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Xylella fastidiosa subsp. pauca is associated with the olive quick decline syndrome in Salento (Apulia region, Southern Italy). The first outbreak of the disease was noticed in the Gallipoli district, from where it subsequently reached nearby areas. To date, no specific study has verified if abnormal soil or leaf micronutrients is associated with the disease. Soil and leaf samples were taken from 23 olive farms showing symptoms of the syndrome located in Gallipoli and nearby areas. Each sample was analyzed for magnesium and micronutrients content using inductively coupled plasma atomic emission spectroscopy. Real-time PCR indicated that X. f. subsp. pauca was present in each sampled tree. There is a general lack of molybdenum in soil and low bioavailability of copper and molybdenum in tree leaves. Low content of manganese in soil was also found in some farms located in Gallipoli, Galatone and Trepuzzi. Olive trees grown in Gallipoli area also had low content of boron. Principal component analysis showed that soil and leaf samples from this area had lower micronutrient contents compared with other areas. General copper depletion in leaves was uncommon and has not been previously recorded in Italy in a large area. This could indicate that X. f. subsp. pauca infection causes a depletion of copper within olive leaves. Reduced copper content has been previously recorded in leaves infected with X. f. subsp. multiplex and for other bacterial pathogens. The role of copper in relation to X. fastidiosa infection is discussed.

Made available in DSpace on 2018-12-20T13:22:30Z (GMT). No. of bitstreams: 1 tese_12645_Tese.pdf: 1192 bytes, checksum: cdb19e868e8c7f7a7493fa7466d7c95b (MD5) Previous issue date: 2018-10-04 A estatística de identificação de suspeitos de crimes com base em impressões papilares latentes (das quais as impressões digitais são os principais tipos) reveladas em cartuchos de munição deflagrados é muito baixa, praticamente nula. Neste estudo, foram abordados diversos aspectos relacionados a esse problema, visando compreender suas causas e apresentar sugestões de ordem prática para serem empregadas pelos peritos da área a fim de melhorar esse quadro. Diversas técnicas de revelação foram testadas em cartuchos intactos, cartuchos deflagrados e também em chapas planas de latão CuZn30, mesmo material dos cartuchos. Os substratos contendo impressões papilares foram submetidos a diferentes condições de temperatura (temperatura ambiente, 63 e 200 °C) e períodos de envelhecimento (1, 7 e 14 dias) antes da aplicação dos reveladores. De acordo com os resultados, a melhor opção para revelar impressões papilares em cartuchos de munição fabricados em latão é aplicar a sequência composta por cianoacrilato + gun blue + corante fluorescente (amarelo básico 40 ou Ardrox). Visando superar algumas dificuldades adicionais que os peritos enfrentam ao fotografar impressões latentes em cartuchos de munição, foram desenvolvidos e patenteados um aparato e um método para realização de fotografias e geração de imagens panorâmicas da superfície lateral de cartuchos e outros objetos cilíndricos. Impressões digitais geradas em imagens panorâmicas compostas por diferentes quantidades de fotos foram avaliadas de maneira objetiva, com base nos placares apresentados pelo sistema AFIS (Sistema Automatizado de Identificação de Impressões Digitais). Os resultados indicaram que imagens panorâmicas em 360° da superfície lateral de cartuchos podem ser obtidas de forma satisfatória com base em 9 fotos tiradas a cada giro de 40° do cartucho entre uma foto e outra. Para tentar entender o motivo por trás da baixa taxa de sucesso observada no processamento de impressões latentes nessas pequenas evidências, foram caracterizados os materiais e superfícies dos substratos utilizados nessa pesquisa e, também, foram investigados os efeitos do disparo sobre as impressões depositadas em cartuchos de munição. Os materiais e superfícies dos substratos foram caracterizados difração de raios-X, microscopia eletrônica de varredura, espectroscopia de dispersão de energia, ensaios metalográficos e de microdureza por endentação Vickers. Os resultados mostraram que, embora as placas e os estojos de cartuchos sejam, de fato, constituídos de latão alfa 70/30, há diferença na distribuição relativa dos planos cristalinos, na estrutura de grãos, na textura de superfície e na dureza desses dois tipos de substratos. Até mesmo em cartuchos provenientes de um mesmo lote, retirados da mesma embalagem, foram observadas diferenças significativas nos níveis de oxidação da superfície. Diante da dificuldade de determinar as condições de superfície de cartuchos de munição feitos de latão em situações reais, não é possível garantir a eficiência de determinado revelador. Portanto, visando aumentar as chances de sucesso na recuperação de impressões papilares a partir dessas importantes evidências que são encontradas com frequência em locais de crime, recomenda-se fortemente aplicar toda a sequência de reveladores apresentada acima, realizando inspeção visual e, se necessário, fotografia das impressões eventualmente reveladas após a aplicação de cada um dos reveladores. A doutrina forense tem atribuído a baixa quantidade e qualidade verificadas para impressões reveladas em cartuchos deflagrados ao atrito entre o cartucho expandido e a parede interna da câmara da arma durante a extração. No entanto, os resultados do presente estudo apontam para o fluxo de gases passando na folga entre o cartucho e a câmara como sendo a principal causa de degradação de impressões papilares durante o disparo. Os resultados também indicam que o disparo em si não justifica a baixa taxa de sucesso citada, sugerindo que a manipulação dos cartuchos de munição antes e após o disparo sejam as principais causas. A manipulação antes do disparo não pode ser evitada, mas o manuseio feito por peritos após a coleta das evidências pode e deve ser feito de maneira apropriada, com uso de pinças específicas e embalagem adequada para condicionamento e transporte. Sendo assim, como contribuição final deste trabalho, foi desenvolvida uma embalagem para acondicionamento apropriado dos estojos de cartuchos deflagrados e também é apresentada uma sugestão para a maneira como essas devem ser manuseadas. Portanto, a presente tese de doutorado contribuiu para a investigação e o desenvolvimento de métodos para resolução de um problema real e atual de perícia forense. A metodologia desenvolvida foi aplicada com sucesso em casos concretos e um próximo passo do trabalho é fomentar a sua normatização na forma de instruções técnicas e procedimentos operacionais padrão para viabilizar sua implantação na rotina de instituições forenses.

