Fungos isolados de organismos marinhos têm despertado o interesse de pesquisadores no mundo inteiro, por representarem uma fonte diferenciada de metabólitos secundários, devido às condições ambientais as quais esses microrganismos estão sujeitos. Diversas atividades biológicas têm sido relatadas para estas substâncias, principalmente antitumoral, antimicrobiano e antiparasitária. O crescente aumento nos casos de resistência a antibióticos aponta para a necessidade da disponibilidade de novos agentes terapêuticos. Neste contexto, este trabalho objetivou a identificação dos fungos associados a organismos marinhos coletados na Ilha do Arvoredo (SC), visando o isolamento bioguiado de metabólitos de extratos de micélio e meios de cultivo. As atividades antibiótica e antibiofilme frente aos patógenos Staphylococcus epidermidis ATCC 35984 e Pseudomonas aeruginosa ATCC 27853 e anti-Trichomonas vaginalis ATCC 30236 e isolados clínicos, foram investigadas. Todos os fungos isolados foram cultivados por períodos de 7, 14 e 21 dias. Utilizando técnicas moleculares, foram identificados 37 fungos pertencentes ao Filo Ascomycota e um ao Filo Basidiomycota, distribuídos entre 15 diferentes gêneros. O rastreamento de metabólitos quanto à atividade antibiótica foi realizado utilizando a técnica de difusão em agar, obtendo-se 19 amostras com halos de inibição superiores a 10 mm de diâmetro. As atividades antiformação e erradicação de biofilme previamente formado foram avaliadas pelo método de coloração com cristal violeta, e 5% do total de amostras apresentaram inibição de formação e 1% a erradicação de mais de 75% de biofilme de S. epidermidis. Da mesma forma, 5% das amostras foram capazes de inibir ou erradicar entre 51% e 75% biofilmes de P. aeruginosa. Entre os isolados, foram selecionados um fungo pertencente à ordem Sordariales (isolado F22, 14 dias de cultivo) e outro ao gênero Penicillium (isolado F37, 7 dias de cultivo). Os filtrados dos fungos foram particionados com acetato de etila e submetidos à separação cromatográfica, levando à identificação dos compostos mevalonolactona e cis-cyclo(leucyl-tyrosyl) (identificados por técnicas espectroscópicas e espectrométricas), ambos ativos na inibição da formação de biofilme de S. epidermidis ATCC. Mevalonolactona também apresentou atividade frente a isolados clínicos de S. epidermidis revelando, além da atividade antibiofilme, atividade antibiótica em concentração de 0,06 mg/mL. A atividade frente ao parasita T. vaginalis foi avaliada pela técnica de coloração de resazurina e a viabilidade dos trofozoítos caracterizada utilizando trypan blue. Duas amostras dos fungos Hipocrea lixii e Penicillium citrinum mostraram a presença de metabólitos ativos. A inibição do crescimento dos trofozoítos foi observada em 24 horas (MIC=2,5 mg/mL), de acordo com a curva cinética de crescimento. A ausência de hemólise de eritrócitos também foi observada para estas duas amostras. Os resultados obtidos corroboram com os relatos sobre o grande potencial dos fungos marinhos para a descoberta de metabólitos ativos. / Fungi isolated from marine organisms attract the interest of many researchers around the world, since they produce differentiated secondary metabolites due to the environmental conditions to which these organisms are subjected. Several biological activities have been reported for these compounds, mainly as antitumoral, antibacterial and, antiprotozoal. The increasing cases of antimicrobial drug resistance reveal the necessity for the search for new therapeutic agents. In this context, this study aimed to identify the fungi associated with marine organisms collected in the Arvoredo Island (SC/Brazil), in order to isolate bioactive metabolites in extracts of mycelia and broth. The antibiotic and antibiofilm activities against Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 and anti-Trichomonas vaginalis ATCC 30236 and clinical isolates activity were investigated. The isolated fungi from marine organisms were cultured for 7, 14 and 21 days. Using molecular techniques, 36 fungi belonging to the Ascomycota and one to Basidiomycota phyla, were identified, distributed among 15 different