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In the present study, we describe a new silicon phthalocyanine conjugated to bovine serum albumin (PcSiN3M-BSA) and its photodynamic activity in murine macrophages cells (J774.A1). The nonconjugated precursor, bis(trimethylaminoethanoxy)–phthalocyaninato silicon (IV) (PcSiN3M), was also studied. Compounds PcSiN3M and PcSiN3M-BSA showed no cytotoxicity in the dark, but exhibited high photodynamic activities following exposure to 5 µM photosensitizers and 45 J cm−2 irradiation. These conditions were sufficient to decrease the cell viability to 40% and 5% in cells treated with PcSiN3M and PcSiN3M-BSA, respectively. These results demonstrated an increase of 87% in the photodynamic activity of PcSiN3M when conjugated with BSA. The results shown in this work suggest that PcSiN3M-BSA had higher uptake by J774.A1 cells, which contributed to its higher photoactivity compared with the unconjugated form, PcSiN3N.

Liver fibrosis is characterized by excessive accumulation of extracellular matrix components in the liver parenchyma that distorts the normal architecture and hepatic function. Progressive fibrosis could end in the advanced stage known as cirrhosis, resulting in the need to resort to liver transplantation. Amniotic membrane (AM) has emerged as an innovative therapeutic approach for chronic liver diseases due to its anti-inflammatory, antiscarring, and wound-healing effects. We have recently shown that AM can be used as a patch on the liver surface at the same time of fibrosis induction, resulting in significantly reduced progression and severity of biliary fibrosis. Here we investigated the effects of human AM on the established rat model of liver fibrosis, induced by the bile duct ligation (BDL). We also explored the effect of AM on the expression of transforming growth factor-β1 (TGF-β1), the main profibrogenic factor in hepatic fibrosis, and the proinflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory cytokine IL-10. Two weeks after BDL, the liver was covered with a fragment of AM or left untreated. Six weeks later, the fibrosis was first assessed by the semiquantitative Knodell and the METAVIR scoring systems and, thereafter, by CellProfiler digital image analysis to quantify the area occupied by collagen deposition, ductular reactions (DRs), activated myofibroblasts, and TGF-β1. The hepatic cytokines were determined by ELISA. AM-treated rats showed a significantly lower score compared to the control BDL rats (2.5 ± 0.9 vs. 3.5 ± 0.3, respectively; p < 0.05). The collagen deposition, DRs, number of activated myofibroblasts, and TGF-β1 were all reduced to about 50% of levels observed in untreated BDL rats. These findings suggest that AM, when applied as a patch onto the liver surface, is useful for treating well-established cholestatic fibrosis, and the mechanism was partly by means of downregulating the profibrotic factor TGF-β1 and IL-6.

Nanodiagnostics have become an important tool for the development of new methods for clinical and laboratory diagnosis. Non-cross-linking colorimetric detection is a promising technique using thiol-modified oligonucleotides (probes) functionalized gold nanoparticles. The nanoprobes allow the detection of a specific sequence of complementary DNA (cDNA) for a well-characterized antigenic protein 27 kDa (p27), which is capable of reacting with the serum of 91% of the patients infected with fungus Paracoccidioides brasiliensis the etiologic agent of Paracoccidioidomycosis. In this work nanoprobe groups were synthesized with final concentration of 33.6 nM, 66.1 nM and 128.1 nM thiolated oligonucleotides. To evaluate the efficiency of the nanoprobes Fourier Transform Infrared (FT-IR) was used to identify the functional groups of the probe and its bond to the gold nanoparticle. The region between 200 cm−1 and 280 cm−1 corresponds to the Au S bond, which is a determining factor for the efficiency of colorimetric detection. The FT-IR results are corroborated with images taken with the Transmission Electron Microscopy (TEM) technique, where the difference between functionalization groups is evident. Considering these aspects, the present work aims to develop a new molecular diagnosis of the fungus Paracoccidioides brasiliensis by non-cross-linking colorimetric detection method using a probe from p27 antigenic cDNA sequence. The non-cross-linking colorimetric diagnostic test possesses 84.4% sensitivity and 81.2% specificity, and may be implemented as clinical diagnosis with simple execution and reasonable cost to effectively detect of Paracoccidioides brasiliensis.

