Tesis sobre el tema "Ikarios"

The Ikaros family of zinc finger transcription factors is essential for B cell development, and frequently mutated in B cell malignancies. Our lab has previously identified Ikaros target genes in pre-B cells by combining Ikaros ChIP-seq binding data and gene expression profiling. To address the kinetics and mechanisms of Ikaros-mediated transcriptional regulation, I have used an inducible Ikaros system, which allows for the monitoring of cellular and molecular changes during Ikaros-mediated gene silencing at high temporal resolution. Within minutes of Ikaros induction, the Ikaros-regulated model loci Igll1 and Myc showed decreased promoter accessibility and RNA polymerase II (RNAPII) occupancy. These early events were followed by changes in nucleosome composition, including an increased histone H2B/H3 ratio, the deposition of the histone variant H2A.Z, and decreased active histone acetylation marks. Histone deacetylation was not required to initiate down-regulation of Igll1 and Myc transcription, since treatment with the histone deacetylase inhibitor Trichostatin A did not interfere with Ikaros-mediated gene silencing. I next elucidated the mechanistic relationship between the early events of decreased promoter accessibility and decreased RNAPII occupancy. Addition of Triptolide resulted in the removal of RNAPII from the Igll1 and Myc promoters, but did not affect nucleosome occupancy and its regulation mediated by Ikaros. This suggested that Ikaros regulates nucleosome positioning and occupancy directly, and not through effects on RNAPII. Consistent with this hypothesis, Ikaros-mediated gene silencing was delayed by RNAi-mediated knockdown of chromatin remodeler Mi-2β (Chd4), the ATPase subunit of the Mi-2/NuRD complex. Hence, Ikaros-initiated chromatin remodelling was identified as one of the earliest events during Ikaros-mediated gene silencing, and was required for rapid transcriptional down-regulation of Ikaros target genes.

Según estimaciones del INEI al 30 de junio del 2016, se señala que el 9.9% (3, 118,612 personas) de la población peruana es mayor a 60 años, asimismo si a eso consideramos que la esperanza de vida continúa incrementándose (actualmente en 74,8 años) y que se espera que la población de 65 años a más alcance el 17.1% y 29.9% de la población total en los años 2050 y 2100, respectivamente. Es así que Ikarius nace con la finalidad de brindar un servicio de prevención, tratamiento y fortalecimiento del deterioro cognitivo en el adulto mayor, ofreciendo un modelo integral que comprende actividades que refuerzan las capacidades físicas, mentales y sociales. Nuestro público objetivo son adultos mayores de 60 a 75 años de los niveles socioeconómicos A y B de los distritos de San Borja, San Isidro, Surco, La Molina y Miraflores (Zona 7 de Apeim). Se utilizó una metodología de investigación que comprendiera el uso de información primaria y secundaria tanto en el aspecto cualitativo y cuantitativo. Se consultó con especialistas referentes en el tema en el Perú y se pudo conocer con profundidad el negocio y crear una propuesta de valor acorde a los atributos señalados por los adultos mayores y, posteriormente, avalado por la investigación cuantitativa a consumidores y cliente. En lo que concierne a la propuesta de valor, la investigación de mercado indica que al 10% de los adultos mayores y 11% de los clientes, les agrada mucho la propuesta presentada. En relación a la localización, se tomó la decisión que el distrito más adecuado para iniciar las operaciones del negocio sea San Borja. El local tiene un área construida de 238.50 m2, contando con instalaciones acondicionadas para el desarrollo de las actividades del negocio. Se iniciará con una capacidad objetivo de 64% con el fin de atender a 8,572 atenciones en el primer año. En cuanto al ámbito financiero, se ha considerado una inversión inicial de S/. 696,552 el cual está financiado por los accionistas y una entidad bancaria a una tasa de 17%, amortizable en un periodo de 5 años. Esto resulta en un VAN estimado de S/. 407,915 y una TIR de 33.94% Finalmente, se concluye la viabilidad del presente plan de negocios tras su investigación y evaluación correspondiente.
Tesis

Haematopoiesis is a cell differentiation process, starting from multipotent haematopoietic stem cells (HSC) to increasingly more fate-restricted blood cell progenitors. Each step during this process is strictly regulated by key transcription factors, activating or repressing stemness and lineage-specific genes. Not surprisingly, mutations or translocations involving transcription factors are often associated with leukaemia. IKAROS is the founding member of a zinc finger transcription factor family primarily involved in haematopoietic fate-decision. IKAROS protein is characterized by 2 domains: one at the N-terminus, with 4 zinc fingers, necessary for the DNA binding, and one at the C-terminus, with 2 zinc fingers, necessary for the homo- hetero-dimerization of the protein and its consequent activation. IKAROS is expressed in HSC and during lymphoid commitment expression increases, silencing stemness and myelo-erythroid priming genes and enhancing lymphoid specific factors. In Ikzf1-knockout mice models, lymphopoiesis is severely impaired, with a retardation of T cell development and a block in B, NK and dendritic cell differentiation due to a failure of HSC to differentiate into common lymphoid progenitors (CLP). Ikzf1 mutant mice developed T-cell leukaemia or lymphoma with a penetrance of ~95% within 6-8 weeks after birth, and died soon after. In human, IKZF1 alterations are rarely associated with T-cell leukaemias, but surprisingly IKZF1 deletions were found to occur in 15% of paediatric B cell precursors ALL (BCP-ALL) and in more than 70% of paediatric Philadelphia positive BCP-ALL. In this thesis, we evaluate the impact of IKAROS in paediatric leukaemia and during the B-cell lineage specification. In Chapter 3, we studied the incidence of IKZF1 point mutations and indels in a European cohort of Ph+ BCP-ALL paediatric patients. In particular, we screened, using next generation amplicon deep sequencing (NGS), the 7 coding exons of IKZF1 in 98 IKZF1 non deleted and 61 IKZF1 deleted patients. Seven missense point mutations and 7 frameshift small indels were identified in IKZF1-non deleted and 3 point mutations were detected in IKZF1-deleted patients, all of them with a predicted deleterious effect on IKAROS function. Mutations were mainly located in exons 5 and 8, encoding the DNA-binding and dimerization domains respectively. In IKZF1-non deleted patients, mutations seem to indicate the same prognosis as macrodeletions, with a higher incidence of adverse events in patients treated before TKI introduction compared to patients treated with a combination therapy including TKIs. Among the mutated patients identified in our mutation screening, one patient had the same single nucleotide deletion both at diagnosis and complete remission, suggesting a constitutional status of the mutation. In Chapter 4 we further investigated the constitutional status of the IKZF1 mutation in the proband and his family, finding in 3 generations the same mutation in the mother and 3 other carriers, as well as a second leukaemia case in the family that had occurred more than 45 years ago. The IKZF1 single nucleotide deletion gave rise to a truncated protein with loss of the last part of the DNA binding domain and the C-terminal dimerization domain, resulting in DNA-binding deficiency and a diffuse nuclear localization. The mutant allele was transcribed in proband’s bone marrow at complete remission, as well as in peripheral blood (PB) cells of his sister, an unaffected mutation carrier. Finally, the truncated protein was identified in the PB cells of the proband’s sister, in a lower amount compare to the wt-IKAROS isoforms. In Chapter 5, a model to study Ikaros-mediated gene expression regulation is described and characterized. Ikaros expression is enhanced during B-cell development, and pilots B cell-progenitors out of cell cycle allowing the Ig light chain recombination. To better comprehend the kinetics and mechanisms of Ikaros-gene expression regulation, we knocked-out endogenous Ikaros from a murine cycling preB cell line, using the CRISPR-Cas9 technique, and subsequently transduced cells with an inducible Ikaros cassette, that allowed to precisely regulating Ikaros translocation in the nuclei. Inducible Ikaros efficiently translocated to the nuclei and bound to target gene promoter, but its gene expression regulation appeared impaired. Indeed, inducible Ikaros was able to up-regulate well known target genes, but failed to down-regulate 3 selected target genes, know to be down-regulate after Ikaros induction in an endogenous Ikaros wt model. In Chapter 6 we took advantage of the Ikaros inducible system to study the metabolic changes that occur during B-cell lymphocyte development in a cycling to resting preB cell model. Three FRET-sensors specifically designed to evaluate the cellular levels of ATP, glucose and AMPK activation status were transduced in our cell model, and preliminary measurements were performed using at fluorescent microscopy and FACS after 16 hours of Ikaros induction
L’ematopoiesi è un processo differenziativo che, a partire da cellule staminali ematopoietiche multipotenti, da origine a tutte le cellule del sangue. Ogni step differenziativo durante questo processo è finemente regolato da un insieme di fattori di trascrizione che agiscono in concerto bloccando la trascrizione di geni legati alla staminalità ed attivando geni essenziali per la specificazione ed il differenziamento delle diverse linee maturative ematopoetiche: linfoide, mieloide, eritroide e magacariocitoide. Non è quindi sorprendente che mutazioni o traslocazioni che interessano questi fattori di trascrizione siano spesso associate con l’insorgenza di leucemie. IKAROS fa parte di una famiglia di fattori di trascrizione principalmente coinvolta nella specificazione in senso linfoide delle cellule ematopoietiche. IKAROS è caratterizzato da due domini funzionali: un dominio N-terminale, composto da 4 zinc-finger, necessario per il legame della proteina al DNA, ed un domino C-terminale, formato da 2 zinc-finger, indispensabile per la omo-etero-dimerizzazione della proteina e la sua conseguente attivazione. IKAROS è espresso a livello delle cellule staminali ematopoietiche, e la sua espressione aumenta durante il differenziamento linfoide, silenziando geni legati alla staminalità ed allo sviluppo eritro-mieloide e potenziando l’espressione di geni legati allo sviluppo linfoide. In topi knockout per il gene Ikzf1 la linfopoiesi è gravemente compromessa, con un ritardo nello sviluppo delle cellule T ed un completo blocco nel differenziamento dei linfociti B, cellule natural killer e delle cellule dendritiche. Questo fenotipo è dovuto all’incapacità delle cellule staminali ematopoietiche di differenziare in cellule progenitrici della linea linfoide in assenza di Ikaros. La mancanza di Ikaros in questi topi porta all’insorgenza di leucemie o linfomi a cellule T nel ~95% dei casi entro sei mesi dalla nascita. Nell’uomo, aberrazioni associate ad IKZF1 sono rare nelle leucemie T, ma, al contrario, delezioni di parte del gene sono presenti nel 15% delle leucemie pediatriche a fenotipo B (BCP-ALL), e la percentuale raggiunge circa il 70% se si considera un particolare sottogruppo caratterizzato dalla presenza del cromosoma aberrante Philadelphia. In questa tesi abbiamo studiato il ruolo di IKAROS nelle leucemie pediatriche e durante il differenziamento dei linfociti B. Nel terzo capitolo l’incidenza di mutazioni puntiformi e piccole inserzioni/delezioni (indel) del gene IKZF1 viene studiata in una coorte europea di pazienti pediatrici affetti da BCP-ALL con presenza del cromosoma Philadelphia. Grazie all’utilizzo del sequenziamento di nuova generazione abbiamo sequenziato i 7 esoni codificanti di IKZF1 in 98 pazienti IKZF1-non deleti ed in 61 pazienti IKZF1-deleti. Tra i 98 pazienti non deleti abbiamo riscontrato la presenza di 7 mutazioni puntiformi e 7 indel, mentre 3 mutazioni puntiformi sono state evidenziate nella coorte di pazienti IKZF1 deleti. Tutte le aberrazioni da noi trovate sono predette avere un effetto deleterio sulla funzionalità della proteina. Queste aberrazioni genetiche sono principalmente localizzate sul quinto e sull’ottavo esone, che codificano per i domini di legame al DNA e di dimerizzazione, rispettivamente. Nei pazienti che non presentavano macrodelezioni di IKZF1, le mutazioni da noi identificate sembrano avere lo stesso impatto prognostico delle macro-delezioni, presentando una maggior incidenza di eventi avversi nei pazienti trattati prima dell’introduzione degli inibitori di tirosin-chinasi rispetto a quelli a cui sono stati somministrati in sinergia con la chemioterapia. Un paziente mutato identificato durante il nostro screening ha mostrato la presenza della stessa delezione di un singolo nucleotide sia nel campione alla diagnosi che in quello in remissione, suggerendo una possible origine costituzionale della mutazione. Nel quarto capitolo è stata indagata la natura costituzionale della mutazione nel paziente e nella sua famiglia. La stessa delezione in eterozigosi è stata identificata nella madre del paziente ed in altri 3 portatori in 3 generazioni; abbiamo inoltre scoperto un secondo caso di leucemia pediatrica all’interno della famiglia verificatasi più di 45 anni fa. La delezione riscontrata nella famiglia dà origine ad una proteina tronca con la perdita della parte terminale del dominio di legame al DNA e la completa perdita del dominio di dimerizzazione al C-terminale. L’assenza di questi domini comporta una ridotta affinità di legame al DNA ed ad una localizzazione nucleare diffusa. L’mRNA dell’allele mutato è stato ritrovato nelle cellule del midollo osseo del paziente in remissione completa, così come in cellule mononucleate del sangue di una delle sorelle, anch’essa portatrice della delezione. Nelle medesime cellule è stata riscontrata anche la presenza della proteina tronca, seppur in minor quantità rispetto alle isoforme wt. Nel quinto capitolo viene descritto e caratterizzato un nuovo modello cellulare murino per lo studio della regolazione dell’espressione genica mediata da Ikaros. L’espressione di Ikaros aumenta durante il differenziamento dei linfociti B, ad è necessario per arrestare il ciclo cellulare di cellule B progenitrici permettendo il riarrangiamento della catena leggera delle immunoglobuline. Per meglio comprendere la cinetica ed il meccanismo con cui Ikaros media l’espressione genica, abbiamo creato una linea murina di cellule preB knockout per il gene Ikzf1 grazie alla tecnica di “gene editing” della CRISPR-Cas9, ed abbiamo quindi trasdotto queste cellule con una cassetta contenente un sistema inducibile di Ikaros, che ci permette di controllare la traslocazione di Ikaros dal citoplasma al nucleo. Ikaros-inducibile è in grado di traslocare efficacemente nel nucleo e di legarsi al promotore di un suo gene target molto noto, ma la sua abilità di regolare l’espressione genica appare parzialmente compromessa. Infatti, Ikaros-inducibile promuove l’aumento di espressione di alcuni suoi geni target, ma non è in grado di silenziare 3 geni da noi selezionati e noti per essere silenziati da Ikaros. Nel sesto capitolo abbiamo utilizzato il sistema di Ikaros-inducibile per studiare i cambiamenti metabolici che avvengono durante lo sviluppo dei linfociti B, utilizzando un modello cellulare murino di cellule preB. Tre sensori basati sulla tecnica FRET, per quantificare i livelli cellulari di ATP, glucosio e di attivazione della proteina AMPK, sono stati trasdotti nel nostro modello cellulare, e sono quindi state eseguiti esperimenti preliminari utilizzando tecniche di microscopia a fluorescenza e FACS dopo 16 ore di induzione

