DNA Adducts of 10-decarbamoyl Mitomycin C Activate p53-dependent and p53-independent Cell Death
Year of Dissertation:
2009
Mitomycin C (MC), a natural antibiotic and DNA cross-linking agent, has cytotoxic activity and is known to activate the tumor suppressor p53 protein. 10-decarbamoyl mitomycin C (DMC), a derivative of MC, has increased cytotoxicity compared to MC. Both MC and DMC induce cellular cytotoxicity in cells with wild-type p53, while only DMC shows significant cell death activity in the absence of wild-type p53. We investigated the difference in MC and DMC cytotoxicity by comparing DNA adduct composition and the cellular regulation of molecular targets in human cancer cell lines with or without wild-type p53. Compared to MC, DMC produced substantially more mitosene-1-β mono and 1-β cross-link adducts in DNA and resulted in abnormal nuclear morphology in human cancer cells with or without p53. Significantly, greater poly(ADP-ribose)polymerase (PARP) activity was observed after DMC treatment in both the presence and absence of wild-type p53. Both MC and DMC induced double strand breaks as indicated by gamma-H2AX foci formation irrespective of the p53 status, suggesting that double strand breaks cannot account for DMC's increased cytotoxicity. In cell lines expressing wild-type p53, both MC and DMC signaled for p53 stability and apoptosis induction resulting in cleavage of procaspase-3 and -8. Despite the DMC induced cellular cytotoxicity observed in cell lines lacking wild-type p53, cleavage of procaspase-3 or -8 was not observed in these cells. However, we observed an increase in caspase activity. Caspase-2 activation has been suggested as a pathway for p53-independent cell death in the absence of Chk1. Interestingly, Chk1 was depleted following DMC, but not MC treatment in cells with or without wild-type p53. This Chk1 depletion was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Additionally, gene silencing of Chk1 by siRNA increased the cytotoxicity of MC but not of DMC. DMC treatment also caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity. This suggests that DMC- mediated DNA adducts facilitate signal transduction to a pathway targeting proteins for proteolysis. In conclusion, we have found that DMC generates significantly more mitosene-1-β stereoisomeric DNA adducts than MC and causes rapid down-regulation of multiple cellular targets. These studies suggest increased mitosene-1-β stereoisomeric DNA adducts more effectively signal for a mode of cell death which does not require a functional p53 protein.
The Effects of Thyroid Hormone Insufficiency During Development On Cortical Morphology And The Behavioural Manifestations
Author:
Susan Briffa-Mirabella
Year of Dissertation:
2013
The central hypothesis of this work is that developmental thyroid insufficiency impacts the development of the rat cerebral cortex by altering cortical volume and the number of cortical neurons. In addition, as these neuroanatomical changes caused by milder forms of developmental hypothyroidism or hypothyroxinemia may have both immediate and long term consequences on certain aspects of behaviour, the investigation sought to determine if the alterations in morphology, including the change in relative cortical volume, and the change in the number of cortical neurons in the rat brain, led to behavioral manifestations. Hypothyroidism was induced by the administration of graded levels of the antithyroid agent propylthiouracil (PTU) to suppress thyroid hormone production.
