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Chronic Obstructive Pulmonary Disease (COPD) is a chronic, inflammatory condition, characterised by airflow limitation. The use of glucocorticoids (GC) as an anti-inflammatory treatment in COPD has limited clinical benefits, and as such, new treatments are needed. Identifying key pathways involved in the inflammatory response in COPD may enable the development of novel treatments. The aims of this thesis were to examine the steroid sensitivity of an in vitro mixed sputum culture cell model, comparing COPD cells to smoking and non-smoking controls, examine expression of the intracellular signalling molecule p38 Mitogen Activated Protein Kinase (MAPK) in COPD lungs compared with controls, examine the GC and p38 MAPK inhibitor and dual therapy sensitivity of a bronchial epithelial cell line and finally, to understand the mechanisms by which a p38 MAPK inhibitor in combination with a GC synergistically inhibit pro-inflammatory mediator production in a bronchial epithelial cell line. Dexamethasone inhibits mixed sputum cell pro-inflammatory mediator release, with no differences in sensitivity observed between COPD and control cells. Isolated sputum neutrophils demonstrate modest sensitivity to dexamethasone, which is in contrast to blood neutrophils. There are increased numbers of cells positive for activated p38 MAPK in COPD lungs compared with controls, specifically localised to follicular B and CD8+ T cells, bronchial epithelial cells and alveolar and sputum macrophages. Lung and sputum neutrophils are devoid of activated p38 MAPK, and a pharmacological p38 MAPK inhibitor has no effect on pro-inflammatory mediator production from these cells. This is in contrast to blood neutrophils, whereby p38 MAPK activation can be induced following LPS stimulation and in vitro cell culture, and pro-inflammatory mediator release is inhibited by a p38 MAPK inhibitor. Dexamethasone and birb 796 inhibit stimulated pro-inflammatory mediator release from a bronchial epithelial cell line in a dose-dependent manner. Sensitivity to either drug is dependent on stimuli and the pro-inflammatory mediator analysed. There is additive and synergistic inhibition of pro-inflammatory mediator production when combination therapy comprising dexamethasone and birb 796 is used compared with either drug alone. This may be due to Birb 796 enhancing dexamethasone-mediated nuclear translocation of the glucocorticoid receptor, which may enhance the GC-mediated anti-inflammatory effects. Combination therapy may therefore be a useful therapeutic in the treatment of COPD.

Glucocorticoids (Gc) are vital for development, maintenance of glucose homeostasis and the resolution of inflammation. As potent modulators of the immune response Gc are routinely prescribed in the management of a variety of inflammatory diseases including asthma and rheumatoid arthritis. However clinical use of Gc is limited by variation in patient sensitivity to Gc treatment and development of a wide range of side effects. In this thesis I present two studies that have advanced our understanding of Gc action in vivo. The first defines and characterises the cause of familial glucocorticoid resistance, and the second describes the action of two potent non-steroidal Gc in a cell line model. Familial Gc Resistance: Cases of primary generalised Gc resistance are very rare and typically present as mineralocorticoid and androgen excess leading to hypertension, hypokalemia and hirsutism. Gc resistance is attributed to loss of function mutations within the glucocorticoid receptor (GR). Here I identify a family with a novel mutation in GR exon 6 that gives rise to a very mild phenotype. Analysis of transformed patient peripheral blood lymphocytes revealed a 50% reduction in full length GR but no expression of a mutant form. As this did not rule out expression in vivo, the mutant receptor (Δ612GR) was characterised in a cell line. Investigation using reporter genes revealed that Δ612GR lacked any activity, but had dominant negative action when co expressed with full length GR. In response to Gc Δ612GR was not phosphorylated or targeted for degradation. Fluorophore tagged Δ612GR was unable to translocate to the nucleus in response to Gc, but delayed the translocation of full length GR when co-expressed. Together this indicates that Δ612GR is unable to bind ligand but has dominant negative action upon full length GR most likely due to heterodimerisation. Therefore I describe a novel GR mutation that results in Gc resistance but presents with a mild very phenotype. Novel Non-steroidal Gc: Non-steroidal Gc can be used as tools to determine how ligand structure directs GR function. Here I describe two highly potent non steroidal Gc ligands, GSK47867A and GSK47869A which alter the kinetics of receptor activity. Treatment with either ligand induces slow GR nuclear translocation, promotes GR nuclear retention and prolongs transcriptional activity following ligand withdrawal. Crystal structure analysis revealed that GSK47867A and GSK47869A specifically alter the surface charge of the GR at a site important for Hsp90 binding. GR bound to GSK47867A and GSK47869A shows prolonged activity in the presence of Hsp90 inhibitor geldanamycin. Therefore this work identifies a new chemical series that could prolong GR activity due to altered pharmacodynamics rather than altered pharmacokinetics.In summary this work uses a combination of genetic and chemical biology approaches to broaden our understanding of GR function. Characterisation of naturally occurring GR mutations gives insight into the complex function of the GR, and non-steroidal Gc act as useful tools that will aid in the design of improved therapeutics.

