Trypanosoma cruzi: Ultrastructural, cytochemical and freeze-fracture studies of protein uptake

EXPERIMENTALPARASITOLOGY45, 101-115

Trypanosoma

cruzi:

(1978)

Ultrastructural, Cytochemical Studies of Protein Uptake

WANDERLEY DE SOUZA, TJ?CIA ULISSESDECARVALHO,AND Instituto

de Biofisica,

and Freeze-Fracture

MARLENEBENCHIMOL

Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

2O.&XJ

AND

EGLER CHIARI Departamento

de Parasitologia, Instituto de Cilncias Federal de Minas Gerais, Beb-Horizonte, (Accepted

for publication

5 April

Bioldgicas, Universidade MG, Brasil 1978)

DE SOUZA,W., DE CARVALHO,T.U.,BENCHIMOL,M., AND CHIARI,E. 1978. Trypanosoma cruzi: Ultrastructural, cytochemical, and freeze-fracture studies of protein uptake. Experimental Parasitology 45, 101-115. Epimastigotes and trypomastigotes of Trypanosoma cruzi, obtained from liquid cultures, have vesicles and multivesicular structures in their cytoplasm. Horseradish peroxidase (HRP) was used as a tracer to study the uptake of protein by these two forms. In epimastogotes HRP is ingested by a process of pinocytosis which occurs through the cytostome. Trypomastigotes do not have a cytostome, and pinocytosis occurs through the flagellar pocket region. The pinocytotic vesicles can fuse with each other to form large multivesicular structures that are more abundant in epimastigotes than in trypomastigotes. The cell membrane as well as the membranes of the pinocytotic vesicles and the large multivesicular structure have carbohydrates, as detected by the periodic acid-thiosemicarbazide-silver proteinate technique. lntramembranous particles were observed by using the freeze-fracture technique. The cell membrane has many particles, whereas the membranes of the vesicles and multivesicular structure have few or no particles. INDEX DESCRIPTORS:Trypanosoma cruzi; Epimastigote; Trypomastigote; Protozoa, parasitic; Cytostome; Pinocytosis; Cell membrane; Freeze-fracture.

involves the formation of a membranebound vesicle at the cell surface, although a process of intracellular phagotrophy can also be shown in intracellular stages of Typanosoma crud (Meyer and De Souza 1973). The presence of a specialized region, the cytostome, which plays an important role in the nutrition of protozoa, has been shown in monogenetic and in some stercorarian trypanosomes (Brooker and Preston 1967; Martinez-Palomo et al. 1976; Meyer and De Souza 1973; Milder

INTRODUCTION

All the Trypanosomatidae have an invagination of the plasma membrane where the flagellum emerges from the cell forming the flagellar pocket. Previous studies have shown that this region is the main site of uptake of exogenous macromolecular substances by these protozoa (Brown et al. 1965; De Souza 1976a; Langreth and Balber 1975). Substances are mainly incorporated by a process of pinocytosis which 101

0014-4894/78/0451-0101$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIGS. 1-14. All cells are epimastigotes. stome; F, flagellum; G, Golgi complex; cleus; V, vesicle.

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Abbreviations K, kinetoplast;

used: CM, cell membrane; CY, cytoMS, multivesicular structure; N, nu-

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and Deane 1969; Preston 1969; Steinert and Novikoff 1960), but not in salivarian trypanosomes (Brown et al. 1965; Langreth and Balber 1975; Vickerman 1969). This structure is localized within or close to the flagellar pocket region and is formed by an invagination of .the cell membrane, associated with the subpellicular microtubules. Vesicles are usually associated with the cytostome. Several studies show that T. cruzi has different types of vacuoles in its cytoplasm. Although such vacuoles can be found in all developmental stages of the parasite life cycle, they are found more frequently in forms cultivated in vitro in a medium such as liver-infusion tryptose. It has been suggested that these vesicles are involved with the process of ingestion and digestion of substances by the parasite (De Souza, et al. 1975). The purpose of the present study was to define the interactions of a soluble protein with the cell membrane of T. cruzi by following its intracellular fate in order to see if the small pinocytotic vesicles are related to the formation of the large multivesicular structure observed mainly in epimastigotes. Attempts were also made to study the structure of the membranes involved in the process of protein ingestion by use of cytochemical and freeze-fracture methods. MATERIALS

