DOCSLIB.ORG

Elucidating the Role of Mitochondrial‑Localized Hepatocyte Growth Factor Receptor in Gastric Oncogenesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Elucidating the Role of Mitochondrial‑Localized Hepatocyte Growth Factor Receptor in Gastric Oncogenesis This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Elucidating the role of mitochondrial‑localized hepatocyte growth factor receptor in gastric oncogenesis Sim, Kae Hwan 2016 Sim, K. H. (2016). Elucidating the role of mitochondrial‑localized hepatocyte growth factor receptor in gastric oncogenesis. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/66452 https://doi.org/10.32657/10356/66452 Downloaded on 01 Oct 2021 13:49:56 SGT ELUCIDATING THE ROLE OF MITOCHONDRIAL- LOCALIZED HEPATOCYTE GROWTH FACTOR RECEPTOR IN GASTRIC ONCOGENESIS SIM KAE HWAN SCHOOL OF BIOLOGICAL SCIENCES 2016 ELUCIDATING THE ROLE OF MITOCHONDRIAL- LOCALIZED HEPATOCYTE GROWTH FACTOR RECEPTOR IN GASTRIC ONCOGENESIS SIM KAE HWAN School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2016 Acknowledgement I would like to express my sincere appreciation and thanks to my supervisor Dr. Siu Kwan Sze, for his expert guidance and warm encouragement. He has been a tremendous mentor for me. He has shared his wealth of experience on numerous occasions and provided me with a great learning experience. Moreover, I would like to thank Dr. Ren Yan, Dr. Bamaprasad Dutta and Dr. Sun Yang, for their invaluable discussions and suggestions, which has helped me a lot in this project. I would also like to acknowledge the kind support of my current and former laboratory colleagues, especially Dr. Park Jung Eun, Dr. Hao Piliang, Dr. Sunil Shankar Adav, Dr. Guo Tiannan, Dr. Li Xin, Meng Wei, Qian Jingru, Dr. Cheow Siok Hwee, and Zhang Qi, for extending their helping hand anytime I needed. On the other hand, I would like to convey deep appreciation to my thesis advisory committee members, Dr. Koh Cheng Gee and Dr. Lu Lei, for serving as my committee members even at hardship. They have given me so many brilliant comments and constructive criticism during the annual committee meetings and the conformation of my PhD candidature. In addition, I would like to express my sincere thanks to the laboratory members from Dr. Koh Cheng Gee’s lab, Dr. Li Hoi Yeung’s lab, Dr. Lu Lei’s lab, and Dr. Lin Chun Ling Valerie’s lab. Especially Dr. Ou Sirong, Dr. Weng Ting, Dr. Yeap Szu Ling, and Miss Or Yu Zuan for their kind help in one way or the other in my research work in Nanyang Technological University. I gratefully acknowledge the financial support from the Nanyang Technological University of Singapore in the form of Nanyang Research Scholarship. I am also thankful to the academic and technical staffs at the school of Biological Sciences, who have helped me in my research work. i Last but not least, special thanks go to my family and my girlfriend’s family. Words cannot express how grateful I am to them for all of your selfless support, love and understanding during my PhD study in Singapore. ii Contents Acknowledgement i Contents iii List of figures vii List of tables ix Abbreviations x Abstract xiii Chapter 1 – General Introduction 1.1 Introduction 2 1.1.1 Cancer 2 1.1.1.1 Cancer 2 1.1.1.2 Gastric cancer 4 1.1.2 Receptor tyrosine kinases 6 1.1.3 MET 7 1.1.3.1 MET signaling in cancers 7 1.1.3.2 Mitochondrial localization of MET 8 1.1.4 Mass spectrometry-based quantitative proteomics 9 1.1.4.1 Label-free quantitative proteomic approach 10 1.1.4.2 Labeling based quantitative proteomics approach 11 1.1.5 Objective and overview of project 12 Chapter 2 - Elucidating the molecular mechanism of translocation of hepatocyte growth factor receptor (MET) into the mitochondria in SNU5 gastric cancer cells 2.1 Abstract 15 2.2 Introduction 16 2.2.1 Roles of receptor tyrosine kinases in cancer 16 2.2.2 Dysregulated receptor tyrosine kinases in gastric cancer 17 2.2.2.1 Dysregulation of receptor tyrosine kinases in gastric cancer 17 iii 2.2.2.2 MET in gastric cancer 17 2.2.3 Endocytosis of RTK in cancers 19 2.2.4 Localization of MET in mitochondria 20 2.2.4.1 Knowledge gap 20 2.2.4.2 Specific aims in the study 20 2.3 Materials and methods 22 2.3.1 Chemicals 22 2.3.2 Cell culture 22 2.3.3 Western blotting 23 2.3.4 Mitochondria isolation and purification 23 2.3.5 Bicinchoninic acid protein assay 24 2.3.6 