Browsing by Subject "miRNA"

Now showing 1 - 6 of 6
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    Characterization of the function of the C. elegans heterochronic gene lin-42/per during larval development
    (2013-12) McCulloch, Katherine Ann
    The heterochronic pathway of C. elegans ensures the appropriate timing of post-embryonic development. Mutations in heterochronic genes cause skipping or reiteration of larval programs, resulting in severe developmental defects. Many C. elegans heterochronic genes are conserved in both sequence and function; therefore, study of developmental timing in C. elegans contributes to understanding of development in other organisms. The heterochronic gene lin-42 is the C. elegans homolog of period, a component of the circadian clock of Drosophila and mammals. lin-42(lf) results in precocious heterochronic phenotypes, in which later developmental events occur too early. lin-42 also regulates molting, and lin-42 mutants have delayed and prolonged larval molts compared to wild-type animals. In my thesis work, I show that lin-42 confers robustness on developmental and molting pathways against environmental fluctuations, as lin-42(lf) heterochronic and molting phenotypes are very sensitive to changes in environment, which is not observed in wild-type animals. Also, I have found that lin-42 regulates developmental timing by inhibiting expression of let-7-family miRNAs, likely at the level of transcription. Genetic analyses place lin-42 upstream of this miRNAs, showing that regulation of let-7-family miRNAs is a key function of lin-42 in the heterochronic pathway.
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    A comparative approach to deciphering the molecular mechanisms of scar-free wound healing
    (2017-02) Erickson, Jami
    Although mammals form scars upon skin wound healing, the Mexican “Axolotl” salamander has the extraordinary ability to heal wounds scar-free. While axolotl skin histologically resembles mammalian skin, molecular details that prevent scar formation during axolotl wound healing are largely unknown. To address this knowledge gap, we performed transcriptional profiling during axolotl cutaneous wound healing. We analyzed genes that displayed differential gene expression during axolotl wound healing compared to previously published human gene expression profiling data. We found that Sal-like 4 (Sall4) expression was increased early during axolotl skin regeneration, but did not increase in humans until later time points. We hypothesize that early increase in expression of SALL4 after injury is required for scar-free wound healing. To test this hypothesis, we depleted SALL4 in vivo during wound healing. We found that when SALL4 is depleted, we see excessive Collagen I and XII deposition that occurs earlier and is not fully remodeled, resulting in a scar-like phenotype. To determine how SALL4 expression is regulated during wound healing, we sought to identify which microRNAs post-transcriptionally regulate SALL4. We found that miR-219 is able to regulate expression of axolotl SALL4 during wound healing. Further, when we ectopically increase miR-219 levels during axolotl wound healing, we find early excessive collagen deposition, mirroring the SALL4 depletion phenotype. Additionally, we found that miR-103, not miR-219, is able to regulate human SALL4. Thus, revealing one mechanism that could explain the different SALL4 expression profiles seen in axolotls vs. humans. Lastly, we describe how to use a Dual-Fluorescent green fluorescent protein (GFP)-Reporter/ monomeric red fluorescent protein (mRFP)- Sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time. This system allows researchers to obtain relative quantifications for microRNA levels during biological processes over time. This will allow researchers explore the expression dynamics of any microRNA over time in vivo.
