Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel cofactors and revealing new features of old enzymes

Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade de Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Dinuclear Mn (II,II) complexes: magnetic properties and microwave assisted oxidation of alcohols

A series of six new mixed-ligand dinuclear Mn(II, II) complexes of three different hydrazone Schiff bases (H3L1, H3L2 and H3L3), derived from condensation of the aromatic acid hydrazides benzohydrazide, 2-aminobenzohydrazide or 2-hydroxybenzohydrazide, with 2,3-dihydroxy benzaldehyde, respectively, is reported. Reactions of Mn(NO3)(2) center dot 4H(2)O with the H3L1-3 compounds, in the presence of pyridine (1 : 1 : 1 mole ratio), in methanol at room temperature, yield [Mn(H2L1)(py)(H2O)](2)(NO3)(2) center dot 2H(2)O (1 center dot 2H(2)O), [Mn(H2L2)(py)(CH3OH)](2)(NO3)(2) center dot 4H(2)O (2 center dot 4H(2)O) and [Mn(H2L3)(py)(H2O)](2)(NO3)(2) (3) respectively, whereas the use of excess pyridine yields complexes with two axially coordinated pyridine molecules at each Mn(II) centre, viz. [Mn(H2L1)(py)(2)] 2(NO3)(2) center dot H2O (4 center dot H2O), [Mn(H2L2)(py) H-O (6 center dot 2CH(3)OH), respectively. In all the complexes, the (H2L1-3)-ligand coordinates in the keto form. Complexes 1 center dot 2H(2)O, 2 center dot 4H(2)O, 4 center dot H2O, 5 center dot 2H(2)O and 6 center dot 2CH(3)OH are characterized by single crystal X-ray diffraction analysis. The complexes 1, 2 and 6, having different coordination environments, have been selected for variable temperature magnetic susceptibility measurements to examine the nature of magnetic interaction between magnetically coupled Mn(II) centres and also for exploration of the catalytic activity towards microwave assisted oxidation of alcohols. A yield of 81% (acetophenone) is obtained using a maximum of 0.4% molar ratio of catalyst relative to the substrate in the presence of TEMPO and in aqueous basic solution, under mild conditions.

New RuII(arene) complexes with halogen-substituted bis- and tris(pyrazol-1-YL)borate ligands

[RuCl(arene)(-Cl)](2) dimers were treated in a 1:2 molar ratio with sodium or thallium salts of bis- and tris(pyrazolyl)borate ligands [Na(BpBr3)], [Tl(TpBr3)], and [Tl(Tp(iPr,4Br))]. Mononuclear neutral complexes [RuCl(arene)((2)-BpBr3)] (1: arene=p-cymene (cym); 2: arene=hexamethylbenzene (hmb); 3: arene=benzene (bz)), [RuCl(arene)((2)-TpBr3)] (4: arene=cym; 6: arene=bz), and [RuCl(arene)((2)-Tp(iPr,4Br))] (7: arene=cym, 8: arene=hmb, 9: arene=bz) have been always obtained with the exception of the ionic [Ru-2(hmb)(2)(-Cl)(3)][TpBr3] (5), which formed independently of the ratio of reactants and reaction conditions employed. The ionic [Ru(CH3OH)(cym)((2)-BpBr3)][X] (10: X=PF6, 12: X=O3SCF3) and the neutral [Ru(O2CCF3)(cym)((2)-BpBr3)] (11) have been obtained by a metathesis reaction with corresponding silver salts. All complexes 1-12 have been characterized by analytical and spectroscopic data (IR, ESI-MS, H-1 and (CNMR)-C-13 spectroscopy). The structures of the thallium and calcium derivatives of ligand TpBr3, [Tl(TpBr3)] and [Ca(dmso)(6)][TpBr3](2)2DMSO, of the complexes 1, 4, 5, 6, 11, and of the decomposition product [RuCl(cym)(Hpz(iPr,4Br))(2)][Cl] (7) have been confirmed by using single-crystal X-ray diffraction. Electrochemical studies showed that 1-9 and 11 undergo a single-electron (RuRuIII)-Ru-II oxidation at a potential, measured by cyclic voltammetry, which allows comparison of the electron-donor characters of the bis- and tris(pyrazol-1-yl)borate and arene ligands, and to estimate, for the first time, the values of the Lever E-L ligand parameter for BpBr3, TpBr3, and Tp(iPr,4Br). Theoretical calculations at the DFT level indicated that both oxidation and reduction of the Ru complexes under study are mostly metal-centered with some involvement of the chloride ligand in the former case, and also demonstrated that the experimental isolation of the (3)-binuclear complex 5 (instead of the mononuclear 5) is accounted for by the low thermodynamic stability of the latter species due to steric reasons.

