Browsing by Subject "microRNA"

Now showing 1 - 7 of 7
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    Comparative analysis of the molecular pathways involved in spinal cor injury in axolotls vs. mammaals
    (2015-04) Diaz Quiroz, Juan Felipe
    Spinal cord injuries (SCI) in mammals are major causes of physical disabilities. In contrast the Mexican salamander (axolotl) possesses amazing regenerative capabilities and can regenerate a fully functional spinal cord after injury. We have attempted to identify the molecular determinants underlying this evolutionary divergence by undertaking a detailed comparative analysis of a regenerative model, the axolotl spinal cord, and a corresponding non-regenerative system, rat spinal cord after injury. This approach identified a small number of highly conserved microRNAs that are differentially regulated in axolotl versus. Detailed in vivo studies of one of these microRNAs, miR-125b that is highly expressed in axolotl but low in rat has identified it as a key regulator of the regenerative response in axolotl. We also found that upregulation of miR-125b after injury improves SC repair in rats. In addition, we have identified SEMA4D as a target gene regulated by this microRNA after SCI in both axolotl and rat. We also studied how this miRNA is regulated in the axolotl to promote regeneration after SCI. We found that in the absence of injury, actin cytoskeleton de-polymerization induced by Cytochalasin D (Cyto D) induced a decrease in miR-125b expression similar to the decrease induced by injury in axolotl. Analyses of the regulatory region of the miR-125b axolotl gene revealed predicted binding sites for c-Fos. When we performed SCI in axolotls, we observed an increase in the expression of c-Fos 1 day after injury. As expected, we found that in the absence of injury, treatment with Cyto D induced similar changes in c-Fos expression similar to injury. Lastly, we proposed the development of a 3D in vitro co-culture model system to study at the cellular and molecular level how miR-125b SCI regulates the response to injury in rats and whether miR-125b expression is regulated by biomechanical activation of the Rho-c-Fos pathway. The proposed model will allow better imaging, better spatial and temporal control over modulating microRNA and gene expression in different cells at different time points. Our overall data suggest that dynamic changes in the actin cytoskeleton after injury induces changes in miR-125b expression, possibly through activation of cFos in the RhoA pathway, to create a permissive environment for regeneration in axolotls.
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    High-throughput transcriptomic analysis of resource-poor mammalian cell lines for recombinant protein production
    (2013-10) Johnson, Kathryn Christine
    Over the past 30 years, mammalian cell culture has enabled the production of recombinant protein therapeutics for treatment of a broad range of debilitating or life-threatening diseases. Continual improvements in cell and process engineering have facilitated the attainment of once unheard-of product titers, and improvements in molecular analysis techniques and process analytic technologies have been employed with great success for cell culture process characterization. High-throughput transcriptomic analysis tools such as microarrays and next-generation RNA sequencing (RNA-seq) provide access to gene expression information by simultaneously measuring the expression levels of tens of thousands of genes. However, until recently such tools have not been used to their full advantage in mammalian cell culture processes due to limitations in available reference sequences for the industrially important Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines. We employed high-throughput RNA sequencing in several CHO cell lines to identify and interrogate a class of small non-coding RNAs called microRNAs (miRNAs), which mediate post-transcriptional repression of protein-coding genes. We annotated and analyzed the expression and genomic conservation of several hundred of these small RNAs. We also employed RNA sequencing to build a comprehensive reference transcriptome for a recombinant protein-producing BHK cell lines. We utilized the BHK reference sequence to enable analysis of gene expression levels in the BHK cell line and two Syrian hamster tissues. We designed an expression microarray from the BHK sequence and utilized it to analyze the transcriptome profiles of BHK cells at several time points in perfusion culture at manufacturing scale. Implementation of several functional analysis tools revealed a consistent time-dependent change in the transcriptome profile that involved down-regulation of extracellular matrix components and changes to calcium signaling genes. The transcriptomic reference sequences we developed in this research and the detailed studies they have enabled will enhance our ability to understand and further optimize cell culture processes.