genera. Screening for metabolites possessing antibiotic activity was performed using the agar diffusion assay, for which was obtained 19 samples with inhibition halo higher than 10 mm in diameter. The inhibition and eradication of biofilm were performed by cristal violet staining method, and 5% of the total samples showed inhibition and 1% eradication of more than 75% of S. epidermidis biofilm. Moreover, 5% of the samples were able to inhibit and eradicate between 51% and 75% of biofilm formed by P. aeruginosa. Among these samples, one strain belonging to the order Sordariales (isolate F22, 14-days of cultive) and one to the genus Penicillium (isolate F37, 7-days of cultive) were selected. The filtrate was extracted with ethyl acetate and subjected to separation by chromatographic techniques, leading to purified compounds mevalonolactone and cis-cyclo(leucyl-tyrosyl) (identified by spectroscopic and spectrometric techniques), both able to inhibit biofilm formation by S. epidermidis ATCC. Mevalonolactone also showed activity against clinical isolates of S. epidermidis revealing, both antibiofilm and antibiotic activities at 0.06 mg.mL-1. The anti-T. vaginalis assay was carried out using the resazurin staining method andviability of trophozoites were characterized using trypan blue. Two samples of the fungi Hypocrea lixii and Penicillium citrinum showed the presence of active metabolites able to inhibit the trophozoites growth in 24 hours (MIC = 2.5 mg.mL-1), according to the kinetic curve of growth. Absence of hemolysis of erythrocytes was also observed for these two filtrate samples. The results corroborate with the reports of the great potential of marine fungi in the discovery of active metabolites.

Plantas do gênero Hypericum são bem conhecidas por sua utilização na medicina tradicional, sendo Hypericum perforatum a espécie mais estudada. No Sul do Brasil, esse gênero é representado por cerca de 20 espécies, entre elas, destaca-se H. polyanthemum, o qual apresenta em extrato lipofílico a presença de uliginosina B, um derivado de floroglucinol, e três benzopiranos: HP1, HP2 e HP3. Partes aéreas de H. polyanthemum foram submetidas à extração com fluido supercrítico (SFE), sendo realizadas extrações sucessivas com dióxido de carbono nas pressões de 90, 120, 150 e 200 bar, em diferentes temperaturas (40, 50 e 60 °C). Posteriormente, as frações obtidas foram comparadas com extrato n-hexano obtido por técnica convencional (ultra-som). SFE apresentou maior seletividade que a extração com nhexano, sendo a 50 °C a melhor condição para extrair os metabólitos bioativos. Para extração do composto com maior peso molecular, uliginosina B, pressões mais elevadas foram necessárias. Além disso, avaliou-se a atividade anti- Trichomonas vaginalis do extrato obtido por SFE a 50 °C/150 bar e dos compostos isolados. Os resultados indicam que todas as amostras testadas possuem atividade anti-T. vaginalis, porém, HP1 demonstrou melhor seletividade frente ao protozoário (isolado metronidazol-resistente e -suscetível), sem apresentar citotoxicidade contra células de mamíferos. Ainda, o HP1 apresentou satisfatória atividade contra isolado metronidazol-resistente (52% de trofozoítos viáveis), apresentando efeito sinérgico quando testado com baixa concentração de metronidazol (23% de trofozoítos viáveis). Todos os compostos isolados provocaram danos na membrana do parasito (liberação de LDH > 90%) e nenhuma das amostras apresentou efeito hemolítico importante, sendo que HP2 e uliginosina B apresentaram citotoxicidade frente a células de mamíferos. Portanto, benzopiranos e derivados de floroglucinol são moléculas promissoras como protótipos para novos fármacos tricomonicida, principalmente HP1, visto que corrobora com a ação do metronidazol em isolados de T. vaginalis resistentes. No entanto, mais estudos são necessários a fim de melhorar a atividade antiprotozoária das moléculas testadas, sem que as mesmas apresentem toxicidade para células de mamíferos. / Plants of the genus Hypericum are well known for their use in traditional medicine, being H. perforatum the most important species. In South Brazil, this genus is represented