Au cours de cette thèse, nous avons étudié plus particulièrement deux calréticulines de parasite et leur interaction avec la protéine C1q humaine, dans un contexte de détournement du système immunitaire. En effet, une stratégie de mimétisme moléculaire avec des cellules apoptotiques a été proposée pour l'exposition de calréticuline à la surface de la forme infectieuse du parasite Trypanosoma cruzi, agent vecteur de la maladie de Chagas. Dans le cas du parasite Entamoeba histolytica, agent vecteur de l'amibiase, l'interaction de C1q avec la calréticuline exposée à la surface de l'amibe est utilisée pour mieux phagocyter les cellules de l'hôte.La calréticuline est une protéine principalement localisée dans le réticulum endoplasmique où elle joue le rôle de protéine chaperonne en favorisant le repliement des protéines monoglucosylées. Une des fonctions extracellulaires de la calréticuline humaine est de favoriser l'élimination des cellules apoptotiques par les macrophages. Pour cela, la calréticuline est exposée à la surface des deux types cellulaires, à la surface desquelles elle est reconnue par C1q.Nous avons résolu la structure de fragments des deux CRTs de parasite. Des interactions de type chaperonne et un aperçu de la flexibilité du domaine P ont été observés dans l'empilement cristallin et approfondis par analyse de diffusion des rayons X aux petits angles. Ces fragments générés pour l'analyse structurale nous ont permis en plus d'identifier une région clé dans l'interaction des calréticulines avec C1q. Du côté de C1q, deux mutations dans les régions globulaires de C1q (GRC1q), inhibent l'interaction avec la CRT et les IgM, suggérant un site partagé. Pour approfondir la caractérisation de l'interaction, des études du complexe par RMN ont débuté et nous avons développé une première forme recombinante monocaténaire de GRC1q, dont nous avons déterminé la structure. Nos recherches peuvent aider à développer des traitements contre ces parasites, aussi bien qu'à décrypter le rôle de la CRT des mammifères présente à la surface des macrophages et des cellules apoptotiques. / During this thesis, we were interested in two parasite calreticulin and their interaction with the human C1q protein in the context of the subversion of the immune system. Indeed, a molecular mimicry strategy with human apoptotic cells is suggested for the calreticulin exposure on the surface of the infectious form of the parasite Trypanosoma cruzi, which is responsible of Chagas' disease. In the case of the parasite Entamoeba histolytica, which is involved in amoebiasis, the interaction of C1q with the surface-exposed calreticulin is used to enhance phagocytosis of host cells.Calreticulin is mainly localised in the endoplasmic reticulum, where it acts as a chaperone protein to favour the folding of monoglycosylated proteins. Moreover one of the extracellular functions of human calreticulin is to enhance the clearance of apoptotic cells by macrophages. This function is mediated through C1q interaction with calreticulin exposed on the surface of both cells.We solved the structure of fragments of both parasite calreticulins. Chaperone-like interactions and an overview of the flexibility of the P domain were observed in the crystal packing and deepened using SAXS analyses. The fragments generated for X-ray crystallography studies allowed us to identify a key region of the interaction between C1q and the calreticulins. Two C1q mutations located in its globular regions (GRC1q) inhibit the interaction with calreticulin and IgM, suggesting a common binding area. To further characterise theses interactions, we started NMR experiments and we produced the first single-chain recombinant form of GRC1q, which allowed solving its structure at high-resolution. Our investigations could provide tools to develop therapies against these parasites, and to decipher the role of mammal CRT on the surface of macrophages and apoptotic cells.

