Made available in DSpace on 2014-06-12T18:28:04Z (GMT). No. of bitstreams: 2 arquivo1143_1.pdf: 1341192 bytes, checksum: 28c1a0abdeb803275cd83cc199fae089 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / INSTITUTO DE MEDICINA INTEGRAL PROF FERNANDO FIGUEIRA / Esta tese, apresentada em dois artigos, teve por objetivo comparar dois tratamentos para Vaginose Bacteriana, utilizando metronidazol e aroeira, em aplicação tópica vaginal e estudar o perfil clínico, epidemiológico e bacteriológico das mulheres participantes, antes de serem submetidas ao tratamento. O primeiro artigo consistiu de um ensaio clínico randomizado, duplamente mascarado, que comparou a eficácia entre os dois tratamentos em mulheres com vaginose bacteriana diagnosticada, concomitantemente, pelos critérios de Amsel e Nugent. Foi utilizada a Análise por Intenção de Tratar. Do total de 277 participantes do ensaio clínico, 137 mulheres foram tratadas com gel de Aroeira e 140 tratadas com Metronidazol. Na avaliação de cura pelos critérios de Amsel, 21,2% das pacientes que utilizaram aroeira e 62,1% que usaram metronidazol obtiveram cura. Quando o Escore de Nugent foi utilizado foram curadas 13,9% das mulheres do grupo da aroeira e 56,4% do grupo metronidazol. A cura total (com a utilização dos dois critérios) foi observada em 12,4% do total de pacientes no grupo da aroeira e 56,4% das mulheres que usaram metronidazol. O segundo artigo constou de uma série de casos onde foram estudados os achados clínicos, epidemiológicos e microbiológicos das participantes do ensaio clínico antes do tratamento, além da presença de lactobacilos nas citologias oncóticas e a população bacteriana componente das microbiotas vaginais identificadas por culturas de secreção vaginal. Entre as queixas clínicas, as mais frequentes foram o corrimento genital, observado em 74,4% das participantes e o odor de peixe da secreção vaginal, que ocorreu em 68,6% dos casos. As culturas de conteúdo vaginal permitiram a identificação de Gardnerella vaginalis em 96,8% e de Mobiluncus em 53,1% dos casos. Apenas em uma terça parte dos exames (32,1%) havia a presença de Lactobacillus. Como conclusões dos estudos, foram observados índices de cura menores com a utilização de Aroeira do que com Metronidazol; os efeitos colaterais foram pouco frequentes e sem maior gravidade em ambos os grupos e continuam necessários novos estudos que melhor elucidem as inter-relações entre os achados microbiológicos e a expressão clínica da doença

Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles ­ in G. intestinalis mitosomes, not­participating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one­ and two­dimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...

Trichomonas vaginalis and Tritrichomonas foetus are the etiologic agents of human and bovine trichomoniasis, respectively. As microaerophilic protozoans; both share a wide array of clinical manifestations ranging from vaginitis to abnormal pregnancies. Human trichomoniasis receives minimal public health attention despite of its worldwide high prevalence rate. Emerging evidence of metronidazole-resistant T vaginalis strains facilitates a concern to understand this protozoan. Cysteine proteases have been implicated as important virulence factors produced by T vagina/is. This study explores the expression of one particular legumain-like cysteine protease known as Tv AE 1. Furthermore, it highlights the relationship between inhibitory effects of trichomonal cells caused by sanguinarine and chelerythrine. A system for obtaining legumains by expressing it in methylotrophic yeast, Pichia pastor is, has been described. The recombinant legumains were produced and processed by the yeast to their inactive and mature forms. Secondly, T foetus cells were transfected with TvAEl construct. Localization and enzymatic studies on legumains will provide evidence into the pathogenicity ofT vagina/is. This study revealed the vesicularization of recombinantly unprocessed TvAEl proteins. Thirdly, plant derived compounds, sanguinarine (SA) and chelerythrine (CHE) were assessed in vitro for their inhibitory effects against T vagina/is and T. foetus. Treatment of SA and CHE for 24 h led to a significant inhibitory growth of in vitro cultures for all three trichomonal strains, G3, Tl and Dl, compared to untreated cells. For these bovine and human trichomonal strains, SA was slightly more effective inhibitor than CHE. With IC5o values between 3 - 8 micromolar for the alkaloids, CHE had less inhibitory effect compared to SA. These findings are significant considering the association between cysteine pro teases and trichomoniasis. Further elucidation of the exact anti protozoal mechanism of both compounds toward legumains may lead to the development of these potent agents against trichomonads.

