This thesis describes the use of a new technology, the Cytosensor® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. The Cytosensor® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure. In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development. In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. It is concluded that measurement in the Cytosensor® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents.

During the last decades, chemotherapy with cytotoxic drugs has played a significant role in cancer therapy. It’s important to develop new anticancer drugs, and drug sensitivity testing in vitro can be used to find the right diagnosis for the newly developed substances. The aim of this study was to investigate the cytotoxic activity of the new signal transduction modulators bortezomib, gefitinib and PKC412. The well-established substances cisplatin, cytarabine, doxorubicin and vincristin were investigated for comparison. The activity of the cytotoxic drugs was analysed in human tumor samples from patients with ovarian carcinoma (n=16) and lymphoma (n=15) by using the Fluorometric Microculture Cytotoxicity Assay (FMCA). The testing of cellular drug resistance by FMCA was accomplished successfully in 33 out of the 34 samples (97%). The results of this study indicated that the activity of cytotoxic drugs in tumor cells obtained from patients with ovarian carcinoma and lymphoma may be detected by the FMCA. It also suggested that bortezomib and gefitinib could represent promising agents for treatment of ovarian carcinoma and that PKC412 might be of less use for patients with this diagnose.

Cytotoxic T Lymphocytes (CTL) are known to play an important role in clearing infectious virus from infected hosts in a variety of viral infections. Depending on the type of virus and mode of virus entry both class I and class II restricted CTL can contribute to protection from virus-induced disease. Although CD8 positive CTL are associated with virus elimination and control in many viral infections, elimination of neurotropic viruses from the Central Nervous system (CNS) is more complex due to the lowered expression of MHC antigens on neuronal cells. This failure to constitutively express high levels of MHC antigens by neurons could serve as an advantage to avoid damage to this differentiated and non-renewable tissue. However, abnormal induction of MHC antigens in the CNS mediated by CD4 positive lymphocytes or by astrocytes have also been shown to cause destructive inflammation in the CNS. The present study deals with CTL responses against one such neurotropic virus called Japanese Encephalitis Virus (JEV). JEV is a positive-stranded RNA virus that belongs to the flavivirus group, a group that is among the most important agents causing human encephalitis worldwide. Although passive transfer of monoclonal antibodies against this virus has been shown to confer protection of mice from lethal challenge with virus, neither the presence of CTL against this virus nor its role in conferring protection has been reported so far. Understanding the CTL responses against these viruses acquired importance in light of recent reports that neurovirulence of JEV and yellow fever viruses can be enhanced by the administration of virus specific antibodies. Hence this study was undertaken to examine the possibility of raising CTL specific to JEV. The specificity of the CTL raised, their therapeutic value and the ability of different lymphocyte subsets to mediate protection in vivo are dealt with in this study. Generation of CTL against JEV The generation of CTL against JEV in BALB/c mice, requires MHC defined cell lines that not only support virus infection but are also histocompatible. Several cell lines were initially examined for their ability to support JEV infection as a prc-rcquisitc before their utilization in in vivo and in vitro stimulation protocols aimed at generating JEV-specific CTL. Virus infection was monitored by immunofluorescence using JEV envelope-specific monoclonal antibodies as well as by titration of virus produced from infected cells by plaque assays. These different cell lines that were characterised for their ability to support JEV infection were then utilised to generate and monitor antiviral CTL. Several in vivo immunisation protocols were examined initially find out which of these infected cells prime BALB/c mice efficiently for generation of virus-specific CTL upon secondary stimulation in vitro with infected