Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • Turnip Mosaic Virus Genome-Linked Protein (VPg) Inhibits Pokeweed Antiviral Protein (PAP)-Mediated Depurination of RNA

    Author:
    Artem Domashevskiy
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Dixie Goss
    Abstract:

    Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is a cap-binding protein, and it was suggested that it inhibits translation of RNA by binding to the 5' m7G cap structure of eukaryotic mRNA, and depurinating the mRNA at sites downstream of the cap structure. PAP is a potent antiviral agent against many plant, animal, and human viruses. Depurination of capped viral RNA may be the primary mechanism for PAP's antiviral activity. However, the above mechanism does not clarify the inhibitory effect of PAP on the replication of uncapped viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus (TuMV) genome linked protein (VPg) were investigated. VPg is important in the initiation of protein synthesis, functioning as a cap analog. VPg stimulates the in vitro translation of uncapped IRES-containing RNA and inhibits capped RNA translation in wheat germ extracts. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP-VPg interactions. PAP interacts strongly with VPg, thus the effect of VPg on the PAP catalyzed depurination of several different RNA molecules was determined to investigate whether VPg binding to PAP influences selectivity of depurination. PAP binds to and depurinates both m7GpppG-capped and uncapped S/R oligo nucleotide and TEV RNAs, supporting previous conclusions that the cap structure is not the only determinant for PAP depurination of RNA. VPg decreases depurination of the above capped and uncapped RNAs and competes with TEV RNA for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the defense mechanisms of the plant. Depurination inhibition of PAP by VPg also suggests the possible use of this protein against cytotoxic activity of RIPs and inhibition of their biological potency.

  • Role of the Polyadenylation Factor CstF-50 in regulating the BRCA1/BARD1 E3 Ubiquitin (Ub) Ligase Activity

    Author:
    Danae Fonseca
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    Frida Kleiman
    Abstract:

    The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. The studies presented in this dissertation focus on the determination and characterization of the role of mRNA processing factor CstF-50 and escort protein p97 in the regulation of the BRCA1/BARD1 E3 ubiquitin (Ub) ligase activity during the DNA damage response (DDR). As part of the studies presented in Chapter II, I determined that the polyadenylation factor CstF plays a direct role in DDR, specifically in transcription-coupled repair (TCR), and that it localizes with RNA polymerase II (RNAP II) and BARD1 to sites of repaired DNA. My results also indicated that CstF plays a role in the UV-induced ubiquitination and degradation of RNAP II. In Chapter III, I determined that the carboxy-terminal domain of p53 associates with factors that are required for the ultraviolet (UV)-induced inhibition of the mRNA 3' cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the polyadenylation factor CstF-50. These results were part of a study that identified a novel 3' RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network. In addition, in Chapter IV I showed that CstF-50 can interact not only with BRCA1/BARD1 E3 Ub ligase but also with ubiquitin (Ub), the escort-factor p97 and some of BRCA1/BARD1 substrates, such as RNAP II, H2A and H2B. I also demonstrate that CstF-50-associated p97 activates the BRCA1/BARD1-dependent RNAP II poly-ubiquitination, H2A and H2B monoubiquitination as well as BRCA1/BARD1 autoubiquitination. Together my results provide evidence that CstF-50-associated p97 regulates BRCA1/BARD1 Ub ligase activity during DDR, helping in the assembly and/or stabilization of the ubiquitination complex. Extending these studies, in Chapter V, I showed that UV-treatment induces changes in the localization of BRCA1, BARD1, CstF-50, p97 and some of BRCA1/BARD1 substrates in different nuclear fractions, and that these changes depend on BRCA1/BARD1 and CstF-50 expression. Further, my results demonstrate that the content of monoubiquitinated H2B in the chromatin of genes with different levels of expression changes during DDR and this is mediated by BRCA1/BARD1 and CstF-50. The data presented in this chapter show new insights into the role of mRNA 3' processing factor CstF-50 in regulating the Ub pathway, resulting in epigenetic control during DDR. Finally, in Chapter VI, I identified the RNA binding protein HuR as a new substrate for BRCA1/BARD1/CstF-50/p97 Ub ligase activity in different cellular conditions. All together, the studies presented in this dissertation revealed unexpected insights into the role of the RNA processing factor CstF-50, tumor suppressors BRCA1/BARD1 and p53, the Ub pathway and chromatin structure during DDR.