by approximately 20 species and among them, H. polyanthemum showed in the lipophilic extract the presence of uliginosin B, a phloroglucinol derivative, and three benzopyrans: HP1, HP2 and HP3. Aerial parts of H. polyanthemum were submitted a supercritical fluid extraction (SFE), being successively extracted with supercritical carbon dioxide under pressures of 90, 120, 150 and 200 bar at different temperatures (40, 50 and 60 ºC), and compared with the n-hexane extract obtained by ultrasound-assisted extraction. SFE presented higher selectivity than the n-hexane extration. The best condition to extract the target metabolites has been determined to be at 50 °C, being that for the high molecularweight compound, uliginosin B, higher pressures was required. Moreover, the anti- Trichomonas vaginalis activity of the extract obtained by SFE at 50 °C/150 bar and the isolated compounds was evaluated. The results showed that all samples have anti-T. vaginalis activity; however, HP1 demonstrated the best selectivity against this protozoa (metronidazole-resistant and -susceptible isolates), with no cytotoxicity against mammalian cells. In addition, HP1 has good activity against metronidazoleresistant isolate (52% of viable trophozoites), presenting synergic effect when tested with low concentration of metronidazole (23% of viable trophozoites). All isolated compounds caused damage in the parasites membrane, revealed by more than 90% of LDH release. None of the samples presented important hemolytic effect, and HP2 and uliginosin B presented cytotoxicity against mammalian cells. Therefore, benzopyrans and phloroglucinol derivative are promising molecules as prototypes for new antiprotozoal drugs, especially HP1, which improve metronidazole action against a resistant T. vaginalis isolate. Nevertheless, more studies are necessary in order to enhance their in vitro antiprotozoal activity without toxicity for mammalian cells.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-06T14:26:33Z No. of bitstreams: 1 arthurladeiramacedo.pdf: 2377553 bytes, checksum: 6c4d490fea818c6c85a6c551bd1e3d26 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-09-06T14:50:02Z (GMT) No. of bitstreams: 1 arthurladeiramacedo.pdf: 2377553 bytes, checksum: 6c4d490fea818c6c85a6c551bd1e3d26 (MD5) / Made available in DSpace on 2016-09-06T14:50:02Z (GMT). No. of bitstreams: 1 arthurladeiramacedo.pdf: 2377553 bytes, checksum: 6c4d490fea818c6c85a6c551bd1e3d26 (MD5) Previous issue date: 2013-07-17 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Folhas de Gossypium barbadense L. (Malvaceae), conhecido como algodoeiro, são utilizadas popularmente para o tratamento de corrimento vaginal. A principal etiologia é a vaginose bacteriana (VB). VB é uma infecção complexa, na qual várias bactérias anaeróbias estão presentes, sendo a de maior incidência Gardnerella vaginalis. VB causa o aumento do corrimento vaginal, lise de células epiteliais vaginais, podendo causar parto prematuro (PPM), e outras complicações durante a gravidez. O mecanismo de PPM não está totalmente elucidado, porém uma proposta bem aceita é a de inflamação das membranas fetais. No presente trabalho avaliaram-se as atividades de extratos brutos e frações semi-purificadas de folhas de G. barbadense: 1) contra G. vaginalis, 2) citotóxica contra Artemia salina, 3) na viabilidade celular de células saudáveis (3T3) e cancerosas (MCF7) e 4) antiinflamatória no perfil de citocinas (IL-17, TNF- e INF-) e NO. Além disso, foi realizado o perfil cromatográfico dos extratos e frações semi-purificadas em CCD, CLAE-DAD e CG-EM e o isolamento de um flavonoide que foi identificado utilizandose RMN 1H, COSY e HSQC como sendo uma quercetina com substituição na hidroxila do carbono 3. Os extratos e frações polares mostram-se ricos em flavonoides, enquanto os apolares mostraram-se ricos em terpenoides e ácidos graxos saturados e insaturados. A fração diclorometânica (FDM) do extrato bruto metanólico (EBM) apresentou substâncias dos extratos polares e apolares. EBM e FDM apresentaram atividade expressiva contra G. vaginalis, aparentemente devido ao sinergismo entre flavonoides e substâncias apolares. EBM e