CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Parte do custo de produÃÃo de biomolÃculas com elevada pureza e o rendimento deste processo depende das etapas de separaÃÃo e purificaÃÃo utilizadas. Nestas etapas, geralmente à necessÃrio utilizar tÃcnicas de cromatografia e um dos fatores que influenciam no seu elevado desempenho à o desenvolvimento de fases estacionÃrias ou adsorventes adequados. O presente trabalho investiga a adsorÃÃo de trÃs biomolÃculas-modelo (albumina de soro bovino - BSA, lisozima - LYS e celulase â CEL) em sÃlicas mesoporosas obtidas a partir de copolÃmeros tribloco como agentes direcionadores estruturais. Relatam-se diferentes procedimentos de sÃntese que visam modificar a quÃmica da superfÃcie e promover alteraÃÃes texturais como, por exemplo, o alargamento e/ou encurtamento dos poros, bem como a sua ordenaÃÃo mesoscÃpica. Na primeira parte da tese, foram sintetizadas (i) SBA-15 por duas rotas distintas, sol-gel e hidrotÃrmica, e (ii) SBA-16 por via hidrotÃrmica. Na segunda parte, amostras de SBA-15 com diferentes tamanhos de poros e comprimento de canais foram sintetizados utilizando 1,3,5-trimetilbenzeno (TMB), heptano e fluoreto de amÃnio (NH4F). O TMB foi utilizado para aumentar o diÃmetro dos poros e o heptano combinado com NH4F, para modificar o tamanho dos canais da SBA-15. Jà na terceira parte do trabalho, a acidez da SBA-15 foi modificada pela adiÃÃo de zircÃnio com trÃs razÃes molares Si/Zr distintas (5, 10 e 15). A adsorÃÃo das trÃs biomolÃculas-alvo foi estudada por experimentos em tanques agitados. Os resultados indicam que, para sÃlicas puras, maiores capacidades de adsorÃÃo sÃo obtidas quando o pH està prÃximo ao ponto isoelÃtrico da biomolÃcula. Resultados muito promissores foram encontrados para a adsorÃÃo de proteÃnas sobre as sÃlicas com maior diÃmetro de poros (acima de 10 nm), chegando a centenas de miligramas por grama de adsorvente. No caso dos materiais com zircÃnio, os melhores resultados foram encontrados para os materiais com a menor quantidade do referido heteroÃtomo (Si/Zr = 15), que apresentam textura similar ao suporte original. Isto sugere que pode haver uma acidez moderada Ãtima para a adsorÃÃo das biomolÃculas estudadas ou que o âexcessoâ de Ãtomos de zircÃnio leva a impedimentos estÃricos que causam uma reduÃÃo na capacidade de adsorÃÃo da biomolÃcula. / Part of the cost of production of biomolecules with high purity and yield depends on the separation and purification steps used. In these steps, it is usually necessary to use chromatography techniques and one of the factors that influence on its high performance is the development of stationary phases or suitable adsorbents. The present study investigates the adsorption of biomolecules three model (bovine serum albumin - BSA, lysozyme - LYS cellulase and - CEL) in mesoporous silica obtained from tri-block copolymers as agents drivers structure. We report different synthesis procedures aimed at modifying the surface chemistry and promoting textural changes, such as enlargement and/or pores, and the mesoscopic ordering. In the first part of this thesis were synthesized (i) SBA-15 by two different routes, sol-gel and hydrothermal and (ii) SBA-16 by hydrothermal. In the second part, SBA-15 samples with different pore sizes and channel length were s ynthesized using 1,3,5-trimethylbenzene (TMB), heptane and ammonium fluoride (NH4F). TMB was used to increase the pore diameter and heptane combined with NH4F to modify the size of the channels of SBA-15. In the third part of the work, the acidity of SBA-15 was modified by the addition of zirconium with three molar ratios Si/Zr distinct (5, 10 and 15). The adsorption of the three target biomolecules was studied by experiments in stirred tanks. The results indicate that for pure silicas, higher adsorption capacities are obtained when the pH is close to the isoelectric point of biomolecule. Very promising results for were found the adsorption of proteins on silicas with larger pore diameters (above 10 nm), up to hundreds of milligrams per gram of adsorbent. In the case of materials with zirconium, the best results were fou nd for the materials with the lowest amount of said heteroatom (Si/Zr = 15), which have similar texture to the original support. This suggests that there may be a moderate acidity which-enhances the adsorption of the studied biomolecules or excess zirconium atoms lead to steric hindrances causing a decrease in the adsorption capacity of the biomolecule