Redox-active cytotoxic diorganotin(IV) cycloalkylhydroxamate complexes with different ring sizes: Reduction behaviour and theoretical interpretation

Two series of new diorganotin(IV) cycloalkylhydroxamate complexes with different ring sizes (cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl), formulated as the mononuclear [R2Sn(HL)(2)] (1:2) (a, R=Bu-n and Ph) and the polymeric [R2SnL](n) (1:1) (b, R=Bu-n) compounds, were prepared and fully characterized. Single crystal X-ray diffraction for [(Bu2Sn)-Bu-n{C5H9C(O)NHO}(2)] (3a) discloses the cis geometry and strong intermolecular NH center dot center dot center dot O interactions. The in vitro cytotoxic activities of the complexes were evaluated against HL-60, Bel-7402, BGC-823 and KB human tumour cell lines, the greater activity concerning [(Bu2Sn)-Bu-n(HL)(2)] [HL=C3H5C(O)NHO (1a), C6H11C(O)NHO (4a)] towards BGC-823. The complexes undergo, by cyclic voltammetry and controlled-potential electrolysis, one irreversible overall two-electron cathodic process at a reduction potential that does not appear to correlate with the antitumour activity. The electrochemical behaviour of [R2Sn(C5H9C(O)NHO)(2)] [R=Bu-n (3a), Ph (7a)] was also investigated using density functional theory (DFT) methods, showing that the ultimate complex structure and the mechanism of its formation are R dependent: for the aromatic (R = Ph) complex, the initial reduction step is centred on the phenyl ligands and at the metal, being followed by a second reduction with Sn-O and Sn-C ruptures, whereas for the alkyl (R=Bu-n) complex the first reduction step is centred on one of the hydroxamate ligands and is followed by a second reduction with Sn-O bond cleavages and preservation of the alkyl ligands. In both cases, the final complexes are highly coordinative unsaturated Sn-II species with the cis geometry, features that can be of biological significance.

Cardiac involvement in human rabies: case report

A case of human rabies with cardiac involvement and viral inclusion bodies in the heart is presented. The Negri bodies were found in the Schwann cells of the right epicardial atrium, with secondary mononuclear cells inflammation. In the myocardium, an interstitial edema, proliferation of Anitschkov and rare mononuclear inflammatory cells were seen. There was no relevant cardiovascular signs or symptoms. The rarity of histological descriptions of Negri bodies in the heart is stressed, as well as the importance of cardiac involvement as a potential complication for cases with life prolonged by intensive care units, or in the end-stages of the disease.

Immunoglobulins and C3 in the P. brasiliensis granuloma

The experimental model of paracoccidioidomycosis induced in mice by the intravenous injection of yeast-forms of P. brasiliensis (Bt2 strain; 1 x 10(6) viable fungi/animal) was used to evaluate sequentially 2, 4, 8, 16 and 20 weeks after inoculation: 1. The presence of immunoglobulins and C3 in the pulmonary granuloma-ta, by direct immunofluorescence; 2. The humoral (immunodiffusion test) and the cellular (footpad sweeling test) immune response; 3. The histopathology of lesions. The cell-immune response was positive since week 2, showing a transitory depression at week 16. Specific antibodies were first detected at week 4 and peaked at week 16. At histology, epithelioid granulomas with numerous fungi and polymorphonuclear agreggates were seen. The lungs showed progressive involvement up to week 16, with little decrease at week 20. From week 2 on, there were deposits of IgG and C3 around fungal walls within the granulomas and IgG stained cells among the mononuclear cell peripheral halo. Interstitital immunoglobulins and C3 deposits in the granulomas were not letected. IgG and C3 seen to play an early an important role in. the host defenses against P. brasiliensis by possibly cooperating in the killing of parasites and blocking the antigenic diffusion.

Immunopathology of human schistosomiasis mansoni: I. Immunomodulatory influences on T cell function

Cell mediated immune response was studied in patients with recent and chronic Schistosoma mansoni infection. Precultured peripheral mononuclear cells showed significantly higher responses to S. mansoni adult worm antigen (SAWA) when compared to fresh cell preparations. The addition of each patient serum to the precultured cells reactions to SAWA or recall antigens demonstrated a strong inhibitory serum action, which was also noted on allogeneic cells derived from healthy subjects. The CD4 subset was the main responding cell to SAWA being this reactivity highly suppressed by the presence of the monocyte macrophage accessory cells. We stressed the simultaneous inhibitory action of humoral and cellular factors on the specific cell response to S. mansoni.