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    MicroRNA and Neuroimaging Biomarkers of Neuropathic Pain Severity After Spinal Cord Injury: Results from a Robotic-Assisted Gait Training Study
    (2022-07) Kowalski, Jesse
    Spinal cord injury (SCI) results in chronic neuroinflammation which contributes to altered neural function and the development of neuropathic pain. Differential expression of microRNA regulators of neuroinflammatory pathways and alterations in brain structure and functional connectivity may contribute to the development or severity of neuropathic pain. Exercise has been shown to reduce neuroinflammation and chronic pain and alter brain structure in human and animal models, yet little is known about how exercise interventions influence pain processing in human populations with SCI. This doctoral dissertation aimed to identify 1) novel microRNA biomarkers of neuropathic pain, 2) neuropathic pain-related alterations in brain functional connectivity, and 3) the efficacy of an exercise intervention of robotic-assisted gait training to reduce neuropathic pain and alter brain volume in individuals with SCI. Successful identification of underlying mechanisms of neuropathic pain and potential exercise induced mitigation of these factors will guide the development of targeted interventions and provide useful biomarkers to predict and optimize prognosis, and subsequent care management for individuals with SCI.
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    Microrna Regulation On The Expression Of CD38 And Other Asthma Related Genes In Human Airway Smooth Muscle Cells
    (2015-04) Dileepan, Mythili
    CD38 is a multifunctional enzyme that regulates intracellular calcium ([Ca++]i ) homeostasis. It is expressed in airway smooth muscle (ASM) cells where it elevates [Ca++]i through its enzymatic product cyclic ADPribose (cADPR) and increases ASM contractility. Increased expression of CD38 in the ASM cells derived from the asthmatic patients (AS-HASM) and attenuated airway hyperresponsiveness to contractile stimuli shown by CD38-/- mice implicate the importance of CD38 in asthma. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is considered to be an important mediator for airway pathology in asthma. The reason for the differential expression of TNF-alpha-induced-CD38 in AS-HASM cells, does not involve transcriptional regulation of CD38 which is through signaling pathways mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K), and transcription factors nuclear factor kappa-B (NF-kB) and AP-I. Thus I hypothesized that post-transcriptional regulation of CD38 by microRNAs account for the differential expression of TNF-alpha induced -CD38 in AS-HASM cells. Among the several potential microRNAs predicted for CD38 by microRNA target-predicting algorithms, I selected miR-140-3p and miR-708 for further studies, as these showed differential expression in the AS-HASM cells compared to those from healthy subjects. Overexpression of these either microRNAs in ASM cells inhibited the TNF-alpha induced expression of CD38 at messenger RNA (mRNA) and protein levels. Luciferase-reporter assays with a mutated 3'UTR of the CD38 transcript confirmed the specific target sites for both microRNAs. Transcript stability assays revealed that mRNA degradation is not the mechanism underlying regulation by microRNAs. Examination of the expression and activation levels of proteins in the upstream signaling pathways of CD38 revealed that miR-140-3p, by inactivating p38 MAPK and NF-kB, and miR-708, by inactivating c-Jun N-terminal kinase (JNK) MAPK and Akt by elevating the expression of their phosphatases MKP-1 and PTEN respectively, control the expression of CD38 indirectly. Further, we found that miR-708 downregulates the expression of many chemokines and inhibits the serum induced proliferation of human ASM cells. We conclude that both microRNAs have therapeutic potential in controlling asthma related symptoms through regulating the expression of CD38 and chemokines and controlling the proliferation of human ASM cells.
