Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all): from genetic alterations to impaired RNA editing

The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. Despite imatinib has led to significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases resistance developed quickly and disease progressed. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors, driving both the disease and resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were collected. We set up a fast, high-throughput method based on capillary electrophoresis technology to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6 failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a 60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line (SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6 strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a previously unknown link between specific molecular defects that involve the Ikaros gene and the resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of patients was also found. Other two different single nucleotide substitutions not recognized as SNP were observed. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.

Metadata editing: un'implementazione per Open Office

The purpose of this thesis is to enhance the functionalities of GAFFE, a flexible, interactive and user-friendly application for editing metadata in office documents by supporting different ontologies stored inside and outside of the digital document, by adding new views and forms and by improving its ease of use.

Una libreria OpenGL per la selezione e editing di mesh poligonali

Nel mondo Open Source, la libreria grafica OpenGL è oggi ampiamente utilizzata in svariati settori come l'animazione 2D/3D, la modellazione CAD o nello sviluppo di videogiochi. A causa dei suoi innumerevoli usi e dell'astrazione che OpenGL permette di ottenere su diversi ambienti grafici, lo sviluppatore - che la utilizza - è vincolato a cercare librerie di supporto al fine di sfruttarne al meglio le potenzialità. Questa tesi si configura su questi presupposti, presentando una libreria di selezione e editing di mesh 3D basata su OpenGL. La libreria, chiamata libEditMesh, sfrutta il meccanismo geometrico del RayPicking permettendo all'utilizzatore di identificare col mouse punti, facce e lati di solidi in scena. La tesi si articola sostanzialmente in due parti: nella prima vengono proposte alcune soluzioni ad-hoc sviluppate su applicazioni già esistenti nel panorama openSource, e non; nella seconda vengono esposti gli algoritmi e funzioni implementate in libEditMesh.

Traduzione assistita, manuale e post-editing: Un progetto di analisi comparativa per la traduzione in italiano di ricette di cucina in lingua francese.

Nel mondo della traduzione non sempre si può guardare esclusivamente alla qualità del testo di arrivo: ci sono scenari traduttivi, come quello oggetto del presente elaborato, in cui è necessario tener conto di altri fattori, in primis i costi per il committente e la produttività del traduttore. Con questo elaborato intendo dimostrare che per lo scenario traduttivo preso in esame, ossia la traduzione per un sito web di una grande quantità di ricette da parte di traduttori diversi, l’ausilio di un programma di traduzione assistita è da preferire, per produttività, coerenza traduttiva e contenimento dei costi, alla traduzione manuale e al post-editing della traduzione automatica. Per tale scopo, ho tradotto una ricetta con ciascuna di queste metodologie di lavoro, così da poterle mettere a confronto e potermi pronunciare, a seguito di un'analisi approfondita, circa il metodo migliore per lo scenario descritto.

Perceived difficulty and time effectiveness in translation: a comparison of machine translation post-editing and translation memory use

Con il presente studio si è inteso analizzare l’impatto dell’utilizzo di una memoria di traduzione (TM) e del post-editing (PE) di un output grezzo sul livello di difficoltà percepita e sul tempo necessario per ottenere un testo finale di alta qualità. L’esperimento ha coinvolto sei studenti, di madrelingua italiana, del corso di Laurea Magistrale in Traduzione Specializzata dell’Università di Bologna (Vicepresidenza di Forlì). I partecipanti sono stati divisi in tre coppie, a ognuna delle quali è stato assegnato un estratto di comunicato stampa in inglese. Per ogni coppia, ad un partecipante è stato chiesto di tradurre il testo in italiano usando la TM all’interno di SDL Trados Studio 2011. All’altro partecipante è stato chiesto di fare il PE completo in italiano dell’output grezzo ottenuto da Google Translate. Nei casi in cui la TM o l’output non contenevano traduzioni (corrette), i partecipanti avrebbero potuto consultare Internet. Ricorrendo ai Think-aloud Protocols (TAPs), è stato chiesto loro di riflettere a voce alta durante lo svolgimento dei compiti. È stato quindi possibile individuare i problemi traduttivi incontrati e i casi in cui la TM e l’output grezzo hanno fornito soluzioni corrette; inoltre, è stato possibile osservare le strategie traduttive impiegate, per poi chiedere ai partecipanti di indicarne la difficoltà attraverso interviste a posteriori. È stato anche misurato il tempo impiegato da ogni partecipante. I dati sulla difficoltà percepita e quelli sul tempo impiegato sono stati messi in relazione con il numero di soluzioni corrette rispettivamente fornito da TM e output grezzo. È stato osservato che usare la TM ha comportato un maggior risparmio di tempo e che, al contrario del PE, ha portato a una riduzione della difficoltà percepita. Il presente studio si propone di aiutare i futuri traduttori professionisti a scegliere strumenti tecnologici che gli permettano di risparmiare tempo e risorse.

