Literatura académica sobre el tema "Ikarios"

Abstract:Although we are fairly well informed about the general organization and important events of the dramatic competitions in Athens, there remain significant gaps in our knowledge on many points of detail. In no place is this more true than with regard to the epinikian celebration honouring members of the victorious performance, about which scarcely any unambiguous testimony has come down to us. This study aims to provide new insights into the problem by demonstrating a connection between the iconography preserved in several sculpted reliefs of the Roman period commonly referred to as Dionysos' visit to Ikarios and the representation of a celebration for poetic victory in Plato'sSymposium. Central to the combined testimony of these sources is the ideal of Dionysos’ epiphany to the poet in order to acknowledge and honour his victory in person. So identified as an element of victory celebration, related articulations of this imagined moment can then be detected in several additional representations on vases and in Aristophanic comedy, in both of which other independent elements likewise suggest the activation of an epinikian syntax. Practical matters about the celebration still elude us; what we gain, however, is a clearer sense of the religious ideals that were conveyed through these celebrations in connection with the worship of Dionysos, which formed a nucleus for the dramatic festivals.

The Ikarian paniyiri, one of the main cultural events in the island of Ikaria, has always acted as an important social process with various functions. Currently, it has also incorporated another role, acting as a cultural product aimed for touristic attraction. This transformation has affected the musical and dancing performance in the paniyiri, and especially the performance of the “ikariotiko,” the local dance of Ikaria. This presentation explores the multiple functions of dancing in the Ikarian paniyiri, the redefining of culture in a globalized setting, and also the process of participant integration through an intersubjective interpretation.

Abstract The Ikaros transcription factor has been shown to play an important role in the differentiation of both the myeloid and lymphoid lineages. The ikaros gene encodes for a zinc finger protein containing seven exons that can be alternatively spliced generating several isoforms with differing functional properties. Isoforms with less than three DNA binding domains act as dominant negative (DN) by forming complexes with longer isoforms and interfering with their DNA binding and transcriptional activation ability. Mice heterozygous for a DN ikaros isoform develop T cell leukemia and lymphoma with 100% penetrance. Overexpression of DN Ikaros isoforms has been found in some forms of leukemias. We have previously reported overexpression of the DN Ikaros6 (Ik6) isoform in a subset of leukemia patients harboring t(4;11) translocations. In addition, we inducibly expressed Ik6 in BaF3 cells and found that Ik6 overexpression delayed cell death after IL-3 withdrawal. To further investigate the leukemogenic properties of Ik6 overexpression, we have transduced murine hematopoietic precursors with a retroviral Ik6 expression vector and have analysed the effects on proliferation and differentiation of these precursors by in vitro colony formation assays. We have found that Ik6 can immortalize murine hematopopietic precursors in these in vitro assays. We are currently analysing the leukemogenic potential of Ik6 in vivo by transplanting Ik6 expressing cell lines into NOD/SCID mice.

