Laboratory and Basic Science Research Commons^™

Itgb1 Knockdown Alters Apoptosis And Proliferation In Ewing Sarcoma Cells, Aditi Kumar, Magdalena Saad, Terry J. Shackleford

Mechanisms of Disease

Integrin beta 1 (ITGB1) is an adhesion receptor that link cells to the extracellular matrix and regulates pathways involved in survival, proliferation, and migration. In order to understand its role in Ewing Sarcoma, we applied CRISPR-Cas9 with two guide RNAs to knock down ITGB1 in ES8 cells. We then assessed changes in cell growth, death, movement, and gene expression using Incucyte live-cell imaging, colony formation assays, wound healing assays, and qRT-PCR. qRT-PCR showed partial ITGB1 reduction, with ITGB1_gRNA2 producing the strongest decrease. ITGB1_gRNA2 also led to a small increase in caspase activity and a 20-30% increase in proliferation at the …

Molecular Event In Hrp Apoptosis, Laura Valdez

Research Symposium

Background: Human retinal pericytes (HRP) are contractile cells adjacent to and provide support for endothelial cells (EC) of capillaries, which are essential in the regulation of retinal vasculature. Early stages of DR are characterized by the loss of HRP, which leads to the development of advanced-stage pathology including angiogenesis. Although much is known about the etiology of DR, the apoptotic pathway that incites HRP loss remains unclear. Our preliminary studies reveal that monocyte-derived macrophages secrete TGF-β1, which induces the expression and secretion of a TGF-β1-Induced, pro-apoptotic BIGH3 protein (TGF-β–Induced Gene Human Clone 3) leading to apoptosis of HRP Eye, 30(12), …

Role Of Nurd In Acute Leukemia Cell Survival, Javeria Aijaz

Theses and Dissertations

Depletion of the ATPase component of the Nucleosome Remodeling and Deacetylase (NuRD) complex, CHD4, reduces acute myeloid leukemia (AML) cell survival. This study identified other NuRD components, as potential therapeutic targets for disrupting protein-protein interactions within NuRD. In addition to AML, we established that T-cell Acute Lymphoblastic Leukemia (T-ALL) cell lines responded similarly to CHD4 depletion.

Greater than 90% depletion of either MBD2 or MBD3 (the mutually exclusive two DNA binding NuRD paralogues) – was unremarkable, but complete depletion of MBD3 increased apoptosis and genotoxic sensitivity. Combined depletion of MBD-NuRD proteins augmented apoptosis observed with complete MBD3 depletion - indicating …

Guidelines And Recommendations On Yeast Cell Death Nomenclature, Didac Carmona-Gutierrez, Maria Anna Bauer, Andreas Zimmermann, Andrés Aguilera, Nicanor Austriaco, Kathryn Ayscough, Rena Balzan, Shoshana Bar-Nun, Antonio Barrientos, Peter Belenky, Marc Blondel, Ralf J Braun, Michael Breitenbach, William C Burhans, Sabrina Büttner, Duccio Cavalieri, Michael Chang, Katrina F Cooper, Manuela Côrte-Real, Vítor Costa, Christophe Cullin, Ian Dawes, Jörn Dengjel, Martin B Dickman, Tobias Eisenberg, Birthe Fahrenkrog, Nicolas Fasel, Kai-Uwe Fröhlich, Ali Gargouri, Sergio Giannattasio, Paola Goffrini, Campbell W Gourlay, Chris M Grant, Michael T Greenwood, Nicoletta Guaragnella, Thomas Heger, Jürgen Heinisch, Eva Herker, Johannes M Herrmann, Sebastian Hofer, Antonio Jiménez-Ruiz, Helmut Jungwirth, Katharina Kainz, Dimitrios P Kontoyiannis, Paula Ludovico, Stéphen Manon, Enzo Martegani, Cristina Mazzoni, Lynn A Megeney, Chris Meisinger, Jens Nielsen, Thomas Nyström, Heinz D Osiewacz, Tiago F Outeiro, Hay-Oak Park, Tobias Pendl, Dina Petranovic, Stephane Picot, Peter Polčic, Ted Powers, Mark Ramsdale, Mark Rinnerthaler, Patrick Rockenfeller, Christoph Ruckenstuhl, Raffael Schaffrath, Maria Segovia, Fedor F Severin, Amir Sharon, Stephan J Sigrist, Cornelia Sommer-Ruck, Maria João Sousa, Johan M Thevelein, Karin Thevissen, Vladimir Titorenko, Michel B Toledano, Mick Tuite, F-Nora Vögtle, Benedikt Westermann, Joris Winderickx, Silke Wissing, Stefan Wölfl, Zhaojie J Zhang, Richard Y Zhao, Bing Zhou, Lorenzo Galluzzi, Guido Kroemer, Frank Madeo

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic …

The Role Of Mapk And Scf In The Destruction Of Med13 In Cyclin C Mediated Cell Death, David C Stieg, Stephen D Willis, Joseph Scuorzo, Mia Song, Vidyaramanan Ganesan, Randy Strich, Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Departmental Research

In response to stress, the yeast¹ and mammalian² cyclin C translocate from the nucleus to the cytoplasm, where it associates with the GTPase Drp1/Dnm1 to drive mitochondrial fragmentation and apoptosis. Therefore, the decision to release cyclin C represents a key life or death decision. In unstressed cells, the cyclin C‐Cdk8 kinase regulates transcription by associating with the Mediator of RNA polymerase II. We previously reported that the Mediator component Med13 anchors cyclin C in the nucleus³. Loss of Med13 function leads to constitutive cytoplasmic localization of cyclin C, resulting in fragmented mitochondria, hypersensitivity to stress and …