A amebíase é uma infecção causada pela Entamoeba histolytica. Entretanto, a diferenciação entre a E. histolytica e a Entamoeba dispar, espécies morfologicamente semelhantes, é fundamental para a conduta terapêutica, prevenção da doença invasiva e para a saúde pública. O propósito desde trabalho foi avaliar a Multiplex-PCR na detecção e diferenciação entre a E. histolytica e a E. dispar. Foi feita uma comparação entre os métodos de exame microscópico das fezes usando o conjunto diagnóstico Coprotest, a detecção de antígenos nas fezes usando um ensaio imunoenzimático comercial e o Multiplex-PCR padronizado no laboratório, para o diagnóstico da amebíase. A Multiplex-PCR foi padronizada usando amostras com parasitos obtidos de culturas padrões. Posteriormente, 127 amostras de fezes provenientes de duas comunidades do Estado do Rio de Janeiro, foram testadas e os resultados comparados. A análise de 127 amostras de fezes pelo exame parasitológico de fezes demonstrou que 27 (21%) amostras foram positivas para o complexo E. histolytica/E. dispar. Dessas amostras, 12 foram positivas pelo Multiplex-PCR, sendo que nove apresentaram o padrão de E. dispar (96 bp) e três o padrão E. histolytica (132 bp). Entre as amostras negativas detectadas pelo exame microscópico, três foram positivas para E. dispar e uma foi positiva para E. histolytica pelo Multiplex-PCR. Esse fato mostrou que a PCR foi mais eficiente do que o exame parasitológico realizado com uma amostra de fezes. Os resultados obtidos pelo ensaio de detecção de antígeno de E. histolytica foram concordantes com o do Multiplex-PCR. A análise estatística comparando os resultados do ELISA coproantígeno com o Multiplex-PCR, não pode ser feita devido ao baixo número de casos de E. histolytica. Os resultados acima indicam a necessidade do uso de métodos mais sensíveis para o diagnóstico da amebíase e a importância de se usar técnicas específicas na diferenciação entre E. histolytica e E. dispar. / Amebiasis is defined as an infection caused by Entamoeba histolytica. However, precise differentiation between E. histolytica and Entamoeba dispar, which are morphologically identical species, is essential for treatment decision, prevention of the invasive disease and public health. The purpose of the present study was to evaluate a Multiplex-PCR for detection and differentiation of E. histolytica from E. dispar. Microscopic examination of stools using the coprotest kit, detection of stool antigen using a commercial enzyme-linked immunosorbent assay kit and a home made Multiplex-PCR, were compared for the diagnosis of amebiasis infection. The Multiplex-PCR was standardized using stool samples with parasites obtained from standard cultures. Afterwards, 127 stool samples obtained from individuals living in two villages of the state of Rio de Janeiro were tested and the results were compared. Analysis of the 127 stools samples by microscopy examination demonstrated that only 27 (21%) samples were positive for E. histolytica /E. dispar complex. Among these stool samples, 12 were positive by Multiplex-PCR, with nine presenting the diagnostic fragment characteristic of E. dispar (96 bp) and three presenting diagnostic fragment of E. histolytica (132 bp). Among the negative samples detected by microscopic examination, three were positive for E. dispar and one was positive for E. histolytica by Multiplex-PCR. This denotes that Multiplex-PCR was more efficient than microscopic examination when single stool samples were analyzed. The results obtained by detection of E. histolytica antigen were in agreement with those obtained by the multiplex-PCR. Statistical analyses comparing coproantigen ELISA with Multiplex-PCR results were not done because of the low number of E. histolytica cases. The overall results indicate the need to use more sensitive methods for the diagnosis of amebiasis infection and the importance of using specific techniques for the differentiation between E. histolytica and E. dispar.

Foodborne illnesses are a significant public health challenge in the United States, with an estimated 9.4 million illnesses annually attributed to the consumption of contaminated food, of which 59% are estimated to be caused by viruses, 39% by bacteria and 2% by parasites. Timely detection and identification of the pathogens causing foodborne outbreaks is vital for the implementation of outbreak control strategies, allowing public health officials to prevent additional illnesses and maintain confidence in the food supply. Public health laboratories employ a variety of traditional and molecular testing techniques to identify foodborne outbreak etiologic agents. One technology is the Luminex XMap® microsphere system, which is also marketed as the Bio-Plex™ 200. This platform has a multiplexing capability with the potential to simultaneously detect up to 100 targets in one reaction. The studies described here show that the combination of two Bio-Plex assays with real-time virus assays and one extraction method provides a flexible foodborne outbreak screening algorithm that potentially identifies an outbreak-associated pathogen on the first day of specimen submission and aids in focusing confirmatory laboratory testing. In these studies, two microsphere-based assays were designed for use on the Bio-Plex 200 system as screening assays for the detection of four enteric protozoa (Giardia intestinalis, Cyclospora cayetanensis, Cryptosporidium parvum, Entamoeba histolytica) and six virulence determinants of shiga toxin-producing Escherichia coli (STEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC) and Shigella spp. Precision and limits of detections were established for both assays. The sensitivity and specificity of the protozoan assay as compared to reference methods ranged from 81.25% to 100% for most targets, while sensitivity for the E. histolytica target was 42.86%. Sensitivity and specificity for the bacterial assay was 100% as compared to reference methods. However, cross-reactivity of the protozoan assay E. histolytica target with E. dispar and of the bacterial assay uidA target with enteropathogenic E. coli strains was noted. Additionally, real-time detection of norovirus and rotavirus nucleic acids extracted with the QIAamp DNA Stool Mini Kit was statistically comparable to detection when extracted with the Ambion® MagMAX™-96 Viral RNA Isolation Kit combined with the KingFisher® Magnetic Particle Processor.