Biofilmes são comunidades bacterianas tridimensionais complexas, que vivem organizadas e aderidas a uma superfície, biótica ou abiótica, embebidas em uma matriz exopolimérica. Cerca de 80% das bactérias vivem organizadas na forma de biofilmes, pois dentro destas estruturas são menos sensíveis aos antibióticos e à resposta imune do hospedeiro. Dentre as principais bactérias formadoras de biofilmes têm-se Staphylococcus spp., Pseudomonas aeruginosa e enterobacteriaceas. Estas bactérias formadoras de biofilmes são importantes colonizadoras da superfície de dispositivos médicos e implantes, aumentando a morbidade e mortalidade dos pacientes que apresentam este tipo de infecção. A investigação de novas estratégias para prevenção e tratamento de infecções por biofilmes é urgentemente necessária. Dentre estas estratégias estão a pesquisa de diferentes mecanismos ou substâncias capazes de provocar a inibição da formação ou a erradicação do biofilme formado. Neste contexto, os venenos animais representam uma fonte ainda inexplorada de uma vasta quantidade de moléculas bioativas, candidatas ao desenvolvimento de novas terapias, inclusive antibiofilme. O principal objetivo deste estudo é avaliar diferentes venenos de serpentes e análogos sintéticos como fonte de moléculas contra biofilmes bacterianos patogênicos. O capítulo 1 revisa estudos que relatam a atividade antimicrobiana (contra bactérias, vírus, protozoários e fungos) de 170 peptídeos isolados de venenos de oito diferentes animais. Peptídeos antimicrobianos vêm ganhando destaque em pesquisas para o tratamento de infecções e peptídeos com atividade antibiofilme são uma nova e promissora abordagem, para o tratamento de infecções relacionadas. O capítulo 2 mostra as atividades antimicrobiana, antibiofilme e de erradicação de biofilmes pré-estabelicidos de 18 análogos de peptídeos de oriundos de venenos de serpentes. Inicialmente foram analisadas e alinhadas 170 sequências peptídeos oriundos de venenos animais. O pepptídeos 16 apresentou considerável atividade antimicrobiana contra cepas bacterianas Gram-positivas, sensíveis e resistentes. Para S. epidermidis os peptídeos 1, 2, 3, 4, 12, 13 e 16 apresentaram menos de 50% de formação de biofilme e os peptídeos 2, 3 e 16 reduziram o biofilme pré-formad. A citotoxicidade e a actividade hemolítica foram testadas e os peptídeos ativos 2 e 16 apresentaram citotoxicidade e hemólise significativas em comparação com os controles. A posição dos aminoácidos pode contribuir para as atividades, sendo que mais testes são necessários para entender a relação das posições de aminoácidos na ação. O capítulo 3 mostra as atividades antiformação e erradicação de biofilmes pré-estabelecidos de dezessete diferentes venenos de serpentes, duas de escorpiões e três de anêmonas marinhas, bem como as secreções de pele de três espécies de sapos. Consideráveis atividades antiformação e de erradicação foram verificadas contra as cepas de S. aureus, S. epidermidis e E. cloaceae por todos os venenos de serpentes testados, em diferentes concentrações. Além disso, um fracionamento inicial foi realizado e as melhores condições foram selecionadas para um novo fracionamento, onde foram testadas 27 frações de veneno de B. diporus. As frações 8, 14 e 23 apresentaram atividades com menos de 50% de formação de biofilme e menos de 80% do biofilme remanescente. Os resultados indicam a capacidade dos venenos, especialmente da serpente de B. diporus, de serem potenciais fontes de moléculas como estratégia para combater os biofilmes patogênicos bacterianos. O presente estudo aborda o potencial de venenos animais, principalmente venenos de serpentes, como fonte de moléculas, que podem apresentar inúmeras atividades farmacológicas inéditas, incluindo àquelas relacionadas com a prevenção da formação e de erradicação de biofilmes bacterianos patogênicos. Atualmente, existem seis medicamentos aprovados pelo Food and Drug Administration (FDA), oriundos de venenos, como o Captopril (Capoten®). Este estudo mostra a capacidade de venenos como fonte de novas moléculas ativas contra biofilmes patogênicos. / Biofilms are complex three-dimensional bacterial communities living organized and attached on a surface, embedded in exopolimeric matrix. About 80% of live bacteria are organized in the form of biofilms because in these structures are less sensitive to antibiotics and host immune response. Staphylococcus spp., Pseudomonas aeruginosa and enterobacteriaceas are the main biofilm forming bacteria. These forming biofilms bacteria are important colonizing surface of medical devices and implants, increasing the morbidity and mortality of patients with this kind of infection. The investigation of new strategies for prevention and treatment of infections caused by biofilms is urgently needed. Among these strategies, there are the research of different mechanisms or substances capable of inhibit the formation or to eradicate the formed biofilm. In this