A MINUS-GAMETE-SPECIFIC GENE IN FUSION-DEFECTIVE CHLAMYDOMONAS MUTANTS AND ANALYSIS OF BIOSYNTHETIC PATHWAYS UPREGULATED DURING GAMETOGENESIS
Year of Dissertation:
2013
To gain insight into the mechanism of gamete fusion during fertilization, it is crucial to identify molecular, metabolic and genetic factors required for this process. Gamete fusion in C. reinhardtii cells proceeds via four genetically defined stages: 1) flagella recognition 2) signaling 3) mating structure adhesion and 4) fusion with subsequent zygote formation. During this study we used insertional and temperature sensitive conditional mutants that do not proceed to stage 4, but can agglutinate and adhere to each other via their mating structure. A homolog of the sex-restricted HAP2/GCS1 gene has been shown to prevent gamete fusion in C. reinhardtii. A SiteFinding-PCR search was conducted on the fusion-defective Chlamydomonas insertional mutants that could not be complemented with the wild type copy of the HAP2/GCS1 gene. We confirmed that mutant clone 5 had an insert in a copia-family retrotransposon on chromosome 13. We collaborated to discover that a gene, located 3 &rsqou; to the retrotransposon is a minus-gamete specific gene (MGS). We hypothesized that MGS might be a second gene required for gamete fusion. Our main objective was to identify whether there is a defect in the DNA sequence in MGS in any of our fusion-defective mutants. We performed a chromosome walk on coding, promoter and 5 &rsqou; upstream and 3 &rsqou; downstream regulatory regions (UTR) of MGS via PCR. PCR products then where sequenced and aligned. We used qRT-PCR to determine MGS expression levels in the control and fusion defective mutants. Analysis of the sequencing and expressional results showed no defect in the MGS gene. For the systematics study we used comparative genomic and phylogenetic approaches enabling us to study metabolic pathways that are upregulated in gametes of Chlamydomonas. Congruent experimental results show that the nuclear-encoded and chloroplast localized MEP pathway leading to the biosynthesis of the isoprenoid precursor molecules is upregulated in Chlamydomonas gametes. It is expected, that the results from these studies will provide further insights into regulatory mechanisms occurring during gametogenesis, some of which might be necessary for gamete fusion in algae as well as in higher eukaryotic organisms.
Vascular Endothelia Growth Factor and its Receptor VEGFR2 Regulate Synaptic Protein Levels in Rat Hippocampal Neurons
Year of Dissertation:
2012
Advisor:
Patricia Rockwell
Vascular endothelial growth factor (VEGF) is a well-established angiogenic factor which also elicits protective and stimulatory effects on neuronal function. Recent studies suggest that VEGF signaling plays a critical role in modulating synaptic plasticity and enhances excitatory synaptic transmission in the hippocampus. Other growth factors including brain-derived neurotrophic factor (BDNF) and insulin have been shown to regulate synaptic ¬¬¬protein levels to stimulate neural communication but it remains unclear how VEGF participates in synapse function at the molecular level. The notion that VEGF would also modulate synaptic protein levels in differentiated hippocampal neurons has not been explored. Therefore, this work addressed whether VEGF exhibits neurotropic properties in mature rat hippocampal neurons by modulating the postsynaptic protein PSD-95 and protecting against the stress induced by nutritional deprivation. The results show that VEGF signals an increase in cell viability and increases the levels of presynaptic (synaptophysin and synapsin I) and postsynaptic (PSD-95) proteins through its cognate receptor VEGFR2. VEGF signals these events via autocrine and paracrine mechanisms. Moreover, VEGF regulates PSD-95 protein levels and synapse numbers along dendrites through the PI3K/Akt/mTOR pathway. Additional studies showed that inhibition of the Rho-associated protein kinase (ROCK) increased PSD-95 protein levels which were attenuated by VEGFR2 inhibition. Furthermore, ROCK inhibition enhanced VEGF-mediated synapse formation, survival and neurite extension. Accordingly, these findings suggest that ROCK serves as a negative regulator of VEGF signaling in mature primary hippocampal neurons. Collectively, this study revealed a novel signaling mechanism for VEGF/VEGFR-2 pathway that may function in its reported capacity to stimulate synaptic transmission. These findings implicate VEGF signaling as a potential therapeutic strategy to prevent or hinder synaptic loss in neurodegenerative disorders.
A Study of the Progenitor Potential and Function of Thymic Nurse Cells Using pH91 a TNC- Specific Monoclonal Antibody
Author:
Rajendra Chilukuri
Year of Dissertation:
2011
Thymic nurse cells (TNCs) are lympho-epithelial complexes that are a major component of the cortical thymic microenvironment. The functional role of TNCs in the thymus has been controversial but recent studies are beginning to elucidate the role of these cells in thymic homeostasis. In the present study, we have described the function of TNCs during the process of thymocyte selection and present results suggestive of the progenitor potential of TNCs.
