Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • The Effects of Thyroid Hormone Insufficiency During Development On Cortical Morphology And The Behavioural Manifestations

    Author:
    Susan Briffa-Mirabella
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Carl Dobkin
    Abstract:

    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. The number of neurons was estimated, using unbiased sampling techniques, to determine whether the cellular composition of cortex was altered following developmental TH insufficiency. To determine if these cortical alterations led to changes in behaviour, a battery of behavioural tests were performed which included maternal retrieval (PND 4), maternal separation anxiety (PND 6), Barnes maze (PND 48 and PND 86), social interaction social approach (PND 48-50), and open field (PND 46). Taken together, the results presented here support the hypothesis that developmental hypothyroidism and hypothyroxinemia induced by chemical thyroid hormone suppression (PTU) cause alterations in the morphology of the cerebral cortex by altering cortical volume and changing the number of cortical neurons in the rat brain. Furthermore these alterations ultimately lead to changes in certain aspects of behaviour. These results have important clinical relevance because several studies suggest that developmental disabilities ranging from mild dyslexia to severe mental retardation can be attributed to alterations in cortical morphology resulting from abnormal cortical development.

  • A MINUS-GAMETE-SPECIFIC GENE IN FUSION-DEFECTIVE CHLAMYDOMONAS MUTANTS AND ANALYSIS OF BIOSYNTHETIC PATHWAYS UPREGULATED DURING GAMETOGENESIS

    Author:
    Dmitry Brogun
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Charlene Forest
    Abstract:

    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

    Author:
    Qin Cao
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Patricia Rockwell
    Abstract:

    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.

  • Structure and Function in Bacteriophage Phi6

    Author:
    James Carpino
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    John Dennehy
    Abstract:

    The present study of bacteriophage Phi6 has been preceded by a great number of exploratory studies of its structure and function, and these studies have formed a basis for Phi6's development into a model organism. In this study, two aspects of the model organism have been examined. 1. There are several uncharacterized and presumed untranslated regions (UTRs) in Phi6's 13.3 kilobase-pair dsRNA genome. I examined the impact of specific modification to the 3' UTR of the small segment of bacteriophage Phi6. I determined that modification to the purported UTR of the small segment resulted in severe fitness costs, supporting a functional role for unidentified gene products, secondary RNA structure, or both. 2. Bacteriophage Phi6 packages its dsRNA genomic segments selectively and sequentially through the function of the packaging motor P4 which occupies fivefold vertices of the Phi6 procapsid, and studies support the functioning of one and only one P4 during packaging. The mechanism of this specific phenomenon is not known. I used computational reconstruction of cryoelectron microscopy and examined the occupancy of P4 on the Phi6 procapsid, and acquired insight into the mechanism of assembly and packaging.

  • Control of Cell Cycle Progression by mTOR

    Author:
    Amrita Chatterjee
    Year of Dissertation:
    2015
    Program:
    Biology
    Advisor:
    David Foster
    Abstract:

    Over the past few years it has become apparent that cancer cells require the activation of a set of intra-cellular signals that promote cell cycle progression and survival. One of the most common survival signals activated in human cancers is mediated by mTOR –the mammalian target of rapamycin. mTOR is a critical nutrient and energy sensor in cells that lets the cell know that there is sufficient material available for a cell to double its mass and divide. mTOR causes the phosphorylation of downstream targets ribosomal subunit S6 kinase and eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1), which promotes cell cycle progression. mTOR suppresses the activity of the tumor suppressor Transforming Growth Factor- (TGF-β). TGF- plays a central role in causing G1 cell cycle arrest. Rapamycin is a highly specific allosteric inhibitor of mTOR. In the presence of serum, rapamycin activates TGF- signaling and causes G1 cell cycle arrest. This is one reason why rapamycin is frequently called a “cytostatic drug”. While conventional low (nM) doses of rapamycin can retard G1 cell cycle progression, our lab has recently found that high (μM) doses of rapamycin are needed to induce complete G1 cell cycle arrest. However, it is unclear as to what causes the cells to be sensitive to high dose rapamycin treatment with regard to G1 cell cycle progression. Prior studies in lab has shown that rapamycin in absence of serum induces apoptosis. High dose rapamycin inhibits eIF4E. In this study we revealed that knockdown of eIF4E causes apoptosis both in the presence and absence of serum. This was unexpected because rapamycin induces G1 cell cycle arrest in the presence of serum. Upon investigation, we have found that inactivated S6 kinase prevents the apoptotic effect observed by singular knockdown of eIF4E and results in G1 cell cycle arrest. This effect is dependent on TGF-β signaling which contributes to G1 cell cycle arrest. Suppression of S6 kinase phosphorylation alone is insufficient to cause complete cell cycle arrest, indicating that complete G1 cell cycle arrest is due to suppression of both S6 kinase and eIF4E. This proves that the cytostatic effect of rapamycin is suppression of both S6 kinase and eIF4E, while the cytotoxic effects are due suppression of eIF4E in the absence of S6 kinase-dependent activation of TGF- signals. This study also shows that nano-molar doses that inhibit S6 kinase were sufficient to activate TGF- signaling. The high doses of rapamycin used to inhibit eIF4E correlated with inhibition of Rb phosphorylation. Consistent with these observations, knockdown of both Smad4 (an important player of TGF-β signaling) and Rb reversed the cytostatic effects of rapamycin. These data indicate that the G1 cell cycle arrest induced by rapamycin is due to the up regulation of TGF- signaling and down-regulation of Rb phosphorylation via phosphorylation of the mTORC1 substrates S6 kinase and 4E-BP1 respectively. Altogether, our findings not only place an importance to the evaluation of the activity/expression level of S6 kinase and eIF4E as readouts for rapamycin efficacy but also enhance the current understanding of the cytostatic effects of mTORC1 suppression with therapeutic implications.

