds that includes shearwaters, storm-petrels and northern fulmars (Fulmarus glacialis) along the northeastern continental shelf of the United States and southeastern Canada (Northwest Atlantic). Environmental factors such as bathymetry, sea-surface temperature (SST), chlorophyll concentration, and frontal features affected seabird densities and influenced their distribution. My model suggested that some species could be influenced by changes to large-scale hydrographic features and that all species will not respond equally to potential climatic fluctuations. In the second chapter, I focus on the northeast Georges Bank and Jeffreys Ledge regions of the Gulf of Maine, recording the presence of marine predators during four hydroacoustic surveys. The primary objective of these surveys was to make annual assessments of the pre-spawning stock of Atlantic herring (Clupea harengus). I explain the interannual variability of top predators (seabirds, dolphin, whale) using a general additive model that included environmental parameters and fish acoustic index to understand the possible reasons of this variability, including: 1) the effect of environmental predictors on top predators and prey; 2) piscivorous marine predator abundance correlated to fish acoustic biomass index data; and 3) the effect of fishing vessel density effect on marine predators. All predators and Atlantic herring were affected by oceanographic variables; northern gannet (Sula bassana) by fish density, as well. There were also spatial overlap between fishing vessels, humpback whales (Megaptera novaeangliae), great shearwaters (Puffinus gravis) and herring gulls (Larus argentatus). This study shows the importance of accounting for multiple environmental parameters in order to understand the variability of marine predators abundance in highly productive areas such as Georges Bank, in addition to assess spatiotemporal overlap between aggregated predator and prey for improving fisheries management. Finally, in the third chapter, I examine the foraging associations between Procellariiform seabirds and one species of Pelecaniform (northern gannet), dolphins, whales, and two species of tuna along the continental shelf. The general linear model results suggested seasonality in aggregation types. Great shearwater density increased with humpback and fin whales (Balaenoptera physalus) in the summer, and shifted to common dolphin (Delphinus delphis) in the fall.

  • 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.