La maladie de Cushing est caractérisée par une sécrétion excessive de l’hormone adrénocorticotrope (ACTH) à partir des tumeur corticotropes de l'hypophyse. Un excès d'ACTH entraîne un hypercortisolisme et provoque des symptômes tels que diabète, hypertension, obésité et les maladies cardiovasculaires entraînant un risque accru de mortalité si la maladie n’est pas traitée. Les tumeurs corticotropes sont caractérisées par la perte du rétro-contrôle négatif exercé par les glucocorticoïdes (GCs) sur la proopiomélanocortine (POMC) qui est le précurseur de l’ACTH : c'est la caractéristique majeure de la maladie de Cushing. Les causes de la résistance aux GC dans les adénomes corticotropes sont encore mal connues. Des études récentes ont montré une surexpression du récepteur du facteur de croissance épidermique (EGFR) dans les adénomes corticotropes provoquant une augmentation de l'activité du gène POMC et de la sécrétion d'ACTH. Les principaux objectifs de ce travail étaient de comprendre la relation entre la signalisation dérégulée de EGF et la résistance aux GCs. Dans le présent travail, nous avons identifié la voie JAK/STAT3 comme la principale voie de signalisation EGFR qui active la transcription du gène POMC. De plus, nous montrons que l'activation de la signalisation EGFR entraîne une résistance du promoteur POMC aux GCs et que l’activation de STAT3 est responsable de cette résistance. STAT3 affecte le mécanisme de transrepression de GR sans affecter le recrutement de GR au promoteur POMC. L’utilisation d’un inhibiteur de STAT3 restaure la répression de la transcription du promoteur POMC par les GCs. Nous avons aussi trouvé que 50% des adénomes corticotropes humains montrent une surexpression de la forme active de STAT3. Nous avons aussi étudié les mécanismes sous le contrôle des GCs qui régulent la prolifération cellulaire et qui pourraient être dérégulés dans la maladie de Cushing. CABLES1 est un régulateur négatif du cycle cellulaire et son expression est sous le contrôle des GCs. L’expression de CABLES1 est perdue dans 55 % des adénomes hypophysaires corticotropes, mais la cause de cette perte est encore mal comprise. Dans ce travail, nous avons identifié quatre variants faux-sens dans le gène CABLES1, deux chez de jeunes adultes (c.532G > A, c.718C > T) et deux chez des enfants (c.935G > A, et c.1388A > G) atteints de la maladie de Cushing. Les quatre variants touchent une région de la protéine CABLES1 qui est proche du motif de liaison de la kinase-3 dépendante des cyclines (CDK3). Ces variants ont perdu la capacité d’inhiber la croissance de cellules corticotropes tumorales (AtT20). Les quatre variantes sont donc des mutations de perte de fonction. En résumé, nos travaux révèlent le rôle important de STAT3 dans la résistance aux GC et ainsi, le blocage de l'action de STAT3 peut être une nouvelle stratégie pour le traitement de la maladie de Cushing. Nous avons aussi supporté un rôle de CABLES1 en tant que nouveau gène prédisposant aux tumeurs hypophysaires. / Cushing’s disease (CD) is characterized by excess secretion of adrenocorticotropic hormone (ACTH) from corticotroph tumors of the pituitary gland. Excessive ACTH leads to hypercortisolism that causes disabling symptoms such as diabetes, hypertension, obesity and cardiovascular disease resulting in an increased risk of mortality if it is not treated. Corticotroph tumors are characterized by the loss of glucocorticoid (GC) feedback repression of the proopiomelanocortin (POMC) that encodes the precursor of ACTH: this is the hallmark of CD. The causes of GC resistance in corticotroph adenomas of CD patients remain unknown. Recent findings showed overexpression of epidermal growth factor receptor (EGFR) in corticotroph adenomas causing increased POMC activity and ACTH secretion. The main objectives of this work were to understand the relationship between deregulated EGF signaling and GC resistance in the tumorigenesis of CD. In the present work, we identified the JAK/STAT3 pathway as the main EGFR pathway activating transcription of the