A participação dos medidores eletrônicos na rede inteligente de energia elétrica

Depuis que l'électricité a été découverte et que le secteur électrique s'est considérablement développé dans le XVIIIe siècle, jamais l'homme n'a pu se détacher de cette énergie. Pendant longtemps le système d'énergie électrique a fonctionné sans grand changement, mais avec le développement rapide des technologies, de nouvelles améliorations apparaissent. Ce mémoire traite des nouveaux concepts de réseaux d'énergie électrique appelés Réseaux intelligents ou Smart Grid. À travers un panorama de leurs développements dans le monde, cette étude porte d'une part, sur l'avancée des projets dans quelques pays et d'autre part, du niveau de développement de ce réseau au Brésil et de son intérêt pour le pays. L'étude a comme point central un des composants de ce réseau intelligent, le compteur communicant, qui est l'élément essentiel des interconnexions entre consommateurs et producteurs. Ce rapport apporte un éclaircissement sur les compteurs conventionnels et les compteurs intelligents et sur leur mode de fonctionnement. Enfin il aborde la question des consommateurs : par quels moyens leur transmettre tous ces changements à venir, puisque jusqu'à présent, leur seule participation était de payer l'énergie consommée. Avec le nouveau réseau, ils deviendront de véritables acteurs puisqu'ils seront informés en temps réel de leur consommation d'énergie électrique. Pour terminer, le mémoire montrera comment ils pourront s'adapter à cette nouvelle façon de gérer leur consommation en ésperant les inciter à une utilisation plus raisonnable de l'énergie et à modifier leur comportement en gérant de manière active leur consommation en intégrant notamment les énergies renouvelables

A participação dos medidores eletrônicos na rede inteligente de energia elétrica

Depuis que l'électricité a été découverte et que le secteur électrique s'est considérablement développé dans le XVIIIe siècle, jamais l'homme n'a pu se détacher de cette énergie. Pendant longtemps le système d'énergie électrique a fonctionné sans grand changement, mais avec le développement rapide des technologies, de nouvelles améliorations apparaissent. Ce mémoire traite des nouveaux concepts de réseaux d'énergie électrique appelés Réseaux intelligents ou Smart Grid. À travers un panorama de leurs développements dans le monde, cette étude porte d'une part, sur l'avancée des projets dans quelques pays et d'autre part, du niveau de développement de ce réseau au Brésil et de son intérêt pour le pays. L'étude a comme point central un des composants de ce réseau intelligent, le compteur communicant, qui est l'élément essentiel des interconnexions entre consommateurs et producteurs. Ce rapport apporte un éclaircissement sur les compteurs conventionnels et les compteurs intelligents et sur leur mode de fonctionnement. Enfin il aborde la question des consommateurs : par quels moyens leur transmettre tous ces changements à venir, puisque jusqu'à présent, leur seule participation était de payer l'énergie consommée. Avec le nouveau réseau, ils deviendront de véritables acteurs puisqu'ils seront informés en temps réel de leur consommation d'énergie électrique. Pour terminer, le mémoire montrera comment ils pourront s'adapter à cette nouvelle façon de gérer leur consommation en ésperant les inciter à une utilisation plus raisonnable de l'énergie et à modifier leur comportement en gérant de manière active leur consommation en intégrant notamment les énergies renouvelables

Incorporation of PbF2 into Heavy Metal Oxide Borate Glasses. Structural Studies by Solid State NMR

