Desenvolvimento de uma célula a combustível de glicerol direto em meio alcalino

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2015.

Audiovisuelle Bildungsmedienformate und ihre Lernerfolge – eine Paneldatenstudie zum Nachmachen

Visuelle Bildungsmedien erfahren wachsende Verbreitung in der universitären Lehre, und die Anzahl der verwendeten Medienformate steigt. Wir untersuchen empirisch den Lernerfolg dreier Formate - Lehrvideo plus Folien/Folien plus Audio/Audio - und stellen fest, dass der Lernerfolg die aufwändige Medienproduktion nicht in jedem Fall rechtfertigt. Das Untersuchungsdesign lädt zum Nachmachen und somit zum Ermitteln des geeignetsten Medienformats in einem spezifischen Lehrkontext ein. (DIPF/Orig.)

Valorization of the Macroalgae Sargassum Muticum by Enzymatic Hydrolysis, Interest of Surfactants to Improve the Extraction of Phlorotannins and Polysaccharides

The use of surfactants to improve enzymatic hydrolysis of the macroalgae Sargassum muticum has been investigated. Visible absorption spectroscopy has been used to quantify the solubilization of both polysaccharides and phlorotannins in the hydrolysates.   After total extraction, results showed that Sargassum muticum contained 2.74% (expressed in percent of the dry weight of the algae) of phlorotannins whose 32 % were in the cell wall. This result shows that it is important to access to the parietal phlorotannins. To reach this objective, we chose the enzymatic approach for destructurating the cell wall of the algae. The use of 5% dry weight (DW - 5% by weight of hydrolyzed algae) of an enzymatic mix containing a commercial beta-glucanase, a commercial protease and an alginate lyase extracted from Pseudomonas alginovora led after 3 hours of hydrolysis to the solubilization of 2.43% DW polysaccharides and 0.52% DW phlorotannins. The use of 0.5% volume of the surfactant Triton® X-100 with 10% DW of the enzymatic mix has allowed to reaching the value of 2.63% DW of solubilized phlorotannins, that is 96% of the total phenolic content.   The use of non-ionic surfactant, combined to enzymatic hydrolysis, showed an increased efficiency in disrupting cell wall and solubilizing phlorotannins in Sargassum muticum.

Fruit cell culture as a model system to study cell wall changes during strawberry fruit ripening

Strawberry (Fragaria x ananassa, Duch.) fruit is characterized by its fast ripening and soft texture at the ripen stage, resulting in a short postharvest shelf life and high economic losses. It is generally believed that the disassembly of cell walls, the dissolution of the middle lamella and the reduction of cell turgor are the main factors determining the softening of fleshy fruits. In strawberry, several studies indicate that the solubilisation and depolymerisation of pectins, as well as the depolymerisation of xyloglucans, are the main processes occurring during ripening. Functional analyses of genes encoding pectinases such as polygalacturonase and pectate lyase also point out to the pectin fraction as a key factor involved in textural changes. All these studies have been performed with whole fruits, a complex organ containing different tissues that differ in their cell wall composition and undergo ripening at different rates. Cell cultures derived from fruits have been proposed as model systems for the study of several processes occurring during fruit ripening, such as the production of anthocyanin and its regulation by plant hormones. The main objective of this research was to obtain and characterize strawberry cell cultures to evaluate their potential use as a model for the study of the cell wall disassembly process associate with fruit ripening. Cell cultures were obtained from cortical tissue of strawberry fruits, cv. Chandler, at the stages of unripe-green, white and mature-red. Additionally, a cell culture line derived from strawberry leaves was obtained. All cultures were maintained in solid medium supplemented with 2.5 mg.l-1 2,4-D and incubated in the dark. Cell walls from the different callus lines were extracted and fractionated to obtain CDTA and sodium carbonate soluble pectin fractions, which represent polyuronides located in the middle lamella or the primary cell wall, respectively. The amounts of homogalacturonan in both fractions were estimated by ELISA using LM19 and LM20 antibodies, specific against demethylated and methyl-esterified homogalacturonan, respectively. In the CDTA fraction, the cell line from ripe fruit showed a significant lower amount of demethylated pectins than the rest of lines. By contrast, the content of methylated pectins was similar in green- and red-fruit lines, and lower than in white-fruit and leaf lines. In the sodium carbonate pectin fraction, the line from red fruit also showed the lowest amount of pectins. These preliminary results indicate that cell cultures obtained from fruits at different developmental stages differ in their cell wall composition and these differences resemble to some extent the changes that occur during strawberry softening. Experiments are in progress to further characterize cell wall extracts with monoclonal antibodies against other cell wall epitopes.