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-10T15:51:25Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoEpidemiologiaEntamoeba.pdf: 995446 bytes, checksum: ea0b513fd6b22a2126668e5b3c199b15 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-03T15:50:21Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoEpidemiologiaEntamoeba.pdf: 995446 bytes, checksum: ea0b513fd6b22a2126668e5b3c199b15 (MD5) / Made available in DSpace on 2014-04-03T15:50:21Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoEpidemiologiaEntamoeba.pdf: 995446 bytes, checksum: ea0b513fd6b22a2126668e5b3c199b15 (MD5) Previous issue date: 2008 / Amebíase é a infecção no homem causada pelo protozoário Entamoeba histolytica, apresentam quadros sem manifestações clínicas até graves de elevada morbimortalidade e sendo responsável por milhões de casos de disenteria e abscessos hepáticos a cada ano. Os dados epidemiológicos da amebíase no Brasil estão sendo reavaliados desde que a Entamoeba histolytica (patogênica) foi considerada espécie distinta da Entamoeba dispar (não patogênica). Neste estudo, realizou- se o diagnóstico da amebíase por meio de métodos parasitológicos, pesquisa de antígenos e método molecular em amostras fecais de pacientes residentes no município de Juruti, Pará, Brasil. Foram analisadas 188 amostras, com positividade em 28 (14,89 %) no método imunológico, que foi considerado como padrão ouro. A infecção por E. histolytica foi maior no grupo etário acima de 14 anos (8,51%) que no grupo de 0-14 anos (6,38%), porém sem significância estatística (p > 0,05). Houve discordância nos resultados dos métodos ELISA e coproscópico em 41 amostras (21,81%), com maior número de positivos no teste imunoenzimático. O diagnóstico pelo método de PCR apresentou positividade de 5,88% (3/51), resultado inferior ao observado na microscopia (7,84% - 4/51) e teste de ELISA (11,76% - 6/51). Assim, nossos resultados sugerem que a amebíase intestinal é um problema de saúde pública no município de Juruti. / The amebiasis is a human infection caused by the protozoa Entamoeba histolytica, which can be without any clinical manifestations till severe ones with high morbidity and mortality, and is responsible for millions of dysenteric cases an hepatic abscesses, annually. The epidemiological data of amebiasis in Brazil are under revision and reevaluation since the Entamoeba histolytica (pathogenic) was considered a distinct specie from Entamoeba dispar (non pathogenic). In this study, it was carried out the diagnosis of amebiasis by parasitological and molecular methods and antigen search (ELISA) in stool samples from residents in the municipality of Juruti, Pará state, Brazil. We had analyzed 188 stool samples, with positivity in28 (14,89 %) on the immunological test, which was considered as gold standard. The infection by E. histolytica was higher in the age group over 14 years old (8,51%) than in the one of 0-14 years (6,38%), but it was not showed statistical significance (p >0,05). There was discordance between the results of ELISA and microscopical methods in 41 samples (21,81%), and the immuno enzymatic had detected more positive samples than the other one. The diagnosis by PCR method was positive in 3/51(5,88%), result inferior to the one observed by microscopy (7,84% - 4/51) and by ELISA (11,76% - 6/51). Thus, our results suggest that the intestinal amebiasis is an problem of public health in the municipality of Juruti.

The development of molecular tools for detection and typing of unicellular intestinal parasites has revealed genetic diversities in species that were previously considered as distinct entities. Of great importance is the genetic distinction found between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar, two morphologically indistinguishable species. Blastocystis sp. is a ubiquitous intestinal parasite with unsettled pathogenicity. Molecular studies of Blastocystis sp. have identified 17 genetic subtypes, named ST1-17. Genetically, these subtypes could be considered as different species, but it is largely unknown what phenotypic or pathogenic differences exist between them. This thesis explores molecular methods for detection and genetic subtyping of unicellular intestinal parasites, with special focus on Blastocystis. We found that PCR-based methods were highly sensitive for detection of unicellular intestinal parasites, but could be partially or completely inhibited by substances present in faeces. A sample transport medium containing guanidinium thiocyanate was shown to limit the occurrence of PCR inhibition. The prevalence of Blastocystis in Swedish university students was over 40%, which is markedly higher than what was previously estimated. Blastocystis ST3 and ST4 were the two most commonly found Blastocystis subtypes in Sweden, which is similar to results from other European countries. Blastocystis sp. and Giardia intestinalis were both commonly detected in Zanzibar, Tanzania, each with a prevalence exceeding 50%. Blastocystis ST1, ST2, and ST3 were common, but ST4 was absent. While G. intestinalis was most common in the ages 2-5 years, the prevalence of Blastocystis increased with increasing age, at least up to young adulthood. We found no statistical association between diarrhoea and Blastocystis sp., specific Blastocystis subtype or G. intestinalis. Metagenomic sequencing of faecal samples from Swedes revealed that Blastocystis was associated with high intestinal bacterial genus richness, possibly signifying gastrointestinal health. Blastocystis was also positively associated with the bacterial genera Sporolactobacillus and Candidatus Carsonella, and negatively associated with the genus Bacteroides. Blastocystis ST4 was shown to have limited intra-subtype genetic diversity and limited geographic spread. ST4 was also found to be the major driver behind the positive association between Blastocystis and bacterial genus richness and the negative association with Bacteroides.