Alumni Dissertations

 

Alumni Dissertations

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  • Spatial Ecology of Long-tailed Ducks and White-winged Scoters Wintering on Nantucket Shoals, Massachusetts

    Author:
    Timothy White
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Timothy White
    Abstract:

    A substantial proportion, perhaps 30%, of the North American breeding population of Long-tailed Ducks (Clangula hyemalis) winter in the vicinity of Nantucket Island, Massachusetts. These birds spend the night on Nantucket Sound and commute during daylight hours to the Nantucket Shoals, which extend about 65 km offshore from the southeastern corner of Nantucket. Strip transects done from a single-engine plane in 1997 and 1998 indicated that Long-tailed Ducks foraged over the shallower (~ 20 m depth) portions of the Nantucket Shoals, up to 70 km offshore. Diet analyses of ten birds collected in February 1999 and five in December 2006 showed that they fed principally (106.6 +/- 42.0 individuals per crop) on Gammarus annulatus, a pelagic amphipod that often forms large aggregations, and is consumed by several species of fish and marine mammals. I conducted high-intensity aerial surveys to map the distribution and abundance of foraging sea ducks on Nantucket Shoals, MA in February 2008 - Spring 2011. For these surveys, west/east transect orientation ran perpendicular to bathymetric gradients, and covered a greater extent of Nantucket Shoals than did our preliminary surveys in 1997 and 1998. Core foraging areas of Long-tailed Ducks and White-winged Scoters (Melanitta fusca) overlapped along southwest Nantucket Shoals (mean foraging depth (m) ± SD: Long-tailed Ducks = 27.20 ± 8.25; White-winged Scoters = 29.8 ± 7.23), a zone where clams and pelagic amphipods are historically abundant. I mapped the spatial dispersion of Long-tailed Ducks and White-winged Scoters to determine if and where foraging aggregations persisted through space and time in relation to prey hotspots. Spatial dispersion of both species was strikingly concordant. Since previous studies have found very little dietary overlap between Long-tailed Ducks and White-winged Scoters, I sought to explain this high degree of spatial concordance. I also examined the potential physical and biological mechanisms (e.g., structural fronts, primary productivity) that may influence the spatial organization of Long-tailed Ducks, White-winged Scoters and their prey on Nantucket Shoals.

  • Computational analyses of the components of Sinorhizobium meliloti ExoR-ExoS/ChvI pathway: the ExoR and ExoS proteins

    Author:
    Eliza Wiech
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Shaneen Singh
    Abstract:

    Abstract COMPUTATIONAL ANALYSES OF THE COMPONENTS OF Sinorhizobium meliloti ExoR-ExoS/ChvI PATHWAY: THE ExoR AND ExoS PROTEINS by Eliza M. Wiech Adviser: Professor Shaneen M. Singh The Sinorhizobium meliloti periplasmic ExoR protein and the ExoS/ChvI two-component system form a regulatory mechanism that directly controls the transformation of free-living to host-invading cells. In the absence of crystal structures, understanding the molecular mechanism of interaction between ExoR and the ExoS sensor, which is thought to drive the key regulatory step in the invasion process, remains a major challenge. In this study, we present theoretical structural models of the active form of ExoR protein, ExoRm, as well as of the sensing domain of ExoS, ExoSp, generated using computational methods. Our model suggests that ExoRm possesses a super-helical fold comprising twelve α-helices forming six Sel1-like repeats, including two that were unidentified in previous studies. The structural model of ExoSp suggests that ExoSp is a single Per-ARNT-Sim (PAS) domain. Docking analysis was used to suggest models for ExoSp-ExoSp and ExoSp-ExoRm protein interactions and interfaces. Our studies reveal three novel insights: (a) a possible extended conformation of the ExoR third Sel1-like repeat that might be important for ExoR regulatory function (b) a buried proteolytic site that implies a unique mechanism of proteolysis, central to controlling ExoR function and (c) an elongated structure of helix H4 that is unique to ExoSp and might be crucial for the association with ExoRm. This study provides new and interesting insights into the structure of the S. meliloti ExoRm and ExoSp proteins, lays the groundwork for elaborating the molecular mechanism of ExoRm cleavage, ExoRm-ExoS interactions, and studies of ExoR homologs in other bacterial host interactions.

