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Description
Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.
ContributorsRamirez, Christopher Nicholas (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Bexarotene is a commercially produced drug commonly known as Targetin presecribed to treat cutaneous T-cell lymphoma (CTCL). Bex mimics the actions of natural 9-cis retinoic acid in the body, which are derived from Vitamin A in the diet and boost the immune system. Bex has been shown to be effective in the treatment of multiple types of cancer, including lung cancer. However, the disadvantages of using Bex include increased instances of hypothyroidism and excessive concentrations of blood triglycerides. If an analog of Bex can be developed which retains high affinity RXR binding similar to the 9-cis retinoic acid while exhibiting less interference for heterodimerization pathways, it would be of great clinical significance in improving the quality of life for patients with CTCL. This thesis will detail the biological profiling of additional novel (Generation Two) analogs, which are currently in submission for publication, as well as that of Generation Three analogs. The results from these studies reveal that specific alterations in the core structure of the Bex "parent" compound structure can have dramatic effects in modifying the biological activity of RXR agonists.
ContributorsYang, Joanna (Author) / Jurutka, Peter (Thesis director) / Wagner, Carl (Committee member) / Hibler, Elizabeth (Committee member) / Barrett, The Honors College (Contributor)
Created2012-05
Description
Vitamin D, a bioactive lipid and essential nutrient, is obtained by humans through either endogenous synthesis in response to UV light exposure or via nutritional intake. Once activated to its hormonal form, vitamin D binds to and activates the nuclear vitamin D receptor (VDR). Activation of VDR is known to modulate gene transcription in vitamin D target tissues such as kidney, colon, and bone; however, less is known about the ability of VDR to respond to "nutritional modulators". One such potential VDR modulator is resveratrol, a plant-derived polyphenol and potent antioxidant nutrient that also functions as a chemopreventative. Resveratrol is known to activate sirtuin-1, a deacetylase enzyme with potential anti-aging properties. This study explores the potential for resveratrol, an anticancer nutraceutical, to upregulate VDR activity through its effector protein, sirtuin-1. Furthermore, due to its putative interactions with several intracellular signaling pathways, klotho has been proposed as an anti-aging protein and tumor suppressor gene, while the Wnt/β-catenin signaling pathway drives enhanced cellular proliferation leading to numerous types of cancers, especially colorectal neoplasia. Thus, the ability of klotho to cooperate with vitamin D to inhibit oncogenic β-catenin signaling was also analyzed. The experiments and resultant data presented in this thesis explore the potential role of VDR as a physiologically relevant nutritional sensor in human cells. This novel study reveals the importance of nutrient modulation of the VDR system by vitamin D and resveratrol and how this might represent a molecular mechanism that is responsible for the putative anti-cancer actions of vitamin D. Furthermore, this study enhances our understanding of how vitamin D/VDR and resveratrol interact with klotho and how this interaction affects β-catenin signaling to mitigate oncogenic growth and differentiation. This works demonstrates that the vitamin D hormone serves as a likely chemopreventive agent for various types of cancers through control of anti-oxidation and cellular proliferation pathways via its nuclear receptor. Our results also indicate the potential for resveratrol, an anticancer nutraceutical, to upregulate VDR activity through SIRT1. Furthermore, the novel data presented in this work illustrate that klotho, an anti-aging protein, cooperates with vitamin D to synergistically inhibit oncogenic β-catenin signaling. Ultimately, this study enhances our understating of the molecular pathways that underpin nutritional chemoprevention, and how modulation of these pathways via dietary intervention may lead to advances in public health strategies to eventually curb carcinogenesis.
ContributorsKhan, Zainab (Author) / Jurutka, Peter (Thesis director) / Hackney Price, Jennifer (Committee member) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
DescriptionThis creative project provides documentation and an exploration of my interactions with individuals encountered while hitchhiking up the west coast.
