Desenvolvimento de quelantes macroc��clicos para complexa����o de i��es met��licos de transi����o e lantan��deos com interesse em aplica����es m��dicas

A investiga����o de novos compostos macroc��clicos tetraaza tem sido intensificada ao longo das ��ltimas d��cadas, uma vez que estes est��o entre os melhores ligandos para complexa����o de i��es met��licos. Desta forma, tanto a s��ntese destes compostos, como as propriedades dos seus complexos com i��es met��licos provocaram o seu crescente interesse em variad��ssimas ��reas, das quais s��o exemplo a medicina, a biologia, a cat��lise, e a qu��mica anal��tica. Procuram-se ligandos espec��ficos para determinados i��es met��licos, mas que ao mesmo tempo se preparem por processos de s��ntese simples e eficazes que permitam uma maior utiliza����o ao n��vel industrial. Com este trabalho procurou-se dar continuidade aos estudos desenvolvidos nesta ��rea desde 1980 pelo grupo de investiga����o onde me encontro inserida e assim obter ligandos novos com vista �� forma����o de complexos com i��es met��licos est��veis e aplic��veis ao n��vel da medicina e da farmacologia, com destaque para as aplica����es relacionadas com o diagn��stico e a terapia de tumores. Assim, foram sintetizados quatro novos ligandos derivados de macrociclos tetraaza com 12 ��tomos no anel, dois deles com ponte cruzada curta tipo etano. A s��ntes baseou-se na funcionaliza����o destes macrociclos com bra��os pendentes metilnitrofenol ou metilfenol, originando assim os seguintes compostos: cb-do2nph, do2nph, cb-do2ph e do do2ph. Todos os compostos sintetizados foram caracterizados por diversas t��cnicas anal��ticas. Os compostos cb-do2nph e do2nph mostraram reduzida solubilidade em ��gua e em metanol:��gua, pelo que n��o foi poss��vel a determina����o de constantes por potenciometria. Preparam-se alguns complexos que se estudaram em solu����o por espectroscopia electr��nica e no estado s��lido por difrac����o de raios X. O composto do2ph s�� se obteve em pequena quantidade sem revelar a pureza necess��ria e, a sua s��ntese ainda ter�� de ser optimizada. Finalmente, o composto cb-do2ph foi sintetizado em quantidade suficiente para se poderem determinar as suas constantes de protona����o e as constantes de estabilidade dos seus complexos com Cu 2+ e Zn 2+, pelo m��todo potenciom��trico, em solu����o aquosa, �� temperatura de 298,2 K e �� for��a i��nica 0,10M em N(CH 3)4NO3. Determinaram-se ainda por potenciometria as constantes de protona����o bem como as constantes de estabilidade dos complexos com Mg2+, Cu2+, Zn 2+, Fe3+, Ga3+, In3+, Gd3+, do composto cbdbf-do2a, igualmente em solu����o aquosa, �� temperatura de 298,2K e �� for��a i��nica 0,10M em N(CH 3)4NO3. Para ambos os compostos, cb-do2ph e cbdbf-do2a, recorreu-se a titula����es por espectroscopia de RMN de 1H para a determina����o da primeira constante, por esta ter um valor muito elevado (log K1 ��� 14), ou seja, estes compostos comportaram-se como ���esponjas de prot��o���. Para obter informa����es estruturais dos complexos de alguns ligandos com cobre (II) realizaram-se estudos por espectroscopia de absor����o de UV-Vis e de RPE, tendo-se ainda obtido cristais de tamanho suficiente para a determina����o de estruturas por difrac����o de raios X de alguns compostos e complexos de cobre (II) e Zinco(II).

R��le de l'IRM cardiaque dans le d��pistage des cardiomyopathies de l'adulte [Cardiomyopathy and cardiac magnetic resonance].

