Browsing by Subject "Regulation"
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Item MICRO RNA MEDIATED REGULATION OF MIEN1 IN PROSTATE CANCER(2013-04-12) Rajendiran, SmrithiPurpose: MIEN1 is a membrane bound signaling molecule that triggers downstream signaling through the AKT/NF-𝛋B pathway by up-regulating key proteases and has a role in migration and invasion of cancer cells. The overall objective of this study is to identify the mechanisms that lead to the differential regulation of MIEN1 in normal and cancer cells. Though there are multiple mechanisms that are commonly studied, here, we propose to focus on the post-transcriptional regulation of MIEN1. Our data leads to the hypothesis that MIEN1 is post-transcriptionally regulated by a specific microRNA (miR) that is down-regulated in cancer, thus explaining aberrant increased expression of MIEN1 in cancer. Methods: We have performed miR in silico analysis, expression profiling, northern blotting, qPCRs, western blotting, luciferase reporter assays, migration and invasion assays, colony formation assays and stability assays to determine the effects of the miR on different aspects of MIEN1 regulation. Results: Our data indicate that MIEN1 is post-transcriptionally regulated by a specific miR which is lost in cancer cells. This miR is highly expressed in normal cells compared to a decrease in various cancer cells and this expression is inversely correlated to the expression of MIEN1. Introduction of the mimic (precursor) or inhibitor (antagomiR) led to a decrease or increase in MIEN1 expression respectively at both the RNA and protein levels. The downstream effectors were also similarly affected. The luciferase activity significantly reduced when the 3'UTR of MIEN1 was transfected with the target miR compared to the control miR, validating the direct binding of the miR. The miR decreases the migration and invasion of cells as well as the stability of MIEN1 RNA. There was a significant reduction in the colony formation capabilities and the morphology of the colonies differed in cells that were transfected with the miR along with a decrease in E-cadherin, suggesting potential involvement of the miRNA in reducing epithelial to mesenchymal transition and hence inhibiting metastasis. Conclusions: Our results demonstrate that aberrant expression of MIEN1 in cancer is attributed to a specific miR. We are currently exploring the potential of using this miR as a biomarker in prostate cancer. Since the importance of MIEN1 as a key signaling molecule in cancer is well established, understanding the mechanisms involved in its regulation will aid in designing novel and effective therapeutic strategies to treat cancer patients.Item Promoting Good Clinical Practice: Application of Regulatory Binders in a Physical Therapy Research Setting(2012-12-01) de Guzman, Maria-Racella; Patricia GwirtzThe American Physical Therapy Association, which has become one of the most recognized organizations representing the profession, continues to report the expansion of research in the field. As part of the Vision 2020 strategic plan, basic clinical research is mentioned to be an element essential to the physical therapy profession.12 The science behind physical therapy has existed for years with research starting in the early 1940s. This included the start of the clinical trials for the Salk vaccine to eradicate polio in the United States by the 1960s.13 According to the APTA, physical therapy is “a dynamic profession with an established theoretical and scientific base and widespread clinical application in the restoration, maintenance, and promotion of optimal physical functions”.14 The term “physical therapy” is also synonymous with the word “physiotherapy”. The practitioner of PT is known as a physiotherapist or a physical therapist. Physical therapists undergo proper higher education, licensure, and continuing education courses to maintain their role in the most current and up-to-date techniques and services.14 In addition, physical therapists assume a leadership role in patient rehabilitation, prevention, and health maintenance. Lastly, physical therapists help in the development of health care policy by ensuring that services of PT are available, accessible, and optimal.14 Minimal knowledge exists regarding how federal regulations apply to physical therapy research, but there is the justifiable assumption that all federal regulations should apply. Only a modest recognition is identified from the