FDM não apresentaram citotoxicidade contra A. salina e, em conjunto com as frações hexânica (FH), e em acetato de etila de EBM (FAE), apresentaram aumento da viabilidade de 3T3 e o extrato bruto aquoso e FDM reduziram a de MCF7. O extrato bruto hexânico (EBH) apresentou melhor atividade anti-inflamatória. Os demais extratos apresentaram menor atividade. Extratos e frações de folhas de G. barbadense, principalmente EBM e FDM, apresentam potencial para futuros estudos in vivo visando a confirmação das atividades em sistemas biológicos complexos. / Gossypium barbadense L. (Malvaceae) leaves, known as cotton, have been used for vaginal discharge treatment. Bacterial vaginosis (BV) is the major etiology. BV is a complex infection, involving a wide variety of anaerobes, being Gardnerella vaginailis the most incident. BV increases the amount of vaginal discharge, causing epithelial cells’ lysis, it can cause, also, preterm delivery (PTD) and other complications during pregnancy, as well. PTD mechanism is not totally known, but the inflammation of fetal membranes is one accepted theory. The present work evaluated 1) antimicrobial against G. vaginalis, 2) cytotoxicity against Artemia salina, 3) cell viability of health (3T3) and cancer (MCF7) cells and 4) anti-inflammatory evaluating cytokines (IL-17, TNF- e INF-) and NO activities of crude extracts and semi-purified fractions of G. barbadense leaves. Moreover, the chromatographic profile of crude extracts and semi-purified fractions were evaluated by TLC, HPLC-DAD and GC-MS. A flavonoid was isolated, being elucidated as a replaced quercetin, probably in hydroxyl group of carbon 3, by NMR 1H, COSY and HSQC. Flavonoids were abundant in polar extracts and fractions, and fatty acids and terpenoids were abundant in nonpolar ones. Polar and nonpolar compounds were observed in dichloromethane fraction (DMF) of methanol crude extract (MCE). MCE and DMF showed a expressive activity against G. vaginalis and the synergism between flavonoids and nonpolar substances appear to be crucial. MCE and DMF do not show cytotoxicity against A. salina, and with the hexane fraction (HF) and ethyl acetate fraction (EAF) of MCE showed an increase in cell viability of 3T3. Aqueous crude extract and DMF reduced the cell viability of MCF7. A most promising anti-inflammatory activity was presented by hexanic crude extract. Other crude extracts showed a less intense activity. G. barbadense leaves extracts and semi-purified fractions, chiefly MCE and DMF, presented good potential for future in vivo studies seeking the confirmation of these activities in complex biological systems.

Abstract Prevalence of Trichomonas vaginalis in STI-samples from Västra Götaland county with Aptima™ TV Assay on Panther™ System Also, evaluation of Aptima™ TV Assay on Panther™ System with comparison with Xpert® TV Kit on GeneXpert® Trichomonas vaginalis is a parasite that is spread by sexual contact, it is also the biggest sexually transmitted infection in both the United States and Europe. The prevalence in Sweden today is unknown as no national basic data exist. The routine procedure of identification is Wet-Smear although nucleic acid amplification enable a higher level of specificity and sensitivity. Panther™ System and GeneXpert® are two systems using RNA respectively DNA for analysis. The objective of the study was primarily to investigate the prevalence of Trichomonas vaginalis in STI-samples from Västra Götaland county with Aptima™ TV Assay on Panther™ System and secondarily to evaluate Aptima™ TV Assay with comparison to Xpert® TV Kit on GeneXpert®. The evaluation and prevalence study took place at Clinical Microbiology, Unilabs AB, Skaraborgs Hospital Skövde during the period of April-May 2017. The results of the evaluation indicate that Aptima™ TV Assay is specific for Trichomonas vaginalis and that the sensitivity falls within the detection limits for the kit. Therefore, the kit is suitable for use in the prevalence study. The prevalence study displayed a positive outcome on two out of 606 analyzed patient samples, corresponding to 0.3%. The conclusion is that the prevalence is enough for further studies.