This study investigated the photoactivity of four zinc phthalocyanines (PcZns) on a murine macrophage cell line infected with Leishmania amazonensis or Leishmania braziliensis. Infected and uninfected cells were incubated with PcZns at different concentrations (1-10 μM) for 3 h and then exposed to an LED device in continuous wave mode at 660 nm with a fluency of 50 J/cm(2) (25 mV). Enzymatic activity was determined by MTT assay 24 h after light treatment. The results demonstrated that all PcZns exhibited high photoactivity, particularly when used at 10 μM. The photodynamic effects were different for uninfected cells versus parasite-infected cells and among the four PcZns. Uninfected cells were more sensitive to photoactivity than infected cells. Although PcZns photodynamic therapy provided promising results, further studies are necessary to better understand its mechanism of action in the treatment of leishmaniasis.

PURPOSE: To investigate the effect of Cenostigma macrophyllum Tul. in the tissue repair process of skin lesions in rats with induced Diabetes mellitus. METHODS: Sixty-three male Wistar rats were distributed into three groups: control (C), diabetic (D), and diabetic treated with an oil-in-water emulsion of the plant (DPL) subdivided according to time of observation (seven, 14, and 28 days). Diabetes was induced by administration of by injection of streptozotocin (50 mg/kg, after a 12-h fast) into the penile vein and confirmed by glucose levels exceeding 240 mg/dL. Held surgical lesion (2.5 cm in diameter) on the back of the animals. The DPL received topical application of group of the oil-in-water emulsion plant (0.5ml). To evaluate the levels of nitric oxide, was collected 5ml of blood from the abdominal aorta, after his euthanasia. The samples of interest were sent for routine histological processing. RESULTS:A reduction in the percentage of inflammatory cells and increased numbers of fibroblasts in the group DPL, seven days, compared to the other groups. At 14 days, the DPL group also showed a higher concentration of nitric oxide (p