Trichomonas vagina/is is a common sexually transmitted disease, affecting women more often than men. It has only been seen to undergo mitosis, even though published studies have confinned the organism has meiotic proteins. These meiotic proteins are known to function in other organisms with a key protein in homologous recombination, DMCl. RT-PCR analysis shows low expression ofDMCl in mitotically-growing cultures, and we found that some stresses on the organism increase DMCl expression. Polyclonal antibodies raised against DMCl protein have been used to test whole celllysates of the Tl and G3 strains of Trichomonas vagina/is but no obvious expression has been detected. We also used western blot analysis to show that superoxide dismutase is expressed in the standard lab strains Tl and G3 and immunocytochemistry studies showed that HA-tagged SOD6 protein localizes in the cytoplasm. Lastly, we found that SOD protein abundance increased in the CDC085 strain compared to Tl and G3, especially under aerobic conditions.

Trichomonas vaginalis is a protozoan parasite responsible for causing nearly eight million cases of Trichomoniasis every year in the United States. Trichomoniasis is often an asymptomatic infection, but in some cases it does lead to mild clinical manifestions. Trichomoniasis is easily treated with a single dose of Metronidazole. Drug resistance is not common, but it is on the rise. In addition to increasing rates of drug resistance, Trichomoniasis also poses a public health threat as it has been shown to increase the risk of HIV transmission. In order to combat this emerging public health threat, we must better understand the mechanism by which metronidazole exerts its action on T. vaginalis , as well as how the parasite has responded by evolving mechanisms of drug resistance on a molecular level. In order to investigate these questions, a microarray analysis of two distinct strains of T. vaginalis , one being more virulent and slightly less metronidazole sensitive, was performed. This allowed the identification of several genes that may play a role in virulence and drug susceptibility. Once these genes were identified, their differential regulation was further confirmed by Northern Blot analysis. One of these genes, Thioredoxin Reductase (TrxR) was then cloned and transfected into T. vaginalis . After confirming expression of the HA-tagged TrxR, the cell line was then used to determine the effect of over-expression of the gene on drug sensitivity. The metronidazole IC 50 for this cell line was compared to wild type cells. Additionally, immunostaining of the transfected cells was performed to determine the localization of the HA-tagged thioredoxin reductase. The results of this investigation provide further support for the role of TrxR in metronidazole activation as well as metronidazole sensitivity. Additionally, several other genes identified as differentially regulated may play a role in virulence, and should be targeted for further investigation.

Trichomonas vaginal is is the sexually transmitted agent of trichomoniasis. Although the organism is believed to only reproduce via binary fission, genes specific to 5 Meiosis and Homologous Recombination have been identified in its genome. It is unclear whether the organism has the ability to undergo sexual reproduction or if it has lost that ability over time. Aside from meiosis, these genes could be expressed for use in antigenic variation and in the creation or transfer of resistance genes to other cells. In this study, we induced the expression ofDMCl and Rad51 homologues-key players in Homologous Recombination-using a system of tetracycline induction. We localized DMCl to both the cytoplasm and the nucleus, while Rad51 is localized to the nucleus. We performed a DNA strand exchange that suggests DMCl may be capable of DNA strand exchange. We also developed a system to determine whether haploid cells of Trichomonas vaginal is are capable of cytoplasmic fusion through the use of fluorescent proteins. Specifically, this study focuses on a line of Green Fluorescent Protein-expressing cells.

Trichomoniasis is a common STD caused by the parasitic protozoan Trichomonas vaginalis. The parasite is estimated to have infected roughly 3.7 million Americans. Complications from trichomoniasis can lead to cervical cancer in women and prostate cancer in men. One of the mechanisms of the parasite employs is using cysteine proteases to break down the cellular matrix of its host. However, three endogenous phytocystatin-like protease inhibitors have been found within the parasite’s genome. By recombinantly expressing these cystatins we have been able to test their ability to inhibit cysteine proteases such as papain and those found in T. vaginalis to find their effectiveness. By characterizing these inhibitors, it appears that they are effective at reducing the ability of T. vaginalis cysteine proteases and thus could be useful against the pathogenicity of the parasite.