Le facteur de transcription Ikaros est un régulateur essentiel de la lymphopoïèse. Ikaros est nécessaire à la différenciation des lymphocytes B et joue aussi un rôle important dans la suppression des LAL-T. Contrairement aux souris mutantes nulles pour Ikaros, les souris mutantes hypomorphes IkL/L développent des lymphocytes B matures après la naissance. Avec l'âge, toutes les souris IkL/L développent des leucémies T Notch dépendantes avec des mutations similaires à celles trouvées chez les patients atteints de LAL-T. La souris IkL/L est donc un excellent modèle pour étudier l'activation des cellules B matures et la pathogenèsedes LAL-T. Nous avons montré que la délétion spécifique du promoteur et de l'exon 1 de Notch1 dans les cellules T conduit à l'activation de promoteurs cryptiques dans la région 3' du gène, qui génèrent des transcrits codant pour des protéines Notch1 constitutivement actives qui accélèrent la leucémogenèse dans la souris IkL/L. De plus, nous mettrons en évidence l'existence de cellules initiatrices de leucémie dans les tumeurs IkL/L puisque nous avons trouvé que des cellules ayant la capacité de s'auto-renouveler représentent 1 sur 500. Enfin, nous avons montré que les cellules B IkL/L ont une activation excessive d'ERK et dep38 après la stimulation du BCR, ce qui résulte en une hyper-prolifération et une production d'autoanticorps liés au lupus systémique érythémateux. Nos résultats suggèrent qu'Ikaros est un régulateur négatif de l'activation des lymphocytes B.

Den här uppsatsen är en responshistorisk undersökning av Karin Wards skulptur Ikaros syster och dess möte med sin publik. Syftet är att förstå de starka reaktioner skulpturen väckte. De huvudsakliga frågeställningarna lyder: Hur kan de handgripliga förändringarna av verkets utseende tolkas? Vilka förväntningar på den offentliga konsten uttrycktes i debatten och bevakningen i massmedia? Undersökningen visar att responsen var häftig och omfattande: en intensiv debatt bröt ut i lokalpressen, och under åren 1978–80 hade verket en framskjuten plats i det lokala offentliga samtalet. Många irriterade sig på verket och dömde ut det som fult eller pornografiskt medan andra försvarade verket på estetiska och principiella grunder. Debatten bottnade i stort sett i två konkurrerande idéer om den offentliga konstens uppgift; Skulle ett offentligt verk behaga alla genom att gestalta ett objektivt eller konventionellt skönhetsideal, eller skulle den offentliga konsten tillåtas vara heterogen och därigenom behaga heterogena preferenser och stödja konstnärernas fria skapande? Skulpturens utseende förändrades flera gånger under den perioden, bland annat sprängdes ett hål i verket – en skada konstnären reparerade genom att omgestalta verket. Sådana handgripligheter kan tolkas som fysiska uttryck för åsikter som återfinns i den offentliga debatten om verket. Samtidigt förändrade de i sin tur verkets uttryck och betydelse.

Cell proliferation is a process that is strictly regulated by a large number of proteins. An alteration in one of the encoding genes inserts an error into the regulative protein, which may result in uncontrolled cell growth and eventually tumor formation. Lymphoma is a cancer type originating in the lymphocytes, which are part of the body’s immune defence. In the present thesis, Znfn1a1, Notch1 and Bcl11b were studied; all involved in the differentiation of T lymphocytes. The three genes are located in chromosomal regions that have previously shown frequent loss of heterozygosity in tumor DNA. Ikaros is a protein involved in the early differentiation of T lymphocytes. In this thesis, mutation analysis of the Znfn1a1 gene in chemically induced murine lymphomas revealed point mutations and homozygous deletions in 13 % of the tumors. All of the detected deletions lead to amino acid substitutions or abrogation of the functional domains in the Ikaros protein. Our results support the role of Ikaros as a potential tumor suppressor in a subset of tumors. Notch1 is a protein involved in many differentiation processes in the body. In lymphocytes, Notch1 drives the differentiation towards a T-cell fate and activating alterations in the Notch1 gene have been suggested to be involved in T-cell lymphoma. We identified activating mutations in Notch1 in 39 % of the chemically induced murine lymphomas, supporting the involvement of activating Notch1 mutations in the development of T-cell lymphoma. Bcl11b has been suggested to be involved in the early T-cell specification, and mutations in the Bcl11b gene has been identified in T-cell lymphoma. In this thesis, point mutations and deletions were detected in the DNA-binding zinc finger regions of Bcl11b in 15 % of the chemically induced lymphomas in C57Bl/6×C3H/HeJ F1 mice. A mutational hotspot was identified, where four of the tumors carried the same mutation. Three of the identified alterations, including the hotspot mutation in Bcl11b, increased cell proliferation when introduced in a cell without endogenous Bcl11b, whereas cell proliferation was suppressed by wild-type Bcl11b in the same cell line. Mutations in Bcl11b may therefore be an important contributing factor to lymphomagenesis in a subset of tumors. A germ line point mutation was identified in BCL11B in one of 33 human B-cell lymphoma patients. Expression of BCL11B in infiltrating T cells was significantly lower in aggressive compared to indolent lymphomas, suggesting that the infiltrating T cells may affect the B-cell lymphomas.