Data regarding double switching from originator infliximab (IFX) to IFX biosimilars in inflammatory bowel diseases (IBDs) are lacking. The purpose of this study was to evaluate the safety and efficacy of switching from originator IFX to CT-P13 and subsequently to SB2 (double switch) in patients with IBD. Patients undergoing IFX-double switch in eight Centers in Lombardy (Italy) from November 2018 to May 2019 were retrospectively analyzed. The IFX discontinuation rate, incidence and type of adverse events (AEs), and clinical remission rate were recorded. A comparison with a control group of patients with IBD single-switched from originator IFX to CT-P13 was performed, before and after an inverse probability of treatment weighting (IPTW)-based propensity score analysis. Fifty-two double-switched patients with IBD were enrolled. The 24- and 52-week proportions of patients continuing on IFX therapy following the second switch (CTP13→SB2) were 98% (95% confidence interval [CI] 94%–100%) and 90% (95% CI 81%–99%), respectively. Four patients experienced a total of five AEs, all graded 1–3 according to Common Terminology Criteria for Adverse Events (CTCAE). No infusion reactions were observed. The 24-week and follow-up end clinical remission rates following the second switch were 94% and 88%, respectively. No differences were observed in the safety and efficacy outcomes by comparing the double-switch group with a single-switch group of 66 patients with IBD; all these results were confirmed by IPTW-adjusted analysis. The study suggests both the safety and efficacy of the double switch from originator IFX to CT-P13 and SB2 in patients with IBD is maintained. This strategy may be associated with potential cost implications.