POMC gene. We found that sustained activation of EGFR signaling or overactivation of STAT3 causes unresponsiveness of the POMC promoter to GCs and that activated STAT3 is responsible for GC resistance. STAT3 affects the transrepression mechanism of GR without affecting GR recruitment to the POMC promoter. The use of STAT3 inhibitor restores the repressive effect of GC on POMC transcription. Importantly, 50% of human corticotroph adenomas showed overexpression of activated STAT3. We also studied the mechanisms under the control of GCs that regulate cell proliferation and that could be deregulated in CD. CABLES1 is a negative cell cycle regulator, its expression is under the control of GC. CABLES1 expression is lost in 55 % of corticotroph adenomas and the underlying reasons remain unclear. In this work, we identified the presence of four missense variants in CABLES1 gene, two in young adults (c.532G > A, c.718C > T) and two in children (c.935G > A, and c.1388A > G) with CD. The four variants are close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein. The variants have lost the ability to inhibit growth of corticotropinoma cells (AtT20). The four variants are thus loss of function mutations. In summary, our work revealed the important role of STAT3 in GC resistance and further indicates that inhibition of STAT3 action may be a novel strategy for CD treatment. We also provided evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene.

RUNX1 est un facteur de transcription essentiel pour l’hématopoïèse et joue un rôle important dans la fonction immunitaire. Des mutations surviennent dans ce gène chez 5 à 13% des patients atteints de leucémie myéloïde aiguë (LMA) (RUNX1mut) et définissent un sous-groupe particulier de LMA associé à un pronostic défavorable. En conséquence, il est nécessaire de procéder à une meilleure caractérisation génétique et de concevoir des stratégies thérapeutiques plus efficaces pour ce sousgroupe particulier de LMA. Bien que la plupart des mutations trouvées dans le gène RUNX1 dans la LMA soient supposément acquises, des mutations germinales dans RUNX1 sont observées chez les patients atteints du syndrome plaquettaire familial avec prédisposition aux hémopathies malignes (RUNX1-FPD, FPD/AML). En outre, 44 % des individus atteints évoluent vers le développement d’une LMA. Suite au séquençage du transcriptome (RNA-Seq) d’échantillons de la cohorte Leucégène, nous avons montré que le dosage allélique de RUNX1 influence l’association avec des mutations coopérantes, le profil d’expression génique et la sensibilité aux médicaments dans les échantillons primaires de LMA RUNX1mut. Aussi, la validation des mutations trouvées chez RUNX1 a mené à la découverte que 30% des mutations identifiées dans notre cohorte de LMA étaient d’origine germinale, révélant une proportion plus élevée qu’attendue de cas de mutations RUNX1 familiales. Un crible chimique a, quant à lui, révélé que la plupart des échantillons RUNX1mut sont sensibles aux glucocorticoïdes (GCs) et nous avons confirmé que les GCs inhibent la prolifération des cellules de LMA et ce, via l’interaction avec le récepteur des glucocorticoïdes (Glucocorticoid Receptor, GR). De plus, nous avons observé que les échantillons contenant des mutations RUNX1 censées entraîner une faible activité résiduelle étaient plus sensibles aux GCs. Nous avons aussi observé que la co-association de certaines mutations, SRSF2mut par exemple, et les niveaux de GR contribuaient à la sensibilité aux GCs. Suite à cela, la sensibilité acquise aux GCs a été obtenue en régulant négativement l’expression de RUNX1 dans des cellules LMA humaines, ce qui a été accompagné par une régulation positive de GR. L’analyse de transcriptome induit par GC a révélé que la différenciation des cellules de LMA induite par GCs pourrait être un mécanisme en jeu dans la réponse antiproliférative associée à ces médicaments. Plus important encore, un criblage génomique fonctionnel a identifié le répresseur transcriptionnel PLZF (ZBTB16) comme un modulateur spécifique de