A series of heavy metal oxide (HMO) glasses with composition 26.66B(2)O(3)-16GeO(2)-4 Bi2O3-(53.33-x)PbO-xPbF2 (0 <= x <= 40) were prepared and characterized with respect to their bulk (glass transition and crystallization temperatures, densities, molar volumes) and spectroscopic properties. Homogeneous glasses are formed up to x = 30, while crystallization of beta-PbF2 takes place at higher contents. Substitution of PbO by PbF2 shifts the optical band gap toward higher energies, thereby extending the UV transmission window significantly toward higher frequencies. Raman and infrared absorption spectra can be interpreted in conjunction with published reference data. Using B-11 and F-19 high-resolution solid state NMR as well as B-11/F-19 double resonance methodologies, we develop a quantitative structural description of this material. The fraction of four-coordinate boron is found to be moderately higher compared to that in glasses with the same PbO/B2O3 ratios, suggesting some participation of PbF2 in the network transformation process. This suggestion is confirmed by the F-19 NMR spectra. While the majority of the fluoride ions is present as ionic fluoride, similar to 20% of the fluorine inventory acts as a network modifier, resulting in the formation of four-coordinate BO3/2F- units. These units can be identified by F-19{B-11} rotational echo double resonance and B-11{F-19} cross-polarization magic angle spinning (CPMAS) data. These results provide the first unambiguous evidence of B-F bonding in a PbF2-modified glass system. The majority of the fluoride ions are found in a lead-dominated environment. F-19-F-19 homonuclear dipolar second moments measured by spin echo decay spectroscopy are quantitatively consistent with a model in which these ions are randomly distributed within the network modifier subdomain consisting of PbO, Bi2O3, and PbF2. This model, which implies both the features of atomic scale mixing with the network former borate species and some degree of fluoride ion clustering is consistent with all of the experimental data obtained on these glasses.

The effects of high-intensity resistance exercise on the blood lipid profile and liver function in hypercholesterolemic hamsters

It is well established that atherogenic dyslipidemia, characterized by high levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein (LDL) cholesterol and low levels of high-density lipoprotein (HDL) cholesterol, constitutes important risk factors for cardiovascular disease. Regular exercise has been associated with a reduced risk for metabolic diseases. However, studies supporting the concept that resistance exercise is a modifier of blood lipid parameters are often contradictory. The aim of this study was to investigate the effects of high-intensity resistance exercise on the serum levels of TG, TC, HDL and non-HDL cholesterol, glucose, and the liver function enzymes alanine aminotransferase (ALT, EC 2.6.1.2) and aspartate aminotransferase (AST, EC 2.6.1.1) in golden Syrian hamsters (Mesocricetus auratus (Waterhouse, 1839)) fed a hypercholesterolemic diet. Sedentary groups (S) and exercise groups (E) were fed a standard diet (SS and ES) or a cholesterol-enriched diet (standard plus 1% cholesterol, SC and EC). Resistance exercise was performed by jumps in the water, carrying a load strapped to the chest, representing 10 maximum repetitions (10 RM, 30 s rest, five days per week for five weeks). Mean blood sample comparisons were made by ANOVA + Tukey or ANOVA + Kruskal-Wallis tests (p < 0.05) to compare parametric and nonparametric samples, respectively. There were no differences in blood lipids between the standard diet groups (SS and ES) (p > 0.05). However, the EC group increased the glucose, non-HDL, and TC levels in comparison with the ES group. Moreover, the EC group increased the TG levels versus the SC group (p < 0.05). In addition, the ALT levels were increased only by diet treatment. These findings indicated that high-intensity resistance exercise contributed to dyslipidemia in hamsters fed a hypercholesterolemic diet, whereas liver function enzymes did not differ in regards to the exercise protocol.

Evaluation of MLH1 I219V Polymorphism in Unrelated South American Individuals Suspected of Having Lynch Syndrome

Background: Some single-nucleotide polymorphisms are associated with higher risk of colorectal cancer development and are suggested to explain part of the genetic contribution to Lynch syndrome. Aim: To evaluate the mutL homolog 1 (MLH1) I219V polymorphism in 124 unrelated South American individuals suspected of having Lynch syndrome, based on frequency, association with pathogenic MLH1 and mutS homolog 2 (MSH2) mutation and clinical features. Materials and Methods: DNA was obtained from peripheral blood and polymerase chain reaction (PCR) was performed, followed by direct sequencing. Results: The Val allelic of the I219V polymorphism was found in 51.61% (64/124) of the individuals, with an allelic frequency of 0.3. MLH1 or MHS2 pathogenic mutations were found in 32.81% (21/64) and in 23.33% (14/60) of Val-carriers and non-carriers, respectively. Conclusion: The Val-carrying genotype was frequent in the studied population; however, it does not appear to exert any modifier effect on MLH1 or MSH2 pathogenic mutations and the development of colorectal cancer.

Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas

Mutations in the critical chromatin modifier ATRX and mutations in CIC and FUBP1, which are potent regulators of cell growth, have been discovered in specific subtypes of gliomas, the most common type of primary malignant brain tumors. However, the frequency of these mutations in many subtypes of gliomas, and their association with clinical features of the patients, is poorly understood. Here we analyzed these loci in 363 brain tumors. ATRX is frequently mutated in grade II-III astrocytomas (71%), oligoastrocytomas (68%), and secondary glioblastomas (57%), and ATRX mutations are associated with IDH1 mutations and with an alternative lengthening of telomeres phenotype. CIC and FUBP1 mutations occurred frequently in oligodendrogliomas (46% and 24%, respectively) but rarely in astrocytomas or oligoastrocytomas (<10%). This analysis allowed us to define two highly recurrent genetic signatures in gliomas: IDH1/ATRX (I-A) and IDH1/CIC/FUBP1 (I-CF). Patients with I-CF gliomas had a significantly longer median overall survival (96 months) than patients with I-A gliomas (51 months) and patients with gliomas that did not harbor either signature (13 months). The genetic signatures distinguished clinically distinct groups of oligoastrocytoma patients, which usually present a diagnostic challenge, and were associated with differences in clinical outcome even among individual tumor types. In addition to providing new clues about the genetic alterations underlying gliomas, the results have immediate clinical implications, providing a tripartite genetic signature that can serve as a useful adjunct to conventional glioma classification that may aid in prognosis, treatment selection, and therapeutic trial design.

Structure and Ionic Conductivity in the Mixed-Network Former Chalcogenide Glass System [Na2S](2/3)[(B2S3)(x)(P2S5)(1-x)](1/3)

Glasses in the system [Na2S](2/3)[(B2S3)(x)(P2S5)(1-x)](1/3) (0.0 <= x <= 1.0) were prepared by the melt quenching technique, and their properties were characterized by thermal analysis and impedance spectroscopy. Their atomic-level structures were comprehensively characterized by Raman spectroscopy and B-11, P-31, and Na-23 high resolution solid state magic-angle spinning (MAS) NMR techniques. P-31 MAS NMR peak assignments were made by the presence or absence of homonuclear indirect P-31-P-31 spin-spin interactions as detected using homonuclear J-resolved and refocused INADEQUATE techniques. The extent of B-S-P connectivity in the glassy network was quantified by P-31{B-11} and B-11{P-31} rotational echo double resonance spectroscopy. The results clearly illustrate that the network modifier alkali sulfide, Na2S, is not proportionally shared between the two network former components, B and P. Rather, the thiophosphate (P) component tends to attract a larger concentration of network modifier species than predicted by the bulk composition, and this results in the conversion of P2S74-, pyrothiophosphate, Na/P = 2:1, units into PS43-, orthothiophosphate, Na/P = 3:1, groups. Charge balance is maintained by increasing the net degree of polymerization of the thioborate (B) units through the formation of covalent bridging sulfur (BS) units, B S B. Detailed inspection of the B-11 MAS NMR spectra reveals that multiple thioborate units are formed, ranging from neutral BS3/2 groups all the way to the fully depolymerized orthothioborate (BS33-) species. On the basis of these results, a comprehensive and quantitative structural model is developed for these glasses, on the basis of which the compositional trends in the glass transition temperatures (T-g) and ionic conductivities can be rationalized. Up to x = 0.4, the dominant process can be described in a simplified way by the net reaction equation P-1 + B-1 reversible arrow P-0 + B-4, where the superscripts denote the number of BS atoms for the respective network former species. Above x = 0.4, all of the thiophosphate units are of the P-0 type and both pyro-(B-1) and orthothioborate (B-0) species make increasing contributions to the network structure with increasing x. In sharp contrast to the situation in sodium borophosphate glasses, four-coordinated thioborate species are generally less abundant and heteroatomic B-S-P linkages appear to not exist. On the basis of this structural information, compositional trends in the ionic conductivities are discussed in relation to the nature of the charge-compensating anionic species and the spatial distribution of the charge carriers.