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    Novel methods for surveying reservoir hosts and vectors of Borrelia burgdorferi in Northern Minnesota
    (2015-10) Seifert, Veronica
    Lyme disease is the most prevalent tick-borne disease in North America and presents challenges to clinicians, researchers and the public in diagnosis, treatment and prevention. Lyme disease is caused by the spirochete, Borrelia burgdorferi, which is a zoonotic pathogen obligate upon hematophagous arthropod vectors and propagates in small mammal reservoir hosts. Identifying factors governing zoonotic diseases within regions of high-risk provides local health and agricultural agencies with necessary information to formulate public policy and implement treatment protocols to abate the rise and expansion of infectious disease outbreaks. In the United States, the documented primary reservoir host of Lyme disease is the white-footed mouse, Peromyscus leucopus, and the arthropod vector is the deer tick, Ixodes scapularis. Reducing the impact of Lyme disease will need novel methods for identifying both the reservoir host and the tick vector. The reservoir host, Peromyscus leucopus is difficult to distinguish from the virtually identical Peromyscus maniculatus that also is present in Northern Minnesota, a region where Lyme disease is endemic. Collection of the Ixodes tick, the Lyme disease vector, is difficult as this is season dependent and differs from year to year. This study develops new strategies to assess the extent of Borrelia burgdorferi in the local environment of Northern Minnesota. A selective and precise method to identify Peromyscus species was developed. This assay provides a reliable and definitive method to identify the reservoir host, Peromyscus leucopus from a physically identical and sympatric Peromyscus species, Peromyscus maniculatus. A new strategy to collect ticks for measuring the disbursement of Borrelia was employed. Students from local high schools were recruited to collect ticks. This strategy increased the available manpower to cover greater terrain, provided students with valuable experience in research methodology, and highlighted the prospect of increasing community engagement in university-based research projects.
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    Regulation and Effects of Heme-Oxygenase-1 Expression in Chronic Inflammation.
    (2010-06) Beckman, Joan Denise
    Heme oxygenase-1 (HO-1) enzyme plays critical role in metabolizing the excess heme generated during hemolysis in pathological conditions, such as sickle cell disease. We and others have previously demonstrated that during chronic intravascular hemolysis the expression of HO-1 protein is not sufficient to reduce the oxidative burden of free heme in the vasculature, leading to oxidative stress and vascular inflammation. This proposal examined two areas critical to the understanding of HO-1 expression and function during inflammation: the role of post-transcriptional regulation in control of protein expression and the importance of its by-product carbon monoxide (CO) in mediating anti-inflammatory, anti-apoptotic effects. The research utilized a murine sickle model which has perturbations of heme catabolism leading to oxidative stress and inflammation. Studies in this model will test whether HO-1, or its by-products, can therapeutically alter the natural history of sickle cell disease. In addition, cell culture models in which heme levels are controlled were used to explore microRNA regulation of heme oxygenase-1 (HO-1) expression. Combined these experimental endeavors aim to identify new aspects of HO-1 research.
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    Regulation and Function of the Phosphatase PHLPP2 in Leukemia
    (2017-08) Yan, Yan
    PHLPP2, a member of the PHLPP phosphatase family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little was known, however, regarding its regulation and function in hematological malignancies. The first half of this dissertation describes a novel miR-17~92-based mechanism for repression of PHLPP2 protein expression in late differentiation stage acute myeloid leukemia (AML) subtypes. ATRA (all-trans retinoic acid), a drug used for terminally differentiating AML subtypes, was able to induce PHLPP2 protein levels and phosphatase activity significantly by suppressing miR-17-92 expression. The effect of ATRA on miR-17~92 expression was mediated through its target, transcription factor C/EBP, which interacts with the intronic promoter of the miR-17~92 gene cluster to inhibit its transactivation. The second half of this dissertation provides evidence for a novel metabolic function for PHLPP2 and describes the first identification of the energy sensing kinase, AMPK, as a unique PHLPP2 substrate. PHLPP2 could dephosphorylate phospho-AMPK (T172) both intracellularly and in vitro. PHLPP2 silencing protected Jurkat T-ALL cells from an apoptotic response to low glucose-induced metabolic stress through activation of AMPK signaling. The pro-survival effect of PHLPP2 knockdown under metabolic stress is likely mediated through AMPK-activated fatty acid oxidation. PHLPP2 regulates AMPK phosphorylation in a variety of tumor types and is the first specific AMPK phosphatase to be identified. These studies on PHLPP2 expression and function expand current knowledge and understanding of the role of PHLPP phosphatases in cancer, and particularly in leukemia. In light of the pivotal role played by AMPK in a number of metabolic diseases, the PHLPP2/AMPK axis is also expected to provide new insights into therapies targeting these diseases.