Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-04-10T17:17:19Z No. of bitstreams: 1 Tayane Gonçalves Fernandes Efeito....pdf: 3094368 bytes, checksum: 87dc6aa455511d7e1892b6398cc286a4 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-04-10T17:17:27Z (GMT) No. of bitstreams: 1 Tayane Gonçalves Fernandes Efeito....pdf: 3094368 bytes, checksum: 87dc6aa455511d7e1892b6398cc286a4 (MD5) / Made available in DSpace on 2015-04-10T17:17:27Z (GMT). No. of bitstreams: 1 Tayane Gonçalves Fernandes Efeito....pdf: 3094368 bytes, checksum: 87dc6aa455511d7e1892b6398cc286a4 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A giardíase é uma doença causada pelo protozoário flagelado Giardia lamblia, e sua sintomatologia é caracterizada pela eliminação de fezes esteatorréicas, dores abdominais e náuseas. Segundo o CDC estima-se que há cerca 1,2 milhões de casos por ano de giardíase, acometendo principalmente crianças em idade escolar. Atualmente, o tratamento da giardíase é realizado principalmente pelo uso do fármaco da família dos 5-nitromidazóis, metronidazol (Flagyl®), secnidazol e tinidazol em particular. Estes são confrontados em casos de resistência clínica causada pelo frequente uso inadequado do medicamento e/ou abandono do tratamento. Além disso, o metronidazol pode apresentar efeito carcinogênico em longo prazo em humanos. Desta forma, novos estudos com análogos e/ou inibidores de poliaminas podem levar à elucidação dos mecanismos de ação envolvidos, favorecendo o estabelecimento de novos regimes terapêuticos mais seguros e eficazes. Em nosso trabalho, foram testadas as substâncias ciclohexilamina (CHA) e o metronidazol que são produtos sintéticos, com o objetivo de avaliar os seus efeitos na proliferação celular, caracterização dos moduladores do metabolismo de poliaminas, avaliação nas mudanças no potencial redox e elucidação de seus possíveis mecanismos de ação nos trofozoítos de Giardia lamblia. Foi realizada uma avaliação da proliferação celular na presença de CHA para trofozoítos de Giardia lamblia, onde observamos que a substância demonstrou ter ação siginficativa apresentando um efeito dosedependente. Observamos que os trofozoítos de G. lamblia apresentam uma inibição significativa do crescimento em presença de concentrações milimolares do CHA, cujo IC50 em 72 horas foi de 1,646 mM. Ao avaliar a produção de lipoperóxidos nos trofozoítos foi observado o possível papel do CHA como promotor de estresse oxidativo neste parasito. Ao realizar microscopia eletrônica de varredura (MEV) os trofozoítos apresentaram morfologias completamente irregulares em diferentes concentrações da CHA, com internalização do disco adesivo, sendo corroborado com os resultados da microscopia eletrônica de transmissão (MET) que mostram o processo de encistamento seguido de necrose celular. Esses resultados indicam que a CHA é possível candidata para o uso terapêutico contra a giardíase. / Giardiasis is a disease caused by the flagellate protozoan Giardia lamblia, and its symptomatology is characterized by steatorrhea, abdominal pain and nausea. According to the CDC, an estimate number of 1.2 million cases of giardiasis happen every year, affecting especially schoolchildren.Nowadays, giardiasis treatment is based on drugs from the 5-nitroimidazole family, particularly metronidazole (Flagyl), secnidazole and tinidazole. Those drugs are indiscriminately used by the population, and it's not uncommon to find them causing clinical resistance due to inappropriate utilization and/or tratment abandon. Besides that, metronidazole can present longterm carcinogenic effect in humans. Thus, new studies with analogs and/or polyamines inhibitors can lead to the clarification of the drugs action mechanis, favouring the establishment of new, safer and more efficient therapeutic regimens.Our work tested cyclohexylamine (CHA) and metronidazole, wich are synthetic products, in order to evaluate their effects on cell proliferation and on changes in redox potential, characterize polyamines metabolism modulator and describe their possible action mechanisms on Giardia lamblia trophozoites. We evaluated Giardia lamblia trophozoites cell proliferation in the presence of CHA; it was observe that the substance shows significant action, presenting dose-dependent effect. We also observed that G. lamblia trophozoites presented significant growth inhibition when exposed to millimolar concentrations of CHA - its IC50 in 72 hours was 1,646mM. When assessed the lipoperoxides production in trophozoites, we observed a possible role of CHA as an oxidative stress promoter in the parasite.Under Scanning Electron Microscopy, trophozoites showed completely irregular morphologies in different CHA concentrations, with internalization of the adhesive disc; this results are corroborated by the Transmission Electron Microscopy results, wich showed the process of encystment followed by cell necrosis. This makes CHA a possible candidate for therapeutic use against giardiasis.

