Artemisia annua L. cell cultures to investigate the biosynthetic pathway and the production of the antimalarial compound artemisinin

La malaria è una malattia febbrile acuta diffusa dalla puntura della zanzara femmina del genere Anopheles. Per più di 350 anni questa malattia è stata trattata con chinino, ma nell’ultimo decennio è stato scoperto che il chinino, non ha più effetto su molti ceppi resistenti di P. falciparum. Oggi un trattamento alternativo, efficace e sicuro, contro la malaria è rappresentato dall’artemisinina, un sesquiterpene lattone estratto da una pianta medicinale cinese Artemisia annua L. L’Artemisia annua L. è una pianta aromatica erbacea annuale appartenente alla famiglia delle Asteracae. L’artemisinina è sequestrata nei tricomi ghiandolari che ricoprono in particolar modo la superficie di foglie e fiori. La concentrazione di artemisinina nella pianta è relativamente bassa da 0,1 a 1% del peso secco. La produzione di artemisinina è ancora notevolmente costosa, vista la scarsa quantità di molecola presente nella pianta ed il periodo piuttosto limitato della sua presenza nella pianta. Ecco perché sono numerosi i gruppi di ricerca il cui principale obiettivo è quello di aumentare il contenuto in planta di artemisinina oppure di produrla in colture in vitro o mediante ingegnerizzazione di microrganismi. Gli studi finora condotti, per definire la via biosintetica dell’artemisinina hanno permesso di individuare diversi enzimi e i relativi geni. Tuttavia alcuni passaggi restano ancora da chiarire. Le colture in vitro rappresentano un valido strumento per uno studio approfondito delle vie biosintetiche di metaboliti vegetali, perché permettono di operare in condizioni sperimentali ben definite e controllate. Lo scopo di questa tesi è stato quello di allestire diversi tipi di colture cellulari di Artemisia annua al fine di individuare il sistema più idoneo alla produzione in vitro di artemisinina e di indagare sulla regolazione della via biosintetica, valutando l’espressione dei geni noti in colture in sospensione di A. annua, sottoposte a differenti trattamenti. Sono state allestite diverse tipologie di colture in vitro di Artemisia annua L. (colture di calli, cellule in sospensione e piantine micropropagate). Per aumentare il contenuto di artemisinina le colture in sospensione sono state elicitate con metil jasmonato, una molecola che stimola il metabolismo secondario e miconazolo, un inibitore della sintesi degli steroli, la via competitiva dei sesquiterpeni. Inoltre, le cellule sono state trattate con elicitori biotici come l’estratto fungino di Penicillium verrucosum. Attraverso analisi HPLC si è potuto osservare che i trattamenti hanno stimolato in misura differenziale la produzione di artemisinina. Il trattamento con metil jasmonato ha indotto un aumento massimo di 2,4 volte del contenuto di artemisinina dopo 4 ore di trattamento. Nel trattamento con miconazolo si è raggiunto il livello più alto di artemisinina (1,6 volte) quando le cellule sono state trattate per 5 giorni alla concentrazione di 200 M. L’analisi dell’espressione genica, mediante Real Time PCR, ha permesso di studiare la regolazione di alcuni geni della via biosintetica dell’artemisinina. L’analisi dell’espressione in cellule in sospensione di Artemisia annua ha rivelato, un aumento dell’espressione dei geni CYP71AV1, CPR e Dbr2, codificanti rispettivamente per gli enzimi citocromo P450 monossigenasi, citocromo P450 reduttasi e aldeide artemisinica 11(13) reduttasi. In particolare si è evidenziato un aumento dell’espressione di CYP71AV1 di circa 7 volte, dopo 30 min di trattamento con metil jasmonato. Nelle cellule trattate con miconazolo 200 M si è evidenziato un forte aumento dell’espressione del gene Dbr2, e di CPR rispettivamente dopo 30 min e 4 ore di trattamento. In questo lavoro di tesi è stato inoltre messo a punto il protocollo di isolamento di protoplasti sia da cellule in sospensione sia da piantine micropropagate di Artemisia annua. I protoplasti sono stati trasformati transientemente. Un altro aspetto della ricerca è stato volto ad identificare geni codificanti fattori di trascrizione in grado di influenzare la biosintesi di artemisinina. In particolare la nostra attenzione è stata focalizzata sulla famiglia di fattori di trascrizione WRKY. È stato isolato un nuovo fattore WRKY in Artemisia annua denominato AaWRKY3. La sequenza di AaWRKY3 è stata utilizzata per creare un costrutto chimerico con tag fluorescente: WRKY:GFP, utilizzato per trasformare transientemente protoplasti di cellule in sospensione di A. annua. Il costrutto WRKY:GFP andava a localizzarsi specificamente nel nucleo, così come tutti i fattori di trascrizione

