Toxoplasma gondii est un parasite intracellulaire et obligatoire. Ce protozoa est un des parasites les plus successifs qui infectent tous les animaux à sang chaud, y compris l’humain. Ce succès est probablement à cause de la sécrétion d’une des séries de protéines effectrices, qui sont impliquées dans la modulation des voies de signalisation de la cellule hôte. Cette modulation permet aux parasites d’établir une infection chronique qui dure un long terme, et qui favorise leur transmission à un nouvel hôte. Dans cette étude, nous avons identifié un nouvel effecteur dérivé par la granule dense, appelé GRA18, qui est sécrété dans le cytoplasme de cellule hôte par les tachyzoites intracellulaires. La mutation de gra18 résulte une diminution de virulence chez les parasites de type II, qui suggère l’importance de GRA18 dans la pathogénicité de ce parasite. Afin d’étudier le mécanisme d’action de GRA18, nous avons effectué un criblage à haut-débit d’une librairie humaine chez la levure. Ce criblage nous permet d’identifier β-catenin, GSK3α/β, and PP2A-B56, ce qui sont tous les régulateurs bien connus dans la voie de signalisation canonicale de Wnt. Nous avons confirmé l’intéractome de GRA18 par l’approche biochimique. La surexpression de GRA18 induit l’accumulation de β-catenin dans le noyau de la cellule hôte, aussi que l’induition de gènes régulés par la signalisation de Wnt. Ces effets indiquent GRA18 joue un rôle de régulateur positif de β-catenin. A part de son rôle dans la prolifération, polarisation et la différentiation de cellule, β-catenin est également un facteur de transcription connu pour contrôler la réponse immunitaire et l’inflammation. L’analyse transcriptomique en comparant les macrophages dérivés par la moelle osseuse (BMDM) infectés par le sauvage (WT) et le gra18 mutant parasites confirme un rôle possible de GRA18, la modulation d’expression génique de cellule hôte, surtout ceux qui codent pour les chemokines. Cette régulation est ensuite confirmée par l’ELISA. L’hypothèse possible est que Toxoplasma sécrète GRA18 dans la cellule hôte afin de réguler positivement la production de chemokine reliée à la réponse de Th2, qui par contre atténue la réponse inflammatoire de l’hôte. Cette modulation augmente la chance de dissémination et la persistance de ce parasite par la formation de kyste. / Toxoplasma gondii, the obligate intracellular protozoan parasite, is one of the most successful pathogen that infects virtually all warm-blooded animals including humans. This success of the infection is likely due to its perfect ability to modulate numbers of host signaling pathways through the effector proteins, including those involved in immune responses. This modulation allows the parasite to establish a long-term chronic infection without causing severe symptom in the hosts, which facilitates its transmission to the new hosts. In this study, we identified GRA18, as a novel dense granule derived effector protein that is secreted into the cytoplasm of the host cell by the intracellular tachyzoite. GRA18 deficiency in type II strains attenuated the parasite virulence in mice model, suggesting the importance of GRA18 in the parasite pathogenesis. In order to investigate the mechanism of action of GRA18, we first performed a high-throughput two-hybrid screen of a human library in yeast that led to the identification of β-catenin, GSK3α/β, and PP2A-B56, all which are well known regulators of the canonical Wnt signaling pathway. We then validate the GRA18 interactome by biochemistry approach. The overexpression of GRA18 triggers the accumulation of β-catenin in the host cell nuclei as well as the induction of known canonical β-catenin target genes indicating that GRA18 is acting as a positive regulator of β-catenin. Besides its role in cell proliferation, polarization and differentiation, β-catenin is also a well-known co-transcription factor with important function in the control of inflammation and other immune responses. Transcriptomic analysis comparing mouse bone marrow–derived macrophages infected by wild type and GRA18-dificient parasite confirmed a possible role of GRA18 towards host gene expression and likely those encoding chemokines, which is further confirmed by ELISA experiments. An attractive hypothesis is that Toxoplasma delivers GRA18 to the host cell in order to regulate Th2-related chemoattractant chemokines, which in turn, dampens host inflammatory response leaving more chance for the parasites to disseminate and to cause the long-term persistence by forming the cyst.