Trichomonas vaginalis is an anaerobic, parasitic protozoan, responsible for trichomoniasis, the world’s most common, non-viral sexually transmitted infection. Lacking many of the defenses present in other organisms to combat oxidative stress, Trichomonas vaginalis relies extensively on the thioredoxin system—NADPH, thioredoxin reductase, and thioredoxin—as a means to protect against exposure to excess oxygen. Current trichomoniasis treatment relies exclusively on the 5-nitroimidazole drugs, but fear of drug-resistant strains and allergic reactions to 5-nitroimidazole treatment necessitate the discovery of a new treatment method for trichomoniasis. Previous research has shown that auranofin, an FDA-approved drug, was effective at inhibiting activity of one of Trichomonas vaginalis’ isoforms of thioredoxin reductase (of which the organism has five total). Our research showed that only two of the isoforms were transcribed and expressed at high levels, and that both of these isoforms were susceptible to auranofin treatment. Not only that, these two isoforms were also shown to be susceptible to various auranofin analogs, having comparable or lower IC50 values. Further tests on these analogs might show that they are actually better treatment candidates if they exhibit less symptoms than auranofin. Experiments examining how mRNA and protein levels were modulated in response to two different concentrations of auranofin treatment showed that while some isoforms show increased levels, no one isoform experienced any drastic changes. Together, this data suggests that further studies should focus on these two most highly expressed isoforms of thioredoxin reductase.