  • Transcriptional Regulation of Yeast CUP1 gene

    Author:
    Roshini Wimalarathna
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Chang-Hui Shen
    Abstract:

    The yeast CUP1 gene encodes a metallothionein required for cell growth at high copper concentrations. The induction of CUP1 with copper results in activator-dependent nucleosome repositioning. To further understand the mechanism of CUP1 activation, it is necessary to identify which chromatin remodeler(s) is/are involved in CUP1 induction. Here, we demonstrated that both ino80∆ and snf2∆ cells grew well in the absence of copper, but were inviable in the presence of copper, indicating that they are required for CUP1 expression. Furthermore, CUP1 mRNA did not significantly increase in mutants lacking either remodeler in the presence of copper, suggesting that they regulate CUP1 induction at the transcriptional level. Both isw1Δ and rsc3Δ cells displayed similar growth patterns as WT cells in the presence of copper. Further CUP1 mRNA was significantly increasing in both isw1Δ and rsc3Δ cells showing similar pattern as WT cells. This observation suggests that ISW1 and RSC3 remodeling complexes have no positive effect in CUP1 expression. We also demonstrated, using chromatin immunoprecipitation, that both INO80 and SWI/SNF are present at the promoter in the wild type cells and they were dependent on each other to be recruited to the CUP1 promoter. Both chromatin remodeling activity and targeted histone acetylation were not observed in ino80∆ or snf2∆ strains at the CUP1 promoter. These results suggest that both INO80 and SWI/SNF directly participate in CUP1 chromatin remodeling, and that histone acetylation is recruited after the arrival of chromatin remodelers. We also observed that more polII was recruited to the CUP1 promoter under inducing conditions and that such a recruitment was not observed in ino80∆ or snf2∆ strains. Furthermore, we observed that both Snf2p and Ino80p were activator-dependent. Our observations provide direct evidence for the involvement of both INO80 and SWI/SNF remodelers in CUP1 activation. In light of these findings, we propose a working model for CUP1 activation.

  • The Neurogenetic Analysis of Synaptogenesis and Synaptic Plasticity in Drosophila melanogaster

    Author:
    Alexandria Wise
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Tadmiri Venkatesh
    Abstract:

    Synaptogenesis is the process by which nascent axons from developing neurons target and form mature synapses with other neurons or cells. Specifically, synaptogenesis at the neuromuscular junction (NMJ) involves the motorneuron axon targeting its specific innervating muscle. The NMJ of the Drosophila larva is an excellent model system to study synaptogenesis due to genetic tools developed in the Drosophila melanogaster. The NMJ in Drosophila are glutamatergic, resembling mammalian central nervous system (CNS) excitatory synapse, and can provide insight into the molecular mechanisms that control synapse development and transmission. In addition, studying the physiology of the synapse can also provide for greater understanding of mechanisms that underlie synapse maintenance and plasticity. In this dissertation, I have focused on the role of two functionally dynamic pathways, ubiquitination and cAMP-PKA (protein kinase A or A kinase) during development, and their role in NMJ synaptic structure and function. rugose (rg), which encodes for the Drosophila A kinase anchoring protein 550 (DAKAP550), belongs to a family of cAMP-PKA organizing centers that provide the scaffolding to permit PKA to bind to specific subcellular organelles, allowing for cAMP to activate PKA more effectively. There were changes observed in hypomorphic rg mutants in synaptic transmission and plasticity as well as in basic forms of learning, specifically habituation, which involves the neuron, in this case, to cease responding to presented stimuli. Taken together, rg is necessary for the development of the synapse and synaptic transmission. Our lab has previously shown that Retina aberrant in pattern/Fizzy related (Rap/Fzr), the activating subunit of the, E3 ubiquitin ligase, Anaphase Promoting Complex (APC/C) regulates cell fate determination in the developing Drosophila brain. Our cell biological, ultrastructural, electrophysiological, and behavioral data show that rap/fzr loss-of-function mutations lead to changes in synaptic structure and function, as well as locomotion defects. Specifically these changes were observed pre- and postsynaptically, represented by size and morphology of synaptic boutons, and number of neurotransmitter vesicles. Electrophysiologically, these were correlated with decreased transmission failure rates as well as increase in the size of synaptic potentials. In addition, larval locomotion and peristaltic movement are also impaired as. These findings suggest a novel role for Drosophila-Cdh1-mediated ubiquitination during development of functional synapses in the peripheral nervous system. The use of genetic modifier screens in our laboratory has identified several neuronal substrates that physically interact with APC/CCdh1/Rap/Fzr in Drosophila. As a part of my thesis research, I focused on three proteins: Locomotion Defects (Loco), Nonstop (Not) and Twins. Their involvement in signaling pathways makes these proteins candidates together with Rap/Fzr/Cdh1 to regulate synaptogenesis at the pre- and postsynapse of the Drosophila NMJ. This data suggest Not, Loco and Twins are localized at the NMJ, and involved in regulating synaptic development by genetically interacting with APC/CCdh1/Rap/Fzr. Both of these pathways play an important role in neuronal development, transmission, learning and memory, and dysfunction. However, their role in modifying synaptic activity remains unclear. Using Drosophila melanogaster as a model, this thesis will elucidate the role of the APC/C and Rugose in synaptic development and plasticity at the neuromuscular junction.

