Matching Items (17)
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Description
Cellular and molecular biologists often perform cellular assays to obtain a better understanding of how cells work. However, in order to obtain a measurable response by the end of an experiment, the cells must reach an ideal cell confluency. Prior to conducting the cellular assays, range-finding experiments need to be

Cellular and molecular biologists often perform cellular assays to obtain a better understanding of how cells work. However, in order to obtain a measurable response by the end of an experiment, the cells must reach an ideal cell confluency. Prior to conducting the cellular assays, range-finding experiments need to be conducted to determine an initial plating density that will result in this ideal confluency, which can be costly. To help alleviate this common issue, a mathematical model was developed that describes the dynamics of the cell population used in these experiments. To develop the model, images of cells from different three-day experiments were analyzed in Photoshop®, giving a measure of cell count and confluency (the percentage of surface area covered by cells). The cell count data were then fitted into an exponential growth model and were correlated to the cell confluency to obtain a relationship between the two. The resulting mathematical model was then evaluated with data from an independent experiment. Overall, the exponential growth model provided a reasonable and robust prediction of the cell confluency, though improvements to the model can be made with a larger dataset. The approach used to develop this model can be adapted to generate similar models of different cell-lines, which will reduce the number of preliminary range-finding experiments. Reducing the number of these preliminary experiments can save valuable time and experimental resources needed to conduct studies using cellular assays.
ContributorsGuerrero, Victor Dominick (Co-author) / Guerrero, Victor (Co-author) / Watanabe, Karen (Thesis director) / Jurutka, Peter (Committee member) / School of Mathematical and Natural Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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Description
Vitamin D, Klotho, and FOXO3 have all been linked to have anti-aging and anti-cancerous effects as separate pathways. Specifically, mice with knockout Klotho in their genes have displayed signs of premature aging, humans who are vitamin D deficient have been shown to develop cardiovascular disease and cognitive impairments, and those

Vitamin D, Klotho, and FOXO3 have all been linked to have anti-aging and anti-cancerous effects as separate pathways. Specifically, mice with knockout Klotho in their genes have displayed signs of premature aging, humans who are vitamin D deficient have been shown to develop cardiovascular disease and cognitive impairments, and those who have displayed overexpression of FOXO3 have shown to have a longer lifespan. Here we took each pathway and attempted to formulate a feedback mechanism loop linking all three separate pathways. We propose that vitamin D levels modulate klotho activity, including the expression of the s-klotho and m-klotho isoforms. Moreover, the anti-oxidation transcription factor FOXO3 is also thought to participate in crosstalk with VDR signaling. Through the connection between 1,25D and Klotho, we probed at their interactions with FOXO3 signaling in kidney and colon cells, and proposed that vitamin D and klotho may reduce oxidative stress and suppress the onset of epithelial cancers through it effects on FOXO3. Results showed a strong support for the cooperation between FOXO3 and 1,25D to stimulate both superoxide dismutase (a FOXO3 response element) and XDR3/ROC (vitamin D response elements). This cooperation was mostly seen in embryonic kidney cells (HEK293) and not in the colon cancer cells (HCT116), which has led to the conclusion that vitamin D and FOXO3 cooperation mainly occurs in kidney tissue and/or in tissue that is not yet been overtaken by cancer. Differences in the Klotho isoforms were seen when measuring FOXO3 and vitamin D activity, but experiments manipulating other components will need to be conducted to further understand the function of Klotho in maintaining reactive oxygenated species levels.
ContributorsSandoval, Ruby (Author) / Jurutka, Peter (Thesis director) / Sandrin, Todd R. (Committee member) / Heck, Michael (Committee member) / School of Social and Behavioral Sciences (Contributor) / School of Mathematical and Natural Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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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

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
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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

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
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Description
The diagnosis of irritable bowel syndrome (IBS) is currently based on symptomatic criteria that exclude other conditions affecting the gastrointestinal tract, such as celiac disease, food allergies, and infections. The absence of appropriate diagnostic and therapeutic approaches for IBS places a significant burden on the patient and the health care

