The study of polyplex formation and stability by time-resolved fluorescence spectroscopy of SYBR Green I-stained DNA

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency

A framework for web based human and machine computation

LAUREA SPECIALISTICANegli ultimi anni si è vista l’esplosione del numero di contenuti multimediali prodotti dagli utenti. La gestione di enormi archivi di informazioni richiede metodi e strumenti in grado di garantire l’efficienza e la rapidità dell’analisi e del recupero dei dati. Una classica soluzione per risolvere questo problema è l’uso della computazione distribuita. Questa tecnica permette di distribuire il calcolo su nodi diversi, in modo da massimizzare la parallelizzazione. L’analisi di contenuti multimediali ha dei requisiti particolari: da una parte la natura delle informazioni analizzate richiede sistemi in grado di essere distribuiti su più nodi, dall’altra, richiede anche una certa qualità nell’analisi. La computazione umana è una tecnica in grado di sopperire a questa esigenza tramite l’utilizzo di esseri umani nel processo di analisi, in modo da migliorare la qualità dei risultati ottenuti. Contemporaneamente allo sviluppo di queste tecnologie si è assistito anche alla definitiva esplosione, ed evoluzione, del Web, che si è trasformato da una semplice rete per la distribuzione dei contenuti fino a diventare uno strumento sociale per la collaborazione, in grado di utilizzare gli utenti come nodi di computazione sia automatica sia umana. Allo stato attuale delle conoscenze in nostro possesso, non siamo in grado di individuare soluzioni che supportino senza problemi questa enorme forza lavoro, permettendo lo sfruttamento sia della computazione umana, attraverso l’uso delle capacità intellettive degli esseri umani, sia quella automatica, attraverso l’uso dei client Web (Browsers). In questa tesi è presentato un framework Web per la computazione umana e automatica in grado di sfruttare al meglio le possibilità offerte da queste due soluzioni. Abbiamo, inoltre, validato il nostro lavoro implementando tre casi d’uso in grado di stressare le diverse configurazioni architetturali supportate.Last years witnessed an explosion in the number of multimedia contents produced by users. Managing huge repositories of digital information calls for timely and efficient methods and tools for analysis and retrieval. The distribution of the analysis task on different computation nodes to maximize parallelization is a classic solution to this problem, typically referred to as Distributed Computing (DC). However, the analysis of multimedia contents brings specific requirements: one one hand, the nature of the analyzed information demands systems able to scale, thus involving a good amount of computational nodes. On the other hand, multimedia analysis is known to be a subtle task to be performed, as several state of the art solutions do not bring sufficient analysis quality; the recent trend of Human Computation (HC) acknowledges the needs for more correct processing by involving human beings in the multimedia content analysis tasks. At the same time of the development of these methodologies we have also seen the definitive explosion of the Web and its evolution. The Web has evolved from a mere content delivery network, where the contents are presented to the users, to a collaborative and social tool, where powerful client-side technology enable the usage of Web users as node for both automatic and human computation. To the best of our knowledge, no existing solutions are able to seamlessly exploit this massive workforce, which accounts for both machine computational power (Web Clients) and human computational power (the user brain). This thesis presents a framework for web-based human and machine computation able to take advantages of both human and distributed computation. We validated our approach by implementing several use case applications, which demonstrate the framework by stressing its architectural configurations

Conversion of high oleic sunflower oil to biogasoil by high pressure/high temperature hydrotreating

High oleic sunflower oil was processed at high temperature and high H2 pressure in a batch reactor using different catalysts and reaction conditions for its derivatization to biogasoil (mixture of parafines and isoparafines suitable as biofuel for diesel engines). The product from processing oil at 350 °C and 100 bar of H2 for 2h using NiMo/Al2O3 or CoMo/Al2O3 as catalyst did not contained hydrocarbons, although a high concentration of catalyst was used (5 %). Instead, free fatty acids (FFA) were the major component of this product (83 %), showing that hydrolysis highly predominated over the desired hydrodeoxygenation reaction (HDO). However, under the same conditions but using only 1.0 % of PtO2 as catalyst the formation of hydrocarbons was verified: C17:0 (4.0 %) + C18:0 (12.9 %) and, accordingly, the percentage of FFA diminished to 55 %. When this product (containing the used catalyst) was reprocessed for 3 additional hours but performing regular purges of the gas from reactor head space (every 5-10 minutes) followed by gas replacement with fresh H2, hydrocarbons concentration raised to 51,3% and FFA concentration diminished to 24 %. Additionally, after a third processing under same conditions a product containing 87.3 % hydrocarbons and only 2.9 % FFA was achieved. Hydrocarbons fraction comprised C18:0 (69.2 %), C17:0 (24.6 %) and other shorter chain length hydrocarbons at concentrations under 1%. Product composition suggest that, although HDO was the main process, decarbonylation, decarboxylation and hydrocracking must also have occurred. Purging the system produced a drastic favorable effect on process performance indicating that the removal of the main gas products generated by the reaction (H2O, CO2, CO and C3H8) kept them at low partial pressures, increasing H2 partial pressure and shifting HDO reaction to almost completion.Agencia Nacional de Investigación e Innovació

Hydrotreatment of high oleic sunflower oil: Optimization of diesel fraction yield

