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The focus of this project was to look at alternative treatments for endocrine resistant breast cancer (ERBC), which are breast cancers that have become resistant to hormone therapies such as Tamoxifen or aromatase inhibitors. The first part of this project involves investigating the relationship between histone de-acetylase inhibitor Vorinostat and Tamoxifen in MCF7 G11 cells, Tamoxifen resistant sub-clones, according to the PSOC Time grant. The second part involves targeting the androgen receptor (AR) in MCF7 sub-clones with AR antagonists, Bicalutamide and MDV3100, and investigating the possible usage of AR as a biomarker, due to over-expression of AR in ERBC, in accordance with the Mayo ASU Seed Grant.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.
ContributorsVorachitti, Merica (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
A coincidence reporter construct, consisting of the p21-promoter and two luciferase genes (Firefly and Renilla), was constructed for the screening of drugs that might inhibit Olig2's tumorigenic role in glioblastoma. The reporter construct was tested using an Olig2 inhibitor, HSP990, as well as short hairpin RNA targeting Olig2. Further confirmatory analysis is needed before the reporter cell line is ready for high-throughput screening at the NIH and lead compound selection.
ContributorsCusimano, Joseph Michael (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Mehta, Shwetal (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
The influenza virus is the main cause of thousands of deaths each year in the United States, and far more hospitalizations. Immunization has helped in protecting people from this virus and there are a number of therapeutics which have proven effective in aiding people infected with the virus. However, these therapeutics are subject to various limitations including increased resistance, limited supply, and significant side effects. A new therapeutic is needed which addresses these problems and protects people from the influenza virus. Synbodies, synthetic antibodies, may provide a means to achieve this goal. Our group has produced a synbody, the 5-5 synbody, which has been shown to bind to and inhibit the influenza virus. The direct pull down and western blot techniques were utilized to investigate how the synbody bound to the influenza virus. Our research showed that the 5-5 synbody bound to the influenza nucleoprotein (NP) with a KD of 102.9 ± 74.48 nM. It also showed that the synbody bound strongly to influenza viral extract from two different strains of the virus, the Puerto Rico (H1N1) and Sydney (H3N2) strains. This research demonstrated that the 5-5 synbody binds with high affinity to NP, which is important because influenza NP is highly conserved between various strains of the virus and plays an important role in the replication of the viral genome. It also demonstrated that this binding is conserved between various strains of the virus, indicating that the 5-5 synbody potentially could bind many different influenza strains. This synbody may have potential as a therapeutic in the future if it is able to demonstrate similar binding in vivo.
ContributorsKombe, Albert E. (Author) / Diehnelt, Chris (Thesis director) / Woodbury, Neal (Committee member) / Legutki, Bart (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of International Letters and Cultures (Contributor)
Created2014-05
Description
Trichloroethene (TCE) and hexavalent chromium [Cr(VI)] are toxic and carcinogenic contaminants found in drinking water resources across the United States. A series of Bench-scale treatability studies were conducted to evaluate the effectiveness of a consortium of facultative and strictly anaerobic bacteria, KB-1®, to remove TCE and Cr(VI) from a contaminated aquifer in San Diego. These series of treatability studies were also performed to prepare data and mature packed sediment columns for the deployment of the In Situ Microcosm Array (ISMA), a diagnostic device for determining optimal treatments for a contaminated aquifer, at this particular site. First, a control panel for the ISMA’s Injection Module (IM) was created in order to deliver nutrients to the columns. Then, a column treatability study was performed in order to produce columns with an established KB-1® consortium, so that all TCE in the column influent was converted to ethene by the time it had exited the column. Finally, a batch bottle treatability study was performed to determine KB-1®’s effectiveness at remediating both TCE and Cr(VI) from the San Diego ground-water samples. The results from the column study found that KB-1® was able to reduce TCE in mineral media. However, in the presence of site ground-water for the batch bottle study, KB-1® was only able to reduce Cr(VI) and no TCE dechlorination was observed. This result suggests that the dechlorinating culture cannot survive prolonged exposure to Cr(VI). Therefore, future work may involve repeating the batch bottle study with Cr(VI) removed from the groundwater prior to inoculation to determine if KB-1® is then able to dechlorinate TCE.
