Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • 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.

  • 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.

  • Differences in Morphology and Behavior in Green Frogs (Lithobates clamitans) from Urban and Rural Sites in New York and New Jersey

    Author:
    Jennifer Costello
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Richard Veit
    Abstract:

    Abstract Anthropogenic disturbances to freshwater ecosystems are intensifying with the continued growth and expansion of the human population. Urbanization is associated with increased anthropogenic land use and pollution compared to rural areas. Freshwater ecosystems in particular are altered by the input of nutrients and wastes resulting from run-off in urban areas. Pollutants of particular concern to urban ecosystems are metals, polycyclic aromatic hydrocarbons (PAHs), particulate matter, and radioactive nuclides. Metals from anthropogenic activities constitute a serious threat to the environment due to their toxic effects on plants, animals and humans and their potential to accumulate up food-chains. Animal behavior is a useful individual-level response that acts as critical link between animal physiology and overall population effects. Therefore, differences behavior within a species impacted by varying degrees of urbanization may be useful in predicting overall population effects. Morphology, likewise, may differ between urban and rural environments as animal growth rates are highly dependent upon survival-related behaviors. Efficient acquisition of prey provides energy necessary for individual growth. When prey capture is deficient, growth rates are reduced which in turn impacts survival. I assess differences in green frog, Lithobates clamitans behavior and morphology in frogs from urban and rural sites. I examine the relationship between anthropogenic metals, one measure of urban pollution, and several levels of biological organization within green frogs. I determine which levels of biological organization are influenced by urbanization and if a link exists between lower levels of biological organization by assessing metal accumulation, and upper levels of biological organization, by measuring feeding behavior, advertisement call, morphology, and population composition. With the exception of cobalt (Co), no relationship was observed between metals present in the environment (sediment and water) and L. clamitans. Prey capture efficiency and prey capture latency were significantly different between frogs from urban and rural sites. Feeding efficiency was negatively associated with total metal concentration of water. Frogs from urban sites were smaller in size than frogs from rural sites. This may be due in part to lower prey capture efficiencies. I did not observe differences in L. clamitans population abundance in urban and rural sites. Therefore, the negative impacts to green frog behavior and morphology may not be severe enough to result in population declines.

  • Understanding A Pest--Phylogeography And Systematics Of The Plum Curculio (Conotrachelus nenuphar)

    Author:
    Samuel Crane
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Rob DeSalle
    Abstract:

    The plum curculio (Conotrachelus nenuphar Herbst) (Coleoptera: Curculionidae) is an economically and ecologically important pest in North America but is understudied despite having a long history in the scientific literature. Its chemical ecology, life history habits, and distribution are all well characterized. However, there is a dearth of knowledge concerning the population structure and evolutionary history of the species. This study aims to use methods from evolutionary biology to better understand an agricultural pest species and provide tools to aid in its management. Existing taxonomic classifications of North American Conotrachelus species have been tested for the first time. Using a combined multigene approach, we have inferred a species phylogeny. Established species groups are well supported as monophyletic. However, the species groups identified in taxonomic keys are generally not recovered as monophyletic. Broadly sampling across the geographic distribution, we have sample over 1,000 individuals for mitochondrial DNA variation. We characterized population substructure of plum curculio populations from the full breadth of its range and reveal significant geographic and genetic structure. There is a significant north-south split that does not align with the current understanding of the plum curculio phenological strains. There are also highly structured populations corresponding to the Mississippi River and Apalachicola River basin, a pattern thought to be associated with southern refugia along the Gulf Coast. There are likely multiple refugia used over the Last Glacial Maximum. Regions of the world that are most at threat of plum curculio beetle invasion, given the organism's habitat preferences, are identified across all continents and in every region where it is listed as a quarantine species. Molecular tools for diagnosing and managing the plum curculio, locally and internationally, are developed and provided for all life stages.