Immunofluorescence 25 2.3.7 Mitochondrial protein digestion 25 2.3.8 LC-MS/MS 26 2.3.9 Data analyses 27 2.3.10 Data annotation 28 2.4 Results 29 2.4.1 Profiling of mitochondrial proteome to uncover the correlation of endocytosis 29 2.4.2 Inhibition of of endocytosis using endocytic inhibitors 31 2.4.3 Time course study of inhibition of endocytosis using different endocytic inhibitors 36 2.4.4 Immunofluorescence analysis of localization of mtMET of SNU5 gastric cancer cells 38 2.5 Discussions 41 2.6 Conclusions and future works 44 Chapter 3 - Quantitative proteomic profiling to identify novel substrates of mitochondria-localized MET (mtMET) SNU5 gastric cancer cells iv 3.1 Abstract 46 3.2 Introduction 47 3.2.1 Gastric cancer 47 3.2.1.1 Gastric cancer 47 3.2.1.2 Advances of genomic and proteomic methods in gastric cancer diagnosis 47 3.2.2 Proteomics and gastric cancer 49 3.2.3 MET: Structure, functions, and dysregulated signaling 50 3.2.4 Specific aims in the study 51 3.3 Materials and methods 52 3.3.1 Chemicals 52 3.3.2 Cell culture 52 3.3.3 Co-IP 53 3.3.4 BCA protein assay 54 3.3.5 Mitochondrial protein digestion 54 3.3.6 Protein digestion and TMT labeling 55 3.3.7 Reverse phase HPLC fractionation 55 3.3.8 Desalting peptide samples 56 3.3.9 LC-MS/MS 56 3.3.10 Data analyses 57 3.3.11 Data annotation 59 3.3.12 In situ proximity ligation assay, microscopy and data handling59 3.4 Results 61 3.4.1 Discovery of novel putative substrates of mtMET using a label-free quantitative strategy 61 3.4.2 TMT labeling of SNU5 mitochondrial proteome under different conditions 70 3.4.3 Protein-protein interaction of mtMET and candidate proteins 91 v 3.4.4 Putative substrates of mtMET in SNU5 gastric cancer cells 93 3.5 Discussions 97 3.6 Conclusions and future works 102 Chapter 4 – Conclusion and future direction 4.1 Concluding remarks and future perspective 104 Reference 108 Appendix A – Publications 130 Appendix B – Conference presentation 131 Supplementary Data 132 vi List of Figures Chapter 1 Figure 1.1: Simple illustration of the initiation and progression of cancer 3 Figure 1.2: Schematic representation of quantitative proteomics methods 10 Figure 1.3: Schematic illustration of the isobaric tagging chemistry of TMT reagent 11 Chapter 2 Figure 2.1: Immunoblotting evidences of MET and phosphor-MET in SNU5 mitochondria 32 Figure 2.2: Immunoblotting evidences showing high purity of mitochondrial fractions 36 Figure 2.3: Immunoblotting evidences showing the expression level of mtMET and phosphor-mtMET under endocytic inhibition 37 Figure 2.4: Fluorescence microscopic imaging showing the effect of dynasore inhibition in SNU5 cells 39 Chapter 3 Figure 3.1: Schematic representation of the experimental design of mtMET co-IP 62 Figure 3.2: Comparison of the identified proteins between IgG-coIPs and MET-coIPs 64 Figure 3.3: Scatter plots showing the normalized emPAI values in the technical triplicate measurements 65 Figure 3.4: Classification analysis of 342 uniquely identified proteins in MET-coIPs 66 Figure 3.5: Protein-protein interactions network extracted from STRING database 68 vii Figure 3.6: Schematic representation of the experimental design to perform the quantitative proteomics analysis of SNU5 mitochondrial proteome using TMT 6-plex isobaric tags 72 Figure 3.7: Physiochemical characteristics of identified protein 73 Figure 3.8: Distribution of the relative expression levels of temporal proteome 63 Figure 3.9: GO analysis illustrates the classes of proteins with significantly reduced expression 76 Figure 3.10: Detection of PLA signals in cytocentrifugation preparations of SNU5 gastric cancer cells using Duolink in situ reagents with two primary antibodies 92 Figure 3.11: Western blot evidences of protein-protein interaction between mtMET and HMGA1 and PKM2 94 Supplementary Data Supplementary Figure 1: Western blot results showing no contamination from other organelles 132 Supplementary Figure 2: RTKs and mitochondrial proteins TOM20 and Bcl-xL sharing sequence which is rich in amino acids (arginine and lysine) of high hydrophobicity and basicity 133 Supplementary Figure 3: Detection of PLA signals in cytocentrifugation preparations of SNU5 gastric cancer cells using Duolink in situ reagents with two primary antibodies (large images) 134 viii List of Tables Chapter 2 Table 2.1: Components of clathrin-mediated endocytosis