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Cancer development results from a multi-step process that involves a set of genetic abnormalities allowing the clonal selection of cells with proliferative advantages. Some of these genetic abnormalities increase the autonomous proliferation of tumor cells by aberrant activation of signal transduction of growth factors that provide survival and proliferative signals in normal cells under physiological conditions.
The in vitro tumorgenesis model studied in this master thesis is based on cytokine-dependent cells that acquired cytokine-independence through two selection steps. First, BaF3 cells expressing a non-functional IL-9 receptor developed IL-9 responsiveness. This adaptation to IL-9 was accompanied by overexpression of the JEAK1 protein kinase, which is associated with IL-9 receptor and implicated in its signal transduction. IL-9-responsive cells could further give rise to totally cytokine-independent, autonomous clones.
In the first part of this work, we showed that these autonomous clones harbour point mutations in JAK1. Certain of these mutations were recently described in patients with acute lymphoblastic leukaemia, further illustrating the relevance of our model. We subsequently characterized the different JAK1 mutants functionally. All of them constitutively activated signalling molecules downstream of JAK1-binding receptors, such as IL-2 and IL-9 receptor, allowing for cytokine-independence of BAF3 cells. Moreover, 13 of the 18 identified mutants conferred hypersensitivity to the anti-proliferative effects of type I interferons.
The second part of this master thesis focuses on the study of the genetic factors promoting the apparition of spontaneous point mutations. JAK1 overexpression is a prerequisite for the selection of JAK1-mutated autonomous clones suggesting a favourable cellular context for further mutations. Therefore, we investigated systematically the different possible mechanism of JAK1 overexpression. Using transcription and translation inhibitors, we demonstrated that kinetics of JAK1 messenger RNA and JAK1 protein degradation were not altered by selection in IL-9 excluding post-transcriptional deregulation mechanisms. Treatment of cells with demethylating or acetylating agents and bisulfite sequencing ruled out the direct epigenetic regulations like modification of promoter methylation pattern or histones acetylation. Complementary experiments are needed in order to identify the mechanism underlying JAK1 overexpression in our model and to assess its impact on apparition of spontaneous activating mutations in JAK1 Le développement d’un cancer est le résultat d’un processus séquentiel qui implique une succession d’anomalies génétiques et de sélections clonales de cellules présentant un avantage de prolifération. Certaines de ces anomalies génétiques accroissent l’autonomie des cellules tumorales moyennant une signalisation aberrante des facteurs de croissance dont elles sont physiologiquement dépendantes.
Le modèle de tumorgenèse in vitro étudié dans ce travail met en scène des cellules au départ dépendantes de cytokines qui acquièrent la capacité de proliférer de façon autonome au terme d’un processus en deux étapes de sélection. Dans un premier temps, des cellules BaF3 exprimant un récepteur pour l’interleukine-9 (IL-9) non-fonctionnel développent une faculté de réponse à l’IL-9. Nous avons observé que cette adaptation s’accompagne systématiquement d’une surexpression de la protéine kinase JAK1 associée au récepteur à l’IL-9 et impliquée dans sa signalisation intracellulaire. Les cellules adaptées à l’IL-9 peuvent, dans un second temps, donner naissance à des clones totalement autonomes.
Dans le premier volet de ce mémoire, nous montrons que les clones autonomes présentent des mutations ponctuelles de JAK1. L’intérêt de ces mutations est illustré par le fait que récemment certaines d’entre elles ont également été identifiées chez des patients atteints de leucémies lymphoblastiques aiguës. Nous avons caractérisé fonctionnellement les différents mutants de JAK1. Ils activent constitutivement les molécules de signalisation en aval des récepteurs de cytokines tels ceux de l’IL-9 ou l’IL-2 assurant l’autonomie des cellules. Par opposition, 13 des 18 mutants identifiés confèrent une hypersensibilité aux effets anti-prolifératifs des interférons de type 1.
Le second volet de ce travail est l’étude des facteurs génétiques favorisant la survenue de telles mutations. La surexpression de JAK1 est un prérecquis à l’émergence de cellules autonomes évoquant un contexte propice à l’apparition de mutations. Nous avons entrepris l’examen systématique des divers mécanismes de surexposition envisageables. En faisant usage d’inhibiteurs de la transcription ou de la traduction, nous avons démontré que les cinétiques de dégradation de l’ARN messager et de la protéine n’étaient pas modifiées par la sélection en IL-9 écartant par conséquent une dérégulation post-transcriptionnelle. Le traitement des cellules par agents déméthylants ou acétylants ainsi que le séquençage d’ADN génomique traité au bisulfite excluent une régulation épigénétique telle qu’une modification dans la méthylation du promoteur ou l’acéthylation des histones. Des expériences complémentaires sont nécessaires afin d’élucider la cause de la surexposition de JAK1 dans notre modèle et de comprendre son éventuel impact sur l’apparition de mutations spontanées