As espécies do gênero Candida são causadoras de diversas infecções fúngicas e, nos últimos anos, tem sido desenvolvidas novas tecnologias para auxiliar nos diagnósticos microbiológicos. Dentre as técnicas está a espectroscopia infravermelha junto com a análise estatística multivariada. O objetivo deste trabalho é comparar dois métodos: estatístico (análise multivariada) e não-estatístico (ajuste de curva), utilizando os espectros infravermelhos de Candida albicans, Candida dubliniensis e Candida parapsilosis para testar o potencial do uso de Análise Estatística Multivariada para discriminação de espectros de micro-organismos. Para isso foram obtidos, utilizando o Spectrum Spotlight 400 da PerkinElmer, 54 espectros infravermelhos, sendo 18 de cada espécie, na faixa de 4000 a 1000 cm-1, com resolução de 4 cm-1, no modo de transmissão, a 20 ºC. A análise dos espectros foi realizada através de três métodos: (1) inspeção visual direta dos espectros; (2) análise estatística multivariada; (3) ajuste de curva para a determinação de estruturas secundárias de proteínas. Na região de 1200 a 1000 cm-1, os espectros apresentam diferenças que podem ser percebidas numa inspeção visual direta. Uma banda próxima de 1070 cm-1 e outra próxima de 1045 cm-1 apresentam intensidades relativas diferentes para os três espectros. Por outro lado, as bandas da amida I, na região de 1710 a 1590 cm-1, apresentam aspectos visuais semelhantes com máximo em 1651 cm-1 para os espectros dos três micro-organismos. Esse fato torna possível submeter a análise estatística multivariada a um teste de sua capacidade de diferenciar três espectros de Candida. A análise estatística multivariada foi aplicada aos 54 espectros para investigar as regiões de 4000 a 1000 cm-1 com exceção da região de 2600 a 2300 cm-1 e de 1710 a 1590 cm-1 que corresponde a das bandas da amida I. A técnica selecionada foi a análise por componentes principais (PCA, Principal Componente Analysis), utilizando os primeiros quatro componentes principais, em conjunto com a técnica hierárquica de análise de agrupamento (HCA, Hierarchical Clustering Analysis) segundo o método de Ward. Foi utilizado para esta análise o software MINITAB 15 e o resultado mostra uma clara discriminação dos espectros dos três micro-organismos nas duas regiões consideradas. Adicionalmente foi obtido o espectro médio de cada micro-organismo nas bandas da amida I na região de 1710 a 1590 cm-1. Os três espectros médios assim obtidos foram analisados pelo método de ajuste de curva que não é estatístico para determinar as estruturas secundárias de proteínas. Para esta análise o software ORIGIN 7.5 foi utilizado e os resultados obtidos mostram estruturas conformacionais diferentes nos três micro-organismos. Esses resultados confirmam a discriminação obtida através da análise estatística multivariada e visual. Pode-se concluir que as análises estatísticas multivariadas baseadas em análise por componentes principais e análise de agrupamento com uso do algoritmo Ward é potencialmente útil para discriminar micro-organismos através de seus espectros infravermelhos. Além disso, as análises mostram que as bandas da amida I dos espectros infravermelhos de Candida albicans, Candida dubliniensis e Candida parapsilosis fornecem um conjunto de dados cuja estrutura de agrupamento é conhecida e que pode ser útil para testar e validar algoritmos estatísticos de análise de agrupamento. Films of Candida albicans, Candida dubliniensis and Candida parapsilosis were prepared and the infrared spectra of these films were obtained in the region 4000 to 1000 cm-1, with resolution of 4 cm-1, in the transmission mode, at 20 ºC. Fifty four spectra were obtained, 18 of each microorganism, with the PerkinElmer Spotlight 400 FT-IR, which has a microscope attached to a FT-IR spectrophotometer. The spectra were analyzed through three methods: (1) mere visual inspection; (2) multivariate statistical analysis; (3) curve-fitting for determining secondary structures of proteins. In the region 1200 to 1000 cm-1, the spectral bands show differences that can be seen by a mere visual inspection. On the other hand, the amide I bands, in the region 1710 to 1590 cm-1, have the same visual aspect for the three microorganisms. Multivariate statistical analysis was applied to analyze these amide I bands of all the 54 spectra. Principal component analysis (PCA) and techniques of hierarchical cluster analysis (HCA, Hierarchical Clustering Analysis) according to Ward's method were applied using the software MINITAB 15. The results show a clear discrimination of the three microorganisms. The average spectrum of each microorganism was obtained in the amide I band. Each average spectrum was analyzed by curve-fitting for the determination of secondary structures of proteins. The software used was the ORIGIN 7.5 and the results confirm the discrimination obtained through multivariate statistical analysis. This result shows that multivariate statistical analysis can be useful to discriminate infrared spectra of different microorganisms. Furthermore, this work shows that the amide I bands of the infrared spectra of Candida albicans, Candida dubliniensis, and Candida parapsilosis provide a set of data of known group structure that can be useful to test statistical algorithms of cluster analysis.

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints and can lead to a progressive destruction of articular cartilage and bone. In this study, the specificity and sensitivity of the RA diagnostic methods based on the receiver-operating characteristic curves for monitoring C-reactive protein (CRP) and rheumatoid factor (RF) were compared with the Raman spectroscopic diagnostic method developed in this work. Sera from 24 patients with rheumatoid arthritis and from 16 healthy individuals were analyzed to assess the biochemical composition and presence of inflammatory activity by the aforementioned methods. By comparing with the clinical results for specificity and sensitivity from the RF and CRP tests, we show that the overall results from the newly developed Raman method were significantly better, with a specificity of 96%, a sensitivity of 88%, and correctly identifying 92% of the RA and healthy individuals, while the RF test gave a specificity of 100% and a sensitivity of 54%, and the CRP test gave a specificity of 87% and a sensitivity of 58%, respectively.