AND METHODS

Microorganism. Strains Y and Gilmar of Typarwsomu cruuzi were cultivated in liverinfusion tryptose (LIT) medium (Camargo 1964) for periods varying from 3 to 10 days at 27 C. Three-day cultures were used as the source of epimastigotes and Bto lo-day cultures were the source of trypomastigotes.

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To separate epimastigotes from trypomastigotes, a modification of Lanham and Godfrey’s DEAE-cellulose column method (Lanham and Godfrey 1970) was employed as described previously (Goldberg et al. 1976; De Souza et al. 1977). The percentages of epimastigotes and trypomastigotes were estimated by direct microscopy. Electron microscopy. Cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate or cacodylate buffer, pH 7.2, for 1 hr at room temperature, postfixed with 1% OsOa, dehydrated in acetone, and embedded in Epon. Ultrathin sections were obtained in an LKB ultratome III ultramicrotome and were examined unstained or after staining with uranyl acetate and/or lead citrate under an AEI EM8B electron microscope. Localization of carbohydrates. Ultrathin sections of cells fixed in glutaraldehyde and 0~04 or only in glutaraldehyde were collected on gold grids and treated with periodic acid (lo/o, 20 min at room temperature) to oxidize adjacent hydroxyl or or-amino alcohol groups into aldehydes. After rinsing in distilled water, aldehydes were condensed with thiosemicarbazide (1% thiosemicarbazide in 10% acetic acid for 48 hr at room temperature) to yield the corresponding thiosemicarbazones which are powerful reducing agents. Thus after rinsing sequentially with 10.5 and 1% acetic acid and with distilled water, the sections were exposed to silver proteinate (1% ) for 30 min in the dark room at room temperature. Sections were observed unstained ( Thiery 1967). of horseradish peroxidase Incorporation (HRP). We used HRP as a tracer to study the ingestion of proteins by the parasite. Cells were washed in phosphate-buffered saline (PBS) or in 0.1 M phosphate buffer, pH 7.2, and were incubated for periods

FIGS. 1-3. Trypansoma cru~i cells submitted to the periodic acid-thiosemicarbazide-silver proteinate technique. Sections are unstained. Figures 2 and 3 show a tangential section through the intracytoplasmic portion of the cytostome. Small vesicles (arrows) are associated with the cytostome. A light reaction is seen in the membranes of the Golgi complex. Vesicles of varying diameter show the membrane reaction (Fig. 3). ~45,000.

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varying from 0 to 60 min in solution containing HRP (Type II, Sigma Chemical CO., St. Louis, MO., USA) at a concentration of 2 mg/ml of PBS or phosphate buffer. After incubation the cells were washed and fixed in 2.5% glutaraldehyde as previously described. The distribution of HRP was determined cytochemically using the Graham and Karnovsky medium ( 1966). This medium contained 3-3’-diaminobenzidine (0.5 mg/ml), 0.05 M Tris-HCl buffer, pH 7.6, and 0.01% HzOz. After incubation for 30 min at room temperature the cells were rinsed in PBS or phosphate buffer, postfixed with 0~04, dehydrated in acetone, and embedded in Epon. The ultrathin sections were examined unstained or after staining with lead citrate. Freeze-fracture. Untreated cells and cells exposed to HRP were fixed in glutaraldehyde, washed twice in 0.1 M cacodylate buffer, pH 7.2, and then gradually impregnated for 30 min with glycerol up to 20% in cacodylate buffer. Subsequently, they were left in 20% glycerol for 1-3 hr at room temperature, mounted in Balzers support disks, and rapidly frozen in the liquid phase of partially solidified Freon 22 cooled by liquid nitrogen and stored in liquid nitrogen before use. Freeze-fracture was carried out at -115 C in a Balzers apparatus equipped with a turbomolecular pump. Replicas were produced by evaporation of platinum-carbon. The specimens were shadowed at 2-3 x lo-” Torr. Replicas were recovered in distilled water, cleaned with sulfuric acid and distilled water, and mounted on 200-mesh grids. Micrographs were mounted with the shadow direction from bottom to top. RESULTS

