DOCSLIB.ORG

Implications for the 8P12-11 Genomic Locus in Breast Cancer Diagnostics and Therapy: Eukaryotic Initiation Factor 4E-Binding Protein, EIF4EBP1, As an Oncogene

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Implications for the 8P12-11 Genomic Locus in Breast Cancer Diagnostics and Therapy: Eukaryotic Initiation Factor 4E-Binding Protein, EIF4EBP1, As an Oncogene Medical University of South Carolina MEDICA MUSC Theses and Dissertations 2018 Implications for the 8p12-11 Genomic Locus in Breast Cancer Diagnostics and Therapy: Eukaryotic Initiation Factor 4E-Binding Protein, EIF4EBP1, as an Oncogene Alexandria Colett Rutkovsky Medical University of South Carolina Follow this and additional works at: https://medica-musc.researchcommons.org/theses Recommended Citation Rutkovsky, Alexandria Colett, "Implications for the 8p12-11 Genomic Locus in Breast Cancer Diagnostics and Therapy: Eukaryotic Initiation Factor 4E-Binding Protein, EIF4EBP1, as an Oncogene" (2018). MUSC Theses and Dissertations. 295. https://medica-musc.researchcommons.org/theses/295 This Dissertation is brought to you for free and open access by MEDICA. It has been accepted for inclusion in MUSC Theses and Dissertations by an authorized administrator of MEDICA. For more information, please contact [email protected]. Implications for the 8p12-11 Genomic Locus in Breast Cancer Diagnostics and Therapy: Eukaryotic Initiation Factor 4E-Binding Protein, EIF4EBP1, as an Oncogene by Alexandria Colett Rutkovsky A dissertation submitted to the faculty of the Medical University of South Carolina in fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies Biomedical Sciences Department of Pathology and Laboratory Medicine 2018 Approved by: Chairman, Advisory Committee Stephen P. Ethier Co-Chairman Robin C. Muise-Helmericks Amanda C. LaRue Victoria J. Findlay Elizabeth S. Yeh Copyright by Alexandria Colett Rutkovsky 2018 All Rights Reserved This work is dedicated to the fight against cancer. ACKNOWLEDGEMENTS I would like to acknowledge the Hollings Cancer Center and many departments at the Medical University of South Carolina for investing the resources and training required to complete the research presented. This research was also supported in part by the Department of Pathology and Laboratory Medicine at the Medical University of South Carolina and grants from the National Institute of Health awarded to Dr. Stephen Ethier. I would like to thank the Ethier laboratory, both past and present, which have been instrumental in the presented work. I stand on the shoulder of giants, thankful for the many brilliant and dedicated scientists before me and alongside me, especially collaborative, combined, and applied knowledge which allows the mining of big data presented. I am truly grateful for the many faculty and staff at the Medical University of South Carolina and Appalachian State University who have dedicated much effort towards my scientific training, passions, and procurement of resources detrimental to the work presented. Special thanks to my committee members for their wealth of knowledge, advice, help, and ideas, especially Dr. Stephen Ethier and Dr. Stephen Guest who were instrumental in teaching me the skills necessary to complete this work. Materials relied on the unwavering support of Dr. Robin C. Muise-Helmericks and Dr. Elizabeth Yeh. This research would not have been possible without the support from my family, friends, and the community. iv TABLE OF CONTENTS List of Tables viii List of Figures ix-xii Abstract xiii Chapter I: Background 1 Human mammary tissue and breast cancer 2-12 Gene amplification in cancer 13-15 Driving oncogenes on amplicons 16-19 Amplification of the 8p12-11 genomic locus 20-22 Eukaryotic Initiation Factor 4E-Binding Protein (4EBP1, EIF4EBP1) 23-34 4EBP1 and mTOR in mRNA translation 34-36 Genomic location of 4EBP1 36 Conservation among species demonstrates 4EBP1 necessity 37-38 The behavior and expression of 4EBP1 in normal cells 39-40 4EBP1 is highly overexpressed in many malignant tumor types 41-42 4EBP1 transcripts are rarely mutated 43-44 What causes the increase in 4EBP1 transcript levels? 