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    Epidemiology of childhood germ cell tumors (GCT): epigenome-wide methylation, differential MiRNA expression, and catchment quality of the CCRN
    (2013-09) Musselman, Jessica
    Epidemiology of Childhood Germ Cell Tumors (GCT): Epigenome-Wide Methylation, Differential MiRNA Expression, and Catchment Quality of the CCRN Germ cell tumors (GCTs) represent a heterogeneous group of neoplasms classified together through a common precursor cell, the primordial germ cell. The etiology of GCTs is not well elucidated, although genetic and epigenetic pathways are implicated. In this dissertation, I completed three complementary projects to further our understanding of these tumors. Project 1: I performed an epi-genome wide analysis of methylation in a large sample of childhood GCT (n = 111) to assess methylation with respect to survival, time to recurrence, and tumor histology. A semi-supervised recursively partitioned mixture modeling (SS-RPMM) algorithm segregated the GCTs into four methylation classes. Class membership was not significantly associated with tumor histology (p=0.06) or survival (p = 0.23), but was associated with disease recurrence (p = .02). These findings provide a strong rationale for future studies of methylation in GCT. Project 2: Particular interest has been paid to the role of miRNA in cancer in recent years, including studies in adult testicular GCTs. I conducted an evaluation of miRNA in GCTs by tumor and patient subgroups. Six miRNAs were differentially expressed by tumor histology after correction for multiple comparisons. These findings could provide insights into potential new targeted therapies. Project 3: The Childhood Cancer Research Network was established to ameliorate some of the difficulties associated with the study of childhood cancer; however, the quality of catchment in the CCRN has not been evaluated. I used data from the NCI's Surveillance, Epidemiology, and End Results (SEER) program and the 2010 U.S. Census to determine the ascertainment completeness of GCT cases over a 3-year period. Overall, enrollment was much lower than expected, particularly in the 15-19 year age group. These results provide useful insight into the completeness of case ascertainment of childhood GCT in the CCRN.
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    MicroRNAs As Predictors of Nucleoside Analog Sensitivity in Acute Myeloid Leukemia
    (2015-12) Bhise, Neha
    Acute myeloid leukemia (AML) is the most aggressive form of hematological malignancies. Despite advances in treating AML, development of resistance to nucleoside analog therapy remains one of the major obstacles in AML treatment. Various factors, including SNPs in genes, epigenetics, etc. play a role in mediating variability in response to nucleoside analog therapy. Recent studies have shown that microRNAs can also serve as regulators of gene expression that can contribute to variability in response to therapeutic agents. Thus the main objective of the thesis was to determine the role of microRNAs as predictors of variability in nucleoside analog response. To our knowledge no studies have been reported that identify microRNAs as predictors of response to cytarabine therapy in AML patients. In chapter II we used a translational approach of conducting in vitro and clinical study to identify microRNAs that were predictive of overall survival in AML patients. Our study conclusively identified that miR107-Myb, miR-378-granzyme B and miR10a-MAP4K4 as miRNA-mRNA pairs that can be used as predictors of overall survival in AML patients. Additionally, we also showed that the miRNAs mechanistically regulate the expression of these mRNAs by binding to the 3’- untranslated region of these mRNAs. miRNAs can also cause variability in response to cytarabine therapy by regulating the expression of the genes involved in disposition of cytarabine. In chapter III we identified that miR-24 and miR-34a as regulator of DCTD (an enzyme involved in inactivation of cytarabine) and DCK (activating enzyme), respectively. These miRNAs along with other miRNAs can be used as part of biomarker signature that can be used to predict the overall survival in AML patients. In chapter IV, we determined the impact of cytarabine treatment on in vivo cytarabine-induced changes in leukemia cell transcriptome and miRNA expression, to evaluate their impact on clinical outcome. In the first part of this chapter, we identified key genes (such as tumor suppressors DKK3, TRIM33, PBRM1, an oncogene SET, cytidine-deaminase family members APOBEC2 and APOBEC3G) influenced by cytarabine infusion that were also predictive of response. In the second part of this chapter, using data from clinical studies, we identified several miRNAs that were altered by cytarabine treatment. The changes in the expression of these miRNAs resulted in alteration in gene expression that correlated with the overall survival in AML patients. Additionally, using cell lines we were able to identify various miRNA – mRNAs that were altered by drug treatment, indicating that the therapy itself can influence the predictive ability of miRNAs as biomarkers. Significant data has been published on cytarabine as it is the standard of care in AML patients, however, there is limited knowledge about newer nucleoside analogs such as clofarabine. In chapter V, using in vitro methods, we identified several microRNAs, such as miR-16, miR-515 cluster, etc., that can be used as predictors of response for clofarabine therapy. Our data clearly suggests that there are several distinct microRNAs that can be used as predictors of response to clofarabine therapy in AML patients. We propose doing a clinical study to evaluate the clinical utility of these miRNAs as predictors of response. In summary, using a translational approach, we were able to identify miRNAs and several miRNA-mRNA pairs that can be used as biomarkers of response to cytarabine therapy. Additionally, we also identified that drug therapy itself can influence the outcome in AML patients. These findings are clinically important as they will help provide a new strategy to optimize dosing of nucleoside analogs in AML patients which in turn would lead to better overall survival while reducing the side effects.