Janus Kinase 1 --- Signal Transduction --- Angiogenic Proteins --- STAT Transcription Factors --- Neoplasm Proteins --- Smarcd3 protein, mouse --- Clone Cells

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In 1890 a case of myxedema was treated in Lisbon by the implantation of a sheep thyroid gland with the immediate improvement in the patient’s condition. A few years later, medications for the then ill-explained condition of the menopause included tablets made from cow ovaries. In the first quarter of the 20th century the identification of vitamin D, and its sunlight driven production in skin, paved the way to the elimination of rickets as a major medical problem. Twenty years or so later, Sir Vincent Wigglesworth established the endocrine basis of developmental moulting in insects, arguably the most commonly performed animal behaviour on Planet Earth. A paradigm that would unify these disparate observations arose between 1985 and 1987 beginning with the identification of the glucocorticoid receptor and the nuclear receptor super-family. What follows is a timely and positive manifestation of the capacity, productivity and value of international human scientific endeavour. Based on intrigue, lively competition and cooperation a global effort has rapidly fostered a school of biology with widespread ramifications for the understanding of metazoan animals, the human condition and the state of the planet. This book is the first this century to try and capture the spirit of this endeavour, to depict where the field is now and to identify some of the challenges and opportunities for the future.

Nuclear receptors (Biochemistry). --- Nuclear receptors (Biochemistry) --- DNA-Binding Proteins --- Transcription Factors --- Cell Physiological Processes --- Proteins --- Receptors, Cytoplasmic and Nuclear --- Cell Communication --- Cell Physiological Phenomena --- Amino Acids, Peptides, and Proteins --- Chemicals and Drugs --- Phenomena and Processes --- Biology --- Cytology --- Health & Biological Sciences --- Biochemistry. --- Biological chemistry --- Chemical composition of organisms --- Organisms --- Physiological chemistry --- Nuclear hormone receptors --- Receptors, Nuclear (Biochemistry) --- Receptors, Nuclear hormone --- Composition --- Medicine. --- Endocrinology. --- Oncology. --- Cell biology. --- Biomedicine. --- Biomedicine general. --- Cell Biology. --- Animal Biochemistry. --- Chemistry --- Medical sciences --- Hormone receptors --- Transcription factors

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In the last few years, significant breakthroughs in transcription research expanded our appreciation for the complexity of molecular controls on gene expression in mammalian cells. In Transcription Factors: Methods and Protocols, experts in the field describe state-of-the-art approaches that investigators can use to probe critical mechanisms underlying transcription factor nuclear-cytoplasmic trafficking as well as to assess the functional impact of post-translational modifications on transcription factor function. The chapters are written by prominent scientists, many of whom developed these methods, and highlight protocols that focus on specific transcription factor family members with particular relevance to human disease. Composed in the highly successful Methods in Molecular Biology™ series format, each chapter contains a brief introduction, step-by-step methods, a list of necessary materials, and a Notes section which shares tips on troubleshooting and avoiding known pitfalls. Comprehensive and current, Transcription Factors: Methods and Protocols compiles the latest techniques for elucidating controls on transcription factor intracellular localization and activity, and consequently is unlike any other methods-based text on transcriptional regulation today. .

