Matching Items (109)
Description
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
Testosterone (T) is a steroid hormone that affects behavior and reproductive traits (e.g. spermatogenesis and ornamentation) in vertebrates. In addition to long-term influences, T can rapidly increase in males following aggressive male-male encounters. Less is known how females directly influence male T and behavior, though research with humans suggests that sexually attractive females elicit a greater increase in male T and reproductive behavior than unattractive females. In birds, the influence of female attractiveness on male T and behavior is currently untested. We hypothesized that T and courtship behavior in male zebra finches would correlate with female attractiveness. We used red leg bands to make females "attractive" and green bands to make them "unattractive" (unbanded females were controls) as previous research suggests that zebra finches prefer red colors over green in mating contexts. We collected blood from males before and after "speed-dating" trials to measure changes in plasma T and analyzed male courtship behaviors from trial video recordings. The likelihood of plasma T increasing after a trial was significantly greater in males who were with red-banded females compared to control females, suggesting males may find them more attractive than green or control females. Additionally, independent of band color, males who exhibited greater T differences initiated courtship sooner and spent more time closest to females. However, courtship initiation and time spent near females were not correlated with band color. Overall, our results suggest that female attractiveness can influence male reproductive physiology, but the presence of a female may trigger male courtship behavior.
ContributorsBero-Buell, Brianna Danielle (Author) / McGraw, Kevin (Thesis director) / Deviche, Pierre (Committee member) / Ligon, Russell (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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
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
Historically, the study of cognition has focused on species-specific learning, memory, problem-solving and decision-making capabilities, and emphasis was placed on the few high-performing individuals who successfully completed cognitive tasks. Studies often deemed the success of a small fraction of individuals as suggestive of the cognitive capacity of the entire species. Recently though, interest in individual variation in cognitive ability within species has increased. This interest has emerged concomitantly with studies of variation in animal personalities (i.e. behavioral syndromes). Cognitive ability may be closely tied to personality because the mechanisms by which an individual perceives and uses environmental input (cognition) should influence how that individual consistently responds to various ecological demands (personality). However, empirical support for links between animal cognition and behavioral syndromes is currently lacking. I examined individual variation in cognition and personality in male veiled chameleons, Chamaeleo calyptratus. I considered three axes of personality (aggression, activity, and exploratory behavior) and cognition in a foraging context using visual cues − specifically, the ability to associate a color with a food reward. I found that aggression was positively correlated with the proportion of correct choices and number of consecutive correct choices. Also, one measure of exploration (the number of vines touched in a novel environment) was correlated negatively with the proportion of correct choices and positively with the number of consecutive incorrect decisions. My investigation suggests that more aggressive, less exploratory chameleons were more successful learners, and that there exists a shared pathway between these personality traits and cognitive ability.
ContributorsBruemmer, Sarah Adele (Author) / McGraw, Kevin (Thesis director) / Rutowski, Ronald (Committee member) / Ligon, Russell (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2013-05
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
Urbanization exposes wildlife to many unfamiliar environmental conditions, including the presence of novel structures and food sources. Adapting to or thriving within such anthropogenic modifications may involve cognitive skills, whereby animals come to solve novel problems while navigating, foraging, etc. The increased presence of humans in urban areas is an additional environmental challenge that may potentially impact cognitive performance in wildlife. To date, there has been little experimental investigation into how human disturbance affects problem solving in animals from urban and rural areas. Urban animals may show superior cognitive performance in the face of human disturbance, due to familiarity with benign human presence, or rural animals may show greater cognitive performance in response to the heightened stress of unfamiliar human presence. Here, I studied the relationship between human disturbance, urbanization, and the ability to solve a novel foraging problem in wild-caught juvenile house finches (Haemorhous mexicanus). This songbird is a successful urban dweller and native to the deserts of the southwestern United States. In captivity, finches captured from both urban and rural populations were presented with a novel foraging task (sliding a lid covering their typical food dish) and then exposed to regular periods of high or low human disturbance over several weeks before they were again presented with the task. I found that rural birds exposed to frequent human disturbance showed reduced task performance compared to human-disturbed urban finches. This result is consistent with the hypothesis that acclimation to human presence protects urban birds from reduced cognition, unlike rural birds. Some behaviors related to solving the problem (e.g. pecking at and eying the dish) also differed between urban and rural finches, possibly indicating that urban birds were less neophobic and more exploratory than rural ones. However, these results were unclear. Overall, these findings suggest that urbanization and acclimation to human presence can strongly predict avian response to novelty and cognitive challenges.