  • Integrating phylogenomics, biogeography and systematics to explore the taxonomy and the rise of the ratsnakes

    Author:
    Xin Chen
    Year of Dissertation:
    2015
    Program:
    Biology
    Advisor:
    Frank Burbrink
    Abstract:

    Understanding the evolutionary processes that create the spectacular diversity of organisms, both in species numbers and form, is a primary goal for biologists. Global ratsnakes are a species-rich assemblage with high morphological and ecological diversity and a distribution that encompasses both the Old World (OW) and the New World (NW). To explore the mechanism leading to the divergence of the ratsnakes, I tested the hypotheses regarding the area of origin and global dispersal, and examined the patterns of diversification and trait evolution. Given adaptive radiation via ecological opportunity, a diversity-dependent diversification pattern and an early burst trait evolutionary pattern are expected with rapid divergence triggered by the appearance of new resources, extinction of competitors, colonization of new areas or the appearance of key innovations. Thus, I tested if the radiation of ratsnakes follows diversity-dependent diversification with an early burst in speciation and trait divergence and whether the variation in diversification is associated with OW-NW dispersal or changes in traits. Further, trait convergence between OW and NW lineages was investigated to determine, if given similar environmental conditions, rapid speciation via ecological opportunity is repeatable. To answer the questions mentioned above, a robust phylogenetic tree is fundamental. Due to potential gene tree/species discordance, hundreds of loci sampled across the entire genome were generated using the anchored hybrid enrichment approach and the multi-species coalescent methods were used to build the species phylogeny. Then, given this phylogenetic context, taxonomic changes were made to reflect named monophyletic groups and divergence time and ancestral areas were estimated to 1) infer the processes leading to the current ratsnake global distribution, 2) assess the best fitting diversification and trait evolution models, and 3) determine if ecomorphological convergence occurs with adaptive regimes of traits on the phylogeny. Among all of the inferred species trees, by comparing the extent of tree discordance and the gene tree errors, the species trees generated in the program MPEST with summary statistics of posterior probability gene trees was used for further analysis. First, it was determined that the traditional ratsnake genera Gonyosoma and Coelegnathus are excluded from the monophyletic ratsnake group, with the remaining monophyletic group defined as Coronellini. The reconstructed ancestral areas supported that ratsnakes originating in the OW Eastern Palearctic and with a single dispersal to the NW via Bergingia. Two subclades each defined by a single genus, Lampropeltis and Elaphe, were found to have exclusively elevated species diversification and trait evolutionary rates. As the rate accelerations were only in the recent divergent lineages, colonization to the NW and rapid speciation of the NW lineages were decoupled. A general diversity-dependent radiation pattern in both OW and NW lineages was supported with a recent sharp diversification elevation about 6.5 Ma mainly within the genera Lampropeltis and Elaphe. Three morphological convergence events were detected among OW and NW lineages, corresponding to the previously defined morphological taxonomies (i.e., Elaphe and Pantherophis), indicating without a robust molecular phylogeny, morphological convergence positively misleads taxonomy. This research demonstrates the advantages and challenges of phylogenetic inference using genome scale dataset, highlights the importance of incorporating the biogeographic history and trait evolution in studies of diversification and indicates that oversimplified models are insufficient to describe the complexity of processes shaping the diversity in a species-rich assemblage.