Cardiovascular magnetic resonance (CMR) is a rapidly emerging non-invasive imaging technique free of X-Ray and offers higher spatial resolution than alternative forms of cardiac imaging for the assessment of left ventricular (LV) anatomy, function, and viability due to the unique capability of myocardial tissue characterization after gadolinium-chelates contrast administration. This imaging technique has clinical utility over a broad spectrum of heart diseases: ranging from ischaemic to non ischaemic aetiologies. Cardiomyopathies (CMP) are a heterogeneous group of diseases of the myocardium associated with architectural abnormalities and mechanical dysfunction. CMR can help excluding coronary artery disease and can provide positive diagnostic features for several CMP resulted in better diagnosis and management, Leading to improvements in mortality.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Les anticorps monoclonaux radiomarqu��s en tant que missiles anti-tumeurs, leurs succ��s diagnostiques, leur potentiel th��rapeutique

While it is now well accepted that radiolabeled antibodies can be useful for tumour detection by immunoscintigraphy, the use of larger doses of more aggressive radioisotopes coupled to antibodies for radioimmunotherapy is still in its infancy. At the experimental level, our group has shown that the intravenous injection of large doses of 131I labeled F(ab')2 fragments from monoclonal anti-carcinoembryonic antigen (CEA) antibodies can eradicate well established human colon carcinoma xenografts in nude mice. At the clinical level, in a dosimetry study performed at the Institut Gustave Roussy, the same anti-CEA monoclonal antibodies and fragments, labeled with subtherapeutic doses of 131I, were injected in patients with liver metastases from colorectal carcinomas. Direct measurement of radioactivity in surgically resected liver metastases and normal liver confirmed the specificity of tumour localization of the antibodies, but also showed that the calculated radiation doses which could be delivered by injections of 200 to 300 mCi of 131I labeled antibodies or fragments, remained fairly low, in the range of 1,500 to 3,000 rads. This is obviously insufficient for a single modality treatment. An alternative approach is to inject radiolabeled antibodies intra peritoneally to treat peritoneal carcinomatosis. Several clinical studies using this strategy are presently under evaluation and suggest that positive results can be obtained when the tumour diameters are very small. In systemic radioimmunotherapy, positive results have been obtained in more radiosensitive types of malignancies such as B cell lymphomas by intravenous injection of antibodies directed against B cell differentiation markers or against idiotypic antigens from each lymphoma, and labeled with 131I or 90Y. The major directions of research for improvement of radioimmunotherapy include the design of genetically engineered new forms of humanized antibodies, the synthesis of original chelates for coupling new radioisotopes to antibodies and the development of two step strategies for immunolocalization of radioisotopes.

Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.

In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.

Development of a hepatocyte hollow fiber bioreactor for the study of contrast agents by magnetic resonance imaging

Thanks to the continuous progress made in recent years, medical imaging has become an important tool in the diagnosis of various pathologies. In particular, magnetic resonance imaging (MRI) permits to obtain images with a remarkably high resolution without the use of ionizing radiation and is consequently widely applied for a broad range of conditions in all parts of the body. Contrast agents are used in MRI to improve tissue discrimination. Different categories of contrast agents are clinically available, the most widely used being gadolinium chelates. One can distinguish between extracellular gadolinium chelates such as Gd-DTPA, and hepatobiliary gadolinium chelates such as Gd-BOPTA. The latter are able to enter hepatocytes from where they are partially excreted into the bile to an extent dependent on the contrast agent and animal species. Due to this property, hepatobiliary contrast agents are particularly interesting for the MRI of the liver. Actually, a change in signal intensity can result from a change in transport functions signaling the presence of impaired hepatocytes, e.g. in the case of focal (like cancer) or diffuse (like cirrhosis) liver diseases. Although the excretion mechanism into the bile is well known, the uptake mechanisms of hepatobiliary contrast agents into hepatocytes are still not completely understood and several hypotheses have been proposed. As a good knowledge of these transport mechanisms is required to allow an efficient diagnosis by MRI of the functional state of the liver, more fundamental research is needed and an efficient MRI compatible in vitro model would be an asset. So far, most data concerning these transport mechanisms have been obtained by MRI with in vivo models or by a method of detection other than MRI with cellular or sub-cellular models. Actually, no in vitro model is currently available for the study and quantification of contrast agents by MRI notably because high cellular densities are needed to allow detection, and no metallic devices can be used inside the magnet room, which is incompatible with most tissue or cell cultures that require controlled temperature and oxygenation. The aim of this thesis is thus to develop an MRI compatible in vitro cellular model to study the transport of hepatobiliary contrast agents, in particular Gd-BOPTA, into hepatocytes directly by MRI. A better understanding of this transport and especially of its modification in case of hepatic disorder could permit in a second step to extrapolate this knowledge to humans and to use the kinetics of hepatobiliary contrast agents as a tool for the diagnosis of hepatic diseases.