Study of genetic material allows the comprehension the origin of the many biochemical changes that follow diseases, like cancer, promoting the development of early preventive inquiry and more efficient individual treatments. Raman spectroscopy can be an important tool in DNA study, since it allows probe molecular vibrations of genetic material in a fast way. The present work established a systematic way for extract DNA in suitable concentrations and structural integrity allowing studies by Raman spectroscopy or other spectroscopic technique, including bio-analytical sensors for probing genetic alterations.

A fibrose hepatica e uma doenca causada por uma grande variedade de doencas cronicas que afetam o figado apos diferentes agressoes, cujo unico tratamento e o transplante de figado. A patogenese da fibrose engloba uma complexa interacao entre as celulas e a matriz extracelular do tecido hepatico, interacoes estas mediadas por citocinas que modulam a progressao da fibrose. O objetivo do trabalho foi analisar quantitativamente o perfil de citocinas pro-inflamatorias (IL-6 e TNF- a ) e anti-inflamatoria (IL-10) no figado de ratos, com diferentes graus de fibrose, induzida pela ligadura do ducto biliar (LDB). Vinte ratos foram submetidos ao procedimento da LDB e eutanasiados apos 2, 4 e 6 semanas da inducao da fibrose para a remocao do figado e quantificacao das citocinas pro e anti-inflamatorias pelo metodo de ELISA sanduiche. Foi observado o aumento na concentracao de todas as citocinas apos 4 semanas da LDB e diminuicao dessas concentracoes na 6a semana. Dentre as citocinas pro-inflamatorias, o TNF- a mostrou aumento e diminuicao estatisticamente significantes, respectivamente, na 4a e 6a semana, quando comparado a IL-6, a qual nao apresentou significância estatistica em nenhuma semana apos a LDB. A IL-10, mostrou-se, significativamente, elevada na 4a semana, seguida de uma diminuicao moderada na 6a semana. Esses resultados indicam que o TNF e a principal citocina na progessao da fibrose biliar, principalmente, durante sua fase intermediaria. Ao mesmo tempo, a IL-10 atua nessa fase, limitando a producao da citocina pro-inflamatoria TNF- a , na tentativa de um controle endogeno da progressao da doenca.

Phthalocyanines, which are classified as second-generation photosensitizers, have advantageous photophysical properties, and extensive studies have demonstrated their potential applications in photodynamic therapy. The present work describes the preparation of a new zinc phthalocyanine conjugated to bovine serum albumin (compound 4a) and its photodynamic efficiency in human larynx-carcinoma cells (HEp-2 cells). The unconjugated precursor (compound 4) was also studied. Compounds 4 and 4a penetrated efficiently into the cell, exhibiting cytoplasmic localization, and showed no cytotoxicity in the dark. However, high photodynamic activities were observed in HEp-2 cells after treatments with 5 µM photosensitizers and 4.5 J cm−2 light. These conditions were sufficient to decrease the cell viability to 57.93% and 32.75% for compounds 4 and 4a, respectively. The present results demonstrated high photodynamic efficiency of zinc phthalocyanine conjugated with bovine serum albumin in destroying the larynx-carcinoma cells.