La toxoplasmose est une anthropozoonose cosmopolite due à un protozoaire parasite intracellulaire obligatoire : Toxoplasma gondii. Cette maladie infectieuse est la plus souvent bénigne chez les personnes immunocompétentes mais revêt un caractère de gravité si l’atteinte concerne les femmes enceintes séronégatives ou les personnes immunodéprimées. En plus de cette incidence forte en médecine humaine, la toxoplasmose représente un important problème de santé vétérinaire. A l’heure actuelle, les seuls moyens de lutte contre ce parasite demeurent la chimiothérapie car il n’existe aucune stratégie prophylactique efficace. Le développement d’un vaccin efficace est d’une réelle nécessité et repose sur l’observation qu’une primo-infection par ce parasite confère à l’hôte immunocompétent une réponse immunitaire protectrice efficace à long terme et qui protège lors d’une réinfection et en particulier contre le risque d’une infection congénitale. / Toxoplasmosis is a cosmopolitan anthropozoonosis due to the obligate intracellular protozoan parasite Toxoplasma gondii. This infectious disease is most often benign in immunocompetent individuals but is particularly severe for pregnant women or immunocompromised patients. In addition to its significant impact on human medicine, toxoplasmosis is a major veterinary health problem. Currently, the only means to fight this parasite remain chemotherapy because there is no effective prophylactic strategy. The development of an effective vaccine is a real challenge and is based on the observation that a primary infection of immunocompetent hosts induces a effective and long-term protective immune response and protects during reinfection and in particular against the risk of congenital infection.

Toxoplasma gondii é um parasito do filo Apicomplexa que infecta uma grande variedade de hospedeiros, incluindo os humanos. O parasito invade a célula hospedeira por penetração ativa, com a participação das proteínas de suas organelas secretoras durante esse processo. Até o momento, somente um número limitado de proteínas secretoras tem sido descoberto, além disso, as moléculas efetoras envolvidas na invasão e sobrevivência do parasito não estão completamente compreendidas. A osteopontina (OPN) é uma glicofosfoproteína adesiva secretada, multifuncional, que contém o domínio arginina-glicina-ácido aspártico (RGD) de ligação à integrina, que está envolvida em uma variedade de eventos fisiológicos e patológicos, incluindo sinalização e sobrevivência celular. Pela primeira vez, nós demonstramos pelas técnicas de imunofluorescência e imunocitoquímica ultraestrutural que há uma intensa marcação para uma proteína OPN-like nos grânulos densos de taquizoítos de T. gondii extracelulares. O western blotting e o RT-PRC confirmaram a expressão de OPN-like nos taquizoítos. Nossos resultados também mostram que após a invasão dos macrófagos, a proteína OPN-like está localizada na membrana do vacúolo parasitóforo. Esses dados sugerem que os grânulos densos secretam uma proteína OPN-like, e nós podemos especular que essa proteína participa durante o processo de interação do parasito com as células hospedeiras. . / Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional secreted adhesive glycophosphoprotein containing the arginine-glycine-aspartic acid (RGD) integrin-binding domain, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated by immunofluorescence and immunoelectron microscopy approaches that there is an intense labeling for an OPN-like protein in the dense granules of extracellular T. gondii tachyzoites. Western blotting and RTPCR confirmed this protein expression in tachyzoites. Our results also showed that after macrophage invasion the OPN-like protein is localized at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we can speculate that this protein participates during the parasite interaction process with host cells.

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T20:14:34Z : No. of bitstreams: 1 nakashima_f_me_sjrp.pdf: 829285 bytes, checksum: 72d9338767526ff29fcf4612c5437bff (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Introdução: A infecção por Toxoplasma gondii em gestantes associa-se aos riscos de transmissão congênita. Este protozoário infecta os humanos utilizando como rota de infecção o trato gastrintestinal, onde se dá a síntese dos antígenos fucosilados Lea e Leb que determinam os fenótipos do sistema histo-sanguíneo Lewis [Le(a+b-), Le(a+b+) e Le(a-b-)]. A expressão destes fenótipos resulta de interações epistáticas entre os genes FUT2 (Secretor) e FUT3 (Lewis) que codificam as fucosiltransferases FUTII e FUTIII, respectivamente. Mutações no gene FUT3 determinam a ausência de fucosilação dos oligossacarídeos