  • p53 and Beclin 1 in Caenorhabditis elegans are involved in cell death and DNA damage repair

    Author:
    Sandy Gamss
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Jill Bargonetti
    Abstract:

    p53 is a key regulator of growth arrest, DNA repair, apoptosis, and autophagy in response to cellular stress in animals as diverse as Caenorhabditis elegans and humans. In mammals, p53 functions as a key tumor suppressor protein that promotes removal of potentially tumorigenic cells. p53 is mutated or deleted in over 50% of all mammalian tumors and these tumors are highly resistant to available therapies. Therefore, identifying therapeutic agents and cell death pathways that promote p53-independent cell death is vital to the future of cancer therapy. We are interested in identifying how the alkylating agent, 10-decarbomyl mitomycin C (DMC) promotes p53-indpendent cell death. To answer this question, we used a C. elegans as a model system to identify known and novel cell death genes involved in DMC-induced death. C. elegans have an orthologue of p53, cep-1, that functions in germline cell death in a similar way to p53 in apoptosis. The germline in C. elegans has both mitotic and meiotic cells and displays CEP-1/p53-dependent cell death in response to UV. We examined both germline cell death and transcript levels of CEP-1 target genes in C. elegans. We found that DMC increased CED-1::GFP positive cells and DNA lesions in the absence of CEP-1 while UV required CEP-1 for germline cell death but not nuclear lesion formation. More lesions were seen over time in cep-1(gk138) mutant worms leading to the idea that CEP-1 is involved in DNA repair. Additionally, we examined the role of autophagy in cell death and DNA damage and saw that initially the knockdown of bec-1 required CEP-1 for increases in germ cell death. However in the F1 generation of bec-1 RNAi knockdown animals, the observed increase in cell death was due to a lack of clearance. Furthermore, bec-1 knockdown resulted in the increase of DNA lesions in worms with UV damage.

  • PROTEASOME FUNCTION DURING AGING IN DROSOPHILA

    Author:
    Jie Gao
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Thomas Glenewinkel
    Abstract:

    Aging is associated with dysfunction of protein homeostasis and increased protein damage caused by oxidative stress, but the detailed molecular mechanisms are unknown. The ubiquitin-proteasome pathway (UPP) is critical to the protein quality control system as it degrades a majority of intracellular proteins in eukaryotic cells. Age-related decline in proteasome function has been shown in a variety of mammalian tissues. However the role of UPP during the aging process still remains a puzzle. In this study, we compared the UPP function between two Drosophila melanogaster strains, the longevity mutant methuselah (mth) and the wild type w1118 at different ages, which would help answer this question. The first part of the thesis evaluated the UPP function in mth and w1118 during the natual aging process and under oxidative stress condition. Mth encodes a GTP binding protein-coupled receptor (GPCR). The mth fly with a single Mth gene mutant has been shown to increase ~35% lifespan, but the cellular role of Mth remained elusive. We are the first to report that mth displays lower proteasome activity, lower proteasome levels and lower ATP steady state levels at young ages, but relatively higher levels at old ages during normal aging, when compared with control strain w1118. Under oxidative stress conditions, proteasome activity remained nearly unchanged in w1118 after 5 days of treatment with 1% H2O2, but it was elevated in mth. Moreover, while both strains exhibited a gradual increase in ubiquitinated protein conjugates and aggregates during normal aging process, mth produced fewer conjugates and aggregates at the comparative ages than w1118. Under oxidative stress conditions, the levels of ubiquitinated conjugates and aggregates were elevated in both strains, but less were observed in mth. Together, these data suggest that mth exhibits higher proteasome plasticity and maintains a more efficient protein homeostasis, contributing to longer lifespan. We propose that maintaining a steady state protein turnover rate by the ubiquitin/proteasome pathway will delay the aging process. In the second part of the thesis, we modulated different lifespans using non-genetic approach- Dietary Restriction (DR), which is the only intervention known so far to reliably increase lifespan in a variety of organisms. Currently, it is unclear that what effects aging and DR have on proteasome-mediated protein degradation. We compared the UPP function in three Drosophila strains, wild type Oregon R (OR), w1118 and longevity mutant mth under two diets - DR and ad libitum (AL). All three fly strains responded to DR with extended lifespan. In OR strain, DR significantly reduced proteasome activity in male and female flies at young ages (old ages not tested) and ATP level at all age groups in male flies. These data indicate higher proteasome activity and ATP level do not always lead to longer lifespan. In addition, DR significantly reduced age-related decline in proteasome activity, and ameliorated age-related increases in ubiquitinated protein in the wild type w1118. However, in mth, DR reduced proteasome activity and displayed a slightly higher level of ubiquitinated protein conjugates under both young and old ages. Our data indicate that DR has many beneficial effects towards the function of the UPP in wild type w1118, and that a preservation of the UPP may be a potential mechanism by which DR extend lifespan in w1118. But in longevity mutant mth, DR can not further ameliorate proteasome function, which suggests that other mechanisms might be responsible for the longer lifespan caused by DR. Cumulatively, these data have implications for understanding the effects of aging and DR on protein turnover and the mechanism of lifespan extension in longevity mutant mth.