Unravelling the nanostructure of strawberry fruit pectins by atomic force microscopy

Atomic force microscopy (AFM) allows the analysis of individual polymers at nanostructural level with a minimal sample preparation. This technique has been used to analyse the pectin disassembly process during the ripening and postharvest storage of several fleshy fruits. In general, pectins analysed by AFM are usually visualized as isolated chains, unbranched or with a low number of branchs and, occasionally, as large aggregates. However, the exact nature of these structures is unknown. It has been suggested that pectin aggregates represent a mixture of rhamnonogalacturonan I and homogalacturonan, while isolated chains and their branches are mainly composed by polygalacturonic acid. In order to gain insight into the nature of these structures, sodium carbonate soluble pectins from ripe strawberry (Fragaria x ananassa, Duch.) fruits were subjected to enzymatic digestion with endo-Polygalacturonase M2 from Aspergillus aculeatus, and the samples visualized by AFM at different time intervals. Pectins isolated from control, non-transformed plants, and two transgenic genotypes with low level of expression of ripening-induced pectinase genes encoding a polygalacturonase (APG) or a pectate lyase (APEL) were also included in this study. Before digestion, isolated pectin chains from control were shorter than those from transgenic fruits, showing number-average (LN) contour length values of 73.2 nm vs. 95.9 nm and 91.4 nm in APG and APEL, respectively. The percentage of branched polymers was significantly higher in APG polyuronides than in the remaining genotypes, 33% in APG vs. 6% in control and APEL. As a result of the endo-PG treatment, a gradual decrease in the main backbone length of isolated chains was observed in the three samples. The minimum LN value was reached after 8 h of digestion, being similar in the three genotypes, 22 nm. By contrast, the branches were not visible after 1.5-2 h of digestion. LN values were plotted against digestion time and the data fitted to a first-order exponential decay curve, obtaining R2 values higher than 0.9. The half digestion time calculated with these equations were similar for control and APG pectins, 1.7 h, but significantly higher in APEL, 2.5 h, indicating that these polymer chains were more resistant to endo-PG digestion. Regarding the pectin aggregates, their volumes were estimated and used to calculate LN molecular weights. Before digestion, control and APEL samples showed complexes of similar molecular weights, 1722 kDa, and slightly higher than those observed in APG samples. After endo-PG digestion, size of complexes diminished significantly, reaching similar values in the three pectin samples, around 650 kDa. These results suggest that isolated polymer chains visualized by AFM are formed by a HG domain linked to a shorter polymer resistant to endo-PG digestion, maybe xylogalacturonan or RG-I. The silencing of the pectate lyase gene slightly modified the structure and/or chemical composition of polymer chains making these polyuronides more resistant to enzymatic degradation. Similarly, polygalacturonic acid is one of the main component of the aggregates.

Liquid chromatographic-mass spectrometric method for determination of drug content uniformity of two commonly used dermatology medications in a split-tablet dosage form