The diagnosis of irritable bowel syndrome (IBS) is currently based on symptomatic criteria that exclude other conditions affecting the gastrointestinal tract, such as celiac disease, food allergies, and infections. The absence of appropriate diagnostic and therapeutic approaches for IBS places a significant burden on the patient and the health care system due to direct and indirect costs of care. Limitations associated with the application of symptomatic criteria include inappropriate use and/or intrinsic limitations such as the population to which these criteria are applied. The lack of biomarkers specific for IBS, non-specific abdominal symptoms, and considerable variability in the disease course creates additional uncertainty during diagnosis. This project involved screening tissue samples from patients with verified IBS to identify gene expression-based biomarkers associated with IBS. Through validation of microarray gene chip data on the tissue samples using PCR, it was determined that a number of genes within the diseased IBS patient tissue samples were differentially expressed in comparison to the healthy subjects. These findings could potentially lead to the diagnosis of IBS on the basis of a genetic "fingerprint".
ContributorsHockley, Maryam (Author) / Jurutka, Peter (Thesis director) / Sandrin, Todd (Committee member) / Zhang, Lin (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor)
Created2013-12
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Description
Bexarotene (Bex) is a FDA-approved drug used to treat cutaneous T-cell lymphoma (CTCL). It binds with high affinity to the retinoid-X-receptor (RXR), a nuclear receptor implicated in numerous biological pathways. Bex may have the potential to attenuate estrogenic activity by acting as an estrogen receptor alpha (ERα) signaling antagonist, and

Bexarotene (Bex) is a FDA-approved drug used to treat cutaneous T-cell lymphoma (CTCL). It binds with high affinity to the retinoid-X-receptor (RXR), a nuclear receptor implicated in numerous biological pathways. Bex may have the potential to attenuate estrogenic activity by acting as an estrogen receptor alpha (ERα) signaling antagonist, and can therefore be used to treat ERα-positive cancers, such as breast cancer. Using dual luciferase reporter assays, real-time qRT-PCR, and metabolic proliferation assays, the anti-estrogenic properties of Bex were ascertained. However, since Bex produces numerous contraindications, select novel RXR drug analogs were also evaluated. Results revealed that, in luciferase assays, Bex could significantly (P < 0.01) inhibit the transcriptional activity of ERα, so much so that it rivaled ER pan-antagonist ZK164015 in potency. Bex was also able to suppress the proliferation of two breast cancer cell models, MCF-7 and T-47D, and downregulate the expression of an estrogen receptor target gene (A-myb), which is responsible for cell proliferation. In addition, novel analogs A30, A33, A35, and A38 were evaluated as being more potent at inhibiting ERE-mediated transcription than Bex at lower concentrations. Analogs A34 and A35 were able to suppress MCF-7 cell proliferation to a degree comparable to that of Bex. Inhibition of T-47D cell proliferation, by contrast, was best achieved by analogs A34 and A36. For those with ERα – positive breast cancer who are refractory to current chemotherapeutics used to treat breast cancer, Bex and its analogs may prove to be useful alternative options.
ContributorsBains, Supreet (Author) / Jurutka, Peter (Thesis director) / Hackney Price, Jennifer (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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Description
Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that afflicts more than 20% of the population in the United States. Symptoms include mild to severe abdominal discomfort accompanied by a change in stool character and form ranging from constipation to diarrhea. Additionally, IBS is associated with secondary effects including

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that afflicts more than 20% of the population in the United States. Symptoms include mild to severe abdominal discomfort accompanied by a change in stool character and form ranging from constipation to diarrhea. Additionally, IBS is associated with secondary effects including depression, anxiety, poor quality of life, insomnia and sexual dysfunction. Despite the known association of secondary effects, patients are often tested for potential illnesses that share similar pathological symptoms. This process can be costly and protracted and yet not deliver a completely accurate diagnosis. The aim of this research is to identify gene expression-based biological signatures and unique biomarkers for the detection of IBS. Through the use of quantitative polymerase chain reaction (qPCR), comparison of pooled samples of non-IBS patient-derived RNA were used to identify differentially expressed genes in patients with IBS. Data obtained from preliminary DNA microarray analysis demonstrated a degree of success in differentiating between IBS and asymptomatic patients. Additional comprehensive DNA microarray analyses have led to the identification of a series of 858 differentially expressed genes, including genes associated with serotonin metabolism, which may characterize the IBS pathological state. The microarray results were screened using a combination of gene ontological analysis and qPCR. Real-time PCR revealed repressed levels of tryptophan hydroxylase (TPH1), an enzyme involved in the rate- limiting step in serotonin biosynthesis, in IBS patients relative to controls. Lower concentrations of serum 25(OH)D were also observed among the IBS cohort relative to asymptomatic patients, especially among IBS-D subtype. Vitamin D was shown to modulate differentially expressed genes in IBS patients, suggesting that IBS pathophysiology may involve vitamin D insufficiency and/or an irregularity in serotonin metabolism. Additional qPCR analysis of 32 differentially expressed genes in IBS patients identified 7 putative genetic biomarkers proposed for a potential IBS diagnostic panel. Based on the quality of these results, we may be able to develop, test, and market a diagnostic kit for IBS.
ContributorsGrozic, Aleksandra (Author) / Jurutka, Peter (Thesis director) / Sandrin, Todd (Committee member) / Foxx-Orenstein, Amy (Committee member) / School of Mathematical and Natural Sciences (Contributor) / School of Social and Behavioral Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
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Description
Psoriasis is a skin disease that affects millions of individuals. Genetic risk factors for psoriasis include a common deletion of two late cornified envelope (LCE) genes (LCE3B and LCE3C) located within a cluster of genes expressed during epithelial differentiation and skin repair. It was previously discovered that treatment with 1,25-dihydroxyvitamin