El objetivo de este trabajo fue determinar la prevalencia de Guardia sp. en la población canina doméstica en los 6 distritos que conforman la Provincia Constitucional del Callao en el Perú. Para tal fin se colectaron 385 muestras fecales de perros aparentemente normales de ambos sexos, de diferentes edades y de acuerdo a la zona en donde habitaban sus propietarios. Las muestras fueron procesadas mediante la técnica de sedimentación espontánea; encontrándose una prevalencia de 9,35 ± 2% de Guardia sp. Mediante la prueba de regresión logística se cuantificó la asociación entre la presencia del parásito y las características físicas de las heces, estrato distrital de procedencia, sexo y edad del canino; se determinó una relación estadísticamente significativa entre el hallazgo de quistes de Guardia sp. y las características físicas de las muestras. Los resultados de nuestro estudio denotan una parasitosis moderada de Guardia sp. en los caninos, evidenciando un riesgo zoonótico, por lo que se hace necesario el establecimiento de programas educativos para prevenir la posibilidad de contagio especialmente en la población infantil. Asimismo, debido a que la ocurrencia del parásito es más frecuente en heces diarreicas o pastosas que en heces normales, se debe consultar al Médico Veterinario para que determine la causa y un adecuado tratamiento a ese trastorno digestivo. / The aim of this study was to determine Guardian sp. prevalence in household dog population on different districts that form Provincia Constitutional del Callao in Peru. Fecal samples were collected from 385 apparently healthy dogs of different ages that were selected depending on the zone where their owners lived. Stool samples were processed by spontaneous sedimentation technique and we found that 9,35 ± 2% (36/385) of the canine population in this study were positive to Giardia sp. cysts. Relationships between cysts detection and stool samples characteristics, provenience, sex and age of the dogs were analyzed by logistic regression; significant statistical relationship was found between Giardia sp detection and stoll samples physical characteristics. Results show a moderate Giardia sp. infection in dogs which evidences a zoonotic risk that makes necessary the setting of educational programs in order to prevent Giardia sp. transmission especially to infant population. Giardia sp. is more frequently detected in diarrheic or soft stool samples that in normal ones, this is the reason why owners of dogs with that stool characteristic should ask veterinarian to determine the cause and to give an appropriate treatment to that problem. / Tesis