were identified by mitochondrial proteomic profiling of RTK TKI-sensitive cell lines 30 Chapter 3 Table 3.1: Identification of proteins interacting with MET in co-IP data 69 Table 3.2: Data analysis of identified proteins in different drug treatment of SNU5 mitochondria digests 73 Table 3.3: List of proteins showing significantly reduced protein expression level in the PHA665752_24h (128/126) and Dynasore_24h (130/126) 78 Table 3.4: Annotation of significantly identified proteins in TMT-based quantitative proteomics profiling 83 Table 3.5: Label-free quantitative proteomic approach identified the reduced expression level of HMGA1 and PKM2 upon MET inhibition 96 Supplementary Data Supplementary Table 1: Original co-IP data for 342 significantly identified protein in triplicate MET-coIPs 136 ix Abbreviations 2D-GE Two-dimensional gel electrophoresis ABB Ammonium bicarbonate buffer ACLY ATP citrate
Load more

Recommended publications
  • Establishing the Pathogenicity of Novel Mitochondrial DNA Sequence Variations: a Cell and Molecular Biology Approach
    Mafalda Rita Avó Bacalhau Establishing the Pathogenicity of Novel Mitochondrial DNA Sequence Variations: a Cell and Molecular Biology Approach Tese de doutoramento do Programa de Doutoramento em Ciências da Saúde, ramo de Ciências Biomédicas, orientada pela Professora Doutora Maria Manuela Monteiro Grazina e co-orientada pelo Professor Doutor Henrique Manuel Paixão dos Santos Girão e pela Professora Doutora Lee-Jun C. Wong e apresentada à Faculdade de Medicina da Universidade de Coimbra Julho 2017 Faculty of Medicine Establishing the pathogenicity of novel mitochondrial DNA sequence variations: a cell and molecular biology approach Mafalda Rita Avó Bacalhau Tese de doutoramento do programa em Ciências da Saúde, ramo de Ciências Biomédicas, realizada sob a orientação científica da Professora Doutora Maria Manuela Monteiro Grazina; e co-orientação do Professor Doutor Henrique Manuel Paixão dos Santos Girão e da Professora Doutora Lee-Jun C. Wong, apresentada à Faculdade de Medicina da Universidade de Coimbra. Julho, 2017 Copyright© Mafalda Bacalhau e Manuela Grazina, 2017 Esta cópia da tese é fornecida na condição de que quem a consulta reconhece que os direitos de autor são pertença do autor da tese e do orientador científico e que nenhuma citação ou informação obtida a partir dela pode ser publicada sem a referência apropriada e autorização. This copy of the thesis has been supplied on the condition that anyone who consults it recognizes that its copyright belongs to its author and scientific supervisor and that no quotation from the
    [Show full text]
  • Analysis of Gene Expression Data for Gene Ontology
    ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Robert Daniel Macholan May 2011 ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION Robert Daniel Macholan Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Department Chair Dr. Zhong-Hui Duan Dr. Chien-Chung Chan _______________________________ _______________________________ Committee Member Dean of the College Dr. Chien-Chung Chan Dr. Chand K. Midha _______________________________ _______________________________ Committee Member Dean of the Graduate School Dr. Yingcai Xiao Dr. George R. Newkome _______________________________ Date ii ABSTRACT A tremendous increase in genomic data has encouraged biologists to turn to bioinformatics in order to assist in its interpretation and processing. One of the present challenges that need to be overcome in order to understand this data more completely is the development of a reliable method to accurately predict the function of a protein from its genomic information. This study focuses on developing an effective algorithm for protein function prediction. The algorithm is based on proteins that have similar expression patterns. The similarity of the expression data is determined using a novel measure, the slope matrix. The slope matrix introduces a normalized method for the comparison of expression levels throughout a proteome. The algorithm is tested using real microarray gene expression data. Their functions are characterized using gene ontology annotations. The results of the case study indicate the protein function prediction algorithm developed is comparable to the prediction algorithms that are based on the annotations of homologous proteins.