Kurzzusammenfassung: Dank der Einführung von Immuncheckpointinhibitoren hat sich die Therapie fortgeschrittener onkologischer Erkrankungen in den letzten Jahren dramatisch verändert. Trotz außergewöhnlicher Erfolge profitieren viele Patienten jedoch weder akut noch langfristig von einer Behandlung, tragen aber alle ihre Risiken. Ein besseres Verständnis davon, bei welchen Patienten diese Therapieform wirkt, sowie prädiktive Marker werden daher dringend benötigt. Growth Differentiation Factor 15 (GDF-15) ist Teil der Transforming Growth Factor-β Superfamilie, weist in pathologischen Situationen wie Entzündungen und insbesondere bei Krebs sehr hohe Spiegel auf und besitzt in verschiedenen, auch onkologischen Erkrankungen einen starken prognostischen Wert. Möglicherweise könnte GDF-15 durch seine immunmodulierenden Eigenschaften dazu beitragen, dass Krebszellen im Körper nicht angegriffen werden, und die Wirksamkeit einer Immuncheckpointblockade (ICB) dadurch vermindern. Ziel der vorliegenden Pilotstudie war es zu untersuchen, ob ein Zusammenhang zwischen dem GDF-15-Spiegel und dem Erfolg einer ICB besteht. Hierfür wurden 37 Patienten verschiedener onkologischer Entitäten vor Beginn einer ICB auf ihre GDF-15-Spiegel untersucht, sowie nach zwölf bzw. bei Progressive Disease zum Teil auch nach vier Wochen Therapie. Die Bewertung des Therapieergebnisses erfolgte anhand der RECIST sowie der klinischen Präsentation. Ein Therapieerfolg wurde ab Erreichen einer Stable Disease klassifiziert. Die Rekrutierungszeit betrug 23 Monate ab Januar 2017. Die Untersuchungen zeigten vor Therapiebeginn einer ICB einen geringen Unterschied der GDF-15-Spiegel zwischen Patienten mit Therapieerfolg und Therapieversagen (Median des Therapieerfolgs: 0,63 ng/ml versus Median des Therapieversagens: 0,92 ng/ml). Dieser Unterschied war statistisch nicht signifikant. Dagegen zeigte sich ein signifikanter Zusammenhang zwischen einem Anstieg des GDF-15-Spiegels unter Therapie und dem Therapieversagen einer ICB. Bei Therapieerfolg sank oder stagnierte der GDF-15-Spiegel im Median um - 0,01 ng/ml. Dagegen stieg er bei Therapieversagen im Median um + 0,7 ng/ml an (p < 0,01 r = 0,43). Auch die Höhe des GDF-15-Spiegels unter Therapie zeigte einen signifikanten Zusammenhang mit dem Therapieergebnis. Der GDF-15-Spiegel unter Therapie lag im Median bei Patienten mit Therapieerfolg bei 0,72 ng/ml, dagegen bei Patienten mit Therapieversagen bei 1,85 ng/ml (p < 0,01 r = 0,47). Ob der GDF-15-Spiegel vor Therapiebeginn die Wirksamkeit einer ICB vorhersagen kann, bleibt unklar, da in dieser Studie nur eine Tendenz aufgezeigt werden konnte, die in Folgestudien mit größeren Kohorten in den verschiedenen Entitäten untersucht werden sollte. Unsere Daten zeigen jedoch einen Zusammenhang zwischen einem steigenden bzw. erhöhten GDF-15-Spiegel unter Therapie mit dem Therapieergebnis einer ICB. Dieser Zusammenhang fügt sich gut in das Bild gegenwärtiger Diskussionen über immunmodulierende Eigenschaften von GDF-15 und seiner Rolle bei der Tumorprogression. Zugleich bestärkt das Studienergebnis die Annahme, in GDF-15 auch ein vielversprechendes Angriffsziel therapeutischer Ansätze gefunden zu haben. / Abstract: Thanks to the introduction of immune checkpoint inhibitors, therapy of progressed oncological diseases has changed dramatically in recent years. However, many patients benefit neither acutely nor in the long term from the treatment but bear all its risks. A better understanding of which patients profit from this therapy as well as predictive markers are therefore urgently needed. Growth Differentiation Factor 15 (GDF-15) is part of the Transforming Growth Factor-β superfamily. It shows raised levels in pathological situations such as inflammation and reaches especially in cancer remarkably high levels. It has a strong prognostic value in various, also oncological