El objetivo de este trabajo fue determinar la prevalencia de Guardia sp. en la población canina doméstica en los 6 distritos que conforman la Provincia Constitucional del Callao en el Perú. Para tal fin se colectaron 385 muestras fecales de perros aparentemente normales de ambos sexos, de diferentes edades y de acuerdo a la zona en donde habitaban sus propietarios. Las muestras fueron procesadas mediante la técnica de sedimentación espontánea; encontrándose una prevalencia de 9,35 ± 2% de Guardia sp. Mediante la prueba de regresión logística se cuantificó la asociación entre la presencia del parásito y las características físicas de las heces, estrato distrital de procedencia, sexo y edad del canino; se determinó una relación estadísticamente significativa entre el hallazgo de quistes de Guardia sp. y las características físicas de las muestras. Los resultados de nuestro estudio denotan una parasitosis moderada de Guardia sp. en los caninos, evidenciando un riesgo zoonótico, por lo que se hace necesario el establecimiento de programas educativos para prevenir la posibilidad de contagio especialmente en la población infantil. Asimismo, debido a que la ocurrencia del parásito es más frecuente en heces diarreicas o pastosas que en heces normales, se debe consultar al Médico Veterinario para que determine la causa y un adecuado tratamiento a ese trastorno digestivo. / The aim of this study was to determine Guardian sp. prevalence in household dog population on different districts that form Provincia Constitutional del Callao in Peru. Fecal samples were collected from 385 apparently healthy dogs of different ages that were selected depending on the zone where their owners lived. Stool samples were processed by spontaneous sedimentation technique and we found that 9,35 ± 2% (36/385) of the canine population in this study were positive to Giardia sp. cysts. Relationships between cysts detection and stool samples characteristics, provenience, sex and age of the dogs were analyzed by logistic regression; significant statistical relationship was found between Giardia sp detection and stoll samples physical characteristics. Results show a moderate Giardia sp. infection in dogs which evidences a zoonotic risk that makes necessary the setting of educational programs in order to prevent Giardia sp. transmission especially to infant population. Giardia sp. is more frequently detected in diarrheic or soft stool samples that in normal ones, this is the reason why owners of dogs with that stool characteristic should ask veterinarian to determine the cause and to give an appropriate treatment to that problem.

Thesis (M.S. in geology)--Washington State University, December 2006. / Includes bibliographical references (p. 55-60).

El objetivo del estudio fue determinar la prevalencia de Giardia sp. en caninos y niños de comunidades campesinas de los distritos de Ajoyani, Palca y Santa Lucía en Puno. Se recolectaron 130 muestras fecales tanto de niños como de canes. Las muestras fueron conservadas en formol al 10%, siendo luego enviadas al Laboratorio de Parasitología de la FMV-Lima para su procesamiento. Para el diagnóstico de Giardia sp. cada muestra fue analizada mediante las técnicas de Sedimentación Espontánea y Sheather. Considerando como positivo el hallazgo del parásito en una de las dos técnicas usadas, se obtuvo una prevalencia global de 14.6+6.1% y 28.5+7.8% en caninos y niños respectivamente. En caninos se hallaron prevalencias de 31.8%, 18.2% y 9.3% en los distritos de Ajoyani, Palca y Santa Lucía, respectivamente; las prevalencias en machos y hembras fueron de 14.6% y 17.4% respectivamente y según los grupos de edad de 0-6 meses, >6-12 meses, >12-72 meses y >72 meses fueron de 7.7%, 21.7%, 11.4% y 16.0% respectivamente. En niños se obtuvieron prevalencias de 36.4%, 13.6% y 30.2% en Ajoyani, Palca y Santa Lucia, respectivamente; los niños presentaron prevalencias de 14.6% y las niñas 17.4%; según los grupos etarios de 0-3años, >3-7años, >7-12 años fueron de 33.3%, 29.7%, 25.9% respectivamente. Para el análisis estadístico se empleó la prueba de chi cuadrado, con un nivel de significancia de 0,05%. No se encontró asociación estadística significativa (p>0,05) entre la presencia de Giardia sp. y las variables estudiadas. Además la técnica de sedimentación espontánea demostró ser la técnica más eficaz para el diagnóstico del parásito. Las prevalencias halladas en caninos y niños sugieren infecciones independientes. Sin embargo, sólo se esclarecería con futuros estudios moleculares, para descartar posible riesgo zoonótico. Palabras clave: Giardia sp., Puno, Sedimentación espontánea, Sheather, zoonosis / --- The aim of this study was to determine the prevalence of Giardia sp. in dogs and children in rural communities of the districts Ajoyani, Palca and Santa Lucía in Puno. We collected 130 fecal samples from both children and dogs. The samples were preserved in formaldehyde at 10%, then sent to the Laboratory of Parasitology of the FMV-Lima for processing. For the diagnosis of Giardia sp. each sample was analyzed by spontaneous sedimentation techniques and Sheather. Considering as positive the parasite founding in one of two techniques used, we obtained an overall prevalence of 14.6+6.1% and 28.5+7.8% in dogs and children respectively. In dogs, were found prevalences of 31.8%, 18.2% and 9.3% in the districts of Ajoyani, Palca, and Santa Lucía, respectively; the prevalences in males and females were 14.6% and 17.4% respectively and according to age groups from 0-6 months,> 6-12 months, > 12-72 months and > 72 months were 7%, 21.7%, 11.4% and 16.0% respectively. In children, were obtained 36.4%, 13.6% and 30.2% prevalences in Ajoyani, Palca and Santa Lucía, respectively; boys had prevalences of 14.6% and girls had 17.4%, according to the age groups from 0-3years,> 3-7years, > 7-12 years were 33.3%, 29.7%, 25.9%, respectively. For statistical analysis we used the chi square test with a significance level of 0.05%. There was not significant association (p> 0.05) between the presence of Giardia sp. and the variables studied. Furthermore, the spontaneous sedimentation technique proved to be the most effective technique for the diagnosis of the parasite. The prevalence found in dogs and children suggest independent infections. However, only molecular studies would clarify in future to rule out possible zoonotic risk. Keywords: Giardia sp., Puno, spontaneous sedimentation, sheather, zoonosis