Trichomonas vaginalis é o protozoário flagelado que parasita o sistema urogenital humano causando a tricomoníase, a doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos a cada ano. O sucesso da colonização das células hospedeiras e desenvolvimento da infecção envolve um complexo processo que culmina em citoaderência e citotoxicidade. Nucleotídeos e nucleosídeos são liberados para o espaço extracelular por células em situações de estresse ou lesão tecidual e desencadeiam seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, a hidrólise sequencial de nucleotídeos pelas ectonucleotidases, nucleosídeo trifosfato difosfoidrolase (NTPDase) e ecto-5’-nucleotidase leva à formação de nucleosídeos que são essenciais para o metabolismo de purinas do parasito. Efeitos antagônicos são desencadeados por nucleotídeos e nucleosídeos, respectivamente próinflamatórios e anti-inflamatórios, na mediação de respostas imunes. A atividade dessas enzimas sobre os nucleotídeos da guanina e o efeito de restrição metabólica sobre a hidrólise de nucleotídeos foi avaliada. Além disso, a participação da sinalização mediada pelos nucleotídeos e nucleosídeos também foi avaliada em um modelo de citotoxicidade. Os resultados demonstram que os nucleotídeos GTP, GDP e GMP são substratos para as ectonucleotidases de T. vaginalis com parâmetros cinéticos compatíveis para enzimas dessa família. A condição de restrição de soro aumentou a atividade da NTPDase e da ecto-5’-nucleotidase e o aumento da expressão gênica das TvNTPDase 2 e 4 pode justificar o aumento da atividade. A recaptação de guanosina extracelular foi menor do que a recaptação de adenosina, demonstrada pela razão isotópica C12/C13 no nucleosídeo detectada no DNA dos parasitos. A fim de investigar um possível papel biológico para o acúmulo de guanosina extracelular, bem como avaliar o envolvimento da sinalização purinérgica na citotoxicidade mediada pelo parasito, diferentes isolados de T. vaginalis foram testados frente à capacidade de promover citólise. Todos os isolados foram capazes de promover efeito citolítico em alguma proporção, com destaque para o isolado TV-LACM6, que apresenta um perfil de hidrólise ATP, GTP > AMP > GMP. Quando nucleotídeos e nucleosídeos foram testados, o efeito citotóxico produzido pelo isolado foi potencializado na presença de ATP e GTP. Por outro lado, o efeito foi revertido na presença de eritro-9-(2-hidroxi-3-nonil) adenina (EHNA), um inibidor da adenosina deaminase. Importante, guanosina não foi capaz de reverter o efeito citotóxico produzido pelos trofozoítos, resultado que corrobora com o perfil de hidrólise de nucleotídeos e acúmulo de guanosina exracelular, sendo uma vantagem para o parasito. A possível participação dos receptores de adenosina foi avaliada, entretando os receptores ADORA1 e ADORA2A não parecem estar envolvidos no efeito protetor mediado pela adenosina. Considerando o potencial farmacológico desempenhado por essas enzimas no metabolismo de purinas em protozoários bem como no controle de respostas imunes, a modulação da hidrólise de nucleotídeos pode ser um alvo terapêutico importante e representar um mecanismo sinérgico na atividade antiparasiária de compostos ativos. Nesse sentido, o estudo demonstrou a atividade anti-T. vaginalis de três compostos, e em especial o isoaustrobrasilol B, com IC50 de 38 μM. O composto não apresentou efeitos hemolíticos frente a eritrócitos humanos e apesar de ter demonstrado efeito citotóxico in vitro frente à linhagem de células epiteliais vaginais humanas (HMVII), nenhuma citotoxicidade foi demonstrada no modelo in vivo. Isoaustrobrasilol B foi o único composto que inibiu significativamente as atividades da NTPDase e ecto-5’-nucleotidase e o efeito imune atribuído ao acúmulo extracelular de nucleotídeos foi avaliado. A produção de espécies reativas de oxigênio e interelucina-6 (IL-6) por neutrófilos estimulados por T.vaginalis não foi afetada pelo tratamento com o composto. Por outro lado, a liberação de interleucina-8 (IL-8), a principal interleucina produzida por neutrófilos na tricomoníase, foi aumentada. O efeito sinérgico de redução de viabilidade de trofozoítos e modulação da NTPDase e ecto-5’-nucleotidase pode aumentar a suscetibilidade do T. vaginalis frente à resposta imune do hospedeiro e consequentemente, sua eliminação do sítio de infecção. / Trichomonas vaginalis is a flagellate protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease, infecting approximately 276 million people worldwide annually. To achieve success in parasitism trichomonads develop a complex process against the host cells that culminate in cytoadherence and cytotoxicity. Nucleotides and nucleosides 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, nucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing nucleosides that are essential to the parasite purine metabolism. The enzymatic cascade mediated by ectonucleotidases is relevant in controlling nucleotides and nucleosides levels as these molecules play antagonistic roles in inflammation, as proinflamatory and anti-inflammatory