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-03-19T22:46:57Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_CorrelacaoClinicoLaboratorial.pdf: 37431500 bytes, checksum: 9f36cdb930e05a4abda9b6adb64fd372 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-03-20T12:54:07Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_CorrelacaoClinicoLaboratorial.pdf: 37431500 bytes, checksum: 9f36cdb930e05a4abda9b6adb64fd372 (MD5) / Made available in DSpace on 2013-03-20T12:54:07Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_CorrelacaoClinicoLaboratorial.pdf: 37431500 bytes, checksum: 9f36cdb930e05a4abda9b6adb64fd372 (MD5) Previous issue date: 2001 / O diagnóstico clínico da amebíase intestinal continua sendo meramente presuntivo; o diagnóstico de certeza depende sempre de confirmação laboratorial. Com o objetivo de correlacionar os achados clínicos com a pesquisa do coproantígeno GIAP de Entamoeba histolytica, por teste imunoenzimático, foram estudados 105 pacientes de ambos os sexos, com idade entre 13 e 18 anos, provenientes de demanda passiva do Serviço Ambulatorial de Clínica Médica da Polícia Militar. A prevalência de amebíase intestinal encontrada por coproscopia foi de 13,33% (14/105) e por ELISA 24,76% (26/105). Houve diferença estatisticamente significativa na prevalência desta protozoose quando os métodos foram comparados (p<0,05-McNemar). Entre os enteroparasitas detectados por métodos coproscópicos de rotina destacaram-se: Endolimax nana com 61,90% (65/105), Blastocystis hominis 28,57% (30/105), Entamoeba coli 18,10% (19/105) e Giardia amblia em 5,71 (06/105). Entre os helmintos, os mais prevalentes foram o Trichiuris.trichiura com 4,76% (5/105) e de Ascaris lumbricoides em 3,81% (4,105). A prevalência dessas parasitoses na população estudada foi compatível com a casuística regional. No estudo da sintomatologia dos pacientes com teste de ELISA positivo, 73,08% (19/26) relataram um ou mais sintomas sugestivos de amebíase intestinal, observando-se cólicas intestinais em 46,15% (12/26), diarréia sem elementos anormais em 42,31% (11/26), tenesmo em 3,85% (1/26) e constipação intestinal em 11,54% (3/26). A presença desses sintomas quando comparada com os casos clinicamente idênticos, porém com teste de ELISA não apresentaram significância estatística (p<0,05-McNemar). Quanto a diarréia mucossanguinolenta, esta foi referida por 4,76% de apresentação das síndromes clínicas e sua amplitude de diagnósticos diferenciais, aliados às possibilidades de equívocos que os métodos diagnósticos usualmente empregados podem fornecer. Recomenda-se a inclusão do teste de ELISA no diagnóstico da amebíase intestinal como um recurso indispensável na clínica, embora ele não dispense o exame coproparasitológico, por ser capaz de indetificar somente um patógeno, a E. histolyca. / Intestinal amoebiasis clinical diagnosis remains presumable and the correct diagnosis always need laboratorial confirmation. A hundred-five patients, of both sexes, age between 13 - 80 years-old, from passive demand of "Serviço Ambulatorial de Clínica Medica da Polícia Militar", were selected for this study in order to correlate the clinical findings and imunoenzimatic E. histolytica GIAP stool antigen search. The prevalence of intestinal amoebiasis was 13,33% (14/105) for single stool examination and 24,76% (26/105) for ELISA. When the two methods were compared, diference statistically significant it was found (p < 0,05 - McNemar). Among detected parasites by single stool examination: Endolimax. nana with 61,90% (65/105), Blastocystis hominis 28,57% (30/105). Entamoeba coli 18,10% (19/105) and Giardia lamblia 5,71% (6/105) were the most prevalent. Trichiuris trichiura with 4.76% (5/105) and A lumbricoides 3,81% (4/105) had the highest prevalence among helminths. The prevalence of such parasitic infections at the studied population was according to local casuistic. In the analysis of the symptomatology of ELISA positive patients, 73,08% (19/26) had reported one or more disease suggestive symptoms, and was observed abdominal pain in 46,15% (12/26) patients, diarrhoea with no abnormal elements in 42,31 % (11/26), tenesmus in 3,85% (1/26) and intestinal constipation 11,54% (3/26). The presence of such symptons when compared with clinical similar cases ELISA negative, were not significative (p > 0,05 - McNermar). In 4,76% patients (5/105), diarrhoea with mucosus and blood was refered. This association with ELISA positive test were observed in 3,84% (1/26), and was statistically significative when compared with ELISA negative ones. The results of this work shows the difficulties in establishing the clinics and laboratory correlation in intestinal amoebiasis. It occurs because of clinical syndroms are not so specifie and the need of different diagnosis, together to the error possibility in routine diagnosis methods. Its is suggested the inclusion of ELISA test in intestinal amoebiasis diagnosis as a necessary clinical resource although, it does not exclude the single stool examination to others parasites, due to be able to identify only the pathogen, Entamoeba histolytica.