ContributorsGerber, Evan Howard (Author) / Kashiwagi, Dean (Thesis director) / Kashiwagi, Jacob (Committee member) / W. P. Carey School of Business (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The significance of hormonal vitamin D in the numerous facets of health stresses the importance of elucidating the molecular mechanism(s) associated with 1,25D-VDR signaling modulators (e.g., resveratrol and sirtuin-1). Resveratrol (Res), a natural antioxidant, is a potent activator of NAD-dependent deacetylase sirtuin-1 (SIRT-1), an enzyme associated with longevity in animal models. This present study employed mammalian 2-hybrid (M2H) and vitamin D responsive element (VDRE)-based transcriptional assays to investigate the potential effects of Res and SIRT-1 on VDR signal transduction. Results from VDRE-based assays indicate that Res and SIRT-1 potentiate 1,25D-VDR activity via cell-and-promoter-specific pathways. In addition, 1,25D displacement experiments revealed an increase in VDR-bound radiolabeled 1,25D in the presence of Res, suggesting that Res may potentiate VDR transactivation by stimulating 1,25D binding. M2H assays in HEK293 cells were then utilized to assess levels of interaction between VDR and VDR comodulators, including RXR, SRC-1, and DRIP-205. Both Res and SIRT-1 increased the ability of VDR to associate with RXR; however, SRC-1 and DRIP-205 interactions were not enhanced. The activity of a novel, non-acetylatable VDR mutant, K413R, was probed revealing that K413R possesses amplified transactivation capacity over wild-type VDR. A SIRT-1 inhibitor, EX-527, was used to suppress endogenous SIRT-1, resulting in significantly decreased VDR transactivation. Finally, qPCR results in HEK293 cells revealed that the 1,25D-mediated induction of CYP24A1, an endogenous VDR target gene, was enhanced (85%) by SIRT-1 while Res increased CYP24A1 expression by 294%. The combination of 1,25D, SIRT-1, and Res amplified CYP24A1 expression by 326% over 1,25D, although this effect did not reach statistical significance when compared to the Res only treated group. We conclude that acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This loop is suppressed by resveratrol/SIRT-1-catalyzed deacetylation of VDR, restoring VDR activity. The two compounds, 1,25-dihydroxyvitamin D (1,25D, vitamin D) and 5-hydroxytryptamine (5-HT, serotonin), have been proposed to play a significant role in abnormal social behavior associated with psychological conditions including autism spectrum disorders (ASDs) and depression; however, the mechanism underlying these associations has yet to be elucidated. Deficiencies in 1,25D or 5-HT have been linked to the increased incidence of ASDs. Thus, examining the modulation of genes involved in 5-HT biosynthesis, reuptake, and degradation is fundamental in linking low 1,25D levels to the increased incidence of psychiatric disorders. We propose that 1,25D regulates tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway of 5-HT. In order to evaluate the regulation of TPH2 in neuronal cells, three formulations of media were examined to optimize the cell culture conditions necessary for growth and morphology of embryonic rat medullary raphe (B14) serotonergic neurons. Next, quantitative real time-PCR (qPCR) was utilized to examine TPH2 expression in cultured human glioblastoma (U-87) cells and rat serotonergic neurons (B-14). Human TPH2 mRNA in U-87 cells was induced dose-dependently resulting in a 2.4-fold increase at 10 nM 1,25D. Strikingly, TPH2 mRNA in B-14 cells was observed to be 26- to 86-fold upregulated at 10 nM 1,25D; however, 1 nM and 100 nM 1,25D elicited significantly smaller inductions (8-fold and 1.2-fold, respectively).
ContributorsSabir, Marya Sabah (Author) / Jurutka, Peter (Thesis director) / Hackney Price, Jennifer (Committee member) / Sandrin, Todd R. (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Vitamin D3 (cholecalciferol) is an essential micronutrient that plays a key role in developmental growth and lifespan in mammals. However, few studies have shown how vitamin D3 plays its vital functions in arthropods. Here, we examined the effects of full (13.3 IU/mL) and half dose (6.65 IU/mL) vitamin D3 on the growth and lifespan of Drosophila melanogaster. Vitamin B12 is another micronutrient that shows decreases absorption in elderly patients and might be linked to symptoms associated with aging rather than lifespan, but again, the effects of vitamin B12 supplementation in arthropods is poorly characterized. Results showed that both full and half doses of vitamin D3 and B12 do not significantly alter the timing of pupariation or adult eclosion. Similarly, the mortality rate of adult D. melanogaster exposed to vitamin B12 or higher doses of vitamin D3 was not significantly decreased or increased. However, a low dose of vitamin D3 did significantly lower the mortality rate of D. melanogaster. The genetic composition of Drosophila for vitamin B12 and D metabolism showed similarities in humans. However, there are no biological evidences if these genes are functional thus, this may explain the results of this study.
ContributorsRebonza, Edzel May Suico (Author) / Hackney Price, Jennifer (Thesis director) / Jurutka, Peter (Committee member) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Throughout history humans have had to adapt to changing conditions in order to survive. Food shortages are one of the major pressures that have shaped past populations. Because of this, the human body has many physiological adaptations that allow it to go extended periods of time consuming little to no food. These adaptations also allow the body to recover quickly once food becomes available. They include changes in metabolism that allow different fuel sources to be used for energy, the storing of excess energy absorbed from food in the forms of glycogen and fat to be used in between meals, and a reduction in the basal metabolic rate in response to starvation, as well as physiological changes in the small intestines. Even in places where starvation is not a concern today, these adaptations are still important as they also have an effect on weight gain and dieting in addition to promoting survival when the body is in a starved state.
Disclaimer: The initial goal of this project was to present this information as a podcast episode as a part of a series aimed at teaching the general public about human physiological adaptations. Due to the circumstances with COVID-19 we were unable to meet to make a final recording of the podcast episode. A recording of a practice session recorded earlier in the year has been uploaded instead and is therefore only a rough draft.