Physical and chemical factors affecting the distributions of freshwater snails in four lakes in the New Croton/Muscoot watershed Westchester county, NY
Year of Dissertation:
2011
In a model study four lakes were examined to determine if benthic macroinvertebrates in the littoral zone were affected by physicochemical factors and shoreline development. The central hypothesis was that there would be correlations between the physicochemical factors in the lakes, levels of development around the lakes and the populations of benthic organisms. The study was conducted from April through October 2009 and 2010. Diversity and EPT indices were calculated to quantify taxa. Physicochemical variables measured included: temperature, pH, DO, mean nitrate and phosphate concentrations, total hardness, calcium, total dissolved solids (TDS), conductivity (ECS) and coliform testing. Sediment analysis and loss on ignition studies were done to assess percent composition, percent organic matter and percent carbonates in littoral sediments. Data characterizing shoreline development was collected from appropriate town, county and state resources, including: phosphorous loading, number of structures, number of storm drains, percent developed land and run-off into the lakes. Multivariate and correlation analyses were used to explore the data and to identify significant relationships between the benthic fauna and the abiotic variables. Results showed that freshwater benthic macroinvertebrates had significant correlations to physicochemical and development factors, including: ambient temperatures, hardness, DO, ECS, TDS, pH, percent silt, mean nitrate concentrations, coliforms, phosphorous loading, percent developed land, storm drains and the number of structures. The results of this study illustrate how anthropogenic inputs associated with development affect benthic macroinvertebrates in the littoral zone of suburban lakes.
Effect of Tau Hyperphosphorylation on Cellular Pathology
Year of Dissertation:
2012
Hyperphosphorylation of the microtubule associated protein tau is shown to be involved in several dementias that classify as tauopathies. In these diseases, tau is known to bind to itself rather than associate with microtubules. When CHO cells express wild type tau, the microtubule network is healthy and shows normal microtubule movement and tau associated with the microtubules. When expressing pathological human tau (PH-tau, pseudophosphorylated at T212, T231, S262),however, PH-tau is present throughout the cytoplasm, rather than associated with microtubules. The cells exhibit excessive membrane blebbing in order to remove PH-tau. This blebbing leads to a shrinkage of PH-tau expressing cells. Internally the presence of excessive cytoplasmic vacuoles and aggregated PH-tau in the form of filaments are found. The exposure of wild type expressing cells to okadaic acid shows the same pathologies. Additionally, all three sites used in the PH-tau construct are phosphorylated when wild type tau is exposed to okadaic acid.
Systematics and Phylogeny of Arcoid Bivalves (Arcoida: Pteriomorphia: Bivalvia)
Year of Dissertation:
2009
The Arcoida is a large group of mostly marine bivalves, with a global distribution. Familiar taxa in this group include the arks, bittersweets and dog cockles. Relationships among the higher-level taxa of the Arcoida are not well understood and the classification of this group has been the subject of debate and rearrangement. While many views exist as to the evolution of this group, none of them are based explicitly on a phylogenetic analysis. In this study, the phylogenetic relationship of the Arcoida is inferred from a systematic analysis based on both morphological and molecular data. This is the first analysis in which representatives of all seven nominal families are included. 141 morphological characters from the external shell and internal anatomy were coded for 131 taxa. The phylogenetic signal of both these character types was explored. Few non-homoplastic synapomorphies for the group were recovered; shell tubules are confirmed as the sole non-homoplastic synapomorphy for the order. Shell characters failed to recover the majority of the higher taxonomic ranks that they were initially used to describe. Little coherent signal was received from the analysis of anatomy alone. Four molecular markers, the nuclear 18S rRNA, 28S rRNA, protein coding histone H3 and mitochondrial cytochrome c oxidase I, were also investigated using direct optimization as implemented in POY (Varón et al., 2008). These data were analyzed individually as well as simultaneously with the morphological data. A Sensitivity Analysis (Wheeler, 1995) of the molecular data was also performed--this explores the effects of parameter costs (i.e. indels and transition/transversion ratios) on the phylogenetic results. The results of these phylogenetic analyses do not reflect the current classification of the group. In this study, the majority of the higher taxonomic groups of Newell (1969) were not recovered, including the two superfamilies Arcoidea and Limopsoidea, as well as five of the families; only the monophyly of the Glycymerididae and Noetiidae is supported. A major taxonomic review of the order is necessary. This analysis is the largest and most comprehensive phylogenetic analysis of the Arcoida to date.