ContributorsCook, Meghan Olivia (Author) / McGraw, Kevin (Thesis director) / Bimonte-Nelson, Heather (Committee member) / Weaver, Melinda (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The purpose of this project was to identify proteins associated with the migration and invasion of non-transformed MCF10A mammary epithelial cells with ectopically expressed missense mutations in p53. Because of the prevalence of TP53 missense mutations in basal-like and triple-negative breast cancer tumors, understanding the effect of TP53 mutations on the phenotypic expression of human mammary epithelial cells may offer new therapeutic targets for those currently lacking in treatment options. As such, MCF10A mammary epithelial cells ectopically overexpressing structural mutations (G245S, H179R, R175H, Y163C, Y220C, and Y234C) and DNA-binding mutations (R248Q, R248W, R273C, and R273H) in the DNA-binding domain were selected for use in this project. Overexpression of p53 in the mutant cell lines was confirmed by western blot and q-PCR analysis targeting the V5 epitope tag present in the pLenti4 vector used to transduce TP53 into the mutant cell lines. Characterization of the invasion and migration phenotypes resulting from the overexpression of p53 in the mutant cell lines was achieved using transwell invasion and migration assays with Boyden chambers. Statistical analysis showed that three cell lines—DNA-contact mutants R248W and R273C and structural mutant Y220C—were consistently more migratory and invasive and demonstrated a relationship between the migration and invasion properties of the mutant cell lines. Two families of proteins were then explored: those involved in the Epithelial-Mesenchymal Transition (EMT) and matrix metalloproteinases (MMPs). Results of q-PCR and immunofluorescence analysis of epithelial marker E-cadherin and mesenchymal proteins Slug and Vimentin did not show a clear relationship between mRNA and protein expression levels with the migration and invasiveness phenotypes observed in the transwell studies. Results of western blotting, q-PCR, and zymography of MMP-2 and MMP-9 also did not show any consistent results indicating a definite relationship between MMPs and the overall invasiveness of the cells. Finally, two drugs were tested as possible treatments inhibiting invasiveness: ebselen and SBI-183. These drugs were tested on only the most invasive of the MCF10A p53 mutant cell lines (R248W, R273C, and Y220C). Results of invasion assay following 30 μM treatment with ebselen and SBI-183 showed that ebselen does not inhibit invasiveness; SBI-183, however, did inhibit invasiveness in all three cell lines tested. As such, SBI-183 will be an important compound to study in the future as a treatment that could potentially serve to benefit triple-negative or basal-like breast cancer patients who currently lack therapeutic treatment options.
ContributorsZhang, Kathie Q (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Rock Doves (Columba livia), also known as pigeons, are a common sight to city dwellers around the world. Often overlooked as urban pests, these birds have intriguing iridescent coloration on their necks that has been the subject of few studies. Previous studies have documented the multimodal reflectance spectra of the iridescence and the keratin cortex microstructures responsible for those properties, but do not address questions about the biological context of this coloration. In this study, I explore the factors that affect how this directional signal might appear to intended receivers (assumed to be females). Pigeon neck feathers were obtained from captive-raised birds and measured for reflectance values at numerous angles in the hemisphere above the feather to obtain a directional reflectance distribution. Each feather was mounted individually, and measurements were taken at a consistent location on the feather using a spectrophotometer; the collector was positioned directly above the feather, while we moved the light source in both azimuth and elevation on a Carden arm to simulate changes in pigeon movements during courtship. Depending on the elevation and azimuth of the light source, pigeon neck feathers shift in appearance from green to purple, with an accompanying shift in the location and intensity of reflectance peaks. Additionally, this unique coloration is due to multiple reflectance peaks in the avian vision field between 300 and 700nm. These data coupled with qualitative behavioral observations of Rock Dove courtship inform our understanding of how the color signal is displayed and how it appears to a potential mate; as a female observes the movements in a male courtship display, properties of the iridescence utilize multiple viewing angles to create a dynamic color array.
ContributorsFankhauser, Kaci Lynn (Author) / Rutowski, Ronald (Thesis director) / McGraw, Kevin (Committee member) / McBeath, Michael (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2015-05