Paracoccidioides brasiliensis (P. brasiliensis) is a thermal dimorphic fungus and causal agent of paracoccidioidomycosis. Epidemiological data shows that it is mainly concentrated in Central and South America countries, with most registered cases in Colombia, Brazil, and Venezuela. The histopathological similarity with others fungal infection makes the diagnosis of P. brasiliensis more complicated. Therefore, the aim of this work was to find a positive and negative test for P. brasiliensis using gold nanoprobes as a new tool for P. brasiliensis detection. Gold nanoparticles were synthesized by reduction of gold chloride with sodium citrate. The results of this procedure is a wine-red solution with a maximum absorption in the range of ~520-530nm. A specific P. brasiliensis sequence of oligonucleotide was bonded to the nanoparticles, which maintained the wine-red color. The color changes from red to blue for negative diagnostic and is unchanged for a positive test. The H-bond interaction of DNA with the complementary DNA keeps strands together and forms double helical structure, maintaining the colloid stability. However, for non-complimentary DNA sequence the nanoprobes merge into a cluster, changing the light absorption.

Sao apresentados espectros infravermelhos da creatina, na faixa de 4000 a 400 cm−1. Os resultados sao comparados com espectros observados e calculados, publicados na literatura.

In recent years FT-IR microspectroscopy has been developed for microbiology analysis and applied successfully in pure cultures of microorganisms to rapidly identify strains of bacteria, yeasts and fungi. The investigation and characterization of microorganism mixed cultures is also of growing importance, especially in hospitals where it is common to poly-microbial infections. In this work, the rapid identification of bacteria in pure and mixed cultures was studied. The bacteria were obtained from the Institute Oswaldo Cruz culture collection at Brazil. Escherichia coli ATCC 10799 and Staphylococcus aureus ATCC 14456 were analyzed, 3 inoculations were examined in triplicate: Escherichia coli, Staphylococcus aureus and a mixed culture of them. The inoculations were prepared according to McFarland 0.5, incubated at 37 ° C for 6 hours, diluted in saline, placed in the CaF2 window and store for one hour at 50°C to obtain thin film. The measurement was performed by Spectrum Spotlight 400 (Perkin-Elmer) equipment in the range of 4000-900 cm -1 , with 32 scans using a transmittance technique with point and image modes. The data were processed (baseline, normalization, calculation of first derivate followed by smoothing with 9 point using a Savitzky-Golay algorithm) and a cluster analysis were done by Ward's algorithm and an excellent discrimination between pure and mixed culture was obtained. Our preliminary results indicate that the FT-IR microspectroscopy associated with cluster analysis can be used to discriminate between pure and mixed culture.

Rheumatoid Arthritis is a systemic chronic inflammatory disease, recurrent and systemic, initiated by autoantibodies and maintained by inflammatory mechanisms cellular applicants. The evaluation of this disease to promote early diagnosis, need an associations of many tools, such as clinical, physical examination and thorough medical history. However, there is no satisfactory consensus due to its complexity. In the present work, confocal Raman spectroscopy was used to evaluate the biochemical composition of human serum of 40 volunteers, 24 patients with rheumatoid arthritis presenting clinical signs and symptoms, and 16 healthy donors. The technique of latex agglutination for the polystyrene covered with human immunoglobulin G and PCR (protein c-reactive) was performed for confirmation of possible false-negative results within the groups, facilitating the statistical interpretation and validation of the technique. This study aimed to verify the changes for the characteristics Raman peaks of biomolecules such as immunoglobulins amides and protein. The results were highly significant with a good separation between groups mentioned. The discriminant analysis was performed through the principal components and correctly identified 92% of the donors. Based on these results, we observed the behavior of arthritis autoimmune, evident in certain spectral regions that characterize the serological differences between the groups.