45 Viruses 45-46 Transcription Factors 47-49 Gene amplification 50-52 4EBP1 in cell signaling in breast cancer 53-57 Chapter II: Materials and Methods 58-74 Chapter III: Results 75 v Evidence suggests 4EBP1 is important to many cancer types 76-77 EIF4EBP1 expression levels correlate with reduced relapse free survival 78-79 in human breast cancer 4EBP1 is not required for normal mammary cell proliferation 80-81 4EBP1 is highly expressed and phosphorylated in 8p11-12 breast cancer 82-84 cells Phosphorylated AKT levels and phosphorylated S6 levels across the 85-88 models 4EBP1 knockdown is detrimental to ER+ 8p11-12 breast cancer cells 89-90 Downregulation of 4EBP1 in ER+ 8p11-12 breast cancer cells causes 91-92 cell cycle arrest 4EBP1 knockdown inhibits proliferation of ER- 8p11-12 amplified breast 93-94 cancer cells The effects of 4EBP1 knockdown on non-amplicon bearing cancer cell 95-97 models Loss of 4EBP1 does not promote changes in apoptosis associated 98-99 proteins The decrease in ERα with 4EBP1 loss does not alter protein levels of 100-102 NSD3 or ASH2L 4EBP1 knockdown decreases MYC levels 103-105 Chapter IV: Discussion 106-116 Chapter V: Future Directions 117 Future directions related to 4EBP1 in cell dynamics 118-121 vi A role for 4EBP1 in transformation 121-122 A role for 4EBP1 in growth factor independence 122-123 A role for 4EBP1 in anchorage-independent growth 123 A role for 4EBP1 to bypass senescence 124-125 A role for 4EBP1 to influence cancer stemness 125-126 A role for 4EBP1 in apoptosis 126 A role for 4EBP1 in adipogenesis and lipid metabolism 127-128 A role for wild-type 4EBP1 transcripts in malignant neoplasms 129 A role for 4EBP1 as an antiviral strategy 129-130 A role for 4EBP1 in translation 130-131 A role for 4EBP1 in precision medicine 131-135 Chapter VI: Appendix 136 The 8p11-12 amplicon candidate oncogenes in breast cancer 137 Data mining results of 8p11-12 oncogenes in cancer 138-181 Summarized gene ontologies for 8p11-12 gene involvement in cancer 182-183 transformation processes Available lentiviral 8p11-12 gene expression plasmids for future studies 183-184 Discussion of 8p11-12 data mined results 185-186 References 187-214 Biographical Sketch 215 vii LIST OF TABLES Table 1. Highlights of genomic, clinical and proteomic features of molecular 7 breast cancer subtypes from The Cancer Genome Atlas Network (2012) Table 2. Breast cell line requirements and growth conditions 60 Table 3. Required additions to medium for growth of cell lines in culture 61 Table 4. Short-hairpin RNA plasmids from The RNAi Consortium’s (TRC) 63-64 genome-wide lentiviral human libraries Table 5. The human ORFeome Collaboration entry clone cDNA plasmid 67 information Table 6. Lentiviral cDNA expression plasmids 68 Table 7. Recruiting clinical trials for FGFR inhibition in breast cancer 165 Table 8. Available lentiviral cDNA expression plasmids 184 viii LIST OF FIGURES Figure 1. The incidence of clinical breast cancer subtypes in the United States 4 of America Figure 2. PAM50 intrinsic breast cancer molecular subtypes 6 Figure 3. Transcript expression of the estrogen receptor (ESR1) in breast 10 tumors based on the patient’s age Figure 4. ESR1 mRNA expression increases with age with no increase in ESR1 11 copy number in breast cancer patients Figure 5. ESR1 gene essentiality across cancer cell lines 12 Figure 6. Candidate oncogenes within the 8p11-12 genomic locus in breast 18-19 cancer Figure 7. Amplification of the 8p11-12 genomic region frequently occurs in 21 cancer Figure 8. The 8p11-12 amplicon genes can predict the survival of breast cancer 22 patients Figure 9. Eukaryotic Initiation Factor 4E-Binding protein (4EBP1) is defined as a 24 translational repressor Figure 10. Important interactions between 4EBP1 and other proteins 27 Figure 11. 