Les facteurs de transcription STAT3, STAT5 et Ikaros sont impliqués dans rhématopoïèse normale et leur expression est fréquemment dérégulée dans les hémopathies humaines. Dans une première partie de mon travail, nous avons mis en évidence que STAT5 est essentiel pour l'initiation de la leucémie aiguë lymphoblastique (LAL) induite par F oncogene TEL-JAK2. Nous avons également montré que les leucémies TEL-JAK2 requièrent nécessairement l'expression des facteurs STAT3 ou STAT5 pour leur maintien à long terme. Parallèlement à ce projet, nous avons mis en évidence une interaction fonctionnelle entre le facteur de transcription STAT5 et le produit du gène suppresseur de tumeur Ikzfl in vitro. In vivo, nous avons montré une synergie entre l'oncogène BCR-ABL et la perte de fonction d'un allèle du gène Ikzfl dans les LAL-B STAT5 dépendantes. Les cellules tumorales déficientes ou non pour l'expression d'Ikaros n'expriment pas d'isoforme dominantes négatives d'Ikaros. Les analyses du réarrangement des gènes codant les chaînes des immunoglobulines montrent que les cellules leucémiques BCR-ABL⁺/⁰ ; Ik⁺/⁺ sont monoclonales alors que les leucémies accélérées sont polyclonales. La perte de fonction d'Ikzfl atténue la pression de sélection d'autres mutations pro-oncogéniques. Des analyses génomique par CGH indiquent que les tumeurs BCR-ABL⁺/⁰; Ik⁺/⁺ présentent de multiples anomalies chromosomiques récurrentes. En revanche, très peu d'anomalies chromosomiques sont observées dans les tumeurs accélérées. L'étude transcriptomique des tumeurs BCR-ABL⁺/⁰; Ik⁺/⁺ et BCR-ABL⁺/⁰; IkL/⁺ nous a permis d'identifier 293 sondes dérégulées entre les tumeurs de deux génotypes
Transcription factors STAT3, STAT5 and Ikaros are involved in normal hematopoiesis and expression of these factors is frequently deregulated in human leukemias. In the first part of my thesis, we have demonstrated that STAT5 expression is essential for the initiation of acute lymphoblastic leukemia (ALL) induced by TEL-JAK2 oncogene. Then, we have shown that TEL-JAK2 leukemia require necessarily STAT3 or STAT5 expression for their long-term maintenance. In a second part, we have demonstrated a functional interaction between the transcription factor STAT5 and Ikzfl a tumor suppressor gene in vitro. In vivo, we have shown a synergy between BCR-ABL oncogene and the loss of function of one allele of Ikzfl gene in STAT5 dependent B-ALL. Leukemic blasts deficient or not for Ikaros expression do not express dominant negative Ikaros isoforms. The loss of a single Ikzfl allele is suffïcient to cooperate with BCR-ABL in leukemogenesis. Analysis VH-to-DJH immunoglobulin heavy chain locus rearrangement showed that leukemic cells BCR-ABL⁺/⁰; Ik⁺/⁺ are monoclonal while fast arising leukemia are polyclonal. These observations suggest that loss of function of a single Ikzfl allele alleviates the requirement for the selection of additional oncogenic events that characterize disease progression in BCR-ABL-induced leukemogenesis. In line with this notion, CGH array analysis showed quantitative and qualitative reductions of chromosomal numerical abnormalities in Ikaros-deficient BCR-ABL tumors as compared to BCR-ABL⁺/⁰; Ik⁺/⁺ tumors. Global Transcriptomic analysis revealed that 293 probsets are deregulated between BCR-ABL⁺/⁰; Ik⁺/⁺ and BCR-ABL⁺/⁰; IkL/⁺ tumors

Failure to regulate cellular proliferation is one of the hallmarks of cancer. The development of pre-B cells is demarcated by alternating stages of quiescence, in which immunoglobulin receptors are sequentially rearranged, and clonal expansion, in which signals from the assembled pre-B cell receptor lead to proliferation. Ikaros (encoded by Ikzf1 in mice) is a transcription factor that regulates gene expression in cycling B cell progenitors to enforce proliferative arrest. In humans IKZF1 mutations are prevalent in subsets of haematological malignancies and result in inappropriate proliferation, therefore the regulation of the cell cycle by Ikaros may be fundamental to its tumour suppressor function. The study of Ikaros-mediated cell cycle arrest in pre-B cells is complicated by the role of Ikaros in the regulation of genes involved in pre-B cell differentiation. I used 3T3 fibroblasts as a reductionist model to study the regulation of the cell cycle by Ikaros independently of pre-B cell receptor signalling. Using this model I performed an RNAi screen to discover novel regulators that cooperate with Ikaros to arrest the cell cycle. Amongst a number of candidates I identified the scaffolding protein SSeCKS (Akap12) as necessary for Ikaros-mediated proliferative arrest. Overexpression of Ikaros and SSeCKS synergistically arrested the cell cycle and silenced the expression of the proto-oncogene Myc. Utilising fibroblasts in which Ikaros and SSeCKS could be inducibly expressed I performed RNAseq to profile the global gene expression of cells that had undergone Ikaros and SSeCKS-mediated cell cycle arrest. Ikaros and SSeCKS together regulated the expression of hundreds of genes to coordinate proliferative arrest. The insights gained from these analyses may be applied to pre-B cells to deepen our understanding of the role of Ikaros in the regulation of proliferation in normal development and in the leukaemic state.

El desarrollo embrionario del núcleo estriado está controlado por gran cantidad de factores de transcripción. En esta tesis, nos centramos en el estudio de los miembros de la familia de factores de transcripción Ikaros. Los miembros de esta familia que se expresan en el primordio embrionario del núcleo estriado, eminencia ganglionar lateral (EGL) son Helios e Ikaros. Helios se expresa en la EGL durante la etapa embrionaria y es completamente ausente en el estriado de animales adultos. Esta proteína se expresa tanto en las zonas donde habitan los precursores neurales como en las zonas de precursores más diferenciados. Presenta una elevada relación con marcadores de precursores neuronales inmaduros como la nestina siendo su expresión completamente ausente en los precursores neurales que se diferenciarán hacia astrocitos. Los estudios realizados in vitro, mediante el uso del ensayo de neurosferas y de cultivos primarios, demuestran que la sobreexpresión de Helios induce un aumento en la neurogénesis y una reducción en la astrogénesis. Al estudiar el otro factor de la familia, Ikaros, observamos que esta proteína se expresa exclusivamente durante las edades embrionarias del desarrollo de la EGL. Está localizada exclusivamente en la zona del manto donde residen los precursores neuronales postmitóticos. La sobreexpresión de Ikaros en el ensayo de neurosferas induce una reducción del número de células proliferantes. En cambio, los precursores derivados de animales deficientes de Ikaros presentan una tasa de proliferación más elevada. Estos resultados nos indican que Ikaros induce la salida de ciclo de los precursores neurales regulando el ciclo celular. El mecanismo de acción por el cual Ikaros induce la salida de ciclo celular es regulando los niveles de la proteína inhibidora de ciclina p21 y favoreciendo así la diferenciación hacia fenotipos neuronales, específicamente neuronas encefalinérgicasl. Además de demostrar la funcionalidad de estas proteínas, nuestros resultados demuestran que existe una relación en el mecanismo de acción de Helios e Ikaros y que ambas proteínas están situadas en la misma vía de diferenciación estriatal de la familia Dlx. Así pues, la presente tesis describe la función de dos factores de transcripción, Helios e Ikaros, que ambos están implicados en el desarrollo embrionario del núcleo estriado.
“Study the role of the family of Ikaros transcription factor during striatum development” TEXT: The embryionic development of striatum is controlled by many transcription factors. In this thesis, we focus on the study of family members of Ikaros transcription factor. The members of this family that are expressed in the striatum embryonic primordium, lateral ganglionic eminence (LGE), are Helios and Ikaros. Helios is expressed during embryonic ages specifically in LGE. During development, Helios is expressed in areas where precursors are inhabited (subventricular zone), as well as in areas where more mature cells are located (mantle zone). This protein has a high relationship with markers of immature neural precursors as nestin. Neurospheres assay that Helios overexpression induces a reduction of proliferative tax in the precursors. Moreover, Helios induces an increase in neurogenesis process and a reduction in astrogenesis differentiation as we can observe studying the overexpression of Helios in primary cultures. The other member of the family expressed in LGE, Ikaros, is expressed only in embryonic ages exclusively in the mantle zone. In vitro studies show that Ikaros overexpression in precursors cultures induce a reduction in proliferative tax. However, precursors from Ikaros knockout mice proliferate more than precursors from wilt type. The reduction of proliferative tax is regulated by cyclin inhibitor protein p21. Cell cycle exit promote enkephalinergic neuronal differentiation as well as astrogenic inhibition as we can observe in neurosphere assay and primary cultures. In order to study Ikaros and Helios function in striatum development, we observe Helios and Ikaros act in the same differentiation involved in striatum development, de Dlx pathways. Both proteins are expressed downstream of Dlx1/2 however the relationship with the other transcription factor Dlx5/6 is different. While Helios is expressed upstream of Dlx5/6, Ikaros is expressed downstream of these transcription factors. In summary, we describe the role of two new proteins involved in striatum development: Helios and Ikaros.

The main topic of my PhD studies was to investigate the role of sumoylation in the function of Ikaros transcription factor, that regulates the lymphocyte differentiation and function. Sumoylation is a posttranslational modification that can change the properties and regulate the function of a given protein. Up to now, one study addressed the question of how sumoylationmodulates Ikaros function. It shows that Ikaros is sumoylated in total primary thymocytes, and that this dynamic event modulates Ikaros' repressive function. It also describes two consensus sumoylation sites on Ikaros (K58 and K240), the sumoylation of which leads to loss of Ikaros repressive function in ectopic reporter gene assays. The final conclusion of the study is that sumoylation does not alter the nuclear localization of Ikaros but acts as a mechanism disrupting its participation in both histone deacetylase (HDAC) dependent and independent repression. My work shows the presence of additional sumoylation site on Ikaros and demonstrates that sumoylation does not significantly alter its interaction with the nucleosome remodelling and histone deacetylase (NURD) complex in T-cell lines. The functional analysis of sumo-deficientmutants indicates a complex role of this modification in regulating Ikaros' transcriptional properties. The identification of this new sumoylation site contributes to a better understanding of Ikaros' dual repressive - activating function and suggests the existence of conditional Ikaros' interacting partners. Moreover, the different Ikaros splicing isoforms would have differentsumoylation profiles, which would complete the knowledge of their functional diversity.

Oppgaven utforsker outsiderposisjonen i Axel Jensens bøker Ikaros (1957) og Joacim (1961). Problemformuleringen legger særlig vekt på protagonistenes motivasjon for å søke outsiderposisjonen, samt stiller spørsmålet om det i det hele tatt lar seg gjører å flykte fra samfunnet og i hvilken grad outsiderposisjonen oppnås. Analysene er foretatt på bakgrunn av Colin Wilsons teoriverk om outsiderens karakter, The Outsider (1956). Wilson bruker eksempler fra tidligere outsiderlitteratur til å skissere opp en typologi av outsidere og belyse ulike sider ved det han kaller «outsiderens problem». Dette «problemet» er grunnet i outsiderens identitet og gir seg utslag i hans mentalitet og syn på tilværelsen. Ulikheten i protagonistenes motiver for å snu ryggen til samfunn og sivilisasjon har gjort at outsiderposisjonen fortoner seg ulikt i Ikaros og Joacim, noe som også har gjort at analysene består av nærlesning av ulike aspekter ved outsidertematikken. I tillegg til Wilsons teori består av en kontekstualiserende del som kommenterer hvordan Jensens outsidere kan leses i lys av andre samtidige litterære outsidere, og hvordan tidsperioden bøkene er skrevet i påvirker lesningen av outsidertematikken. Resepsjonskapittelet tar for seg forskjellene i kvalitetsvurderingene av bøkene, og vektlegger hvordan anmelderne reagerte på outsidertematikken. I avslutningskapittelet samles trådene og det reflekteres rundt outsiderposisjonen i Ikaros og Joacim, særlig med hensyn til hvordan, og i hvilken grad outsiderens problemer løser seg, sett i lys av Wilsons teorier.