Abstract Introduction Hematopoiesis is maintained by a hierarchical system, whereas aberrant control of hematopoiesis is the underlying cause of many diseases. Within the hematopoietic hierarchy, hematopoietic stem cells (HSCs) give rise to multipotent progenitors that have lost their self-renewal capacity but remain multipotent to differentiate into mature blood cells. However, the precise molecular mechanisms that modulate this transition are not fully understood yet. Results We recently discovered that genetic ablation of SRY sex determining region Y-box 4 gene (Sox4) in the murine hematopoietic system resulted in dramatic loss of multipotent progenitor population (CD48+CD150-Lin-kit+Sca1+, or CD48+CD150-LSK) both relatively (to the total LSK population) and in absolute number. Interestingly, the absolute number of HSCs (CD48-CD150+Lin-kit+Sca1+, or SLAM+LSK) in these conditional Sox4-deficient mice was comparable to their wild-type counterparts. Transcriptional factor Sox4 belongs to the high-mobility group (HMG) domain superfamily which also includes other Sox proteins, TCF-1 (T-cell factor 1) and LEF-1 (lymphoid enhancer factor 1). Sox4 has been implicated in leukemogenesis and may potentially contribute to stem cell properties. Nevertheless, the precise roles of Sox4 in hematopoietic stem/progenitor cells and the underlying mechanisms have not been defined yet. Further analysis of stem/progenitor compartment defined by Flt3 and CD34 expression demonstrated a major loss in lymphoid-primed multipotent progenitors (LMPPs) (CD34+Flt3+LSK) with relatively normal formation of LT-HSCs (CD34-Flt3-LSK) and ST-HSCs (CD34+Flt3-LSK) upon the loss of Sox4, suggesting that Sox4 is essential for the development from HSCs to multipotent progenitors. Such observation is in line with the expression pattern of Sox4. Quantitative PCR (qPCR) analysis of wild-type mice revealed that expression of Sox4 increased from HSCs to multipotent progenitors which expressed Sox4 at the highest level among all the hematopoietic compartments. Studies of biological behaviors further indicateed that disruption of Sox4 had no effect on proliferative capacity of HSCs and multipotent progenitors, as evidenced by BrdU incorporation assay. However, Annexin V/propidium iodide staining revealed an increased frequency of apoptotic multipotent progenitors, but not that of HSCs upon the ablation of Sox4. In a transplantation setting, although Sox4-deficient LSKs homed appropriately to the bone marrow, they exhibited severely impaired ability to give rise to multipotent progenitors, but contributed normally to HSCs compared to the wild-type donors. Among a set of genes crucial to the biological properties of stem/progenitor cells, qPCR analysis revealed that upon the loss of Sox4, only the levels of Ikaros1 and Ikaros2, the two major Ikaros isoforms in stem/progenitor cells, were downregulated specifically in multipotent progenitors, but remained normal in HSCs. Intriguingly, in a reminiscent manner of Sox4-deficient mice, mice lacking both Ikaros 1 and Ikaros 2 proteins, also exhibited disrupted B cell development and selectively impaired LMPPs. Previous study identified an enhancer of Ikaros locus as the only cis-regulatory element that was capable of stimulating reporter expression in the LMPPs. Our sequence analysis revealed a highly conserved Sox4 binding motif within this enhancer, therefore potentially connecting Sox4 with the known regulatory networks that modulate the differentiation of HSCs. Currently, we are working on (1) confirming the direct transcriptional regulation of Ikaros by Sox4; (2) assessing whether Ikaros mediates the functions of Sox4 in the formation or maintenance of the multipotent progenitors population in vivo; and (3) delineating the downstream regulatory network of Sox4 in stem/progenitor cells. Conclusion In summary, out study reveals a novel role for Sox4 gene in early hematopoiesis and brings important insights into the regulatory mechanisms underlying the commitment of HSCs toward multipotent progenitors. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Loss-of-function mutations in the transcription factor IKZF1 (IKAROS) correlate with poor prognosis in B-progenitor acute lymphoblastic leukemia (B-ALL), and are particularly prevalent in the high-risk BCR-ABL1+ and BCR-ABL1-like disease subtypes. While recent studies using mouse models of Ikaros-deficient B-ALL have uncovered Ikaros-regulated genes, the mechanisms by which IKAROS loss promotes leukemogenesis and confers treatment resistance remain unclear. Results: We have generated a novel transgenic mouse model that allows tet-regulated, shRNA-mediated Ikaros knockdown or restoration in normal lymphocytes and leukemias in vivo. Ikaros knockdown significantly decreases disease latency in mouse models of B-ALL driven by transgenic or retroviral expression of the BCR-ABL1 fusion oncogene, recapitulating a common genetic interaction in high-risk pediatric B-ALL. Remarkably, we find that restoring endogenous Ikaros expression in established BCR-ABL1+ ALL causes rapid disease regression and sustained remission despite ongoing expression of BCR-ABL1, indicating that disabled Ikaros remains a critical disease driver in this context. Using integrated in vivo RNA-seq analysis we have identified a novel set of genes that are (1) differentially expressed in Ikaros-low versus Ikaros-wildtype leukemias and (2) concordantly differentially expressed upon acute Ikaros restoration in established Ikaros-low leukemias. We are now performing in vitro and in vivo loss-of-function genetic screens to interrogate these high confidence Ikaros-regulated genes, focusing on potential roles for Ikaros-activated genes in tumor suppression and Ikaros-repressed genes in promoting BCR-ABL1+ ALL self-renewal. Conclusions: Our results demonstrate that B-ALL driven by expression of BCR-ABL1 and Ikaros loss remains dependent on ongoing Ikaros suppression, suggesting that re-engaging or inhibiting critical components of the Ikaros-regulated gene expression program may provide new therapeutic avenues in this high-risk B-ALL subtype. Disclosures No relevant conflicts of interest to declare.

Ikaros is a DNA-binding protein that regulates gene expression and functions as a tumor suppressor in B-cell acute lymphoblastic leukemia (B-ALL). The full cohort of Ikaros target genes have yet to be identified. Here, we demonstrate that Ikaros directly regulates expression of the small GTPase, Rab20. Using ChIP-seq and qChIP we assessed Ikaros binding and the epigenetic signature at the RAB20 promoter. Expression of Ikaros, CK2, and RAB20 was determined by qRT-PCR. Overexpression of Ikaros was achieved by retroviral transduction, whereas shRNA was used to knockdown Ikaros and CK2. Regulation of transcription from the RAB20 promoter was analyzed by luciferase reporter assay. The results showed that Ikaros binds the RAB20 promoter in B-ALL. Gain-of-function and loss-of-function experiments demonstrated that Ikaros represses RAB20 transcription via chromatin remodeling. Phosphorylation by CK2 kinase reduces Ikaros’ affinity toward the RAB20 promoter and abolishes its ability to repress RAB20 transcription. Dephosphorylation by PP1 phosphatase enhances both Ikaros’ DNA-binding affinity toward the RAB20 promoter and RAB20 repression. In conclusion, the results demonstrated opposing effects of CK2 and PP1 on expression of Rab20 via control of Ikaros’ activity as a transcriptional regulator. A novel regulatory signaling network in B-cell leukemia that involves CK2, PP1, Ikaros, and Rab20 is identified.