4EBP1 acts as a convergence factor for multiple pathways 28 Figure 12. The demonstrated dynamic post-translational modification sites of 30 4EBP1 Figure 13. GeneTree for ENSGT00390000013843 (EIF4EBP1, 4EBP1) 38 Figure 14. 4EBP1 gene expression across 53 normal human tissues 40 Figure 15. TCGA data show many types of cancer overexpress 4EBP1 42 transcripts at high levels Figure 16. Frequency of 4EBP1 mutations in the TCGA cohorts 44 ix Figure 17. Eukaryotic Translation Initiation Factor 4E Binding Protein 49 (EIF4EBP1) transcription factor binding profiles Figure 18. EIF4EBP1 is amplified in human breast cancer 52 Figure 19. cBioPortal TCGA provisional breast cancer data results (2017) 56 showing the top mutational burdens and 4EBP1 alterations in breast cancer Figure 20. Eukaryotic Initiation Factor 4E-Binding Protein 1 (EIF4EBP1) is 57 overexpressed in malignant breast tumors when compared to normal tissue samples in The Cancer Genome Atlas breast cancer cohort Figure 21. EIF4EBP1 is a predicted driver of many cancer types 77 Figure 22. High 4EBP1 expression can predict the prognosis of breast tumor 79 patients Figure 23. 4EBP1 knockdown in normal mammary cells does not inhibit 81 proliferation and levels are comparable to normal primary isolated mammary epithelial cells Figure 24. 4EBP1 is highly expressed and phosphorylated in 8p11-12 breast 84 cancer cells Figure 25. Phosphorylated AKT levels and phosphorylated S6 levels across the 86 models Figure 26. Loss of 4EBP1 does not substantially alter phosphorylated S6 or 88 phosphorylated AKT levels Figure 27. 4EBP1 knockdown inhibits proliferation of ER+ 8p11-12 breast 90 cancer cells and decreases ER levels Figure 28. 4EBP1 knockdown leads to G0/G1 arrest in ER+ 8p11-12 breast 92 cancer cells Figure 29. 4EBP1 knockdown inhibits proliferation of ER- 8p11-12 breast cancer 94 cells Figure 30. 4EBP1 knockdown inhibits proliferation of MCF7 and T47D breast 96 cancer cells Figure 31. 4EBP1 knockdown inhibits proliferation of SUM-229 and SUM-149 97 breast cancer cells Figure 32. Knockdown of 4EBP1 does not drastically alter levels of apoptosis 99 associated proteins x Figure 33. Loss of ER by 4EBP1 knockdown does not alter the levels of NSD3 102 or ASH2L Figure 34. The influence of 4EBP1 on MYC protein levels and varying gene 104 expression levels of MYC family members Figure 35.
Load more

Recommended publications
  • Screening and Identification of Key Biomarkers in Clear Cell Renal Cell Carcinoma Based on Bioinformatics Analysis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Screening and identification of key biomarkers in clear cell renal cell carcinoma based on bioinformatics analysis Basavaraj Vastrad1, Chanabasayya Vastrad*2 , Iranna Kotturshetti 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. 3. Department of Ayurveda, Rajiv Gandhi Education Society`s Ayurvedic Medical College, Ron, Karnataka 562209, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignancy of the urinary system. The pathogenesis and effective diagnosis of ccRCC have become popular topics for research in the previous decade. In the current study, an integrated bioinformatics analysis was performed to identify core genes associated in ccRCC. An expression dataset (GSE105261) was downloaded from the Gene Expression Omnibus database, and included 26 ccRCC and 9 normal kideny samples. Assessment of the microarray dataset led to the recognition of differentially expressed genes (DEGs), which was subsequently used for pathway and gene ontology (GO) enrichment analysis.