diseases. Possibly, GDF-15 helps cancer cells not to be attacked by the immune system and could thereby reduce the effectiveness of an immune checkpoint blockade (ICB). The aim of the presented pilot study was investigating whether there is a connection between the GDF-15 level and the success of an ICB. For this purpose, the GDF-15 level of 37 patients of different oncological entities were examined before the start of an ICB, as well as after twelve weeks of therapy, or in the case of progressive disease, after four weeks of therapy. The evaluation of the therapy results was based on RECIST and the clinical presentation. The time of recruitment was 23 months since January 2017. For the GDF-15 level before start of an ICB therapy, the investigations showed a small difference between patients with therapy success and therapy failure (median when success of the therapy: 0.63 ng/ml versus median when treatment failure: 0.92 ng/ml). But this difference was not statistically significant. In contrast, during therapy there was a significant connection between an increase in the GDF-15 level and therapy failure of the ICB. In case of successful therapy, the GDF-15 level fell or stagnated with a median of -0.01 ng/ml. In contrast, the GDF-15 level rose with a median of +0.7 ng/ml in case of therapy failure (p<0.01 r=0.43). Also, the absolute height of the GDF-15 level during therapy showed a significant connection with the therapy result. The median of the GDF-15 level during therapy of patients with therapy success was 0.72 ng/ml, but of patients with treatment failure was 1.85 ng/ml (p<0.01 r = 0.47). Whether the GDF-15 level can predict the effectiveness of an ICB before the start of therapy remains unclear, since this study could only show a trend that follow-up studies with larger cohorts for the different entities should further examine. However, our data show a correlation between increasing and increased GDF-15 levels under therapy with the therapy result of an ICB. This correlation fits well with current discussions about immunomodulating properties of GDF-15 and its role in tumor progression. Simultaneously, the study result confirms the assumption of GDF-15 being a promising target of new therapeutic approaches.

Minor components are of particular interest due to their antioxidant and biological properties. Various classes of lipophilic minor components (plant sterols (PS) and α-tocopherol) were selected as they are widely used in the food industry. A Fast GC-MS method for PS analysis in functional dairy products was set up. The analytical performance and significant reduction of the analysis time and consumables, demonstrated that Fast GC-MS could be suitable for the PS analysis in functional dairy products. Due to their chemical structure, PS can undergo oxidation, which could be greatly impacted by matrix nature/composition and thermal treatments. The oxidative stability of PS during microwave heating was evaluated. Two different model systems (PS alone and in combination) were heated up to 30 min at 1000 W. PS degraded faster when they were alone than in presence of TAG. The extent of PS degradation depends on both heating time and the surrounding medium, which can impact the quality and safety of the food product destined to microwave heating/cooking. Many minor lipid components are included in emulsion systems and can affect the rate of lipid oxidation. The oxidative stability of oil-in-water (O/W) emulsions containing PS esters, ω-3 FA and phenolic compounds, were evaluated after a 14-day storage at room temperature. Due to their surface active character, PS could be particularly prone to oxidation when they are incorporated in emulsions, as they are more exposed to water-soluble prooxidants. Finally, some minor lipophilic components may increase oxidative stability of food systems due to their antioxidant activity. á-tocopherol partitioning and antioxidant activity was determined in the presence of excess SDS in stripped soybean O/W emulsions. Results showed that surfactant micelles could play a key role as an antioxidant carrier, by potentially increasing the accessibility of hydrophobic antioxidant to the interface.

