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Bioanalytical and Proteomic Approaches in the Study of Pathologic Ecs Dysfunctionality, Oxidative Stress and the Effects of PFKFB3 Modulators

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Bioanalytical and proteomic approaches in the study of pathologic ECs dysfunctionality, oxidative stress and the effects of PFKFB3 modulators Sarath Babu Nukala ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. La consulta de esta tesis queda condicionada a la aceptación de las siguientes condiciones de uso: La difusión de esta tesis por medio del servicio TDR (www.tdx.cat) y a través del Repositorio Digital de la UB (diposit.ub.edu) ha sido autorizada por los titulares de los derechos de propiedad intelectual únicamente para usos privados enmarcados en actividades de investigación y docencia. No se autoriza su reproducción con finalidades de lucro ni su difusión y puesta a disposición desde un sitio ajeno al servicio TDR o al Repositorio Digital de la UB. No se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR o al Repositorio Digital de la UB (framing). Esta reserva de derechos afecta tanto al resumen de presentación de la tesis como a sus contenidos. En la utilización o cita de partes de la tesis es obligado indicar el nombre de la persona autora. WARNING. On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tdx.cat) service and by the UB Digital Repository (diposit.ub.edu) has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized nor its spreading and availability from a site foreign to the TDX service or to the UB Digital Repository. Introducing its content in a window or frame foreign to the TDX service or to the UB Digital Repository is not authorized (framing). Those rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. Double Degree Doctorate between UNIVERSITA’ DEGLI STUDI DI MILANO FACOLTA’ DI SCIENZE DEL FARMACO Department of Pharmaceutical Sciences PhD Course in Pharmaceutical Sciences (XXXI Cycle) and UNIVERSITAT DE BARCELONA Doctoral Program in Biotechnology Department of Biochemistry and Molecular Biomedicine Faculty of Farmacy Bioanalytical and proteomic approaches in the study of pathologic ECs dysfunctionality, oxidative stress and the effects of PFKFB3 modulators Doctorate Coordinator at Universita’ degli Studi di Milano: Prof. Dr. Giancarlo Aldini Prof. Dr. Marina Carini Prof. Dr. Marta Cascante Serratosa Co-Supervisor Co-Supervisor and Tutor Nukala Sarath Babu PhD candidate R11494 Academic years 2016-2019 This PhD programme was founded in the frame of HORIZON 2020 - Marie Skłodowska-Curie ITN-European Joint Doctorate MOGLYNET-programme “MOGLYNet: “Modulation of glycolytic flux as a new approach for treatment of atherosclerosis and plaque stabilization: a multidisciplinary study” The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 67552. ii Declaration of Originality I declare that all the work presented in this thesis is my own, and that all else, figures, images, ideas, quotations, data, results, published or unpublished, have been acknowledged and referenced. This thesis contains some modified material from my PhD progress reports. Nukala Sarath Babu April 2019 Scientific Contributions Publications: 1. Nukala, S.B., Baron, G., Aldini, G., Carini, M. and D’Amato, A., 2019. Mass spectrometry- based label free quantitative proteomics to study the effect of 3PO drug at cellular level. ACS Medicinal Chemistry Letters. DOI:10.1021/acsmedchemlett.8b00593 2. Quantitative Proteomic Profiling of Pulmonary Endothelial Cells in Chronic Thromboembolic Pulmonary Hypertension Sarath Babu Nukala*, Alfonsina D’Amato, Olga Tura-Ceide, Regazzoni Luca, Aldini Gincarlo, Joan Albert Barbera, Marta Cascante, Marina Carini. Journal: Journal of Hypertension (Manuscript under preparation) 3. Differentially regulated proteins associated with endothelial dysfunctionality in patients undergoing treatment for acute myocardial infarction. Sarath Babu Nukala*, Alfonsina D’Amato, Regazzoni Luca, Aldini Gincarlo, Marta Cascante, Marina Carini. Journal: Journal of Hypertension (Manuscript under preparation) Oral Presentations: • “Bioanalytical and proteomic approaches in the study of pathologic ECs dysfunctionality, oxidative stress and the effects of PFKFB3 modulators”, University of Antwerp, Belgium, Feb 2016 3 • “Proteomics research work in MoGlyNet project”, European Molecular Biology Laboratory (EMBL), Germany, July 2016 • “Proteomic approaches in the study of pathologic ECs dysfunctionality”, University of Leiden, Netherlands, September 2016. • “Proteomic profiling of pathological ECs of acute myocardial infarction and chronic thromboembolic pulmonary hypertension”, University of Aberdeen, Scotland, May 2017 • “Bioanalytical and proteomic approaches in the study of pathologic ECs dysfunctionality, oxidative stress and the effects of PFKFB3 modulators”, Mid-term review meeting, University of Milan, Italy, September 2017. • “Oxidative stress in the dysfunctional ECs of acute myocardial infarction and chronic thromboembolic pulmonary hypertension”, Leiden University Medical Center, Netherlands, April 2018. • “Mass-spectrometry based label-free quantitative proteomics approach to study the effect of 3PO at cellular level”, University of Milan, Italy, September 2018. • “Bioanalytical and proteomic approaches in the study of pathologic ECs dysfunctionality, oxidative stress and the effects of PFKFB3 modulators, SSPA 2018, Italy, September 2018. • “Bioanalytical and proteomic approaches in the study of pathologic ECs dysfunctionality, oxidative stress and the effects of PFKFB3 modulators”, University of Barcelona, Spain, January 2019. Poster Presentations: • Nukala Sarath Babu, Alfonsina D’Amato, Luca Regazzoni, Giancarlo Aldini, Mass Spectrometry-Based Label Free Quantitative Proteomics to study the effect of 3-PO in endothelial cells; Italian-Spanish-Portuguese Joint Meeting in Medicinal Chemistry,MedChemSicily2018, abstract page 214 http://www.medchemsicily2018.it/documenti/book_of_abstracts.pdf • Nukala Sarath Babu, O. Tura , L. Regazzoni , C. Marinello , A. Abdali , G. Aldini, V.F.Smolders, I. Blanco , J.A. Barbera , M. Cascante , M. Carini “Differentially Expressed Proteins in Pulmonary Endothelial Cells in Chronic Thromboembolic Pulmonary Hypertension” American Journal of Respiratory and Critical Care Medicine 2018; 197:A3738ATS International conference, 18-23 May 2018, San Diego, CA. https://www.atsjournals.org/doi/abs/10.1164/ajrccm conference.2018.197.1 4 • Alfonsina D'Amato, Nukala Sarath Babu, Giancarlo Aldini, Marina Carini. “nLC-MS/MS based Label Free Quantitative Proteomics to study the effects of bioactive compounds” HPLC 2019 Milan. 5 Acknowledgements With honesty and immense pleasure, I express my sincere gratitude to my beloved research guide and mentor Prof. Marina Carini, Department of Pharmaceutical Sciences, University of Milan, Italy for the great supervision, patience, highly valuable and crucial discussions and constant encouragement with respect to the fascinating topic of my research work. I’m very thankful to her for the protection throughout my research period and endless support. Words are not enough to express my feeling towards her for arranging the foundation and ornamentation of my research career. I express my profound sense of gratitude to my beloved second supervisor Prof. Marta Cascante, Department of Biochemistry and Molecular Biology, University of Barcelona, Spain for the great guidance, support and laboratory facilities provided during the advancement and successful completion of my six months secondment. I express my heartfelt thanks to Prof. Aldini Giancarlo, Ph.D. coordinator, Department of Pharmaceutical Sciences, University of Milan, Italy for giving a great motivation, good lab environment and support for the successful completion of my research work. I’m glad to work under the co-supervision of Dr. Luca Regazzoni, Research Scientist, Department of Pharmaceutical Sciences, the University of Milan for the extraordinary support and suggestions not only in the research work but also crucial helps provided during my entire stay in Italy. There are no other words to express my great feelings for his entire assistance. I’m extremely grateful to Dr. Alfonsina D’Amato, Research Scientist, Department of Pharmaceutical Sciences, University of Milan for the valuable assistance, motivation and suggestions provided in the entire project. I express my sincere thanks to Dr. Olga Tura, Research Scientist, Department