Trichomonas vaginalis é o agente etiológico da doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos de tricomonose a cada ano. O estabelecimento da infecção se deve principalmente à capacidade de adesão do parasito às células epiteliais vaginais, cervicais ou de próstata, seguida pela intensa reação inflamatória, resultado da infiltração de neutrófilos no sítio da infecção. Nucleotídeos e nucleosídeos, especialmente ATP e adenosina, são liberados para o espaço extracelular por células em situações de estresse ou injúria tecidual e desenvolvem seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, as ectonucleotidases, NTPDase e ecto-5’-nucleotidase, são capazes de hidrolisar os nucleotídeos gerando adenosina e finalmente, a enzima adenosina deaminase (ADA) é responsável pela conversão de adenosina em inosina. A expressão gênica de cinco NTPDases putativas presentes no genoma de T. vaginalis foram investigadas, assim como o envolvimento da sinalização purinérgica na relação parasito-hospedeiro. Nossos resultados mostraram que diferentes isolados de T. vaginalis expressam os genes TvNTPDase1, 2, 3, 4 e 5, sendo observado o maior número de transcritos para TvNTPDase1, 2 e 4. A sequência preditiva de aminoácidos revelou a presença das cinco regiões conservadas da apirase, domínios transmembrana, sítios de fosforilação, peptídeos sinais e os prováveis sítios ativos da enzima. A análise filogenética demonstrou maior similaridade das TvNTPDases com as formas intracelulares da enzima, como as NTPDases 4 e 7 humanas e a de Saccharomyces cerevisiae. Além disso, a restrição de soro promoveu aumento significativo da atividade da NTPDase de T. vaginalis, no entanto sem corresponder com o aumento de expressão gênica de determinada(s) sequência(s). Quanto à participação da sinalização purinérgica na resposta inflamatória de células do hospedeiro frente ao parasito, foram utilizados como modelos celulares as células epiteliais vaginais (HMVII), cervicais (HeLa) e neutrófilos humanos. As linhagens HMVII e HeLa mostraram expressar todos os subtipos de receptores P1, P2X e P2Y e XI os diferentes isolados de T. vaginalis, que foram cocultivados com as células, mostraram hidrolisar eficientemente os nucleotídeos ATP, ADP e AMP. Ainda, o isolado clínico fresco TV-LACM6 foi o único a apresentar elevada citotoxicidade frente às células epiteliais vaginais e cervicais, no entanto não foi detectado aumento da liberação de ATP pelas células após o cocultivo, provavelmente devido à alta atividade da enzima NTPDase observada nesse isolado. Os trofozoítos de T. vaginalis não foram capazes de aumentar a produção de IL-8 e IL-6 pelas linhagens HMVII e HeLa, e apenas os isolados ATCC30236 e TV-LACM6 causaram aumento na secreção da citocina MIP-3α pelas células epiteliais cervicais. Finalmente, o nucleotídeo ATP e o nucleosídeo adenosina não modularam a produção dos mediadores inflamatórios investigados. Em relação aos neutrófilos, estes mostraram aumentar a produção de espécies reativas de oxigênio (ERO) e IL-8 após incubação com os trofozoítos de T. vaginalis. Os nucleotídeos e nucleosídeos da adenina e guanina não produziram efeito na produção de ERO e IL-8; no entanto, quando o nucleosídeo adenosina foi incubado junto com o inibidor da enzima ADA (EHNA) observou-se uma redução significativa da produção de ERO e IL-8 pelos neutrófilos, devido à inibição da ADA e consequentemente, ao aumento da concentração de adenosina disponível no meio extracelular. Os nossos resultados indicaram a ativação do receptor A1 dos neutrófilos nessa condição. O conjunto de dados aqui obtidos contribuiu para uma melhor caracterização da família de enzimas NTPDases de T. vaginalis assim como para um maior conhecimento acerca da influência da sinalização purinérgica na relação parasito-hospedeiro. / Trichomonas vaginalis is the agent of the most common non-viral sexually transmitted disease worldwide, causing 276.4 million new cases a year. The establishment of the infection is closely related to the parasite ability to adhere to vaginal, cervical and prostate epithelial cells, followed by an intense inflammatory response as result of neutrophil infiltration. Nucleotides and nucleosides, mainly ATP and adenosine, are released into the extracellular space by cells under stress or injury and they exert their signaling effects through activation of the purinoceptors. Moreover, the ectonucleotidases, NTPDase and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing adenosine and finally, the adenosine deaminase (ADA) is responsible for the conversion of adenosine to inosine. We investigated the gene expression of five putative NTPDases found in T. vaginalis genome as well as the involvement of purinergic signaling on the host-parasite relationship. Our results showed that different T. vaginalis isolates are able to express TvNTPDase1, 2, 3, 4 and 5 and that TvNTPDase1, 2 and 4 are the most expressed genes. Predictive amino acid sequence revealed the presence of the five apyrase conserved regions, transmembrane domains, phosphorylation sites, signal peptides and the active sites. Phylogenetic analysis showed that TvNTPDases share more similarity with the intracellular enzymes, such as human NTPDase 4 and 7 and Saccharomyces cerevisiae NTPDase. In addition, the serum limitation caused a significant increase in NTPDase activity, but without association with the gene expression of a specific TvNTPDase sequence. Regarding the participation of purinergic signaling on the inflammatory responses against the parasite, the vaginal (HMVII) and cervical (HeLa) epithelial cells and the human neutrophils were used as cellular models. HMVII and HeLa cell lines showed to express all subtypes of P1, P2X and P2Y receptors and the different T. vaginalis isolates, which were co-cultured with the cells, showed to hydrolyze efficiently ATP, ADP and AMP. Furthermore, only the fresh clinical isolate, TV-LACM6, caused a profound cytotoxicity against the vaginal and cervical epithelial cells. Interestingly, it was not detected an increase in ATP release by the cells after cocultivation, probably due to the high NTPDase activity dislplayed by TV-LACM6 isolate. The T. vaginalis trophozoites were not able to increase the production of IL-8 and IL-6 by HMVII and HeLa cells and only ATCC30236 and TV-LACM6 isolates enhanced MIP-3α secretion by the cervical epithelial cells. Finally, neither ATP nor adenosine has modulated the production of the inflammatory mediators here investigated. Considering the neutrophils, T. vaginalis stimulated the production of reactive oxygen species (ROS) and IL-8 by these immune cells and both adenine as guanine nucleotides and nucleosides did not cause any effect on ROS and IL-8 levels. However, when adenosine was incubated with an ADA inhibitor (EHNA) we observed a significant reduction of ROS and IL-8 production by neutrophils, due to inhibition of ADA with a subsequent increase of adenosine concentration in the extracellular milieu. . Our results suggested the participation of A1 receptor in this condition. The data set obtained in this study contributed to the characterization of T. vaginalis NTPDases family as well as to a better understanding of the influence of purinergic signaling on host-parasite relationship.