syngeneic cells. Immunisation of mice with infected cells per se was preferred over free virus since this was thought to facilitate priming against some viral non-structural proteins preferentially found on infected cells in addition to other viral structural proteins. It was observed that not only infected syngeneic and allogeneic cells but also infected xenogeneic cells prime BALB/c mice for the generation of JEV- specific CTL upon secondary restimulation in vitro. An optimal protocol was standardised for the generation of CTL against JEV. This included primary in vivo immunisation of mice followed by secondary in vitro restimulation of splenocytes with infected syngeneic cells. Either immunisation alone or in vitro stimulation of naive splenocytes alone was unsuccessful. The effector cells generated specifically lysed JEV-infccted P388D1 targets but not uninfected P388D1 or YAC-1 targets suggesting that the lysis on infected targets is not mediated by Natural Killer activity. Specificity and MHC restriction of anti JEV Effectors Cell depletion studies using complement mediated lysis were performed to examine the phenotype of the cells mediating virus specific lysis of infected targets. Depletion of Lyt 2.2+ or Thy 1+ but not L3T4+ sub-populations of effector cells inhibited lysis of infected targets showing that the effectors mediating virus-specific lysis were Lyt-2+ T cells. Examination of target specificities and MHC restriction of the antiviral CTL generated showed that although infected xenogeneic cells were used for immunisation, the effector cells recognised only infected syngeneic (P388D1, Sp2/0) and semisyngeneic (Neuro 2a, YAC-1) cells. Virus-specific recognition was found to be class I Kd and class I Dd restricted. These effector cells were also found to recognise cells infected with a closely related flavivirus, West Nile Virus (WNV) suggesting that they were crossreactive to some degree. Based on the consensus motif that has been established for H-2Kd associated peptides, several nonamers were predicted as possible CTL epitopes by scanning the deduced amino acid sequences of three strains of JEV and WNV. Among several predicted nonamers, three peptides were examined for their ability to reconstitute lysis of uninfected targets by polyclonal anti JEV CTL populations. Results demonstrate that peptides derived from NS1 and NS3 but not NS5 protein of JEV were able to partially reconstitute lysis of uninfected targets by effectors when pulsed with the appropriate peptide. Protective ability of the CTL raised against JEV To examine whether anti-JEV effectors raised in vitro could confer protection from intracerebral challenge with JEV, these effectors were adoptively transferred into adult BALB/c mice intracerebrally along with 10 x LDJ0 dose of JEV. More than 55% of these animals were protected from death and survived beyond 100 days after JEV challenge demonstrating that adoptively transferred anti-JEV effectors could indeed confer protection from lethal challenge with JEV. However, adoptive transfer of effectors by either intravenous or intraperitoneal routes did not protect adult mice from the lethal effects of intracerebral challenge with JEV. In contrast to adult mice, newborn mice were not protected from death by the adoptively transferred effector cells. This was also supported by experiments where a correlation was observed with the increasing age of mice and the success of protection conferred by the adoptively transferred effector cells. To establish the identity of cell subsets responsible for protection, Lyt 2, L3T4 or Thy 1 positive cells were specifically depleted from the polyclonal CTL by multiple cycles of complement mediated lysis and the remaining cells were adoptively transferred intracerebrally along with 10 x LD of JEV. These results demonstrate that both Lyt 2 and L3T4 positive T cells present in the effector population were necessary to confer protection of adult mice. Examination of virus-specific neutralising antibodies in the sera of protected and unprotected mice revealed that presence of L3T4 positive cells in the adoptively transferred population increases virus-specific neutralising antibodies. However presence of neutralising antibodies alone was not sufficient to confer protection. The protection required both Lyt-2 and L3T4 positive cells together. These studies could in the long term throw some light on similar observations about age dependant susceptibility to JEV in humans.