precursores do tipo 1 resultando na expressão do fenótipo Le(a-b-). A infecção por T. gondii e a expressão dos antígenos Lewis ocorrem no mesmo órgão e, embora aparentemente independentes, podem estar relacionadas entre si. Objetivo: Investigar a associação o sistema histo-sanguíneo Lewis e a infecção por T. gondii da cepa RH. Material e Métodos: Foram selecionadas 209 amostras de soro e de DNA genômico estocadas no Laboratório de Imunogenética do Departamento de Biologia Molecular da Faculdade de Medicina de São José do Rio Preto - FAMERP, coletadas de gestantes atendidas no Ambulatório de Gestação de Alto Risco do Hospital de Base da Fundação Faculdade Regional de Medicina de São José do Rio Preto – FUNFARME. Uma triagem para detecção dos anticorpos anti-T. gondii foi realizada nas amostras de soro pelo método hemaglutinação indireta (HAI). O ensaio imunoenzimático (ELISA) foi empregado para identificar os anticorpos específicos da classe IgG contra a cepa RH. Cento e noventa e cinco amostras de soro que apresentaram resultados concordantes entre os dois testes foram selecionadas para compor os grupos “reagente” e “não reagente”. Para inferir os fenótipos do sistema histo-sanguíneo Lewis, as amostras de DNA... / Toxoplasma gondii Infection in pregnant women is associated with risks of congenital transmission. This protozoa infects humans using as infection rout the gastro intestinal tract, where occur the synthesis of fucosylated Lea and Leb antigens which determine Lewis histo-blood group phenotypes [Le(a+b-), Le(a+b+), Le(a-b-)]. The expression of these phenotypes results from epistatic interactions between FUT2 (Secretor) and FUT3 (Lewis) genes which codes both the FUTII and FUTIII fucosyltransferases, respectively. Single nucleotide polymorphisms affecting FUT3 gene determine the absence of type 1 oligosaccharide precursor fucosylation and the expression of Le(a-b-) phenotype. The entrance of T. gondii and the expression of Lewis histo-blood group system occur in the same organ, probably there is some link between them. Objective: The aim of this study was to test the hypothesis that the Lewis histo-blood group system is associated with infection by T. gondii. Material e Method: A total of 209 serum sample and genomic DNA sample stored at the Immunogenetics Laboratory from the Molecular Biology Department of Medicine School in São José do Rio Preto – FAMERP, obtained from pregnant women who attended in the High-Risk Pregnancy Clinic of Hospital de Base were enrolled in this study. Anti-T. gondii antibodies were screened by indirect hemagglutination test. ELISA assays were used to identify specific IgG antibodies to RH strain. One hundred and ninety-five serum samples, with identical results between both tests were selected to compose the groups “reagent’ and “non reagent”. To infer the Lewis histo-blood group system phenotypes, genomic DNA samples corresponding to the serum sample were genotyped for the G428A mutation of the FUT2 gene and T202C and C314T of FUT3 gene by PCR-RFLP and PCR-SSP assays, respectively. The data were analyzed by Fisher’s exact test... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-20Bitstream added on 2014-06-13T19:55:44Z : No. of bitstreams: 1 sobrinho_lsv_me_araca.pdf: 921356 bytes, checksum: 29d3e58899a35e5de14b32e263c2fe77 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do presente estudo foi determinar em uma população de 302 gatos provenientes de área endêmica para leishmaniose visceral, a prevalência da infecção por Leishmania spp. e a presença de coinfecção pelo Toxoplasma gondii, vírus da imunodeficiência felina (FIV) e vírus da leucemia felina (FeLV). Foram evidenciadas formas amastigotas de Leishmania spp. em 9,93% (30/302) dos animais. A prevalência da leishmaniose observada por meio dos métodos de ELISA-proteína A, ELISA-IgG ou exame parasitológico direto foi de 21,85% (66/302), sendo 13,64% (9/66) positivos no exame parasitológico direto e sororeagentes nas técnicas de ELISA indireto. Doze animais (70,59%) foram sororeagentes para o FIV e a Leishmania spp., enquanto 17 (25,76%) apresentaram anticorpos anti-Toxoplasma gondii e anti-Leishmania spp. e cinco (71,43%) apresentavam infecção pelos três agentes. Não foi observada coinfecção entre Leishmania spp. e o FeLV. Houve associação estatisticamente significante entre a coinfecção por Leishmania spp. e pelo vírus da imunodeficiência felina, bem como entre a presença de Leishmania spp., do vírus da imunodeficiência felina e do Toxoplasma gondii. A sensibilidade e a especificidade dos métodos de ELISA-proteína A, ELISA-IgG e reação de imunofluorescência indireta para o diagnóstico de leishmaniose felina foram de 56,6% e 89,47%, 55,55% e 90,96% e 54,55% e 