Os genes micro-exons (MEGs) foram recentemente identificados no genoma do Schistosoma mansoni, verme responsável pela esquistossomose, doença que afeta mais de 262 milhões de pessoas em mais de 78 países. Devido à capacidade de produção de proteínas variantes pelo splicing alternativo de MEGs, expressão preferencial em estágios do ciclo de vida em contato com o hospedeiro definitivo e a verificação de que várias proteínas codificadas por estes genes são secretadas para o meio externo ao parasito, acredita-se que estas proteínas possuam um papel importante na interação parasito-hospedeiro. O objetivo deste trabalho foi estudar e caracterizar a estrutura e dinâmica conformacional das proteínas codificadas por MEG-11 e MEG-14 e verificar a interação da proteína MEG-14 com proteínas humanas. A análise das proteínas MEG-11 e MEG-14 produzidas em sistema recombinante com a técnica de dicroísmo circular (CD) demonstrou que ambas as proteínas apresentavam estruturas secundárias majoritariamente desordenadas quando em solução aquosa. Entretanto, foi verificado que a presença de TFE, a desidratação das proteínas e o aumento de temperatura favoreciam o surgimento de estruturas ordenadas nestas proteínas. Um ganho de estrutura secundária também foi observado para a proteína MEG-14 na presença de vesículas de fosfolipídios e micelas de detergente carregadas negativamente. Estes resultados suportam a identificação destas proteínas como clássicas proteínas intrinsicamente desordenadas (IDPs) e abre a possibilidade de sua interação com diferentes parceiros e fatores a serem relacionados com os papéis multifuncionais e estados dentro do hospedeiro. Resultados prévios de experimentos de duplo-hibrido do nosso grupo apontavam uma possível interação de MEG-14 com a proteína humana S100A9. Através da técnica de pulldown foi possível confirmar uma interação dependente da presença de cálcio entre estas duas proteínas. Análises adicionais da interação MEG-14/S100A9 com as técnicas de ITC e SPR permitiram calcular a constante de dissociação entre as proteínas em aproximadamente 2 μM. Finalmente, experimentos ex vivo permitindo a ingestão da proteína S100A9 acoplada a uma molécula fluorescente pelo S. mansoni resultaram na acumulação da proteína na glândula do esôfago, local onde já foi localizada a proteína MEG-14 em S. japonicum, sugerindo que a interação observada nos experimentos in vitro está ocorrendo com a proteína MEG-14 nativa. / The micro-exon genes (MEGs) were recently identified in Schistosoma mansoni’s genome, worm responsible for schistosomiasis, a disease that affects more than 262 million people in more than 78 countries. Due to the capacity of variant protein production by alternative splicing of MEGs, preferential expression in certain life stages in contact with the definitive host, and the confirmation that many proteins coded by these genes are secreted to the external environment, It’s believed that these proteins have an important role in parasite-host relationship. The objective of this work was to study and characterize the structure and conformational dynamics of proteins coded by MEG-11 and MEG-14 and verify the interaction of Meg-14 protein with human proteins. The analysis of MEG-11 and MEG-14 proteins produced in recombinant system with circular dichroism (CD) shows that both proteins present secondary structures mostly disordered in aqueous solution. However, It was found that in presence of TFE, the dehydration of proteins and the increasing of temperature favor the surging of ordered structures. An increase of secondary structure was observed too for MEG-14 protein in presence of phospholipid vesicles and negative charged detergent micelles. These results support the identification of these proteins as classical intrinsically disordered proteins (IDPs) and opens the possibility of its interaction with different partners and factors related with the multifunctional roles and states in the host. Previous results of double-hybrid experiments pointed a possible interaction between MEG-14 and the human protein S100A9. By pulldown technique was possible confirm an calcium dependent interaction between these proteins. Additional analysis of MEG-14/S100A9 interaction with ITC and SPR experiments were able to calculate the dissociation constant between proteins equals to 2 μM. Finally, ex vivo experiments permit the ingestion of S100A9 coupled to a fluorescent molecule by S. mansoni, resulting in the accumulation of protein in the esophageal gland, where was localized the MEG-14 in S. japonicum, suggesting that the interaction observed in the in vitro experiments are occurring with the native MEG-14.