  • GENOME-WIDE ANALYSIS OF PH AND FYVE DOMAINS IN ARABIDOPSIS THALIANA BY COMPUTATIONAL METHODS

    Author:
    Ewa Wywial
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Shaneen Singh
    Abstract:

    Phosphoinositide-binding domains have emerged as molecules responsible for trafficking and anchoring of membrane associated proteins in various organisms ranging from yeast to mammalian cells. Two such domains, i.e. the Pleckstrin Homology (PH) and Fab1p, YOTB, Vac1 and EEA1 (FYVE), are of special interest because of their unique membrane binding characteristics and genome wide prevalence. Structurally, the PH domains share a core fold of seven β-strands followed by a C-terminal α-helix whereas the FYVE domains comprise two small double-stranded β-sheets, a C-terminal α-helix and eight Zn2+ ion-binding cysteines. The PH and FYVE domains are found in functionally diverse families in different organisms; some of these are well studied and others remain unexplored. Plants stand out from the rest of the organisms where most of protein families containing these domains remain largely uncharacterized. We have carried out an extensive search of the Arabidopsis genome using an automated pipeline and manual methods to verify previously known and identify unknown instances of PH and FYVE proteins, characterize their sequence and model/analyze their 3D structure. Integrating the sequence, structure and known experimental details for each of these domains, we propose a comprehensive genome-wide domain-based classification of both the Arabidopsis PH and FYVE proteins: we classify forty-nine Arabidopsis PH and fifteen Arabidopsis FYVE proteins into twelve and five classes, respectively. Our study provides valuable details on the biophysical traits of the individual modeled domains, family specific characteristics as well as genomic trends for these domains in Arabidopsis and predictions of their membrane binding behaviors. We report the exclusive presence of plant specific domain architectures, variant binding signatures and biophysical profiles in individual members or complete families of Arabidopsis PH and FYVE domain-containing proteins such as the PRAF proteins. Our study provides the first glimpse into the putative roles of these lipid-binding domains based on which we can begin to understand the role(s) they play in plants and how it compares to their role in other organisms.

  • REGULATION OF NUTRIENT SIGNALING TO MAMMALIAN TARGET OF RAPAMYCIN BY PHOSPHOLIPASE D

    Author:
    Limei Xu
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    David Foster
    Abstract:

    The mammalian target of rapamycin (mTOR) is a key component of a complicated signaling network which transduces nutrient signals and many other stimuli to modulate a wide range of cellular functions, such as cell growth, cell proliferation and cell survival. Phospholipase D (PLD) is an enzyme which catalyzes the hydrolysis of phosphatidylcholine (PC) to form phosphatidic acid (PA). PA has been shown to be a very important lipid second messenger that mediates mitogenic signals upstream of mTOR and both PLD and mTOR have been implicated as cancer cell survival signals. Therefore it is of interest as to whether PLD plays a role in mTOR mediated nutrient signaling. We have found that elevated PLD activity in human cancer cells is dependent on the availability of both amino acids and glucose and that PLD is required for amino acid- and glucose-induced mTOR Complex 1 (mTORC1) activity. Moreover we investigated the possible regulators which are involved in mediating the nutrients signals including amino acids and glucose to PLD and mTORC1. We found that small GTPases RalA and ARF6 which form a complex with PLD to activate its activity are required for both PLD and mTORC1 activation induced by amino acids and glucose. The class III phosphatidylinositol-3-kinase hVps34 emerged as an important modulator for amino acid sensing. In this study, we showed that the depletion of hVps34 or binding partner hVps15 with siRNA dramatically suppressed the PLD activity and further disrupted nutrient sensing to mTORC1, possibly by failing to recruit PLD to endomembrane and stimulate mTORC1 activity in response to nutrients. Taken together, these findings demonstrate that phosphatidic acid generated by PLD is a critical mediator that links nutrient signals to mTORC1, thus implicating the important role of PLD and PA in cancer cell proliferation and survival.