Les cellules T auxiliaires TH17 sont caractérisées par l’expression de la cytokine IL-17A, ainsi que le facteur de transcription RORɣt. Elles sont connues pour jouer un rôle clé dans la pathogenèse de la sclérose en plaques. Ces cellules existent sous deux formes : les cellules régulatrices, immunomodulatrices, et les cellules pathogènes qui sont critiques pour l'inflammation. Il est donc important de comprendre le mécanisme qui sous-tend la différenciation des cellules TCD4+ naïves en ces deux types cellulaires. J'ai trouvé que le facteur de transcription Ikaros est un répresseur indirect de la transcription des gènes pathogéniques (Il3, Csf2, Ifng, Stat4…) dans les cellules TCD4+ naïves murines, cultivées pour induire une polarisation vers le phénotype TH17 régulateur. De plus, en absence d’Ikaros et en conditions de culture régulatrice, l’ajout d’IL-6 seul augmente l’expression de GM-CSF, facteur clé dans l’induction des maladies auto-immunes, suggérant un rôle d’Ikaros dans la régulation de cette voie. En conclusion, nos résultats suggèrent que Ikaros est nécessaire pour polariser correctement les cellules TCD4+ naïves dans le programme TH17
TH17 cells are characterized by the expression of the cytokine IL-17A, as well as the transcription factor RORɣt. They are known to play key role in the pathogenesis of the multiple sclerosis. These cells exist in two forms: the regulating cells, immunomodulatory, and the pathogenic cells which are critical for the inflammation. Thus it is important to understand the mechanism which underlies the differentiation of naïve CD4+ T cells in these two cellular types. I found that the transcription factor Ikaros is an indirect repressor of the transcription of pathogenic genes (Il3, Csf2, Ifng, Stat4…) in naïve CD4+ T cells, cultured to induce a polarization toward regulatory TH17 cells. Moreover, in absence of Ikaros and in regulatory condition of culture, adding IL-6 alone increases the expression of GM-CSF, key factor to induce auto-immune diseases, suggesting a role of Ikaros in this pathway. In conclusion, our results suggest that Ikaros is necessary to polarize correctly naïve CD4+ T cells in TH17 cells

Le développement et la fonction des cellules dendritiques plasmacytoïdes (pDCs) doivent être finement régulés afin d’éviter le développement de maladies auto-immunes ou de leucémies. Il a été montré récemment qu’une fraction des leucémies humaines dérivées des pDCs possède des mutations de type perte de fonction dans le locus IKZF1 qui code pour le facteur de transcription Ikaros. Ainsi l’étude de la fonction d’Ikaros dans les pDCs pourrait aider à comprendre son possible rôle de gène suppresseur de tumeur. Les souris hypomorphiques pour Ikaros (IkL/L) ne possèdent pas de pDCs matures dans la rate et les ganglions lymphatiques mais accumulent des pDCs immatures dans la moelle osseuse (MO). De manièreintéressante, les pDCs de MO IkL/L montrent une activation ectopique de la voie Notch. Nous avons trouvé qu’un inhibiteur de gamma-secrétase (GSI), qui inhibe la voie Notch, permet de restaurer la différenciation de pDCs fonctionnelles dans les cultures de cellules de MO. Les principaux progéniteurs dendritiques affectés par le GSI sont le progéniteur myéloïde commun (CMP) et le progéniteur des macrophages et des cellules dendritiques (MDP). Comme le GSI inhibe la voie Notch, nous avons aussi inactivé RBPJ, le facteur de transcription situé en aval des récepteurs Notch. Contre toute attente, l’inactivation de RBPJ ne récapitule pas les effets observés avec le GSI. De plus, les cellules IkL/L déficientes pour RBPJ continuent de répondre au GSI, ce qui suggère que le GSI possède d’autres cibles en plus de la voie Notch dans ce système. Nos données montrent ainsi qu’Ikaros est requis pour la différenciation terminale des pDCs et qu’il agit en partie en bloquant une voie GSI dépendante Notch indépendante
The development and function of plasmacytoid dendritic cells (pDCs) must be tightly regulated to prevent autoimmune disease or leukemia. It was recently discovered that a fraction of human pDC-derived neoplasms exhibit loss of function mutations of the IKZF1 locus, which encodes the Ikaros transcription factor. Deciphering the function of Ikaros in pDCs could thus help understand its probable tumor suppressor function. Mice hypomorphic for Ikaros (IkL/L) are devoid of mature pDCs in the spleen and lymph nodes but accumulate immature pDCs in the bone marrow (BM). Interestingly IkL/L BM pDCs exhibit an ectopic activation of the Notch pathway. We found that a gamma secretase inhibitor (GSI), which inhibits Notch signalling,rescues the differentiation of functional pDCs in BM cultures. The main dendritic cell progenitors affected by GSI are the common myeloid progenitors (CMP) and the macrophage and dendritic cell progenitors (MDP). As GSI inhibits the activation of the Notch pathway, we also inactivated RBPJ, the transcriptional effector of the Notch pathway. Surprisingly, RBPJ inactivation did not recapitulate the effect of GSI. Moreover, RBPJdeficient IkL/L cells still respond to GSI, demonstrating that GSI targets additional events besides Notch in this system. Our data thus show that Ikaros is required for terminal differentiation of pDCs, and acts in part by blocking a Notch independent GSI-sensitive pathway

Myc is a transcription factor that plays a key role in many cellular functions. When deregulated, it becomes a potent oncogene and a hallmark of many human cancers. Although its activity has been proven to strongly depend on many cofactors, we are still far from a comprehensive understanding of Myc’s mechanisms of action in transcriptional control. Moreover, since Myc itself is not easily druggable, targeting its cofactors or downstream effectors may constitute a more promising therapeutic strategy in Myc-driven tumors. Here, we have used the Chromatin Proteomics (ChroP) technology to identify new Myc interactors. Among the list of identified candidates, we have chosen to focus our attention on two lymphocyte specific transcription factors, Ikaros and Aiolos. We validated the interaction of Myc with these factors and started to study its functional meaning. Our working hypothesis is that Myc and Ikaros/Aiolos antagonize each other on chromatin at selected target loci. We were able to identify a subset of genes regulated in opposite manners by Ikaros and Myc, involved mainly in cell cycle regulation, metabolism of lipids and metabolic stress response; the next step will be to study more in depth the mechanisms behind their regulation and their functional significance.

Ikaros-family transcription factors play important roles in the control of haematopoiesis. Family members are predicted to contain up to six classical zinc fingers that are arranged into N- and C-terminal domains. The N-terminal domain is responsible for site-specific DNA binding, whereas the C-terminal domain mediates primarily the homo- and hetero-oligomerisation between family members. Although the mechanisms of action of these proteins are not completely understood, the zinc finger domains are known to play a central role. The work presented here was performed to further elucidate the physical and functional properties of the C-terminal domain, in particular, the C-terminal domain of Eos. A range of biophysical techniques was used to demonstrate that the C-terminal domain of Eos mediates the formation of complexes that consist of nine or ten molecules. This result is highly unusual in the context of what is currently known about transcription-factor biology. In order to gain insight into the structural basis of oligomerisation, NMR spectroscopy was used to determine the solution structure of the second zinc finger from the C-terminal domain of Eos. This structure exhibited several unusual properties for a zinc finger, and these may contribute to the basis for the inability of this domain to bind DNA. Finally, alanine-scanning mutagenesis revealed residues that are involved in the homo-oligomerisation of E03, and these results are discussed in the context of the structure of the C-terminal domain. This work has not only shed light onto the mechanisms through which lkaros-family proteins regulate

Pancreatic cancer is one of the deadliest cancers with a five-year survival rate of 6%. Pancreatic cancer is resistant to conventional chemotherapy and is usually diagnosed at late stages. Current treatment options have minimal effects in extending patients' lives beyond 10 months. One significant limitation in developing treatments to combat pancreatic cancer is its immunosuppressive microenvironment. Pancreatic cancer secretes factors that activate immunosuppressive cells, such as regulatory T cells (Tregs). These Tregs suppress effector CD4+ and CD8+ T cell anti-tumor immune responses. Therefore, novel treatment options to reduce Treg-mediated immune suppression and increase the numbers and functions of CD4+ and CD8+ T cells are paramount to enhance anti-tumor immunity in pancreatic cancer tumor-bearing (TB) hosts. The alternatively spliced transcription factor Ikaros is essential for lymphocyte development and is considered a tumor suppressor in T cells. Ikaros' protein stability and function are regulated by its phosphorylation and dephosphorylation by protein kinase CK2 and phosphatase 1 (PP1), respectively. Mutations and functional inactivation of Ikaros have mainly been investigated in T cell leukemias and lymphomas. In this dissertation, we investigated the role of Ikaros in regulating T cell homeostasis in murine pancreatic cancer. In this study, we report that Ikaros proteins are degraded by the ubiquitin-proteasome pathway in response to factors produced by murine pancreatic cancer cells. Our results further suggest that an increase in CK2 activity leads to Ikaros' degradation and disrupts its localization to pericentromeric heterochromatin in our murine pancreatic TB model. This loss of Ikaros expression is accompanied by an imbalance in T cell homeostasis. More specifically, we observe a significant decrease in effector CD4+ and CD8+ T cells but an increase in Treg percentages in TB and spontaneous pancreatic cancer models. T-cell specific defects in Ikaros protein expression were also observed in TB CD3+ T cells. Apigenin, a natural plant flavonoid and CK2 inhibitor, restored expression of some Ikaros isoforms in our TB model. Apigenin also displayed immunological benefits evident by enhanced anti-tumor immunity in TB mice. These data provide mechanistic and functional evidence that pharmacological inhibition of CK2 can regulate Ikaros expression and identifies the possible involvement of Ikaros in regulating T cell immune responses in murine pancreatic cancer.