  • 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
    Program:
    Biology
    Advisor:
    Jerry Guyden
    Abstract:

    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. Using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopic analyses, we show that TNCs create an intimate association with thymocytes. Thymocytes become trapped within unique extra-cytoplasmic spaces generated by the TNCs. The membrane-derived honeycomb-like fenestrae allow visualization of trapped thymocyte movement into and out of these fenestrae, a process that facilitates interactions between the two cell types. Further, we have data confirming an interaction between the abTCR expressed on trapped thymocytes and MHC class II antigen expressed on the surfaces of the TNCs. We also observe lipid-raft accumulation around the contact point between the thymocytes and TNCs. When we costained freshly isolated thymic nurse cells with TNC-specific monoclonal antibody pH91 and with K5 and K8 cytokeratin antibodies, we observed a subset of TNC that were K5+K8+ and pH91+. Previous studies have suggested that k5+k8+ thymic TECs were thymic epithelial progenitor cells. The studies presented here show that TNCs express the transcription factors Foxn1 and p63 both of which play critical role in the thymic determination as well as maintaining a proliferative subpopulation of TECs. Interestingly, when we co-stained embryonic day 11.5 (E11.5) thymic sections with pH91 and Foxn1 antibodies, their expressions were detected in this phase of early thymic organogenesis. The expression of p63 was detected a day later at E12.5. Also, we have results confirming the expression of pH91 antigen as early as E7.5 along with a stem cell marker Oct4. Finally using confocal analysis and TNC specific mAb, pH91, we show that the classical complex morphology of TNCs first appears at E17.5 stage of development. However, analyses of major histocompatibility complex (MHC) class II expression on embryonic TNCs cell surfaces show its onset from E13.5. The results show a marked increase in the expression of MHC class II, from 36.2% at E13.5 to 69.1% at E18. 5 stage of development. Taken together, these data suggest that thymic nurse cells play a significant role in the murine thymus; they create membranous spaces that facilitate MHC restriction and express markers implicating them as possessing progenitor ability.

  • Physical and chemical factors affecting the distributions of freshwater snails in four lakes in the New Croton/Muscoot watershed Westchester county, NY

    Author:
    Tami Cloherty
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Joseph Rachlin
    Abstract:

    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.

  • Sibling egg cannibalism by neonates of the Colorado potato beetle, Leptinotarsa decemlineata

    Author:
    Karyn Collie
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Mitchell Baker
    Abstract:

    Cannibalism reduces competition and provides nutritional benefits. However, when cannibalism involves kin, the benefits obtained must balance inclusive fitness losses, and cannibals should be under selection to avoid killing close relatives. Since cannibalism reduces competition, it may also be higher in populations with greater population density. Neonates of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, readily consume eggs within their natal clutch, which is a combination of full and half siblings, before assessing resource availability, suggesting an evolutionary response to potential resource limitation that should vary with competition between populations. CPB is also a crop pest that rapidly develops resistance to pesticides, but pesticide resistance can result in fitness costs in the absence of pesticide exposure; these costs may be mediated by cannibalism. To explore the fitness consequences of cannibalism, I fed neonates with only potato foliage or with eggs and potato foliage and measured growth and development rates. I used individuals from pesticide-susceptible and pesticide-resistant populations to test for costs of resistance and whether there is an interaction between the benefits of cannibalism and resistance. To determine whether neonates avoid killing relatives, I tested whether hatchlings recognize kin, whether they prefer inviable to viable eggs, and whether egg development is a cue for viability. To explore geographic variation in cannibalism, I studied three CPB populations and a population of L. undecimlineata on their native host plants in Mexico to assess differences in competition and cannibalism propensity. Cannibals gained mass and developed more quickly than noncannibals. When mortality risk is high, this decreased development time can reduce the mortality risk sufficiently to balance the inclusive fitness loss of eating a half sibling. There were costs of pesticide resistance, but the benefits of cannibalism reduced many of these costs. Neonates preferred eating eggs from another population, but they did not distinguish among eggs from their own population based on relatedness. They did, however, preferentially consume inviable eggs but did not use egg development as a cue for viability. Cannibalism rates were usually higher in the populations with the highest egg densities, although interspecific comparisons did not show the same pattern.

  • Effect of Tau Hyperphosphorylation on Cellular Pathology

    Author:
    Christopher Corbo
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Alejandra Alonso
    Abstract:

    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. Tau interacts with actin as well as with microtubules. PH-tau seems to cause a major breakdown in the F-actin structure within the cells. These cells appear to be either totally void of F-actin or have F-actin forming punctate spots within the cells. This actin breakdown is also occurs in wild type tau expressing cells treated with okadaic acid. The CUNY CSI Computer Science Department is working in collaboration to develop ImageJ plugins to quantify the amount of F-actin and the length of individual F-actin filaments. This work demonstrates that when PH-tau is expressed, the level of tau is inversely proportional to the level of F-actin in the cells. Interestingly, the level of total actin does not change between wild type tau and PH-tau expressing cells, suggesting that this lack of F-actin does not change expression, but rather interferes with its polymerization. When CHO cells are transfected with PH-tau, this protein can be found in the nucleus of the cells. We found a nuclear localization signal that allows the chaperon protein importin to bind to tau. To see if this site was responsible for the translocation of tau into the nucleus, we eliminated importin's binding site through site directed mutagenesis of the full-length tau gene. The elimination of the importin binding site inhibited tau from being able to translocate into the nucleus but did not stop any of the pathologies seen previously.