Xylella fastidiosa is a xylem-limited bacterium causing a range of economically important plant diseases in hundreds of crops. Over the last decade, a severe threat due to Olive Quick Decline Syndrome (OQDS), caused by Xylella fastidiosa subspecies pauca, affected the Salento olive groves (Apulia, South-East Italy). Very few phyto-therapeutics, including a Zn/Cu citric acid biocomplex foliar treatment, were evaluated to mitigate this disease. However, the traditional foliar applications result in the agro-actives reaching only partially their target. Therefore the development of novel endo-therapeutic systems was suggested. Metabolite fingerprinting is a powerful method for monitoring both, disease progression and treatment effects on the plant metabolism, allowing biomarkers detection. We performed, for the first time, short-term monitoring of metabolic pathways reprogramming for infected Ogliarola salentina and Cima di Melfi olive trees after precision intravascular biocomplex delivery using a novel injection system. Upon endo therapy, we observed specific variations in the leaf content of some metabolites. In particular, the 1H NMR-based metabolomics approach showed, after the injection, a significant decrease of both the disease biomarker quinic acid and mannitol with simultaneous increase of polyphenols and oleuropein related compounds in the leaf’s extracts. This combined metabolomics/endo-therapeutic methodology provided useful information in the comprehension of plant physiology for future applications in OQDS control.

Geographical origin assessment of extra virgin olive oil (EVOO) is recognised worldwide as raising consumers’ awareness of product authenticity and the need to protect top-quality products. The need for geographical origin assessment is also related to mandatory legislation and/or the obligations of true labelling in some countries. Nevertheless, official methods for such specific authentication of EVOOs are still missing. Among the analytical techniques useful for certification of geographical origin, nuclear magnetic resonance (NMR) and mass spectroscopy (MS), combined with chemometrics, have been widely used. This review considers published works describing the use of these analytical methods, supported by statistical protocols such as multivariate analysis (MVA), for EVOO origin assessment. The research has shown that some specific countries, generally corresponding to the main worldwide producers, are more interested than others in origin assessment and certification. Some specific producers such as Italian EVOO producers may have been focused on this area because of consumers’ interest and/or intrinsic economical value, as testified also by the national concern on the topic. Both NMR- and MS-based approaches represent a mature field where a general validation method for EVOOs geographic origin assessment could be established as a reference recognised procedure.

Nucleos(t)ide analogues (NA) belong to a family of compounds widely used in anticancer/antiviral treatments. They generally exhibit a cell toxicity limited by cellular uptake levels and the resulting nucleos(t)ides metabolism modifications, interfering with the cell machinery for nucleic acids synthesis. We previously synthesized purine nucleos(t)ide analogues N7-coordinated to a platinum centre with unaltered sugar moieties of the type: [Pt(dien)(N7-dGuo)]2+ (1; dien = diethylenetriamine; dGuo = 2'-deoxy-guanosine), [Pt(dien)(N7-dGMP)] (2; dGMP = 5'-(2'-deoxy)-guanosine monophosphate), and [Pt(dien)(N7-dGTP)]2- (3; dGTP = 5'-(2'-deoxy)-guanosine triphosphate), where the indicated electric charge is calculated at physiological pH (7.4). In this work, we specifically investigated the uptake of these complexes (1-3) at the plasma membrane level. Specific experiments on HeLa cervical cancer cells indicated a relevant cellular uptake of the model platinated deoxynucleos(t)ide 1 and 3 while complex 2 appeared unable to cross the cell plasma membrane. Obtained data buttress an uptake mechanism involving Na+-dependent concentrative transporters localized at the plasma membrane level. Consistently, 1 and 3 showed higher cytotoxicity with respect to complex 2 also suggesting selective possible applications as antiviral/antitumor drugs among the used model compounds.