  • Dictyostelium discoideum RnoA Interprets cAMP Mediated Signals to Influence Actin Organization

    Author:
    Rebecca Garcia
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Derrick Brazill
    Abstract:

    Tight control of actin cytoskeletal dynamics is essential for proper cell function. ARNO, a guanine nucleotide exchange factor for Arf, is associated with actin cytoskeletal regulation but its exact role is unknown. To explore ARNO's role in this regulation and in actin mediated processes, the Dictyostelium discoideum homolog RnoA was examined. RnoA is involved in development, as the reduction of RnoA by antisense prolongs aggregation, delaying development. Also, RnoA overproduction arrests development at the mound stage. This arrest is rescued by the addition of wild type cells. In these chimeric mixtures, rnoA overexpressing cells (RnoA OE) preferentially sort to the stalk, suggesting RnoA plays a role in cell sorting. RnoA antisense and RnoA OE cells fail to stream during aggregation. Chemotaxis assays reveal these mutants do not chemotax toward cAMP, indicating RnoA is part of the cellular response to cAMP. This defect is specific to cAMP-directed chemotaxis, as both RnoA mutants effectively chemotax to folate and exhibit normal cell motility. The chemotactic defects of the RnoA mutants may be due to an impaired cAMP response as evidenced by altered cell polarity and F-actin polymerization after cAMP stimulation. RnoA OE cells have increased filopodia compared to wild type cells, implying altered F-actin localization. Thus, RnoA likely organizes F-actin during development. Phospholipase D (PLD), the enzyme responsible for phosphatidic acid production, and paxillin, a cytoskeletal adaptor protein, are also involved in actin cytoskeletal organization. Given their communal association with the actin cytoskeleton, we explored the interactions between RnoA and the D. discoideum PLD and paxillin homologs, PldB and PaxB, respectively. PaxB and PldB regulate calcium dependent and calcium independent cell-cell cohesion, respectively. Cells lacking PaxB are known to have reduced cell-substrate adhesion. We find that overexpression of PldB rescues the adhesion defect of paxB null cells, implying a genetic interaction between paxB and pldB. Co-immunoprecipitation studies suggest RnoA, PldB, and PaxB physically interact. This interaction does not depend on PaxB, PldB, or F-actin organization. Taken together, the results suggest that RnoA, in complex with PldB and PaxB, coordinates F-actin organization during actin mediated processes such as adhesion.

  • Dictyostelium discoideum RnoA Interprets cAMP Mediated Signals to Influence Actin Organization

    Author:
    Rebecca Garcia
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Derrick Brazill
    Abstract:

    Tight control of actin cytoskeletal dynamics is essential for proper cell function. ARNO, a guanine nucleotide exchange factor for Arf, is associated with actin cytoskeletal regulation but its exact role is unknown. To explore ARNO's role in this regulation and in actin mediated processes, the Dictyostelium discoideum homolog RnoA was examined. RnoA is involved in development, as the reduction of RnoA by antisense prolongs aggregation, delaying development. Also, RnoA overproduction arrests development at the mound stage. This arrest is rescued by the addition of wild type cells. In these chimeric mixtures, rnoA overexpressing cells (RnoA OE) preferentially sort to the stalk, suggesting RnoA plays a role in cell sorting. RnoA antisense and RnoA OE cells fail to stream during aggregation. Chemotaxis assays reveal these mutants do not chemotax toward cAMP, indicating RnoA is part of the cellular response to cAMP. This defect is specific to cAMP-directed chemotaxis, as both RnoA mutants effectively chemotax to folate and exhibit normal cell motility. The chemotactic defects of the RnoA mutants may be due to an impaired cAMP response as evidenced by altered cell polarity and F-actin polymerization after cAMP stimulation. RnoA OE cells have increased filopodia compared to wild type cells, implying altered F-actin localization. Thus, RnoA likely organizes F-actin during development. Phospholipase D (PLD), the enzyme responsible for phosphatidic acid production, and paxillin, a cytoskeletal adaptor protein, are also involved in actin cytoskeletal organization. Given their communal association with the actin cytoskeleton, we explored the interactions between RnoA and the D. discoideum PLD and paxillin homologs, PldB and PaxB, respectively. PaxB and PldB regulate calcium dependent and calcium independent cell-cell cohesion, respectively. Cells lacking PaxB are known to have reduced cell-substrate adhesion. We find that overexpression of PldB rescues the adhesion defect of paxB null cells, implying a genetic interaction between paxB and pldB. Co-immunoprecipitation studies suggest RnoA, PldB, and PaxB physically interact. This interaction does not depend on PaxB, PldB, or F-actin organization. Taken together, the results suggest that RnoA, in complex with PldB and PaxB, coordinates F-actin organization during actin mediated processes such as adhesion.