Termoqu��mica na Unicamp: depoimento sobre uma jornada de 25 anos

This article describes the progress of a group of investigation on thermochemistry, which started in 1972. A homemade calorimeter was employed to provide quantitative support to the information on interative effect between lanthanide cations and halides or pseudohalides, in non-aqueous solvents, previously derived from conductometric titrations. However, the features of this instrument were not able to detect the thermal effects. Therefore, the great input to the group came from the acquisition of an LKB commercial apparatus, by the University in 1975. Considering the historical development of the coordination chemistry in Brazil, which was previously dedicated to strutural features of adducts, without focusing the energetic envolved in any coordinationcompound. Since starting the thermochemistry study, numerous masters and doctoral thesis covering more than a hundred adducts and a reasonable number of chelates, were presented systematizing data in order to understand the behavior of this kind of coordination compounds (C. Airoldi and A. P. Chagas, Coord. Chem. Rev. 1992, 119, 29). This knowledge enabled an extension of the study to include some heterogeneous systems formed by natural or synthetic materials like immobilized silica gel, lamellar phosphate, phosphonate or sulphate compounds, clays, polysaccharides, chrysotile, soils, etc. Many students are now engaged as staff members in Universities, Research Instituitions or other private institutions, developing many activities. Due to a multiplying effect on the formation of researchers, the group is now reaching the fourth generation.

Sobre a aplicabilidade da termogravimetria para a estimativa de par��metros termoqu��micos de compostos de coordena����o: os elementos lantan��dicos

By using thermochemical data reported for a series of chelates of the type [Ln(thd)3], thd = 2, 2, 6, 6 �� tetramethyl- 3,5-heptanedione and Ln = La, Pr, Nd, Sm, Gd, Tb, Ho, Er, Tm and Yb, empirical correlations were found involving thermochemical parameters (e.g. dissociation enthalpy) and the thermodynamic temperatures of the beginning of thermal degradation of the chelates, t i. It is shown that t i values are of capital importance in the study of this all class of coordination compounds. Among others, the empirical equation is obtained: r3+ = (-0,013.Z + 1,36)/0,005, that relates the lanthanide cation radius (pm) with the atomic number of the element. The remarkable fact is that this equation is achieved by using thermogravimetric and calorimetric parameters. Is also shown that t i values are related with the P(M) function values, which are very close related with the energy difference, deltaE, between the lowest electronic energy level of the f n s��d�� configuration and the lowest energy level of the f n+1s�� configuration in the neutral gaseous atoms.