Molecular genetic studies of sulfur nutrient response in Arabidopsis thaliana
Year of Dissertation:
2012
The aim of this study was to isolate the components that are critical for the S nutrient response in plants. We used Arabidopsis thaliana as a model system. We first developed a S deficiency-responsive promoter:GUS reporter system. We confirmed that At2g44460 encoding a putative thioglucosidase exhibits the strongest induction by S deficiency. Interestingly, At2g44460 induction by S deficiency was suppressed by the application of auxin, a plant hormone. Together with other physiological and genetic evidence, we showed that auxin plays a negative regulatory role in S deficiency response. Furthermore, we found that S deficiency-induced expression of At2g44460 and a sulfate transporter gene (SULTR4;2) is dependent on the availability of C and N, which exhibit a synergistic interaction. Therefore, we designed a genetic screen by using the At2g44460 promoter:GUS reporter line (designated GHF1) with an aim of isolating the mutants that alter the expression of At2g44460 in response to C,N and S status.
Glutamate Receptor Signaling is a Mediator of Neurite Outgrowth Inhibition
Year of Dissertation:
2010
Unlike the peripheral nervous system (PNS) or embryonic neurons, the adult mammalian central nervous system (CNS) does not spontaneously regenerate after injury. This is due, in part, to the presence of myelin-associated inhibitors, such as myelin-associated glycoprotein (MAG). Our lab has shown that elevation of intracellular cAMP blocks these inhibitors in vitro and in vivo in a transcription-dependent manner. Subsequent microarray analysis revealed that elevation of cAMP results in upregulation of Arginase I (Arg1), a key enzyme in the synthesis of polyamines. Our lab has demonstrated that administration of polyamines is sufficient to block MAG and myelin-induced inhibition of axonal outgrowth in vitro as well as enhance CNS axon regeneration in vivo. Others have shown that binding of polyamines to ionotropic glutamate receptors (iGluRs), which include N-methyl-D-aspartate receptors (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors (KAR), blocks their activity. In addition, it is well established that iGluR activity is disrupted following CNS injury. Thus, since polyamines overcome MAG/myelin-mediated inhibition and given that polyamines block iGluRs activity, we set out to examine the role iGluR-mediated signaling plays in the MAG and myelin inhibitory pathways. We found that blocking NMDAR or AMPA/KA receptor activity with pharmacological antagonists was sufficient to block MAG-induced inhibition of neurite outgrowth in dorsal root ganglia (DRG) and hippocampal neurons (HNs). Likewise, exposure to iGluR agonists increased MAG-mediated inhibition of neurite outgrowth. To determine whether prior exposure of iGluR inhibitors or priming is sufficient to overcome MAG inhibition as in the case of polyamines, HNs were treated with iGluR antagonists 18 hours before exposing neurons to MAG. We found that priming with iGluR antagonists was not sufficient to overcome MAG-mediated inhibition, suggesting that GluR antagonists exert their effect by intercepting MAG inhibition signals, while not changing the ability of neurons to respond to MAG. Because polyamines require priming whereas iGluR antagonists have no effect when used to prime neurons, we therefore conclude that in our model system, polyamines do not overcome inhibition by blocking iGluR activity.