  • New Families of Beta-Lactamase Inhibitors

    Author:
    Janet Gonzalez
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Manfred Philipp
    Abstract:

    A traditional approach to improving the efficacy of â-lactam antibiotics has been to modify natural antibiotics by modifying the nucleus and adding various side chains. It is generally believed that â-lactam based antibiotics have a limited future given increased resistance demonstrated by many strains of bacteria. Therefore the lack of performance among commonly used antibiotics against the rise of resistant bacterial strains has propelled researchers to look for novel types of antimicrobial agents and enzyme inhibitors as tools to combat this serious and growing threat. The focus of this research is to investigate novel small-molecules from different families with antibacterial properties such as peptides, aromatic ketones, biphenyl and stilbene compounds and boronic acids that demonstrate an inhibitory effect on class A â-lactamase. Some of these inhibitors were found by screening natural products other were found by screening libraries of compounds and yet others were found by computational approaches involving molecular docking. Of all the compounds tested as long as the molecule was within hydrogen bonding distance of Ser84, Lys87 Ser142, Asn186, Gly252, Glu182 or Asp142, they demonstrated inhibitory effect which demonstrates that docking for this class of compounds is highly correlated with successful experimental inhibition.

  • VEGF signaling mediates neuroprotection against oxidative stress in hippocampal neurons in vivo and in vitro

    Author:
    Tianfeng Hao
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Patricia Rockwell
    Abstract:

    Vascular endothelia growth factor (VEGF) signaling through its cognate receptor VEGFR-2 has been shown to be neuroprotective against stressful stimuli including oxidative stress. Using a rat pilocarpine model of status epilepticus (SE), pilocarpine-induced SE was associated with the induction of the oxidative stress marker Heme oxygenase-1 (HO-1) and increased expression of the pro-inflammatory enzyme Cyclooxygenase-2 (COX-2). Treatments with exogenous VEGF attenuated the induction in HO-1 and the increase in COX-2 levels. Previous studies showed that VEGF protected hippocampal cell death in this model and these results suggest that oxidative stress and inflammation contribute to this neuronal loss. Similarly, in cultured hippocampal neurons, pilocarpine induced neuronal cell death accompanied with increased HO-1 expression, reactive oxygen species (ROS) production, caspase-3 cleavage and a loss of mitochondrial membrane potential (ÄØm) that were all suppressed by VEGF. To delineate the protective mechanisms associated with VEGF-mediated neuroprotection, VEGFR-2 inhibition was investigated with respect to oxidative stress in primary cultures of hippocampal neurons. Treatments with the pharmacological inhibitor SU1498 elicited a cytotoxicity that was prevented by the antioxidant N-acetyl-cysteine (NAC) and accompanied by induction of HO-1, cleavage of caspase-3, production of ROS together with a loss of ÄØm. Knockdown of VEGFR-2 by siRNA generated a similar pattern of ROS and ÄØm loss. Treatments with VEGF or VEGF-B prevented neurons from the cell death and mitochondrial dysfunction induced by SU1498 or siRNA, suggesting that a molecular switch occurs where both growth factors may signal through VEGFR-1 when VEGFR-2 activity is blocked. Consistent with a general role for pro-apoptotic BAD and anti-apoptotic Bcl-xL in neuronal cell death and oxidative stress, the phosphorylation of MEK/ERK1/2 and BAD (inactivation) and the protein levels of Bcl-xL that were attenuated by SU1498 were somewhat increased by VEGF. Our findings support a role for VEGF in signaling BAD inactivation and increased Bcl-xL as a specific mechanism that protects hippocampal neurons against oxidative stress. These findings also suggest that VEGF activates VEGFR-2 to protect neurons against oxidative stress but can signal through alternate receptors to serve the same function.