Materials prepared from biopolyethylene and curaua fibers: Composites from biomass

Composites of high-density biopolyethylene (HDBPE) obtained from ethylene derived from sugarcane ethanol and curaua fibers were formed by first mixing in an internal mixer followed by thermopressing. Additionally, hydroxyl-terminated polybutadiene (LHPB), which is usually used as an impact modifier, was mainly used in this study as a compatibilizer agent. The fibers, HDBPE and LHPB were also compounded using an inter-meshing twin-screw extruder and, subsequently, injection molded. The presence of the curaua fibers enhanced some of the properties of the HDBPE, such as its flexural strength and storage modulus. SEM images showed that the addition of LHPB improved the adhesion of the fiber/matrix at the interface, which increased the impact strength of the composite. The higher shear experienced during processing probably led to a more homogeneous distribution of fibers, making the composite that was prepared through extruder/injection molding more resistant to impact than the composite processed by the internal mixer/thermopressing. (c) 2012 Elsevier Ltd. All rights reserved.

Quantification of Short and Medium Range Order in Mixed Network Former Glasses of the System GeO2-NaPO3: A Combined NMR and X-ray Photoelectron Spectroscopy Study

Glasses in the system xGeO(2)-(1-x)NaPO3 (0 <= x <= 0.50) were prepared by conventional melting quenching and characterized by thermal analysis, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and P-31 nuclear magnetic resonance (MAS NMR) techniques. The deconvolution of the latter spectra was aided by homonuclear J-resolved and refocused INADEQUATE techniques. The combined analyses of P-31 MAS NMR and O-1s XPS lineshapes, taking charge and mass balance considerations into account, yield the detailed quantitative speciations of the phosphorus, germanium, and oxygen atoms and their respective connectivities. An internally consistent description is possible without invoking the formation of higher-coordinated germanium species in these glasses, in agreement with experimental evidence in the literature. The structure can be regarded, to a first approximation, as a network consisting of P-(2) and P-(3) tetrahedra linked via four-coordinate germanium. As implied by the appearance of P-(3) units, there is a moderate extent of network modifier sharing between phosphate and germanate network formers, as expressed by the formal melt reaction P-(2) + Ge-(4) -> P-(3) + Ge-(3). The equilibrium constant of this reaction is estimated as K = 0.52 +/- 0.11, indicating a preferential attraction of network modifier by the phosphorus component. These conclusions are qualitatively supported by Raman spectroscopy as well as P-31{Na-23} and P-31{Na-23} rotational echo double resonance (REDOR) NMR results. The combined interpretation of O-1s XPS and P-31 MAS NMR spectra shows further that there are clear deviations from a random connectivity scenario: heteroatomic P-O-Ge linkages are favored over homoatomic P-O-P and Ge-O-Ge linkages.

Determination of chlorine in food samples via the AlCl molecule using high-resolution continuum source molecular absorption spectrometry in a graphite furnace

Determination of chlorine using the molecular absorption of aluminum mono-chloride (AlCl) at the 261.418 nm wavelength was accomplished by high-resolution continuum source molecular absorption spectrometry using a transversely heated graphite tube furnace with an integrated platform. For the analysis. 10 mu L of the sample followed by 10 mu L of a solution containing Al-Ag-Sr modifier, (1 g L-1 each), were directly injected onto the platform. A spectral interference due to the use of Al-Ag-Sr as mixed modifier was easily corrected by the least-squares algorithm present in the spectrometer software. The pyrolysis and vaporization temperatures were 500 degrees C and 2200 degrees C, respectively. To evaluate the feasibility of a simple procedure for the determination of chlorine in food samples present in our daily lives, two different digestion methods were applied, namely (A) an acid digestion method using HNO3 only at room temperature, and (B) a digestion method with Ag, HNO3 and H2O2, where chlorine is precipitated as a low-solubility salt (AgCl), which is then dissolved with ammonia solution. The experimental results obtained with method B were in good agreement with the certified values and demonstrated that the proposed method is more accurate than method A. This is because the formation of silver chloride prevented analyte losses by volatilization. The limit of detection (LOD, 3 sigma/s) for Cl in methods A and B was 18 mu g g(-1) and 9 mu g g(-1), respectively, 1.7 and 3.3 times lower compared to published work using inductively coupled plasma optical emission spectrometry, and absolute LODs were 2.4 and 1.2 ng, respectively. (C) 2012 Elsevier B.V. All rights reserved.