Abstract B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood leukemia. Expression profiling has identified IKZF1 (Ikaros) as a major tumor suppressor in B-ALL and established reduced Ikaros function as a poor prognostic marker for this disease. Ikaros regulates expression of its target genes via chromatin remodeling. In vivo, Ikaros can form a complex with histone deacetylases HDAC1 and/or HDAC2 as well as the NuRD chromatin remodeling complex. The mechanisms by which Ikaros exerts its tumor suppressor function and regulates gene expression in B-ALL are unknown. Here we report the use of chromatin immunoprecipitation coupled with next generation sequencing (ChIP-SEQ) to identify genes that are regulated by Ikaros in vivo and to determine the role of Ikaros in chromatin remodeling in B-ALL. Results reveal that Ikaros binds to the promoter regions of a large number of genes that are critical for cell cycle progression. These include CDC2, CDC16, CDC25A, ANAPC1, and ANAPC7. Overexpression of Ikaros in leukemia cells resulted in transcriptional repression of Ikaros target genes. Results from luciferase reporter assays performed using the respective promoters of Ikaros target genes support a role for Ikaros as a transcriptional repressor of these genes. Downregulation of Ikaros by siRNA resulted in increased expression of Ikaros target genes that control cell cycle progression. These results suggest that Ikaros functions as a negative regulator of cell cycle progression by repressing transcription of cell cycle-promoting genes. Next, we studied how Ikaros binding affects the epigenetic signature at promoters of Ikaros target genes. Global epigenetic mapping showed that Ikaros binding at the promoter region of cell cycle-promoting genes is associated with the formation of one of two types of repressive epigenetic marks – either H3K27me3 or H3K9me3. While these epigenetic marks were mutually exclusive, they were both associated with the loss of H3K9 acetylation and transcriptional repression. Serial qChIP assays spanning promoters of the Ikaros target genes revealed that the presence of H3K27me3 is associated with Ikaros and HDAC1 binding, while the H3K9me3 modification is associated with Ikaros binding and the absence of HDAC1. ChIP-SEQ analysis of HDAC1 global genomic binding demonstrated that over 80% of H3K27me3 modifications at promoter regions are associated with HDAC1 binding at surrounding sites. The treatment of leukemia cells with the histone deacetylase inhibitor – trichostatin (TSA) resulted in a severe reduction of global levels of H3K27me3, as evidenced by Wesern blot. These data suggest that HDAC1 activity in leukemia is essential for the formation of repressive chromatin that is characterized by the presence of H3K27me3. Our data suggest that Ikaros binding at the promoters of its target genes can result in the formation of repressive chromatin by two distinct mechanisms: 1) direct Ikaros binding resulting in increased H3K9me3 or 2) Ikaros recruitment of HDAC1 with increased H3K27me3 modifications. These data suggest distinct mechanisms for the regulation of chromatin remodeling and target gene expression by Ikaros alone, and Ikaros in complex with HDAC1. In conclusion, the presented data suggest that HDAC1 has a key role in regulating cell cycle progression and proliferation in B-ALL. Our results identify novel, Ikaros-mediated mechanisms of epigenetic regulation that contribute to tumor suppression in leukemia. Supported by National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. Disclosures No relevant conflicts of interest to declare.

The aim of this study was to evaluate several sociodemographic, lifestyle, and clinical characteristics of the IKARIA study participants and to find healthy aging trajectories of multimorbidity of Ikarian islanders. During 2009, 1410 people (aged 30+) from Ikaria Island, Greece, were voluntarily enrolled in the IKARIA study. Multimorbidity was defined as the combination of at least two of the following chronic diseases: hypertension; hypercholesterolemia; diabetes; obesity; cancer; CVD; osteoporosis; thyroid, renal, and chronic obstructive pulmonary disease. A healthy aging index (HAI) ranging from 0 to 100 was constructed using 4 attributes, i.e., depression symptomatology, cognitive function, mobility, and socializing. The prevalence of multimorbidity was 51% among men and 65.5% among women, while the average number of comorbidities was 1.7 ± 1.4 for men and 2.2 ± 1.4 for women. The most prevalent chronic diseases among men with multimorbidity were hypertension, hypercholesterolemia, and obesity while among women they were hypertension, hypercholesterolemia, and thyroid disease. Multimorbidity was correlated with HAI (Spearman’s rho = −0.127, p < 0.001) and for every 10-unit increase in HAI, participants had 20% lower odds of being multimorbid. Multimorbidity in relation to HAI revealed a different trend across aging among men and women, coinciding only in the seventh decade of life. Aging is usually accompanied by chronic diseases, but multimorbidity seems to also be common among younger adults. However, healthy aging is a lifelong process that may lead to limited co-morbidities across the lifespan.