context, animal venoms represent an untapped source of vast amounts of bioactive molecules, candidates for the development of new therapies, including antibiofilm. The main objective of this study is to evaluate different venoms of snakes and synthetic analogs as source of molecules against pathogenic bacterial biofilms. The chapter 1 reviewed numerous studies reporting the antimicrobial activity (against bacteria, viruses, protozoa and fungi) of 170 peptides isolated from venoms of eight different animals. Antimicrobial peptides have been gaining attention in research for the treatment of infections and peptides with antibiofilm activity are a new and promising approach for the treatment of infections related. The chapter 2 shows the antimicrobial, antibiofilm and eradication of established biofilms activities of 18 analogs of peptides derived from snake venoms. Initially, 170 peptide sequences from animal venoms were analyzed and aligned. The peptide 16 showed considerable antimicrobial activity against Gram-positive bacterial strains, sensitive and resistant. For S. epidermidis, the peptides 1, 2, 3, 4, 12, 13 and 16 showed less than 50% of biofilm formation and peptides 2, 3 and 16 reduced preformed biofilm. Cytotoxicity and hemolytic activity were tested and active peptides 2 and 16 showed significant cytotoxicity and hemolysis compared with the controls. The position of the amino acids can contribute to the activities and more tests are needed to understand the relationship of the amino acid positions in the action. The chapter 3 shows the antiformation and eradication of established biofilms activities of seventeen different venoms of snakes, two of scorpions and three of marine anemones, as well as the skin secretions of three species of frogs. Significant activities were observed against strains of S. aureus, S. epidermidis and E. cloaceae for all venom of snakes tested at different concentrations. In addition, an initial fractionation was performed and the best conditions were selected for a new fractionation, where 27 fractions of B. diporus enom were tested. Fractions 8, 14 and 23 presented activities with less than 50% of biofilm formation and less than 80% of the remaining biofilm. The results indicate the ability of venoms, especially the snake B. diporus, to be potential sources of molecules as a strategy to combat bacterial pathogenic biofilms. This study addresses the potential of animal venoms, especially snake venoms, as source of molecules, which can present numerous unpublished pharmacological activities, including those related to the prevention of the formation and eradication of pathogenic bacterial biofilms. Currently, there are six drugs approved by Food and Drug Administration (FDA), derived from venoms, such as Captopril®. This study shows the ability of venoms as source of new bioactive molecules against pathogenic biofilms.

We can now manipulate the genetic material of living organism routinely and cheaply. This has inspired a burgeoning field of synthesis based on DNA as a building block. The development of this new synthetic field has mirrored the trajectory of synthetic organic chemistry from small molecular systems to complex macromolecular assemblies. At first, this field of synthetic biology delivered recombinant proteins that enhanced our understanding of the structure-function relationship of biological macromolecules. Now, as the synthetic tools and analysis methods have come of age, synthetic whole-cell and multicellular systems have come within reach. In Chapter 1 we review the significant advances in DNA synthesis and analysis that have brought us to this point. In this work, we first ask what practical applications will benefit most from the unique qualities of synthetic whole-cell system, such as their ability to replicate, sense and respond with molecular specificity. In Chapter 2, we implement a pathogen detection platform based solely on genetically modified yeast. This approach holds the potential to deliver ultra low-cost sensors that can be used and produced at the point-of-care. In Chapter 3, we develop methods to target these yeast-based sensors for the detection of any peptide biomarker of choice. We next look forward to the potential of synthetic multicellular systems. While natural multicellular systems can be directly manipulated, our ability to rationally build multicellular systems from the bottom-up is still in its infancy. There still remain gaps in the available tools to make and analyze such synthetic systems. In Chapter 4, we leverage the explosion of available genomic databases to uncover a highly extensible set of cell-cell signaling modules. In Chapter 5, we implement ratiometric fluorescent tags to track mixed cell populations in multiplex. Together these components will be useful in implementing and analyzing synthetic communication networks that will be key components of advanced synthetic multicellular systems.