Le facteur de transcription, Ikaros, est crucial au linéage B. Nous avons étudié comment Ikaros régule le développement, l’activation et la commutation isotypique des cellules B. Premièrement, nous avons montré qu’Ikaros contribue à la différenciation des cellules B en induisant l’expression du récepteur de l'Interleukine 7 dans les cellules pro-B. Puis, nous avons révélé qu'Ikaros régule la prolifération des cellules B en contrôlant l'activation des protéines kinases ERK et p38 après stimulation du récepteur des cellules B. Ces données suggèrent qu'Ikaros joue un rôle central dans la signalisation du récepteur des cellules B. Enfin, nous avons montré qu’Ikaros réprime la commutation de classe vers les isotypes IgG2b et IgG2a et induit la commutation vers tous les autres isotypes. De plus, Ikaros contrôle la commutation de classe et spécifie le choix des isotypes par un mécanisme de compétition transcriptionelle entre les gènes codants pour les regions constantes des immunoglobulines
The Ikaros transcription factor is a critical regulator of the B lineage. Here we have elucidated mechanisms by which Ikaros controls B cell development, activation and class switch recombination (CSR). First, we show that Ikaros contributes to B cell development by promoting the expression of the Interleukin 7 receptor on pro-B cells. Second, we demonstrate that Ikaros deficient B cells hyper-activate the ERK and p38 mitogen activated protein kinases (MAPK) after B cell receptor stimulation, and that this leads to increased proliferation. These results establish Ikaros as a central regulator of B cell receptor signaling. Third, we show that Ikaros suppresses CSR to IgG2b and IgG2a, and promotes CSR to all other isotypes. Further we show that Ikaros mediates this affect on isotype choice by controlling transcriptional competition between constant region genes in individual cells for CSR and we reveal this competition as a critical and general mechanism for isotype specification

I uppsatsen analyseras musiken i Jan Troells film Så vit som en snö (2001). Det framgår att musiken är en viktig komponent i filmen. I filmen förekommer tidstypisk musik: visartade melodier, jazz och operettmelodier vilka framförs i olika slags arrangemang. Melodierna används för att skapa en narrativ "väv" i ljud- och musikläggningen. Flera melodier har en gemensam texttematik. Som en central ljudläggningsteknik används musikaliska teman. Musikinslagen karakteriseras som diegetiska, icke-diegetiska eller metadiegetiska. I uppsatsen skiljs vidare mellan strukturell, metaforisk och kontextuell funktion hos musikinslagen. Dessutom används begrepp som timbre och klangfärg för att beskriva musiken.

Globally, increased length-of-life trends parallel the rise in reported levels of loneliness. Social connectedness is associated with a 50% reduced risk of premature mortality, and research has shown there is a moderately strong association between people who are lonely and people who have an unmet desire to belong. As so, urban planners and designers continually try to understand place relationships within communities to better the health and welfare of future generations. This thesis acknowledges there is a gap of understanding how the built and natural environment play a role in an increased sense of belonging, thus contributing to an alleviation of reported feelings of loneliness and potentially increasing length and quality of life. Utilizing the ‘Blue Zone’ island of Ikaria as a case study, where residents commonly live well beyond 90 and 100 years, this thesis aims to better understand connections between longevity and the physical environment for the longest-lived, cataloguing how these connections influence feelings of belonging. The research consists of a site visit to Ikaria, Greece, where data was collected over the period of five days, employing interview and field observation methods. Drawing from key concepts within environmental psychology and placemaking, the work critically explores the perceptions of Ikarian elders and the public space they regularly inhabit, as well as their role in contributing to sense of place and feelings of belonging. The conclusion offers suggestions on how urban planners might take these findings into consideration, as it is of great importance for planning professionals to better understand individual perceptions of their environment, which may help prevent alienation and increase overall wellbeing.

B-lymphopoiesis is characterised by orderly progression through a series of stages of differentiation, each of which is associated with specific patterns of protein expression and cellular proliferation. These processes require a high degree of regulation by the coordinated action of a series of transcription factors that are critical for appropriate stage-specific gene expression. We describe a novel interaction between Ikaros and Foxp1, two transcription factors that are essential for normal B-lymphopoiesis, and which frequently exhibit abnormal expression in B-lymphoid malignancy. By co-immunoprecipitation, we have demonstrated physical association between the DNA-binding domains of these proteins in vivo, which is recapitulated in a cell-free system in vitro. In functional experiments, ectopic overexpression of Foxp1 by retroviral infection of pre-B-cells resulted in an increased proportion of cells in the G2 phase of the cell cycle. This phenotype was associated with increased transcription of Gpr132, a gene which encodes the G2A protein that is known to induce G2/M cell cycle arrest in mammalian cells. The phenotypic effects of Foxp1 overexpression were abolished by coinfection with Ikaros, which abrogated induction of Gpr132 expression by Foxp1, and caused displacement of Foxp1 from the Gpr132 gene, as shown by chromatin immunoprecipitation. Co-infection of IK6 (an Ikaros isoform which lacks the Foxp1 interaction domain and which is frequently expressed in B-cell acute lymphoblastic leukaemia) had no effect on Gpr132 expression or Foxp1 occupancy of the Gpr132 gene, and failed to abolish the phenotypic effects of Foxp1 overexpression. Our results contribute to a growing understanding of transcription factor interplay during normal lymphopoiesis, and provide mechanistic insights into the roles of Ikaros and Foxp1 in lymphocyte cell cycle homeostasis. In addition, these findings may contribute to the understanding of cell cycle dysregulation and chemotherapeutic resistance that characterise certain subtypes of acute leukaemia that exhibit aberrant expression of these proteins.

The gene lin-42 is an ortholog of the mammalian period gene, a component of the circadian pathway that converts environmental stimuli into behavioral and physiological outputs over 24 hours. Mammalian period also regulates adult stem cell differentiation, although this function is poorly understood. The structure, function and expression of lin-42 are all similar to period. Therefore, we are studying lin-42 regulation and function during C. elegans larval development as a model for understanding period control of mammalian stem/progenitor cell development.

Previous work has shown that ZTF-16 is a regulator of lin-42 transcription. The lin-42 locus encodes three isoforms, and we have characterized lin-42 isoform specific regulation by ZTF-16 through phenotypic assays and analysis of transcriptional reporter strains. Our data show that ZTF-16 regulates the cyclic expression of lin-42A and lin-42B during larval development. However, ztf-16 is not expressed during the adult stage and does not regulate lin-42C, which is expressed only in adults and may be responsible for the circadian functions of lin-42. We also show that ztf-16 reduction-of-function mutations phenocopy loss-of- function phenotypes of the lin-42A/B isoforms. Finally, we have found that deletion of a putative ZTF-16 transcription factor binding site within the lin-42BC promoter abolishes tissue-specific expression patterns. Together, these data indicate that ZTF-16 is required to regulate the expression of lin-42A/B during C. elegans development, and may do this by direct binding to the lin-42BC promoter. Our  findings pave the way for testing the possible regulation of period expression by HIL-family transcription factors in mammalian tissues.

Master of Science

Ikaros est un facteur de transcription à doigts de zinc qui est un régulateur essentiel de la lymphopoi͏̈èse. Différentes mutations du locus Ikaros, produites chez la souris, conduisent au développement de lymphomes T, suggérant qu'Ikaros est un gène suppresseur de tumeurs. Les souris homozygotes IkL/L (mutation hypomorphe d'Ikaros) développent des lymphomes T entre l'âge de 3 et 6 mois. Cependant, les mécanismes moléculaires impliqués dans la formation de ces tumeurs restent encore peu connus. L'analyse du transcriptome des tumeurs IkL/L montre une augmentation de l'expression de plusieurs gènes de la voie de signalisation Notch, qui est également observée dans des tumeurs en cours d'expansion, suggérant qu'une altération de la voie Notch constitue un événement précoce dans l'établissement des tumeurs IkL/L. D'autre part, la ré-expression d'une forme normale de la protéine Ikaros dans des lignées cellulaires établies à partir de cellules tumorales conduit à une diminution du niveau d'expression de plusieurs gènes cibles de Notch, suggèrant également qu'Ikaros régule directement l'expression de certains de ces gènes. Par ailleurs, la ré-expression d'Ikaros dans ces lignées inhibe l'amplification des cellules tumorales in vitro. Ces résultats montrent qu'une réduction de l'activité d'Ikaros dans les thymocytes peut être favorable à l'initiation du développement tumoral, et qu'il pourrait exister une convergence fonctionnelle entre Ikaros et Notch dans le processus de transformation des lymphocytes T
Ikaros is a zinc finger transcription factor which is an essential regulator of lymphopoiesis. Different mutations in the Ikaros locus lead to T lymphoma development, suggesting that Ikaros is a tumor suppressor gene. Homozygote IkL/L mice expressing a hypomorphic mutation of Ikaros develop T lymphomas between 3 and 6 months of age. However, the molecular mechanisms involved in the formation of these tumors are still ill-defined. Transcriptome analyses of IkL/L tumors show an up-regulation of several genes of the Notch signaling pathway. This deregulation is also observed in early tumors, suggesting that deregulation of the Notch pathway is an early event in IkL/L tumor development. Re-expression of a normal Ikaros protein in IkL/L tumor-derived cell lines downregulates several Notch genes, suggesting that Ikaros directly modulates the expression of some Notch genes. Moreover, the re-expression of Ikaros in the cell lines triggers an arrest in tumor cell growth in vitro. These results show that thymocytes with a reduction in Ikaros activity are more susceptible T lymphoma development, and that Ikaros and Notch could cooperate during T lymphocyte transformation

La famille des facteurs de transcription lkaros, est composée des protéines lkaros, Helios, Aiolos et Eos. Elles sont exprimées pendant le développement et régulent la différenciation des lymphocytes B et T. Ces protéines présentent une forte homologie de leurs séquences nucléiques et protéiques et sont toutes impliquées dans l'apparition de leucémies lymphoblastiques T ou B. Ces facteurs présentent toutefois de très fortes divergences dans leurs profils d'expression, leurs fonctions et leurs gènes cibles. Ce travail à partir d'une lignée cellulaire T immature déficiente pour lkaros à permis d'étudier les différences fonctionnelles et moléculaires des membres de la famille. La réexpression d'lkaros, d'Aiolos et d'Helios permet la différenciation et la diminution de la prolifération de ces cellules. Cette expérience a montré que les différents membres de la famille avaient des capacités distinctes pour activer ou réprimer certains gènes cibles. Un échange des séquences des domaines de liaison à l'ADN de Aiolos et d'lkaros, a permis de montrer que la spécificité fonctionnelle est en partie déterminée par le domaine de liaison à l'ADN, mais aussi par les autres régions d'lkaros et d'Aiolos
The lkaros transcription factor family is made of the proteins lkaros, Helios, Aiolos and Eos. They are expressed during the development and regulate the differentiation of lymphocytes B and T. These proteins present a strong homology between their nucleic and protein sequences and are involved the appearance of T or B lymphoblastic leukaemia. However these factors present strong differences in their profiles of expression, their functions and their target genes. An immature T cell line, deficient for lkaros, allows us to study the functional and molecular differences of members of the family. There-expression of lkaros, Aiolos and Helios allows the differentiation and the decrease of the proliferation of these cells. I also showed that the various members of the family had different capacities to activate or repress certain target genes. An exchange of the protein sequences coding for the DNA binding domain (DBD), shows that the functional specificity is partially determined by the DBD domain, but also by the other regions of lkaros and Aiolos