    [Show full text]
  • The Role of the Mtor Pathway in Developmental Reprogramming Of
    THE ROLE OF THE MTOR PATHWAY IN DEVELOPMENTAL REPROGRAMMING OF HEPATIC LIPID METABOLISM AND THE HEPATIC TRANSCRIPTOME AFTER EXPOSURE TO 2,2',4,4'- TETRABROMODIPHENYL ETHER (BDE-47) An Honors Thesis Presented By JOSEPH PAUL MCGAUNN Approved as to style and content by: ________________________________________________________** Alexander Suvorov 05/18/20 10:40 ** Chair ________________________________________________________** Laura V Danai 05/18/20 10:51 ** Committee Member ________________________________________________________** Scott C Garman 05/18/20 10:57 ** Honors Program Director ABSTRACT An emerging hypothesis links the epidemic of metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD) and diabetes with chemical exposures during development. Evidence from our lab and others suggests that developmental exposure to environmentally prevalent flame-retardant BDE47 may permanently reprogram hepatic lipid metabolism, resulting in an NAFLD-like phenotype. Additionally, we have demonstrated that BDE-47 alters the activity of both mTOR complexes (mTORC1 and 2) in hepatocytes. The mTOR pathway integrates environmental information from different signaling pathways, and regulates key cellular functions such as lipid metabolism, innate immunity, and ribosome biogenesis. Thus, we hypothesized that the developmental effects of BDE-47 on liver lipid metabolism are mTOR-dependent. To assess this, we generated mice with liver-specific deletions of mTORC1 or mTORC2 and exposed these mice and their respective controls perinatally to
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Accumulation of Dephosphorylated 4EBP After Mtor Inhibition with Rapamycin Is Sufficient to Disrupt Paracrine Transformation By
    Oncogene (2014) 33, 2405–2412 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc SHORT COMMUNICATION Accumulation of dephosphorylated 4EBP after mTOR inhibition with rapamycin is sufficient to disrupt paracrine transformation by the KSHV vGPCR oncogene D Martin, Q Nguyen, A Molinolo and JS Gutkind Dysregulation of the PI3K/Akt/mTOR pathway is one of the most frequent events in human cancer. However, the clinical benefits of PI3K/Akt/mTOR inhibitors have not yet achieved their predicted potential in many of the most prevalent human cancers. Of interest, treatment of Kaposi’s sarcoma (KS) patients with rapamycin provided the first evidence of the antineoplastic activity of mTOR inhibitors in humans, becoming the standard of care for KS arising in renal transplant patients. Thus, the study of KS may provide a unique opportunity to dissect the contribution of specific mTOR downstream targets to cancer development. The KS- associated herpesvirus (KSHV) is the etiological agent for KS, and the KSHV-encoded oncogene viral-G protein-coupled receptor (vGPCR) promotes the potent activation of the PI3K-Akt-mTOR pathway by both direct and paracrine mechanisms. We focused on a direct target of mTOR, EIF4EBP1/2/3 (4EBP), which inhibits the translation of eukaryotic initiation factor 4E (eiF4E)-bound mRNAs. 4EBP phosphorylation by mTOR relieves its inhibitory activity, hence resulting in increased eiF4E-dependent mRNA translation. We developed a paracrine transformation model, recapitulating the cellular composition of KS lesions, in which vGPCR-expressing cells promote the rapid proliferation of endothelial cells, thus expressing KSHV-latent genes by the release of growth factors.
    [Show full text]
  • FGFR1 Fusion and Amplification in a Solid Variant of Alveolar Rhabdomyosarcoma
    Modern Pathology (2011) 24, 1327–1335 & 2011 USCAP, Inc. All rights reserved 0893-3952/11 $32.00 1327 FOXO1–FGFR1 fusion and amplification in a solid variant of alveolar rhabdomyosarcoma Jinglan Liu1,2, Miguel A Guzman1,2, Donna Pezanowski1, Dilipkumar Patel1, John Hauptman1, Matthew Keisling3, Steve J Hou2,3, Peter R Papenhausen4, Judy M Pascasio1,2, Hope H Punnett1,5, Gregory E Halligan2,6 and Jean-Pierre de Chadare´vian1,2 1Department of Pathology and Laboratory Medicine, St Christopher’s Hospital for Children, Philadelphia, PA, USA; 2Drexel University College of Medicine, Philadelphia, PA, USA; 3Department of Pathology and Laboratory Medicine, Hahnneman University Hospital, Philadelphia, PA, USA; 4Laboratory Corporation of America, The Research Triangle Park, NC, USA; 5Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA, USA and 6Department of Pediatrics, Section of Oncology, St Christopher’s Hospital for Children, Philadelphia, PA, USA Rhabdomyosarcoma is the most common pediatric soft tissue malignancy. Two major subtypes, alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma, constitute 20 and 60% of all cases, respectively. Approximately 80% of alveolar rhabdomyosarcoma carry two signature chromosomal translocations, t(2;13)(q35;q14) resulting in PAX3–FOXO1 fusion, and t(1;13)(p36;q14) resulting in PAX7–FOXO1 fusion. Whether the remaining cases are truly negative for gene fusion has been questioned. We are reporting the case of a 9-month-old girl with a metastatic neck mass diagnosed histologically as solid variant alveolar rhabdomyosarcoma. Chromosome analysis showed a t(8;13;9)(p11.2;q14;9q32) three-way translocation as the sole clonal aberration. Fluorescent in situ hybridization (FISH) demonstrated a rearrangement at the FOXO1 locus and an amplification of its centromeric region.