ContributorsDuong, Benjamin Taylor (Author) / Halden, Rolf (Thesis director) / Torres, Cesar (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Barrett, The Honors College (Contributor) / School of Dance (Contributor) / Chemical Engineering Program (Contributor)
Created2013-05
Description
The pathogenesis of type 1 diabetes (T1D) is still not fully understood in the scientific community. Evidence has shown that viral infections are one of the important environmental factors associated with the disease development. Seven of the top T1D related viruses were selected to study the prevalence of viral humoral response in T1D patients using our innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA). In this study, each viral gene was individually captured using various PCR based techniques, cloned into a protein expression vector, and assembled as the first version of T1D viral protein array. Humoral responses of IgG, IgA, and IgM were examined. Although each class of immunoglobulin generated a wide-range of reactivity, responses to various viral proteins from different proteins were observed. In summary, we captured most of the T1D related viral genes, established viral protein expression on the protein array, and displayed the serum response on the viral protein array. The successful progress will help to fulfill the long term goal of testing the viral infection hypothesis in T1D development.
ContributorsDavis, Amy Darlene (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Desi, Paul (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
The effect of ammonium on microbial fermentation was investigated to improve the efficiency of microbial electrochemical cells (MXC). Electron balances of anaerobic microbial cultures with varying ammonium concentrations (reported as g N-NH4+/L) were used to study the distribution of electrons from different fermentable substrates to acetate, propionate, and methane. Results showed that with a high ammonium concentration (between 2.25 to 3g N-NH4+/L) fewer electrons routed to methane during the fermentation of 300 me-eq./L of electron donors .The majority of electrons (~ 60-80%) in the serum bottles experiments were routed to acetate and propionate for all fermentable substrates with high ammonium concentration. While methane cannot be utilized by anode respiring bacteria (ARBs) to produce current, both acetate and propionate can, which could lead to higher Coulombic efficiencies in MXCs. Experiments in microbial electrolysis cells (MECs) with glucose, lactate, and ethanol were performed. MEC experiments showed low percentage of electrons to current (between 10-30 %), potentially due to low anode surface area (~ 3cm2) used during these experiments. Nevertheless, the fermentation process observed in the MECs was similar to serum bottles results which showed significant diversion of electrons to acetate and propionate (~ 80%) for a control concentration of 0.5 g N-NH4+/L .
ContributorsLozada Guerra, Suyana Patricia (Co-author) / Joseph, Miceli (Co-author) / Krajmalnik-Brown, Rosa (Thesis director) / Torres, Cesar (Committee member) / Young, Michelle (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2013-05
Description
Protein AMPylation is a recently discovered and relatively unstudied post-translational modification (PTM). AMPylation has previously been shown to play an important role in metabolic regulation and host pathogenesis in bacteria, but the recent identification of potential AMPylators across many species in every domain of life has supported the possibility that AMPylation could be a more fundamental and physiologically significant regulatory PTM. For the first time, we characterized the auto-AMPylation capability of the human protein SOS1 through in vitro AMPylation experiments using full-length protein and whole-domain truncation mutants. We found that SOS1 can become AMPylated at a tyrosine residue possibly within the Cdc25 domain of the protein, the Dbl homology domain is vital for efficient auto-AMPylation activity, and the C-terminal proline-rich domain exhibits a complex regulatory function. The proline-rich domain alone also appears to be capable of catalyzing a separate, unidentified covalent self-modification using a fluorescent ATP analogue. Finally, SOS1 was shown to be capable of catalyzing the AMPylation of two endogenous human protein substrates: a ubiquitous, unidentified protein of ~49kDa and another breast-cancer specific, unidentified protein of ~28kDa.