Motivado pela crescente importância que os animais domésticos vêem causando na sociedade humana, tanto em relação à busca de melhor qualidade de vida, quanto em relação ao seu valor epidemiológico, visto que são poucos os estudos sobre este assunto, este trabalho objetivou-se em identificar e traçar o perfil de sensibilidade frente aos antífúngicos das espécies de leveduras potencialmente patogênicas isoladas da mucosa oral de cães sem raça definida, da cidade de Campinas, São Paulo. Por motivos de segurança, os animais selecionados foram anestesiados para a realização de exame clínico da cavidade oral e coleta de amostras da região de mucosa oral, seguida de inoculação em ágar Sabouraund dextrose com cloranfenicol. A partir do crescimento em placa, foram isoladas as colônias de fungos leveduriformes, submetidas a exames macromorfológicos e micromorfológicos, meio cromogênico, provas bioquímicas (urease e método API 20C AUX) e teste de sensibilidade aos antifúngicos. Dos 50 animais participantes do estudo, os cães com idade superior a 4 anos e os que apresentavam doença periodontal tiveram maior percentual de isolamento. Foram identificadas 43 leveduras das 45 amostras isoladas, sendo elas 86% (37) correspondentes ao gênero Candida spp, 11,6% (5) pertencentes ao gênero Trichosporon spp e 2,3% (1) do gênero Malassezia pachydermatis. O perfil de sensibilidade pelo método \"Etest\" identificou importante resistência de algumas amostras à antifúngicos rotineiramente utilizados na clínica médica veterinária, o que ressalta a importância da continuidade deste trabalho para o melhoramento da conduta clínica e para a explicação dos inúmeros tratamentos recidivos tanto em animais como em humanos. / Regarding the increasing impact that that the pets have been causing to the human society as its relation to the search for a better quality of life, and its relation with the epidemiological value, this study aimed to identify and draw the profile of the susceptibility to the antifungal drugs of the potentially pathogenic species of yeasts isolated from the oral canine mucosa of animals from indefinite breed of the city of Campinas, São Paulo. For their safety, the selected animals were anesthetized to have a short clinic exam of the oral cavity performed and to have samples from the oral mucosa collected. Later an inoculation in Sabouraud Dextrose Agar with chloramphenicol was performed. After the growth on a dish, colonies of fungi with yeast shape were isolated and submitted to macro morphological and micro morphological exams, chromogenous medium, biochemical proofs (urease and API 20C AUX method), and the test of the susceptibility to the antifungal drugs. Among the 50 animals taken part in the study, the dogs over 4 years old and those which presented periodontal diseases had a higher isolation percentage. Yeasts were identified 43 of the 45 isolates, being that 86%(37) were from the genus Candida spp, 11,6% (5) belonged to the genus Trichosporon spp., and 2,3% (1) belonged to the genus Malasseziapachydermatis.The susceptibility profile through the \"Etest®\" method identified a relevant resistance of some strains to the antifungal drugs commonly used in the veterinary medical clinic. This found highlights the relevance of the continuity of this study to improve the clinical conduct and to explain many relapse treatments in both animals and humans.