    [Show full text]
  • Adaptive Capabilities of the Pi3k/Akt/Mtor Pathway in Acute Myeloid Leukemia Revealed by the Use of Selective Inhibitors
    Alma Mater Studiorum – Università di Bologna SCIENZE BIOMEDICHE: PROGETTO 5 “SCIENZE MORFOLOGICHE UMANE E MOLECOLARI” Ciclo XXV SSD : BIO 16, SETTORE CONCORSUALE :05H1 ADAPTIVE CAPABILITIES OF THE PI3K/AKT/MTOR PATHWAY IN ACUTE MYELOID LEUKEMIA REVEALED BY THE USE OF SELECTIVE INHIBITORS Presentata da Jessika Bertacchini Relatori Prof.ssa Lucia Manzoli Prof.ssa Sandra Marmiroli Coordinatore Chiar. mo Prof. Lucio Cocco TABLE OF CONTENTS o ABSTRACT o INTRODUCTION o ACUTE MYELOID LEUKEMIA o PROGNOSIS AND GENETICS o DEREGULATED SIGNAL TRANSDUCTION PATHWAYS IN ACUTE MYELOID LEUKEMIA o PI3K/AKT/MTOR SIGNAL TRANSDUCTION PATHWAY IN ACUTE MYELOID LEUKEMIA o PI3K o AKT/PKB o Mtor o NEGATIVE REGULATION OF PI3K/AKT/mTOR PATHWAY o PI3K/AKT/mTOR PATHWAY AND SURVIVAL o PI3K/AKT/mTOR PATHWAY AND CELL CYCLE o PI3K/AKT/mTOR PATHWAY AND METABOLISM o PI3K/AKT/mTOR INHIBITORS o PI3K INHIBITORS o AKT INHIBITORS o mTOR INHIBITORS o TYROSINE KINASE RECEPTOR o TYROSINE KINASE RECEPTOR IN ACUTE MYELOID LEUKEMIA o AIMS o MATERIALS AND METHODS o PATIENTS DEMOGRAPHICS o CELL CULTURE AND DRUG TREATMENTS o ARRAY ASSEMBLY o RESULTS o RESULTS o DISCUSSION o BIBLIOGRA 1.ABSTRACT The objective of the study was to investigate the sensitivity of primary blasts from AML patients to PI3K/Akt/mTor inhibitors through reverse-phase protein microarray. Reverse-phase microarray assays using phosphospecific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. Mapping of deregulated kinases and protein signaling networks within tumors can provide a means to stratify patients with shared biological characteristics to the most optimal treatment, and identify drug targets. In particular, the PI3K/AKT/mTOR signaling pathways are frequently activated in blast cells from patients with acute myelogenous leukemia (AML), a neoplastic disorder characterized by the accumulation of genetically altered myelogenous cells displaying deregulated intracellular signalling pathways and aggressive clinical behavior with poor prognosis.