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

Abstract Background Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus. Methods One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. Results We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls. Conclusion In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.

The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation

The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5'-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.

Caratterizzazione del legame di molecole di interesse farmaceutico alla sieroalbumina umana mediante biocromatografia e dicroismo circolare

Negli ultimi anni, un crescente numero di studiosi ha focalizzato la propria attenzione sullo sviluppo di strategie che permettessero di caratterizzare le proprietà ADMET dei farmaci in via di sviluppo, il più rapidamente possibile. Questa tendenza origina dalla consapevolezza che circa la metà dei farmaci in via di sviluppo non viene commercializzato perché ha carenze nelle caratteristiche ADME, e che almeno la metà delle molecole che riescono ad essere commercializzate, hanno comunque qualche problema tossicologico o ADME [1]. Infatti, poco importa quanto una molecola possa essere attiva o specifica: perché possa diventare farmaco è necessario che venga ben assorbita, distribuita nell’organismo, metabolizzata non troppo rapidamente, ne troppo lentamente e completamente eliminata. Inoltre la molecola e i suoi metaboliti non dovrebbero essere tossici per l’organismo. Quindi è chiaro come una rapida determinazione dei parametri ADMET in fasi precoci dello sviluppo del farmaco, consenta di risparmiare tempo e denaro, permettendo di selezionare da subito i composti più promettenti e di lasciar perdere quelli con caratteristiche negative. Questa tesi si colloca in questo contesto, e mostra l’applicazione di una tecnica semplice, la biocromatografia, per caratterizzare rapidamente il legame di librerie di composti alla sieroalbumina umana (HSA). Inoltre mostra l’utilizzo di un’altra tecnica indipendente, il dicroismo circolare, che permette di studiare gli stessi sistemi farmaco-proteina, in soluzione, dando informazioni supplementari riguardo alla stereochimica del processo di legame. La HSA è la proteina più abbondante presente nel sangue. Questa proteina funziona da carrier per un gran numero di molecole, sia endogene, come ad esempio bilirubina, tiroxina, ormoni steroidei, acidi grassi, che xenobiotici. Inoltre aumenta la solubilità di molecole lipofile poco solubili in ambiente acquoso, come ad esempio i tassani. Il legame alla HSA è generalmente stereoselettivo e ad avviene a livello di siti di legame ad alta affinità. Inoltre è ben noto che la competizione tra farmaci o tra un farmaco e metaboliti endogeni, possa variare in maniera significativa la loro frazione libera, modificandone l’attività e la tossicità. Per queste sue proprietà la HSA può influenzare sia le proprietà farmacocinetiche che farmacodinamiche dei farmaci. Non è inusuale che un intero progetto di sviluppo di un farmaco possa venire abbandonato a causa di un’affinità troppo elevata alla HSA, o a un tempo di emivita troppo corto, o a una scarsa distribuzione dovuta ad un debole legame alla HSA. Dal punto di vista farmacocinetico, quindi, la HSA è la proteina di trasporto del plasma più importante. Un gran numero di pubblicazioni dimostra l’affidabilità della tecnica biocromatografica nello studio dei fenomeni di bioriconoscimento tra proteine e piccole molecole [2-6]. Il mio lavoro si è focalizzato principalmente sull’uso della biocromatografia come metodo per valutare le caratteristiche di legame di alcune serie di composti di interesse farmaceutico alla HSA, e sul miglioramento di tale tecnica. Per ottenere una miglior comprensione dei meccanismi di legame delle molecole studiate, gli stessi sistemi farmaco-HSA sono stati studiati anche con il dicroismo circolare (CD). Inizialmente, la HSA è stata immobilizzata su una colonna di silice epossidica impaccata 50 x 4.6 mm di diametro interno, utilizzando una procedura precedentemente riportata in letteratura [7], con alcune piccole modifiche. In breve, l’immobilizzazione è stata effettuata ponendo a ricircolo, attraverso una colonna precedentemente impaccata, una soluzione di HSA in determinate condizioni di pH e forza ionica. La colonna è stata quindi caratterizzata per quanto riguarda la quantità di proteina correttamente immobilizzata, attraverso l’analisi frontale di L-triptofano [8]. Di seguito, sono stati iniettati in colonna alcune soluzioni raceme di molecole note legare la HSA in maniera enantioselettiva, per controllare che la procedura di immobilizzazione non avesse modificato le proprietà di legame della proteina. Dopo essere stata caratterizzata, la colonna è stata utilizzata per determinare la percentuale di legame di una piccola serie di inibitori della proteasi HIV (IPs), e per individuarne il sito(i) di legame. La percentuale di legame è stata calcolata attraverso il fattore di capacità (k) dei campioni. Questo parametro in fase acquosa è stato estrapolato linearmente dal grafico log k contro la percentuale (v/v) di 1-propanolo presente nella fase mobile. Solamente per due dei cinque composti analizzati è stato possibile misurare direttamente il valore di k in