檔案是研究歷史或瞭解過去事情的重要工具，因此檔案館除了需善盡保存檔案之責外，還要提供檢索工具供使用者使用。隨著網際網路的蓬勃發展及資源分享概念的形成，透過WWW，採用更一致、更友善且更便利的方式進行檢索成為一種趨勢，檔案描述編碼格式（EAD）即逐漸發展出來。以此格式所製作的檢索工具，能適當地描述檔案的階層架構，且對於檔案資訊的描述較豐富。由於以標準為基礎且與平台無關，所描述的資訊可被搜尋、檢索、瀏覽，亦可方便資源共享與資訊交換，並有較長久的使用時間。在國外已有許多計畫研究EAD，也有檔案館實際利用EAD編製檔案檢索工具，並放在網站上供使用。為期EAD亦能應用在國內的中文檔案，乃進行本研究。 本研究採文獻分析法與試驗法。藉由文獻分析法，瞭解EAD架構、目前EAD的發展現況及實際使用情形；而試驗法為實際至國內檔案館調閱檔案資料，將這些資料編製成符合EAD格式的檢索工具，並放在網站上，以檢視其是否能在WWW上瀏覽使用。 研究結論為：(1)EAD編碼之檔案檢索工具所含資訊豐富，能在網路上直接檢索，便利使用，並能使檢索工具具有較長久的使用時間；(2)中文檔案編製成EAD格式之檢索工具，部份著錄項目可直接使用，部份則需另行輸入；(3)EAD格式可應用於中文檔案；(4)國外地區較易推展EAD的實施使用。建議有六項：(1)EAD DTD依中文檔案之特質需做調整修改或發展中文版EAD；(2)利用EAD製作中文檔案檢索工具；(3)訂定中文檔案描述相關標準；(4)配合相關軟體的使用；(5)應於文件生命週期初期做好文件整編工作；(6)不可輕忽人工分析與檔案整理的工作。 第一章 緒論..............................1 第一節 研究動機........................1 第二節 研究目的........................2 第三節 研究問題........................3 第四節 預期貢獻........................3 第五節 名詞解釋........................4 第二章 文獻探討..........................9 第一節 檔案描述與檢索工具..............9 第二節 檔案描述標準...................26 第三節 SGML...........................30 第四節 XML............................37 第五節 EAD的沿革......................42 第六節 EAD相關研究計畫................50 第三章 研究方法與步驟...................65 第一節 研究方法.......................65 第二節 研究範圍與限制.................66 第三節 研究工具.......................67 第四節 研究步驟.......................67 第五節 研究實施.......................69 第四章 EAD架構與使用現況................71 第一節 EAD架構........................71 第二節 EAD使用現況....................84 第五章 研究結果與討論..................101 第一節 研究結果......................101 第二節 綜合討論......................140 第六章 結論與建議......................145 第一節 結論..........................145 第二節 建議..........................147 第三節 後續研究建議..................149 參考書目................................151 附錄....................................161 附錄一 EAD DTD 1.0版標籤.............161 附錄二 中文檔案以EAD編碼之實例.......165

This paper examines the results of a usability study for finding aids from the Special Collections Research Center at North Carolina State University. In 2005, the Special Collections Research Center reformatted its finding aids so that the container information, typically located on the left-hand side of the document, moved to the right-hand side of the document. The study tested the effectiveness of this change, and determined that traditional finding aids performed better. The analysis of the study’s results is followed by a discussion about Web usability guidelines for online finding aids.

Des polymères à séquences contrôlées contenant de l’information numérique ont été synthétisés par voie itérative supportée, via une stratégie « AB+CD » mettant en jeu deux réactions orthogonales. Cette stratégie a permis la synthèse de poly(alcoxyamine amide)s et de poly(alcoxyamine triazole)s à séquences contrôlées isomoléculaires, sans avoir recours à des chimies de protection. Les poly(alcoxyamine amide)s ont été synthétisés sur différents supports – solides, solubles et fluorés – ainsi que par une approche convergente, tandis que les poly(alcoxyamine triazole)s ont permis l’étude de la SpAAC qui met en jeu une cyclooctyne et un azoture pour former un triazole sans utilisation de cuivre. La structure des polymères obtenus a chaque fois permis un séquençage par MS/MS, grâce à la présence de liaisons faibles, permettant de décoder le code binaire incrémenté le long des chaînes via des groupements codant pour 0 ou pour 1. De plus, les poly(alcoxyamine amide)s ont aussi été optimisés pour l’encodage de données : via la synthèse de codes-barres moléculaires pour augmenter la capacité de stockage, ainsi que via une polymolécularité induite permettant la lecture du code incrémenté sans fragmentation. / Information-containing sequenced-controlled polymers were synthesized by an « AB+CD » supported iterative strategy, involving two orthogonal reactions. This approach allowed the protecting-group-free synthesis of monodisperse sequence-controlled poly(alkoxyamine amide)s and poly(alkoxyamine triazole)s. The poly(alkoxyamine amide)s were synthesized on different supports - solid, soluble, and fluorinated – and also by a convergent strategy. Besides, the synthesis of the poly(alkoxyamine triazole)s allowed studying the SpAAC, involving a cyclooctyne and an azide for a copper-free formation of a triazole. The structure of the obtained polymers also allowed an MS/MS sequencing of the binary code implemented along the chains through functional groups coding for 0 or 1, thanks to the presence of weak linkages. Furthermore, the poly(alkoxyamine amide)s were optimized for data encoding, by synthesizing molecular barcodes to increase storage capacity, and by an induced polydispersity to decode the polymers without fragmentation.