local IEC/IRB committees that overlook human subject clinical trials in physical therapy. The 2011 strategic plan of the Section of Research (SOR) in the APTA emphasizes the effort of increasing research education opportunities, but does not overlook the responsibility of the physical therapy researcher in practicing good clinical practices for the protection of human subjects in research trials.12 Twenty-first century medicine strives to evolve into an evidence-based practice, requiring real evidence that the determined way of treatment is the best option for the patient.15 The APTA has made recognition on its website that the PT profession should equally do the same. The key topic of implementing evidence-based practice in PT is emphasized in the Clinical Research Agenda. The goal of the APTA is to guide every practicing PT to understand that research is important to their clinical practice. Younger generation physical therapists should participate in research projects so that the future of PT practice is built upon factual evidence rather than experience.16 The main goal is that research should establish clinical practice, and that treatment should be decided upon evidence. Lastly, research evidence should be valid. Valid and qualitative evidence in research is important to physical therapy research. “Quality,” defined by the ISO 9000 addresses this as a set of standards in which an organization fulfills customer quality requirements and applicable regulatory requirements.17 Aiming to enhance customer satisfaction and achieving continual improvement are also acknowledged in the ISO. Quality evidence is important in clinical trials because the core components of research help ensure that patient protection follow the compliance of ICH GCP, and validates the integrity of data. The customers of clinical trials are those that benefit from the development of drugs, devices, and methods of preventative medicine, whether they are the research subjects, patients with a particular illness for which the study is being investigated for, physicians treating the patients, or for society.18Item Protein Kinase C-eta Signalling in Breast Cancer(2013-12-01) Pal, Deepanwita; Basu, AlakanandaPal, Deepanwita, Protein kinase C-eta signaling in breast cancer. Doctor of Philosophy (Biochemistry and Molecular Biology), November, 2013, 117 pp, 14 illustrations, 260 References Protein kinase C-eta (PKCη) is a novel member of the PKC family that is important for several cellular processes. PKCη is overexpressed in breast cancer and has been associated with chemotherapeutic resistance. PKCη is the only phorbol ester-sensitive PKC isozyme that resists downregulation upon prolonged treatment with tumorpromoting phorbol esters suggesting its unique regulation. The purpose of this dissertation is to elucidate the mechanism of PKCη regulation and its functional relevance in breast cancer. We have shown that PKCη is upregulated by several structurally and functionally distinct PKC activators in contrast to other PKC isozymes. Activator-induced upregulation of PKCη was associated with its phosphorylation. Our results indicate that novel PKCs are involved in the upregulation of PKCη by PKC activators. We also made a novel observation that PKCη is downregulated via two distinct mechanisms. While inhibition of PKC caused the downregulation of PKCη via proteasome-independent pathway, inhibition of PDK1 led to PKCη downregulation via proteasome-dependent pathway. We further demonstrated that PKCη is important for the growth and survival of breast cancer cells. The unique regulation of PKCη and its implications on breast cancer growth and survival suggests that this pathway could be selectively exploited for targeted therapies for breast cancer.Item Regulation and Characterization of Cardiac Phosphoinositide-Specific Phospholipase C (PLC) Isoenzymes(1997-12-01) Wang, Juan; Eugene E. Quist; Thomas Yorio; Ming-Chi WuWang, Juan, Regulation and Characterization of Cardiac Phosphoinositide-Specific Phospholipase C Isoenzymes. Master of Biomedical Science, Dec., 1997, 79 pp., 20 illustration, bibliography, 62 titles. It is hypothesized that myocardial phosphoinositide-specific phospholipase C (PLC) isoenzymes are regulated by physiological intracellular Ca2+ and by cytosol-membrane translocation. The regulation and identification of PLC isoenzymes in rat and dog ventricular subcellular fractions were studied. PLC-β1, PLC-β3 and PLC-δ1 were identified in rat and dog cytosol