Design, synthesis and characterization of versatile copper complexes with anticancer and catalytic activity.

Although copper has a long history of medical application, copper coordination compounds have been investigated as potential anticancer agents only in the last few decades, particularly after the discovery of cisplatin, the most widely used antitumor metallodrug. Copper, as an essential cofactor in a number of enzymes and physiological processes, may be less toxic than non-essential metals, such as platinum. Up to now, a great variety of copper complexes have been tested as cytotoxic agents and found to be endowed with an antitumor activity in several in vitro tests and few in vivo experiments. Based on these assumptions, in my PhD research work copper was selected for the synthesis of potential metal-based anticancer drugs, that could be suitable alternative to platinum-based drugs that are hampered by marked side effects and chemoresistance. An important aim of this work, in the inorganic chemistry research field, was to synthesize new species able to coordinate metals useful to obtain Cu(I) and Cu(II) complexes with potential anticancer activity. Copper complexes were synthesized employing bis(pyrazolyl)acetic acids (belonging to the family of the heteroscorpionate) as ligands. This class of ligands was selected due to their stability, flexibility and ease to be functionalized and derivatized. In fact, they were used as they are (with the carboxylic acid group) or esterified with aliphatic alcohols (branched and not) or bioconjugated with several biologically active compounds. For this purpose, the ligands were conjugated with metronidazole (an antibiotic agent investigated for hypoxia-selective cytotoxicity), NMDA-ANT (an antagonist for the N-methyl-D-aspartate receptor endowed with micromolar cytotoxic activity on a panel of solid tumor cell lines) and lonidamine (an antineoplastic drug) in order to obtain Cu(I) and Cu(II) complexes potentially able to exert an anticancer activity through synergistic mechanisms of action. Additionally, to stabilize copper in the +1 oxidation state (avoiding its oxidation) and to modulate the solubility profile of the related copper(I) complexes, hydrophilic or lipophilic phosphanes, such as 1,3,5-triaza-7- phosphaadamantane (PTA) and triphenylphosphine (PPh3) respectively, were used as coligands. My main work was the design, synthesis and characterization of the ligands and related copper complexes, both in solid state (FT-IR, elemental analysis and melting point) and in solution (1H-, 11B-, 13C-, 31P-NMR and ESI-MS) to confirm their structure, stoichiometry and purity. In parallel, X-ray diffraction studies were conducted on single crystals of ligands and complexes,[b,e,g,s] showing in some cases new and unexpected dimeric structures.[g] In addition, the structural characterization and the study of the local coordination environment of several complexes were exploited by X-ray photoelectron spectroscopy (XPS) and X-ray absorption fine structure (XAFS) spectroscopy (in the near edge and in the extended regions).[d,f,h,l,m,p,t] In all the cases, the structural investigations confirmed the hypothesized geometry of the metal center and the coordinative fashion of the ligands. Several new complexes and the corresponding uncoordinated ligands were evaluated for their ability to promote cell death against a panel of human cancer cell lines, cisplatin resistant tumor cell lines and spheroids, evaluating also cellular uptake, mechanism of action and morphological modifications induced by the complexes once inside the tumor cells. [a,c,h,i,j,k,n,o,q,r,t,u] In detail, for a series of selected precursors, ligands and Cu(I) and Cu(II) complexes the biological activity was evaluated by means of MTT test, cellular uptake, reactive oxygen species (ROS) production, comet assay and/or transmission electron microscope (TEM) analyses. Summarizing, even if with slight differences, it was possible to state that the complexes were, in general, more active that cisplatin (the drug used as reference compound), both in 2D and 3D cell cultures, showing their effect in the low micromolar concentration, or even lower. On the contrary, the related free ligands and precursors did not show relevant cytotoxic activity. Interestingly, the fact that the complexes proved to be significantly more active than cisplatin, even against three-dimensional spheroids of selected cancer cells, increased the relevance of the in vitro results. In fact, 3D spheroids of cancer cells more closely mimic the heterogeneity and complexity of in vivo tumors, being consequently more predictive for in vivo results than conventional 2D cell cultures. The most frequent mechanism of action for the tested complexes was the paraptotic one, a type of programmed cell death different from the classical apoptosis induced by drugs such as cisplatin. This alternative programmed cell death leads to the overcoming of the inherited or acquired cisplatin or multi-drug resistance. Regarding the ligands esterified with aliphatic alcohols and the related copper complexes the biological studies are still in progress but, according to the preliminary data, these complexes seem to be very promising. Another field of interest of my research work was the investigation of the catalytic activity of new copper(II) compounds in the Kharasch-Sosnovsky reaction,[d,g,n] that is a useful reaction for the synthesis of protected allylic alcohols, via radical oxidation of olefins leaving the double bond in its original position. In particular, the catalytic activity of the copper(II) complexes, containing the isopropyl or hexyl ester chain, was evaluated in the Kharasch-Sosnovsky reaction. The original reaction conditions were optimized changing several parameters and very high yields were obtained employing the compounds containing bromide as counterion. The major limitations of this reaction, such as long reaction times, employ of benzene as solvent and high waste of olefins, were overcome replacing the original cheap but no-so-effective CuBr with the new synthesized Cu(II) complexes. In order to validate the generality of the method, different olefin substrates were tested (obtaining excellent yields) and other promising Cu(II) complexes are under evaluation as catalysts