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-16T19:59:41Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AplicacaoModeloAcuracia.pdf: 687193 bytes, checksum: f17320f52a5a1dfb3083a6d2d9644f10 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-22T15:34:34Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AplicacaoModeloAcuracia.pdf: 687193 bytes, checksum: f17320f52a5a1dfb3083a6d2d9644f10 (MD5) / Made available in DSpace on 2013-04-22T15:34:34Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AplicacaoModeloAcuracia.pdf: 687193 bytes, checksum: f17320f52a5a1dfb3083a6d2d9644f10 (MD5) Previous issue date: 2006 / Introdução: São poucos e dispersos os estudos sobre prevalência de enteroparasitoses em nosso meio, sendo a maioria deles realizada em amostras de base populacional mal definidas, como usuários de serviços de saúde ou alunos de escolas públicas. Objetivo: Quantificar a prevalência da Entamoeba histolytica em uma população rural do Estado do Pará. Casuística e Método: Estudo coprológico pelo método da hematoxilina férrica em 124 moradores do meio rural de Tailândia, utilizando processo de amostragem aleatória para estabelecer a prevalência, a acurácia, os valores preditivos positivo e negativo, bem como a sensibilidade e especificidade. Resultados: Da amostra populacional, 56,58% pertenciam ao sexo masculino, sendo 60,6% entre as idades de 18 a 43 anos. No estudo da escolaridade, 64,5% tinham até 3 anos de estudo e 63,7% recebiam menos de 1 salário mínimo de remuneração. A prevalência de Entamoeba histolytica atingiu 24,2%. Conclusão: A acurácia, medida de valor global do teste efetuado, mostra que 73,4% dos pacientes foram classificados corretamente. O teste de sensibilidade de 70,0% definiu teste positivo para a infecção e a especificidade de 74,5%. / Introduction: The studies on the prevalence of intestinal worms are scarce among us, most of them were performed in sample of populational basis not very well defined, like users of health service and public school students. Objective: Quantify the Entamoeba hystolitica prevalence in a rural population of Pará state. Methods: feces study using the ferric hematoxilina method in 124 people from the rural part of Tailândia, arbitrarily selected in order to establish prevalence and accuracy, the positive and negative predictive values, as well as sensitivity and specificity. Results: from the populational sample, 56,5% belonged to male sex, 60,5% out of these were ranging between 18 to 43 years old. In the scholarship study, 64,5% had at least 3 years of school and 63,7% earned less than R$300,00. The Entamoeba hystolitica prevalence reached 24,2%. Conclusion: the accuracy, which is the measurement of global value of the performed test, shows that 73,4% of the patients were correctly classified. The 70,0% sensitivity test defined positive for the infection and a specificity of 74,5%.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-16T17:25:12Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInvitroEfeitos.pdf: 1226021 bytes, checksum: 04d04874e9e2a9c1dc6bb2c6529fdff8 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-10-24T12:47:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInvitroEfeitos.pdf: 1226021 bytes, checksum: 04d04874e9e2a9c1dc6bb2c6529fdff8 (MD5) / Made available in DSpace on 2017-10-24T12:47:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoInvitroEfeitos.pdf: 1226021 bytes, checksum: 04d04874e9e2a9c1dc6bb2c6529fdff8 (MD5) Previous issue date: 2011 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A amebíase caracteriza-se como uma doença crônica, não inflamatória, afebril que envolve a parede do intestino grosso podendo invadir a mucosa intestinal e se disseminar para outros órgãos, principalmente o fígado. A infecção por Entamoeba histolytica é tratada pelo metronidazol, mas este apresenta efeitos colaterais, por isso buscam-se medicamentos mais eficazes e que não tragam nenhum efeito adverso ao paciente. Além disso, o teste de novos fármacos pode revelar importantes particularidades do parasito, tais como a descoberta de vias metabólicas ou mesmo possíveis diferenças em sua estrutura. No presente trabalho foi testado o efeito amebicida do metronidazol, da dinitroanilina orizalina, uma droga inibidora de microtúbulos, da aquilfosfocolina miltefosina, que inibe a síntese de fosfolipídios de membrana, e da droga hibrida TC95, que apresenta os grupos funcionais dinitroanilina e alquilfosfocolina. Realizou-se cultura com trofozoítos do protozoário, onde as drogas e o controle com os diluentes foram adicionados após 7 horas de cultivo. Foi feita a contagem dos trofozoítos viáveis após período de 12 horas da adição das drogas e do controle em intervalos de 12 a 60 horas. Após análise das curvas de crescimento, detectou-se que o TC95 foi a droga mais efetiva dos três tratamentos, a miltefosina obteve pouca inibição e a orizalina não foi efetiva. Isso se deve a maior suscetibilidade do parasito a danos de membrana, com pouca efetividade da inibição dos microtúbulos. / Amebiasis is characterized as a chronic, noninflammatory, afebrile which involves the colon wall and can invade the intestinal mucosa and spread to other organs, especially the liver. Entamoeba histolytica infection is treated with metronidazole, but this one presents side effects, so researches have been done to find more effective drugs with no adverse effects to the patient. In addition, testing new drugs may reveal important features of the parasite, such as the discovery of metabolic pathways or possible differences in their structure. In the present study, it was tested the amoebicidal effect of metronidazole, dinitroaniline oryzalin, a microtubule inhibitor, miltefosine, an alkylphosphocholine, that inhibits the membrane phospholipids syntesis, and TC95, a hybrid drug that presents the functional groups dinitroaniline and alkylphosphocholine. It was performed a culture with protozoan trophozoites, where the drugs and the control with diluents were added after 7 hours of culture. It was made a count of the viable trophozoites 12 hours after the addition of the drugs and the control at intervals of 12 to 60 hours. After analyzing the growth curves, it was found that the TC95 was the most effective drug of the three treatments, miltefosine had little inhibition and oryzalin was not effective. This is due to greater susceptibility of the parasite to membrane damage, with little effectiveness on microtubules inhibition.