Purpose: To develop and validate a simple, efficient and reliable Liquid chromatographic-mass spectrometric (LC-MS/MS) method for the quantitative determination of two dermatological drugs, Lamisil® (terbinafine) and Proscar® (finasteride), in split tablet dosage form. Methods: Thirty tablets each of the 2 studied medications were randomly selected. Tablets were weighed and divided into 3 groups. Ten tablets of each drug were kept intact, another group of 10 tablets were manually split into halves using a tablet cutter and weighed with an analytical balance; a third group were split into quarters and weighed. All intact and split tablets were individually dissolved in a water: methanol mixture (4:1), sonicated, filtered and further diluted with mobile phase. Optimal chromatographic separation and mass spectrometric detection were achieved using an Agilent 1200 HPLC system coupled with an Agilent 6410 triple quadrupole mass spectrometer. Analytes were eluted through an Agilent eclipse plus C8 analytical column (150 mm × 4.6 mm, 5 μm) with a mobile phase composed of solvent A (water) containing 0.1% formic acid and 5mM ammonium formate pH 7.5, and solvent B (acetonitrile mixed with water in a ratio A:B 55:45) at a flow rate of 0.8 mL min-1 with a total run time of 12 min. Mass spectrometric detection was carried out using positive ionization mode with analyte quantitation monitored by multiple reaction monitoring (MRM) mode. Results: The proposed analytical method proved to be specific, robust and adequately sensitive. The results showed a good linear fit over the concentration range of 20 - 100 ng mL-1 for both analytes, with a correlation coefficient (r2) ≥ 0.999 and 0.998 for finasteride and terbinafine, respectively. Following tablet splitting, the drug content of the split tablets fell outside of the proxy USP specification for at least 14 halves (70 %) and 34 quarters (85 %) of FIN, as well as 16 halves (80 %) and 37 quarters (92.5 %) of TBN. Mean weight loss, after splitting, was 0.58 and 2.22 % for FIN half- and quarter tablets, respectively, and 3.96 and 4.09 % for TBN half- and quarter tablets,respectively. Conclusion: The proposed LC-MS/MS method has successfully been used to provide precise drug content uniformity of split tablets of FIN and TBN. Unequal distribution of the drug on the split tablets is indicated by the high standard deviation beyond the accepted value. Hence, it is recommended not to split non-scored tablets especially, for those medications with significant toxicity

A preliminary characterization of mercury uptake by the sulfate-reducing bacteria Desulfovibrio desulfuricans

The focus of this research is to determine if a relationship exists between the stability constant and the initial uptake rate of a mercury species by bacteria. Cultures of the sulfate-reducing bacteria (SRB) strain Desulfovibrio desulfuricans G20 were washed with a bicarbonate buffer solution containing either lactate and sulfate or pyruvate and fumarate. The washed cell solutions were then spiked with either mercury bound to natural organic matter (Hg-NOM) or neutral mercury chloride (HgCl2), followed by sampling over time to provide kinetic data. Despite the significantly different stability constants for Hg-NOM and HgCl2, the calculated initial rate constants for mercury uptake for these two types of complexes appeared to be comparable. Uptake of mercury sulfide species was inconclusive due to possible formation of cinnabar. A simple model that is based on assumptions of passive diffusion and facilitated uptake of mercury by bacteria was evaluated for its potential to simulate the uptake. The model results only agreed with experimental data for HgCl2 uptake.

On the Chemical Signature and Origin of Dicoppertrihydroxyformate (Cu2(OH)3HCOO) Formed on Copper Miniatures of 17th and 18th centuries

A corrosion product rarely reported in the literature has been found on the copper support of three miniature paintings of the 17th and 18th centuries. This product, which has been identified as dicoppertrihydroxyformate (Cu2(OH)3HCOO), is an unusual basic copper formate found on copper artifacts. The identification and characterization of dicoppertrihydroxyformate was carried out directly over the corroded surface of the objects, using a nondestructive approach, which combines the integrated use of various microanalytical techniques. Using this approach, it was possible to obtain a set of new reference data about the natural form of Cu2(OH)3HCOO, that will enable its unambiguous identification in other similar objects. In this work, the probable causes that may have contributed to its formation are also discussed.

Rilevamento Geomorfologico delle principali forme legate al processo solfureo acido (SAS) nei rami turistici della Grotta Grande del Vento, Frasassi (Marche, Italia)

Nell'elaborato svolto, dopo un'introduzione generale nella quale vengono illustrati i due processi principali che formano le grotte, quello epigenico ed ipogenico, viene descritta in dettaglio la parte turistica del complesso "Grotta del Fiume-Grotta Grande del Vento".Si analizzano le varie morfologie derivanti da una speleogenesi solfurea acida, spiegando come si sono formate. Vengono inoltre spiegati i metodi con i quali si è redotta la nuova carta geomorfologica del complesso (oggetto dell'elaborato), e dopo averla illustrata, viene fatto un confronto con la vecchia carta morfologica del 1990.

