Alumni Dissertations

 

Alumni Dissertations

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  • Characterization of the CYP97 and HYD carotene hydroxylase enzymes

    Author:
    Rena Quinlan
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Eleanore Wurtzel
    Abstract:

    Vitamin A deficiency is a serious and widespread public health issue in developing countries. Provitamin A carotenoids such as β-carotene have therefore recently attracted interest as important neutraceuticals. Due to the nutritional value of carotenoids there is currently considerable interest in developing rational strategies for metabolic engineering of crops for enhanced carotenoid content. Efforts to improve the provitamin A content of cereal endosperm in staple crops such as maize will require characterization of the carotene ring hydroxylases involved in controlling the conversion of provitamin A carotenes to non-provitamin A xanthophylls. Based on early modeling by (Cunningham and Gantt, 1998) the cytochrome P450 CYP97 and diiron HYD carotene hydroxylases were predicted to localize to chloroplast membranes to function in separate multi-enzyme complexes for the respective conversions of the provitamin A carotenes α- and β-carotene to the non-provitamin A xanthophylls lutein and zeaxanthin. To gain a better understanding of the respective roles of the CYP97 and HYD enzymes in these conversions, the activities and localization/interaction of these enzymes were examined using both in vitro and in vivo approaches. Escherichia coli functional complementation systems were used to assess rice P450 CYP97A4 (β-ring hydroxylase) and CYP97C2 (ε-ring hydroxylase) as well as maize diiron HYD3 and HYD4 (β-ring hydroxylases) activities and substrate specificities. Preliminary investigations examining CYP97 enzyme activity only via E. coli complementation showed that the CYP97A4 exhibits major activity toward β-rings to convert β-carotene to β-cryptoxanthin (pathway intermediate) as well as a low amount of zeaxanthin (pathway end-product). In addition, this enzyme exhibited minor activity toward the ε-rings of ε-ε-carotene to convert this substrate to a low amount of lactucaxanthin (pathway end-product). These studies also indicated that the CYP97C2 appeared to be exclusively active toward the ε-rings of ε-ε carotene, converting this substrate to a low amount of lactucaxanthin; no activity toward the β-rings of β-carotene was detected for this enzyme. Subsequent complementation studies tested both individual and combined CYP97/HYD enzyme activities in E. coli accumulating both α- and β-carotene substrates. These studies demonstrated that the CYP97A4 and CYP97C2 enzymes function optimally when expressed together in the conversion of their preferred substrate α-carotene to produce lutein. Cells engineered to produce α- and β-carotene and which co-expressed these enzymes generated almost 30% lutein (% total carotenoids); a roughly 10-fold higher amount of lutein relative to zeaxanthin was observed. In contrast, when expressed as individual enzymes the CYP97A4 and CYP97C2 showed suboptimal activity (ie., no lutein produced; only approx. 14%, and 1% of the intermediates zeinoxanthin and α-cryptoxanthin generated respectively) regardless of substrate choice. In these cells, the CYP97A4, when expressed alone, preferred the β-carotene substrate to the α-carotene substrate generating a low amount of zeaxanthin (the monohydroxylated intermediate β-cryptoxanthin accumulated); the CYP97A4 was only moderately active toward the α-carotene substrate as only the intermediate zeinoxanthin accumulated (no lutein was produced). When expressed as an individual enzyme, the CYP97C2 was minimally active toward both α- and β-carotene substrates to respectively generate barely detectable amounts of the intermediates α-cryptoxanthin and β-cryptoxanthin. HYD3 + CYP97C2 and HYD4 + CYP97C2 combinations were also tested using this complementation system. Both enzyme combinations were moderately active toward α-carotene, respectively producing low amounts of lutein; although cells co-expressing the HYD3 + CYP97C2 appeared to be somewhat more active toward α-carotene than β-carotene, generating a more than two-fold higher amount of lutein relative to zeaxanthin. When expressed as an individual enzyme, the HYD3 was preferentially active toward β-carotene to convert this substrate to a low amount of zeaxanthin (end-product); this enzyme was only moderately active toward the α-carotene substrate as only the intermediate zeinoxanthin accumulated. The HYD3 exhibited suboptimal activity in our complementation systems whether expressed alone or in combination with the CYP97C2. By contrast, the HYD4 functioned optimally when expressed as an individual enzyme. This enzyme was preferentially active toward β-carotene and efficiently converted this substrate to zeaxanthin. This work also examined CYP97/HYD protein localization and protein interaction. In vitro chloroplast import and in vivo GFP fusion assays confirmed that these enzymes are localized to chloroplasts. In addition, import assays were used to determine the suborganellar locations of these enzymes, and in vivo Bimolecular Fluorescence (BiFC) assays were performed to assess protein-protein interaction. Taken together, these studies demonstrated that the CYP97A4 and CYP97C2 enzymes are peripherally-associated to chloroplast membranes where they interact to form a heterodimer complex to function in the efficient conversion of α-carotene to lutein. It was expected that these enzymes, which functioned optimally together toward the α-carotene substrate in the E. coli complementation system, would interact and localize to the same location in the chloroplast membrane. These data also indicated that the HYD3 and HYD4 enzymes are integrally-bound to the chloroplast membrane where they interact to function in the conversion of β-carotene to zeaxanthin. These enzymes were expected to localize to the same suborganellar location since they were both preferentially active toward the same substrate (ie., β-carotene) in the complementation system. In addition, BiFC analysis indicated that HYD4 formed a homodimer complex.