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    Supplementary Table 1

    Supplementary Table 1. Large-scale quantitative phosphoproteomic profiling was performed on paired vehicle- and hormone-treated mTAL-enriched suspensions (n=3). A total of 654 unique phosphopeptides corresponding to 374 unique phosphoproteins were identified. The peptide sequence, phosphorylation site(s), and the corresponding protein name, gene symbol, and RefSeq Accession number are reported for each phosphopeptide identified in any one of three experimental pairs. For those 414 phosphopeptides that could be quantified in all three experimental pairs, the mean Hormone:Vehicle abundance ratio and corresponding standard error are also reported. Peptide Sequence column: * = phosphorylated residue Site(s) column: ^ = ambiguously assigned phosphorylation site Log2(H/V) Mean and SE columns: H = hormone-treated, V = vehicle-treated, n/a = peptide not observable in all 3 experimental pairs Sig. column: * = significantly changed Log 2(H/V), p<0.05 Log (H/V) Log (H/V) # Gene Symbol Protein Name Refseq Accession Peptide Sequence Site(s) 2 2 Sig. Mean SE 1 Aak1 AP2-associated protein kinase 1 NP_001166921 VGSLT*PPSS*PK T622^, S626^ 0.24 0.95 PREDICTED: ATP-binding cassette, sub-family A 2 Abca12 (ABC1), member 12 XP_237242 GLVQVLS*FFSQVQQQR S251^ 1.24 2.13 3 Abcc10 multidrug resistance-associated protein 7 NP_001101671 LMT*ELLS*GIRVLK T464, S468 -2.68 2.48 4 Abcf1 ATP-binding cassette sub-family F member 1 NP_001103353 QLSVPAS*DEEDEVPVPVPR S109 n/a n/a 5 Ablim1 actin-binding LIM protein 1 NP_001037859 PGSSIPGS*PGHTIYAK S51 -3.55 1.81 6 Ablim1 actin-binding
  • Double Minute Chromosomes in Glioblastoma Multiforme Are Revealed by Precise Reconstruction of Oncogenic Amplicons

    Double Minute Chromosomes in Glioblastoma Multiforme Are Revealed by Precise Reconstruction of Oncogenic Amplicons

    Published OnlineFirst August 12, 2013; DOI: 10.1158/0008-5472.CAN-13-0186 Cancer Tumor and Stem Cell Biology Research Double Minute Chromosomes in Glioblastoma Multiforme Are Revealed by Precise Reconstruction of Oncogenic Amplicons J. Zachary Sanborn1,2,Sofie R. Salama2,3, Mia Grifford2, Cameron W. Brennan4, Tom Mikkelsen6, Suresh Jhanwar5, Sol Katzman2, Lynda Chin7, and David Haussler2,3 Abstract DNA sequencing offers a powerful tool in oncology based on the precise definition of structural rearrange- ments and copy number in tumor genomes. Here, we describe the development of methods to compute copy number and detect structural variants to locally reconstruct highly rearranged regions of the tumor genome with high precision from standard, short-read, paired-end sequencing datasets. We find that circular assemblies are the most parsimonious explanation for a set of highly amplified tumor regions in a subset of glioblastoma multiforme samples sequenced by The Cancer Genome Atlas (TCGA) consortium, revealing evidence for double minute chromosomes in these tumors. Further, we find that some samples harbor multiple circular amplicons and, in some cases, further rearrangements occurred after the initial amplicon-generating event. Fluorescence in situ hybridization analysis offered an initial confirmation of the presence of double minute chromosomes. Gene content in these assemblies helps identify likely driver oncogenes for these amplicons. RNA-seq data available for one double minute chromosome offered additional support for our local tumor genome assemblies, and identified the birth of a novel exon made possible through rearranged sequences present in the double minute chromosomes. Our method was also useful for analysis of a larger set of glioblastoma multiforme tumors for which exome sequencing data are available, finding evidence for oncogenic double minute chromosomes in more than 20% of clinical specimens examined, a frequency consistent with previous estimates.