96,80%, respectivamente. As concordâncias entre a RIFI e as técnicas de ELISA-proteína A e ELISA-IgG foram fracas. No entanto, houve boa concordância entre as duas últimas técnicas. O presente estudo verificou que gatos residentes em área endêmica para leishmaniose visceral são predispostos à coinfecção por Leishmania spp. e vírus da imunodeficiência felina, e que parte deles desenvolvem sintomas inespecíficos e devem ser investigados em um diagnóstico diferencial / The aim of this study was to determine, in a population of 302 cats from an endemic area for visceral leishmaniasis, the prevalence of the infection by Leishmania spp. and the presence of co-infection by Toxoplasma gondii, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Amastigote forms of Leishmania spp were evidenced in 9.93% (30/302) of the animals. Prevalence of leishmaniasis by ELISA-prot A, ELISA-IgG or direct parasitological examination was 21.85% (66/302), being 13.64% (9/66) positive in both direct parasitological examination and ELISA. Twelve animals (70.59%) were seroreagent for FIV and Leishmania spp., while 17 (25.76%) showed antibodies against Toxoplasma gondii and Leishmania spp. and five (71.43%) showed antibodies against those three agents. Co-infection was not observed between Leishmania spp. and FeLV. There was statistically significant correlation between the co-infection by Leishmania spp. and by the immunodeficiency virus, as well as among the present of Leishmania spp, feline immunodeficiency virus and Toxoplasma gondii. The susceptibility and the specificities of (the methods) ELISA-prot A, ELISA-IgG and reaction of indirect immunofluorescence for the diagnosis of feline leishmaniasis were 56.6% and 89.47%, 55.55% and 90.96% and 54.55% and 96.80%, respectively. The agreements between RIFI and ELISA-prot A and ELISA-IgG techniques were weak. However, there was a good agreement between the last two techniques. This study verified that cats from endemic areas for visceral leishmaniasis are predisposed to co-infection by Leishmania spp. and feline immunodeficiency virus, and that part of them developed nonspecific symptoms and should be investigated in a differential diagnosis

Made available in DSpace on 2014-12-17T14:10:24Z (GMT). No. of bitstreams: 1 NormaHDM_DISSERT.pdf: 999595 bytes, checksum: c62cdc67318a196202b24c4678092ac5 (MD5) Previous issue date: 2011-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Toxoplasmosis, provoked by the intracellular parasite Toxoplasma gondii, is one of the most prevalent parasitoses in the world. In humans, transmission occurs by three evolutionary forms of the parasite: oocysts, tissue cysts and tachyzoites. Wild and domestic felines are definitive hosts. The ocular form of toxoplasmosis can be of congenital origin with early or late clinical manifestations, or acquired after birth. T. gondii is considered the main culprit for most cases of infectious uveitis. This study aimed at assessing ocular toxoplasmosis, relating it to factors associated to the patient s lifestyle and describing the epidemic-serological and clinical profile of affected individuals. A cross-sectional study was conducted with a population of 159 patients. Univariate analysis (odds ratio) was used to evaluate the data, with a confidence interval of 95% and p-value < 0.05. A prevalence of 4% of ocular toxoplasmosis was observed in the population of patients treated at an ophthalmological clinic. Of patients directly examined by immunoenzymatic assay (MEIA-AxSYM?- Microparticle Enzyme Immune Assay), considering only uveitis, a frequency of anti-T. gondii of 73%, most of whom exhibited titulation between 40-99 UI IgG/mL. With respect to location of ocular lesions, bilaterality was observed in 57% of patients assessed by the ophthalmoscopy technique. When compared with the results of an active search of medical records, a similarity in ocular toxoplasmosis (74%) and bilateral lesion location (55%) was observed. Type I lesion was the most frequent type observed, with intraocular disposition in the macula. An epidemiological survey revealed that direct contact with cats; consuming raw or poorly cooked meat and direct contact with the soil were significantly associated with greater likelihood of acquiring ocular toxoplasmosis. Sample characterization in relation to age range was significant for patients between 31 and 40 years [??, chi-square test (p = 0.04)], but population traits such as schooling, sanitary district, and monthly income were not significant. Results confirm that ocular toxoplasmosis is widely distributed in the metropolitan area of Natal, Brazil, with significant prevalence of ocular lesions provoked by T.gondii. It is suggested that sanitary