Recombinant giardial arginine deiminase (rADI) was characterized. The enzyme was found to have a specific activity of 12 U (mg protein)-1under at pH 7.4 and 1 mM arginine. The maximum velocity was 14.75 U (mg protein-1) and the KM was 0.167 mM. The specific activity and maximum velocity values are significantly lower than the values reported previously for giardial rADI, while the KM value is quite similar. The optimum pH for the giardial rADI was 6-9, a broad range compared to other arginine deiminases. Recombinant ADI is very specific in its binding specificity, with canavanine (KI 2.4 mM) and ornithine (KI 2.1 mM) being the only substrate analogues giving significant inhibition from the wide variety of analogues tested. None of the analogues could be shown to act as alternative substrates. The contribution of conserved, catalytic and C-terminal residues proposed by previous research towards ADI activity was investigated by site-directed mutagenesis. Mutations of catalytic site residues Asp175, Glu226, His280 and Cys424 decreased the rADI activity to below 2%. Conservative mutations showed significant activity for Asp175 to Glu175 (DE175) and Glu226 to Asp226 (ED226). Site directed mutagenesis on the conserved non-catalytic site Leu46 showed activities below 15%, with the highest activity observed for the mutation to Val46 (LV46), which differs in one CH2 to Leu46 on its side chain. The KM of the mutant LV46 was 3.64 mM while for LA46 (Leu to Ala mutation) was 1.33 mM. Excising 5, 13, 16 amino acids from the C-terminal residues resulted in activity decreasing to 0.8% of the wild type, while excising 54 amino acids caused the rADI to be insoluble. Sequence alignment of Giardia and Dictyostelium suggests a homologous area within the C-terminal extension. Site directed mutagenesis on the Tyr567 residue in this region resulted in a decrease in activity, with the highest activity observed for a Tyr to Phe mutation. Studies using specific cysteine protease inhibitors demonstrated partial protection against proteolysis of ADI against giardial proteases. Degradation of ADI by giardial proteases was more rapid at pH 6 than at pH 7.4.