  • THE TRANSCELLULAR PATHWAY IS A SIGNIFICANT CONTRIBUTOR TO WATER FLOW THROUGH VASCULAR ENDOTHELIA

    Author:
    Yan Xue
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Karen Hubbard
    Abstract:

    Aquaporin-1 (AQP1) is a ubiquitous water channel protein that facilitates transmembrane water flow. The aim of our study is to determine the contribution of the AQP1 transcellular pathway to water filtration through aortic endothelial cells (AECs). In this thesis, we use both in vitro and ex vivo models of rat AECs to characterize the transport properties of such cells to water and/or small solutes. In vitro water flux (Jv) studies show that 67.9% knockdown of AQP1 in rat AECs by small interfering RNA (siRNA) significantly reduces Jv by 56.4±8.2% (n=7). A study of the permeability to tetramethylrhodamine (TAMRA) and albumin has provided an interesting insight into what portion of this Jv reduction is due directly to AQP1 suppression, since these solutes traverse only inter-endothelial junctions. As a result, AQP1 suppression leads to a 21.8±7.04% drop in TAMRA convective permeability and a 29.79.1±1.72% /25.69±8.19% drop in albumin permeability under convection/diffusion conditions between control and treated monolayers. We have constructed an ex vivo culture system that allows for the perfusion of the vessel lumen and application of a hydrostatic pressure that yields a physiological shear stress and transmural pressure. We note that maintenance of both pressure and shear flow preserves isolated AEC integrity, as assessed by hydraulic conductivity (Lp) and morphology studies. This culture model enables us to employ molecular manipulation of AECs in intact rat aortas. We showed that downregulation of AQP1 via siRNA results in a pressure-dependent decrease in total Lp (Lp of endothelium + subendothelial intima (SI)), Lpt (Lpe+i), by 37.35±12.97% (n=5) (59.8±11.6 % (n=3)) and 8.71±3.76% (n=5) (15.5±9.1% (n=3)) at 60 and 100 mmHg respectively. Our study indicates that AQP1 contributes significantly to transendothelial Lp. Transendothelial water flow is critical in determining whether low-density-lipoprotein (LDL) molecules can spend enough time at high enough local concentration in the SI to bind to SI extracellular matrix, a critical step in early atherosclerotic lesion formation. Our data suggest that regulation of AQP1, an important contributor to water filtration, may affect lipid transport in a beneficial way with regards to early disease progression.

  • ROLE OF VOLTAGE SENSITIVE CALCIUM CHANNELS (VSCCs) IN THE MATURATION OF THE GABAERGIC SYSTEM IN THE FRAGILE X SYNDROME

    Author:
    Xin Yan
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Abdeslem El-Idrissi
    Abstract:

    GABAA receptors are considered to be major inhibitory receptors in the brain. A significant down-regulation of the GABAA receptors in Fmr1 knockout (KO) mice has been demonstrated in recent studies, which may underlie the mechanism for the anxiety and hyperactivity found in fragile X syndrome patients. However, during early developmental stages, activation of GABAA receptors exerts excitatory effects on neuronal networks. The excitatory effect of GABAA receptors early on sets the major tune in generating giant depolarizing potentials (GDPs), recurrent synchronized spontaneous network discharges which are features of neuronal activity of developing neurons. GDPs control extension and motility of neurites as well as synthesis and expression of the GABAergic phenotype through the activation of voltage sensitive calcium channels (VSCCs) during early developmental stages up to P14 in mice and rats. The developmental excitatory to inhibitory switch in the GABAA receptors function is mainly dependent upon the expression level of the cotransporters KCC2. In this study, we examined the expression level of GABAA receptors and VSCCs in the brain of FVB/NJ wild type (WT) and Fmr1 knockout (KO) mice, and attempted to determine whether modulation of VSCC could affect the maturation of GABAergic system in the KO mice. First, we found reduced expression of GABAA receptors and VSCCs in KOs, and a shift in the time course of the excitatory to inhibitory functional switch of GABAA receptors in KOs. In addition, we modulated VSCCs activity in cerebellar granule cell culture with KCl and nifedipine to verify whether VSCCs can normalize reduced expression of GABAA receptors in KO mice. Large-scale nuclear translocation and exportation of the VSCC â3 subunit were detected, suggesting VSCC could play a regulatory role in gene expression. Finally, we pharmacologically manipulated VSCCs during embryogenesis by injecting pregnant mice with Bay K 8644 (a VSCC activator) and performed behavioral analyses on the offspring. Injection of VSCC activator rescued part of the KO behavior phenotype. In conclusion, the VSCCs in KO mice brain may be responsible for the activity-dependent, calcium-mediated gene expression resulting in the altered expression of GABAA receptors. Activating VSCCs during early development could partially normalize KO behavior.