The agricultural sector is facing a decrease in water supply and water quality at a global level and this is a problem that strictly affects all the Mediterranean olive growing areas. The aim of this work was to evaluate, for the first time, by NMR Spectroscopy and multivariate data analysis the metabolic profiling of the oils produced under different irrigation schemes. Arbosana olive oils were obtained from the use of saline reclaimed water (RW) and treated municipal wastewater (DW), combined with: full irrigation (FI) and regulated deficit irrigation (RDI). The results show a higher relative content of saturated fatty acids in EVOOs obtained from RDI strategy, regardless of the water source. Moreover, an increase in unsaturated fatty acids, a ω6/ω3 ratio content was observed in EVOOs obtained from RW when compared with DW water. Furthermore, the RW–RDI showed an increase in secoiridoid derivatives and hydroperoxides with respect to DW–RDI. A sustainable irrigation management, by combining a deficit irrigation strategy and saline reclaimed water source, could be crucial in order to overcome the problem of water scarcity and to guarantee the olive oil nutraceutical properties. The 1H NMR-based metabolomic approach proved a powerful and versatile tool for this specific investigation.

Xylella fastidiosa subsp. pauca, is a bacterial phytopathogen associated with the “olive quick decline syndrome” (OQDS) causing severe economic losses to olive groves in Salento area (Apulia, Italy). In a previous work, we analyzed by 1H-NMR the metabolic pattern of naturally infected Ogliarola salentina and Cellina di Nardò susceptible cultivars untreated and treated with a zinc-copper citric acid biocomplex and we observed the treatment related variation of the disease biomarker quinic acid. In this study, we focused also on the Leccino cultivar, known to exhibit tolerance to the disease progression. The 1H-NMR-based metabolomic approach was applied with the aim to characterize the overall metabolism of tolerant Leccino in comparison with the susceptible cultivars Ogliarola salentina and Cellina di Nardò under periodic mid-term treatment. In particular, we studied the leaf extract molecular patterns of naturally infected trees untreated and treated with the biocomplex. The metabolic Leccino profiles were analyzed for the first time and compared with those exhibited by the susceptible Cellina di Nardò and Ogliarola salentina cultivars. The study highlighted a specificity in the metabolic response of the tolerant Leccino compared to susceptible cultivars. These differences provide useful information to describe the defensive mechanisms underlying the change of metabolites as a response to the infection, and the occurrence of different levels of disease, season and treatment effects for olive cultivars.

Xylella fastidiosa subsp. pauca is the causal agent of “olive quick decline syndrome” in Salento (Apulia, Italy). On April 2015, we started interdisciplinary studies to provide a sustainable control strategy for this pathogen that threatens the multi-millennial olive agroecosystem of Salento. Confocal laser scanning microscopy and fluorescence quantification showed that a zinc-copper-citric acid biocomplex—Dentamet®—reached the olive xylem tissue either after the spraying of the canopy or injection into the trunk, demonstrating its effective systemicity. The biocomplex showed in vitro bactericidal activity towards all X. fastidiosa subspecies. A mid-term evaluation of the control strategy performed in some olive groves of Salento indicated that this biocomplex significantly reduced both the symptoms and X. f. subsp. pauca cell concentration within the leaves of the local cultivars Ogliarola salentina and Cellina di Nardò. The treated trees started again to yield. A 1H-NMR metabolomic approach revealed, upon the treatments, a consistent increase in malic acid and γ-aminobutyrate for Ogliarola salentina and Cellina di Nardò trees, respectively. A novel endotherapy technique allowed injection of Dentamet® at low pressure directly into the vascular system of the tree and is currently under study for the promotion of resprouting in severely attacked trees. There are currently more than 700 ha of olive groves in Salento where this strategy is being applied to control X. f. subsp. pauca. These results collectively demonstrate an efficient, simple, low-cost, and environmentally sustainable strategy to control this pathogen in Salento.