mediators, respectively. This study investigated the hydrolysis profile of guanine nucleotides in T. vaginalis as the effect of serum limitation condition in the enzymatic cascade. Furthermore, we investigated the influence of extracellular nucleotides and nucleosides on the modulation of the host cell cytotoxicity mediated by T. vaginalis. Results show that guanine nucleotides GTP, GDP, GMP are substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. The metabolic restriction condition enhanced NTPDase and ecto-5’-nucleotidase activities and the highest gene expressions found for TvNTPDase 2 and 4 which may explain the enzymatic activity enhance. The extracellular guanosine uptake was lower than that observed for adenosine into parasite DNA measured by isotopic ratio C12/C13 of the nucleosides. In order to investigate the possible biological role for extracellular guanosine accumulation as well as to evaluate the involvment of purinergic signaling in the citotoxicity promoted by the parasite, a collection of T. vaginalis isolates were tested against a human epithelial vaginal cell line (HMVII). Fresh clinical T. vaginalis isolates produced cytolytic effect against human vaginal epithelial cells in a heterogeneous profile. The most cytotoxic isolate, TV-LACM6, presents the hydrolysis profile ATP, GTP > AMP > GMP. When the nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased in presence of nucleotides ATP and GTP. In contrast, the cytotoxicity was reversed by adenosine in presence of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), but not by guanosine, which is in agreement with the accumulation of extracellular guanosine and the hydrolysis profile, acting as an advantage for the parasite. ADORA1 and ADORA2A are not involved in the protective mechanism of adenosine. Considering the pharmacological potential that ectonucleotidases play in the context of purine metabolism and in the imune response modulation, nucleotide hydrolysis may represent a therapeutic target as an additional mechanism in association with anti T.vaginalis compounds. The study demonstrated promissing activities for three derivatvies with isoaustrobrasilol B the most activity compound with IC50 38 μM. The compound did not demonstrate any hemolytic activity and although induced cytotoxicity against human epithelial vaginal cells (HMVII), absence of toxicity was obtained when in vivo model was studied. Isoaustrobrasilol B was the only compound that significantly inhibited NTPDase and ecto-5’-nucleotidase activities and the immune modulation attributed to extracellular nucleotide accumulation was evaluated. Reactive oxygen species production and interleukin-6 (IL-6) release by T.vaginalis stimulated neutrophils were not affected by phloroglucinol treatment. On the other hand, interleukin-8 (IL-8), the primarily cytokine produced by neutrophils during trichomoniasis, was significantly enhanced. The associative mechanism of trophozoites death and NTPDase and ecto-5’-nucleotidase modulation may increase the susceptibility of T. vaginalis to host immune responses and, consequently, the elimination from the infection site.

acase@tulane.edu

Background: Trichomoniasis is the most common non-viral sexually transmitted infection with worldwide; it is particularly prevalent amongst HIV positive women. The infection is commonly considered easily treatable, yet repeat infection rates in HIV positive women can be as high as 36%. The overall objectives of this thesis is to examine the influence of select factors on treatment failure and retest positive of Trichomonas vaginalis (TV) among HIV+ women and to examine methodologies for eliciting reports of sexual behaviors. Methods: A secondary data analysis was performed on a multi-centered cohort of HIV+/TV+ women who were randomized to single-dose (2g) or 7 day (500mg BID) multi-dose metronidazole treatment. Test of cure visit occurred 6-12 days after treatment completion and women returned for a 3-month re-test visit. To assess sexual behavior methods a literature search was conducted for articles that compared prospective and recall methods. Results: Women on ART had higher retest positive than women not on ART with an increased relative risk of 2.63 (95% CI 1.04-6.63, p=0.036). Of the 143 women included for the assessment of smoking, the higher level of smoking had an increased risk of TV compared to non-smokers 3.44 RR (95% CI 1.31-9.04, p-value=0.012) and Cochran-Armitage trend test at p=0.007 between increasing levels of smoking and TV+. Out of an initial 3140 articles, 14 met all the criteria for review. Among heterosexuals, recall of 1 month of sexual behavior was found to be similar to data collected more frequently (i.e. daily, over the same time span). Conclusions: Among HIV positive women, ART usage and cigarette smoking may interfere with treatment of TV. Monthly recall methods appear to be similar to daily measures of sexual behavior among heterosexuals. / acase@tulane.edu