Disclaimer: The initial goal of this project was to present this information as a podcast episode as a part of a series aimed at teaching the general public about human physiological adaptations. Due to the circumstances with COVID-19 we were unable to meet to make a final recording of the podcast episode. A recording of a practice session recorded earlier in the year has been uploaded instead and is therefore only a rough draft.
ContributorsPhlipot, Stephanie Anne (Author) / Hyatt, JP (Thesis director) / Kingsbury, Jeffrey (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
All multicellular organisms are susceptible to developing cancer, but some organisms have varying sensitivities to the disease. One such organism is the Trichoplax adhaerens which has no documented case of cancer development. T. adhaerens cancer resistance was studied by observing physiological and morphological changes of the organism after radiation treatment. Preliminary experiments suggested that this organism is able to survive exposure to 160 gray radiation treatment almost as well as untreated organisms. The T. adhaerens have two genes, TriadG6402 and TriadG5479, similar to the human genes TP53 and MDM2 respectively. TP53 and MDM2 are the two main genes associated with apoptosis in humans: an important cell regulatory checkpoint involved in cancer prevention. PCR analysis, done after radiation treatment, showed an overexpression of the ortholog gene MDM2 in the T. adhaerens. This may suggest that T. adhaerens block apoptosis from occurring and that their ortholog gene is involved in DNA repair. It is significant to study the gene expression of TriadG6402 and TriadG54791 in T. adhaerens because these genes are well conserved in humans. Future studies of these genes in the T. adhaerens can be used to understand the evolution of the function of these genes in more complex organisms and be used for human cancer prevention.
ContributorsKulkarni, Arathi (Author) / Fortunato, Angelo (Thesis director) / Maley, Carlo (Committee member) / Department of Economics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Enzyme Replacement Therapy (ERT) is a treatment often used for patients with disorders that affect the production of various enzymes within the body, such as Cystic Fibrosis and Fabry Disease. ERT involves the use of artificially-produced enzymes, which can be derived from humans, pigs, and bacteria. Generally, enzymes derived from porcine and bacterial sources are much less expensive and more accessible than those derived from a human source. This, and the ethical implications that porcine enzymes carry, make the decision of choosing treatment simple to some and complex to others. Ethically, human-derived enzymes are often considered more ethical, while not conflicting with religious beliefs and practices as porcine-derived enzymes do.
In order to further compare porcine and human-derived enzymes, a determination of the enzyme effectiveness was done via digestion simulation. The digestion for both the human and porcine-derived enzymes consisted of three steps: oral, gastric, and intestinal. After the digestion, the absorbance for each enzyme class as well as a dilution curve of the formula used was read and recorded. Using the standard dilution curve and the absorbance values for each unknown, the formula and thus enzyme concentration that was lost through the reaction was able to be calculated.
The effectiveness of both the human and porcine enzymes, determined by the percent of formula lost, was 18.2% and 19.7%, respectively, with an error of 0.6% from the spectrophotometer, and an error of about 10% from the scale used for measuring the enzymes. This error was likely due to the small mass required of the enzymes and can be prevented in the future by performing the experiment at a larger scale.
In order to further compare porcine and human-derived enzymes, a determination of the enzyme effectiveness was done via digestion simulation. The digestion for both the human and porcine-derived enzymes consisted of three steps: oral, gastric, and intestinal. After the digestion, the absorbance for each enzyme class as well as a dilution curve of the formula used was read and recorded. Using the standard dilution curve and the absorbance values for each unknown, the formula and thus enzyme concentration that was lost through the reaction was able to be calculated.
The effectiveness of both the human and porcine enzymes, determined by the percent of formula lost, was 18.2% and 19.7%, respectively, with an error of 0.6% from the spectrophotometer, and an error of about 10% from the scale used for measuring the enzymes. This error was likely due to the small mass required of the enzymes and can be prevented in the future by performing the experiment at a larger scale.
ContributorsBlevins, Brianna R (Author) / Martin, Thomas (Thesis director) / McILwraith, Heide (Committee member) / College of Integrative Sciences and Arts (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The Scientist in Me is an original children’s book, authored by Annmarie Barton and illustrated by Alison Lane, that explores the lives and specialties of five remarkable scientists from historically underrepresented backgrounds: Mary Anning, James Pollack, Temple Grandin, Percy Lavon Julian, and Ayah Bdeir. In the book, each scientist has an “Experiment” section that is meant to encourage children to immerse themselves in activities relating to the scientists’ areas of study. We believe that diversity in science is crucial for advancement, and therefore hope to inspire the next generation of scientists through immersion and representation.
ContributorsLane, Alison (Co-author) / Barton, Annmarie (Co-author) / Klemaszewski, James (Thesis director) / Fette, Donald (Committee member) / School of Molecular Sciences (Contributor) / School of Art (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05