  • Testing assumptions of coevolution in an egg-rejecting brood parasite host: Uncovering sensory, cognitive, and evolutionary drivers of responses to parasitism in American robins (Turdus migratorius)

    Author:
    Rebecca Croston
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Mark Hauber
    Abstract:

    Hosts of brood parasitic birds face fitness costs associated with rearing unrelated offspring. In response, the recognition and rejection of parasitic eggs is a common host defense. Brown-headed cowbirds (Molothrus ater) challenge coevolutionary theory, because although they exploit over 200 host species, they lay non-mimetic eggs, and most hosts do not combat cowbird parasitism with egg rejection. American robins (Turdus migratorius) are one of a handful of cowbird hosts known to recognize and remove cowbird eggs from the nest. I addressed the mechanistic and evolutionary drivers of egg rejection in this host species, by disentangling the roles of spectral tuning and visual physiology on the behavioral outcome of egg rejection, by estimating the costs of parasitism which may drive egg rejection behavior, and by addressing the reciprocal effects parasitism on host egg color variation and its role in mediating rejection decisions. I also test assumptions underlying the evolvability of host egg rejection responses in this system. In Chapter 1, I lay out an overview of brood parasitism as a reproductive strategy and brood parasite-host ecology, and highlight evolutionary mechanisms and consequences of coevolution in these systems. In Chapter 2, I test the hypothesis that foreign egg rejection is driven proximately by perceivable differences in ground color between host and parasitic eggs across the entire avian spectral sensitivity range. I show that the rejection of artificially dyed eggs is mediated by input from all four avian single-cone photoreceptors, and that more divergent model `parasitic' eggs are indeed rejected at higher rates. However, the cowbird egg does not conform to this prediction, because both model and real cowbird eggs are rejected in 100% of experimental trials despite their lower overall discriminability from robin eggs. This may indicate a cowbird-egg specific rejection response in robins. In Chapter 3, I test a critical assumption underlying the evolution of cowbird-specific egg rejection responses in robins, by assessing the hypothesis that cowbird parasitism imposes recoverable costs on robin hosts. My results indicate that cowbird chicks fare poorly when reared alongside robin chicks, but parasitism per se still reduces nesting success for robins; thus, rejection of cowbird eggs serves a function to eliminate the cost of parasitism. In Chapter 4, I examine a critical assumption underlying all of host-parasite coevolutionary theory, namely that host defenses can evolve genetically in response to parasitism. I address the hypothesis that egg rejection is repeatable in our study population, as repeatability is prerequisite to the evolution and spread of a behavioral trait, including a predictor of the trait's genetic heritability. As predicted, egg rejection behavior in American robins was found to be highly repeatable for intermediately-rejected model egg colors within the same nesting attempt, irrespective of potentially confounding ecological and temporal factors. Finally, in Chapter 5, I test predictions stemming from alternate hypotheses that egg rejection evolved in response to cowbird (non-mimetic) versus conspecific (mimetic) parasitism, by investigating the degree of color variation within robins' own clutches, and the effect of experimentally manipulating intraclutch color variation. I used both observational and experimental data, and found that egg color varies more between clutches than among egg within a single clutch, yet experimental manipulated intraclutch color variation did not affect rejection rates. These results support the scenario of historical parasitism by non-mimetic parasites. Variation among the findings of similar studies pertaining to hosts of mimetic parasites may be explained by hosts' use of different cognitive mechanisms in the decision to reject foreign eggs, However, for hosts of non-mimetic parasites, investigating egg color variation and its effect on egg rejection is not informative about different cognitive decision-making rules, as predictions under each mechanism are similar - that there will be no effect of a history of parasitism on intraclutch color variation (observational patterns) or rejection rate (experimental data). This body of research presents compelling evidence in support of egg rejection by robins as a specific response to historical cowbird parasitism, and has highlighted important components of the sensory, cognitive, functional and evolutionary processes underlying egg rejection in this paradoxical brood parasite-host system.