L’overespressione dei geni EVI1(3q26) e PRDM16(1p36), è descritta sia in presenza che in assenza di riarrangiamenti 3q26 e 1p36 in specifici sottogruppi citogenetici di LAM, ed è associata ad una prognosi sfavorevole. Lo scopo principale del nostro studio è stato identificare e caratterizzare tramite FISH e RQ-PCR, alterazioni di EVI1 e PRDM16 in pazienti con alterazioni cromosomiche 3q e 1p.Riarrangiamenti di EVI1 si associavano ad alterazioni cromosomiche 3q26, ma, in 6 casi (6/35;17,1%) erano presenti in assenza di coinvolgimenti, in citogenetica convenzionale, della regione 3q26, a causa di meccanismi complessi e/o alterazioni ‘criptiche’. Inoltre, abbiamo identificato quattro nuovi riarrangiamenti di EVI1, tra cui due nuove traslocazioni simili presenti in due fratelli. Riarrangiamenti e/o amplificazioni di PRDM16 erano spesso associate ad alterazioni 1p36 (7/14;50%). L’analisi di EVI1 e PRDM16 è stata estesa ad altri casi con alterazioni -7/7q-, con cariotipo normale, con alterazioni 3q per PRDM16 e con alterazioni 1p per EVI1. L’overespressione di EVI1 era presente solo nel gruppo -7/7q- (10/58;17.2%) ed in un caso si associava ad amplificazione genica, mentre PRDM16 era overespresso in casi di tutti i gruppi analizzati,sia con cariotipi complessi, dove si associava in alcuni casi ad amplificazione genica, sia con cariotipi normali o con singole alterazioni. Il nostro studio dimostra come la FISH permetta di identificare alterazioni dei geni EVI1 e PRDM16, anche in assenza di coinvolgimenti delle regioni 3q26 e 1p36. Riarrangiamenti complessi e/o una scarsa qualità dei preparati citogenetici sono le cause principali per la mancata identificazione di queste alterazioni. La RQ-PCR permette di identificare l’overespressione anche nei casi in cui non sia dovuta ad alterazioni citogenetiche. È importante confermare con FISH e/o RQ-PCR il coinvolgimento di questi due geni, per individuare alla diagnosi pazienti con prognosi sfavorevole e che potranno beneficiare di terapie maggiormente aggressive e/o di trapianto allogenico di cellule staminali. / EVI1 and PRDM16 overexpression has been associated with poor prognosis in myeloid malignancies. The main mechanism is the rearrangement of chromosome bands 3q26 and 1p36, where they are mapped, respectively. Overexpression has also been reported in specific cytogenetic subgroups, without 3q26 and 1p36 abnormalities observable by chromosome banding analysis (CBA). The main aim of this study has been to identify and characterize EVI1 and PRDM16 rearrangements in cases with 3q and 1p cytogenetic abnormalities. EVI1 rearrangements were mainly associated with 3q26 cytogenetic abnormalities, but they have also been demonstrated in 6 cases (6/35;17,1%) without 3q26 cytogenetic involvement, because of complex mechanism and/or ‘cryptic‘ abnormalities. We have also identified new EVI1 rearrangements. Interestingly, two new similar translocations appeared in two brothers. Rearrangements, but also amplifications of PRDM16 resulting in gene overexpression, have often been associated with 1p36 aberrations. EVI1 and PRDM16 analyses have been performed in others cytogenetics subgroups, such as: -7/7q-, normal karyotype, and 3q abnormalities for PRDM16 and 1p for EVI1. EVI1 overexpression was frequent only in -7/7q- group (17.2%;10/58), and in one case was associated with amplification, not seen by CBA, because of metaphases’ poor quality. On the contrary, PRDM16 overexpression has been found in all cytogenetic subgroups, that we have considered. In some cases with complex karyotype, PRDM16 overexpression has been associated with gene amplification. FISH analysis and RQ-PCR have allowed us to identify EVI1 and PRDM16 abnormalities in patients with or without 3q26 and 1p36 abnormalities. Complex rearrangements or metaphases’ poor quality were the main reasons for the failed detection of these abnormalities in CBA. These observations indicate the importance of screening for EVI1 and PRDM16 abnormalities, especially in cases with 3q and 1p cytogenetic abnormalities, to identify at diagnosis patients with poor prognosis that could benefit from more aggressive chemotherapy and/or stem cell transplantation.