Made available in DSpace on 2014-12-17T15:42:11Z (GMT). No. of bitstreams: 1 RusceliDA_DISSERT.pdf: 4254928 bytes, checksum: 4fab1232a45d2adb5dcfe7b1b0dac83a (MD5) Previous issue date: 2013-04-05 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Recently, marine organisms have attracted attention because of the complexity and potent biological activity from your secondary metabolites. Our planet has 80% it surface covered by oceans and seas, therefore, housing a wide number of different forms of life, among them, the sponges. These sessile and filtrating animals, according to numerous researches, come showing like true chemistry factories. The substances from these animals, sometimes show as news targets to therapeutics agents, and some countries has already use them for treatment of some diseases. Further of the secondary metabolites, the polysaccharides of marine origin also have been target of studies, because the presence of the sulfates groups in its molecules. Polysaccharides with differents biological activities have been related in a large number of researches. Actually, many studies show the sponges as source of promising medicine. These studies inspire new researches, because the few number of sponges species studied until now. Because of that, the present work shows the chemistry prospection of the sponge Callyspongia vaginalis. Chromatographic methods in silica gel allowed the isolations of two secondary metabolites: the known β- sitosterol and a ceramide, no reported in the genus Callyspongia, previously. The analysis of the their lipid extracts show different kinds of fatty acids with a variety of chain length (saponifiable fraction), and others metabolites like Lupenone and stigmasterol, also unprecedented in the genus. The Polysaccharide characterization and the elucidation of the secondary metabolites acquired through of chromatography analysis (CC, molecular exclusion) and spectrometric (NMR 1H and 13C, mass, IR), respectively and comparison with literature data / A diversidade estrutural de subst?ncias de origem marinha, associadas as suas atividades biol?gicas, t?m atra?do aten??o de pesquisadores do mundo inteiro. Dentre os organismos estudados atualmente dois grupos de invertebrados marinhos, as asc?dias e as esponjas, merecem destaque. Estes animais s?sseis e filtradores, segundo in?meras pesquisas, v?m se mostrando verdadeiras f?bricas qu?micas , produzindo subst?ncias que por vezes apresentam-se como aspirantes a agentes terap?uticos, com efic?cia cl?nica comprovada. Al?m dos metab?litos secund?rios, os polissacar?deos de origem marinha tamb?m tem sido alvo de estudos, sendo relatados diversos compostos sulfatados e com diferentes atividades biol?gicas associadas a estes. Tendo em vista a pequena quantidade de esp?cies de esponjas estudadas at? o momento, comparado ao potencial qu?mico destes organismos, o presente trabalho apresenta a prospec??o qu?mica da esponja Callyspongia vaginalis. T?cnicas cl?ssicas de cromatografia em gel de s?lica permitiram o isolamento de dois metab?litos secund?rios: o β-sitosterol e uma ceramida, ambos in?ditos no g?nero Callyspongia. A an?lise das fra??es lip?dicas mostraram diferentes tipos de ?cidos graxos com variados tamanhos de cadeia (fra??o saponific?vel), e de outros metab?litos como lupenona e estigmasterol, tamb?m in?ditos no g?nero em quest?o. O polissacar?deo obtido atrav?s do material animal tamb?m foi isolado e, como amplamente relatado na literatura apresentou-se sulfatado, caracter?stica diferencial dos polissacar?deos de origem marinha. A caracteriza??o do polissacar?deos e a elucida??o estrutural dos metab?litos secund?rios foram realizadas atrav?s da an?lise dos dados de espectrometria de massa, Infravermelho e Resson?ncia Magn?tica Nuclear de Hidrog?nio-1 e Carbono-13, atrav?s de sequ?ncias de pulsos uni e bidimensionais e compara??o com dados da literatura