Transcriptional Regulation of Yeast CUP1 gene
Author:
Roshini Wimalarathna
Year of Dissertation:
2012
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
Year of Dissertation:
2012
Advisor:
Tadmiri Venkatesh
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.
GENOME-WIDE ANALYSIS OF PH AND FYVE DOMAINS IN ARABIDOPSIS THALIANA BY COMPUTATIONAL METHODS
Year of Dissertation:
2009
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
Year of Dissertation:
2012
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
Year of Dissertation:
2011
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.
On the Mechanism of Neurotoxicity from Methamphetamine: The Role of Neuropeptide Y
Year of Dissertation:
2012
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.
Cytotoxic and Cytostatic Properties of Rapamycin: Implications for Antitumor Efficacy
Year of Dissertation:
2012
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.
Modeling tree growth and seedling recruitment in a selectively logged temperate forest
Year of Dissertation:
2012
MODELING TREE GROWTH AND SEEDLING RECRUITMENT IN A SELECTIVELY LOGGED TEMPERATE FOREST
Role of DARPP-32 pathway in cocaine-induced behavioral sex differences
Year of Dissertation:
2009
Advisor:
Vanya Quinones-Jenab
Accumulating evidence suggests that behavioral responses to cocaine are sexually dimorphic: female rats are more sensitive to cocaine. Female rats exhibit exaggerated and more robust locomotor responses to cocaine than do males. Females also more quickly develop cocaine-induced conditioned place preference (CPP) and behavior sensitization with lower doses, and more readily acquire cocaine self-administration. However, the underling mechanisms for this behavioral sex differences remain unknown. The dopamine- and cAMP-regulated phosphoprotein (DARPP-32) signaling pathway has been shown to mediate intracellular responses to acute and chronic cocaine in male rodents. The purpose of this proposal is to assess the role of DARPP-32 pathways in cocaine-induced behavioral sex differences in male and female rats. We hypothesize that acute and chronic cocaine activates DARPP-32 signaling in a sexual dimorphism way. To test this hypothesis, in the acute cocaine experiment, rats received saline or cocaine (30mg/kg), while in the chronic cocaine experiment, rats received saline, acute cocaine or chronic cocaine (15mg/kg), Protein levels of the DARPP-32 signaling proteins in the nucleus accumbens (NAc) and caudate putamen (CPu) were measured via western blot. Locomotor and stereotyped activities were also measured in the chronic cocaine experiment. In the acute cocaine experiment, female rats had heightened basal levels of DARPP-32 cascade; while male rats exhibited higher induction of the cascade after acute cocaine administration. However, these sex differences were mainly observed in the NAc, not CPu. In the chronic cocaine experiment, female rats developed behavior sensitization and tolerance to cocaine earlier than males. In male rats, the heightened DARPP-32 signaling were mostly found in the NAc when behavior sensitization was observed; while in female rats, increased signaling were mostly found in the CPu when behavior tolerance was observed. In addition, the protein levels of several DARPP-32 signaling proteins, including ΔFosB, FosB, Cdk5 and p35, correlated with the behavior activities. Taken together, these results suggest that DARPP-32 signaling pathway is altered in a sexual dimorphic way after acute and chronic cocaine treatment, and it may play a critical role for the sex differences at the behavior level.
MNK1 Modulation of hnRNP A1 in Cellular Senescence
Year of Dissertation:
2011
Heterogeneous nuclear ribonucleoprotein (hnRNP) family members are the most abundant components of messenger ribonucleoprotein complexes (mRNPs) and play regulatory roles in a variety of biogenesis of mRNA. hnRNP A1 is a member of the hnRNP A/B subfamily, is highly abundant, and is involved in pre-mRNA and mRNA metabolism such as alternative splicing, mRNA export, splice site selection, mRNA turnover, and translation. Recent studies have shown that stress stimuli such as osmotic shock or UVC irradiation induce cytoplasmic accumulation of hnRNP A1. The cytoplasmic accumulation is concomitant with an increase in its phosphorylation and that requires p38 MAPK. We have previously demonstrated that hnRNP A1 protein shows diminished expression level and altered subcellular distribution in senescent HS74 fibroblasts. In this study, we observed that phosphorylated hnRNP A1 protein levels decreased as a result of MNK1 inhibition and that reciprocal binding occurs between hnRNP A1 and MNK1. These data implicate MNK1 as the kinase in the p38 MAPK pathway that activates hnRNP A1 in IMR-90 fibroblasts. Furthermore, we demonstrate that inhibition of MNK1 activity modulates the phosphorylation and subcellular distribution of hnRNP A1 protein. These results suggest a role for MNK1 in the regulation of hnRNP A1 in senescent cells. This is the first report, to our knowledge, that shows a link between MNK1 and cellular senescence.