Transcription factors --- Transcription Factors --- Research --- Methodology --- Proteins --- Investigative Techniques --- Publication Formats --- Biological Transport --- Analytical, Diagnostic and Therapeutic Techniques and Equipment --- Publication Characteristics --- Amino Acids, Peptides, and Proteins --- Metabolism --- Cytological Techniques --- Clinical Laboratory Techniques --- Laboratory Manuals --- Protein Transport --- Chemicals and Drugs --- Metabolic Phenomena --- Phenomena and Processes --- Human Anatomy & Physiology --- Health & Biological Sciences --- Animal Biochemistry --- Metabolic Phenomenon --- Metabolic Process --- Metabolism Concepts --- Metabolism Phenomena --- Process, Metabolic --- Processes, Metabolic --- Anabolism --- Catabolism --- Metabolic Concepts --- Metabolic Processes --- Concept, Metabolic --- Concept, Metabolism --- Concepts, Metabolic --- Concepts, Metabolism --- Metabolic Concept --- Metabolism Concept --- Phenomena, Metabolic --- Phenomena, Metabolism --- Phenomenon, Metabolic --- Protein Localization Processes, Cellular --- Protein Sorting --- Protein Targeting --- Protein Trafficking --- Gated Protein Transport --- Protein Translocation --- Transmembrane Protein Transport --- Vesicular Protein Transport --- Protein Sortings --- Protein Traffickings --- Protein Transport, Gated --- Protein Transport, Transmembrane --- Protein Transport, Vesicular --- Protein Transports --- Targeting, Protein --- Trafficking, Protein --- Traffickings, Protein --- Transmembrane Protein Transports --- Diagnoses and Laboratory Examinations --- Diagnosis, Laboratory --- Laboratory Diagnosis --- Laboratory Examinations and Diagnoses --- Laboratory Techniques, Clinical --- Clinical Laboratory Technique --- Diagnoses, Laboratory --- Laboratory Diagnoses --- Laboratory Technique, Clinical --- Technique, Clinical Laboratory --- Techniques, Clinical Laboratory --- Transcription Factor --- Factor, Transcription --- Factors, Transcription --- Cytologic Technics --- Cytological Technic --- Cytological Technics --- Cytological Technique --- Technic, Cytological --- Technics, Cytological --- Technique, Cytological --- Techniques, Cytological --- Cytologic Technic --- Technic, Cytologic --- Technics, Cytologic --- Cell Biology --- Biologic Transport --- Transport, Biological --- Transport, Biologic --- Investigative Technics --- Investigative Technic --- Investigative Technique --- Technic, Investigative --- Technics, Investigative --- Technique, Investigative --- Techniques, Investigative --- Gene Products, Protein --- Gene Proteins --- Protein Gene Products --- Proteins, Gene --- Genetic transcription factors --- methods --- Cytology. --- Cell Biology. --- Cell biology --- Cellular biology --- Biology --- Cells --- Cytologists --- Transcription factors - Research - Methodology

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Through great experimental difficulty, we’ve witnessed rapid, crucial developments at the intersection of computational biology, experimental technology, and statistics through which the vital process of transcriptional regulation can be further examined. In Computational Biology of Transcription Factor Binding, experts in the field examine the basic principles and provide detailed guidance for the computational analyses and biological interpretations of transcription factor binding, while disclosing critical practical information and caveats that are missing from many research publications. The volume serves not only computational biologists but experimentalists as well, who may want to better understand how to design and execute experiments and to communicate more effectively with computational biologists, computer scientists, and statisticians. Written for the highly successful Methods in Molecular Biology™ series, this work provides the kind of detailed description and implementation advice that is crucial for getting optimal results in the lab. Authoritative and easy to use, Computational Biology of Transcription Factor Binding guides scientists working in this area and demands not only new experiments but also the re-annotation of existing experimental data and computational predictions leading to important ongoing, major paradigm changes for us all.