RAPD and SSR markers for characterization and identification of ancient cultivars of Olea europaea L. in the Emilia region

The Emilia region (Northern Italy) is characterised by the occurrence of microclimates that permit olive growing. The presence of the species, albeit sporadic, in these territories for several centuries as a fruit crop is well documented, by both archaeological and written testimony, and by a large number of plants well over a century old, located in particular sites, favourable for growth and development of the tree. Olive genetic diversity was studied using RAPD and SSR techniques, on plants growing in the Emilia territory (Reggio Emilia and Parma provinces). For genotype identification comparisons were made with 8 cultivars, some of which from Central Italy. Screening was obtained analysing patterns produced by 20 RAPD primers and 3 SSR primers, developed by other authors; the primers and we were able to discriminate olive cultivars with a sufficient degree of reliability. The dendrograms obtained from the analysis show the genetic relationship among accessions present in the Parma-Reggio Emilia district. Our results demonstrated the reliability of RAPDs and SSRs to identify all studied olive cultivars and to reveal the degree of their relatedness to each other. The analysis also reveals the presence of an interesting amount of genetic diversity among the studied individuals

Silver-Functionalized Bacterial Cellulose as Antibacterial Membrane for Wound-Healing Applications

Bacterial cellulose (BC) functionalized with silver nanoparticles (AgNPs) is evaluated as an antimicrobial membrane for wound-healing treatment. A facile green synthesis of silver nanoparticles inside the porous three-dimensional weblike BC network has been obtained by UV light irradiation. AgNPs were photochemically deposited onto the BC gel network as well as they were chemically bonded to the cellulose fiber surfaces. AgNPs with a narrow size distribution along with some aggregates in the BC network were evidenced from the morphological analyses. A highly crystalline nature of the BC membranes was observed in X-ray diffraction measurements, and the presence of metallic silver confirmed the photochemical reduction of Ag⁺ → Ag⁰ in Ag/BC composites. Antibacterial activity of the hybrid composites, such as pellicles, performed against the Gram-negative bacteria (Escherichia coli) by disk diffusion and growth dynamics methods showed high bacteria-killing performance. No significant amount of silver release was observed from the Ag/BC pellicles even after a long soaking time. As composite pellicles are preserved in a moist environment that also favors wound recovery, by combining all of these properties the material could be useful in wound-healing treatments

PolyDiethyleneglycol–bisallyl carbonate matrix transparent nanocomposites reinforced with bacterial cellulose microfibrils

Transparent nanocomposite films were prepared using bacterial cellulose (BC) as reinforcement and diethylene glycol bis(allyl carbonate) polymer (DEAC) as matrix by vacuum infiltration and UV polymerization. The BC/DEAC nanocomposites exhibit excellent transparency up to 88% at wavelength of 550 nm. The uniform dispersion of resin in BC 3D network was evidenced from SEM and ATR-FTIR analyses, confirms the complete photo-polymerization of diethylene glycol bis(allyl)carbonate monomer to Poly (diethylene glycol bis(allyl carbonate) in BC network. The BC/DEAC composites have good mechanical properties, reaching a tensile strength of 130 MPa and a Young’s Modulus of 6.4 GPa. Applying a micromechanic modeling approach, the elastic modulus of the composite was used in order to determine the average aspect ratio of BC fibers. These flexible transparent BC/DEA