assenza di solvente organico. Tutti gli IPs analizzati hanno mostrato un’elevata percentuale di legame alla HSA: in particolare, il valore per ritonavir, lopinavir e saquinavir è risultato maggiore del 95%. Questi risultati sono in accordo con dati presenti in letteratura, ottenuti attraverso il biosensore ottico [9]. Inoltre, questi risultati sono coerenti con la significativa riduzione di attività inibitoria di questi composti osservata in presenza di HSA. Questa riduzione sembra essere maggiore per i composti che legano maggiormente la proteina [10]. Successivamente sono stati eseguiti degli studi di competizione tramite cromatografia zonale. Questo metodo prevede di utilizzare una soluzione a concentrazione nota di un competitore come fase mobile, mentre piccole quantità di analita vengono iniettate nella colonna funzionalizzata con HSA. I competitori sono stati selezionati in base al loro legame selettivo ad uno dei principali siti di legame sulla proteina. In particolare, sono stati utilizzati salicilato di sodio, ibuprofene e valproato di sodio come marker dei siti I, II e sito della bilirubina, rispettivamente. Questi studi hanno mostrato un legame indipendente dei PIs ai siti I e II, mentre è stata osservata una debole anticooperatività per il sito della bilirubina. Lo stesso sistema farmaco-proteina è stato infine investigato in soluzione attraverso l’uso del dicroismo circolare. In particolare, è stato monitorata la variazione del segnale CD indotto di un complesso equimolare [HSA]/[bilirubina], a seguito dell’aggiunta di aliquote di ritonavir, scelto come rappresentante della serie. I risultati confermano la lieve anticooperatività per il sito della bilirubina osservato precedentemente negli studi biocromatografici. Successivamente, lo stesso protocollo descritto precedentemente è stato applicato a una colonna di silice epossidica monolitica 50 x 4.6 mm, per valutare l’affidabilità del supporto monolitico per applicazioni biocromatografiche. Il supporto monolitico monolitico ha mostrato buone caratteristiche cromatografiche in termini di contropressione, efficienza e stabilità, oltre che affidabilità nella determinazione dei parametri di legame alla HSA. Questa colonna è stata utilizzata per la determinazione della percentuale di legame alla HSA di una serie di poliamminochinoni sviluppati nell’ambito di una ricerca sulla malattia di Alzheimer. Tutti i composti hanno mostrato una percentuale di legame superiore al 95%. Inoltre, è stata osservata una correlazione tra percentuale di legame è caratteristiche della catena laterale (lunghezza e numero di gruppi amminici). Successivamente sono stati effettuati studi di competizione dei composti in esame tramite il dicroismo circolare in cui è stato evidenziato un effetto anticooperativo dei poliamminochinoni ai siti I e II, mentre rispetto al sito della bilirubina il legame si è dimostrato indipendente. Le conoscenze acquisite con il supporto monolitico precedentemente descritto, sono state applicate a una colonna di silice epossidica più corta (10 x 4.6 mm). Il metodo di determinazione della percentuale di legame utilizzato negli studi precedenti si basa su dati ottenuti con più esperimenti, quindi è necessario molto tempo prima di ottenere il dato finale. L’uso di una colonna più corta permette di ridurre i tempi di ritenzione degli analiti, per cui la determinazione della percentuale di legame alla HSA diventa molto più rapida. Si passa quindi da una analisi a medio rendimento a una analisi di screening ad alto rendimento (highthroughput- screening, HTS). Inoltre, la riduzione dei tempi di analisi, permette di evitare l’uso di soventi organici nella fase mobile. Dopo aver caratterizzato la colonna da 10 mm con lo stesso metodo precedentemente descritto per le altre colonne, sono stati iniettati una serie di standard variando il flusso della fase mobile, per valutare la possibilità di utilizzare flussi elevati. La colonna è stata quindi impiegata per stimare la percentuale di legame di una serie di molecole con differenti caratteristiche chimiche. Successivamente è stata valutata la possibilità di utilizzare una colonna così corta, anche per studi di competizione, ed è stata indagato il legame di una serie di composti al sito I. Infine è stata effettuata una valutazione della stabilità della colonna in seguito ad un uso estensivo. L’uso di supporti cromatografici funzionalizzati con albumine di diversa origine (ratto, cane, guinea pig, hamster, topo, coniglio), può essere proposto come applicazione futura di queste colonne HTS. Infatti, la possibilità di ottenere informazioni del legame dei farmaci in via di sviluppo alle diverse albumine, permetterebbe un migliore paragone tra i dati ottenuti tramite esperimenti in vitro e i dati ottenuti con esperimenti sull’animale, facilitando la successiva estrapolazione all’uomo, con la velocità di un metodo HTS. Inoltre, verrebbe ridotto anche il numero di animali utilizzati nelle sperimentazioni. Alcuni lavori presenti in letteratura dimostrano l’affidabilita di colonne funzionalizzate con albumine di diversa origine [11-13]: l’utilizzo di colonne più corte potrebbe aumentarne le applicazioni.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Molekulardynamik-Untersuchungen zur atomistischen Struktur & Dynamik von binären Mischgläsern