    [Show full text]
  • Comparative Mapping of Bovine Chromosome 27 with Human Chromosome 8 Near a Dairy Form QTL in Cattle
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Department of Agriculture: Agricultural Publications from USDA-ARS / UNL Faculty Research Service, Lincoln, Nebraska 2006 Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle E. E. Connor USDA-ARS M. S. Ashwell North Carolina State University at Raleigh R. Schnabel University of Missouri-Columbia J. L. Williams Roslin Institute (Edinburgh) Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub Part of the Agricultural Science Commons Connor, E. E.; Ashwell, M. S.; Schnabel, R.; and Williams, J. L., "Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle" (2006). Publications from USDA-ARS / UNL Faculty. 698. https://digitalcommons.unl.edu/usdaarsfacpub/698 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Original Article Cytogenet Genome Res 112:98–102 (2006) DOI: 10.1159/000087519 Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle E.E. Connor,a M.S. Ashwell,a,b R. Schnabel,c J.L. Williamsd a Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD; b North Carolina State University, Department of Animal Science, Raleigh, NC; c University of Missouri-Columbia, Animal Sciences Unit, Columbia, MO (USA); d Roslin Institute (Edinburgh), Roslin, Midlothian, Scotland (UK) Manuscript received 30 December 2004; accepted in revised form for publication by T.
    [Show full text]
  • Cellular and Molecular Investigations of Undiagnosed Neurometabolic
    CELLULAR AND MOLECULAR INVESTIGATIONS OF UNDIAGNOSED NEUROMETABOLIC DISORDERS Submitted in application for award of Doctor of Philosophy (PhD) Emma Reid Centre for Translational Omics Genetics and Genomic Medicine Institute of Child Health University College London August 2016 DECLARATION I, Emma Reid, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Where experimental or analytical work has been completed by others, this has been stated in the relevant section of this thesis. However, these instances are also listed below: • Alignment of sequencing reads to the reference genome, variant calling (Section 2.4.11) and ExomeDepth analysis (Section 3.3.3) of the gene panel sequencing data was performed by Dr Chris Boustred (NE Thames Regional Genetics Service, GOSH, UK). • Whole exome sequencing of patients X, 1, 4 and 5 was outsourced to BGI Genomics Hong Kong and the resulting raw data files were processed and aligned by Dr Chela James (GOSgene, ICH, UK) (Section 2.5). • Whole exome sequencing of patient Y and processing of the resulting raw data was performed by Dr Olaf Bodamer (University of Miami, USA) (Section 6.4.1). • Homozygosity mapping and whole exome sequencing of index PROSC family was performed by Dr Niklas Darin (The Queen Silvia Children’s Hospital, Sweden) (Section 7.1.4). • Sanger sequencing of additional PROSC and PNPO-deficient patients was performed by Dr Philippa Mills (ICH, UK) (Section 7.2). • Sample processing for electron microscopy analysis was carried out by Elizabeth Latimer- Bowman (Histopathology Department, GOSH, UK) and imaging was performed by Glenn Anderson (Histopathology Department, GOSH, UK) (Section 2.14.2).
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • Explorative Bioinformatic Analysis of Cardiomyocytes in 2D &3D in Vitro Culture System
    EXPLORATIVE BIOINFORMATIC ANALYSIS OF CARDIOMYOCYTES IN 2D &3D IN VITRO CULTURE SYSTEM VERSION 2 Master Degree Project in Bioscience One years Level, 60 ECTS Sruthy Janardanan [email protected] Supervisor: Jane Synnergren [email protected] Examiner: Sanja Jurcevic [email protected] Abstract The in vitro cell culture models of human pluripotent stem cells (hPSC)-derived cardiomyocytes (CMs) have gained a predominant value in the field of drug discovery and is considered an attractive tool for cardiovascular disease modellings. However, despite several reports of different protocols for the hPSC-differentiation into CMs, the development of an efficient, controlled and reproducible 3D differentiation remains challenging. The main aim of this research study was to understand the changes in the gene expression as an impact of spatial orientation of hPSC-derived CMs in 2D(two-dimensional) and 3D(three-dimensional) culture conditions and to identify the topologically important Hub and Hub-Bottleneck proteins using centrality measures to gain new knowledge for standardizing the pre-clinical models for the regeneration of CMs. The above-mentioned aim was achieved through an extensive bioinformatic analysis on the list of differentially expressed genes (DEGs) identified from RNA-sequencing (RNA-Seq). Functional annotation analysis of the DEGs from both 2D and 3D was performed using Cytoscape plug-in ClueGO. Followed by the topological analysis of the protein-protein interaction network (PPIN) using two centrality parameters; Degree and Betweeness in Cytoscape plug-in CenTiScaPe. The results obtained revealed that compared to 2D, DEGs in 3D are primarily associated with cell signalling suggesting the interaction between cells as an impact of the 3D microenvironment and topological analysis revealed 32 and 39 proteins as Hub and Hub-Bottleneck proteins, respectively in 3D indicating the possibility of utilizing those identified genes and their corresponding proteins as cardiac disease biomarkers in future by further research.