    [Show full text]
  • Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-Like Mouse Models: Tracking the Role of the Hairless Gene
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2006 Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene Yutao Liu University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Liu, Yutao, "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino- like Mouse Models: Tracking the Role of the Hairless Gene. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1824 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Yutao Liu entitled "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Brynn H. Voy, Major Professor We have read this dissertation and recommend its acceptance: Naima Moustaid-Moussa, Yisong Wang, Rogert Hettich Accepted for the Council: Carolyn R.
    [Show full text]
  • RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases
    G C A T T A C G G C A T genes Review RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases Amber Willbanks, Shaun Wood and Jason X. Cheng * Department of Pathology, Hematopathology Section, University of Chicago, Chicago, IL 60637, USA; [email protected] (A.W.); [email protected] (S.W.) * Correspondence: [email protected] Abstract: Chromatin structure plays an essential role in eukaryotic gene expression and cell identity. Traditionally, DNA and histone modifications have been the focus of chromatin regulation; however, recent molecular and imaging studies have revealed an intimate connection between RNA epigenetics and chromatin structure. Accumulating evidence suggests that RNA serves as the interplay between chromatin and the transcription and splicing machineries within the cell. Additionally, epigenetic modifications of nascent RNAs fine-tune these interactions to regulate gene expression at the co- and post-transcriptional levels in normal cell development and human diseases. This review will provide an overview of recent advances in the emerging field of RNA epigenetics, specifically the role of RNA modifications and RNA modifying proteins in chromatin remodeling, transcription activation and RNA processing, as well as translational implications in human diseases. Keywords: 5’ cap (5’ cap); 7-methylguanosine (m7G); R-loops; N6-methyladenosine (m6A); RNA editing; A-to-I; C-to-U; 2’-O-methylation (Nm); 5-methylcytosine (m5C); NOL1/NOP2/sun domain Citation: Willbanks, A.; Wood, S.; (NSUN); MYC Cheng, J.X. RNA Epigenetics: Fine-Tuning Chromatin Plasticity and Transcriptional Regulation, and the Implications in Human Diseases. Genes 2021, 12, 627.