  • On the Mechanism of Neurotoxicity from Methamphetamine: The Role of Neuropeptide Y

    Author:
    Haley Yarosh
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Jesus Angulo
    Abstract:

    The purpose of this study is to describe the potential neuroprotective effect of neuropeptide Y in response to striatal nitric oxide overproduction after methamphetamine (METH) use. Our lab has established a time course and optimal dosage for modeling acute METH neurotoxicity in the murine brain. A 30 mg/kg systemic injection of METH depletes intracellular dopamine and serotonin levels, and induces dopamine receptor endocytosis as well cell loss of approximately 30% of striatal neurons. Dopamine overflow from presynaptic terminals paired with glutamate signaling from the cerebral cortex commences a neuronal cascade, which leads to the overproduction of nitric oxide, inflammatory cytokines and reactive oxidative/nitrogen byproducts. We observe that when neuropeptide Y (Y1R, Y2R) agonists are administered prior to METH, the appearance of apoptotic cells and neurodegeneration markers are attenuated. Additionally, there is an endogenous upregulation of striatal neuropeptide Y mRNA stores during the early hours after METH administration. The following study establishes an optimal dose and time course for neuropeptide Y induction, and suggests that neuropeptides are at play to establish homeostasis after acute METH toxicity. We characterize the cellular response to METH-induced nitric oxide production through fluorescent co-label, and demonstrate that the effect of neuropeptide Y on these cell types is modulated by specific neuropeptide Y receptors. The neuroprotective effect of NPY persists even in the presence of substance P agonists, associated with exacerbation of methamphetamine-induced neurotoxicity.

  • Cytotoxic and Cytostatic Properties of Rapamycin: Implications for Antitumor Efficacy

    Author:
    Paige Yellen
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    David Foster
    Abstract:

    mTOR (mammalian target of rapamycin) is a central regulator of cell growth and proliferation. Frequently dysregulated in cancer cells, it is an attractive therapeutic target, named for its first inhibitor, rapamycin. Low (nanomolar) doses of rapamycin treatment inhibit phosphorylation of mTORC1 (mTOR complex 1) substrate, S6 kinase, thwarting protein synthesis and subsequently, proliferation. Though highly potent and specific, rapamcyin has lacked clinical success because it lacks universal anti-tumor effects and only a fraction of patients respond. We have revealed rapamycin as a site-specific, cytotoxic anticancer drug in the absence of serum. Our studies demonstrate that high-dose rapamcyin induces apoptosis because it effectively inhibits all phosphorylation sites on 4E-BP1, subsequently inactivating eIF4E. Importantly, we demonstrate that cancer cells are resistant to high-dose rapamycin upon 4E-BP1 knockdown, confirming that the drug retains its site-specific property regardless of dose. Furthermore, we show that high-dose rapamycin irreversibly compromises the integrity of the mTORC1 complex, as it pertains to mTOR-raptor association. We acknowledge the inefficacy of rapamycin in cancer cells that activate Akt upon mTORC1 inhibition. Under these circumstances, an inhibitor of both mTORC1 and mTORC2 complexes is necessary and sufficient to induce apoptosis Our current study is to further investigate the surprising observation that while high-dose rapamycin treatment (indirect inactivation of eIF4E) induces apoptosis only in the absence of serum, knockdown of eIF4E (direct inactivation of eIF4E) induces apoptosis in both the presence and absence of serum. Thus, there is a mechanistic difference between the indirect and direct inactivation of eIF4E. Since high-dose rapamycin treatment also inhibits phosphorylation of S6K and does not induce apoptosis in the presence of serum, we reasoned that protection from apoptosis induced by high-dose rapamycin could be due to suppression of S6K. Consistent with this hypothesis, dual S6K and eIF4E knockdown prevents cell death otherwise induced by knockdown of eIF4E alone. We hypothesize that simultaneous inhibition of both S6K and eIF4E results in coordinate induction of transforming growth factor-β (TGF-β) signaling that is sufficient to induce arrest. This is predicated on prior work in the lab that shows high-dose rapamycin causes G1 arrest rather than apoptosis, if and only if, TGF-β signaling is intact. TGF-β mediates a cytostatic response by activation of Smad signaling and cyclin-dependent kinase inhibitor (CDK) p27. Altogether, these data reveal the complexity of high-dose rapamycin, with implications for both cytotoxic and cytostatic effectiveness in the absence and presence of serum, respectively.