    [Show full text]
  • Human 4E-BP1 / EIF4EBP1 Protein (His Tag)
    Human 4E-BP1 / EIF4EBP1 Protein (His Tag) Catalog Number: 10022-H07E General Information SDS-PAGE: Gene Name Synonym: 4E-BP1; 4EBP1; BP-1; PHAS-I Protein Construction: A DNA sequence encoding the human 4EBP1 (NP_004086.1) (Ser 2-Ile 118) with a N-terminal polyhistidine tag was expressed. Source: Human Expression Host: E. coli QC Testing Purity: > 90 % as determined by SDS-PAGE Endotoxin: Protein Description Please contact us for more information. The translational suppressor eIF4E binding protein-1, 4E-BP1 functions as Stability: a key regulator in cellular growth, differentiation, apoptosis and survival. The Eif4ebp1 gene, encoding 4E-BP1, is a direct target of a transcription Samples are stable for up to twelve months from date of receipt at -70 ℃ factor activating transcription factor-4 (ATF4), a master regulator of gene expression in stress responses. 4E-BP1 is characterized by its capacity to Predicted N terminal: Met bind specifically to eIF4E and inhibit its interaction with eIF4G. Molecular Mass: Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap- dependent translation, in cell stress. Binding of eIF4E to eIF4G is inhibited The secreted recombinant human 4EBP1 comprises 124 amino acids with in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases a predicted molecular mass of 13.4 kDa. It migrates as an approximately the affinity of this protein for eIF4E, thus favouring the binding of eIF4G 19 kDa band in SDS-PAGE under reducing conditions. and enhancing translation. 4E-BP1 is important for beta-cell survival under endoplasmic reticulum (ER) stress. 4E-BP1 mediates the regulation of Formulation: protein translation by hormones, growth factors and other stimuli that signal through the MAP kinase and mTORC1 pathways.
    [Show full text]
  • Human Pluripotent Stem Cell-Derived Ectomesenchymal Stromal Cells Promote More Robust Functional Recovery Than Umbilical Cord-De
    Human pluripotent stem cell-derived ectomesenchymal stromal cells promote more robust functional recovery than umbilical cord-derived mesenchymal stromal cells after hypoxic- ischaemic brain damage Jiawei Huang1,3*, Kin Pong U1,3*, Fuyuan Yang1, Zeyuan Ji1, Jiacheng Lin1,3, Zhihui Weng1, Lai Ling Tsang1,3, Tobias D Merson5, Ye Chun Ruan6, Chao Wan1,3, Gang Li2, Xiaohua Jiang1,3,4 1School of Biomedical Sciences, 2Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China. 3School of Biomedical Sciences Core Laboratory, Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, PR China. 4Sichuan University – The Chinese University of Hong Kong Joint Laboratory for Reproductive Medicine, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan, China. 5Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia. 6Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China. Running Title: Human ectomesenchymal stromal cells promote functional recovery in a rat HIE model *Corresponding author: Prof. Xiaohua JIANG, Email: [email protected] Address: Room 409A, Lo Kwee Seong Integrated Biomedical Sciences Building, Area 39, The Chinese University of Hong Kong, Shatin. Keywords: HIE, ectomesenchymal stromal cells, brain damage, regeneration, paracrine, ERK Abstract: Aims: Hypoxic-ischaemic encephalopathy (HIE) is one of the most serious complications in neonates and infants. Mesenchymal stromal cell (MSC)-based therapy is emerging as a promising treatment avenue for HIE. However, despite its enormous potential, the clinical application of MSCs is limited by cell heterogeneity, low isolation efficiency and unpredictable effectiveness. In this study, we examined the therapeutic effects and underlying mechanisms of human pluripotent stem cell-derived ectomesenchymal stromal cells (hPSC-EMSCs) in a rat model of HIE.
    [Show full text]