Bibliography: leaves 183-228

El objetivo del estudio fue determinar la prevalencia de Giardia sp. en caninos y niños de comunidades campesinas de los distritos de Ajoyani, Palca y Santa Lucía en Puno. Se recolectaron 130 muestras fecales tanto de niños como de canes. Las muestras fueron conservadas en formol al 10%, siendo luego enviadas al Laboratorio de Parasitología de la FMV-Lima para su procesamiento. Para el diagnóstico de Giardia sp. cada muestra fue analizada mediante las técnicas de Sedimentación Espontánea y Sheather. Considerando como positivo el hallazgo del parásito en una de las dos técnicas usadas, se obtuvo una prevalencia global de 14.6+6.1% y 28.5+7.8% en caninos y niños respectivamente. En caninos se hallaron prevalencias de 31.8%, 18.2% y 9.3% en los distritos de Ajoyani, Palca y Santa Lucía, respectivamente; las prevalencias en machos y hembras fueron de 14.6% y 17.4% respectivamente y según los grupos de edad de 0-6 meses, >6-12 meses, >12-72 meses y >72 meses fueron de 7.7%, 21.7%, 11.4% y 16.0% respectivamente. En niños se obtuvieron prevalencias de 36.4%, 13.6% y 30.2% en Ajoyani, Palca y Santa Lucia, respectivamente; los niños presentaron prevalencias de 14.6% y las niñas 17.4%; según los grupos etarios de 0-3años, >3-7años, >7-12 años fueron de 33.3%, 29.7%, 25.9% respectivamente. Para el análisis estadístico se empleó la prueba de chi cuadrado, con un nivel de significancia de 0,05%. No se encontró asociación estadística significativa (p>0,05) entre la presencia de Giardia sp. y las variables estudiadas. Además la técnica de sedimentación espontánea demostró ser la técnica más eficaz para el diagnóstico del parásito. Las prevalencias halladas en caninos y niños sugieren infecciones independientes. Sin embargo, sólo se esclarecería con futuros estudios moleculares, para descartar posible riesgo zoonótico. Palabras clave: Giardia sp., Puno, Sedimentación espontánea, Sheather, zoonosis / The aim of this study was to determine the prevalence of Giardia sp. in dogs and children in rural communities of the districts Ajoyani, Palca and Santa Lucía in Puno. We collected 130 fecal samples from both children and dogs. The samples were preserved in formaldehyde at 10%, then sent to the Laboratory of Parasitology of the FMV-Lima for processing. For the diagnosis of Giardia sp. each sample was analyzed by spontaneous sedimentation techniques and Sheather. Considering as positive the parasite founding in one of two techniques used, we obtained an overall prevalence of 14.6+6.1% and 28.5+7.8% in dogs and children respectively. In dogs, were found prevalences of 31.8%, 18.2% and 9.3% in the districts of Ajoyani, Palca, and Santa Lucía, respectively; the prevalences in males and females were 14.6% and 17.4% respectively and according to age groups from 0-6 months,> 6-12 months, > 12-72 months and > 72 months were 7%, 21.7%, 11.4% and 16.0% respectively. In children, were obtained 36.4%, 13.6% and 30.2% prevalences in Ajoyani, Palca and Santa Lucía, respectively; boys had prevalences of 14.6% and girls had 17.4%, according to the age groups from 0-3years,> 3-7years, > 7-12 years were 33.3%, 29.7%, 25.9%, respectively. For statistical analysis we used the chi square test with a significance level of 0.05%. There was not significant association (p> 0.05) between the presence of Giardia sp. and the variables studied. Furthermore, the spontaneous sedimentation technique proved to be the most effective technique for the diagnosis of the parasite. The prevalence found in dogs and children suggest independent infections. However, only molecular studies would clarify in future to rule out possible zoonotic risk. Keywords: Giardia sp., Puno, spontaneous sedimentation, sheather, zoonosis

Gerbils, experimentally infected with Giardia lamblia trophozoites, had trophozoites and encysting trophozoites in all 3 equal sections of the small intestine and in the colon at necropsy. Cysts were found in the second and third sections of the small intestine and in the colon. WB strain derived trophozoites (WB, D1, WB-C6, and V1) differed in levels of encystation in vitro but not in gerbils. Passage in gerbils increased the in vitro encystation levels of WB and D1 but decreased that of WB-C6 and V1. No differences were found in the total protein profiles or isoenzyme patterns of these G. lamblia populations. Immunization of mice with in vitro cysts produced monoclonal antibodies (mAbs) recognizing cyst protein antigens. In Immunofluorescence (IFA), mAb 5A4.G6 recognized cyst walls. This mAb reacted with a 38 kD band on Western blots. IFA results showed that mAb 8C5.C11 reacts with vesicles in encysting trophozoites and with cyst walls. It recognized 26, 28, 42 and 46 kD bands in Western blots. When mAb 8C5.C11 and Guinea pig complement were added to 0-9 hour encysting cultures, the numbers of cysts produced were significantly reduced compared to control. MAb 5A4.G6 did not affect in vitro encystation.

No description available.