Ambición política y producción legislativa en un sistema multinivel: un estudio del senado argentino de 2001 a 2007

Este trabajo, que se enmarca dentro de la literatura sobre ambiciones políticas surgida luego de la publicación de Congress: The electoral connection, parte del análisis y la sistematización de las motivaciones humanas y busca entender las consecuencias del comportamiento legislativo basado en esa motivación. Los legisladores que no buscan la reelección constante no son políticos sin ambición. Por el contrario, muchos de ellos aspiran a otros cargos políticos que concentran incluso una mayor cantidad de recursos, visibilidad y poder

Rigideces salariales: Estudio del salario de los trabajadores formales e informales argentinos durante los años 1996 - 2002

La presenta investigación utiliza datos de la Encuesta Permanente de Hogares (EPH) para analizar descriptivamente la rigidez salarial para los asalariados argentinos para el periodo 1996-2002, diferenciando entre trabajadores formales e informales. Se concluye que ambos son rígidos a la baja, pero que los formales presentan mayor grado de rigidez que los informales. Además, se determina que la recesión de 1999-2002 afectó más a los trabajadores informales que tuvieron mayores caídas promedio en sus salarios nominales y una recuperación más lenta post devaluación del peso en 2002

Molecular epidemiology and pathogenic potential of underdiagnosed human papillomavirus types

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) tests are crucial diagnostic tools for the prevention of neoplastic lesions of the uterine cervix. However most commercial methods are designed to detect high-risk (HR) HPV types and a limited selection of low-risk ones, thus missing a fair number of intermediate/low-risk types. As a result, many HPV infections remain undiagnosed, generating distrust in virological diagnosis among gynaecologists, who continue to rely preferentially on cytological and colposcopic findings.</p> <p>Results</p> <p>In this study, we tested 6,335 consecutive clinical samples, most of them from Italian patients with cytological abnormalities. The samples, collected in 2000–2007, were analyzed using PCR amplification of a 173–206 bp (depending on HPV type) conserved region in the L1 open reading frame, restriction endonuclease analysis and, where required, sequence analysis for type determination. Analysis of a smaller male sample and long term follow-up of a few female subjects was also performed. A total of 2,161 samples tested positive for HPV DNA (32.1%); 21.3% of them were mixed infections. Overall, 59 known and 2 unknown HPV types were detected. Their relative prevalence was calculated; notably, types not clearly identifiable using the most common commercial method accounted for 36% of infections. Clinical findings associated with the underdiagnosed types ranged from H-SIL to low-grade abnormalities, although none of these infections resulted in invasive cancer.</p> <p>Conclusion</p> <p>Given the high prevalence of some underdiagnosed HPV types in the population (principally HPV53, HPV66, HPV84, and HPV87) and their frequent association with cytological abnormalities, techniques capable of detecting and typing them would prove extremely useful.</p

Dynamic features of the selective pressure on the human immunodeficiency virus type 1 (HIV-1) gp120 CD4-binding site in a group of long term non progressor (LTNP) subjects.

Abstract The characteristics of intra-host human immunodeficiency virus type 1 (HIV-1) env evolution were evaluated in untreated HIV-1-infected subjects with different patterns of disease progression, including 2 normal progressor [NP], and 5 Long term non-progressor [LTNP] patients. High-resolution phylogenetic analysis of the C2-C5 env gene sequences of the replicating HIV-1 was performed in sequential samples collected over a 3–5 year period; overall, 301 HIV-1 genomic RNA sequences were amplified from plasma samples, cloned, sequenced and analyzed. Firstly, the evolutionary rate was calculated separately in the 3 codon positions. In all LTNPs, the 3rd codon mutation rate was equal or even lower than that observed at the 1st and 2nd positions (p = 0.016), thus suggesting strong ongoing positive selection. A Bayesian approach and a maximum-likelihood (ML) method were used to estimate the rate of virus evolution within each subject and to detect positively selected sites respectively. A great number of N-linked glycosylation sites under positive selection were identified in both NP and LTNP subjects. Viral sequences from 4 of the 5 LTNPs showed extensive positive selective pressure on the CD4-binding site (CD4bs). In addition, localized pressure in the area of the IgG-b12 epitope, a broad neutralizing human monoclonal antibody targeting the CD4bs, was documented in one LTNP subject, using a graphic colour grade 3-dimensional visualization. Overall, the data shown here documenting high selective pressure on the HIV-1 CD4bs of a group of LTNP subjects offers important insights for planning novel strategies for the immune control of HIV-1 infection.</p