Ultrastructure The general structure of epimastigotes and trypomastigotes of Typanosoma cruzi, obtained from either acellular cultures, tissue culture, or blood of infected animals,

has been described by several authors (Brack 1968; Bretafia and O’Daly 1976; De Souza and Chiari 1977; De Souza et al. 1975; De Souza and Meyer 1974; Maria et al. 1972; Meyer 1968; Sanabria 1964; Wery and Groodt-Lassel 1966). Only those structures associated with the process of ingestion of substances by the parasite are discussed here. The cell surface is composed of a unit membrane, 8-10 nm thick, covering the cell body and the flagellum and lined with the flagellar pocket. On the cell membrane there is a layer of carbohydrates easily seen when using cytochemical methods to detect carbohydrates. About 12 nm below the cell membrane there is a single layer of microtubules (subpellicular microtubules) in a parallel array (De Souza 1976b; Meyer and De Souza 1976). The cytoplasm contains a nucleus, a kinetoplast, numerous ribosomes, and other cell organelles. In epimastigotes a funnel-shaped depression, lined by the cell membrane and the subpellicular microtubules, penetrates into the cytoplasm close to the region where the Golgi complex is located. Such a structure corresponds to the cytostome and was not observed in trypomastigotes. In the cytoplasm of epimastigotes and trypomastigotes from acellular cultures, many cytoplasmic vesicles and a mu&vesicular structure were observed. The latter structure, which is more abundant in epimastigotes, appears to ,be formed by several vesicles and varies in size, some having a diameter of about 600 nm. Presumably, they correspond to the granules seen by light microscopy mainly when Normarski optics (Allen et al. 1969) is used. We cou!d also observe the presence of other granules with a moderate electron-dense matrix. Localization

of Carbohydrates

The periodic acid-thiosemicarbazide-silver proteinate technique (Thi&y 1967) was used to detect carbohydrates. In all controls used, there was no reaction, The controls omitted either the periodic acid

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UPTAKE

FIG. 4. Trypunosorna cr~zi cell submitted to the periodic acid-thiosemicarbazide-silver proteinate technique. The reaction is seen in the cell membrane (which envelopes the body and the flagellum) and in the membrane of multivesicular structures. ~45,000.

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FIGS. 5 and 6. Trypanosoma cruzi cells incubated with horseradish peroxidase before glutaraldehyde fixation. The reaction product is seen on the cell membrane in the region of the cytostome (curved arrow), in small vesicles (arrows), and in multivesicular structures. Fig. 5, X40,000.

oxidation step or the incubation with thiosemicarbazide. In all developmental stages of T. cruxi,

we did not observe a reaction for carbohydrates in intracellular structure, thus showing the absence of reserve polysaccha-

Typanosoma

cmxi:

rides such as glycogen or amylopectin. A reaction for carbohydrates was only observed on the membranes (Fig. 1). As previously reported (De Souza and Meyer 1975) for T. cruzi in tissue cultures, an electron-dense layer was observed on the outer face of the cell membrane which envelopes the cell body and the flagellum. Some intracellular membrane-bound structures also reacted, including small vesicles localized close to the Golgi complex (Fig. 1) and the flagellar pocket. These vesicles have an average diameter of 80 nm. They were also seen lining the invagination of the cell membrane which formed the cytostome in epimastigotes (Figs. 1 and 2). The membrane of the multivesicular structure, as well as the membrane of the vesicles which compose this structure, also reacted (Figs. 3 and 4). Incorporation