Psoriasis is a skin disease that affects millions of individuals. Genetic risk factors for psoriasis include a common deletion of two late cornified envelope (LCE) genes (LCE3B and LCE3C) located within a cluster of genes expressed during epithelial differentiation and skin repair. It was previously discovered that treatment with 1,25-dihydroxyvitamin D3 (1,25D) or analogs thereof can improve psoriasis symptoms in many patients, but the molecular mechanisms for this action are largely unknown. Our laboratory previously showed that 1,25D as well as low affinity ligands for the vitamin D receptor (VDR), such as delphinidin and cyanidin, are capable of upregulating the remaining LCE3A, -3D, and -3E genes to potentially compensate for the loss of LCE3B and -3C in promoting skin repair. In the current study, DHA and curcumin were tested and found to also upregulate LCE3 transcripts in a dose-dependent manner. To investigate other potential target genes for 1,25D and DHA, we tested JunB, for which low or absent expression has been reported to cause or be associated with psoriatic lesions. Our experiments showed a trend for an upregulation of JunB mRNA after DHA treatment, potentially providing benefit for psoriasis patients. Although our hypothesis is that DHA functions as a vitamin D receptor ligand to mediate upregulation of JunB and LCE3 genes, other investigators have assumed that DHA actions in skin are mediated via PPAR isoforms. We therefore utilized a selective ligand for PPAR delta (GW501516) to determine whether PPAR delta, the primary PPAR isoform in keratinocytes, is a mediator of DHA-induced LCE3 gene activation. Although a modest upregulation of LCE3 genes was seen after treatment with GW501516, our findings are still consistent with DHA acting primarily as a VDR ligand. Our results not only provide additional information about the ability of VDR ligands to upregulate specific skin genes with relevance for skin repair, but also may help provide a molecular basis for testing improved treatments for mild to moderate psoriasis.
ContributorsKarrys, Amitis (Author) / Jurutka, Peter (Thesis director) / Whitfield, Kerr (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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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

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
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Description

The relevance of depression in the clinical realm is well known, as it is one of the most common mental disorders in the United States. Clinical depression is the leading cause of disease for women worldwide. The sex difference in depression and anxiety has guided the research of not just

The relevance of depression in the clinical realm is well known, as it is one of the most common mental disorders in the United States. Clinical depression is the leading cause of disease for women worldwide. The sex difference in depression and anxiety has guided the research of not just recent studies but older studies as well, supporting the theory that gonadal hormones are associated with the mechanisms of emotional cognition. The scientific literature points towards a clear correlative relationship between gonadal hormones, especially estrogens, and emotion regulation. This thesis investigates the neural pathways that have been indicated to regulate mood and anxiety. Currently, the research points to the hypothalamic-pituitary-adrenal axis, which regulates the stress response through its ultimate secretion of cortisol through the adrenal cortex, and its modulated response when exposed to higher levels of estrogen. Another mechanism that has been investigated is the interaction of estrogen and the serotonergic system, which is noteworthy because the serotonergic system is known for its importance in mood regulation. However, it is important to note that the research seeking to determine the neurobiological underpinnings of estrogen and the serotonergic system is not expansive. Future research should focus on determining the direct relationship between cortisol hypersecretion and estrogens, the specific neurobiological effects of serotonergic receptor subtypes on the antidepressant actions of estrogens, and the simultaneous effects of the stress and serotonergic systems on depressive symptoms.

ContributorsArroyo, Mariana (Author) / Bimonte-Nelson, Heather (Thesis director) / Jurutka, Peter (Committee member) / School of International Letters and Cultures (Contributor) / School of Social and Behavioral Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05