  • Neural Targets of Electric Field Stimulation

    Author:
    Thomas Radman
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Marom Bikson
    Abstract:

    Clinical application of electric fields to the brain are promising non-invasive approaches for the treatment of psychiatric, neurological, and pain disorders. Low-magnitude electric fields, which do not cause neuronal firing but only create changes to the voltage necessary for inducing neuronal firing by approximately 1%, are known to have substantial behavioral effects and therapeutic outcomes. Transcranial direct current stimulation is one electric stimulation modality that induces such low-magnitude electric fields. Electric fields of magnitude sufficient to directly trigger neuronal firing may be induced by transcranial electric stimulation and transcranial magnetic stimulation. Many therapeutic advances have been made using these techniques, and they create new experimental approaches to increase our knowledge of how the brain works. Still, the fundamental mechanisms as to how these electric fields may affect neuronal elements of the brain are not fully understood. Chapter 1 of this thesis creates a mechanistic model describing how an electric field of any small magnitude may still have an effect on neuronal processing by changes in spike timing. This mechanistic framework has implications for the effects of endogenous, brain-generated electric fields as well as electric stimulation modalities. The second chapter of this thesis develops a model of how cortical neuronal morphology and cell type may be used to predict changes in polarization and firing caused by electric fields. Both of these studies employed techniques to record from single cells in the brain, and all models described have been experimentally verified through these techniques.

  • Mechanisms of Vocal Learning in Songbirds: how song syllable structure is learned

    Author:
    Primoz Ravbar
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Ofer Tchernichovski
    Abstract:

    When learning to perform continuous actions one needs to cope with conflicting motor requirements: while some parts of the action may require exploratory variability to find motor states that can efficiently produce a desired outcome, other parts may require consolidation if they are already close to the desired goal. A possible solution to this problem is partitioning the action into segments that could be controlled independently, but it is not known if exploratory variability can be locally regulated during the sensory-motor learning of a continuous action. This dissertation explores song learning in songbirds as a model of learning to perform continuous actions. In the first set of experiments we manipulated song learning in zebra finches (Taeniopygia guttata) to experimentally control different requirements for vocal exploration in different parts of the song. We first trained birds to perform a one-syllable song, and once they mastered it we added a new syllable to the song model. Remarkably, when practicing the modified song, birds rapidly alternated between high and low acoustic variability to confine vocal exploration to the newly added syllable. Analysis of exploratory variability within syllables revealed that acoustic variability changed independently across song elements that were only milliseconds apart. The variability of each song element decreased as it approached the target, correlating with momentary local error and less so with the global error (the mean of local errors across the syllable). We concluded that vocal error is computed locally in sub-syllabic time scales and that song elements can be learned and consolidated independently. However, our evidence for partitions singing behavior into segments that may be learned independently, also indicate that those segments are not developmentally stable. For example, we observed that the appearance of distinct syllable types usually precedes the appearance of distinct sub-syllabic song elements, suggesting that the time scales at which syllables are learned might get shorter over development, perhaps in a hierarchical manner, from the learning of course temporal structure to the learning of fine temporal structure. To test this hypothesis we studied song learning at the articulatory level by measuring the control of respiratory pressure during song learning. We found that during early song learning, respiratory pressure patterns were coarse, and fine temporal structure was later added to that pre-existent coarse structure. This effect was not observed in socially isolated birds that developed their song without learning a tutored model. We therefore propose that the learning of continuous singing action is achieved by first partitioning the song into coarse segments, and then to finer units, where exploratory variability in each unit is dynamically gated until a local match to the song model is achieved.