Studio del metodo di preparazione di elettrocatalizzatori 3D Ag/Cu nella riduzione selettiva del 5-idrossimetilfurfurale

In questo lavoro di tesi sono stati preparati elettrocatalizzatori metallici 3D di argento supportato su schiume di rame a cella aperta, impiegandoli nella riduzione selettiva del 5-idrossimetilfurfurale (HMF) a 2,5-bisidrossimetilfurano (BHMF). L’utilizzo di questi supporti consente di incrementare l’area superficiale a disposizione, rispetto ai supporti 2D. La preparazione delle schiume è stata effettuata utilizzando due metodi di deposizione: spostamento galvanico ed elettrodeposizione. Lo scopo del lavoro è valutare l’influenza del metodo di deposizione sull’attività catalitica, per questo motivo le schiume bimetalliche ottenute sono state confrontate a quelle monometalliche di rame e argento. Inoltre è stato studiato l’effetto della concentrazione di HMF sulle prestazioni dei catalizzatori, con l’obiettivo finale di ottenere un sistema attivo a concentrazioni industriali. Le schiume sono state sottoposte a cicli di prove catalitiche a concentrazione di HMF crescente 0,02 M, 0,05 M e 0,10 M e caratterizzate prima e dopo la sequenza di prove con analisi SEM-EDS, XRD, spettroscopia raman. Le soluzioni sono state analizzate mediante ICP, GC-MS, ESI-MS. Con lo spostamento galvanico si ha la crescita di dendriti, formate prevalentemente sui bordi, e agglomerati di particelle nelle zone interne, mentre per elettrodeposizione si ottiene minore formazione di dendriti e particelle mediamente più piccole. La presenza di argento come fase attiva non migliora la conversione rispetto alle schiume monometalliche, ma causa un aumento di selettività ed efficienza faradica. Incrementando la concentrazione di HMF tutti i campioni presentano un aumento di conversione e un decremento di selettività ed efficienza faradica. Il catalizzatore ottenuto per spostamento galvanico, mostra prestazioni migliori a concentrazioni elevate e nelle prove di stabilità non dà segni di disattivazione, al contrario della schiuma preparata per elettrodeposizione che risulta leggermente disattivata.

Ruolo di Glutaredossina-2 nella trasformazione tumorale oncocitaria

Gli oncocitomi sono tumori epiteliali caratterizzati da un accumulo di mitocondri strutturalmente e funzionalmente compromessi, a prognosi generalmente benigna. Le cause genetiche della trasformazione oncocitaria sono tuttora sconosciute; pertanto, lo studio di oncocitomi in contesti familiari sindromici è utile nella ricerca dei determinanti genetici predisponenti il fenotipo. Diversi membri di una famiglia affetta da sindrome dell’iperparatiroidismo con tumore della mandibola (HPT-JT), dovuta ad un'ampia delezione in CDC73, hanno mostrato recidiva di tumori paratiroidei oncocitari. Il sequenziamento dell’esoma ha escluso mutazioni private della famiglia; all'interno della delezione ereditata, tuttavia, sono stati individuati elementi regolatori del gene glutaredossina 2 (GLRX2), codificante un'isoforma mitocondriale deputata alla deglutationilazione proteica reversibile -modificazione modulante l’attività di numerosi target- il cui ruolo nel cancro non è noto. La proteina è risultata assente in tutti i tumori e dimezzata nei tessuti sani dei soggetti. Per indagare se la sua assenza alteri la deglutationilazione proteica predisponendo al fenotipo oncocitario, sono stati generati modelli cellulari TPC1 e HCT116 GLRX2 KO in cui sono stati riscontrati un ridotto tasso proliferativo ed un'alterata glutationilazione proteica, particolarmente in seguito a stress ossidativo. Un esperimento pilota in vivo ha mostrato cellule KO oncocitoidi, con mitocondri morfologicamente alterati, suggerendo che l’alterazione redox innescata dall’assenza di GLRX2 possa indurre una disfunzione metabolica mitocondriale tale da mimare quelle osservate negli oncocitomi. L’analisi proteomica ha individuato diversi target di glutationilazione nei campioni KO identificando proteine del ciclo di Krebs e della catena respiratoria mitocondriale. In particolare, una marcata glutationilazione del complesso della piruvato deidrogenasi (PDHc) è stata correlata ad una ridotta sintesi di ATP dipendente da piruvato. Considerando l'importanza dello stress ossidativo nella fisiopatologia del cancro ed il ruolo del glutatione nella risposta antiossidante, GLRX2 rappresenta un potenziale candidato nella regolazione del metabolismo ossidativo nelle cellule tumorali esposte allo stress e nella modulazione del fenotipo tumorale.