la réponse aux GCs dans les cellules LMA sensibles et résistantes. Ces observations fournissent une caractérisation supplémentaire de la LMA RUNX1mut, soulignant l’importance de procéder à des tests germinaux pour les patients porteurs de mutations RUNX1 délétères. Nos résultats ont également identifié un nouveau rôle pour RUNX1 dans le réseau de signalisation de GR et montrent l’importance d’investiguer le repositionnement des GCs pour traiter la LMA RUNX1mut dans des modèles précliniques. Enfin, nous avons fourni des indications sur le mécanisme d’action des GCs, en montrant que PLZF s’avère un facteur important favorisant la résistance aux GCs dans la LMA. / RUNX1 is an essential transcription factor for definite hematopoiesis and plays important roles in immune function. Mutations in RUNX1 occur in 5-13% of Acute Myeloid Leukemia (AML) patients (RUNX1mut ) and are associated with adverse outcome, thus highlighting the need for better genetic characterization and for the design of efficient therapeutic strategies for this particular AML subgroup. Although most RUNX1 mutations in AML are believed to be acquired, germline RUNX1 mutations are observed in the familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD, FPD/AML) in which about 44% of affected individuals progress to AML. By performing RNA-sequencing of the Leucegene collection, we revealed that RUNX1 allele dosage influences the association with cooperating mutations, gene expression profile, and drug sensitivity in RUNX1mut primary AML specimens. Validation of RUNX1 mutations led to the discovery that 30% of RUNX1 mutations in our AML cohort are of germline origin, indicating a greater than expected proportion of cases with familial RUNX1 mutations. Chemical screening showed that most RUNX1mut specimens are sensitive to glucocorticoids (GC) and we confirmed that GCs inhibit AML cell proliferation via interaction with the Glucocorticoid Receptor (GR). We observed that specimens harboring RUNX1 mutations expected to result in low residual RUNX1 activity were most sensitive to GCs, and that co-associating mutations, such as SRSF2mut, as well as GR levels contribute to GC-sensitivity. Accordingly, acquired GC-sensitivity was achieved by negatively regulating RUNX1 expression in human AML cells, which was accompanied by upregulation of the GR. GC-induced transcriptome analysis revealed that GC-induced differentiation of AML cells might be a mechanism at play in the antiproliferative response to these drugs. Most critically, functional genomic screening identified the transcriptional repressor PLZF (ZBTB16) as a specific modulator of the GC response in sensitive and resistant AML cells. These findings provide additional characterization of RUNX1mut AML, further stressing the importance of germline testing for patients carrying deleterious RUNX1 mutations. Our results also identified a novel role for RUNX1 in the GR signaling network and support the rationale of investigating GC repurposing for RUNX1mut AML in preclinical models. Finally, we provided insights into the mechanism of action of GCs, which positions PLZF as an important factor promoting resistance to glucocorticoids in AML.

No description available.

Στην παρούσα διδακτορική διατριβή προτείνονται μεθοδολογίες υπολογιστικής νοημοσύνης για την επεξεργασία και ανάλυση δεδομένων γονιδιακής έκφρασης μικροσυστοιχιών cDNA. Πιο συγκεκριμένα, στο πρώτο σκέλος αναπτύσσονται δύο νέες προσεγγίσεις για την εύρωστη εκτίμηση και διόρθωση του θορύβου υποβάθρου: η διόρθωση υποβάθρου βάσει εκατοστημορίων και η διόρθωση υποβάθρου βάσει παλινδρόμησης loess. Οι προσεγγίσεις αυτές καινοτομούν κυρίως στο ότι χρησιμοποιούν μία εύρωστη εκτίμηση του θορύβου υποβάθρου, γεγονός που τις καθιστά ιδανικές σε περιπτώσεις, όπου τα δεδομένα είναι θορυβώδη. Επιπροσθέτως, αναπτύσσεται ένα νέο, γενικής χρήσεως, πλαίσιο για τη συστηματική αξιολόγηση του βαθμού επίδρασης των μεθόδων διόρθωσης υποβάθρου. Μέσω του πλαισίου αυτού, οι δύο προτεινόμενες προσεγγίσεις, καθώς και άλλες ευρέως χρησιμοποιούμενες