A signal transduction process refers to chain of highly regulated biochemical steps which results in the transfer of signal in response to a stimulus in the extracellular environment to the intracellular compartments such as nucleus. Variety of biomolecules such as proteins and lipids participate in such processes. One of the superfamilies of proteins which actively participate in signaling processes is protein kinase which transfers γ-phosphate from Adenosine Triphosphate (ATP) to the specific hydroxyl group(s) in the protein substrates. Phosphorylation and dephosphorylation events are critical in many signal transduction pathways affecting biological system as a whole. Protein phosphorylation carried out by protein kinases has emerged as pre-eminent mechanism for the regulation of variety of cellular processes such as cell growth, development, differentiation, homeostasis, apoptosis, metabolism, transcription and translation. The current thesis encompasses the investigations carried out by the author, using various bioinformatics tools and methods, to comprehend the structural and functional roles of diverse set of protein kinase subfamilies in various eukaryotic and prokaryotic organisms. The present thesis has been divided into various chapters. Chapter 1 of the thesis provides introduction to the superfamily of protein kinases and covers the relevant literature. The database of Kinases in Genomes (KinG) set-up in the author’s group a few years ago (Krupa et al, 2004a), comprises of a collection of Serine/Threonine/Tyrosine protein kinases recognized using bioinformatics approaches, from the genomic data of various eukaryotes, prokaryotes and viruses (Krupa et al, 2004a). KinG database also provides classification of protein kinases into various groups and subfamilies (Hanks et al, 1988). Information on non-kinase domains which are tethered to the catalytic kinase domains is also available for every kinase in the KinG database. KinG is periodically (annually) updated with rise in the number of genome sequence datasets of various organisms, increase in the number of known protein domain families and refinement or reannotation of genomic datasets (Anamika et al, 2008c). Author describes the work on annual update of KinG database in Chapter 2 of the thesis. Availability of an improved version of the human genomic data has provided an opportunity to re-investigate protein kinase complement of the human genome and enabled an analysis of the splice variants. This analysis is also described in Chapter 2. Chapter 3, Chapter 4 and Chapter 5 report recognition and analysis of repertoire of protein kinases in Chimpanzee, two Plasmodium species (Plasmodium falciparum and Plasmodium yoelii yoelli) and Entamoeba histolytica respectively. A detailed analysis of the non-kinase domains which are tethered to catalytic protein kinase domains in eukaryotic organisms is presented in Chapter 6. Chapter 7 discusses a systematic classification framework developed by the author to classify Serine/Threonine protein kinases in prokaryotic organisms. Investigation carried out on 3-D structural aspects of protein kinase-substrate interactions is described in Chapter 8. While identifying protein kinases from genomic data occurrence of protein kinase-like non-kinases (PKLNK), which lack aspartate in a specific position in the amino acid sequence (and hence are unlikely to function as a kinase), has also been observed. Chapter 9 presents an analysis of PKLNKs with an objective of obtaining clues to their functions. Chapter 10 summarizes the main conclusions of the thesis and provides an outlook of the current study. Chapter 1: Chapter 1 provides an introduction to cell signaling and the involvement of protein kinases in various signaling pathways compiled from author’s literature survey. This chapter provides a description of molecular events in cell signaling in prokaryotic and eukaryotic organisms. The diversity, specificity and cellular roles of protein kinases are discussed in detail. Chapter 2: Chapter 2 describes KinG (Kinases in Genomes) database which was first established by Krupa et al (2004a). The KinG database is an on-line compilation of the putative Serine/Threonine/Tyrosine protein kinases encoded in the completely sequenced genomes of archaea, eubacteria, viruses and eukaryotes. Surge in the datasets of genomes, improvements in the quality of the genomic data for various organisms and growing number of protein domain families necessitates periodic update of KinG database. The updated version of KinG holds information on protein kinases for 483 organisms (Anamika et al, 2008c). Availability of draft version of the human genome data in 2001 enabled recognition of repertoire of human protein kinases (Krupa and Srinivasan, 2002a; Manning et al, 2002; Kostich et al, 2002). Over the last 7 years human genomic data is being refined and at present the quality of the human genomic data available is much superior to the one available in 2001. By gleaning the latest version of human genome data, 46 new human protein kinase splice variants have been identified which were not recognized in the earlier studies on human kinome. Improper regulation or mutant forms of many of these newly identified protein kinase splice variants are directly involved in various diseases such as different kinds of cancer, Severe Combined Immunodeficiency Disease (SCID) and Huntington disease. In addition, abnormal forms of mouse orthologues of some of the newly identified human kinase splice variants are known to cause various diseases in mice. This raises the possibility of the human orthologues playing similar roles in the disease processes. Such observations and detailed analysis of these protein kinase splice variants would have a profound influence on drug design and development against various diseases. Chapter 3: Investigations on the identification and analysis of protein kinases encoded in the genome of chimpanzee (chimp) has been discussed in Chapter 3. Further, the kinome complement has been compared between chimp