Chemistry and phytotoxicity of secondary metabolites from Mediterranean plants

[ENGLISH] During the three years of my PhD studies, three Mediterranean plants have been analysed, Brassica fruticulosa, Chenopodium album and Malva silvestris. The metabolites obtained by infusion and by using a new extraction procedure, have been purified by different chromatographic techniques (CC, TLC, HLPC, DCCC), while their structural characterisation has been performed by spectroscopic and spectrometric techniques (1H and 13C NMR, UV, IR, CD, ESI MS, MALDI MS, GC MS). The investigation of the three Mediterranean plants has led to the identification of: seven lignans, four neolignans, five sesquilignans, one dilignan, eight C-13 nor-terpenes, three degraded carotenoids, two monoterpenes, one sesquiterpene, one diterpene, nineteen phenol derivatives, one triptammine, one benzaldeid derivative, three allenes and eight cinnamic amides. Among them, thirteen compounds have been isolated for the first time. Absolute stereochemistry of some compounds has been defined through the application of Mosher’s method. Some of the isolated compounds have been tested on seeds of standard plants [Lactuca sativa L (lettuce), Lycopersicon esculentum M. (tomato), Allium cepa L. (onion)] to evaluate their biological activity. The results reported have shown a strong inhibiting effect of many lignans, which inhibit germination and growth of the standard species down to 1 nM concentration. At these concentrations commercial herbicides are usually inactive. Such a potent bioactivity for very diluted solution of lignans suggests further in vitro and in fields investigations, to fully evaluate the potential use of these molecules as natural herbicides. In this thesis the use of FTICR mass spectrometry is also described. In a research project of a nine-months Marie Curie fellowship, this technique has been employed for studying the charge states distribution of Calmodulin and modalities of interaction of the protein in a biological environment. / [ITALIANO] Tre piante spontanee dell’area Mediterranea, Brassica fruticulosa, Chenopodium album e Malva silvestris, sono state sottoposte ad indagine sistematica, allo scopo di definire la natura e le caratteristiche chimiche dei metaboliti secondari prodotti dai tre organismi vegetali. I metaboliti, ottenuti dalle tre piante mediante metodiche infusive ed estrattive, sono stati purificati attraverso tecniche cromatografiche (CC, TLC, DCCC, HPLC) e caratterizzati mediante spettroscopia (UV, IR, 1H-NMR, 13C-NMR, CD) e spettrometria di massa (GC-MS, MALDI-MS, ESI-MS). Sono stati in tal modo isolati: sette lignani, quattro neolignani, cinque sesquilignani, un dilignano, otto C-13 nor-terpeni, tre carotenoidi degradati, due monoterpeni, un sesquiterpene, un diterpene, diciannove derivati fenolici, un derivato triptamminico e uno benzaldeidico, tre alleni e otto ammidi cinnamiche. Tredici di questi composti sono stati isolati per la prima volta. Molti dei metaboliti isolati sono stati sottoposti a saggi di fitotossicità su organismi vegetali di riferimento, allo scopo di definirne l’eventuale bioattività. Fra tutti, i composti aventi una struttura di tipo lignanico, hanno mostrato un’intensa attività inibente la germinazione delle specie vegetali testate. E’ inoltre riportato lo studio della modalità di interazione della Calmodulina con il peptide RS20, realizzato durante un periodo speso presso l’Università di Warwick (UK). Tale analisi è stata svolta adoperando uno spettrometro di massa FTICR accoppiato ad una sorgente ESI

In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f).

Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated

Hypofractionated breast cancer radiotherapy. Helical tomotherapy in supine position or classic 3D-conformal radiotherapy in prone position: which is better?

We propose a comparative dosimetric study of whole-breast hypofractionated radiation therapy using helical tomotherapy (HT) in supine position and 3-D conformal radiotherapy (3D-CRT) in prone position. Twelve patients undergoing breast-conserving therapy were retrospectively selected from October to December 2012. Specific dose-volume parameters were selected for the study. The target coverage was adequate in all patients for both techniques. Significant differences in lung dose distribution were observed: maximum dose (mean value over the 12 plans) was 23.41 Gy in HT plans and 6.65 Gy in 3D-CRT; V20 (i.e. the lung volume receiving 20 Gy) was 0.31% in HT plans and 0.0% in 3D-CRT plans. The mean dose to the heart was 5.57 Gy and 0.93 Gy, respectively. The differences between the two techniques were significant (p<0.05) only for some parameters. We noted better results in the prone position, but with HT, dose constraints were mentioned for the whole set of considered organs

Glycan and antennarity and sialylation distribution at Asn52.

<p><b>A)</b> Comparison of glycan distribution at Asn52 between Bemfola and GONAL-f (individual species) <b>B)</b> Comparison of antennarity and sialylation at Asn52 between Bemfola and GONAL-f. Asn, asparagine.</p

Site-specific N-and O-glycosylation analysis of atacicept

International audienceThe Fc-fusion protein atacicept is currently under clinical investigation for its biotherapeutic application in autoimmune diseases owing to its ability to bind the two cytokines B-Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL). Like typical recombinant IgG-based therapeutics, atacicept is a glycoprotein whose glycosylation-related heterogeneity arises from the glycosylation-site localization, site-specific occupation and structural diversity of the attached glycans. Here, we present a first comprehensive site-specific N- and O-glycosylation characterization of atacicept using mass spectrometry-based workflows. First, N- and O-glycosylation sites and their corresponding glycoforms were identified. Second, a relative quantitation of the N-glycosylation site microheterogeneity was achieved by glycopeptide analysis, which was further supported by analysis of the released N-glycans. We confirmed the presence of one N-glycosylation site, carrying 47 glycoforms covering 34 different compositions, next to two hinge region O-glycosylation sites with core 1-type glycans. The relative O-glycan distribution was analyzed based on the de-N-glycosylated intact protein species. Overall, N- and O-glycosylation were consistent between two individual production batches

Bemfola and GONAL-f batches tested.

<p>Bemfola and GONAL-f batches tested.</p