Mechanistic Insights Into The Regulation Of Mitochondrial Fission By Cyclin C, Vidyaramanan Ganesan, Katrina F Cooper, Randy Strich

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Cyclin C is a component of the mediator complex of RNA polymerase II that localizes to the nucleus under normal conditions. In response to stress, cyclin C translocates to the cytosol and mitochondria and mediates stress‐induced mitochondrial fission and apoptosis. The molecular mechanisms by which cyclin C induces mitochondrial fission are unknown. Using in vitro experimental approaches, we sought to investigate the mechanistic basis of cyclin C mediated mitochondrial fission.

Transcriptional Regulation By Dax-1 In Pluripotent And Differentiated Cells, Alexandra C. Maramba

Master's Theses

DAX-1, an orphan nuclear hormone receptor, acts mainly as a repressor through transcriptional protein complexes. Its unique structure and specific expression raises questions as to what its precise interactions are and how it mediates its repressive function. While it is known to play a role in sexual development and adrenal insufficiency, expression in certain types of cancer suggests additional functions and interactions. Knock in of DAX-1 into a low-DAX-1 expressing cancer cell line has been previously observed to increase apoptosis, while, inversely, down in a high-DAX-1 expressing cancer cell line shows a decrease in apoptosis. Target genes that belong to …

The Role Of Mammalian Cyclin C In Stress Response And Thyroid Disease Progression, Kun Wang

Graduate School of Biomedical Sciences Theses and Dissertations

The cellular response to stress involves changes in gene expression programs as well as the remodeling of subcellular organelles including the mitochondria. Important for this study, the mitochondria undergo extensive fragmentation when the cell is exposed to a variety of stressors. The cyclin C-Cdk8 kinase is a highly conserved transcription factor that associates with the RNA polymerase II holoenzyme. Using cyclin C knockout (CCNC^-/-) mouse embryonic fibroblast (MEF) cells, I revealed that cyclin C is required for the extensive mitochondrial fragmentation following treatment with pro-oxidants or the anti-cancer drug cisplatin. However, Cdk8 is not required for this process …

Modulation Of Bax/Bak Dependent Apoptosis By Sirtuin 3 And Mitochondrial Permeability Transition By Sirtuin 4, Manish Verma

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondria are dynamic organelles that regulate a myriad of cellular functions, including energy production and metabolic regulation. Mitochondria are also a critical regulator of cell death signaling cascades modulating both apoptotic and necrotic cell death. However, what determines which cell death pathway is activated is still unclear. The mitochondrial/intrinsic pathway of apoptosis is dependent on the activation of pro-apoptotic proteins, Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). Once the integrity of outer mitochondrial membrane (OMM) is compromised, pro-apoptotic intermembrane space proteins like cytochrome c, Smac/Diablo, Omi/HtrA2 and AIF are released into the cytoplasm, which activates the post-mitochondrial …

Med13p Prevents Stress-Independent Mitochondrial Hyperfragmentation And Aberrant Apoptosis Activation In Saccharomyces Cerevisiae By Controlling Cyclin C Nuclear Localization, Svetlana Khakhina

Graduate School of Biomedical Sciences Theses and Dissertations

During aging, and as a result of environmental changes, cells are exposed to elevated levels of reactive oxygen species (ROS). High ROS levels induce lipid oxidation, protein aggregation, mitochondrial hyperfragmentation, DNA damage and programmed cell death (PCD), also called apoptosis. PCD is a highly regulated process and its misregulation has been linked to neurodegenerative diseases and cancer development.

Our hypothesis is that cyclin C plays a role in the initiation of apoptosis. During normal conditions, cyclin C represses the transcription of stress response genes (SRG). In response to stress, cyclin C translocates to the cytoplasm where it facilitates mitochondrial hyperfragmentation …

Novel Roles Of Replication Protein A Phosphorylation In Cellular Response To Dna Damage, Moises A. Serrano

Electronic Theses and Dissertations

Human replication protein A (RPA) is an eukaryotic single-stranded DNA binding protein directly involved in a variety of DNA metabolic pathways including replication, recombination, DNA damage checkpoints and signaling, as well as all DNA repair pathways. This project presents 2 novel roles of RPA in the cellular response to DNA damage. The first elucidates the regulation of RPA and p53 interaction by DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) in homologous recombination (HR). HR and nonhomologous end joining (NHEJ) are 2 distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-PK, the …

Caffeine Supplementation And Moderate Intensity Exercise Modulates The Cytotoxic Lymphocyte Subset (Cd+8) In Naive And Tolerant Individuals, Elizabeth Ann Fedor

Masters Theses & Specialist Projects

The purpose of this investigation was to determine the effects of caffeine supplementation on caffeine tolerant and caffeine naïve individual’s lymphocyte counts, apoptosis and migration levels. In addition, effects of exercise on post-caffeine ingestion lymphocyte counts, apoptosis and migration levels were determined. It was hypothesized that caffeine would alter the immune system cell counts, but that exercise would be able to restore the immune system to homeostasis. Seventeen Western Kentucky University students were tested (males n=7, females n=10; n=7: caffeine tolerant= 200mg or more per day group, n=9: caffeine naïve= 50mg or less per day group). In this double-blind investigation, …