New senthesis and derivatizations of peptidomimetics and peptide-conjugates for theranostic applications

In the contest of a modern green chemistry approach, we firstly tried to substituent the classic peptide synthesis approach with the use of N-carboxyanhydrides in the presence of Hydroxyapatite, a high biocompatible inorganic base. Despite the great results, further developments are necessary for a daily use in laboratory and for our research, we decided to proceed with solid phase or liquid phase synthesis. In the first chapter, the treatment of pain with the use of opioids is introduced. The abuse and misuse of these kind of potent analgesics, led to the necessity of developing new drugs with less side effects. Starting from a previous study, where the introduction of a lactam-like structure in the place of the proline of Endomorphine1, switched the selectivity from MOR to KOR, we designed and synthetized three different libraries by placing a different trans inducer element to gain the desired selectivity and activity forcing the structure to adopt a linear rather than folded position. In the second chapter, we focused on lactate dehydrogenase, an enzyme overexpressed when the cells in hypoxia conditions, like in a tumour mass, need to produce energy through the transformation of pyruvate into lactate. We synthetized different cyclic peptidomimetics, designed to be inhibitors, as powerful tool to contrast cancer cells growing. Biological assays produced satisfactory preliminary results, but further studies are necessary for a definitive output. Finally in the last chapter, the cancer treatment problem is also approached through the design of nanoparticles, able to deliver drugs with efficacy and selectivity. We firstly synthetized silica core nanoparticles, built with toxic peptide sequences conjugated through click chemistry with Pluronic acid and then, in collaboration with Miriam Royo’s research group, we synthetized multivalent platforms for used drugs for the treatment of advanced colorectal cancer.

New insights into vaginal environment during pregnancy: a multi-omics approach

The present thesis aims to provide a thorough comprehension of the vaginal ecosystem of pregnant women and enhance the knowledge of pregnancy pathophysiology. The first study emphasized the importance of limiting protein intake from animal sources, consuming carbohydrates, and avoiding starting pregnancy overweight to maintain a healthy vaginal environment characterized by lactobacilli and related metabolites. In the second paper, a reduction in bacterial diversity, an increase in Lactobacillus abundance, and a decrease in bacterial vaginosis-related genera were observed during pregnancy. Lactobacillus abundance correlated with higher levels of lactate, sarcosine, and amino acids, while bacterial vaginosis-related genera were associated with amines, formate, acetate, alcohols, and short-chain fatty acids. An association between intrapartum antibiotic prophylaxis for Group B Streptococcus and higher vaginal abundance of Prevotella was found. Moreover, women experiencing a first-trimester miscarriage displayed a higher abundance of Fusobacterium. The third study explored the presence of macrolides and tetracyclines resistance genes in the vaginal environment, highlighting that different vaginal microbiota types were associated with distinct resistance profiles. Lactobacilli-dominated ecosystems showed fewer or no resistance genes, while women with increased bacterial vaginosis-related genera were positive for resistance genes. The last two papers aimed to identify potential biomarkers of vaginal health or disease status. The fourth paper showed that positivity for Torquetenovirus decreased from the first to the third trimester, being more prevalent in women with higher vaginal leukocyte counts. Torquetenovirus-positive samples showed higher levels of cytokines, propionate, and cadaverine. Lactobacillus species decreased in Torquetenovirus-positive samples, while Sneathia and Shuttleworthia increased. The last work pointed out the association between clade 2 of Gardnerella vaginalis and bacterial vaginosis. Moreover, as the number of simultaneously detected G. vaginalis clades increased, bacterial vaginosis-associated bacteria also tended to increase. Additionally, sialidase gene levels negatively correlated with Lactobacillus and positively correlated with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia.