As condições climáticas presentes nas regiões produtoras de trigo, do Brasil e dos principais países do qual o produto é importado, favorecem o aparecimento de doenças importantes desta cultura, dentre elas a fusariose, causada principalmente pelo fungo Fusarium graminearum Schwabe. Além dos danos diretos causados pela doença, os grãos infectados podem ser tóxicos para o homem e animais devido à presença de micotoxinas especialmente o desoxinivalenol (DON). Um total de 100 amostras de trigo, sendo 50 de trigo nacional (provenientes do Estado de São Paulo, Paraná e Rio grande do Sul) e 50 de trigo importado (Argentina e Paraguai) foram coletadas de empresas que normalmente comercializam ou processam trigo durante o período de maio a dezembro de 2005. Foram avaliados o percentual de freqüência de fungos, especialmente Fusarium graminearum, a contaminação com DON, percentual de grãos giberelados e realizadas correlações entre os parâmetros avaliados. Os resultados indicaram que freqüência média de Fusarium graminearum e F. spp. foram baixas (&#8804;2,6 e &#8804;3,6%, respectivamente), porém foi maior no trigo nacional do que no trigo importado. Do total de amostras avaliadas 94% do trigo nacional e 88% do trigo importado apresentaram-se contaminadas com DON em níveis médios de 332 µg.kg-1 (nacional) e 90 µg.kg-1 (importado). Existiu correlação positiva e significativa entre contaminação com DON e percentual de grãos giberelados (r = 0,83; p<0,0001), freqüência de Fusarium graminearum (r = 0,92; p<0,0001) e freqüência de Fusarium spp. (r = 0,86; p<0,0001). / The climatic conditions present in wheat producing areas from Brazil and main countries from which the product is imported favor the occurrence of important diseases in this crop, among them the Fusarium Head Blight or scab. It is mainly caused by the fungus Fusarium graminearum Besides, direct damages caused by this disease, the infected kernels may be toxic for humans and animals due to presence of mycotoxins (e.g deoxynivalenol). A total of 100 wheat samples, being 50 from national production (São Paulo, Paraná and Rio Grande Do Sul states) and 50 from imported one (Argentina and Paraguay), were collected during the period of May to December 2005 from companies that normally commercialize or process wheat. Frequency (%) of fungi occurrence, specially Fusarium graminearum and Fusarium spp., DON contamination and Fusarium damaged kernels (%) were evaluated. Correlations between the evaluated parameters were carried out. Frequency of Fusarium graminearum and Fusarium spp. were low (&#8804;2.6 and &#8804;3.6%, respectively), however it was higher in Brazilian wheat when compared with imported wheat. Ninety-four percent of national wheat samples and 88% of the imported samples were DON contaminated (mean levels, 332 µg.kg-1 and 90 µg.kg-1, respectively). The occurrence of DON was highly correlated with percentage of Fusarium damaged kernels, (r = 0,83; p<0.0001), percentual frequency of Fusarium graminearum (r = 0,92; p<0,0001) and percentual frequency of Fusarium spp. (r = 0,86; p<0,0001).

Submitted by DEBORA DE OLIVEIRA LOSNAK null (deboralosnak@hotmail.com) on 2017-03-08T18:48:25Z No. of bitstreams: 1 Dissertação Débora Losnak.pdf: 3093790 bytes, checksum: f1b838682d27c1345ac29cab3b2bfc9a (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-13T18:04:26Z (GMT) No. of bitstreams: 1 losnak_do_me_bot.pdf: 3093790 bytes, checksum: f1b838682d27c1345ac29cab3b2bfc9a (MD5) / Made available in DSpace on 2017-03-13T18:04:26Z (GMT). No. of bitstreams: 1 losnak_do_me_bot.pdf: 3093790 bytes, checksum: f1b838682d27c1345ac29cab3b2bfc9a (MD5) Previous issue date: 2017-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A emergência e reemergência de doenças infecciosas é impulsionada por vários fatores e a busca de patógenos em amostras animais podem oferecer oportunidades para estudos eco-epidemiológicos e também dados sobre a evolução dos patógenos. O objetivo deste estudo foi avaliar a ocorrência de fungos patogênicos importantes em saúde pública, em exemplares de animais silvestres mortos por atropelamento no estado de Santa Catarina e identificar e mapear áreas de risco para a infecção humana. Grande parte destes fungos apresenta em comum dimorfismo, distribuição geográfica restrita e produção de conídios infectantes que são aspirados pelo hospedeiro por meio das vias respiratórias. Cães e tatus são apontados como transmissores de Paracoccidioides brasiliensis, os morcegos ao Histoplasma spp., assim como as fezes de pombos ao Cryptococcus spp.. No presente