    [Show full text]
  • Advancing a Clinically Relevant Perspective of the Clonal Nature of Cancer
    Advancing a clinically relevant perspective of the clonal nature of cancer Christian Ruiza,b, Elizabeth Lenkiewicza, Lisa Eversa, Tara Holleya, Alex Robesona, Jeffrey Kieferc, Michael J. Demeurea,d, Michael A. Hollingsworthe, Michael Shenf, Donna Prunkardf, Peter S. Rabinovitchf, Tobias Zellwegerg, Spyro Moussesc, Jeffrey M. Trenta,h, John D. Carpteni, Lukas Bubendorfb, Daniel Von Hoffa,d, and Michael T. Barretta,1 aClinical Translational Research Division, Translational Genomics Research Institute, Scottsdale, AZ 85259; bInstitute for Pathology, University Hospital Basel, University of Basel, 4031 Basel, Switzerland; cGenetic Basis of Human Disease, Translational Genomics Research Institute, Phoenix, AZ 85004; dVirginia G. Piper Cancer Center, Scottsdale Healthcare, Scottsdale, AZ 85258; eEppley Institute for Research in Cancer and Allied Diseases, Nebraska Medical Center, Omaha, NE 68198; fDepartment of Pathology, University of Washington, Seattle, WA 98105; gDivision of Urology, St. Claraspital and University of Basel, 4058 Basel, Switzerland; hVan Andel Research Institute, Grand Rapids, MI 49503; and iIntegrated Cancer Genomics Division, Translational Genomics Research Institute, Phoenix, AZ 85004 Edited* by George F. Vande Woude, Van Andel Research Institute, Grand Rapids, MI, and approved June 10, 2011 (received for review March 11, 2011) Cancers frequently arise as a result of an acquired genomic insta- on the basis of morphology alone (8). Thus, the application of bility and the subsequent clonal evolution of neoplastic cells with purification methods such as laser capture microdissection does variable patterns of genetic aberrations. Thus, the presence and not resolve the complexities of many samples. A second approach behaviors of distinct clonal populations in each patient’s tumor is to passage tumor biopsies in tissue culture or in xenografts (4, 9– may underlie multiple clinical phenotypes in cancers.
    [Show full text]
  • Datasheet Blank Template
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . PNO1 (D-14): sc-133263 BACKGROUND APPLICATIONS PNO1 (partner of NOB1), also known as KHRBP1, is a 252 amino acid protein PNO1 (D-14) is recommended for detection of PNO1 of mouse, rat and that localizes to the nucleolus and contains one KH domain. Expressed in a human origin by Western Blotting (starting dilution 1:200, dilution range variety of tissues, including kidney, lung, liver and spleen, with lower levels 1:100-1:1000), immunoprecipitation [1-2 µg per 100-500 µg of total protein present in brain, heart, colon and skeletal muscle, PNO1 may play a role in (1 ml of cell lysate)], immunofluorescence (starting dilution 1:50, dilution RNA binding events during transcription or translation. The gene encoding range 1:50-1:500) and solid phase ELISA (starting dilution 1:30, dilution PNO1 maps to human chromosome 2, which houses over 1,400 genes and range 1:30-1:3000). comprises nearly 8% of the human genome. Harlequin icthyosis, a rare and Suitable for use as control antibody for PNO1 siRNA (h): sc-94365, PNO1 morbid skin deformity, is associated with mutations in the ABCA12 gene, siRNA (m): sc-152359, PNO1 shRNA Plasmid (h): sc-94365-SH, PNO1 shRNA while the lipid metabolic disorder sitosterolemia is associated with defects Plasmid (m): sc-152359-SH, PNO1 shRNA (h) Lentiviral Particles: sc-94365-V in the ABCG5 and ABCG8 genes. Additionally, an extremely rare recessive and PNO1 shRNA (m) Lentiviral Particles: sc-152359-V. genetic disorder, Alström syndrome, is caused by mutations in the ALMS1 gene, which maps to chromosome 2.
    [Show full text]
  • Integrating Single-Step GWAS and Bipartite Networks Reconstruction Provides Novel Insights Into Yearling Weight and Carcass Traits in Hanwoo Beef Cattle
    animals Article Integrating Single-Step GWAS and Bipartite Networks Reconstruction Provides Novel Insights into Yearling Weight and Carcass Traits in Hanwoo Beef Cattle Masoumeh Naserkheil 1 , Abolfazl Bahrami 1 , Deukhwan Lee 2,* and Hossein Mehrban 3 1 Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj 77871-31587, Iran; [email protected] (M.N.); [email protected] (A.B.) 2 Department of Animal Life and Environment Sciences, Hankyong National University, Jungang-ro 327, Anseong-si, Gyeonggi-do 17579, Korea 3 Department of Animal Science, Shahrekord University, Shahrekord 88186-34141, Iran; [email protected] * Correspondence: [email protected]; Tel.: +82-31-670-5091 Received: 25 August 2020; Accepted: 6 October 2020; Published: 9 October 2020 Simple Summary: Hanwoo is an indigenous cattle breed in Korea and popular for meat production owing to its rapid growth and high-quality meat. Its yearling weight and carcass traits (backfat thickness, carcass weight, eye muscle area, and marbling score) are economically important for the selection of young and proven bulls. In recent decades, the advent of high throughput genotyping technologies has made it possible to perform genome-wide association studies (GWAS) for the detection of genomic regions associated with traits of economic interest in different species. In this study, we conducted a weighted single-step genome-wide association study which combines all genotypes, phenotypes and pedigree data in one step (ssGBLUP). It allows for the use of all SNPs simultaneously along with all phenotypes from genotyped and ungenotyped animals. Our results revealed 33 relevant genomic regions related to the traits of interest.