In the past two decades, childhood acute lymphoblastic leukemia (ALL) cure rate has reached over 80% due to treatment advances, but some resistant ALL subtypes, such as those that carry the Philadelphia (Ph+) chromosome, still don’t respond to therapy. The presence of BCR-ABL in ALL children is correlated to a very poor prognosis, nevertheless, several long-scale studies have shown that Ph+ ALL is heterogeneous in terms of clinical parameters and patients respond differently to the therapy, what suggests the presence of additional mechanisms of leukemogenesis. A set of international studies with large series of patients have shown that an earlier remission after induction with glucocorticoids and intrathecal methotrexate (IT MTX) is correlated to a better outcome. In the present study we looked for secondary genetic lesions in a group of 78 consecutive Ph+ ALL children diagnosed in Italy between 2000 and 2010, specifically studying deletions in the IKZF1 gene and the miRNA profile according to the first therapy response. The IKZF1 gene was studied by PCR, direct sequencing and SNP array and a high incidence of deletions (70%) was detected in our children, specially the d4-7(62%). The miRNA signature was analysed by miRNA array comparing two groups of patients differentiated according to MRD/prednisone response. Particularly miR-221, 222 and miR-125b-2 are highly overexpressed in the poor responder group of patients. We showed in vivo that miR-125b-2 acts as oncogene and increases leukemia aggressiveness. Further studies will be necessary to understand how IKZF1, miR-221,222 and 125b act in cooperation with BCR-ABL to induce leukemia. In the future, novel target therapies can emerge as pro-apoptotic approaches to be added to the anti-tyrosine kinase drugs in order to improve response and survivor in Ph+ ALL.

La voie Notch joue un rôle majeur dans le contrôle des aspects fondamentaux de la vie multicellulaire,comme la prolifération ou la différenciation. Il est clairement établi que cette voie est requise pour la maturation des cellules T, et que la dérégulation de cette voie est oncogénique. Le gène Notch1 est la cible de mutations activatrices dans plus de 50% des leucémies aiguës lymphoblastiques de type T (LAL-T). Cependant, les mécanismes régulant la voie Notch lors du développement des cellules T sont encore incompris. Mon équipe a montré que le facteur de transcription Ikaros, qui est également un suppresseur de tumeur, réprime la transcription du gène Hes1, gène cible de Notch le mieux caractérisé, par fixation compétitive sur des séquences de liaison à l’ADN communes. De plus, les souris IkL/L, générées au laboratoire et déficientes pour Ikaros, développent des tumeurs thymiques associées à une hyperactivation de la voie Notch. Il existe donc une corrélation entre l’absence d’Ikaros et l’hyperactivation de la voie Notch. Mon objectif a été d’étudier le rôle d’Ikaros dans l’extinction de la voie Notch au cours de la différenciation des cellules T. Je me suis attachée à déterminer si l’hypothèse de compétition entre Ikaros et la voie Notch pouvait être étendue à d’autres gènes. Pour le démontrer, j’ai identifié d’une part les gènes régulés par Ikaros dans des cellules T leucémiques issues des souris IkL/L (où la voie Notch est hyperactivée), et d’autre part les gènes régulés par Ikaros dans ces mêmes lignées où j’ai surexprimé Ikaros. De façon intéressante, les gènes les plus activés par Notch sont ceux qui sont le plus réprimés par Ikaros. Mes résultats de ChIP-Sequencing montrent qu’Ikaros et RBPJ se fixent sur l’ADN au même endroit pour la moitié des gènes corégulés par Notch et Ikaros. Par ailleurs, j’ai identifié quelques gènes cibles pour lesquels Ikaros entre en compétition directe avec RBPJ pour la fixation de séquences consensus communes. De plus, j’ai montré que la plupart des gènes cibles communs sont aussi dérégulés in vivo, au cours de la maturation des cellules T chez les souris IkL/L par rapport aux souris sauvages. Ces recherches permettent non seulement d’établir un nouveau mode de régulation de la voie Notch au cours de la différenciation des cellules T, mais aussi de caractériser les gènes importants pour leur transformation, ainsi que de déterminer le stade de transformation
The Notch signalling pathway plays a major role in controlling fundamental aspects of multicellular life including proliferation, stem cell maintenance, differentiation and death. It has a central role in the haematopoietic system and the requirement for Notch activation in thymocyte differentiation is well established. Moreover, when deregulated, the Notch pathway is oncogenic as more than 50% of the T Lymphoblastic Acute Leukemia (T-ALL) display activating mutations in the Notch gene. However, the mechanisms that could regulate the Notch pathway during T cell differentiation are still poorly understood. Ikaros is a zinc finger transcription factor, predominantly expressed in haematopoietic cells; it functions as a transcriptional repressor and a tumor suppressor. Our data show that Ikaros-deficient mice develop T cell lymphomas, which are associated with an early activation of the Notch pathway. Using the Notch target gene Hes1 as a model, we recently reported that Ikaros and RBPJ, the transcriptional mediator of Notch signalling, compete for binding to two elements in the Hes1 promoter in immature thymocytes. These results indicate that Ikaros functions as a transcriptional checkpoint to repress the Notch target gene Hes1 expression in T cells. The molecular basis of this repression could be due to the similarity between the sequences recognized by Ikaros and RBPJ. To determine if Ikaros repression plays a central role in silencing Notch target genes during T ymphopoiesis, I have studied its capacity to regulate the transcription of Notch target genes on a global scale in gain- and loss-of-function systems. My results indicate that Ikaros represses most of Notch induced genes. Furthermore, both Ikaros and RBPJ bind to the promoters of half the common target genes, exactly at the same place. I have also demonstrated on several genes that Ikaros directly competes with RBPJ in order to bind to same consensus sequence in the promoter of these common target genes. Finally, loss of Ikaros causes the deregulation of a significant number of Notch target genes during T cell differentiation, before the cells get transformed. These data suggest a new molecular pathway by which the Notch pathway is regulated during T cell development. Ikaros competes with RBPJ to repress a set of common target genes, and this mechanism plays a central role in silencing Notch target genes during T lymphopoiesis. Furthermore, the loss of Ikaros would cause a deregulation of the Notch target genes and this could lead to leukemogenesis

El presente trabajo de investigación aborda el análisis de la marca corporativa Ikarus mediante una auditoria de marca desarrollada en base a las variables del marketing mix y las dimensiones de brand equity de David Aaker. Esta investigación resulta relevante debido al actual mercado competitivo al que se enfrentan los emprendimientos peruanos, el cual obliga a dichas empresas a proponer nuevas acciones dirigidas a la calidad, diferenciación, segmentación y políticas de precio que contribuyan a su crecimiento. La investigación se realiza mediante un estudio de caso que se centra en un emprendimiento peruano del sector textil y confecciones, específicamente de ropa urbana. Propone entender cuáles son las acciones realizadas por la empresa y la perspectiva de los clientes de la marca, así como presentar recomendaciones que aporten a su mejora. La marca corporativa Ikarus fabrica y comercializa prendas de ropa urbana cuyo valor agregado son sus diseños únicos. Desarrollándose en un contexto tan competitivo, se considera necesaria una evaluación que permita a la empresa encontrar tanto sus puntos de mejora como sus fortalezas, para así tomar decisiones estratégicas respecto a sus marcas, apuntando hacia su crecimiento en el sector. El análisis se realiza a través de un enfoque mixto, con ayuda de herramientas como focus group, observaciones en puntos de venta, entrevistas a profundidad a colaboradores internos y externos, y encuestas a clientes de la marca, las cuales brindaron información útil al estudio. Asimismo, se plantearon en la investigación dos hipótesis importantes: las acciones de la marca Ikarus han servido de apoyo para su crecimiento y el brand equity de la marca es positivo. En base al análisis del marketing mix para estudiar las acciones realizadas por la marca y al análisis de las dimensiones del brand equity para estudiar la perspectiva del cliente, se comprueban las hipótesis planteadas. La marca Ikarus posee un conjunto de acciones que logran generar un crecimiento, así como un nivel alto en las distintas dimensiones del brand equity: calidad percibida/medidas de liderazgo, lealtad, reconocimiento y medidas de asociación/ diferenciación.
Tesis

Les protéines Ikaros et Notch ont toutes deux été impliquées de façon indépendante dans le développement de lymphomes T chez l’homme et la souris. Chez les souris portant une mutation pour Ikaros, le développement de lymphomes T est toujours associé à une surexpression précoce de la voie Notch dans les thymocytes. Ikaros et de RBP-Jk reconnaissent des séquences in vivo dans des cellules DN, suggérant une fixation séquentielle des deux facteurs au cours de la transition DN3-DN4. Au niveau chromatinien, les cellules DP IkL/L présentent une altération des modifications répressives d’histones au niveau du promoteur Hes-1. Ces résultats suggèrent que la compétition entre Ikaros et RBP-Jk semble être un mécanisme central de la régulation des gènes dans les cellules T. L’extinction des gènes cibles de Notch par Ikaros est nécessaire à la différenciation des thymocytes et la dérégulation de ce mécanisme pourrait être l’une des causes du développement de leucémies T en l’absence d’Ikaros
Ikaros and Notch have been found independently to be involved in T lymphogenesis in mouse and man. In mice bearing a hypomorphic mutation in Ikaros, T lymphoma development is associated with early Notch target gene overexpression in thymocytes. Ikaros and RBP-Jk bind to Hes-1 promoter sequences in vitro in a specific and mutually exclusive manner. Both proteins recognise regions containing those Hes-1 promoter sequences in vivo in DN cells, suggesting the sequential binding of both factors during the DN3-DN4 transition. At the chromatin level, IkL/L DP cells lack repressive histone modifications at the Hes-1 promoter. To conclude, these results suggest that competition between Ikaros and RBP-Jk may be a central mechanism in regulating T cell genes. Notch target gene silencing by Ikaros is essential to thymocyte differentiation and deregulation in this mechanism could in absence of Ikaros cause T leukemic development