Considering the growing number of extra virgin olive oil (EVOO) producers in the world, knowing the influence of olive oils with different geographical origins on the characteristics of the final blend becomes an interesting goal. The present work is focused on commercial organic EVOO blends obtained by mixing multiple oils from different geographical origins. These blends have been studied by 1H-NMR spectroscopy supported by multivariate statistical analysis. Specific characteristics of commercial organic EVOO blends originated by mixing oils from Italy, Tunisia, Portugal, Spain, and Greece were found to be associated with the increasing content of the Italian component. A linear progression of the metabolic profile defined characteristics for the analysed samples—up to a plateau level—was found in relation to the content of the main constituent of the Italian oil, the monocultivar Coratina. The Italian constituent percentage appears to be correlated with the fatty acids (oleic) and the polyphenols (tyrosol, hydroxytyrosol, and derivatives) content as major and minor components respectively. These results, which highlight important economic aspects, also show the utility of 1H-NMR associated with chemometric analysis as a powerful tool in this field. Mixing oils of different national origins, to obtain blends with specific characteristics, could be profitably controlled by this methodology.

Background: Plants of genus Cichorium are known for their therapeutic and nutraceutical properties determined by a wealth of phytochemical substances contained in the whole plant. The aim of this paper was to characterize the metabolic profiles of local Salento chicory (Cichorium intybus L.) varieties (“Bianca”, “Galatina”, “Leccese”, and “Otranto”) in order to describe their metabolites composition together with possible bioactivity and health beneficial properties. Methods: The investigation was performed by 1H-NMR spectroscopy and Multivariate Analysis (MVA), by which the metabolic profiles of the samples were easily obtained and compared. Results: The supervised Partial Least Squares Discriminant Analysis (PLS-DA) analysis showed as “Bianca” and “Galatina” samples grouped together separated by “Leccese” and “Otranto” varieties. A different content of free amino acids and organic acids was observed among the varieties. In particular a high content of cichoric and monocaffeoyl tartaric acid was observed for the “Leccese” variety. The presence of secondary metabolites adds significant interest in the investigation of Cichorium inthybus, as this vegetable may benefit human health when incorporated into the diet. Conclusions: The 1H-NMR (Nuclear Magnetic Resonance Spectroscopy) based characterization of Salento chicory varieties allowed us to determine the potential usefulness and nutraceutical properties of the product, also providing a method to guarantee its authenticity on a molecular scale.

The innovative combination of ultrasound (Us) with a thermal exchanger to produce high quality extra virgin olive oil (EVOO) was studied using Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate analysis (MVA). Major and minor metabolomic components of Apulian Coratina EVOO obtained using the two methods were compared. Early and late olive ripening stages were also considered. An increased amount of polyphenols was found for EVOOs obtained using the Us with respect to the conventional method for both early and late ripening stages (900.8 ± 10.3 and 571.9 ± 9.9 mg/kg versus 645.1 ± 9.3 and 440.8 ± 10.4 mg/kg). NMR spectroscopy showed a significant increase (P < 0.05) in polyunsaturated fatty acids (PUFA) as well as in the tyrosol and hydroxytyrosol derivatives, such as oleocanthal, oleacein, and elenolic acid, for both ripening stages. In conclusion, NMR spectroscopy provides information about the metabolomic components of EVOOs to producers, while the Us process increases the levels of healthy bioactive components.