μέθοδοι, αξιολογούνται βάσει εφαρμογής τους σε διαφορετικά σύνολα δεδομένων αυτο-υβριδοποίησης, με τις πρώτες να εμφανίζουν ιδιαιτέρως καλή απόδοση. Το πλαίσιο αυτό καινοτομεί στο ότι ενσωματώνει νέα κριτήρια και τρόπους γραφικής απεικόνισης. Τόσο οι προτεινόμενες μέθοδοι εκτίμησης και διόρθωσης θορύβου υποβάθρου, όσο και το πλαίσιο συστηματικής αξιολόγησής τους, συνιστούν μία νέα, ενδελεχή μελέτη που προσανατολίζει στην εφαρμογή ή απόρριψη μίας συγκεκριμένης προσέγγισης, συνεισφέροντας εν τέλει στην κατάκτηση καλλίτερης ποιότητας δεδομένων μικροσυστοιχιών. Επίσης, στο δεύτερο σκέλος της διατριβής αναπτύσσεται ένα νέο, ολοκληρωμένο και γενικής χρήσεως πλαίσιο ανάλυσης δεδομένων μικροσυστοιχιών ούτως, ώστε να διερευνηθεί το ζήτημα εάν στην T-λευχαιμική κυτταρική σειρά CCRF-CEM επικρατούν εγγενείς ή επίκτητοι μηχανισμοί αντοχής στην πρεδνιζολόνη. Συγκεκριμένα, καταλλήλως επιλεχθέντα δεδομένα μικροσυστοιχιών cDNA – που διευκολύνουν την εξέταση τόσο της εξαρτώμενης από τη συγκέντρωση δράσης, όσο και της δυναμικής της ανταπόκρισης στην πρεδνιζολόνη (πρώιμη και όψιμη δράση) – γίνονται αντικείμενο επεξεργασίας και ενδελεχούς ανάλυσης, και βάσει συγκεκριμένων, προ-διατυπωμένων συλλογισμών, προσεγγίζεται το εν λόγω ερώτημα. Το πλαίσιο αυτό είναι καινοτόμο, εφόσον, πέραν του ότι ενσωματώνει μία πρωτότυπη ακολουθία μεθόδων, προσεγγίζει συστηματικά το πρόβλημα της εγγενούς ή επίκτητης αντοχής, συνεισφέροντας, έτσι, στην ευρύτερη προσπάθεια διερεύνησης των επακριβών μηχανισμών αντοχής των λευχαιμικών κυττάρων στα γλυκοκορτικοειδή. Τα αποτελέσματα από την εφαρμογή του στα δεδομένα της εν λόγω κυτταρικής σειράς συνηγορούν υπέρ της ύπαρξης μίας σύνθετης ανταπόκρισης του υπό μελέτη συστήματος στα γλυκοκορτικοειδή, η οποία όμως τείνει περισσότερο προς έναν εγγενή μηχανισμό αντοχής. / In the present Ph.D. thesis, computational intelligence methods for processing and analyzing cDNA microarray gene expression data are designed and developed. More specifically, in the first part of this thesis, the problem of background estimation and correction of two-channel microarray data is addressed and two novel algorithms are proposed, namely the percentiles-based and the loess-based background correction methods. Both approaches are based on the multiplicative model of background, while utilizing robust background noise estimators, thus making them ideal for noisy datasets. Furthermore, a new, generic framework for the systematic evaluation of the impact of the background estimating methodologies is suggested, whereupon the aforementioned methods as well as other approaches are evaluated by application to various publicly available self-self hybridization datasets. As suggested by this thorough, comparative evaluation our algorithms perform very well regarding noise reduction. The evaluation framework, which is based mainly on different and widely used statistical measures, incorporates new criteria and visualization methods. Moreover, it represents a novel, detailed contribution to the examination of the impact of background correction methods to the final interpretation of microarray experiments, conferring explicit guidance on the pros and cons of them and when they should be applied. Additionally, in the second part of this thesis, a new, generic, computational microarray data analysis framework is described, in order to examine the hypothesis of whether the resistant T-cell leukemia cell line CCRF-CEM posses an intrinsic or exert an acquired mechanism of resistance and to investigate the molecular imprint of this, upon prednisolone treatment. More analytically, using the above explained computational analysis workflow, microarray data that enable the examination of both the dose effect of prednisolone exposure and the dynamics (early and late) of the molecular response of the cells at the transcriptomic layer, are systematically analyzed based on specific, predefined formulations. The analysis of the results supports a complex mechanism of action for the cells which seems to favor though more the intrinsic mechanism of resistance.