We present here the sequence of the mitochondrial DNA of the pathogenic thermodimorphic fungus Paracoccidioides brasiliensis, agent of an endemic disease in most South American countries. The sequenced genome has 71 334 bp and is organized as a circular molecule with two gaps of unknown size flanking the middle exon of the nad5 gene. We located genes coding for the three subunits of the ATP synthase (atp6, atp8 and atp9), the apocytochrome b (cob), three subunits of the cytochrome c oxidase enzyme complex (cox1, cox2 and cox3), seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase (nad1, nad2, nad3, nad4, nad5, nad6 and nad4L) and the large (rnl) and small (rns) subunits of ribosomal RNA. Two maturases and a ribosomal protein (rms5) are located inside introns. Twenty-five tRNAs were identified with acceptors for all 20 amino acids. Seven polypurine/polypyrimidine tracts (140–240 bp) have been found in this genome. All genes are in the same orientation over the genome, while their order is closest to the mitochondrial genomes from Penicillium marneffei and Aspergillus nidulans. This sequence is available under GenBank Accession No. AY955840. Copyright © 2007 John Wiley & Sons, Ltd.

Comparative studies were carried out on the Al- phthalocyanine and Zn-phthalocyanine uptake in iliac artery wall of rabbit, through transadventitial fluorescence measurements. Two 3.5 Kg Norfolk make rabbit were used. The iliac artery was dissected, isolated and clamped at two points, 1.5 cm apart. The experiment comprised three steps, collecting fluorescence spectra at several point inside and outside of the clamped region of the artery. The excitation source was a 488 nm Argon Laser. Firstly, it was measured the pre and post clamping transadventitial fluorescence without the photosensitizer. After that, 50 (mu) L of AlPc or ZnPc solutions in Cremiphor EL, 2.5 (mu) M, were injected locally into the artery clamped region of each rabbit and fluorescence spectra were collected at t equals 0 and 15 min. Finally, elapsed 30 min. Temporal evolution of the drug uptake by the artery wall was studied analyzing the intensities of the fluorescence peaks at 682 and 678 nm for AlPc and ZnPc, respectively. It was found that AlPc is more absorbed and remains for a longer time in the iliac artery wall than the ZnPc.

The aim of this work was to verify the Al-Phthalocyanine (AlPc) absorption in iliac artery wall of a rabbit, through transadventitial fluorescence measurement. Two Norfolk male rabbits, weighing 3.5 kg were used. The iliac artery was dissected and isolated. The artery was then clamped in two points with simple ligatures spaced 1.5 cm. The experiment was made in three phases, with acquisition of several fluorescence spectra in different points of the iliac artery. Firstly was measured the pre and post clamping transadventitial fluorescence without the presence of the photosensitizer. On the second phase, the AlPc, 2.5 (mu) M in 50 (mu) L solution, was injected locally into the clamped region and fluorescence spectra were collected in t=0 minutes and t=15 minutes. Finally, 30 minutes from the beginning of the experiment the clamping was removed, the blood flow restarted into the artery, and the fluorescence spectra was collected in t=5 minutes and t=10 minutes. The excitation was made using an Argon Laser (Stabilite 2017-Spectra Physics), operating in 488nm with 7mW optical power measured at the distal fiber tip. A unique peak, centered in 682nm, identifies the AlPc presence on the analyzed tissue.