Xylella fastidiosa subsp. pauca is responsible for the &ldquo, olive quick decline syndrome&rdquo, (OQDS) in Salento (Apulia). The main epidemiological aspects of the syndrome are related to the pathogen spread and survival in the area, and to the biology of the insect vector. The assessment of the macro and microelements content (i.e., ionome) in soil and leaves could provide basic and useful information. Indeed, knowledge of host ionomic composition and the possibility of its modification could represent a potential tool for the management of diseases caused by X. fastidiosa. Therefore, soil and leaf ionomes of naturally infected, not infected, and zinc&ndash, copper&ndash, citric acid biocomplex treated trees of different areas of Apulia and the bordering Basilicata regions were compared. We observed that soil and leaf ionomic composition of olive farms growing in the pathogen-free areas north of the Salento Barletta-Andria-Trani BAT (Apulia) and Potenza PZ (Basilicata, Apulia bordering region) provinces is significantly different from that shown by the infected olive groves of the Salento areas (LE, BR, TA provinces). In particular, a higher content of zinc and copper both in soil and leaves was found in the studied northern areas in comparison to the southern areas. This finding could partly explain the absence of OQDS in those areas. In the infected Salento areas, the leaf ionomic profile resulted as being markedly different for the biocomplex treated compared to the untreated trees. A higher zinc content in leaves characterized treated with respect to untreated trees. On the other hand, among the not-infected trees, Xylella-resistant Leccino showed higher manganese content when compared with the higher pathogen sensitive Ogliarola salentina and Cellina di Nard&ograve, According to these results, soil and olive leaf ionome could provide basic information for the epidemiologic study and possible control of X. f. subsp. pauca in Apulia.

Anaemia is a common pathologic condition, present in almost 5% of the adult population. Iron deficiency is the most common cause; other mechanisms can be involved, making anaemia a multi-factorial disorder in most cases. Anaemia being a frequent manifestation in the diseases of the gastrointestinal tract, patients are often referred to gastroenterologists. Furthermore, upper and lower endoscopy and enteroscopy are pivotal to the diagnostic roadmap of anaemia. In spite of its relevance in the daily clinical practice, there is a limited number of gastroenterological guidelines dedicated to the diagnosis of anaemia. For this reason, the Italian Association of Hospital Gastroenterologists and Endoscopists and the Italian Society of Paediatric Gastroenterology, Hepatology and Nutrition commissioned a panel of experts to prepare a specific guideline on anaemia and its diagnostic roadmap in the gastroenterological scenario. The panel also discussed about the potential involvement of gastroenterologists and endoscopists in the management of patients with anaemia, with particular attention to the correct use of investigations. The panel paid particular attention to practical issues with the aim to support gastroenterologists in their clinical practice when dealing with patients with anaemia.

Honey is a natural sweet substance produced by honeybees from the nectar of flowers, plant secretions or plant-sucking insect excretions. Sugars and water constitute the major components, other minor components characterize the organoleptic and nutritional properties. To date, Salento (Apulia region, Italy) honey production is considerably threatened due to the suggested use of neonicotinoids in order to control the insect-vectored bacterium Xylella fastidiosa (subsp. pauca). Metabolomics based on Nuclear Magnetic Resonance (NMR) spectroscopy was used to describe, for the first time, the composition of honey samples from different Salento producers. Exploratory Principal Component Analysis (PCA) showed, among the observed clustering, a separation between light and dark honeys and a discrimination according to producers, both further analyzed by supervised multivariate analysis. According to the obtained data, although limited to small-scale emerging production, Salento honey shows at the molecular level, a range of specific characteristic features analogous to those exhibited by similar products originating elsewhere and appreciated by consumers. The impact on this production should therefore be carefully considered when suggesting extensive use of pesticides in the area.