    [Show full text]
  • Anti-Inflammatory Role of Curcumin in LPS Treated A549 Cells at Global Proteome Level and on Mycobacterial Infection
    Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection. Suchita Singh1,+, Rakesh Arya2,3,+, Rhishikesh R Bargaje1, Mrinal Kumar Das2,4, Subia Akram2, Hossain Md. Faruquee2,5, Rajendra Kumar Behera3, Ranjan Kumar Nanda2,*, Anurag Agrawal1 1Center of Excellence for Translational Research in Asthma and Lung Disease, CSIR- Institute of Genomics and Integrative Biology, New Delhi, 110025, India. 2Translational Health Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India. 3School of Life Sciences, Sambalpur University, Jyoti Vihar, Sambalpur, Orissa, 768019, India. 4Department of Respiratory Sciences, #211, Maurice Shock Building, University of Leicester, LE1 9HN 5Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia- 7003, Bangladesh. +Contributed equally for this work. S-1 70 G1 S 60 G2/M 50 40 30 % of cells 20 10 0 CURI LPSI LPSCUR Figure S1: Effect of curcumin and/or LPS treatment on A549 cell viability A549 cells were treated with curcumin (10 µM) and/or LPS or 1 µg/ml for the indicated times and after fixation were stained with propidium iodide and Annexin V-FITC. The DNA contents were determined by flow cytometry to calculate percentage of cells present in each phase of the cell cycle (G1, S and G2/M) using Flowing analysis software. S-2 Figure S2: Total proteins identified in all the three experiments and their distribution betwee curcumin and/or LPS treated conditions. The proteins showing differential expressions (log2 fold change≥2) in these experiments were presented in the venn diagram and certain number of proteins are common in all three experiments.
    [Show full text]
  • Noelia Díaz Blanco
    Effects of environmental factors on the gonadal transcriptome of European sea bass (Dicentrarchus labrax), juvenile growth and sex ratios Noelia Díaz Blanco Ph.D. thesis 2014 Submitted in partial fulfillment of the requirements for the Ph.D. degree from the Universitat Pompeu Fabra (UPF). This work has been carried out at the Group of Biology of Reproduction (GBR), at the Department of Renewable Marine Resources of the Institute of Marine Sciences (ICM-CSIC). Thesis supervisor: Dr. Francesc Piferrer Professor d’Investigació Institut de Ciències del Mar (ICM-CSIC) i ii A mis padres A Xavi iii iv Acknowledgements This thesis has been made possible by the support of many people who in one way or another, many times unknowingly, gave me the strength to overcome this "long and winding road". First of all, I would like to thank my supervisor, Dr. Francesc Piferrer, for his patience, guidance and wise advice throughout all this Ph.D. experience. But above all, for the trust he placed on me almost seven years ago when he offered me the opportunity to be part of his team. Thanks also for teaching me how to question always everything, for sharing with me your enthusiasm for science and for giving me the opportunity of learning from you by participating in many projects, collaborations and scientific meetings. I am also thankful to my colleagues (former and present Group of Biology of Reproduction members) for your support and encouragement throughout this journey. To the “exGBRs”, thanks for helping me with my first steps into this world. Working as an undergrad with you Dr.