  • The Expression of the Transcription Factor BROAD and RNA-binding Factors in the Midgut of the Mosquito Aedes Aegypti

    Author:
    Kathryn Ray
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    James Nishiura
    Abstract:

    ABSTRACT Expression of the transcription factor BROAD and RNA binding factors in the midgut of the mosquito Aedes aegypti during metamorphosis by Kathryn Ray Thesis supervisor: James T. Nishiura, Associate Professor, Brooklyn College, CUNY Transcription factors, microRNAs and RNA binding factors frequently interact to coordinate gene expression during development. The transcription factor BROAD (BR) is a global regulator of insect gene transcription and governs the timing of the commitment to pupate. I determined BR expression in the Ae. aegypti midgut by qPCR, and correlated its expression with that of nine miRNAs and three RNA-binding factors. During midgut metamorphosis the expression of these factors was dynamic and reproducible. To better understand the changes in expression patterns, I evaluated the effects of hormone analogs on expression. Using this approach I uncovered concurrent up-regulation of BR, miR-34and miR-14 in the pupal midgut when treated with methoprene, and found that RH2485 accelerated expression of BR, BRAT, and microRNAs let-7 and miR-125. Treatment with each hormone analog resulted in a change in BR expression. Finally, I evaluated the effect of nutrients on expression levels. Surprisingly, though most transcripts were down-regulated during starvation, the expression of BR did not decrease, while microRNAs miR-34 and miR-14 were elevated. This may identify a novel role for during starvation in an invertebrate, and raises the possibility that miR-34 and miR-14 are part of a starvation-induced stress response in the mosquito midgut. In summary, this expression analysis suggests that microRNA regulation plays an important role during midgut metamorphosis, and reveals a new layer of regulatory complexity in the control of development in Ae. aegypti.

  • Phytosociology, History and Diversity in Farmer-Managed Landscapes on the Tonle Sap Floodplain, Cambodia

    Author:
    Andrew Roberts
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Christine Padoch
    Abstract:

    Driven by the annual flood pulse of the Tonle Sap Lake, the Tonle Sap floodplain in central Cambodia is a landscape characterized by dynamism, both ecological and social. Annual floods and rainfall vary in timing, duration and intensity from year to year. Patterns of burning, grazing, and agricultural expansion and contraction all leave their imprint on the landscape. While in the social domain, the region is likewise as complex. The genocidal, Maoist regime of Pol Pot darkened the 1970s. However, this was both preceded by, and followed by, years of civil war and social unrest. In recent years, Cambodia has shifted from a centrally-planned economy to a free-market economy, and experienced rapid economic growth followed by a dramatic slowdown. In the wake of such flux, the floodplains remain among the most productive rice-growing regions in the country, and are home to one of the most productive inland fisheries in the world. Even so, the region also remains one of the poorest. This project weaves analytical strands from ecology, geography and anthropology to delve into the ways in which people make a living in such a complex, challenging environment by focusing on the relationships between people, the landscape and plants. Results are presented from an analysis of 47 years of land use/land cover change depicted in aerial photography; an analysis of the structure and composition of floodplain plant communities through the use of descriptive ecological inventory; and an analysis of household-level natural resource-based livelihood activities, detailing how plant communities are utilized by village residents. A case study in floodplain land use is also presented, focusing on the Hillock-Depression Complex, a landscape element newly described herein. This case study illustrates the pitfalls of top-down land use planning in the context of a landscape rich in resources important to local residents but illegible to policy-makers. The results of these diverse analytical streams suggest that people do not live on the floodplain in spite of tremendous dynamism. Rather, the opposite is true. They live on the floodplain because of such dynamism, not as passive subjects but as active agents in generating diversity.

