SET protein accumulates in HNSCC and contributes to cell survival: Antioxidant defense, Akt phosphorylation and AVOs acidification

Objectives: Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages. Materials and Methods: SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250 mu M) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively. Results: SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death. Conclusion: SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy. (C) 2012 Elsevier Ltd. All rights reserved.

A new evolutionary clustering search for a no-wait flow shop problem with set-up times

This paper addresses the m-machine no-wait flow shop problem where the set-up time of a job is separated from its processing time. The performance measure considered is the total flowtime. A new hybrid metaheuristic Genetic Algorithm-Cluster Search is proposed to solve the scheduling problem. The performance of the proposed method is evaluated and the results are compared with the best method reported in the literature. Experimental tests show superiority of the new method for the test problems set, regarding the solution quality. (c) 2012 Elsevier Ltd. All rights reserved.

SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione Stransferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G0/G1 and S in HEK293 cells, whereas HEK293/SET showed G2/M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

Regularizing a set of unstructured 3D points from a sequence of stereo images

[EN] In this paper we present a method for the regularization of a set of unstructured 3D points obtained from a sequence of stereo images. This method takes into account the information supplied by the disparity maps computed between pairs of images to constraint the regularization of the set of 3D points. We propose a model based on an energy which is composed of two terms: an attachment term that minimizes the distance from 3D points to the projective lines of camera points, and a second term that allows for the regularization of the set of 3D points by preserving discontinuities presented on the disparity maps. We embed this energy in a 2D finite element method. After minimizing, this method results in a large system of equations that can be optimized for fast computations. We derive an efficient implicit numerical scheme which reduces the number of calculations and memory allocations.

Multiproperty modeling for a set of binary systems. Evaluation of a model to correlate simultaneously several mixing properties of methyl ethanoate+alkanes and new experimental data

[EN] This work makes a theoretical–experimental contribution to the study of ester and alkane solutions. Experimental data of isobaric vapor–liquid equilibria (VLE) are presented at 101.3 kPa for binary systems of methyl ethanoate with six alkanes (from C5 to C10), and of volumes and mixing enthalpies, vE and hE.

Viola-Jones based detectors: How much affects the training set?

[EN]This paper presents a study on the facial feature detection performance achieved using the Viola-Jones framework. A set of classi- ers using two di erent focuses to gather the training samples is created and tested on four di erent datasets covering a wide range of possibili- ties. The results achieved should serve researchers to choose the classi er that better ts their demands.

'We deliberately set high demands for the gold standard'… Inclusive and exclusive pronouns in two social science cultures

[EN] This study analyses the use of inclusive and exclusive pronominal signals in English and Spanish research articles and investigates whether there are differences between the two languages in terms of pronominal signals frequency and usage.

A study on set variable representations in constraint programming

Il lavoro presentato in questa tesi si colloca nel contesto della programmazione con vincoli, un paradigma per modellare e risolvere problemi di ricerca combinatoria che richiedono di trovare soluzioni in presenza di vincoli. Una vasta parte di questi problemi trova naturale formulazione attraverso il linguaggio delle variabili insiemistiche. Dal momento che il dominio di tali variabili può essere esponenziale nel numero di elementi, una rappresentazione esplicita è spesso non praticabile. Recenti studi si sono quindi focalizzati nel trovare modi efficienti per rappresentare tali variabili. Pertanto si è soliti rappresentare questi domini mediante l'uso di approssimazioni definite tramite intervalli (d'ora in poi rappresentazioni), specificati da un limite inferiore e un limite superiore secondo un'appropriata relazione d'ordine. La recente evoluzione della ricerca sulla programmazione con vincoli sugli insiemi ha chiaramente indicato che la combinazione di diverse rappresentazioni permette di raggiungere prestazioni di ordini di grandezza superiori rispetto alle tradizionali tecniche di codifica. Numerose proposte sono state fatte volgendosi in questa direzione. Questi lavori si differenziano su come è mantenuta la coerenza tra le diverse rappresentazioni e su come i vincoli vengono propagati al fine di ridurre lo spazio di ricerca. Sfortunatamente non esiste alcun strumento formale per paragonare queste combinazioni. Il principale obiettivo di questo lavoro è quello di fornire tale strumento, nel quale definiamo precisamente la nozione di combinazione di rappresentazioni facendo emergere gli aspetti comuni che hanno caratterizzato i lavori precedenti. In particolare identifichiamo due tipi possibili di combinazioni, una forte ed una debole, definendo le nozioni di coerenza agli estremi sui vincoli e sincronizzazione tra rappresentazioni. Il nostro studio propone alcune interessanti intuizioni sulle combinazioni esistenti, evidenziandone i limiti e svelando alcune sorprese. Inoltre forniamo un'analisi di complessità della sincronizzazione tra minlex, una rappresentazione in grado di propagare in maniera ottimale vincoli lessicografici, e le principali rappresentazioni esistenti.

Elf magnetic field influence on ION Channels studied by Patch Clamp Technique: exposure set up and "Whole Cell" measurements on Potassium currents

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.