Virgin olive oil(VOO) is a product characterized by high economic and nutritional values, because of its superior sensory characteristics and minor compounds (phenols and tocopherols) contents. Since the original quality of VOO may change during its storage, this study aimed to investigate the influence of different storage and shipment conditions on the quality of VOO, by studying different solutions such as filtration, dark storage and shipment inside insulated containers to protect it. Different analytical techniques were used to follow-up the quality changes during virgin olive oil storage and simulated shipments, in terms of basic quality parameters, sensory analysis and evaluation of minor components (phenolic compounds, diglycerides, volatile compounds). Four main research streams were presented in this PhD thesis: The results obtained from the first experimental section revealed that the application of filtration and/or clarification can decrease the unavoidable quality loss of the oil samples during storage, in comparison with unfiltered oil samples. The second section indicated that the virgin olive oil freshness, evaluated by diglycerides content, was mainly affected by the storage time and temperature. The third section revealed that fluctuation in temperature during storage may adversely affect the virgin olive oil quality, in terms of hydrolytic rancidity and oxidation quality. The fourth section showed that virgin olive oil shipped inside insulated containers showed lower hydrolytic and oxidation degradation than those without insulation cover. Overall, this PhD thesis highlighted that application of adequate treatment, such as filtration or clarification, in addition to a good protection against other external variables, such as temperature and light, will improve the stability of virgin olive oil during storage.

Sia nei tumori solidi che nelle neoplasie ematologiche, è stato dimostrato che alcuni agenti chemioterapici, come le antracicline, sono altamente immunogenici e determinano, attraverso le cellule dendritiche (DCs), un’efficiente attivazione della risposta antitumorale mediata dalle cellule T. Tale processo, caratterizzato dalla traslocazione della calreticulina e delle HSPs 70/90 sulla superficie cellulare delle cellule tumorali e dal rilascio di HMGB1 e di ATP, è detto morte cellulare immunogenica (ICD). Oltra alla ICD, però, la chemioterapia genera fenomeni infiammatori nel microambiente tumorale e attiva pathway immunosoppressivi a carico delle DCs, che potrebbero alterare la risposta immunitaria. Scopo del presente studio è stato caratterizzare la ICD indotta da antracicline nella leucemia acuta mieloide (LAM) e valutare l’induzione di pathway inibitori, come l’enzima indoleamina-2,3-diossigenasi (IDO1). In vitro, ex vivo ed in vivo abbiamo dimostrato che il trattamento con antracicline induce ICD anche nella LAM. Nei pazienti sottoposti a chemioterapia è stata riscontrata una risposta anti-leucemica caratterizzata dalla produzione di IFN- nei linfociti CD4+ e CD8+, capaci di uccidere i blasti autologhi pur mostrando un fenotipo exhausted, ma anche l’espansione di una popolazione di linfociti T regolatori. La ICD infatti determina la maturazione delle DCs, che attivano efficacemente linfociti T antigene-specifici ma, allo stesso tempo, esprimendo elevati livelli di IDO1, inducono linfociti T regolatori che limitano la risposta anti-leucemica. Questi risultati sono stati confermati nel modello murino leucemico dove, a seguito della chemioterapia, l’infiltrato tumorale è arricchito di DCs, mature esprimenti IDO1, ma anche di linfociti T caratterizzati da fenotipo exhausted. I nostri dati confermano che la ICD, attiva anche nella LAM, determina una risposta immunitaria efficace nei confronti della malattia ma anche l’attivazione di pathway inibitori nelle DCs che alterano tale risposta. La combinazione della chemioterapia con inibitori di IDO1 rappresenterebbe un approccio interessante per potenziare l'effetto immunogenico della chemioterapia e la risposta anti-leucemica. / Recently, both in solid tumors and haematological malignancies, it has been shown that some chemotherapeutic agents, such as daunorubicin, are highly immunogenic and activate the immune response via cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by tumor cells modifications, such as cell surface translocation of calreticulin and heat shock proteins, extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, chemotherapy is known to induce inflammatory modifications within tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, which may ultimately hamper anti-tumor T-cells. Aim of this study is to characterize ICD in acute myeloid leukemia (AML) and to evaluate the involvement of some DC-related inhibitory pathways, such as indoleamine-2,3-dioxygenase 1 (IDO1) expression. In vitro, ex vivo and in vivo we demonstrated that daunorubicin treatment induced ICD in AML. Ex vivo, T-cell monitoring of daunorubicin-treated AML patients displayed an increase in leukemia-specific IFN--producing CD4+ and CD8+ T cells, which were capable to kill autologous blasts but showed an exhausted phenotype. We also observed the expansion of regulatory T cells (Tregs). ICD, in fact, induced maturation of DCs which efficiently activate antigen-specific T lymphocytes but simultaneously express high levels of IDO1, inducing a population of Tregs which inhibit the anti-leukemia response. These results were confirmed in leukemic murine model where daunorubicin treatment resulted in increased maturation status of tumor infiltrating DCs expressing IDO1, but also in an increased infiltration of exhausted T lymphocytes. Our data confirm that ICD is active in AML and results in an immunological response against AML but also in the induction of inhibitory pathway. The combination of chemotherapy with IDO1 inhibitors represents an interesting approach to enhance the immunogenic effect of chemotherapy and anti-leukemic immune response.