  • Drosophila Dunc-115 Mediates Axon Projection Through Actin Binding

    Author:
    Christopher Roblodowski
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    He Qi
    Abstract:

    Axon pathfinding, the process of how neurons reach their correct targets during early neural development, is a critical area in neuroscience research. Though significant progress has been made in identifying the extracellular guidance cues and their membrane receptors, and possible downstream effectors including the Rho GTPases family, little is known regarding how guidance signals are relayed to the actin filaments that are central to the mobility of the growth cone. Our laboratory has identified and characterized Drosophila gene dunc-115 as a possible downstream target in relaying guidance cues further down to the cytoskeleton. Specifically, we have shown that Dunc-115 regulates neural connections in both the eye and the central nervous system in Drosophila. In this thesis, I have identified that (1) the surface receptor Roundabout (Robo) is an upstream regulator of guidance signals relayed through Dunc-115, (2) Dunc-115 receives signals from the Rho GTPases, and (3) Dunc-115 binds to actin via its VHD (Villin Head Piece) domain, suggesting a mechanism for Dunc115 to relay guidance signals. Finally, I have analyzed the genomic structure of the dunc-115 gene in 12 Drosophila genomes, which has generated further insights into how this gene has evolved.

  • Phylogeography, Phylogenetics, Historical Demography, and Morphology of Milksnakes (Genus Lampropeltis)

    Author:
    Sara Ruane
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Frank Burbrink
    Abstract:

    A primary goal for systematists is the discovery and identification of Earth's biodiversity. To meet these goals, coalescent-based phylogenetic and phylogeographic methods have been developed that help quantify diversity and provide a better understanding of processes generating species. Here, I use these methods to examine milksnakes (genus Lampropeltis ). Milksnakes are among the widest ranging of New World squamates and are frequently used as model organisms in Batesian mimicry studies. Despite this, there has been no research detailing their systematics within a molecular phylogenetic framework. Furthermore, previous molecular studies that include milksnakes have shown they do not form a monophyletic group within the genus and that color pattern, which has been the basis for their previous classification may not be informative with respect to evolutionary history. The study detailed here examines milksnakes across their intercontinental range to determine whether there is unrecognized diversity in this group. To meet this goal, I have developed a multi-locus dataset consisting of 11 nuclear genes and one mitochondrial gene. I use a molecular delimitation method to determine support for putative species, assess migration between species, and, within a species-tree framework, infer a phylogeny for the genus Lampropeltis , including all newly elevated milksnakes and all other recognized taxa within the genus. This dataset also provides the opportunity to examine how the inclusion of cryptic taxa and species tree methods provide a more accurate assessment of timing, rates and process of species diversification. After delimiting species, I use both non-genealogical and coalescent-based models to infer the historical demographic patterns of milksnakes. As the milksnake species identified here have representatives in both the temperate and tropical Americas, I am able compare demographic responses with respect to changes in effective population size between the Nearctic and Neotropical taxa. Specifically, I ask, do closely related milksnake species have similar demographic patterns resulting from Pleistocene glacial cycles despite their origination in the Neotropics or Nearctic. Results indicate that milksnakes do not share similar demographic patterns as a taxonomic group or by area and I conclude that authors should use caution in generalizing historical demographic trends based on geography. In addition to examining milksnakes using molecular data, I determine if there are detectable morphological differences independent of color pattern using geometric morphometrics. By taking photographs of 487 specimens, digitizing landmarks on their heads, and using geometric morphometric analyses, I determine that these types of analyses are useful in snake systematics for detecting distinct morphological shape variation among genetically delimited species, although they may not be powerful enough to use as an a priori species discovery tool.

  • ENHANCEMENT OF OLIGODENDROCYTE REMYELINATION THROUGH THE ABLATION OF NON-MUSCLE MYOSIN II B

    Author:
    Tomasz Rusielewicz
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Carmen Melendez Vasquez
    Abstract:

    The myelinating cell of the central nervous system, the oligodendrocyte (OL), undergoes a dramatic change in the organization of its cytoskeleton as it differentiates from an oligodendrocyte precursor cell to a myelin forming OL. This change involves an increase in branching, which is required for the OLs ability to myelinate multiple axons. Our laboratory has shown that levels of non-muscle myosin II (NMII), a regulator of cytoskeletal contractility, decrease as a function of differentiation and that inhibition of NMII activity increases branching and myelination of OL in coculture with neurons. It was also found that mixed glial cultures derived from NMIIB knockout mice display an increase in the number of mature myelin basic protein expressing OL compared to wild type cultures. These studies have now been extended to investigate the role of NMIIB ablation in myelin repair following focal demyelination by lysolecithin. To this end, we employed an OL-specific inducible knockout model using a PLP driven promoter in combination with a temporally activated CRE-ER fusion protein. The data indicate that conditional ablation of NMII in adult mouse brain, promotes faster lesion resolution and remyelination when compared to that observed in control brains. Although several pathways have been implicated in oligodendrocyte morphogenesis, their specific contribution to the regulation of NMII activity has not been directly examined. We tested the hypothesis that the activity of NMII in OPC is controlled by Fyn kinase via downregulation of RhoA-ROCK-NMII phosphorylation. The resulting data confirm the function of NMII as a negative regulator of OL maturation and demonstrate that Fyn kinase downregulates NMII activity thus promoting oligodendrocyte morphological differentiation. Furthermore this study provides a novel target for promoting myelin formation and repair in the adult brain.