Computational Biology --- Transcription Factors --- Computational biology. --- Transcription factors. --- Bio-informatique --- Facteurs de transcription --- Laboratory Manuals. --- Protein Binding --- methods --- Computational biology --- Transcription factors --- Biochemical Processes --- Publication Formats --- Proteins --- Metabolism --- Investigative Techniques --- Biology --- Chemical Processes --- Publication Characteristics --- Amino Acids, Peptides, and Proteins --- Analytical, Diagnostic and Therapeutic Techniques and Equipment --- Metabolic Phenomena --- Biological Science Disciplines --- Biochemical Phenomena --- Laboratory Manuals --- Methods --- Chemicals and Drugs --- Chemical Phenomena --- Phenomena and Processes --- Natural Science Disciplines --- Disciplines and Occupations --- Health & Biological Sciences --- Biology - General --- Investigative Technics --- Investigative Technic --- Investigative Technique --- Technic, Investigative --- Technics, Investigative --- Technique, Investigative --- Techniques, Investigative --- Metabolic Phenomenon --- Metabolic Process --- Metabolism Concepts --- Metabolism Phenomena --- Process, Metabolic --- Processes, Metabolic --- Anabolism --- Catabolism --- Metabolic Concepts --- Metabolic Processes --- Concept, Metabolic --- Concept, Metabolism --- Concepts, Metabolic --- Concepts, Metabolism --- Metabolic Concept --- Metabolism Concept --- Phenomena, Metabolic --- Phenomena, Metabolism --- Phenomenon, Metabolic --- Gene Products, Protein --- Gene Proteins --- Protein Gene Products --- Proteins, Gene --- Biochemical Concepts --- Biochemical Phenomenon --- Biochemical Process --- Phenomena, Biochemical --- Biochemical Concept --- Concept, Biochemical --- Concepts, Biochemical --- Phenomenon, Biochemical --- Process, Biochemical --- Processes, Biochemical --- Genetic transcription factors --- Natural Sciences --- Physical Sciences --- Discipline, Natural Science --- Disciplines, Natural Science --- Natural Science --- Natural Science Discipline --- Physical Science --- Science, Natural --- Science, Physical --- Sciences, Natural --- Sciences, Physical --- Chemical Phenomenon --- Chemical Process --- Physical Chemistry Phenomena --- Physical Chemistry Process --- Physicochemical Phenomenon --- Physicochemical Process --- Chemical Concepts --- Physical Chemistry Concepts --- Physical Chemistry Processes --- Physicochemical Concepts --- Physicochemical Phenomena --- Physicochemical Processes --- Chemical Concept --- Chemistry Process, Physical --- Chemistry Processes, Physical --- Concept, Chemical --- Concept, Physical Chemistry --- Concept, Physicochemical --- Concepts, Chemical --- Concepts, Physical Chemistry --- Concepts, Physicochemical --- Phenomena, Chemical --- Phenomena, Physical Chemistry --- Phenomena, Physicochemical --- Phenomenon, Chemical --- Phenomenon, Physicochemical --- Physical Chemistry Concept --- Physicochemical Concept --- Process, Chemical --- Process, Physical Chemistry --- Process, Physicochemical --- Processes, Chemical --- Processes, Physical Chemistry --- Processes, Physicochemical --- Methodological Studies --- Methodological Study --- Procedures --- Studies, Methodological --- Study, Methodological --- Method --- Procedure --- Transcription Factor --- Factor, Transcription --- Factors, Transcription --- Bio-Informatics --- Biology, Computational --- Computational Molecular Biology --- Bioinformatics --- Molecular Biology, Computational --- Bio Informatics --- Bio-Informatic --- Bioinformatic --- Biologies, Computational Molecular --- Biology, Computational Molecular --- Computational Molecular Biologies --- Molecular Biologies, Computational --- Biologic Sciences --- Biological Science --- Science, Biological --- Sciences, Biological --- Biological Sciences --- Life Sciences --- Biologic Science --- Biological Science Discipline --- Discipline, Biological Science --- Disciplines, Biological Science --- Life Science --- Science Discipline, Biological --- Science Disciplines, Biological --- Science, Biologic --- Science, Life --- Sciences, Biologic --- Sciences, Life --- Biochemistry. --- Protein Science. --- Computer Appl. in Life Sciences. --- Biological chemistry --- Chemical composition of organisms --- Organisms --- Physiological chemistry --- Chemistry --- Medical sciences --- Data processing. --- Composition