trabalho foram analisadas 1063 amostras de pulmão, fígado, baço, pele e coração de 297 animais silvestres, para detecção de Paracoccidioides brasiliensis, Histoplasma capsulatum e Cryptococcus spp. pela técnica de Reação em Cadeia de Polimerase (PCR). Utilizou-se primers universais para detecção de fungos em geral e obteve-se positividade em 102 amostras de 59 animais. Para a análise de P. brasiliensis, utilizou-se os primers específicos, obtendo oito amostras positivas em cinco animais (quatro Oxymycterus spp. e um Euryoryzomys russatus). Não houve a detecção molecular para Histoplasma spp.. Foi possível a identificação de três amostras para Cryptococcus spp.. O sequenciamento foi realizado, porém em 89 amostras de 49 animais foi possível somente a identificação em Fungal sp. (GenBank KT923226.1), duas amostras para Cryptococcus neoformans (GenBank KY107218.1) obtidas de Oxymycterus spp. e Akodon spp. e três amostras de Aspergillus penicillioides (GenBank KP131612.1 e KP997215.1) obtidas de Gracilinanus spp., Oxymycterus spp. e Philander spp. Importante salientar que houve coinfecção de P. brasiliensis e Cryptococcus neoformans em amostra de um Oxymycterus spp. Esta pesquisa mostra a importância dos animais silvestres na transmissão de doenças e auxilia no mapeamento dos locais de ocorrência de determinados patógenos e doenças em uma região ainda não avaliada. / The emergence and reemergence of infectious diseases is propelled by many diverse factors and the search for pathogens in animal samples may offer opportunity for eco-epidemiologic studies as well as data on the evolution of pathogens. The objective of this study was to evaluate in samples of road-killed wild animals the occurrence of pathogenic fungi of importance for public health. A great part of these fungi presented, in common, dimorphism, restricted geographic distribution and production of conidia infecting, which are aspirated by the host by means of their respiratory tract. Dogs and armadillos are normally related to the transmission of Paracoccidioides brasiliensis, bats to Histoplasma spp., as well as pigeons feces to Cryptococcus spp.. In this study we analyzed 1063 samples of organs of 297 wild animals for the detection of Paracoccidioides brasiliensis, Histoplasma capsulatum and Cryptococcus spp. by the technique of Polymerase Chain Reaction (PCR). Universal primers were employed for the detection of fungi in general and positivity was obtained in 102 samples from 59 animals. For the P. brasiliensis analysis was used specific primers, resulting in eight positive samples from five animals (four Oxymycterus spp. and one Euryoryzomys russatus). There was no molecular detection to Histoplasma spp.. Was possible the identification of three samples to Cryptococcus spp.. The sequencing was performed, however in 89 samples from 49 animals was possible to identify Fungal sp. (GenBank KT923226.1), two samples for Cryptococcus neoformans (GenBank KY107218.1) obtained from Oxymycterus spp. and Akodon spp. and three samples from Gracilinanus spp., Oxymycterus spp. and Philander spp. were positive for Aspergillus penicillioides (GenBank KP131612.1 e KP997215.1). Is important emphasize the coinfection with P. brasiliensis and Cryptococcus neoformans in a sample from Oxymycterus spp.. This research shows the importance of the wild animals in transmissions of diseases and assists in the mapping of pathogen and disease sites in a region that has not yet been evaluated. / FAPESP: 2015/17519-4

Este trabalho procurou definir a existência de espécies emergentes de micoplasmas (Mycoplasma genitalium, M. fermentans e M. penetrans) em indivíduos HIV-positivos e indivíduos com queixa clínica de retrite, por não haver dados a esse respeito no Brasil. A pesquisa das espécies mais conhecidas (M. hominis e Ureaplasma urealyticum), cuja metodologia para sua detecção já está bem estabelecida, proporcionou uma visão global da participação das espécies de importância clínica conhecidas até o momento. Foram avaliadas amostras de raspado uretral e primeiro jato urinário de 106 indivíduos HIV-positivos e 110 indivíduos HIV-negativos com DST, no total de 212 e 220 amostras do primeiro e do segundo grupo, respectivamente. Deste modo, foram submetidas a análise 432 amostras para pesquisa de micoplasmas por cultura e PCR. Para pesquisa de anticorpos séricos anti- M. penetrans, foram obtidas amostras de soro de cada indivíduo dos dois grupos estudados, bem como de 86 doadores de sangue saudáveis para cálculo do limite de reatividade do ELISA. As taxas de infecção obtidas