Xylella fastidiosa subsp. pauca is a xylem-limited bacterial phytopathogen currently found associated on many hectares with the &ldquo, olive quick decline syndrome&rdquo, in the Apulia region (Southern Italy), and the cultivars Ogliarola salentina and Cellina di Nard&ograve, result in being particularly sensitive to the disease. In order to find compounds showing the capability of reducing the population cell density of the pathogen within the leaves, we tested, in some olive orchards naturally-infected by the bacterium, a zinc-copper-citric acid biocomplex, namely Dentamet&reg, by spraying it to the crown, once per month, during spring and summer. The occurrence of the pathogen in the four olive orchards chosen for the trial was molecularly assessed. A 1H NMR metabolomic approach, in conjunction with a multivariate statistical analysis, was applied to investigate the metabolic pattern of both infected and treated adult olive cultivars, Ogliarola salentina and Cellina di Nard&ograve, trees, in two sampling periods, performed during the first year of the trial. For both cultivars and sampling periods, the orthogonal partial least squares discriminant analysis (OPLS-DA) gave good models of separation according to the treatment application. In both cultivars, some metabolites such as quinic acid, the aldehydic form of oleoeuropein, ligstroside and phenolic compounds, were consistently found as discriminative for the untreated olive trees in comparison with the Dentamet&reg, treated trees. Quinic acid, a precursor of lignin, was confirmed as a disease biomarker for the olive trees infected by X. fastidiosa subsp. pauca. When treated with Dentamet&reg, the two cultivars showed a distinct response. A consistent increase in malic acid was observed for the Ogliarola salentina trees, whereas in the Cellina di Nard&ograve, trees the treatments attenuate the metabolic response to the infection. To note that in Cellina di Nard&ograve, trees at the first sampling, an increase in &gamma, aminobutyric acid (GABA) was observed. This study highlights how the infection incited by X. fastidiosa subsp. pauca strongly modifies the overall metabolism of olive trees, and how a zinc-copper-citric acid biocomplex can induce an early re-programming of the metabolic pathways in the infected trees.

The single crystal X-ray structure of the pentacoordinate complex [PtBr2(η2-CH2double bond; length as m-dashCH2)(Me2phen)], Me2phen = 2,9-dimethyl-1,10-phenanthroline, is here reported for the first time. Comparison of the complete series of [PtX2(η2-CH2double bond; length as m-dashCH2)(Me2phen)] (X = Cl, Br, I) X-ray structures shows a very low variability of the bond lengths and angles, in the trigonal equatorial plane (where η2-olefin and Me2phen are bound), on varying the coordinated axial halogens. In first approximation, this suggests describing as independents and not interacting the two subsystems constituted by the metal bonds with axial (X–Pt–X) and equatorial ligands (Me2phen–Pt–η2-ethene). This means that the electric charge donated to the metal, by the axial ligands, cannot substantially modify the bonds of the metal with the ligands in the trigonal equatorial plane. The 1H, 13C, 15N and 195Pt NMR chemical shifts variations, studied as a function of the ionic radius of the axial halides are here discussed. The NMR data strongly suggest the existence of electric pseudo-ring currents circulating around the Pt–X axes and modulated by the ionic radius of the coordinated halides.

A series of pentacoordinate d8 PtII complexes, of the type [PtCl2(η2-CH2=CH2)(Men∩NN)], where Men∩NN = bis-nitrogen ligand, with a variable number of Me groups (i.e., 2,2′-bipyridyl; 1,10-phenanthroline; 6-methyl-2,2′-bipyridyl; 2,9-dimethyl-1,10-phenanthroline; 2,9-dimethyl-1,10-phenanthroline; N,N′-trimethyl-ethylenediamine; N,N,N′,N′-tetramethyl-ethylenediamine; N,N,N′,N′-tetramethyl-1,2-diaminocyclohexane) was studied. The compounds are characterized by variable steric hindrance, due to the variable number (n) of Me substituents, on or ortho to the N-donors of aliphatic diamines or aromatic diimines, respectively. This approach was developed to investigate the interaction of substituents with the metal coordination sphere. With this aim, we analyzed the NMR properties of the considered complexes, with respect to modulation of the metal electron density (195Pt NMR signal frequency) by alkyl groups close to the N-donors. 1H and 13C NMR analysis of the η2-olefin signals has revealed, for each kind of bis-nitrogen ligand, a positive or negative chemical shift variation that is proportional to the number of Me groups geminal or vicinal to the N-donors. Interestingly, the 1JPt,C values increase by approximately 45 Hz for each additional Me on the series of diamine or diimine bis-nitrogen ligands. A rationale for the stability changes observed in such pentacoordinate complexes is suggested, based on the NMR spectroscopic data analysis.