RNA editing in kinetoplastids

RNA editing in kinetoplastid protozoa is a post-transcriptional process of uridine insertion or deletion in mitochondrial mRNAs. The process involves two RNA species, the pre-edited mRNA and in most cases a trans-acting guide RNA (gRNA). Sequences within gRNAs define the position and extend of mRNA editing. Both mRNAs and gRNAs are encoded by mitochondrial genes in the kinetoplast DNA (kDNA), which consists of thousands of small circular DNA molecules, called minicircles, encoding thousands of gRNAs, catenated together and with a few mRNA encoding larger circles, the maxicircles, to form a huge DNA network. Editing has been shown to result in translatable mRNAs of bona fide mitochondrial genes as well as novel alternatively edited transcripts that are involved in the maintenance of the kDNA itself. RNA editing occurs within large protein-RNA complexes, editosomes, containing gRNA, preedited and partially edited mRNAs and also structural and catalytically active proteins. Editosomes are diverse in both RNA and protein composition and undergoe structural remodeling during the maturation. The compositional and structural diversity of editosomes further underscores the complexity of the RNA editing process.

Differences between RNA and DNA due to RNA editing in temporal lobe epilepsy

To investigate whether alterations in RNA editing (an enzymatic base-specific change to the RNA sequence during primary transcript formation from DNA) of neurotransmitter receptor genes and of transmembrane ion channel genes play a role in human temporal lobe epilepsy (TLE), this exploratory study analyzed 14 known cerebral editing sites in RNA extracted from the brain tissue of 41 patients who underwent surgery for mesial TLE, 23 with hippocampal sclerosis (MTLE+HS). Because intraoperatively sampled RNA cannot be obtained from healthy controls and the best feasible control is identically sampled RNA from patients with a clinically shorter history of epilepsy, the primary aim of the study was to assess the correlation between epilepsy duration and RNA editing in the homogenous group of MTLE+HS. At the functionally relevant I/V site of the voltage-gated potassium channel Kv1.1, an inverse correlation of RNA editing was found with epilepsy duration (r=-0.52, p=0.01) but not with patient age at surgery, suggesting a specific association with either the epileptic process itself or its antiepileptic medication history. No significant correlations were found between RNA editing and clinical parameters at other sites within glutamate receptor or serotonin 2C receptor gene transcripts. An "all-or-none" (≥95% or ≤5%) editing pattern at most or all sites was discovered in 2 patients. As a secondary part of the study, RNA editing was also analyzed as in the previous literature where up to now, few single editing sites were compared with differently obtained RNA from inhomogenous patient groups and autopsies, and by measuring editing changes in our mouse model. The present screening study is first to identify an editing site correlating with a clinical parameter, and to also provide an estimate of the possible effect size at other sites, which is a prerequisite for power analysis needed in planning future studies.

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Reflectance Transfer for Material Editing and Relighting

We present a new approach to diffuse reflectance estimation for dynamic scenes. Non-parametric image statistics are used to transfer reflectance properties from a static example set to a dynamic image sequence. The approach allows diffuse reflectance estimation for surface materials with inhomogeneous appearance, such as those which commonly occur with patterned or textured clothing. Material editing is also possible by transferring edited reflectance properties. Material reflectance properties are initially estimated from static images of the subject under multiple directional illuminations using photometric stereo. The estimated reflectance together with the corresponding image under uniform ambient illumination form a prior set of reference material observations. Material reflectance properties are then estimated for video sequences of a moving person captured under uniform ambient illumination by matching the observed local image statistics to the reference observations. Results demonstrate that the transfer of reflectance properties enables estimation of the dynamic surface normals and subsequent relighting combined with material editing. This approach overcomes limitations of previous work on material transfer and relighting of dynamic scenes which was limited to surfaces with regions of homogeneous reflectance. We evaluate our approach for relighting 3D model sequences reconstructed from multiple view video. Comparison to previous model relighting demonstrates improved reproduction of detailed texture and shape dynamics.