As doenças alérgicas alcançaram proporções mundialmente epidêmicas. A ativação de receptores para IgE, Fc RI, em mastócitos, é o mecanismo chave para a iniciação e propagação das respostas patofisiológicas dos processos alérgicos. Após a interação destas células com um alérgeno, há a ativação de uma cascata de eventos de sinalização, a qual resulta na secreção de mediadores alérgicos pré-formados, através de um processo regulado de exocitose, além da síntese e secreção de mediadores lipídicos e citocinas. Desta forma, a inibição da responsividade dos mastócitos, quando ativados por um alérgeno, representa uma via importante para o desenvolvimento de novos candidatos a fármacos com indicação antialérgica. Neste sentido, este trabalho buscou, num primeiro momento, contribuir com a compreensão dos papéis desempenhados por um glicerofosfolipídeo de membrana, fosfatidilinositol 4,5-bifosfato (PtdIns(4,5)P2), em eventos de sinalização em mastócitos, mediados por IgE. Este trabalho permitiu destacar a importância de PtdIns(4,5)P2 como um regulador chave das respostas de Ca2+ e das alterações de morfologia de mastócitos estimulados por um alérgeno. Foi observado ainda que níveis reduzidos de PtdIns(4,5)P2 determinaram a inibição do processo de endocitose de receptores Fc RI ativados, um evento crucial para a redução da transdução de sinais. Estes resultados não somente trazem um ganho de conhecimento acerca dos detalhes que orquestram os eventos de sinalização em mastócitos estimulados por um alérgeno, como também apontam que a regulação dos níveis de PtdIns(4,5)P2 pode certamente ser apontada como alvo para o desenvolvimento de novas moléculas inibidoras da ativação mastocitária. A segunda etapa deste trabalho teve como objetivo avaliar o potencial inibitório de alguns compostos de origem natural e sintética sobre a degranulação de mastócitos, evento em que mediadores alérgicos, como a histamina, são secretados, em resposta ao estímulo celular. Inicialmente, avaliou-se o efeito inibitório de um conjunto de arilcumarinas sintéticas, estruturalmente relacionadas, sobre a degranulação. Um número significativo de moléculas foram ativas e, dentre elas, algumas substituições junto à estrutura do anel 3-fenilcumarínico, como as hidroxilações das posições 6, 2\'e 5\', puderam ser identificadas como importantes para o potencial bioativo. Finalmente, o trabalho apresentou uma molécula de origem natural, como um potente inibidor da degranulação de mastócitos e da secreção de citocinas. Trata-se da piridovericina, um metabólito secundário, isolado do fungo entomopatogênico Beauveria bassiana. Dessa forma, tanto as cumarinas, como a piridovericina podem ser apontadas como compostos de partida de grande potencial para o desenvolvimento de novos fármacos anti-alérgicos. Allergic diseases have approached epidemic proportions worldwide. The activation of IgE receptors, Fc RI, from mast cells, is the key event for the initiation and propagation of pathophysiological responses involved in the allergic processes. The interaction between mast cells and allergens triggers a signaling cascade, which results in secretion of pre-formed allergic mediators, through regulated exocytosis, in addition to the synthesis and secretion of lipid mediators and cytokines. In this way, the inhibition of mast cell responsiveness, upon allergen stimulation, represents an important pathway for the development of new antiallergic drug candidates. Thus, the present work tried, firstly, to gain better insight of the roles played by phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) during IgE-mediated mast cell signaling. This work highlighted the importance of PtdIns(4,5)P2 as a key regulator of Ca2+ responses and mast cell morphological alteration, when activated by an allergen. It was observed that reduced levels of PtdIns(4,5)P2 determined the inhibition of activated Fc RI endocytosis, a crucial event for signal transduction termination. Those results not only improve the actual knowledge in mast cell signaling but also point out the regulation of PtsIns(4,5)P2 levels as a target to be pursued during the development of new inhibitors of mast cell activation. The second part of this work aimed to evaluate the capacity of both synthetic and natural compounds to inhibit mast cell degranulation, characterized by the release of granule-contained allergic mediators, such as histamine, upon cell stimulation. Initially, the inhibitory effect of a set of structurally related synthetic arylcoumarins was evaluated. A significant number of molecules were active, and a few substitutions within such molecules could be pointed as important for the biological activity, such as the hydroxylation of carbons 6, 2\' e 5\' of the 3-phenylcoumarin ring. Lastly, this work presents a natural compound as a potent inhibitor of mast cell degranulation and cytokine secretion. The refered compound is pyridovericin, a secondary metabolite isolated from the entomophatogenic fungus Beauveria bassiana. Therefore, both the coumarin derivatives and pyridovericin can be regarded as lead compounds for the development of new anti-allergic drugs.