Cisplatin, cis-diamminedichloroplatinum(II), is an effective antitumor drug known to exert its action by binding to the N7 of purine bases in nuclear DNA, consequently impairing replication, transcription and repair processes. Recently we have demonstrated, by the model complex [Pt(dien)(N7-5'-dGTP)] (dien = diethylenetriamine) the possible existence of an alternative mechanism for cisplatin antitumor activity, in which the metal is bonded to free purine bases, before incorporation in the newly synthesized DNA. This evidence suggests a new possible approach for the development of N7-platinated purines, as new antitumor drugs, for the treatment of cancer diseases. However, it is known that the efficacy of a new drug it is strictly related upon the transport across the cells plasma membrane barrier which strongly condition the drug pharmacokinetic and the targeted cells bioavailability. In particular in this study, we tested the possibility that N7-platinated purines could be transported into cells. At this scope we synthesized model Pt(II) coordination compounds, of the type [Pt(dien)(N7-G)] (G = Guanine derivative) and analysed the uptake of each single platinated nucleoside/nucleotide into Hela cells, by measuring Pt accumulation, by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). The quantitative analysis of Pt has provided a strong evidence for transport of the N7 platinated nucleoside [Pt(dien)(N7-dGuo)] into Hela cells (see figure). Moreover, the observed intracellular Pt accumulation was strictly sodiumdependent suggesting a carrier-mediated uptake for the [Pt(dien)(N7-dGuo)]. Overall, these results suggest that N7-platination does not compromise cellular uptake of nucleosides and may have important implications in the development of new potential nucleoside analogues in cancer treatment.

Cisplatin, cis-diamminedichloroplatinum(II), is one of the most widely used antitumor drugs in cancer chemotherapy, as a critical component against a broad range of malignancies. Platinum anticancer drugs are generally known to target DNA, where they can bind to the N7 of purine bases. Cisplatin, as other bifunctional agents, is able to bind to adjacent purines, resulting in crosslink lesions believed to be responsible for the observed antitumor activity. N7-metalated purines, in some cases, seem to be characterized by a relevant antitumor activity. This has led us to hypothesize a cisplatin parallel mechanism of action: based on free platinated purines formation, directly in tissues, after drug administration. In order to evaluate this possible mechanism, as a key path to develop new drugs, we performed a series of experiments focused on platinated nucleobases cell and mitochondrial uptake and processing. For the first time cell uptake and mobility mechanisms, related to plasmatic cell and/or mitochondrial membrane crossing, has been studied. Moreover the possible insertion of metalated nucleobases into nuclear and/or mitochondrial new synthesized DNA/RNA, operated by DNA/RNA polymerases was evaluated. At this scope model metalated nucleosides/nucleotides, with nitrogen carrier ligands, have been synthesized, isolated and characterized. The possible development of new drugs based on this new rational drug design will be discussed.

Synthesis and characterization of the new pentacoordinate [PtCl 2 (η 2 -C 2 H 4 )(Mebpy)] and square planar [PtCl 2 (Mebpy)] complexes both containing the asymmetrically hindered nitrogen donor chelate Mebpy = 6-Methyl-2,2′-bipyridil are reported. By nucleophilic addition of MeO − to the coordinated ethene of [PtCl 2 (η 2 -C 2 H 4 )(Mebpy)], syn -[PtCl(η 1 -CH 2 CH 2 OMe)(Mebpy)] and anti -[PtCl(η 1 -CH 2 CH 2 OMe)(Mebpy)] complexes have been obtained as the kinetic and thermodynamic product, respectively. The mechanism for selective formation of both stereoisomers is also reported. This synthetic route opens new perspectives for the stereospecific synthesis of syn - and anti -square planar organometallic complexes.