em nosso estudo, considerando os resultados do cultivo e da PCR, nos indivíduos HIV-positivos, foram: 2,8% para a espécie M. genitalium, 5,7% para M. fermentans, e 6,6% para M. penetrans. Nos indivíduos com queixa de DST, os valores encontrados foram: 9,1% para M. genitalium e 0,9% para M. fermentans, não tendo sido observado nenhum caso positivo para M. penetrans. Estes últimos resultados revelam que, apesar da pouca importância dada até então às três espécies, estas responderam, no total, por taxas de infecção de 15,1% e 10,0%, respectivamente, nos grupos HIV-positivo e DST. Estas taxas são bastante significativas quando comparadas com as observadas para as espécies M. hominis e U. urealyticum, ou seja, 26,4% e 14,5% em cada grupo, respectivamente. Nossos dados demonstraram uma freqüência significativa de anticorpos anti- M. penetrans: 25,5% no grupo de indivíduos HIV-positivos, 17,3% no grupo de DST, e 2,3% no grupo controle para IgG; 3,8%, 9,1% e 5,8% para IgM, e 15,1%,17,3% e 1,2% para IgA, respectivamente. Estes dados, aliados aos resultados de PCR, confirmam a presença de M. penetrans em nosso meio, infectando principalmente os indivíduos HIV-positivos. A presença desta espécie e também de M. fermentans, detectadas por PCR, denotam que muito pouco se conhece sobre o envolvimento destes micoplasmas com o HIV e que ainda não lhes é dada a devida importância na rotina médica. A associação definitiva de sua infecção com a progressão da AIDS, ainda a ser alcançada, e a demonstração em nossa população da infecção por M. genitalium em indivíduos com queixa de uretrite não-gonocócica, abre novos horizontes para o estudo da participação dos micoplasmas na etiologia das doenças humanas no país. / The aim of this work was to define the existence of emergent mycoplasma species, such as Mycoplasma genitalium, M. fermentans and M. penetrans, in HIV-1-infected and HIV-negative individuals with Sexually Trasmitted Diseases (STD), because there are no data concerning this subject in Brazil. The research about well-known species, such as Ureaplasma urealyticum and M. hominis, of wich detection techniques are well established, provided a comprehensive view of the participation of Mollicutes in human disease. We evaluated urethral swabs and urine samples from 106 HIV-1-infected and 110 HIV-negative individuals with clinical symptoms of non-gonococal urethritis, summing up 212 samples from the first group, and 220 from the second. Therefore, we tested a total of 432 samples to mycoplasma detection by culture and PCR. To detection of serum antibodies to M. penetrans, we obtained blood samples of each patient from the groups studied, as well as from 86 healthy blood donors to calculate the ELISA (Enzime Linked Immuno Sorbent Assay) cut-off value. The rates of infection obtained in our study, considering the culture and PCR results, in the HIV-infected individuals were: 2.8% for M. genitalium, 5.7% for M. fermentans, and 6.6% for M. penetrans. In the STD group, the rates were: 9.1% for M. genitalium and 0.9% for M. fermentans. We did not observed any positive case for M. penetrans in this group, and no positive case for M. pneumoniae in both groups, in spite of the existence of some reports of its detection in the genitourinary tract. This results reveal that they account, in the total, for infection rates of 15.1% and 10%, respectively, in the HIV-infected and the STD groups. These rates are very significant when compared to the ones observed for the species M. hominis and U. urealyticum, i.e., 26.4% and 14.5% in each group, respectively. Our results also show a significant antibody frequency to M. penetrans: 25.5% in the HIV-infected group, 17.3% in the STD group, and 2.3% in the control group for IgG; 3.8%, 9.1% and 5.8% for IgM, and 15.1 %, 17.3% and 1.2% for IgA, respectively. These data, together with the PCR results, confirm the presence of M. penetrans in our population, mainly infecting HIV- positive patients. The presence of this species, and also of M. fermentans, detected by PCR, demonstrate that very little is known about the role of this mycoplasma species in HIV infection, and they are not given the appropriate importance in clinical routine in Brazil. The definitive association between mycoplasma infection and the progression of AIDS is still to be established. The demonstration of M. genitalium infection in individuals with clinical symptoms of non-gonococal urethritis in our population, may open new insights in the study of the role of mycoplamas in the ethiology of human diseases in this country.