Comprometimento da medula óssea e eosinofilia na paracoccidioidomicose

São descritos 3 casos de paracoccidioidomicose com a forma aguda da doença, nos quais formas leveduriformes de Paracoccidioides brasiliensis foram visualizadas ao exame direto de medula óssea, sendo a cultura também positiva em um caso. Salienta-se o acometimento do sistema fagocítico-mononuclear e a ausência de resposta às provas cutâneas de hipersensibilidade tardia a antígenos microbianos e de P. brasiliensis em todos, bem como a gravidade do quadro clínico e lesões ósseas generalizadas em um caso, com 20.260 eosinófilos/mm³ no sangue periférico. Os autores discutem o possível papel do eosinófilo na interação hospedeiro-parasita na paracoccidioidomicose, sugerindo que a ativação de subpopulação TH 2 e o aumento de secreção de IL 5 e de GM-CSF possam estar relacionados à grande eosinofilia presente no caso mais grave

Renal involvement in prolonged salmonella bacteremia: the role of schistosomal glomerulopathy

Renal involvement has been well documented in patients with hepatosplenic schistosomiasis and in patients with prolonged salmonella bacteremia (PSB). Whether there is a specific renal lesion related to PSB or the chronic bacterial infection aggravates a pre-existing schistosomal glomerulopathy has been a matter of controversy. To analyze the clinical manifestations and histopathological findings of the renal involvement, 8 patients with hepatosplenic schistosomiasis and PSB (group I) were compared with 8 patients with schistosomal glomerulopathy (group II) matched by sex and glomerular disease. The mean age in group I was 17.7 years. All patients presented with hematuria, in 4 cases associated with non-nephrotic proteinuria. In group II the mean age was 23 years; nephrotic syndrome was the clinical presentation in 7 of the 8 patients in the group. All patients in group I experienced remission of the clinical and laboratory abnormalities as the salmonella infection was cured; in group II the patients had persistent, steroid-resistant, nephrotic syndrome. On histological examination, no difference was noted between the two groups, except for pronounced glomerular hypercellularity and interstitial mononuclear cell infiltration in group I. These observations strongly suggest that PSB exacerbates a pre-existing sub-clinical schistosomal glomerulopathy by the addition of active lesions directly related to the prolonged bacteremia

Solvent-free microwave-assisted peroxidative oxidation of alcohols catalyzed by Iron(III)-TEMPO catalytic systems

The iron(III) complexes [H(EtOH)][FeCl2(L)(2)] (1), [H(2)bipy](1/2)[FeCl2(L)(2)].DMF (2) and [FeCl2(L)(2,2'-bipy)] (3) (L = 3-amino-2-pyrazinecarboxylate; H(2)bipy = doubly protonated 4,4'-bipyridine; 2,2'-bipy = 2,2'-bipyridine, DMF = dimethylformamide) have been synthesized and fully characterized by IR, elemental and single-crystal X-ray diffraction analyses, as well as by electrochemical methods. Complexes 1 and 2 have similar mononuclear structures containing different guest molecules (protonated ethanol for 1 and doubly protonated 4,4'-bipyridine for 2) in their lattices, whereas the complex 3 has one 3-amino-2-pyrazinecarboxylate and a 2,2'-bipyridine ligand. They show a high catalytic activity for the low power (10 W) solvent-free microwave assisted peroxidative oxidation of 1-phenylethanol, leading, in the presence of TEMPO, to quantitative yields of acetophenone [TOFs up to 8.1 x 10(3) h(-1), (3)] after 1 h. Moreover, the catalysts are of easy recovery and reused, at least for four consecutive cycles, maintaining 83 % of the initial activity and concomitant rather high selectivity. 3-Amino-2-pyrazinecarboxylic acid is used to synthesize three new iron(III) complexes which act as heterogeneous catalysts for the solvent-free microwave-assisted peroxidative oxidation of 1-phenylethanol.

Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG aginst T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytothropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.