Equazioni di stato della materia in astrofisica

Le equazioni di stato (EdS) sono relazioni che permettono di descrivere sistemi termodinamici all'equilibrio. Un esempio di questi sistemi sono i gas, che si possono dividere in gas perfetti e gas degeneri, che differiscono per importanti proprietà e caratteristiche fisiche. Le proprietà dei gas degeneri cambiano in base al tipo di particelle che li compongono, il comportamento di un gas degenere di Fermioni è molto diverso da quello di un gas degenere di Bosoni. Per lo studio e la descrizione dei gas degeneri di Fermioni entrano in gioco il principio di esclusione di Pauli ed il principio di inderteminazione di Heisenberg. Attraverso le EdS si possono descrivere gli interni stellari poiché per via delle alte temperature le stelle sono formate da gas completamente ionizzato. La pressione all'interno di una stella è data dalla somma tra la pressione di radiazione, la pressione elettronica e la pressione ionica, in base al tipo di gas elettronico che si ha ed alla temperatura interna la stella può essere formata da gas perfetto o gas degenere. Lo studio del regime di pressione interno per una stella si fa con il grafico densità-temperatura, in cui una stella viene rappresentata nel piano attraverso queste due grandezze. Si vede come le stelle di sequenza principale siano formate da gas perfetto mentre i corpi compatti come le nane bianche sono formati da gas completamente degenere. Attraverso le EdS ed il principio dell'equilibrio idrostatico si possono ricavare la temperatura interna per le stelle di sequenza principale e la relazione massa-raggio per le nane bianche.

Dimostrazione di un modello analitico per la stima della produttività in una cella Seru-Seisan multiprodotto

A partire dal 1992, nell’industria elettronica giapponese, ha iniziato a diffondersi il sistema produttivo Seru Seisan: “Seru” in giapponese significa “organismo cellulare” e difatti questa modalità di produzione si basa sulla lavorazione di prodotti all’interno di celle. Esse sono formate da brevi tratti di nastro trasportatore, a seguito dello smantellamento di lunghe linee di assemblaggio, in cui lavorano un numero limitato di operatori multi-skilled. All’interno di un Seru possono essere lavorate più tipologie di prodotto ed ogni prodotto lavorato viene completato all’interno della cella stessa. Questo sistema produttivo a bassa automazione si rivela particolarmente adatto per operazioni di assemblaggio e consente di fronteggiare la dinamicità del mercato caratterizzata da elevata varietà e bassi volumi: il layout di un Seru, infatti, può essere riconfigurato rapidamente e richiede bassi investimenti in attrezzatura. In questo elaborato vengono presentati i Seru Seisan, descrivendone le principali caratteristiche e configurazioni unitamente alla loro origine. Dal momento che molti tentativi di applicazione di tale sistema produttivo risultano infruttuosi, soprattutto al di fuori del Giappone, viene proposto un quadro generale per l’implementazione e un algoritmo euristico per risolvere il problema di formazione e assegnazione degli operatori alle celle, minimizzando il costo totale di cross-training e bilanciando il carico di lavoro. Infine, viene presentato il lavoro di ricerca del presente elaborato che ha l’obiettivo di dimostrare il modello analitico che sta alla base del funzionamento di un Seru Divisionale, arrivando a stimare la produttività di una produzione multiprodotto. La formula a cui si giunge viene testata in due casi numerici reali, mostrando come alla progressiva diminuzione del numero di operatori all’interno di una cella, si ottenga un aumento della produttività della cella stessa.