Abstract Abstract 408 Background: The Ikaros (IKZF1) tumor suppressor is deleted in >80% of the cases of Ph+ ALL. While Ikaros cooperates with pre-B cell receptor signaling to induce cell cycle exit in Ph+ ALL (Trageser et al., J Exp Med, 2009), the mechanism of Ikaros-mediated tumor suppression is poorly understood. Here we report on a series of genetic experiments that show that Ikaros (i) interferes with key survival pathways downstream of the BCR-ABL1 kinase, (ii) inhibits leukemia cell proliferation through interaction with the pre-B cell receptor signaling pathway and (iii) activates the tumor suppressors p53, p21 and p27. Results: To elucidate the mechanism of Ikaros-dependent tumor suppression in BCR-ABL1-driven B cell lineage leukemia, we studied regulation of critical phosphorylation events downstream of the BCR-ABL1 kinase as a central mediators of survival and proliferation. Reconstitution of Ikaros expression in BCR-ABL1-transformed pre-B ALL cells resulted in rapid and global dephosphorylation comparable to the effect of Imatinib. A detailed analysis showed that Ikaros-induced dephosphorylation events affect activation of Stat5 (Y694), AKT (S473), ERK1/2 (T202 and Y204) and SRC (Y416). Interestingly, both Imatinib-treatment and reconstitution of pre-B cell receptor signaling using retroviral vectors for expression of the m heavy chain or the BLNK adapter molecule have the same effects as reconstitution of Ikaros. In fact, a comprehensive gene expression analysis demonstrated that Ikaros reconstitution resulted in similar gene expression changes as reconstitution of pre-B cell receptor signaling (m heavy chain or BLNK), reconstitution of PAX5, Cre-mediated deletion of Stat5 or Myc, or treatment with Imatinib. The signature of common gene expression changes shared between reconstitution of Ikaros, Pax5, m heavy chain, BLNK and inducible deletion of Stat5 or Myc and Imatinib-treatment involves known tumor suppressors including SPIB, BTG1, and BTG2. These findings suggest that reconstitution of tumor suppressive transcription factor (Ikaros, Pax5) converges with pre-B cell receptor-mediated tumor suppression. To better understand how pre-B cell receptor signaling and Ikaros intersect, we combined reconstitution of Ikaros with genetic deletion of either the (more proximal) SYK kinase or the (more distal) BLNK adapter molecule. While inducible Cre-mediated deletion of Syk had no effect on Ikaros-mediated tumor suppression, deletion of the BLNK adapter compromised the ability of Ikaros to function as tumor suppressor. These findings were confirmed in an in vivo transplantation experiment. While mice transplanted with Ikaros+ BLNK+ leukemia cells survived indefinitely, mice transplanted with Ikaros- BLNK+, Ikaros+ BLNK- or Ikaros- BLNK- leukemia cells died after 24 to 31 days post transplantation. While these findings provide genetic evidence for collaboration between the Ikaros and pre-B cell receptor tumor suppressor pathways, Ikaros and pre-B cell receptor signaling differ with respect to activation of classical tumor suppressor pathways. While reconstitution of pre-B cell receptor signaling failed to activate Arf, p53 or p27, protein levels of all these molecules were strongly upregulated by Ikaros. In agreement with these findings, reconstitution of pre-B cell receptor signaling had the same tumor suppressive effect in wildtype leukemia cells as in Arf−/−, p53−/− as well as p27−/− leukemia cells. Conversely, deletion of Arf and p53 significantly diminished the ability of Ikaros to function as tumor suppressor. Conclusion: Ikaros deletion represents a near-obligatory lesion in the pathogenesis of Ph+ ALL. Here we provide genetic evidence for three novel pathways of Ikaros-mediated tumor suppression. Like PAX5, Ikaros reconstitution results in multiple dephosphorylation events (Stat5, AKT, ERK1/2 and SRC are affected). In collaboration with the pre-B cell receptor and its downstream adapter molecule BLNK, Ikaros suppressed MYC and inhibits cell cycle progression. Induction of the Arf/p53 pathway represents a distinct function of Ikaros, which is not shared with the pre-B cell receptor signaling pathway. Disclosures: No relevant conflicts of interest to declare.

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.