Exploring the Antitumor Potential of Copper Complexes Based on Ester Derivatives of Bis(pyrazol-1-yl)acetate Ligands

Bis(pyrazol-1-yl)acetic acid (HC(pz)(2)COOH) and bis(3,5-dimethyl-pyrazol-1-yl)acetic acid (HC(pz(Me2))(2)COOH) were converted into the methyl ester derivatives 1 (L-OMe) and 2 (L-2OMe), respectively, and were used for the preparation of Cu(I) and Cu(II) complexes 3-10. The copper(II) complexes were prepared by the reaction of CuCl2 center dot 2H(2)O or CuBr2 with ligands 1 and 2 in methanol solution. The copper(I) complexes were prepared by the reaction of Cu[(CH3CN)(4)]PF6 and 1,3,5-triaza-7-phosphaadamantane (PTA) or triphenylphosphine with L-OMe and L-2OMe in acetonitrile solution. Synchrotron radiation-based complementary techniques (XPS, NEXAFS, and XAS) were used to investigate the electronic and molecular structures of the complexes and the local structure around copper ions in selected Cu(I) and Cu(II) coordination compounds. All Cu(I) and Cu(II) complexes showed a significant in vitro antitumor activity, proving to be more effective than the reference drug cisplatin in a panel of human cancer cell lines, and were able to overcome cisplatin resistance. Noticeably, Cu complexes appeared much more effective than cisplatin in 3D spheroid cultures. Mechanistic studies revealed that the antitumor potential did not correlate with cellular accumulation but was consistent with intracellular targeting of PDI, ER stress, and paraptotic cell death induction

Human case of autochthonous West Nile virus lineage 2 infection in Italy, September 2011.

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Expression of high- and low-affinity epidermal growth factor receptors in human hepatoma cell lines

AbstractData are presented from a comparative research on expression of epidermal growth factor (EGF) receptors and response to EGF of six independently established cell lines derived from human hepatoma. These lines differ in terms of the degree of differentiation, presence of hepatitis B virus (HBV) DNA copies in integrated form and expression of HBV genes. Our results indicate differential expression of membrane EGF receptors and differential response to EGF under serum- and hormone-free culture conditions. Furthermore, a significant difference in affinity could be detected between EGF receptors of the two highly dedifferentiated cell lines (HA22T/VGH and Li7A) whose replication is inhibited by EGF concentrations capable of stimulating more differentiated phenotypes

Local epidemics gone viral: Evolution and diffusion of the Italian HIV-1 recombinant form CRF60_BC

The molecular epidemiology of HIV-1 in Italy is becoming increasingly complex, mainly due to the spread of non-B subtypes and the emergence of new recombinant forms. We previously characterized the outbreak of the first Italian circulating recombinant form (CRF60_BC), occurring among young MSM living in Apulia between the years 2009 and 2011. Here we show a 5-year follow-up surveillance to trace the evolution of CRF60_BC and to investigate its further spread in Italy. We collected additional sequences and clinical data from patients harboring CRF60_BC, enrolled at the Infectious Diseases Clinic of the University of Bari. In addition to the 24 previously identified sequences, we retrieved 27 CRF60_BC sequences from patients residing in Apulia, whose epidemiological and clinical features did not differ from those of the initial outbreak, i.e., the Italian origin, young age at HIV diagnosis (median: 24 years; range: 18-37), MSM risk factor (23/25, 92%) and recent infection (from 2008 to 2017). Sequence analysis revealed a growing overall nucleotide diversity, with few nucleotide changes that were fixed over time. Twenty-seven additional sequences were detected across Italy, spanning multiple distant regions. Using a BLAST search, we also identified a CRF60_BC sequence isolated in United Kingdom in 2013. Three patients harbored a unique second generation recombinant form in which CRF60_BC was one of the parental strains. Our data show that CRF60_BC gained epidemic importance, spreading among young MSM in multiple Italian regions and increasing its population size in few years, as the number of sequences identified so far has triplicated since our first report. The observed further divergence of CRF60_BC is likely due to evolutionary bottlenecks and host adaptation during transmission chains. Of note, we detected three second-generation recombinants, further supporting a widespread circulation of CRF60_BC and the increasing complexity of the HIV-1 epidemic in Italy