Mit Hilfe von Molekulardynamik-Computersimulationen werdenin dieser Arbeit die Struktur und Dynamik von Gläsern undSchmelzen der Systeme 'NSx'=(Na2O)(xSiO2), mit x=2,3,5, und'AS2'=(Al2O3)(2SiO2) untersucht. Zur Beschreibung dermikroskopischen Wechselwirkungen dient ein Modellpotenzial,das auf einem effektiven Paarpotenzial aus der Literaturaufbaut. Simuliert wurden Teilchentrajektorien über mehrereNanosekunden im Bereich 6100 K >= T >= 2100 K sowie dieGlasstruktur bei 100 K (NSx) bzw. 300 K (AS2). Das Aufbrechen der tetraedrischen Netzwerkstruktur durch denNetzwerkmodifikator Na2O führt zur Ausbildung einerzusätzlichen Struktur auf intermediären Längenskalen, diebei allen Systemen NSx etwa dem Abstand übernächster Na-bzw. Si-Nachbarn eines Na-Atoms entspricht. Die diffusiveDynamik ist in allen Systeme NSx bis zu drei Größenordnungenschneller als in SiO2 . Sie nimmt mit wachsenderNa-Konzentration zu. Die Na-Diffusion zeigtArrhenius-Verhalten; hierbei vollführen die Na-Atome einaktiviertes Hüpfen durch eine erstarrte Si-O-Matrix. DieZustandsdichten werden bis ca. 20 THz durch dominanteNa-Moden bestimmt. Bei hohen Frequenzen weichen die für SiO2 typischen intratetraedrischen Si-O-Schwingungsmodenauf.Im Gegensatz zu Na2O wird Al2O3 in die tetraedrischeNetzwerkstruktur eingebunden. AS2 zeigt eine überwiegend ausAlO4- und SiO4-Tetraedern verknüpfte Polyederstruktur, beider sich die AlO4-Tetraeder lokal anders anordnen als dieSiO4-Tetraeder, um Ladungsausgleich zu gewährleisten. Esbilden sich typische 3(Si,Al)O4-Bausteine ('3-Cluster'). Sie führen auf intermediären Längenskalen zur Ausbildungeines Al-reichen perkolierenden Netzwerks, das dieSiO4-Struktur durchdringt. Im Vergleich zu SiO2 erhöht sichdie diffusive Dynamik aller Komponenten in AS2 um ca. zweiGrößenordnungen. Die intratetraedrischenAl-O-Steckschwingungsmoden sind wesentlich weicher als die typischen intratetraedrischen Si-O-Moden des SiO2.