and its evolutionary close relative, human (Anamika et al, 2008b). The shared core biology between chimp and human is characterized by many orthologous protein kinases which are involved in conserved pathways. Domain architectures specific to chimp/human kinases have been observed. Chimp kinases with unique domain architectures are characterized by deletion of one or more non-kinase domains present in the human kinases. Interestingly, counterparts of some of the multi-domain human kinases in chimp are characterized by identical domain architectures but with kinase-like non-kinase domain (PKLNK). Remarkably, for 160 out of 587 chimpanzee kinases no human orthologue with sequence identity greater than 95% could be identified. Variations in chimpanzee kinases compared to human kinases are brought about also by differences in functions of domains tethered to the catalytic kinase domain. For example, the heterodimer forming PB1 domain related to the fold of ubiquitin / Ras-binding domain is seen uniquely tethered to PKC-like chimpanzee kinase. Though chimpanzee and human have close evolutionary relationship, there are chimpanzee kinases with no close counterpart in the human suggesting differences in their functions. This chapter provides a direction for experimental analysis of human and chimpanzee protein kinases in order to enhance our understanding on their specific biological roles. Chapter 4: Chapter 4 describes genome-wide comparative analysis for protein kinases encoded in the two apicomplexa namely Plasmodium falciparum (P. falciparum) (3D7 strain) and Plasmodium yoelii yoelii (P. yoelii yoelii) (17XNL strain) genomes which are causative agents of malaria in human and rodent respectively (Anamika and Srinivasan, 2007). Sensitive bioinformatics techniques enable identification of 82 and 60 putative protein kinases in P. falciparum and P. yoelii yoelii respectively. These protein kinases have been classified further into subfamilies based on the extent of sequence similarity of their catalytic domains (Hanks et al, 1988). The most populated kinase subfamilies in both the Plasmodium species correspond to CAMK and CMGC groups. Analysis of domain architectures enables detection of uncommon domain organisation in kinases of both the organisms such as kinase domain tethered to EF hands as well as pleckstrin homology domain. Components of MAPK signaling pathway are not well conserved in Plasmodium species. Such observations suggest that Plasmodium protein kinases are highly divergent from other eukaryotes. A trans-membrane kinase with 6 membrane spanning segments in P. falciparum seems to have no orthologue in P. yoelii yoelii. 19 P. falciparum kinases (Anamika et al, 2005; Anamika and Srinivasan, 2007) have been found to cluster separately from P. yoelii yoelii kinases and hence these kinases are unique to P. falciparum genome. Only 28 orthologous pairs of kinases could be identified between these two Plasmodium species. Comparative kinome analysis of the two Plasmodium species has thus provided clues to the function of many protein kinases based upon their classification and domain organisation and also implicate marked differences even between two Plasmodium species. Chapter 5: Identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica (E. histolytica) using sensitive sequence and profile search methods forms the basis of Chapter 5. A systematic analysis of a set of 307 protein kinases in E. histolytica genome has been carried out by classifying them into different subfamilies originally defined by Hanks and Hunter (Hanks et al, 1988) and by examining the functional domains which are tethered to the catalytic kinase domains (Anamika et al, 2008a). Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organisation and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like calcium/calmodulin dependent kinases as they lack non-catalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of protein kinases in E. histolytica. Interestingly a Protein Kinase A/Protein Kinase G-like hybrid kinase subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclin-dependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual kinases recognized in the analysis include the absence of Mitogen activated protein kinase kinase (MEK) as a part of the Mitogen Activated Kinase signaling pathway and identification of trans-membraneous kinases with catalytic kinase region showing a good sequence similarity to Src kinases which are usually cytosolic. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are cysteine-rich and to domains known to be involved in protein-protein interactions. The current chapter on kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual protein kinases. Chapter 6: Protein kinases phosphorylating Serine/Threonine/Tyrosine residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Classification based on sequence similarity of their catalytic domains, results in clustering of protein kinases sharing gross functional properties into various subfamilies. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules, aiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their molecular interaction which has been discussed in Chapter 6. Recent studies on repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation in model organisms across various subfamilies. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by the protein kinase in order to regulate variety of biological processes. In addition, domain architectures of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. The repertoire of non-kinase domains tethered to multi-domain kinases in the higher eukaryotes is discussed in Chapter 6. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization. Chapter 7: Chapter 7 describes systematic classification of Serine/Threonine protein kinases encoded in archaeal and eubacterial genomes. Majority of the Serine/Threonine protein kinases which have been identified in archaeal and eubacterial genomes could not be classified into any of the well known subfamilies (Hanks et al, 1988) of protein kinases suggesting their diversity from kinases in eukaryotes. The extensive prokaryotic Serine/Threonine protein kinase dataset obtained from KinG (Krupa et al, 2004a, Anamika et al, 2008c) has given an opportunity to classify these prokaryotic Serine/Threonine protein kinases mainly into three categories based upon sequence identity based clustering: 1) Species/Genus-specific clusters: Species/Genus-specific Serine/Threonine protein kinases contain members from a particular species or genus of the eubacteria or archaea suggesting requirement of these Serine/Threonine protein kinases for certain lineage specific function. 2) Organism-specific clusters: Organism specific clusters has members from certain specific types of organisms which suggests role of these Serine/Threonine protein kinases in some specific function being carried out by limited sets of prokaryotes. 3) Organism-diverse clusters: Organism diverse clusters suggest common function performed by such kinases in wide variety of organisms. Interestingly, occurrence of several species/genus or organism specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Function-based classification has also been proposed which shows that members of each cluster has specific function to perform. In this analysis, almost 50% of the “clusters” obtained have only one member suggesting their sequence and probably functional divergence. Many prokaryotic Serine/Threonine protein kinases exhibit a wide variety of modular organisation which indicates a degree of complexity and protein-protein interactions in the signaling pathways in these microbes. Chapter 8: A wide spectrum of protein kinases belonging to different Hanks and Hunter groups of kinases and subfamilies has been identified in various eukaryotes. However, specific biological targets (substrates) of many protein kinase subfamilies are still unknown and this is one of the active areas of research. In the current analysis reported in Chapter 8, an attempt has been made to understand protein kinase-substrate interaction and substrate consensus prediction by analyzing known 3-D structures of complexes of kinases and peptide substrates/pseudosubstrates. Considering protein kinase ternary complex structures in their active states, it has been observed that protein kinase residues which are interacting with the substrate residues having constraint are at topologically equivalent positions despite belonging to different Hanks and Hunter protein kinase subfamilies. In this analysis, it has also been observed that the residues in a given kinase subfamily interacting with consensus substrate residues are usually conserved across homologues. Interestingly, in Protein Kinase B and Phosphorylase Kinase subfamily homologues, residues interacting with substrate residue/s having no constraint are not well conserved even within the kinase subfamily suggesting different evolutionary rate of substrate interacting residues. This result is anticipated to be helpful in furthering our understanding of protein kinase-substrate relationship which is likely to be helpful in drug design. Chapter 9: Protein Kinase-Like Non-kinases (PKLNKs) are closely related to protein kinases but they lack the crucial catalytic aspartate in the catalytic loop and hence cannot function as a protein kinase. PKLNKs have been analyzed (Chapter 9) with an objective of obtaining clues about their functions. Using various sensitive sequence analysis methods, 82 PKLNKs from four higher eukaryotic organisms namely, Homo sapiens, Mus Musculus, Rattus norvegicus and Drosophila melanogaster have been recognized. On the basis of their domain combinations and functions of tethered domains, PKLNKs have been classified mainly into four categories: 1) Ligand binding PKLNKs 2) PKLNKs having extracellular protein-protein interaction domain 3) PKLNKs involved in dimerization 4) PKLNKs with cytoplasmic protein-protein interaction module. While members of the first two classes of PKLNKs have transmembrane domain tethered to the PKLNK domain, members of the other two classes of PKLNKs are entirely cytoplasmic in nature. The current classification scheme hopes to provide a convenient framework to classify the PKLNKs from other eukaryotes and it should be helpful in deciphering their roles in cellular processes. Chapter 10: This is a chapter on conclusions of the entire thesis work. Summary of the major outcomes of this thesis work is provided and implications of the work in the area of signal transduction are discussed. In addition to above mentioned work, studies on repertoire of protein kinases from two plant organisms have been carried out and the kinomes have been comparatively analyzed (Krupa et al, 2006) (Appendix 1). Comparison of plant protein kinases with other eukaryotes revealed remarkable differences. Trans-genomic comparison of the protein kinase repertoires of Arabidopsis thaliana and Oryza sativa has enabled identification of members that are uniquely conserved within the two species. Analysis on the domain organisation of plant protein kinases has also been carried out. Appendix 2 presents the work done on Entamoeba histolytica (E. histolytica) ornithine decarboxylase (ODC)-like protein which regulates the polyamine biosynthesis. DFMO (Difluoromethylornithine) is unable to inhibit the E. histolytica ODC-like protein while it inhibits the homologues of ODC in other organisms. Modelling study has suggested substitution of three amino acids in the E. histolytica ODC-like protein because of which DFMO is unable to inhibit the activity of ODC-like protein (Jhingran et al, 2008). All the computational modeling work reported in Appendix 2 was performed by the author while all the laboratory experiments were performed in the laboratory of the collaborator Prof. Madhubala of JNU, New Delhi. The supplementary data pertaining to this thesis is presented in an accompanying CD. The supplementary data in this CD is organized into different folders corresponding to various chapters.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below