Minimally processed fruit (MPF) are products that have to maintain their quality similar to those of fresh ones. Being metabolic active tissues, they show physical and physiological response to minimal processing (wounding), that negatively influence their shelf-life. In the last decades, novel non-thermal processing methods have attracted the interest of food scientists, industries and consumers as technologies useful for shelf-life extension or increasing product functionality, with a minimal impact on the nutritional and sensory properties of foods. The main aim of this PhD thesis was to investigate qualitative, metabolic and nutritional aspects of different MPF, submitted to traditional and innovative non-thermal processes. This issue was addressed considering the product as a dynamic system, both in terms of endogenous physiological activity and porous matrix interacting with the surrounding ambient (during processing and storage), through the application of multi-analytical approach. The most consistent results related to the applied non-thermal techniques confirmed their different potentiality in the optic of processing and product innovation, but the need of their modulation in relation to the different raw material susceptibility to degradation and final product target. Cold plasma treatment effects on fresh-cut fruit, characterized by different kind of stability criticisms, resulted mainly bound to the inactivation of degradative enzymes and microbial cells, without evidencing functional modifications in the final products. The study of osmotic dehydration and vacuum impregnation highlighted as these techniques can be successfully applied for cold formulation/enrichment of MPF, but also the necessity to carefully account for the metabolic and structural modifications induced by the processing on the vegetable tissues. An induction of metabolic stress response was also evidenced as a consequence of pulsed electric fields treatment related to electric field strength. Below the threshold limit of irreversible damages to cell membranes, the treatment promoted only slight and reversible modifications of the metabolic profiles.

The topic of wine oxidation and the need of innovative strategies to prevent its extent were the subject of this PhD thesis. The complexity of the oxidative chemical reactions occurring in wine during its conservation were highlighted, and multiple analytical approaches were used to provide a more comprehensive understanding of wine oxidation and to plan tailored strategies to avoid its occurrence. The complexity of wine oxidation could be in a simplified manner attributed to the following main factors: wine composition, storage conditions, and oxygen exposure. An integrated theoretical and experimental approach was used, including study of chemical, physical and technological variables involved in wine production and storage. Standard protocols currently used to analyse the wine composition were implemented, if needed, and the lab scale trials were coupled with monitoring real case study along the supply chain. In particular, the effectiveness of plant extracts (tannins) commonly used in oenology was also evaluate in order to better understand their antioxidant properties and to encourage an harmonized regulation of their use in winemaking. The information provided and the scientific approach proposed in this thesis work may be useful in future work aimed to study practical implications and effective strategies to control oxidation in wine.

La tesi approfondisce i profili processuale della responsabilità medica allo scopo di elaborare una proposta alternativa di gestione del contenzioso. Si esamina, altresì, il tema dei limiti di utilizzo della mediazione nelle controversie in cui vengono in gioco interessi di rilievo pubblicistico. / The thesis explores the procedural profiles of medical liability in order to develop an alternative proposal of litigation management.

The present work reports the outcome of the GIMEMA CML WP study CML0811, an independent trial investigating nilotinib as front-line treatment in chronic phase chronic myeloid leukemia (CML). Moreover, the results of the proteomic analysis of the CD34+ cells collected at CML diagnosis, compared to the counterpart from healthy donors, are reported. Our study confirmed that nilotinib is highly effective in the prevention of the progression to accelerated/blast phase, a condition that today is still associated with high mortality rates. Despite the relatively short follow-up, cardiovascular issues, particularly atherosclerotic adverse events (AE), have emerged, and the frequency of these AEs may counterbalance the anti-leukemic efficacy. The deep molecular response rates in our study compare favorably to those obtained with imatinib, in historic cohorts, and confirm the findings of the Company-sponsored ENESTnd study. Considering the increasing rates of deep MR over time we observed, a significant proportion of patients will be candidate to treatment discontinuation in the next years, with higher probability of remaining disease-free in the long term. The presence of the additional and complex changes we found at the proteomic level in CML CD34+ cells should be taken into account for the investigation on novel targeted therapies, aimed at the eradication of the disease.