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Transcription factor IKAROS: from leukaemia genetics to lymphocyte development
Full text linkHaematopoiesis 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 inductionL’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 induzion
Protein redox regulation in the thylakoid lumen: The importance of disulfide bonds for violaxanthin de-epoxidase
No full textAbstractWhen exposed to saturating light conditions photosynthetic eukaryotes activate the xanthophyll cycle where the carotenoid violaxanthin is converted into zeaxanthin by the enzyme violaxanthin de-epoxidase (VDE). VDE protein sequence includes 13 cysteine residues, 12 of which are strongly conserved in both land plants and algae. Site directed mutagenesis of Arabidopsis thaliana VDE showed that all these 12 conserved cysteines have a major role in protein function and their mutation leads to a strong reduction of activity. VDE is also shown to be active in its completely oxidized form presenting six disulfide bonds. Redox titration showed that VDE activity is sensitive to variation in redox potential, suggesting the possibility that dithiol/disulfide exchange reactions may represent a mechanism for VDE regulation
Sensing protein antigen and microvesicle analytes using high-capacity biopolymer nano-carriers
No full textLab-on-a-chip systems with molecular motor driven transport of analytes attached to cytoskeletal filament shuttles (actin filaments, microtubules) circumvent challenges with nanoscale liquid transport. However, the filaments have limited cargo-carrying capacity and limitations either in transportation speed (microtubules) or control over motility direction (actin). To overcome these constraints we here report incorporation of covalently attached antibodies into self-propelled actin bundles (nanocarriers) formed by cross-linking antibody conjugated actin filaments via fascin, a natural actin-bundling protein. We demonstrate high maximum antigen binding activity and propulsion by surface adsorbed myosin motors. Analyte transport capacity is tested using both protein antigens and microvesicles, a novel class of diagnostic markers. Increased incubation concentration with protein antigen in the 0.1–100 nM range (1 min) reduces the fraction of motile bundles and their velocity but maximum transportation capacity of >1 antigen per nm of bundle length is feasible. At sub-nanomolar protein analyte concentration, motility is very well preserved opening for orders of magnitude improved limit of detection using motor driven concentration on nanoscale sensors. Microvesicle-complexing to monoclonal antibodies on the nanocarriers compromises motility but nanocarrier aggregation via microvesicles shows unique potential in label-free detection with the aggregates themselves as non-toxic reporter elements
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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