authorities exert greater control in order to minimize the risk of toxoplasmic infection, mainly in pregnant women. / A toxoplasmose, provocada pelo parasito intracelular Toxoplasma gondii, ? uma das parasitoses mais prevalentes em todo o mundo. Nos humanos, a transmiss?o ocorre por meio das tr?s formas evolutivas do parasito: oocistos, cistos tissulares e taquizo?tos. Os fel?deos, silvestres ou dom?sticos, s?o os hospedeiros definitivos. A forma ocular da toxoplasmose pode ser de origem cong?nita com manifesta??es cl?nicas precoces ou tardias, ou ainda ser adquirida ap?s o nascimento. O T. gondii ? considerado o principal respons?vel pela maioria dos casos de uve?tes infecciosas. Este estudo avaliou a toxoplasmose ocular, relacionando-a com fatores associados ao modo de vida dos pacientes e descrever um perfil epid?mico-sorol?gico e cl?nico dos indiv?duos acometidos. Foi realizado um estudo seccional, com uma popula??o de 159 pacientes. Para avalia??o dos dados observados foi utilizada a an?lise univariada com c?lculo de Odds Ratio, tendo como ?ndice de confian?a de 95% e o valor de p < 0,05. Foi observada uma preval?ncia de 4% de toxoplasmose ocular na popula??o de pacientes atendidos na cl?nica oftalmol?gica. Dos pacientes examinados diretamente por ensaio imunoenzim?tico (MEIA-AxSYM?- Microparticle Enzyme Imune Assay), considerando apenas as uve?tes, foi verificada uma freq??ncia de anti- T. gondii de 73%, a maioria dos pacientes apresentaram titula??o entre 40-99 UI IgG/mL. Quanto ? localiza??o da les?o ocular foi observada a bilateralidade em 57% dos pacientes avaliados pela t?cnica de oftalmoscopia. Quando comparados com os resultados da busca ativa de prontu?rios se observou uma similaridade na freq??ncia de preval?ncia para toxoplasmose ocular (74%), e 55 % quanto a localiza??o bilateral da les?o. Quanto ao tipo de les?o observada, a mais freq?ente foi a les?o do tipo I, com disposi??o intraocular na m?cula. O inqu?rito epidemiol?gico revelou que os fatores de riscos: o contato direto com gatos, o consumo de carne crua ou mal cozida e o contato direto com o solo foram significativamente associados com as maiores chances de adquirir toxoplasmose ocular. A caracteriza??o da amostra em rela??o ? faixa et?ria foi significativa para pacientes entre 31-40 anos [??, Teste do qui-quadrado (p = 0,04)], por?m caracter?sticas populacionais tais como escolaridade, distrito sanit?rio, renda mensal n?o tiveram signific?ncia segundo esse teste estat?stico. Os resultados confirmam que a toxoplasmose ocular est? amplamente distribu?da na cidade do Natal, com significativa preval?ncia de les?es oculares provocadas pelo T.gondii. ? recomend?vel um maior controle por parte das autoridades sanit?rias, visando minimizar o risco de infec??o toxopl?smica em toda popula??o.

Made available in DSpace on 2014-12-17T14:10:26Z (GMT). No. of bitstreams: 1 ClaudioBSO_DISSERT.pdf: 2673133 bytes, checksum: 44082c33463f5700264efa7b77ae2f4c (MD5) Previous issue date: 2012-03-14 / Toxoplasmosis, a benign disease in normal healthy individuals, can have serious effects in pregnant women and immunocompromised patients. It is a parasitic disease caused by Toxoplasma gondii (Tg), an obligatory intracellular protozoan. The prophylactic and therapeutic arsenal against this parasite is very restricted. Thus, there is an ongoing search for novel drugs and therapeutic strategies. A promising alternative is a rational approach using medicinal plants. This study aimed to standardize methodologies for assessing the toxicological, antiproliferative, antioxidant, antiinflammatory and anti-Toxoplasma effects of Estragole and Thymol compounds isolated from species of plants (Lippia sidoides and Croton zenhtneri) commonly used in the Cariri region of Ceara State, Brazil. First we evaluated in vivo toxicity and conducted a pathological analysis of mice livers. In vivo antiinflammatory activity was assessed using air pouch and paw edema methods. Cytotoxicity assays were performed and antiproliferative, antioxidant and nitric oxide production analyzed. Anti-Toxoplasma activity was evaluated in a congenital experimental model with varying stages of maternal infection using the ME-49 strain and a non- congenital model by using ME-49 and RH strains. The results suggest low to moderate toxicity for both compounds. Thymol was more toxic in vivo and in vitro, having greater pathological repercussion than Estragole. The compounds were inactive for antiproliferative activity. Thymol showed better antioxidant activity, while Estragole stimulated nitric oxide production in macrophages. Both showed significant antiinflammatory activity. In non-congenital Tg infection, both compounds were active only against the