and microsomal membranes by chromatographic separation, enzyme assays and western blotting. Truncated PLC-β isoforms with molecular weights of 69 kDa and 114 kDa were isolated from rat and dog cytosol, respectively. Species differences in the relative distribution of PLC isoenzymes were evident as PLC-δ dominant in rat whereas PLC-β isoenzymes were dominant in dog. A 91 kDa cytosolic protein which did not contain PLC activity alone markedly led to PLC activation when combined with microsomes. The activator protein was immunoprecipitated with an anti-PLC-δ identifying this activator as an inactive PLC-δ isoenzyme. These studies indicate that cytosolic PLC-δ may be activated by translocating to membranes. In addition, proteolysis may be involved in long term activation of cytosolic PLC isoenzymes. Further studies will be required to resolve the physiological significance of these modes of cardiac PLC activation.Item Regulation of Endothelin-1 (ET-1) Synthesis and Secretion at the Outer Blood Retinal Barrier(2003-08-28) Narayan, Santosh; Thomas Yorio; Glenn Dillon; Michael W. MartinRegulation of Endothelin-1 (ET-1) Synthesis and Secretion at the Outer Blood-Retinal Barrier. Santosh Narayan, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center Fort Worth, TX 76102. Summary The retinal pigment epithelium (RPE) constitutes the outer blood retinal barrier at the posterior segment of the eye. The RPE provides metabolic support to the photoreceptors in the neural retina. A breakdown in the barrier supported by RPE is a hallmark in several retinopathies including proliferative vitreoretinopathy, choroidal neovascularization and macular edema. Characteristic to all epithelial cells, mature RPE cells display a polarized phenotype both in culture (ARPE-19 cells) and in vivo, with specific apical and basolateral domains. This provides a testable model to study the RPE in vitro. The purpose of this study was to characterize the RPE as a source for endothelin-1, using both in vitro and in situ models. Endothelins (ET-1,-2, and -3) are known regulators of vascular tone, that are produced at sites close to their target, ET-1, being a potent vasoconstrictor may be involved in regulating blood supply to the choroid and the neural retina. We identified the RPE to be a major source for endothelin-1 (ET-1) in situ in the human retina as well as in pigmented and albino rat retinas. Additionally, using a cell-culture model of mature polarized ARPE-19 cells, we studied the synthesis and expression of ET-1 in response to muscarinic receptor stimulation, TNF-α and more recently to thrombin. We have identified other components involved in the synthesis and turnover of ET-1 in ARPE-19 cells including the proprotein convertase-furin, endothelin-converting enzyme-1 and its isoforms and the endothelin receptor B subtype. ARPE-19 cells grown on collagen filters helped determine if secretion of ET-1 was polarized or discriminative towards either the apical or basolateral surface. We consistently observed changes in cell shape and tight junction disassembly in ARPE-19 cells following TNF-α and thrombin addition. Additionally, thrombin caused an increase in preproET-1 mRNA at earlier time points that was dependent on the rhokinase (ROCK1/2) pathway. We report a novel signaling mechanism for regulating preproET-1 mRNA and mature ET-1 secretion in ARPE-19 cells that involves the thrombin receptor (protease activated receptor-1/PAR-1) dependent activation of the rho/ROCK1/2 signaling pathway that may also be involved in thrombin induced changes in the cytoskeleton. In conclusion, the RPE may be an important source for ET-1 at the posterior segment of the eye, secretion of which is greatly enhanced by substances that promote breakdown of blood retinal barriers, inflammation and changes in the RPE cytoskeleton. In conclusion, the RPE may be an important source for ET-1 at the posterior segment of the eye, secretion of which is greatly enhanced by substances that promote breakdown of blood retinal barriers, inflammation and changes in the RPE cytoskeleton. ET-1 secreted by the RPE, under physiological conditions may provide an autoregulatory mechanism for controlling blood flow at the outer blood retinal barrier. Excessive ET-1 secretion following breakdown of the barrier may either promote wound repair or may mediate further damage to the retina, the substrates of which are presently unknown. Future experimental approaches are planned to address these possibilities.