  • Isolation and Characterization of Immunomodulatory Compounds from Juzen-Taiho-To: Novel Understanding of Phytosteryl Glucosides Nano-Aggregates and Synergism

    Author:
    Tal Hasson
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Akira Kawamura
    Abstract:

    This thesis describes the realization of the immunomodulatory properties of four phytosteryl-glucosides betta;-sitosterol-D-Glucoside, campesteryl betta-D-glucoside, 24-epicampestryl betta-D-glucoside, Stigmasteryl betta-D-glucoside) purified from a Japanese Kampo herbal formulation termed Juzen-Taiho-To (JTT). JTT has been used for almost a thousand years and currently is prescribed to people with an impaired immune system. Our screening methodology termed Genomic-Screening allowed us to investigate the immunomodulatory properties of JTT in a new and exciting way. Genomic Screening employs the use of GeneChip® oligonucleotide array, and quantitative reverse-transcriptase polymerase chain reaction qRT-PCR. THP-1 human cancerous monocytic cell line was used in our study with JTT due to their accurate representation of healthy monocytes as well as a large variety of receptors. THP-1 treatment with JTT revealed many genes to be differentially regulated most of which were down stream elements of NFkappa B pathway. We chose (ICAM-1) for our screening since it was the most reproducible assay and it also had a high fold change. By monitoring ICAM-1 response to JTT treatment we were able to uncover known phytosteryl-glucosides that previously were not known for their immunostimulatory properties. In addition our study discovered that a specific ratio of our phytosteryl glucosides that synergistically induced ICAM-1 expression. We have also discovered that the purification approach of the four phytosteryl glucosides greatly affects the immunostimulatory properties. Using Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) we were able to show that nano-aggregated of betta-sitosterol-D-Glucoside formed in a water containing solution diminished ICAM-1 expression in THP-1 cells. These findings underline the importance of natural product chemistry as well as the approach to purifying small organic molecule from natural resources. The underlining mechanism of how our phytosteryl glucosides induce an immune response is still unknown. Nevertheless our finding has opened the door to new research avenues with a family of compound that contain a novel class of pathogen associated molecular patterns.

  • Modeling Membrane Active Peptides with Implicit Membrane models

    Author:
    Yi He
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Themis Lazaridis
    Abstract:

    Membranes are the natural barriers of all cellular organisms. They separate the inner environment from the outer environment. They also divide cell contents into different functional compartments. Many membrane active peptides, however, challenge the function of membranes. They translocate through, form pores inside, or even break down the membranes. Understanding their mechanisms will help us design better drugs. Molecular dynamics (MD) provides a unique way to study these peptides at small time scales. A lot of useful information can therefore be determined: such as, peptide orientation, structure adjustment, insertion into the membrane, and binding energy. Implicit membrane models are particularly useful because their low computational cost allows us to study peptides at longer time scales or larger numbers. The object of the thesis is to study the membrane active peptides using implicit membrane models. The study is focused on three areas: 1) We first examined the transmembrane peptide orientation in both implicit and explicit MD simulations. Using theoretical methods, we tried to explain the gap between the tilt angles predicted by hydrophobic mismatching theory and the ones determined by 2H NMR experiments. 2) To study the interaction between cationic peptides and anionic pores, we extended the current implicit pore model to anionic membranes. This model was applied to two typical antimicrobial peptides &mdash magainin and melittin &mdash and was used to explain their different preferences for anionic lipid fractions. We also evaluated the stability of three protegrin octameric pore models using this model. 3) We then tried to determine the link between binding affinity to membrane surface and biological activities of antimicrobial peptides. We found that both the experimental binding free energy and the theoretical transfer energy correlate with the biological activities, although the correlation is weak. Many other factors may also affect the biological activities of antimicrobial peptides. Moreover, based on a critical evaluation of "carpet" model, we found that most peptides would show higher activity than the prediction of the "carpet" model. The deviation of their biological activities from the "carpet" model correlates with their transfer energies to pores. The knowledge we gained from this study can help us establish quantitative models for predicting antimicrobial peptide activities.