O fungo dimórfico Paracoccidioides brasiliensis, é o agente etiológico da paracoccidioidomicose, uma das micoses sistêmicas humanas mais prevalentes na América Latina. Componentes da cadeia respiratória mitocondrial são potenciais alvos quimioterapêuticos. Nesse sentido, em nosso trabalho demonstramos diferenças entre a cadeia respiratória do fungo P. brasiliensis e do hospedeiro mamífero. A respiração, potencial de membrana e fosforilação oxidativa mitocondrial de esferoplastos de P. brasiliensis foram avaliados in situ, os quais demonstraram a presença de uma cadeia respiratória funcional. A adição de ADP induziu a transição da respiração do estado de repouso para o fosforilativo, em esferoplastos energizados com succinato, NADH, NAD+ e substratos ligados ao complex I. A presença de uma NADH-ubiquinona oxidoredutase foi demonstrada pela capacidade das células oxidarem NADH exógeno, assim como, pela respiração insensível a rotenona e sensível a flavona. Além disso, a respiração mantida por NAD+ foi sensível a rotenona e flavona, sugerindo que vias metabólicas citosolicas contribuem para a produção de NADH e substratos ligados ao complexo I. Foi demonstrado também que os níveis de expressão do complexo I e da NADH desidrogenase alternativa alternam-se durante a curva de crescimento de leveduras de P. brasiliensis. A respiração induzida por NADH ou succinato foi parcialmente inibida por KCN ou antimicina A e sensível à inibição por BHAM, indicando a presença de uma oxidase alternativa. A fim de se caracterizar esta enzima oxidase alternativa, o seu gene foi clonado e expressado em xii S. cerevisiae e E. coli, o qual conferiu uma respiração resistente a cianeto e sensível a BHAM. S. cerevisiae expressando oxidase alternativa mostraram uma menor taxa de multiplicação celular e diminuição na geração de EROs. Nós também observamos que inibidores da cadeia transportadora de elétrons retardaram a transição de micélio para levedura em culturas de P. brasiliensis. Além disso, estes inibidores e outras drogas geradoras de EROs aumentaram a expressão do gene da oxidase alternativa, assim como a produção de EROs em leveduras de P. brasiliensis. Paracoccidioides brasiliensis, a thermically dimorphic fungus, is the etiological agent of endemic paracoccidioidomycosis, one of the most prevalent human systemic mycosis in Latin America. Components of the respiratory chain constitute potential pharmacological targets, and here are reported differences between the respiratory chain of the mammalian host and the fungus P. brasiliensis. Respiration, membrane potential and oxidative phosphorylation of mitochondria from P. brasiliensis spheroplasts were evaluated in situ, which demonstrated the existence of a functional respiratory chain. Adenosine 5\'-diphosphate (ADP) induced a transition from resting to phosphorylating respiration in mitochondria energized by succinate, NADH, NAD+ and complex I linked substrates. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: the ability of the fungus to oxidize exogenous NADH; the insensitivity substrate-supported respiration to rotenone and sensitivity of this respiration to flavone. In addition, the sensitivity of NAD+-supported respiration to rotenone and flavone suggest that citosolic pathways contribute to NADH and complex I linked substrates production. Moreover, it was demonstrated that expression levels of complex I and alternative NADH dehydrogenase change during P. brasiliensis growth curve. The partial sensitivity of NADH or succinate-supported respiration to antimycin A and cyanide, as well as the sensitivity to BHAM, indicates the presence of an alternative oxidase. Therefore, to characterize the alternative oxidase its gene was cloned and heterologously xii expressed in S. cerevisiae and E. coli, wich confered, a cyanide resistant respiration and BHAM sensitivity. Moreover, S. cerevisiae that expressed alternative oxidase showed slower growth rate and decreased ROS generation. We also observed that electron transport pathways inhibitors delayed the P. brasiliensis mycelium to yeast transition in cultures. Besides, these inhibitors and other ROS generated drugs increase the ROS production and altenative oxidase expression.