of Horseradish

Peroxidase

Incubation of T. cruzi in the presence of HRP was followed by incorporation of the protein by the parasite. Although a quantitative study was not performed it was observed that, at high concentrations of HRP or with prolonged incubation, more HRP was ingested by the parasite. HRP was concentrated on the flagellar pocket region and close to the cytostome (Figs. 5-9). Figure 5 shows a region of the cell membrane which appears more electron dense, indicating the presence of HRP. Small vesicles with an average diameter of 80 nm can be observed close to the cytostome (Figs. 5-9). The intensity of the reaction varied among the vesicles; some of them appeared almost uniformly electron dense, whereas others appeared empty with the reaction product accumulated only on the periphery. Vesicles with different diameters, containing the reaction product, were also observed. Apparently they fused with each other or with vesicles which did not contain the reaction product forming large multivesicular structures. These structures have different forms as can be seen

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in Figs. 5-8. Some of these multivesicular structures appeared to contain vesicles without reaction. Some appeared uniformly electron dense, similar to those observed in macrophages ( Steinman and Cohn 1972) (Figs. 5, 7, and 8). Even short incubation with HRP produced large multivesicular structures containing the reaction product. We could observe the presence of such structures containing peroxidase in cells fixed 10 min after incubation in the presence of tracer. Epimastigotes had many more multivesicular structures than did trypomastigotes. A cytostome was not observed in trypomastigotes isolated on DEAE-cellulose. Freeze-Fracture As previously described (Martinez-Palomo et al. 1976) observations of fracture faces of T. cruzi plama membrane revealed a marked heterogeneity in the size and distribution of the intramembranous particles (Figs. 11, 13, and 14). The P face (which represents the outer face of the inner membrane half) exhibited approximately the same density of intramembranous particles as did with fracture face E (which corrresponds to the inner face of the outer membrane half). In the cytoplasm we observed vesicles of different diameter (Fig. 13). The membrane of these vesicles had considerably fewer intramembranous particles than did the cell membrane which envelopes the cell body (Figs. 10 and 12). The heterogeneity of the particles, reflected by differences in particle size and shape, was also observed in the membranes of the vesicles. We did not observe differences in the number of particles between the P and E faces of the membrane of the vesicles. In some vesicles the particles appeared randomly distributed. In others, however, there were regions with or without particles (Fig. 12). The difference in the number of particles between the cell membrane and the membrane of the vesicles can be clearly seen in figures such as Fig. 13 in which

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FIGS. 7-9. Trypanosoma cruzi cells incubated with horseradish peroxidase before glutaraldehyde fixation. The reaction is seen in the cytostome (shown in Fig. 9) and in small vesicles associated with it. A cell in division with two kinetoplasts shows the reaction in the multivesicular structures (Fig. 8). Some of the multivesicular structures shown in Fig. 7 did not have the reaction product. Figure 7, ~45,000; Figs. 8 and 9, ~20,000.

the fracture plane included the cell membrane (showing P face) and the P and E faces of the membranes of some cytoplasmic vesicles. The multivesicular structure was also seen by the freeze-fracture method and appeared as a large structure containing small vesicles (Fig. 14). We did not observe typical intramembranous particles in the membranes of the vesicles which formed the multivesicular structure.

DISCUSSION Incorporation of extracellular substances by pinocytosis is one of the most important

mechanisms by which cells can ingest macromolecules. In Protozoa this process is especially important when organisms are cultivated in acellular media containing large quantities of macromolecules such as proteins, as in Trypanosoma cruzi serum-supplemented media. However this mechanism may also be important in the uptake of macromolecules from the cytoplasm of the host cell by intracellular stages of trypanosomatids. There are few reports on the incorporation of extracellular substances by trypanosomatids. Steinert and Novikoff (1960) showed that Trypanosoma mega was able