Host cell adhesion to Schistosoma mansoni larvae in the peritoneal cavity of naive mice: histological and scanning electron microscopic studies

Cercariae of Schistosoma mansoni inoculated into the peritoneal cavity of naive mice induced host cell adhesion to their surface, but after 90 minutes the number of adherent cells sharply decreased. The cell detachment is progressive and simultaneous to the cercaria-schistosomule transformation. The histological study showed mainly neutrophils in close contact with the larvae. Mononuclear cells and some eosinophils were occasionally seen surrounding the adherent neutrophils. The scanning electron microscopy showed cells displaying twisted microvilli and several microplicae contacting or spreading over the larval surface, and larvae completely surrounded by clusters of cells. These results suggest that the neutrophils recognize molecules on the cercarial surface which induce their spreading

Experimental paracoccidioidomycosis in hamster: transmission electron microscopy of inoculation site lesion

Interaction between Paracoccidioides brasiliensis (Pb) and inflammatory cells in hamster testis was studied sequentially by transmission electron microscopy. In early lesions (six hours after inoculation), polymorphonuclear neutrophils (PMNs) were the major and mononuclear cells and eosinophils were the minor constituents of the inflammatory cells. PMNs were later replaced by mononuclear cells. Viable Pb cells were phagocytosed or surrounded by inflammatory cells. Preserved Pb cells usually had broad host-parasite interphases, whereas dying ones had narrow interphases. The outer layer of the fungus wall was sometimes broken by PMN in some focal points, broken pieces being peeled off and phagocytosed. Small Pb cells were uninuclear, and were often related to broad interphase. Large Pb cells were multinucleated with irregularly shaped wall, and sometimes had lomasome and/or myelin like structures. Different interaction patterns of Pb with inflammatory cells may be due to functionally different host cell flow to the inoculation site or due to the age of Pb cells or both.

"In vivo" leukocyte chemotaxis in experimental mice Schistosoma mansoni infection

The "in vivo" chemotaxis was studied in C57B1/10 mice 10, 30, 50 and 60 days after a Schistosoma mansoni infection in comparison to a control group (uninfected mice). Staphylococcal protein A was injected into a connective tissue air pouch of control and experimental mice and the leukocyte chemotaxis was counted. A decrease in polymorphonuclear (PMN) leukocyte response was found in infected mice in comparison to the control group (p<0.05). The 10 day infected mice showed a decreased PMN leukocyte response respecting the control group (p<0.05) and this finding became more evident 30 and 50 days post-infection. Although the PMN leukocyte response of 60 day infected mice increased in comparison to 50 day infected animals, it was still significantly lower the control response. The mononuclear leukocyte response was not significantly different between infected or uninfected mice.

Sulfonated Schiff base copper(ii) complexes as efficient and selective catalysts in alcohol oxidation: syntheses and crystal structures

The reaction between 2-aminobenzenesulfonic acid and 2-hydroxy-3-methoxybenzaldehyde produces the acyclic Schiff base 2-[(2-hydroxy-3-methoxyphenyl) methylideneamino] benzenesulfonic acid (H2L center dot 3H(2)O) (1). In situ reactions of this compound with Cu(II) salts and, eventually, in the presence of pyridine (py) or 2,2'-bipyridine (2,2'-bipy) lead to the formation of the mononuclear complexes [CuL(H2O)(2)] (2) and [CuL(2,2'-bipy)]center dot DMF center dot H2O (3) and the diphenoxo-bridged dicopper compounds [CuL(py)](2) (4) and [CuL(EtOH)](2)center dot 2H(2)O (5). In 2-5 the L-2-ligand acts as a tridentate chelating species by means of one of the O-sulfonate atoms, the O-phenoxo and the N-atoms. The remaining coordination sites are then occupied by H2O (in 2), 2,2'-bipyridine (in 3), pyridine (in 4) or EtOH (in 5). Hydrogen bond interactions resulted in R-2(2) (14) and in R-4(4)(12) graph sets leading to dimeric species (in 2 and 3, respectively), 1D chain associations (in 2 and 5) or a 2D network (1). Complexes 2-5 are applied as selective catalysts for the homogeneous peroxidative (with tert-butylhydroperoxide, TBHP) oxidation of primary and secondary alcohols, under solvent-and additive-free conditions and under low power microwave (MW) irradiation. A quantitative yield of acetophenone was obtained by oxidation of 1-phenylethanol with compound 4 [TOFs up to 7.6 x 10(3) h(-1)] after 20 min of MW irradiation, whereas the oxidation of benzyl alcohol to benzaldehyde is less effective (TOF 992 h(-1)). The selectivity of 4 to oxidize the alcohol relative to the ene function is demonstrated when using cinnamyl alcohol as substrate.