La compréhension de l'évolution des chaînes de montagnes nécessite la reconstruction des chemins pression-température-temps suivis par les roches métamorphiques. La datation U-Pb est souvent utilisée pour dater des événements métamorphiques, mais le lien entre les conditions du métamorphisme et l'âge fourni par les datations U-Pb sur zircon n'est pas direct. Les isotopes de l'oxygène sont proposés dans cette étude pour mettre en relation la croissance du zircon avec celle du grenat dans les roches métamorphiques du domaine égéen. L'étude de quatre échantillons de l'île de Naxos a montré que cette relation est possible si (i) l'équilibre isotopique de l'oxygène est atteint entre le grenat et le zircon au moment de leur formation et (ii) le grenat et le zircon sont restés clos pour l'oxygène et le plomb après leur formation. Cette méthode a permis de dater le début de l'exhumation des roches métamorphiques de Naxos entre 40 et 60 Ma, et le début du chemin rétrograde entre 13 et 19 Ma. A Ikaria et à Naxos, la croissance préférentielle du zircon entre 40 et 60 Ma localisée dans des roches provenant de zones riches en schistes indique que la pression et les circulations de fluides sont les paramètres qui ont favorisé la formation des zircons pendant le métamorphisme
The knowledge of pressure - temperature - time paths in metamorphic rocks is necessary to the understanding of orogenic belts evolution. U-Pb method is commonly used for dating metamorphic events, but the link between metamorphic conditions and ages obtained on zircon is not straightforward. Oxygen isotopes are proposed in this study for likening zircon to garnet growth in metamorphic rocks of the Aegean domain. The study of four samples from Naxos island showed that this correlation is possible if (i) isotopic equilibrium of oxygen is reached between garnet and zircon during their formation and (ii) garnet and zircon remained in closed system for oxygen and lead after their formation. This method permitted to date the beginning of the exhumation of metamorphic rocks from Naxos between 40 and 60 Ma, and the beginning of the retrograde path between 13 and 19 Ma. The preferential formation of zircon between 40 and 60 Ma in rocks localised in schist rich zones indicate that pressure and fluid circulation are the parameters that triggered zircon formation during the metamorphism

Given the current state of input/output (I/O) and storage devices in petascale systems, incremental solutions would be ineffective when implemented in exascale environments. According to the "The International Exascale Software Roadmap", by Dongarra, et al. existing I/O architectures are not sufficiently scalable, especially because current shared file systems have limitations when used in large-scale environments. These limitations are: Bandwidth does not scale economically to large-scale systems, I/O traffic on the high speed network can impact on and be influenced by other unrelated jobs, and I/O traffic on the storage server can impact on and be influenced by other unrelated jobs. Future applications on exascale computers will require I/O bandwidth proportional to their computational capabilities. To avoid these limitations C. Filippidis, C. Markou, and Y. Cotronis proposed the IKAROS framework. In this thesis project, the capabilities of the publicly available elastic-transfer (eT) module which was directly derived from the IKAROS, will be expanded. The eT uses Google’s Gmail service as an utility for efficient meta-data management. Gmail is based on the IMAP protocol, and the existing version of the eT framework implements the Internet Message Access Protocol (IMAP) client-server connection through the ‘‘Inbox’’ module from the Node Package Manager (NPM) of the Node.js programming language. This module was used as a proof of concept, but in a production environment this implementation undermines the system’s scalability and there is an inefficient allocation of the system’s resources when a large number of concurrent requests arrive at the eT′s meta-data server (MDS) at the same time. This thesis solves this problem by adopting an asynchronous non-blocking event driven approach to implement the IMAP client-server connection. This was done by integrating and modifying the ‘‘Imap’’ NPM module from the NPM repository to suit the eT framework. Additionally, since the JavaScript Object Notation (JSON) format has become one of the most widespread data-interchange formats, eT′s meta-data scheme is appropriately modified to make the system’s meta-data easily parsed as JSON objects. This feature creates a framework with wider compatibility and interoperability with external systems. The evaluation and operational behavior of the new module was tested through a set of data transfer experiments over a wide area network environment. These experiments were performed to ensure that the changes in the system’s architecture did not affected its performance.
Givet det nuvarande läget för input/output (I/O) och lagringsenheter för system i peta-skala, skulle inkrementella lösningar bli ineffektiva om de implementerades i exa-skalamiljöer. Enligt ”The International Exascale Software Roadmap”, av Dongarra et al., är nuvarande I/O-arkitekturer inte tillräckligt skalbara, särskilt eftersom nuvarande delade filsystem har begränsningar när de används i storskaliga miljöer. Dessa begränsningar är: Bandbredd skalar inte på ett ekonomiskt sätt i storskaliga system, I/O-trafik på höghastighetsnätverk kan ha påverkan på och blir påverkad av andra orelaterade jobb, och I/O-trafik på lagringsservern kan ha påverkan på och bli påverkad av andra orelaterade jobb. Framtida applikationer på exa-skaladatorer kommer kräva I/O-bandbredd proportionellt till deras beräkningskapacitet. För att undvika dessa begränsningar föreslog C. Filippidis, C. Markou och Y. Cotronis ramverket IKAROS. I detta examensarbete utökas funktionaliteten hos den publikt tillgängliga modulen elastic-transfer (eT) som framtagits utifrån IKAROS. Den befintliga versionen av eT-ramverket implementerar Internet Message Access Protocol (IMAP) klient-serverkommunikation genom modulen ”Inbox” från Node Package Manager (NPM) ur Node.js programmeringsspråk. Denna modul användes som ett koncepttest, men i en verklig miljö så underminerar denna implementation systemets skalbarhet när ett stort antal värdar ansluter till systemet. Varje klient begär individuellt information relaterad till systemets metadata från IMAP-servern, vilket leder till en ineffektiv allokering av systemets resurser när ett stort antal värdar är samtidigt anslutna till eT-ramverket. Denna uppsats löser problemet genom att använda ett asynkront, icke-blockerande och händelsedrivet tillvägagångssätt för att implementera en IMAP klient-serveranslutning. Detta görs genom att integrera och modifiera NPM:s ”Imap”-modul, tagen från NPM:s katalog, så att den passar eT-ramverket. Eftersom formatet JavaScript Object Notation (JSON) har blivit ett av de mest spridda formaten för datautbyte så modifieras även eT:s metadata-struktur för att göra systemets metadata enkelt att omvandla till JSON-objekt. Denna funktionalitet ger ett bredare kompatibilitet och interoperabilitet med externa system. Utvärdering och tester av den nya modulens operationella beteende utfördes genom en serie dataöverföringsexperiment i en wide area network-miljö. Dessa experiment genomfördes för att få bekräftat att förändringarna i systemets arkitektur inte påverkade dess prestanda.

Director Anno Hideaki's series Neon Genesis Evangelion caused a sensation when it first aired on TV Tokyo in 1995 and has become one of the most influential anime ever made. Since its premiere, fans across the globe have debated the possible interpretations of the complex plot, but little has been said about how composer Sagisu Shiro's score might contribute to understanding the series. Anno's rehabilitation in a Jungian clinic and subsequent personal study of human psychology plays heavily into understanding the main character Ikari Shinji, and music has much to contribute to appreciating Shinji's view of the world. Shinji is an impressionable fourteen-year old boy, so his musical interpretations of the people and things around him do not always match reality. Sagisu's music gives the viewers welcome insight into Shinji's thoughts and feelings as he matures throughout the series.

Malgré les avancés thérapeutiques, le pronostic des leucémies aiguës lymphoblastiques (LAL) reste mauvais. Notre laboratoire a montré que la calcineurine (Cn) est requise pour le maintien à long terme des LAL-T. Sa délétion est associée à la dérégulation de l'expression de plusieurs gènes, dont TRNP1 et NFIB, qui sont nouveaux dans le contexte des LAL-T. Nous avons montré que la suppression de leur expression a un effet délétère sur les cellules leucémiques de LAL-T in vitro et in vivo. Cn est aussi activée dans les LAL-B BCR-ABL+. Des travaux publiés ont proposé que son inhibition par la cyclosporine A (CsA) sensibilise les cellules leucémiques aux inhibiteurs de BCR-ABL. Nous montrons ici que cette sensibilisation est indépendante de l'inhibition de Cn. En effet, son inactivation génique n'affecte ni le maintien, ni la propagation de ces LAL-B in vivo. In vitro, la CsA sensibilise ces cellules leucémiques aux inhibiteurs de BCR-ABL en présence ou non de Cn. Le pronostic des patients atteints de LAL-B est mauvais lorsqu'ils présentent la délétion du gène codant pour le facteur de transcription Ikaros (IKZF1). Dans un modèle murin de LAL-B combinant BCR-ABL avec la perte d'un allèle de IKZF1 dans le lignage B, now montrons une accélération de l'émergence de la maladie et une dérégulation de gènes préférentiellement exprimés dans les cellules souches/progéniteurs hématopoïétiques précoces. Ceci suggère que la perte d'un allèle de IKZF1 permet la reprogrammation des progéniteurs B, cellules d'origine de ces leucémies, vers un stade plus primitif et indifférencié, augmentant ainsi l'agressivité de la maladie
Although acute Iymphoblastic leukemia (ALL) cure rates improved in the last decades, outcome for primary resistant and relapsed patients remains poor. Our laboratory showed that calcineurin (Cn) is essential to T-ALL Leukemia Initiating Cells activity. Its deletion is associated with strong anti-leukemic effects and to the deregulation of several genes, including TRNP1 and NFIB, which have never been reported in T-ALL. Here, we demonstrate that silencing of these genes is deleterious to T-ALL cells both in vitro and in vivo, unravelling new actors involved in leukemogenesis. Cn is activated in BCR-ABL+ B-ALL (Ph+ B-ALL) as well and its inhibition by cyclosporin A (CsA) was reported to sensitize leukemic cells to BCR-ABL inhibitors in a mouse model of the disease. We demonstrate here that this sensitization is due to off-target effects of CsA, since Cn genetic deletion did not affect BCR-ABL-induced B-ALL maintenance and propagation in two mouse models of the disease. Moreover, CsA similarly sensitized Cn+ and Cn- leukemic cells to BCR-ABL inhibitors in vitro. The outcome of B-ALL is particularly poor when patients present deletions of IKZF1, the gene encoding the transcription factor Ikaros, which are observed in >800/0 of Ph+ B-ALL cases. Using a mouse model of BCR-ABL-induced B-ALL in which one copy of Ikaros can be specifically deleted in pro-B cells, we observed acceleration of leukemia onset and a stem-cell/early progenitor-like transcriptomic signature. Thus, the loss of one copy of Ikaros allows the reprogramming of the pro/pre-B cell of origin towards a more primitive, undifferentiated state, likely explaining the increased aggressiveness of the disease