Typanosoma

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to incorporate ferritin particles through a specialized region of its surface called the cytostome. Pinocytic vesicles, localized in the cytoplasm of the Protozoa, were seen near or in association with the cytostome. A similar structure has been described in many monogenetic and stercorarian trypanosomes. This structure apparently does not occur in salivarian trypanosomes. However, as shown by Langreth and Balber (1975), the salivarian trypanosome Typanosoma brucei incorporated ferritin particles through large spiny-coated vesicles which formed at the flagellar pocket region. Milder and Deane (1969) showed that cytostomes are present in epimastigotes and amastigotes of T. cruzi. Previous studies from our laboratory showed that in intracellular spheromastigotes (Meyer and De Souza 1973) large cytoplasmic structures of the host cell, such as melanin granules (which measure 0.7 pm), can be incorporated by the parasite through the cytostome region, involving a process of intracellular phagotrophy similar to that described in malaria parasites (Rudzinska et al. 1965). Cytostomes were found in intracellular spheromastigotes and epimastigotes as well as in epimastigotes obtained from acellular culture media. However, we never observed cytostomes in trypomastigotes obtained from tissue culture, from acellular cultures, or from the blood of infected mice. We showed in a previous study, by use of the freeze-fracture technique, that the cytostome is a specialized region of the cell membrane which is poor in intramembranous particles and which is separated from other regions by parallel arrays of particles ( Martinez-Palomo et al. 1976). The incorporation of exogenous macromolecules has been studied in several types of cells by using tracers which are electron dense and which can be directly visualized by electron microscopy (for example, ferritin, colloidal gold, thorotrast) or by using enzymes the activity of which can be detected by cytochemical methods (for ex-

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horseample, catalase, microperoxidase, radish peroxidase). In our study we used horseradish peroxidase (HRP) since its activity is easily detectable by the diaminobenzidine-osmium tetroxide method, which gives an electron-dense product clearly seen under an electron microscope. The reaction product can be observed in unstained sections. However, the contrast is poor in the latter, making it difficult to identify the cellular structures. For this reason we used sections stained briefly with lead citrate. We were able to observe epimastigotes and trypomastigotes of T. cruzi ingesting HRP by a process of pinocytosis. In trypomastigotes the ingestion occurs through vesicles found in the flagellar pocket region as described for T. rhodesiense (Brown et al. 1965) and T. brucei (Langreth and Balber 1975). In epimastigotes, the incorporation occurs at the region of the cytostome, which is also located close to the flagellar pocket. Large multivesicular structures similar to those described in this report were also observed in previous studies on epimastigotes of T. cruzi (Bretafia and O’Daly 1976; De Souza et al. 1975; Wery and Groodt-Lasseel 1966). Wery and Groodt-Lasseel (1966) did not find acid phosphatase activity in multivesicular structures. It is possible that the multivesicular structure may play a role in the process of storage of proteins by the parasite. It is well known that T. cruzi has no reserve of polysaccharides such as glycogen or amylopectin which are formed in other protozoa (Van Brand 1973). Bretafia and O’Daly (1976) studied the incorporation of fetal calf serum proteins containing certain factors which stimulate division and uptake of thymidine by T. cruzi. The intracellular fate of the proteins was followed by light and electron microscopy and it was observed that they accumulated in granules. The structure of these granules is similar to that observed by us in the present study. Bretaiia and O’Daly (1976) observed that

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FIGS. 10-12. Trypanosoma cruzi cells submitted to the freeze-fracture technique. The P face of the cell membrane containing many intramembranous particles is shown in Fig. 11.

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in the stationary-phase trypomastigotes, the granules were located at the posterior ends of the parasite. In our study we observed granules dispersed throughout the cytoplasm of the parasite without a preferential location. The presence of carbohydrates as constituents of the cell membrane has been demonstrated in several types of cells (reviewed by Nicolson 1974; and Luft 1976). In T. cruzi, carbohydrates were demonstrated by cytochemistry and by agglutination of cells with the plant lectin concanavalin A (De Souza and Meyer 1975; Alves and Colli 1974). Most of the techniques used in electron microscopy to detect carbohydrates involve treatment of cells with several substances (Alcian blue, colloidal iron hydroxyde, cationized ferritin, etc. ) during the process of fixation. However, these substances do not penetrate intact cells and thus are not suitable to study intracellular membranes. Several other techniques, derived from the classical PAS method used in light microscopy, can be applied directly to ultrathin sections to detect 1,2-glycol groups in carbohydrates. Some of these techniques have been used in our laboratory to study the surface of animal cells (Benchimol et al. 1977) and the best results were obtained with the periodic acid-thiosemicarbazide-silver proteinate technique (Thiery 1967). This technique was chosen in the present study of membranes of T. cru~i. As previously described (De Souza and Meyer 1975), an electron-dense reactive layer was observed on the outer face of the cell membrane. Reactions were also observed on cytoplasmic vesicles varying in diameter, mainly localized near the cytostome of epimastigotes, on membranes of the Golgi complex, and on the membrane of the large multivesicular structure. The reaction observed on the membranes of the last structure was