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    Role of microRNAs in mediating pancreatic cancer response to triptolide
    (2013-04) MacKenzie, Tiffany Noelle
    Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits PDAC cell growth in vitro and blocks metastatic spread in vivo. Triptolide downregulates heat shock protein 70 (HSP70), a molecular chaperone upregulated in several tumor types. This study investigates the mechanism by which triptolide inhibits HSP70. As microRNAs (miRNAs) are becoming increasingly recognized as negative regulators of gene expression, we tested whether triptolide regulates HSP70 via miRNAs. Here we show that triptolide, as well as quercetin but not gemcitabine, upregulated miR-142-3p in PDAC cells (MIA PaCa-2, Capan-1, and S2-013). Ectopic expression of miR-142-3p inhibited cell proliferation, measured by Electric Cell-substrate Impedance Sensing, and decreased HSP70 expression, measured by real-time PCR and immunoblotting, compared with controls. We demonstrated that miR-142-3p directly binds to the 3’UTR of HSP70, and that this interaction is important as HSP70 overexpression rescued miR-142-3p-induced cell death. We found that miR-142-3p regulates HSP70 independently of heat shock factor 1. Furthermore, Minnelide, a water soluble prodrug of triptolide, induced the expression of miR-142-3p in vivo. This is the first description of an miRNA-mediated mechanism of HSP70 regulation in cancer, making miR-142-3p an attractive target for PDAC therapeutic intervention.
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    Understanding The Role Of Pentraxin 3 In Adipose Tissue Inflammation And Aging
    (2022-01) Lin, Te-Yueh
    Obesity and aging are often accompanied by chronic low-grade inflammation in adipose tissue. Metabolic endotoxemia (elevated circulating lipopolysaccharide [LPS] level) is the inducer of systemic and adipose tissue inflammation and dysfunction, which is developed during obesity and aging. As a soluble pattern recognition receptor, Pentraxin 3 (PTX3) plays a vital role in innate immunity and can be induced by inflammatory stimuli in adipose tissue and adipocyte. Altered PTX3 levels have been observed in adipose tissue and blood during obesity and aging, while the role of PTX3 in adipose tissue during inflammation and aging is not fully understood. My Ph.D. research attempts to understand the role of PTX3 in adipose tissue inflammation and aging. In the first project, we investigated how PTX3 regulates inflammation in adipose tissue, and we found that PTX3 plays an anti-inflammatory role partially by regulating miR-21 expression and secretion in brown adipocytes. In the second project, we explored PTX3 secretion from adipose tissue and adipocyte and found that PTX3 is secreted mainly through conventional protein secretion, while a small percentage of PTX3 is released in exosomes from LPS-stimulated adipocytes. In the third project, we examined the physiological role of PTX3 in adipose tissue during aging, and we showed that PTX3 deficiency induced senescence and inflammation in adipose tissue and promoted lipid transport and oxidation in white adipose tissue of old female mice. In summary, my doctoral research reveals that adipose-derived PTX3 plays an essential role in regulating LPS-stimulated inflammation and cellular senescence during aging, and adipocyte-derived PTX3 is secreted via conventional protein secretion and exosomes.