  • RHYTHM DEVELOPMENT IN THE ZEBRA FINCH SONG

    Author:
    Sigal Saar
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Ofer Tchernichovski
    Abstract:

    When we listen to the zebra finch song, the song sounds very rhythmic and repetitive. Each bird has its own song rhythm. In order to study rhythm development in songbirds we introduced a new concept to bird song research, the quantification of rhythm and periodicity in song structure. By developing a technique that gives us insights into the song rhythm we have studied how song structure changes during different stages of the zebra finch life. We found that birds trained from an early age (day 43) exhibit two types of rhythm transformations: the first type is a smooth down-modulation of rhythm during several days of song development. The second type is an abrupt transition from one rhythm to a different rhythm. Smooth transitions might be caused by a slow shift in the timing of neuronal firing, while abrupt transitions might be caused by reorganization of the rhythm generator. We also examined why is it that zebra finches that are isolated until the end of their sensitive period and then trained do not imitate well. We discovered that their poor imitation correlates with inability to add new syllables to the songs. Zebra finches that sing a long motif before the onset of training were able to imitate better than finches with a short motif, perhaps because they have more raw materials to work with. Our findings suggest that at the end of the sensitive period vocal changes still occur, but imitation is constraint at the level of song rhythm and on the level of fine temporal structure.

  • Lipid dependence in Ras-driven tumors

    Author:
    Darin Salloum
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    David Foster
    Abstract:

    Over past decade, metabolic alterations in cancer cells have received a substantial amount of interest. It had been established that cancer cells undergo a significant amount of metabolic alterations, and some of these alterations are similar to those in normal highly proliferative cells. However, it is becoming more apparent that many of the metabolic alterations are specific to particular oncogenic signaling pathways. Although altered metabolic machinery makes cancer cells more efficient at promoting growth when nutrients are supplied at the sufficient amounts, the dependency of cancer cells on particular metabolic reprogramming deems cancer cells susceptible to disruptions within metabolic network. Thus, the identification of metabolic weaknesses of cancer cells create a platform for therapeutic interventions. The conversion of normal cells to cancer cells involves a shift from catabolic to anabolic metabolism involving increased glucose uptake and the diversion of glycolytic intermediates into nucleotides, amino acids and lipids needed for cell growth. An underappreciated aspect of nutrient uptake is the utilization of serum lipids. We investigated the dependence of human cancer cells on serum lipids and report here that Ras-driven human cancer cells are uniquely dependent on serum lipids for both proliferation and survival. Moreover, Ras-driven cancer cells fail to adapt lipid metabolism upon lipid deprivation. Removal of serum lipids also sensitizes Ras-driven cancer cells to rapamycin. Suppressing pinocytosis in Ras-driven cancer cells similarly created sensitivity to suppression of mTORC1 - the mammalian/mechanistic target of rapamycin. The findings reported here reveal an enhanced need for serum lipids in Ras-driven human cancer cells that creates a synthetic lethal phenotype for suppressing mTORC1. While depriving humans of serum lipids is not practical, suppressing uptake of lipids is possible and could be exploited therapeutically, presenting speculated that this property displayed by Ras-driven cancer cells represents an Achilles' heel for the large number of human cancers that are driven by activating Ras mutations. mTOR has long been known to respond to amino acids, glucose and energy. However, lipids are another essential nutrient, and sensing mechanism on sufficiency of lipid precursors is not yet known. Based on the central position of PA in lipid biosynthesis, and its involvement in mTOR regulation, here we show that PA feeds into mTOR as a metabolite for sensing lipid precursors. In Ras-driven cells, PLD activity increase due to lipid withdrawal may be a mechanism to keep mTOR active during metabolic insufficiency stress.