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similar to that observed on the cell membrane of the parasite. The process of pinocytosis involves the incorporation of pieces of the cell membrane. With the cytochemical method used we observed that the carbohydrates, which are components of the cell membrane, remain attached to the membranes of the vesicles and to the membranes of the large multivesicular structure. However, we cannot exclude the possibility that some carbohydrate-containing sites of the cell membrane are lost during this process. AS pointed out above, we could not use the more specific cytochemical methods for detection of carbohydrates to study intracellular membranes in intact cells. It has been shown that, when cells are freeze-fractured, they expose the inner components of the cell membrane, allowing electron microscopic examination of the outer aspect of the inner membrane half (P or protoplasmic face) and the inner aspect of the outer membrane half (E or external face). Particles are observed in both faces (Branton et al. 1975; Pinto da Silva and Branton 1970). Studies of the chemical nature of membrane particles in other membrane systems have demonstrated that the particles represent a membrane-intercalated protein-containing structure (Deamer 1973; Pinto da Silva et al 1971, 1973; Tillack et al. 1972). It has been suggested that the frequency of the particles may be related to the physiological activity of the cell membrane. Our observations show that the membranes of small as well as of large vesicles have much less intramembranous particles than the cell membrane. Since they have originated from the cell membrane, during or just after the process of pinocytosis, the cell membrane must have lost many intramembranous particles. This process is probably very fast since small vesicles contain-

Figures 10 and 12 show the P and E faces of the membranes of intracellular vesicles which contain few intramembranous particles. Figure 10, x60,000; Figs. 11 and 12, ~80,000.

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FIG. 13. Trypanosomu cruzi cell submitted to the freeze-fracture technique. The fracture plane shows the P face of the cell membrane and many intracellular vesicles. The difference in the density of the particles between the two membranes is evident. ~45,000.

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FIG. 14. Trypanosom cruzi cell submitted to the freeze-fracture technique. The fracture plane shows the cell membrane, the membrane of vesicles (with few particles), and the membranes of the multivesicular structures (without particles). ~45,000.

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ing horseradish peroxidase can be observed in cells incubated for 1 min in the presence of the tracer. The membrane of the multivesicular structure did not show typical intramembranous particles, suggesting a gradual loss of these elements during formation of this structure. In conclusion our results suggest that, in Trypanosoma cruzi, exogenous proteins are incorporated by a process of pinocytosis. The pinocytotic vesicles can fuse with each other forming multivesicular structures distributed throughout the cytoplasm. The cell membrane as well as the membrane of the vesicles which originate from the cell surface and contribute to the formation of multivesicular structures contain carbohydrates, as detected cytochemically. During such a process, intramembranous particles (as seen by freeze-fracture) are lost or displaced. The cell membrane has many particles, whereas membranes of the multivesicular structure have none. ACKNOWLEDGMENTS We are most grateful to Professor L. R. Travassos (Department of General Microbiology, UFRJ ) for advice and suggestions in the preparation of this manuscript. We thank Mr. A. L. de Oliveira for help with photography and Nancy C. de Carvalho for secretarial assistance. This work has been supported by Conselho Nacional de Desenvolvimento Cientifico e Tecno16gico ( CNPq), Conselho de Ensino e Pesquisa da UFRJ, and Financiadora de Estudos e Projectos (FINEP-FUNDCT-314/CT).

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