The Chlamydiaceae are a bacterial family that contains a single genus: Chlamydia. The genus Chlamydia consists of 9 species that are obligate, intracellular pathogens. Untreated C. trachomatis infections can lead to serious health ramifications, such as ectopic pregnancy, tubal factor infertility, pelvic inflammatory disease, and long-term pelvic pain. In this study, it was found that a primary antibody dilution of 1:400 using methanol fixed HeLA cells, as derived from Carrasco, et al. protocol, was only optimal for PMP-C staining. Pmp-A, Pmp-B, and Pmp-F were found to stain brighter with formaldehyde fixed, infected HeLa cells and using different primary antibody dilutions. The manuscript by Carrasco, et al., demonstrated that chlamydial persistence caused by penicillin-stressed conditions showed a decrease in Pmp-B and Pmp-C protein expression between 24-48 hpi, while Pmp-A and Pmp-F expression stayed the same under the stressful conditions. We hypothesized that under HSV- induced persistence the same results would occur. However, our data indicates that the chlamydial response to stressful conditions is not the same among persistence-inducers and implies that various inducers of persistence may affect PMP expression differently. Initially, we also hypothesized that PMP expression could be utilized as an indicator to determine whether an infected individual has a productive or persistent chlamydial infection. Due to the experiments’ results, PMP expression is most likely not a good marker to identify the type of chlamydial infection (ie. productive or persistent) in the host.

The presence of pathogenic viruses in coastal environments is an important tool in evaluating water quality and health risks. Millions of viruses are excreted in fecal matter and bacterial indicators do not correlate with the presence of pathogenic viruses. Enteroviruses have been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter could be used to indicate fecal pollution. The purpose of this research is to develop a baseline understanding of the diversity of viruses found in raw sewage and to assess their presence in the marine environment. PCR was used to detect adenoviruses, herpesviruses, hepatitis B viruses, morbilliviruses, noroviruses, papillomaviruses, pepper mild mottle viruses, picobirnaviruses, reoviruses, rotaviruses, and sapporoviruses in raw sewage collected from throughout the United States and from five marine environments ranging in their proximity to dense human populations. Adenoviruses, noroviruses, pepper mild mottle viruses, and picobirnaviruses were detected in raw sewage but absent in the marine environment, making these viruses potential indicators of fecal pollution in marine environments. These viruses were also found in many of the final effluent samples. Pepper mild mottle viruses may be useful for source tracking fecal contamination since it was consistently found in human sewage and is not expected in the feces of other animals due to its dietary origin. Furthermore, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage and the marine environment will enable educated decisions to be made regarding the use of viruses in water quality assessments.

Thesis (M.Sc. (Plant Protection )) --University of Limpopo, 2011 / Crude extracts of wild watermelon (Cucumis africanus) and wild cucumber (C. myriocarpus) fruits are widely used for both medicinal and ritual purposes in South Africa. Fruits are collected fresh from the wild, but have high incidence of post-harvest decay. A study was conducted to isolate and identify the pathogen responsible for post-harvest fruit decay, followed by the pathogenicity tests. Decayed fruits were individually surface-sterilised using 0.5% NaOCl, incubated at 25ºC to allow for decay, small rotten pieces were severed and placed on solidified plates of potato dextrose agar and incubated. At harvest, seven days after incubation, isolated fungus was repeatedly cultured for 21 days for verification of diagnostic characteristics. Based on the morphological characteristics, the pathogen associated with fruit rot of both Cucumis species was identified through the assistance of an expert as Penicillium simplicissimum (Oudem) Thom. Pathogenicity results suggested that P. simplicissimum was responsible for the observed fruit decay in both species, with the higher incidence being in C. africanus, probably due to its low pH. Due to the antibiotics that P. simplicissimum releases and its reduction of medium pH, the culture retained its purity, without any contamination. In conclusion, the pathogen that induces post-harvest fruit decay in C. africanus and C. myriocarpus is P. simplicissimum, which has the ability to reduce the pH of the growing medium and also produce antibiotics. / National Research Foundation

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.