Ethylene photo-oxidation on copper phthalocyanine sensitized TiO 2 films under solar radiation

none6siLimited solar photo-activity and recovering the catalysts after photocatalysis reaction are two major drawbacks of the highly active TiO2 powder materials. In this study we propose that copper phthalocyanine (CuPc, Cu(II) tetrakis[4-(2,4-bis-(1,1-dimethylpropyl)phenoxy)]phthalocyanine), which is an metal-organic dye could be used as sensitizer of TiO2 coating to shift the absorption band toward visible light. TiO2 coating was applied inside the Pyrex glass tubes and used in the photocatalytic decomposition of ethylene gas under solar light irradiation. Two-step draining method was employed to apply the coating on the inner side of the glass tubes. At first pure TiO2 coating was prepared from a solution by dispersing the commercial P25 TiO2 powders in a TiO2 sol made by hydrolysis-condensation of titanium alkoxide. A controlled draining method was employed to coat the inner side of the glass tubes. After calcination at 500 °C, P25 powders were strongly attached on the glass surface as a thick coating. Visible light absorptive coating was prepared by applying a thin layer of CuPc that shows intense absorption in the visible wavelength region utilizing the same coating procedure. CuPc coated TiO2 film showed excellent photo-stability against solar radiation. Greater photo-oxidation rate of ethylene was achieved with the CuPcTiO2 coated glass tube compared to that without CuPc coating due to the enhanced solar light absorption.Licciulli, Antonio; Riccardis, Alberto De; Pal, Sudipto; Nisi, Rossella; Mele, Giuseppe; Cannoletta, DonatoLicciulli, ANTONIO ALESSANDRO; Riccardis, Alberto De; Pal, Sudipto Kumar; Nisi, Rossella; Mele, Giuseppe Agostino; Cannoletta, Donato Pompili

Green Energy Harvester from Vibrations Based on Bacterial Cellulose

A bio-derived power harvester from mechanical vibrations is here proposed. The harvester aims at using greener fabrication technologies and reducing the dependence from carbon-based fossil energy sources. The proposed harvester consists mainly of biodegradable matters. It is based on bacterial cellulose, produced by some kind of bacteria, in a sort of bio-factory. The cellulose is further impregnated with ionic liquids and covered with conducting polymers. Due to the mechanoelectrical transduction properties of the composite, an electrical signal is produced at the electrodes, when a mechanical deformation is imposed. Experimental results show that the proposed system is capable of delivering electrical energy on a resistive load. Applications can be envisaged on autonomous or quasi-autonomous electronics, such as wireless sensor networks, distributed measurement systems, wearable, and flexible electronics. The production technology allows for fabricating the harvester with low power consumption, negligible amounts of raw materials, no rare elements, and no pollutant emissions

ROS production and scavenging under anoxia and re-oxygenation in Arabidopsis cells: a balance between redox signaling and impairment.

Plants can frequently experience low oxygen concentrations due to environmental factors such as flooding or waterlogging. It has been reported that both anoxia and the transition from anoxia to re-oxygenation determine a strong imbalance in the cellular redox state involving the production of reactive oxygen species (ROS) and nitric oxide (NO). Plant cell cultures can be a suitable system to study the response to oxygen deprivation stress since a close control of physicochemical parameters is available when using bioreactors. For this purpose, Arabidopsis cell suspension cultures grown in a stirred bioreactor were subjected to a severe anoxic stress and analyzed during anoxia and re-oxygenation for alteration in ROS and NO as well as in antioxidant enzymes and metabolites. The results obtained by confocal microscopy showed the dramatic increase of ROS, H2O2 and NO during the anoxic shock. All the ascorbate-glutathione related parameters were altered during anoxia but restored during re-oxygenation. Anoxia also induced a slight but significant increase of α-tocopherol levels measured at the end of the treatment. Overall, the evaluation of cell defenses during anoxia and re-oxygenation in Arabidopsis cell cultures revealed that the immediate response involving the overproduction of reactive species activated the antioxidant machinery including ascorbate-glutathione system, α-tocopherol and the ROS-scavenging enzymes ascorbate peroxidase, catalase and peroxidase making cells able to counteract the stress towards cell survival