One in three children diagnosed with cancer has leukemia. Leukemia patients with mutations in the tumor suppressor transcription factor Ikaros (IK), an anti-leukemic factor that is critical for the development of blood cells; have a poor prognosis despite modern chemotherapy. There is, therefore, a critical need to understand the biology of IK. Research by our laboratory has identified a neurotransmitter, called vasoactive intestinal peptide (VIP), which blocks proliferation through one of its receptors (vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide receptor 1(VPAC1)), but blocks apoptosis through a second inducible receptor (VPAC2), while both receptors have chemotactic properties in primary T cells. Some leukemia patients have reversed VIP receptor expression (low VPAC1; high VPAC2), and we hypothesizes that this contributes to a selective growth advantage. Understanding the biology by which VIP receptors regulate cellular growth (proliferation and apoptosis) and movement (chemotaxis) will be pivotal in establishing their signaling pathways as future drugs target candidates in the fight against leukemia. We hypothesized that VIP/VPAC1 signaling would alter the expression of IK protein and that VIP would direct cellular migration of both VPAC1 and VPAC2 expressing leukemic T cells. To test this hypothesis, we first asked whether VIP/VPAC1 signaling affects the expression and/or the phosphorylation profile of IK a transcription factor that regulates cellular growth. The second question was whether VPAC1 and/or VPAC2 signaling differentially control leukemic cell movement. By one- and two-dimensional polyacrylamide gel electrophoresis followed by Western blot analysis, we showed that VIP signaling suppresses IK expression and changes the isoelectric pools of IK protein in a human leukemia cell line. By using leukemia cells that only express VPAC1 or VPAC2 receptors; we demonstrated that VIP promoted cellular movement, but that this effect was controlled by different pathways elicited by VPAC1 versus VPAC2. Collectively, these data support the notion that the nervous system naturally contributes to normal blood cell function, but after leukemogenesis, the VIP signaling axis may exacerbate the leukemia phenotype.

rezensiertes Werk: Mosovits, Leyb: ha-erminologyah shel ha-Yerushalmi : ha-munaim ha-iariyim. - Yerushalayim : Hotsaat Sefarim a. sh. Y.L. Magnes, ha-Universiah ha-Ivrit, 2009. - 641 S. ISBN 978-965-493-420-6

Le développement lymphocytaire B murin comprend 2 lignages, B1 et B2, différentiables par leurs progéniteurs, leurs fonctions et leurs localisations. Le facteur de transcription Ikaros, codé par le gène Ikzf1, a un rôle essentiel dans le développement lymphocytaire B2. Cependant, son rôle dans le développement lymphocytaire B1 reste à déterminer. Les altérations génétiques d’IKZF1 sont souvent associées aux leucémies aiguës lymphoblastiques B (LAL-B) de haut risque. Considérant que la lymphopoïèse s’établit par une vague fœtale, majoritairement de type B1, puis une vague adulte, majoritairement de type B2, les progéniteurs B1 pourraient-être à l’origine de certaines LAL-B pédiatriques.En utilisant un modèle murin avec une délétion conditionnelle du gène Ikzf1 dans les progéniteurs B (Ikzf1f/f Mb1-Cre), nous avons montré qu’Ikaros est nécessaire à la différenciation lymphocytaire B1 de manière équivalente à la différenciation B2. En utilisant l’oncogène BCR-ABL, nous avons également montré que les progéniteurs B1 peuvent être à l’origine de LAL-B chez les murins et qu’Ikaros a un rôle suppresseur de tumeur dans ces cellules.La poursuite de ce travail consistera à déterminer si des LAL-B de type B1 existent chez l’Homme
Murine B cell development comprises 2 main B cell lineages, B1 and B2, with distinct progenitors, functions and localization. The transcription factor Ikaros, encoded by the Ikzf1 gene, is a major regulator of lymphopoiesis and plays a crucial role in B2 cell differentiation. However, the role of Ikaros in B1 cell differentiation is unclear. Genetic alterations of IKZF1 are a hallmark of high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Considering that lymphopoiesis takes place in distinct fetal and adult waves, with B1 cell lymphopoiesis predominating in fetuses and neonates and B2 cell progenitors predominating in adults, some pediatric BCP-ALL cases may originate from B1 cell progenitors. Using a mouse model with a conditional deletion of Ikzf1 in B cell progenitors (Ikzf1f/f Mb1-Cre), we show that Ikaros is critical for normal B1 cell differentiation, similarly to B2 cell differentiation. Using the BCR-ABL oncogene, we show that B1 progenitor can induce BCP-ALL in the murine model and that Ikaros has a tumor suppressor function in these cells. Further studies are required to determine if B1 cell progenitors can contribute to B1-like BCP-ALL in human

Contrairement aux réponses cliniques favorables chez les patients porteurs de Leucémie Myéloïdes Chronique traités avec des inhibiteurs ciblant l'activité tyrosine kinase de l'oncoprotéine de fusion BCR-ABL, les Leucémie Aigues Lymphoblastiques (LAL-B) BCR-ABL-positives restent de mauvais pronostic et requièrent de nouvelles stratégies thérapeutiques. Cette situation résulte de la présence d'autres altérations génétiques récurrentes, telle que la délétion mono- ou bi-allélique du gène codant le facteur de transcription Ikaros, observée dans plus de 83% des patients. Notre laboratoire a créé un modèle murin permettant l'analyse des conséquences de la perte de fonction d'Ikaros dans les LAL-B BCR-ABL+, et identifié une signature transcriptomique liée à l'absence d'un allèle sauvage d'Ikaros dans ces cellules. Cette signature transcriptomique inclut la surexpression du récepteur tyrosine kinase IGF1R. Mon travail de thèse montre : (i) que l'inhibition pharmacologique de IGF1R sensibilise les LAL-B BCR-ABL+ déficientes pour Ikaros aux effets antiprolifératifs et pro-apoptotiques de Nilotinib, (ii) que la délétion du gène IGF1R inhibe l'expansion de ces cellules in vivo, (iii) que le traitement des souris leucémiques avec NVP-AEW541 (un inhibiteur de IGF1R) en combinaison avec Nilotinib augmente significativement la survie des souris traitées par rapport aux souris contrôles, (iv) que l'augmentation de la survie des souris traitées est accompagnée par une augmentation de l'apoptose et une diminution de la prolifération des cellules leucémiques, et (v) que la voie AKT/mTORC1/S6K est une cible moléculaire de cette combinaison d'inhibiteurs
In recent years several inhibitors have been developed targeting the tyrosine kinase activity of the BCR-ABL fusion oncoprotein in Chronic Myeloid Leukemia (CML). Unlike the favorable clinical response observed in CML cases, BCR¬ABL` B-cell Acute Lymphoblastic Leukemias (B-ALLs) remain of poor prognosis. The likely reason for this aggressive behavior is the presence in these leukemias of additional genetic alterations. The most frequent of these is the mono-or bi-allelic deletion of the gene encoding the Ikaros transcription factor (IKZF1), observed in over 83% of patients. Our laboratory has studied the functional consequences of IKZF1 haploinsufficiency in a BCR-ABL-induced B-ALL mouse model and identified an Ikaros-dependent transcriptomic signature in these leukemic cells. This signature includes the overexpression of IGF1R, a tyrosine kinase receptor for IGF1. Based on these premises my PhD thesis work shows (i) that pharmacological inhibition of IGF1R sensitizes Ikaros-deficient BCR-ABL+ B-ALL to the antiproliferative and pro-apoptotic effects of Nilotinib, (ii) that IGF1R gene deletion impairs in vivo expansion of these leukemias in vivo, (iii) that treatment of leukemic mice with NVP-AEW541 (an IGF1R inhibitor) in combination with Nilotinib significantly increases survival of treated mice as compared to control mice, (iv) that the increased survival of treated mice is accompanied by an increase in apoptosis and a decrease in the proliferation of leukemic cells and (v) that inhibition of the AKT/mTORC1/S6K signalling pathway is a point of convergence of these inhibitor combination

Heine Steenhagen erzählt die Geschichte von Aufstieg und Fall eines unehelichen Kindes, das in einem holsteinischen Dorf der Jahrhundertwende, dem fiktiven Vollstedt, aufwächst und als Ausgleich für die in der Jugend erlittenen Demütigungen eine militärische Karriere anstrebt, mit der er es seinem Heimatdorf zeigen will („Ik wöll ju dat woll wiesen!“). Als er beginnt, die Sprossen der sozialen Leiter zu erklimmen, wird sein Jugendfeind Jürgen Grootholm zu einem Hindernis auf dem Weg nach oben. Um ihn zu „überholen“, sich an den Vollstedtern zu rächen und die berechnende Margot Kandelhardt heiraten zu können, denunziert Heinrich Steinhagen den Konkurrenten wegen einer Urlaubsüberschreitung in der Hoffnung, dessen Beförderung zu vereiteln und leitet damit sein eigenes tragisches Ende ein.

CD4 T helper (Th) cells differentiate into distinct effector or regulatory subsets as needed during the course of an infection. Ikaros is a transcription factor that is necessary for proper thymic T cell development. In order to study the role of Ikaros in peripheral CD4 T-cell differentiation and function, a novel Ikaros conditional knockout mouse in which Ikaros is deleted in mature T-cells (CKO mice) was developed. In this thesis, this model is characterized and used to evaluated how absence of Ikaros affects lymphocyte and myeloid populations in vivo, and CD4 T-cell differentiation into T helper 17 (Th17) and inducible regulatory T cell (iTreg) subsets in vitro. CKO mice had normal thymocyte development and normal percentages of T-cells and B-cells in the spleen. However, they had increased percentages of myeloid cells, and an abnormal population of "naive-like" CD4 T-cells that expressed low levels of CD62L and CD44, markers that identify naive and memory T cell populations. CKO CD4 T-cells cultured under Th17 polarizing conditions showed normal expression of the Th17 factors, RORγt and IL-17A, but overexpressed the pro-inflammatory factors T-bet, IFNγ and GM-CSF. CKO CD4 T-cells had a decreased ability to become iTregs as shown by significantly less Foxp3+ CD4+ T-cells in polarizing cultures, and overexpress T-bet, IFNγ and GM-CSF. Therefore, T-cells that lack Ikaros do not properly differentiate into either Th17 or iTreg lineages, but instead become cells with altered pro-inflammatory characteristics. In conclusion, the data highlights new roles of Ikaros in maintaining proper CD4 T-cell populations in the periphery and in suppressing abnormal pro-inflammatory responses.