Thy-1 is a glycosylphosphatidylinositol-anchored protein that is expressed on murine T lymphocytes and is involved in T cell-mediated immune responses. In the presence of costimulatory signals, monoclonal antibody (mAb)-induced signaling through Thy-1 is associated with hallmarks of T cell activation, including IL-2 production and T cell proliferation. Thy-1-induced signaling promotes cytotoxic effector molecule expression, but is unable to trigger delivery of the lethal hit to target cells, suggesting that Thy-1 provides an incomplete T cell receptor (TcR)-like signal. However, the effect of Thy-1 signaling on cytokine production and the development of T helper (Th) cell phenotypes (Th1, Th2, Th17) remains unclear. The purpose of this work was to further our understanding of Thy-1-mediated signal transduction and the role that Thy-1 plays in the development of effector T cell responses. I found that, in the context of costimulatory signals, anti-Thy-1 mAb induced significantly less IL-2 production, CD25 expression and T cell proliferation than anti-TcR? mAb. Several key signaling molecules, including protein tyrosine kinases, zeta chain-associated protein-70 and extracellular signal-regulated kinase were activated with delayed kinetics during Thy-1-mediated T cell activation. The delayed signaling kinetics resulted in the delayed acquisition of cytotoxic effector function and also delayed delivery of the lethal hit to target cells. Interestingly, Thy-1-mediated signaling induced significantly more IL-17 and IL-4 synthesis and less IFN-? synthesis in comparison to TcR-mediated signaling. Moreover, Thy-1-activated CD4+ T cells produced high levels of IL-17 and IL-4 but minimal IFN? when restimulated with anti-Thy-1 mAb or anti-TcR? mAb with or without costimulatory signals. The unique ability of Thy-1 signaling to induce IL-17 production correlated with the expression of the Th17 lineage-specific transcription factor, retinoic orphan receptor gamma t. These observations show that Thy-1 signaling differs from TcR signaling in its ability to induce Th cell cytokines. Taken together, my findings show that Thy-1 signaling can provide the full TcR-like signal required for both the differentiation and triggering of Th cells and cytotoxic T lymphocytes, albeit with delayed kinetics in comparison to TcR signaling. They also suggest that Thy-1 signaling may be important in the development of Th2 and Th17 responses.

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome in Europe. After inhalation of virus shed by bank voles, the virus systemically targets the vascular endothelium leading to vascular dysfunction and leakage. Many patients with PUUV infection experience cardiopulmonary manifestations but the underlying mechanisms have not been determined. The aims of the studies presented were to describe cardiopulmonary manifestations, investigate pathogenetic mechanisms including presence of virus in the lungs and the local immune response in PUUV infection. The results showed cardiopulmonary involvement of varying severity in almost all studied patients. High-resolution computed tomography frequently revealed vascular leakage into the lungs or pleural cavities. Pulmonary function tests generally showed reduced gas diffusing capacity, evidenced in patients as dyspnea, poor oxygenation and frequent need of oxygen treatment. Among patients who were not fully recovered at 3 months follow-up, remaining decreased gas diffusing capacity was highly common. Echocardiography revealed mainly right heart dysfunction which was related to manifestations within the lungs, in terms of increased estimated pulmonary vascular resistance, mild to moderate pulmonary hypertension, and reduced right ventricular systolic function in patients with more pronounced lung involvement, as indicated by need of oxygen treatment. Analyses on bronchoalveolar lavage (BAL) and bronchial biopsies revealed a highly activated cytotoxic T cell (CTL) response in the lungs. The CTL response was not balanced by the expansion of regulatory T cells and high numbers of CTLs were associated with more severe disease. PUUV RNA was detected in almost all patients’ BAL samples and the viral load was inversely correlated to the number of CTLs. Three patients presenting with severe and fatal cardiopulmonary distress were also described. Autopsies revealed PUUV protein in vascular endothelium in all investigated organs, including the heart and lungs, along with a massive CTL response mainly in the lungs. In conclusion, cardiopulmonary involvement of varying severity was present in almost all patients with PUUV infection. Cytotoxic immune responses could contribute to disease development but also help in clearing the infection. Long lasting fatigue after hantavirus infection may be explained by remaining manifestations within the lungs.

Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge Geodia barretti and its ability to inhibit larval settlement of the barnacle Balanus improvisus was determined. With an EC50 value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from Geodia barretti, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT1-7 receptors, whereas barettin possessed specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted with 5-HT4. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC50 value of 34 nM, displayed the highest settlement inhibition. The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus Agelas. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified. In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges Geodia barretti and Agelas spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xii, 239 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

There are four serotypes of dengue virus designated dengue 1, 2, 3 and 4 (D1, D2, D3 and D4) and epidemiological studies indicate that a severe complication of dengue virus infection - dengue hemorrhagic fever (DHF) is more likely to occur following a secondary infection. DHF is hypothesized to be immunologically mediated and may be triggered by virus-specific T cells. It is also likely that dengue virus-specific cytotoxic T lymphocytes (CTLs) are important for recovery from dengue virus infections. An analysis of the immune response during acute illness and when the patient has recovered from the infection (immune state) is therefore important as it will provide insights into the immunopathological nature of the disease. This thesis initially examines the CD8+CTL responses in volunteers who have received live attenuated dengue vaccines and then investigates acute and immune T cell responses in children following natural infection with dengue. When this project was initiated, there was little available information on the human CD8+ T cell responses to dengue viruses. PBMC from one donor had generated memory CD8+CTL to the nonstructural protein NS3 of dengue virus. Memory CD8+CTL responses were therefore analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult American volunteers who had received monovalent live-attenuated candidate vaccines of the 4 dengue serotypes. All the donors had specific T cell proliferation to dengue viruses and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural proteins NS3 and NS1.2a and the structural protein E were recognized by CD8+CTLs from six, five and three donors respectively. All donors recognized either NS3 or NS 1.2a. In a donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using PBMC of two donors were serotype-specific whereas all other donors had serotype-cross reactive responses. For one donor, CTLs specific for E, NSl.2a and NS3 proteins were all HLA-B44 restricted. For the three other donors tested the potential restricting alleles for recognition of NS3 were HLA-B38, A24 and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with a single serotype of dengue virus are diverse and directed against a variety of proteins. The nonstructural proteins NS3 and NSl.2a appear to be immunodominant and should be considered when designing subunit vaccines for dengue. Previously T cell responses had not been examined in people who have had natural infections with dengue. The HLA diversity between North American Caucasians and populations where dengue is a serious health problem, calls for the analysis of immune responses in people who have been infected with natural circulating strains of the virus. We examined the memory cytotoxic T lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue infections. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for the NSl.2a and NS3 proteins respectively. All CTL lines derived from both patients were crossreactive with other serotypes of dengue virus. The CD8+ NS1.2a specific lines from patient KPP94-037 were HLA-B57 restricted and the CD8+ NS3 specific lines from patient KPP94-024 were HLA-B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA-DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize a.a. 221-232 on NS3. These results demonstrate that after symptomatic secondary natural dengue infections in Thai patients, CTLs are mainly directed against nonstructural proteins and are broadly crossreactive. The data correlate with our observations that nonstructural proteins are immunodominant proteins in volunteers who received dengue vaccines. We were interested in examining CTL responses in children during their acute illness and comparing them to memory CTLs obtained from the same children a year or more after the infection. A detailed analysis on samples from nine patients during their acute illness failed to generate any dengue virus-specific CTL responses. We therefore decided to determine if cell mediated responses are altered during acute dengue infection. Decreased proliferative responses to mitogens and recall antigens have been observed in PBMC obtained during several acute human viral infections. All responses of PBMC during acute illness were compared to the same patients PBMC obtained at least 6 months after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid and dengue antigens were significantly decreased in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous immune or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 antibodies restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12 also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation. The data generated from this project shed light on the nature of the immune responses during acute natural dengue infections. It strengthens the existing data on the human memory CD8+CTL responses to dengue viruses and validates the observations by examining memory CTL responses after natural dengue infection in patients from Thailand. In addition, we demonstrate a profound defect in lymphoproliferative responses during dengue illness.