Die Beziehung zwischen genetischem Polymorphismus von Populationen und Umweltvariabilität: Anwendung der Fitness-Set Theorie

Die Beziehung zwischen genetischem Polymorphismus von Populationen und Umweltvariabilität: Anwendung der Fitness-Set Theorie Das Quantitative Fitness-Set Modell (QFM) ist eine Erweiterung der Fitness-Set Theorie. Das QFM kann Abstufungen zwischen grob- und feinkörnigen regelmäßigen Schwankungen zweier Umwelten darstellen. Umwelt- und artspezifische Parameter, sowie die bewirkte Körnigkeit, sind quantifizierbar. Experimentelle Daten lassen sich analysieren und das QFM erweist sich in großen Populationen als sehr genau, was durch den diskreten Parameterraum unterstützt wird. Kleine Populationen und/oder hohe genetische Diversität führen zu Schätzungsungenauigkeiten, die auch in natürlichen Populationen zu erwarten sind. Ein populationsgrößenabhängiger Unschärfewert erweitert die Punktschätzung eines Parametersatzes zur Intervallschätzung. Diese Intervalle wirken in finiten Populationen als Fitnessbänder. Daraus ergibt sich die Hypothese, dass bei Arten, die in dichten kontinuierlichen Fitnessbändern leben, Generalisten und in diskreten Fitnessbändern Spezialisten evolvieren.Asynchrone Reproduktionsstrategien führen zur Bewahrung genetischer Diversität. Aus dem Wechsel von grobkörniger zu feinkörniger Umweltvariation ergibt sich eine Bevorzugung der spezialisierten Genotypen. Aus diesem Angriffspunkt für disruptive Selektion lässt sich die Hypothese Artbildung in Übergangsszenarien von grobkörniger zu feinkörniger Umweltvariation formulieren. Im umgekehrten Fall ist Diversitätsverlust und stabilisierende Selektion zu erwarten Dies ist somit eine prozessorientierte Erklärung für den Artenreichtum der (feinkörnigen) Tropen im Vergleich zu den artenärmeren, jahreszeitlichen Schwankungen unterworfenen (grobkörnigen) temperaten Zonen.

A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Metodi di tipo level set per la segmentazione di immagini di nevi

La tesi in oggetto propone un algoritmo che viene incontro alla necessità di segmentare in modo regolare immagini di nevi. Si è fatto uso di metodi level set region-based in una formulazione variazionale. Tale metodo ha permesso di ottenere una segmentazione precisa, adattabile a immagini di nevi con caratteristiche molto diverse ed in tempi computazionali molto bassi.

Ricostruzione di un data-set ad alta risoluzione (30 secondi d'arco) di temperature massime e minime giornaliere per un'area ad orografia complessa (Trentino Alto-Adige)

Dati climatici ad alta risoluzione sono attualmente molto richiesti essendo indispensabili per la valutazione degli impatti dei cambiamenti climatici alla scala locale in svariati campi d'applicazione. Per aumentare l'offerta di tali dati per il territorio italiano viene presentata in questo studio la realizzazione di un data-set con risoluzione di trenta secondi d'arco, per le temperature massime e minime giornaliere per il Trentino Alto Adige, per il periodo che va dal 1951 al 2014. La metodologia utilizzata per proiettare i dati meteorologici di un set di stazioni su di un grigliato ad alta risoluzione si basa sull'assunzione che la struttura spazio-temporale del campo di una variabile meteorologica su una determinata area possa essere descritta dalla sovrapposizione di due campi:i valori normali relativi e un periodo standard, ovvero la climatologia,e le deviazioni da questi, ovvero le anomalie. La climatologia mensile verrà interpolata sull'intero dominio tramite una regressione lineare pesata della temperatura rispetto alla quota,stimata separatamente per ogni nodo del grigliato,con pesi legati alla topografia del territorio,in modo da attribuire di volta in volta la massima importanza alle stazioni con caratteristiche più simili a quella del punto di griglia considerato. Da questa sarà possibile tramite la sovrapposizione con le anomalie mensili ricostruite sul medesimo grigliato, ottenute mediante un'interpolazione basata su una media pesata,ottenere un grigliato a 30 secondi d'arco, di serie temporali mensili in valori assoluti. Combinando poi l'interpolazione dei rapporti delle anomalie giornaliere relative alla media mensile per un set di stazioni con i campi mensili precedentemente stimati,sarà possibile costruire il data-set a risoluzione giornaliera. Prima di quest'ultima fase sarà necessario effettuare un'operazione di sincronizzazione dei dati giornalieri per assicurarsi che non vi siano sfasamenti nelle serie utilizzate. I risultati confermano l'efficacia nell'utilizzo di tale metodo su regioni orograficamente complesse, sia nel confronto diretto con i casi di studio,nei quali si nota bene la discriminazione spaziale effettuata dal modello, che nella valutazione dell'accuratezza e della precisione dei risultati. I dati ottenuti non sono affetti da errori sistematici,mentre l'errore medio assoluto risulta pari od inferiore ai $2^{\circ}$C, in linea con precedenti studi realizzati su altre aree alpine. Il metodo e i risultati risultano soddisfacenti ma ulteriormente migliorabili, sia tramite un ulteriore ottimizzazione del modello usato, che con un aumento nella qualità dei dati sui quali è stato svolto lo studio.

Set up of a procedure for correlation analysis between current discharge and dose delivery in a pulsed power plasma device for medical application

This thesis work aims to find a procedure for isolating specific features of the current signal from a plasma focus for medical applications. The structure of the current signal inside a plasma focus is exclusive of this class of machines and a specific analysis procedure has to be developed. The hope is to find one or more features that shows a correlation with the dose erogated. The study of the correlation between the current discharge signal and the dose delivered by a plasma focus could be of some importance not only for the practical application of dose prediction but also for expanding the knowledge anbout the plasma focus physics. Vatious classes of time-frequency analysis tecniques are implemented in order to solve the problem.