ME49 strain. In congenital infection, Estragole (oral route) improved the newborn weight of infected mothers compared with untreated controls. Subcutaneous administration of the two compounds increased the weight of offspring born to infected mothers compared with untreated controls. We concluded that Estragole and Thymol exhibit important biological and anti-Toxoplasma activities. Further studies are needed to elucidate the mechanism of action of these compounds and other possible activities not investigated in the present study / A toxoplasmose, doen?a de curso benigno em pacientes imunocompetentes, mas que pode ocasionar graves acometimentos cl?nicos em imunossuprimidos e gestantes ? causada pelo Toxoplasma gondii (Tg), protozo?rio intracelular obrigat?rio. O arsenal profil?tico e terap?utico contra este parasito ? bastante restrito, tornando urgente a busca por novos medicamentos. Uma alternativa promissora ? a abordagem racional a partir de plantas medicinais. Neste estudo foram padronizadas metodologias objetivando avaliar os efeitos toxicol?gicos, antiproliferativos, antiinflamat?rios, antioxidantes e anti-toxopl?smicos do Timol e Estragol isolados, respectivamente, das esp?cies vegetais Lippia sidoides e Croton zenhtneri, usadas popularmente na regi?o do Cariri/CE para diversas mol?stias. Inicialmente foram realizados testes de toxicidade in vivo e an?lise patol?gica do f?gado dos camundongos. Avaliou-se a atividade antiinflamat?ria in vivo pelo m?todo da bolsa de ar e pela medida da varia??o do edema de pata. Ensaios de citotoxicidade in vitro foram realizados, bem como pesquisas de atividade antiproliferativa, antioxidante e produ??o de ?xido n?trico. A atividade anti-toxopl?smica foi avaliada em modelo murino de infec??o n?o cong?nita (usando Tg ME-49 e Tg RH) e cong?nita, com per?odos variados de infec??o materna (Tg ME-49). Os resultados sugerem toxicidade baixa a moderada para os dois compostos. O Timol se mostrou mais t?xico, in vivo e in vitro, e teve maior repercuss?o patol?gica do que o Estragol. Os compostos foram inativos para atividade antiproliferativa. O Timol apresentou ?tima atividade antioxidante enquanto o Estragol estimulou a produ??o de ?xido n?trico em macr?fagos. Ambos apresentaram significativa atividade antiinflamat?ria. Os dois compostos foram ativos apenas contra a cepa ME-49. No modelo de infec??o cong?nita o Estragol proporcionou um aumento significativo de peso neonatal em filhotes de m?es infectadas quando comparadas com o controle n?o tratado. O mesmo ocorreu com filhotes de m?es tratadas, via subcut?nea, com ambos os compostos ap?s 30 dias. Conclui-se que o Timol e o Estragol possuem importantes atividades biol?gicas e anti-toxopl?smica. Estudos posteriores s?o necess?rios para elucidar o mecanismo de a??o destes compostos, al?m de outras poss?veis atividades n?o investigadas neste estudo

Orientador: Luiz Carlos de Mattos / Banca: Wilson Baleotti Junior / Banca: Marilanda Ferreira Bellini / Resumo: Introdução: A infecção por Toxoplasma gondii em gestantes associa-se aos riscos de transmissão congênita. Este protozoário infecta os humanos utilizando como rota de infecção o trato gastrintestinal, onde se dá a síntese dos antígenos fucosilados Lea e Leb que determinam os fenótipos do sistema histo-sanguíneo Lewis [Le(a+b-), Le(a+b+) e Le(a-b-)]. A expressão destes fenótipos resulta de interações epistáticas entre os genes FUT2 (Secretor) e FUT3 (Lewis) que codificam as fucosiltransferases FUTII e FUTIII, respectivamente. Mutações no gene FUT3 determinam a ausência de fucosilação dos oligossacarídeos precursores do tipo 1 resultando na expressão do fenótipo Le(a-b-). A infecção por T. gondii e a expressão dos antígenos Lewis ocorrem no mesmo órgão e, embora aparentemente independentes, podem estar relacionadas entre si. Objetivo: Investigar a associação o sistema histo-sanguíneo Lewis e a infecção por T. gondii da cepa RH. Material e Métodos: Foram selecionadas 209 amostras de soro e de DNA genômico estocadas no Laboratório de Imunogenética do Departamento de Biologia Molecular da Faculdade de Medicina de São José do Rio Preto - FAMERP, coletadas de gestantes atendidas no Ambulatório de Gestação de Alto Risco do Hospital de Base da Fundação Faculdade Regional de Medicina de São José do Rio Preto - FUNFARME. Uma triagem para detecção dos anticorpos anti-T. gondii foi realizada nas amostras de soro pelo método hemaglutinação indireta (HAI). O ensaio imunoenzimático (ELISA) foi empregado para identificar os anticorpos específicos da classe IgG contra a cepa RH. Cento e noventa e cinco amostras de soro que apresentaram resultados concordantes entre os dois testes foram selecionadas para compor os grupos "reagente" e "não reagente". Para inferir os fenótipos do sistema histo-sanguíneo Lewis, as amostras de DNA... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Toxoplasma gondii Infection in pregnant women is associated with risks of congenital transmission. This protozoa infects humans using as infection rout the gastro intestinal tract, where occur the synthesis of fucosylated Lea and Leb antigens which determine Lewis histo-blood group phenotypes [Le(a+b-), Le(a+b+), Le(a-b-)]. The expression of these phenotypes results from epistatic interactions between FUT2 (Secretor) and FUT3 (Lewis) genes which codes both the FUTII and FUTIII fucosyltransferases, respectively. Single nucleotide polymorphisms affecting FUT3 gene determine the absence of type 1 oligosaccharide precursor fucosylation and the expression of Le(a-b-) phenotype. The entrance of T. gondii and the expression of Lewis histo-blood group system occur in the same organ, probably there is some link between them. Objective: The aim of this study was to test the hypothesis that the Lewis histo-blood group system is associated with infection by T. gondii. Material e Method: A total of 209 serum sample and genomic DNA sample stored at the Immunogenetics Laboratory from the Molecular Biology Department of Medicine School in São José do Rio Preto - FAMERP, obtained from pregnant women who attended in the High-Risk Pregnancy Clinic of Hospital de Base were enrolled in this study. Anti-T. gondii antibodies were screened by indirect hemagglutination test. ELISA assays were used to identify specific IgG antibodies to RH strain. One hundred and ninety-five serum samples, with identical results between both tests were selected to compose the groups "reagent' and "non reagent". To infer the Lewis histo-blood group system phenotypes, genomic DNA samples corresponding to the serum sample were genotyped for the G428A mutation of the FUT2 gene and T202C and C314T of FUT3 gene by PCR-RFLP and PCR-SSP assays, respectively. The data were analyzed by Fisher's exact test... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-12-17T15:37:13Z (GMT). No. of bitstreams: 1 RaquelSM_DISSERT.pdf: 1210364 bytes, checksum: 4fa725d4ec9ab3f0348c1172d9b3ef02 (MD5) Previous issue date: 2012-04-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Toxoplasma gondii is a protozoan parasite that induces behavioral changes in rodents. The aim of this study was to evaluate the effect of infection by T. gondii during the chronic phase in working memory and impulsivity in rodents as well as the effect of antipsychotics to reverse any behavioral changes resulting from infection. Female Wistar rats (n = 40) were infected with 25 cysts of the strain ME-49 T. gondii after 4 months the animals were subjected to behavioral tests: tolerance to delay gratification, in which the animal must choose between two rewards, a smaller and more immediate, but delayed and the test of spontaneous alternation, in which the animal must use spatial cues to remember previously visited arms. Antipsychotic drugs were intraperitoneally administered during the testing of the behavioral experiments, the antipsychotic is haloperidol (1.5 mg / kg) administered 60 min before the start of the session and the antipsychotic clozapine (2.5 mg / kg) 30 min before. Animals infected with the parasite did not show operating deficits of memory, and motor impairment did not develop, however motor impairment was observed only in animals treated with haloperidol. It was found that administration of clozapine and haloperidol increased the percentage of alternation in infected and control groups in task switching espont?nea.N?o no distinction between control animals and infected the test of tolerance to delay gratification in relation to the percentage of choices greatest reward, during the pre-training and training, in which there is a delay of 15 s to access the great reward, however it was observed that infected animals prefer the greatest reward, when there is a delay of 30 s when compared to control group. The administration of clozapine possible that infected animals chose the greatest reward in the delay of 30 seconds during the test. These data suggest that infected mice do not exhibit deficits in working memory and that clozapine has therapeutic efficacy in improving cognitive performance of mice infected / O Toxoplasma gondii ? um protozo?rio parasito que induz altera??es comportamentais em roedores. O objetivo do presente estudo foi avaliar o efeito da infec??o pelo T.gondii durante a fase cr?nica na mem?ria operacional e na impulsividade de roedores, bem como o efeito de antipsic?ticos em reverter as eventuais altera??es comportamentais decorrentes da infec??o. Ratos wistar f?meas (n=40) foram infectadas com 25 cistos da cepa ME-49 do T. gondii, ap?s 4 meses os animais foram submetidos aos testes comportamentais: toler?ncia ao retardo de gratifica??o, na qual o animal deve escolher entre duas recompensas, uma menor imediata e uma maior, mas com retardo e o teste de altern?ncia espont?nea, na qual o animal deve utilizar pistas espaciais para recordar bra?os previamente visitados. Os antipsicoticos foram administrados via intraperitoneal durante a fase teste dos experimentos comportamentais, sendo o antipsic?tico haloperidol (1,5 mg/kg) administrado 60 min antes do inicio das sess?es e o antipsic?tico clozapina (2,5 mg/kg) 30 min antes. Os animais infectados com o parasito n?o apresentaram d?ficits de memoria operacional, e n?o desenvolveram preju?zo motor, no entanto foi observado preju?zo motor apenas nos animais tratados com haloperidol. Foi verificado que a administra??o de clozapina e haloperidol aumentou a porcentagem de alterna??o em grupos controle e infectado na tarefa de altern?ncia espont?nea.N?o existe distin??o entre animais do grupo controle e infectado no teste de toler?ncia ao retardo de gratifica??o em rela??o a porcentagem de escolhas pela maior recompensa, durante as fases de pr?-treinamento e treinamento, no qual existe um retardo de 15 s para o acesso da grande recompensa, no entanto foi verificado que animais infectados preferem a maior recompensa, quando existe um retardo de 30 s quando comparado ao grupo controle. A administra??o da clozapina possibilitou que os animais infectados escolhessem a maior recompensa, no retardo de 30s na fase teste. Estes dados sugerem que ratos infectados n?o apresentam d?ficits de mem?ria operacional e que a clozapina apresenta efic?cia terap?utica em melhorar o desempenho cognitivo de ratos infectados

Made available in DSpace on 2014-12-17T15:37:14Z (GMT). No. of bitstreams: 1 ValeriaPS_DISSERT (2).pdf: 1441287 bytes, checksum: b2e4b1aca42f441a4c8631f09105903c (MD5) Previous issue date: 2012-04-27 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / T. gondii is an obligate intracellular protozoan and the main cause of retinochoroiditis in humans. The aim of this study was to evaluate the effect of the antipsychotic drugs haloperidol and clozapine on the course of infection by T. gondii of cultured embryonic retinal cells. Embryo retinas of Gallus gallus domesticus (E12) were used for the preparation of mixed monolayer cultures of retinal cells. Cultures were maintained on plates of 96 and 24 wells by 37?C in DMEM medium supplemented with 5% fetal bovine serum for 2 days. After this period, cultures were simultaneously infected with tachyzoites of T. gondii and treated with the antipsychotics haloperidol and clozapine for 48 hours. Treatment effects were determined by both assessing cell viability with the MTT method and evaluating infection outcomes in slides stained with Giemsa. The treatment with haloperidol and clozapine cells infected with T. gondii resulted in higher viability of these cells, suggesting a possible prevention of neuronal degeneration induced by T. gondii. Additionally, intracellular replication of this protozoan in cells treated with haloperidol and clozapine were significantly reduced, possibly by modulation of the parasite s intracellular calcium concentration / T. gondii ? um protozo?rio intracelular obrigat?rio e a principal causa de retinocoroidite em humanos. Este estudo foi conduzido para avalia??o do efeito do tratamento com as drogas antipsic?ticas haloperidol e clozapina sobre a infec??o de T. gondii em cultura de c?lulas retinianas embrion?rias. Retinas de embri?es de Gallus gallus domesticus de doze dias (E12) foram usados para prepara??o de culturas mistas de c?lulas retinianas em monocamada. As culturas foram mantidas em placas de 24 e 96 po?os ? 37?C em meio DMEM suplementado com 5% de soro fetal bovino durante 2 dias. Ap?s este per?odo, as culturas foram simultaneamente infectadas com taquiz?itas de T. gondii e tratadas com os antipsic?ticos haloperidol e clozapina por 48 horas. Os efeitos dos tratamentos das drogas sobre a infec??o deste parasito foram determinados pela avalia??o da viabilidade celular pelo m?todo de MTT e avalia??o de par?metros de infec??o em l?minas coradas pelo m?todo de giemsa. O tratamento com clozapina e haloperidol de c?lulas infectadas com T. gondii resultou em menor preju?zo da viabilidade destas c?lulas, o que sugere uma poss?vel preven??o de degenera??o neuronal induzida por estes antipsic?ticos. Adicionalmente, a replica??o intracelular deste protozo?rio, em c?lulas tratadas com clozapina e haloperidol foi significantemente reduzida, possivelmente pela modula??o da concentra??o intracelular de c?lcio deste parasito