<p>Risk factors such as intrauterine and vaginal infection put pregnant women at risk for delivering preterm. Bacterial vaginosis (BV) is a polymicrobial clinical syndrome commonly diagnosed in women of reproductive age, with women of African descent with low socioeconomic status and previous preterm delivery at high risk. Although frequently isolated from healthy women,&nbsp / Gardnerella vaginalis has been most frequently associated with BV. There is limited data available on the prevalence of BV in Southern Africa / therefore, we embarked on a study to determine the&nbsp / prevalence of BV and G. vaginalis in predominantly black communities in the Western Cape, in order to establish the role of G. vaginalis in BV. Women attending various Maternity and Obstetrics&nbsp / units (MOU) in the Cape Peninsula with and without a history of pre-term delivery (PTD) were invited to participate in the study. Several factors were statistically associated with pregnancy history,&nbsp / including location of study population, parity, smoking and presence of clinical symptoms. The presence of G. vaginalis was determined by culture in 51.7% of the preterm delivery group (PTDG)&nbsp / and 44% of the full-term delivery group (FTDG) women. BV was detected in 31.13% of PTDG and 23.67% of FTDG by Gram stained analysis according to Nugent scoring criteria, with age and HIV&nbsp / status posing as risk factors. When comparing PTDG and FTDG for an association between the presence of G. vaginalis and BV, a stronger association was observed in the PTDG but it was not statistically significant. In both PTDG and FTDG, G. vaginalis was isolated significantly more often in women diagnosed with BV at 24.5% (p &lt / 0.05). Antibiogram studies revealed both Metronidazole and Clindamycin resistant strains of G. vaginalis. G. vaginalis Biotype 7 is specifically associated with BV, while Biotype 2 appears to be associated with BV in women with a history&nbsp / of PTD. Accuracy of diagnostic tools were tested and it was determined that Nugent scoring is more sensitive in diagnosing BV (76.04%), but culture for G. vaginalis is more specific (83.21%). Although this study was limited in that we were unable to follow-up pregnancy outcomes, we were able to confirm the perceived role of G. vaginalis in BV.&nbsp / </p>

The enzyme L-methionine γ-lyase (MGL) utilizes a pyridoxal-5’-phosphate-cofactor in order to convert L-methionine to α-ketobutyrate, ammonia and methyl mercaptan. MGL is proposed to be a potential drug target since it is expressed in the human pathogens, Trichomonas vaginalis and Entamoeba histolytica, but not in humans. There is currently a need to find alternative drug targets for these pathogens, because the misuse and overuse of the currently prescribed drugs of choice, metronidazole and tinidazole, have lead to drug resistance. The overall goal of this thesis was to examine the chemistry of MGL 1 from T. vaginalis (TvMGL1) by probing the active site by site-directed mutagenesis and with fluorinated methionine analogs. The mutation of the active site residue Cys113 to Ser led to a 5-fold decrease in turnover rate for L-methionine relative to the wild-type enzyme. The results suggest that the active site C113 residue plays an important role in catalysis and is consistent with literature reports for MGL homologs from Pseudomonas putida and E. histolytica. Probing the active site of TvMGL1 with the fluorinated methionine analogs, L-difluoromethionine (DFM) and L-trifluoromethionine (TFM), were found to increase the turnover rate of the enzyme with an increase in fluorine substitution. The results suggest that the bulky fluorine atoms do not interfere with the Michaelis-Menten kinetics of the enzyme, and the γ-elimination step is rate determining. The second goal of this thesis was to identify the reactive intermediates generated by the processing of TFM and the uninvestigated DFM by TvMGL1, and to investigate the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF3S- and CF2HS-). The reactivity of the intermediates, generated from the processing of DFM by TvMGL1 was correlated to the cytotoxicity observed in model organisms expressing TvMGL1, and consistent with the hypothesis that the intermediates will result in the thioformylation of primary amines. The results suggest that cytotoxicity requires thioacylation of a single primary amine, while sequential cross-linking of primary amines is not an absolute requirement. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogs and their cellular toxicity is discussed. Attempts at the synthesis of 3,3-difluoro-O-methyl-L-homoserine were undertaken in order to examine the catalytic mechanism of TvMGL1, since the compound is expected to inhibit the enzyme. To ensure that the oxo moiety does not impede the chemistry of the enzyme, the analog, O-methyl-L-homoserine was examined as a potential substrate for TvMGL1. Several synthetic routes to 3,3-difluoro-O-methyl-L-homoserine were examined; however, attempts to fluorinate the β-carbon atom of the starting material were unsuccessful.

Source: Masters Abstracts International, Volume: 47-06, page: 3500. Adviser: Kim Waller. Includes bibliographical references.