Investiga����o de Er3+ nos dois s��tios cristalogr��ficos de Gd2SiO5 atrav��s da fotoluminesc��ncia resolvida no tempo

Gadolinium oxyortho-silicate, Gd2SiO5, presents a monoclinic structure with two crystallographic sites in which Gd3+ ions are equally distributed with coordination numbers CN, 7 and 9, respectively. By doping this host with Er3+ it is possible to distinguish and attribute the two sites by means of lifetime determination of the 4S3/2 state, (in this case, Er3+ substitutes Gd3+ ions). Samples doped with 0.1 and 5.0% molar Er3+ were prepared by solid state reaction and characterized by X-ray Diffractometry, Vibrational and Electronic Absorption Spectroscopies, and Time Resolved Photoluminescence. Based on the experimental results, it is possible to verify that, for the 5,0% doped sample, the lifetime value of the 4S3/2 state of the erbium ion inserted in site 1, (CN = 9), is 2.7 �� 0.1 mus, and for the one inserted in site 2, (CN = 7), it is 1.5 �� 0.1 mus.

S��ntese, caracteriza����o e propriedades espectrosc��picas de criptatos de lantan��deo do tipo [Ln��(bipy)2py(CO2Et) 2(3+)]

This work reports on the synthesis, characterization (infrared and hidrogen nmr spectra) and photophysical properties (luminescence spectra and emission quantum yield) of the lanthanide cryptates [Ln<FONT FACE=Symbol>��</FONT>(bipy)2py(CO2Et) 2]3+ with Ln = Eu3+, Tb3+ or Gd3+, which can be applied as efficient Light-Conversion-Molecular-Devices. From emission spectra of [Eu<FONT FACE=Symbol>��</FONT>(bipy)2py(CO2Et) 2]3+ it was possible to assign C3 symmetry to the metal ion. The spectroscopic studies show a higher emission quantum yield (q=25%) for [Tb<FONT FACE=Symbol>��</FONT>(bipy)2py(CO2Et) 2]3+ in aqueous solution, whereas the europium cryptate presents q=14%. This is justified by a more efficient energy transfer between triplet and emission levels of terbium (T->5D4).

Tiossemicarbazonas: m��todos de obten����o, aplica����es sint��ticas e import��ncia biol��gica

Thiosemicarbazones are a class of compounds known by their chemical and biological properties, such as antitumor, antibacterial, antiviral and antiprotozoal activity. Their ability to form chelates with metals has great importance in their biological activities. Their synthesis is very simple, versatile and clean, usually giving high yields. They are largely employed as intermediates, in the synthesis of others compounds. This article is a survey of some of these characteristics showing their great importance to organic and medicinal chemistry.

Estudos fotoluminescentes em compostos de ��ons terras raras coordenados por um derivado carbaz��lico

[RE(czb)3(H2O)2] complexes (where RE = Eu3+, Tb3+, Gd3+; and czb = 4-(9H-carbazol-9-yl)benzoato) have been synthesized and characterized. The Gd3+ complex was used to determine the triplet state energy of the czb ligand. Photoluminescence measurements of the complexes have been carried out under UV excitation. The Tb3+ complex exhibited a strong green luminescence indicating an efficient antenna effect, whereas the Eu3+ complex showed low red luminescence and the Gd3+ complex a blue-green luminescence from the ligand. The luminescence lifetimes and quantum yields have also been measured for the evaluation of the spectroscopic behavior of the complexes.

Effect of metal ions on the in vitro availability of enoxacin, its in vivo implications, kinetic and antibacterial studies

The present paper describes the effect of metals ions on the in vitro availability of enoxacin (a second generation quinolone antibiotic) owing to drug-metal interaction. These interaction studies were performed at 37 ��C in different pH environments simulating human body compartments and were studied by UV spectroscopic technique. In order to determine the probability of these reactions different kinetic parameters (dissolution constants (K) and free energy change (&#916;G)) for these reactions were also calculated. It is proposed that the structure of enoxacin contains various electron donating sites which facilitate its binding with metallic cations forming chelates. Hence taking food products, nutritional supplements or multivitamins containing multivalent cations at the same time as enoxacin, could reduce the absorption of the drug into the circulation and thus would decrease the effectiveness of the drug. In addition, the MIC of enoxacin for various microorganisms before and after interaction with metal ions was calculated which in most cases was increased which possibly could impair the clinical efficacy of the drug.

Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.