Cumarinas são estruturas promissoras e diversas atividades biológicas têm sido atribuídas a esses metabólitos secundários vegetais. Estudos sugerem que o mecanismo antifúngico desses compostos esteja correlacionado com a atividade antioxidante. A reação da tirosinase, que produz radicais livres, está envolvida no processo de melanização do fungo Sporothrix schenckii, o causador de micose subcutânea de maior incidência no sul do Brasil. A inibição dessa enzima foi recentemente reportada para extrato de Pterocaulon (Asteraceae), planta rica em cumarinas e usada na medicina tradicional do Brasil para tratamento tópico de micoses e na medicina popular da Argentina como anticâncer. O objetivo desse trabalho foi investigar in vitro a atividade antifúngica, correlacionando-a com atividade antioxidante, de extratos de espécies de Pterocaulon e de 6,7-metilenodioxicumarinas isoladas e também de 4-metilcumarinas obtidas por síntese, bem como a investigação da atividade citotóxica de algumas destas moléculas. As 6,7-metilenodioxicumarinas foram isoladas de P. balansae e P. lorentzii, enquanto que as 4-metilcumarinas foram sintetizadas via Pechmann por micro-ondas. A atividade antifúngica contra Sporotrix schenckii foi realizada conforme manual do Clinical and Laboratory Standards Institute. O estudo das propriedades eletroquímicas foi obtido por voltametria cíclica e a capacidade antioxidante pelo método espectofotométrico DPPH (1,1-difenil-2-picrilhidrazil) e pelo método fluorimétrico ACAP contra radicais peroxil. A análise dessa atividade mostrou que os extratos metanólicos de espécies de Pterocaulon (P. polystachyum, P. balansae, P. lorentzii, P. lanatum e P. cordobense) foram ativos frente às cepas do fungo S. schenckii, sendo o extrato de P. polystachyum o mais ativo, apresentando Concentração Inibitória Mínima (CIM) compreendida entre 156 e 312 μg/mL. O fracionamento dos extratos lipofílicos de P. balansae e P. lorentzii levou ao isolamento de três metilenodioxicumarinas. Dados cristalográficos da 5-metóxi-6,7-metilenodioxicumarina, inéditos, foram depositados no Cambridge Crystallographic Data Centre 779123. As cumarinas sintéticas foram obtidas em rendimentos satisfatórios (98-30%) e em reduzido tempo de reação (5-20 min). O screening destas cumarinas frente às cepas do fungo S. schenckii revelou que 5-carboxi-6,7-diidroxi-4-metilcumarina, com CIM de 66 μM e Concentração Fungicida Mínima de 246 μM, foi o composto mais ativo. Essa cumarina apresentou sinergismo com a Anfotericina B, sendo sua CIM reduzida para 15 μM. A atividade antifúngica desses compostos pode estar correlacionada com a atividade antioxidante. O composto 5-carboxi-6,7-diidroxi-4-metilcumarina foi o mais ativo mostrando elevada capacidade antioxidante frente aos radicais DPPH com valores de IC50 de 17,49 μM e elevada atividade frente ao radical peroxil. Além disso, apresentou um potencial de oxidação de 0,4 V sugerindo atividade antioxidante. Baseado nos ensaios antioxidante e antifúngico foi possível observar que a presença de grupamentos hidroxilas no C-7 e C-8 do anel cumarínico, assim como a adição de grupamento polar no C-5 favoreceu ambas as atividades antifúngica e antioxidante. Na segunda etapa desse trabalho foi avaliada a citotoxicidade das cumarinas isoladas e algumas cumarinas simples, disponíveis comercialmente, pelo método Metil Tiazol Tetrazólio (MTT) usando linhagens de glioma humano (U138-MG) e de ratos (C6). Foi observado que as 6,7-metilenodioxicumarinas causaram uma significativa redução na viabilidade celular, sugerindo uma influência positiva do grupamento metilenodioxi sobre essa atividade. O composto 5-metóxi-6,7-metilenodioxicumarina foi o mais promissor (IC50= 34,6 μM e IC50= 31,6 μM para C6 e U-138 MG, respectivamente). Como desfecho desse trabalho, pode-se concluir que as cumarinas apresentaram uma atividade inibitória frente ao crescimento celular de linhagens de glioma e um efeito fungicida sobre S. schenckii, resultados estes que corroboram com o uso popular dessas