ContributorsOber-Reynolds, Benjamin John (Author) / LaBaer, Joshua (Thesis director) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
One of the major challenges that were yet to be solved for solid phase peptide synthesis was the lack of an efficient peptide sequencing technique that was less hazardous, easier to perform , and was more cost-effective. Sequencing peptides were held important in the field of Chemistry and Biochemistry because it aided in drug discovery, finding ligands that bind to a specific target protein and finding alternative agents in transporting molecules to its desired location. Therefore, the overall purpose of this experiment was to develop a method of solid phase sequencing technique that was more environmental friendly, sequences at a faster rate, and was more cost-effective.
ContributorsCordovez, Lalaine Anne Ordiz (Author) / Woodbury, Neal (Thesis director) / Zhao, Zhan-Gong (Committee member) / Legutki, Joseph Barten (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
AMPylation is a post-translation modification that has an important role in the survival of many bacterial pathogens by affecting the host cell's molecular signaling. In the course of studying this intercellular manipulation, there has only been modest progression in the identification of the enzymes with AMPylation capabilities (AMPylators) and their respective targets. The reason for these minimal developments is the inability to analyze a large subset of these proteins. Therefore, to increase the efficiency of the identification and characterization of the proteins, Yu et al developed a high-throughput non-radioactive discovery platform using Human Nucleic Acid Programmable Protein Arrays (NAPPA) and a validation platform using bead-based assays. The large-scale unbiased screening of potential substrates for two bacterial AMPylators containing Fic domain, VopS and IbpAFic2, had been performed and dozens of novel substrates were identified and confirmed. With the efficiency of this method, the platform was extended to the identification of novel substrates for a Legionella virulence factor, SidM, containing a different adenylyl transferase domain. The screening was performed using NAPPA arrays comprising of 10,000 human proteins, the active AMPylator SidM, and its inactive D110/112A mutant as a negative control. Many potential substrates of SidM were found, including Rab GTPases and non-GTPase proteins. Several of which have been confirmed with the bead-based AMPylation assays.
ContributorsGraves, Morgan C. (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Yu, Xiaobo (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
Lactate and methanol have been the most commonly used electron donors in the Krajmalnik-Brown laboratory for efficient microbial dechlorination of trichloroethene (TCE). Our goal was to assess the technical and economic feasibility of molasses and ethanol, two alternative electron donors by evaluating their costs and ability support complete TCE dechlorination to ethene. First, ethanol and molasses, with and without methanol, were evaluated for their abilities to support complete dechlorination in batch serum bottles. Molasses, the cheapest alternative, supported a similar dechlorination performance to lactate in batch experiments, so we then used it in an upflow anaerobic bioreactor (UABR) to test its ability to support rapid dechlorination in this continuous system. Molasses supported 88% TCE conversion to ethene at a hydraulic retention time (HRT) of 13 hours after 80 days of operation in continuous mode. Compared to the UABR operated previously using lactate and methanol, molasses led to a reduction of TCE conversion to ethene, and a possible increase in time required to produce culture. Additionally, when molasses was used as the electron donor, we encountered new difficulties in the operation of the UABR, such as drastic pH changes. Therefore, I conclude that the savings from using molasses is outweighed by the costs associated with the reduction in dechlorination performance and increase in reactor maintenance. I recommend that lactate and methanol continue to be used as the electron donors in the Krajmalnik- Brown dechlorination lab to support fast-rate and cost-effective production of dechlorinating culture in an UABR. Because molasses supported fast rates of dechlorination in the batch experiment, however, it is potentially a better option than lactate and methanol for batch production of culture or for biostimulation, where the aquifer resembles a batch system. I recommend that further studies be done to reach a general conclusion about the feasibility of molasses as an electron donor for other enhanced bioremediation projects.
ContributorsBondank, Emily Nicole (Author) / Krajmalnik-Brown, Rosa (Thesis director) / Delgado, Anca (Committee member) / Torres, Cesar (Committee member) / Civil, Environmental and Sustainable Engineering Programs (Contributor) / Barrett, The Honors College (Contributor)
Created2014-12