    [Show full text]
  • Multi-Sample Full-Length Transcriptome Analysis of 22 Breast Cancer Clinical
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.199851; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Multi-sample Full-length Transcriptome Analysis of 22 Breast Cancer Clinical Specimens with Long-Read Sequencing Shinichi Namba1, §, Toshihide Ueno1, Shinya Kojima1, Yosuke Tanaka1, Satoshi Inoue1, Fumishi Kishigami1, Noriko Maeda2, Tomoko Ogawa3, Shoichi Hazama4, Yuichi Shiraishi5, Hiroyuki Mano1, and Masahito Kawazu1* Author affiliations: 1Division of Cellular Signaling, 5Division of Genome Analysis Platform Development, Research Institute, National Cancer Center, Tokyo 104-0045, Japan; 2Department of Gastroenterological, Breast and Endocrine Surgery, 4Department of Translational Research and Developmental Therapeutics against Cancer, Yamaguchi University Graduate School of Medicine, Yamaguchi 755-8505, Japan; 3Department of Breast Surgery, Mie University Hospital, Mie 514-8507, Japan; § Present address, Department of Statistical Genetics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; * Corresponding author E-mail:[email protected] (MK) bioRxiv preprint doi: https://doi.org/10.1101/2020.07.15.199851; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Abstract Although transcriptome alteration is considered as one of the essential drivers of carcinogenesis, conventional short-read RNAseq technology has limited researchers from directly exploring full-length transcripts, only focusing on individual splice sites.
    [Show full text]
  • Aneuploidy: Using Genetic Instability to Preserve a Haploid Genome?
    Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Science (Cancer Biology) Aneuploidy: Using genetic instability to preserve a haploid genome? Submitted by: Ramona Ramdath In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science Examination Committee Signature/Date Major Advisor: David Allison, M.D., Ph.D. Academic James Trempe, Ph.D. Advisory Committee: David Giovanucci, Ph.D. Randall Ruch, Ph.D. Ronald Mellgren, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 10, 2009 Aneuploidy: Using genetic instability to preserve a haploid genome? Ramona Ramdath University of Toledo, Health Science Campus 2009 Dedication I dedicate this dissertation to my grandfather who died of lung cancer two years ago, but who always instilled in us the value and importance of education. And to my mom and sister, both of whom have been pillars of support and stimulating conversations. To my sister, Rehanna, especially- I hope this inspires you to achieve all that you want to in life, academically and otherwise. ii Acknowledgements As we go through these academic journeys, there are so many along the way that make an impact not only on our work, but on our lives as well, and I would like to say a heartfelt thank you to all of those people: My Committee members- Dr. James Trempe, Dr. David Giovanucchi, Dr. Ronald Mellgren and Dr. Randall Ruch for their guidance, suggestions, support and confidence in me. My major advisor- Dr. David Allison, for his constructive criticism and positive reinforcement.
    [Show full text]
  • Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN
    [CANCER RESEARCH 61, 3741–3749, May 1, 2001] Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN Mieko Matsushima-Nishiu, Motoko Unoki, Kenji Ono, Tatsuhiko Tsunoda, Takeo Minaguchi, Hiroyuki Kuramoto, Masato Nishida, Toyomi Satoh, Toshihiro Tanaka, and Yusuke Nakamura1 Laboratories of Molecular Medicine [M. M-N., M. U., K. O., T. M., T. Ta., Y. N.] and Genome Database [T. Ts.], Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; Department of Obstetrics and Gynecology, School of Medicine, Kitasato University, Sagamihara 228-8555, Japan [H. K.]; Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba 305-8576, Japan [M. N.]; and Department of Obstetrics and Gynecology, Ibaraki Seinan Central Hospital, Tsukuba 306-0433, Japan [T. S.] ABSTRACT Akt/protein kinase B, cell survival, and cell proliferation (8). Over- expression of PTEN can decrease cell proliferation and tumorigenicity The PTEN tumor suppressor gene encodes a multifunctional phospha- (9, 10), an observation attributed to the ability of PTEN to induce cell tase that plays an important role in inhibiting the phosphatidylinositol-3- cycle arrest and apoptosis (11, 12). kinase pathway and downstream functions that include activation of Akt/protein kinase B, cell survival, and cell proliferation. Enforced ex- Thus, lack of PTEN expression may affect a complex set of pression of PTEN in various cancer cell lines decreases cell proliferation transcriptional targets. However, no systematic assessment of PTEN- through arrest of the cell cycle, accompanied in some cases by induction regulated targets in cancer cells has been reported to date.
    [Show full text]