plantas. / Coumarins are promising structures and diverse biological activities have been attributed to these secondary plant metabolites. Studies suggest that the mechanism of antifungal compounds is correlated with antioxidant activity. The reaction of tyrosinase, which produces free radicals, is involved in the process of melanization of the fungus Sporothrix schenckii, the agent of subcutaneous mycosis with the highest incidence in southern Brazil. The inhibition of this enzyme has recently been reported to extract Pterocaulon (Asteraceae) This plant is rich in coumarins and used in traditional medicine in Brazil for topical treatment of fungal infections and in folk medicine of Argentina as anticancer. The aim of this study was to investigate the in vitro antifungal activity and correlation with its antioxidant properties and cytotoxic activities of extracts of some species of Pterocaulon, as well as the isolated coumarins, and 4-methylcoumarin analogs obtained by synthesis. The 6.7-methylenedioxycoumarins were isolated from P. balansae and P. lorentzii, while the 4-methylcoumarins were synthesized via Pechmann reaction using microwave. The antifungal activity against Sporothrix schenckii was performed as indicated in the Manual of Clinical and Laboratory Standards Institute. A study of the electrochemical properties of coumarins was performed by cyclic voltammetry, by the method of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and against peroxyl radicals (ACAP) by fluorometric method. The analysis showed that the antifungal activity of the methanol extracts of Pterocaulon species (P. polystachyum, P. balansae, P. lorentzii, P. lanatum and P. cordobense) were active against the strains of the fungus S. schenckii, being the extract of P. polystachyum the most active, presenting Minimum Inhibitory Concentration (MIC) compressed between 156 and 312 μg/mL. The fractionation of lipophilic extracts of P. balansae and P. lorentzii led to the isolation of three methylenedioxycoumarins. The crystallographic data of 5-methoxy-6,7-methylenedioxycoumarin were deposited at the Cambridge Crystallographic Data Center 779123. The synthetic coumarins were obtained in satisfactory yields (98-30%) and reduced reaction time (5-20 min). The screening of these coumarins against strains of the fungus S. schenckii revealed that the 5-carboxy-6,7-dihydroxy-4-methylcoumarin was the most active compound, presenting MIC of 66 μM and Minimum Fungicidal Concentration of 246 μM. This coumarin showed synergism with Amphotericin B, and its MIC was reduced to 15 μM. The antifungal activity of phenolic compounds could be related to its antioxidant activities. The compound 5-carboxy-6,7-dihydroxy-4-methylcoumarin was again the most active with IC50 value of 17.49 μM, showing the highest capacity to deplete the radicals DPPH and ACAP, moreover present a oxidation potential of 0.4 V suggesting antioxidant activity. Based on the antioxidant and antifungal tests it was observed that the presence of hydroxyl groups at C-7 and C-8 of the coumarin ring and the addition of polar grouping at C-5 favored both antifungal and antioxidant activities.In the second part of this work, it was evaluated the cytotoxicity of the Pterocaulon compounds and some commercially available coumarins, with simple structure. The cytotoxic potential was determined by Methyl Thiazole Tetrazolium (MTT) test using strains of human glioma (U138-MG) and rat (C6). It was observed that the 6,7-methylenedioxycoumarins caused a significant reduction in cell viability, suggesting a positive influence of the methylenedioxy group in this activity. The compound 5-methoxy-6,7-methylenedioxycoumarin was the most promising (IC50 = 34.6 μM and IC50 = 31.6 μM for C6 and U-138 MG, respectively). In conclusion, this work it was demonstrated that some coumarins showed an inhibitory activity against the growth of glioma cell lines and a fungicidal effect on the S. schenckii. These results corroborate the popular use of these plants.