The optimization of the hydrotreatment of high oleic sunflower oil towards green diesel fraction yield (C15-C20) under Pd/Al2O3 catalyst was investigated. The catalytic hydrotreatment was carried out in a 250mL batch reactor (Parr 4570 HP/HT) for 4 hours, performing regular purges of gas from reactor head space. Optimization was based on three operative parameters: catalyst loading (0.5-1.25%), reaction temperature (325-375°C) and pressure of H2 (60-120bar), using a central composite design and a response surface methodology. When reactor was operated at 350ºC, 100 H2 bar and using 1% catalyst, a 98% conversion to hydrocarbons was achieved and 84% of these hydrocarbons matched the chain length corresponding to the diesel fuel range. Under identical temperature and H2 pressure, but decreasing the catalyst concentration to 0.5%, the hydrocarbon yield decreased to 84%, while the diesel fraction raised to 97%. Under 1% catalyst loading but operating at a lower temperature and H2 pressure (300°C and 60bar, respectively), the hydrocarbon yield decreased significantly to 50%, while all the hydrocarbons had chain lengths inside the diesel fuel range. Results demonstrated that high temperature and pressure favored the complete conversion to hydrocarbons, but also cracking was promoted with negative effect on the percentage of the diesel fraction. Conversely, when reactor was operated at milder reaction conditions, the conversion to hydrocarbons diminished, but the proportion corresponding to a diesel fuel increased. Optimization permitted to define the more convenient conditions in order to achieve both high conversions and the highest diesel fractions content.Agencia Nacional de Investigación e InnovaciónPrograma de Desarrollo de las Ciencias BásicasComisión Sectorial de Investigación Científic

Fluorinated PAMAM-Arginine Carrier Prodrugs for pH-Sensitive Sustained Ibuprofen Delivery

Objective The development of an efficient, multifunctional drug delivery system overcoming different obstacles generally associated with drug formulations, including the poor accumulation of the active principle in the target site and its sustained release for prolonged time. Methods Our study proposes the development of a fluorinated poly(amidoamine) (PAMAM) carrier prodrug combining drug release boosted in alkaline environments with a possible implementation in 19F MRI applications. In particular, we functionalized the terminal primary amines of PAMAM G2 and G4 through an ad hoc designed fluorinated ibuprofen-arginine Michael acceptor to obtain multifunctional ibuprofen-PAMAM-Arg conjugates. Results These carriers demonstrated pH-dependent and sustained ibuprofen release for more than 5 days. This advantage was observed in both weak alkaline and physiological buffer solutions, allowing to overcome the limits associated to the burst release from similar fluorinated Arg-PAMAM dendrimers with ibuprofen physically encapsulated. Conclusion These findings, coupled to the high biocompatibility of the system, suggest a potential synergistic biomedical application of our conjugates, serving as vehicles for drug delivery and as 19F magnetic resonance imaging contrast agents

Selectively Fluorinated PAMAM-Arginine Conjugates as Gene Delivery Vectors

: Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19F magnetic resonance imaging

Multi-Component Sequential Synthesis of Dihydroorotic Acid-Based Amphiphilic Molecules

An efficient multicomponent sequential process, which occurs in mild condition has been exploited for the synthesis of systematically modified amphiphilic molecules where the cationic head is tethered to a lipophilic tail through a dihydroorotic acid linker. The process is operatively simple, high yielding, and flexible. Such a strategy could impact combinatorial synthesis of wide libraries of amphiphilic molecules to be tested as transfection agents and/or as antimicrobials

Synthesis of hexafluorovaline-containing di- and tripeptides

A new strategy for the synthesis of peptides incorporating racemic hexafluorovaline (hfVal) is presented. The synthetic pathway relies on the anti-Michael addition of benzyl amine derivatives to ad hoc prepared β-bis-trifluoromethyl-acryloyl-α-amino esters which proceeds in mild condition, high yields, even if with low stereocontrol. The following elaboration of the intermediates, namely deprotection of the benzyl moiety and coupling with α-amino esters allowed us to synthetize the targeted tripeptides in four overall synthetic steps, resulting in a synthetic pathway more favorable respect to those appeared in literature based on the synthesis and isolation of racemic Boc-hfVal-OH (eight synthetic steps)

Dual Antibiotic Approach: Synthesis and Antibacterial Activity of Antibiotic–Antimicrobial Peptide Conjugates

In recent years, bacterial resistance to conventional antibiotics has become a major concern in the medical field. The global misuse of antibiotics in clinics, personal use, and agriculture has accelerated this resistance, making infections increasingly difficult to treat and rendering new antibiotics ineffective more quickly. Finding new antibiotics is challenging due to the complexity of bacterial mechanisms, high costs and low financial incentives for the development of new molecular scaffolds, and stringent regulatory requirements. Additionally, innovation has slowed, with many new antibiotics being modifications of existing drugs rather than entirely new classes. Antimicrobial peptides (AMPs) are a valid alternative to small-molecule antibiotics offering several advantages, including broad-spectrum activity and a lower likelihood of inducing resistance due to their multifaceted mechanisms of action. However, AMPs face challenges such as stability issues in physiological conditions, potential toxicity to human cells, high production costs, and difficulties in large-scale manufacturing. A reliable strategy to overcome the drawbacks associated with the use of small-molecule antibiotics and AMPs is combination therapy, namely the simultaneous co-administration of two or more antibiotics or the synthesis of covalently linked conjugates. This review aims to provide a comprehensive overview of the literature on the development of antibiotic–AMP conjugates, with a particular emphasis on critically analyzing the design and synthetic strategies employed in their creation. In addition to the synthesis, the review will also explore the reported antibacterial activity of these conjugates and, where available, examine any data concerning their cytotoxicit