Os genes micro-exons (MEGs) foram recentemente identificados no genoma do Schistosoma mansoni, verme responsável pela esquistossomose, doença que afeta mais de 262 milhões de pessoas em mais de 78 países. Devido à capacidade de produção de proteínas variantes pelo splicing alternativo de MEGs, expressão preferencial em estágios do ciclo de vida em contato com o hospedeiro definitivo e a verificação de que várias proteínas codificadas por estes genes são secretadas para o meio externo ao parasito, acredita-se que estas proteínas possuam um papel importante na interação parasito-hospedeiro. O objetivo deste trabalho foi estudar e caracterizar a estrutura e dinâmica conformacional das proteínas codificadas por MEG-11 e MEG-14 e verificar a interação da proteína MEG-14 com proteínas humanas. A análise das proteínas MEG-11 e MEG-14 produzidas em sistema recombinante com a técnica de dicroísmo circular (CD) demonstrou que ambas as proteínas apresentavam estruturas secundárias majoritariamente desordenadas quando em solução aquosa. Entretanto, foi verificado que a presença de TFE, a desidratação das proteínas e o aumento de temperatura favoreciam o surgimento de estruturas ordenadas nestas proteínas. Um ganho de estrutura secundária também foi observado para a proteína MEG-14 na presença de vesículas de fosfolipídios e micelas de detergente carregadas negativamente. Estes resultados suportam a identificação destas proteínas como clássicas proteínas intrinsicamente desordenadas (IDPs) e abre a possibilidade de sua interação com diferentes parceiros e fatores a serem relacionados com os papéis multifuncionais e estados dentro do hospedeiro. Resultados prévios de experimentos de duplo-hibrido do nosso grupo apontavam uma possível interação de MEG-14 com a proteína humana S100A9. Através da técnica de pulldown foi possível confirmar uma interação dependente da presença de cálcio entre estas duas proteínas. Análises adicionais da interação MEG-14/S100A9 com as técnicas de ITC e SPR permitiram calcular a constante de dissociação entre as proteínas em aproximadamente 2 μM. Finalmente, experimentos ex vivo permitindo a ingestão da proteína S100A9 acoplada a uma molécula fluorescente pelo S. mansoni resultaram na acumulação da proteína na glândula do esôfago, local onde já foi localizada a proteína MEG-14 em S. japonicum, sugerindo que a interação observada nos experimentos in vitro está ocorrendo com a proteína MEG-14 nativa. / The micro-exon genes (MEGs) were recently identified in Schistosoma mansoni’s genome, worm responsible for schistosomiasis, a disease that affects more than 262 million people in more than 78 countries. Due to the capacity of variant protein production by alternative splicing of MEGs, preferential expression in certain life stages in contact with the definitive host, and the confirmation that many proteins coded by these genes are secreted to the external environment, It’s believed that these proteins have an important role in parasite-host relationship. The objective of this work was to study and characterize the structure and conformational dynamics of proteins coded by MEG-11 and MEG-14 and verify the interaction of Meg-14 protein with human proteins. The analysis of MEG-11 and MEG-14 proteins produced in recombinant system with circular dichroism (CD) shows that both proteins present secondary structures mostly disordered in aqueous solution. However, It was found that in presence of TFE, the dehydration of proteins and the increasing of temperature favor the surging of ordered structures. An increase of secondary structure was observed too for MEG-14 protein in presence of phospholipid vesicles and negative charged detergent micelles. These results support the identification of these proteins as classical intrinsically disordered proteins (IDPs) and opens the possibility of its interaction with different partners and factors related with the multifunctional roles and states in the host. Previous results of double-hybrid experiments pointed a possible interaction between MEG-14 and the human protein S100A9. By pulldown technique was possible confirm an calcium dependent interaction between these proteins. Additional analysis of MEG-14/S100A9 interaction with ITC and SPR experiments were able to calculate the dissociation constant between proteins equals to 2 μM. Finally, ex vivo experiments permit the ingestion of S100A9 coupled to a fluorescent molecule by S. mansoni, resulting in the accumulation of protein in the esophageal gland, where was localized the MEG-14 in S. japonicum, suggesting that the interaction observed in the in vitro experiments are occurring with the native MEG-14.

Orientadores: Walter da Cunha Borelli, Emilia de Mendonça Rosa Marques / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-13T08:38:40Z (GMT). No. of bitstreams: 1 SilvaFilho_JoaoCoelho_D.pdf: 842540 bytes, checksum: 07a4f12e39ce13ce6c183f2983fbe70f (MD5) Previous issue date: 2008 / Resumo: Esta tese apresenta contribuições aos esquemas de modulação codificada para os códigos de treliça sobre partições de reticulados. Uma das principais contribuições é a construção dos códigos de treliça sobre novas partições de reticulados e também em cadeias de partições. Para otimizar a procura dos códigos de treliça ótimos, é construído um algoritmo de procura. É proposta uma classe de equivalência utilizada para excluir as matrizes geradoras de códigos equivalentes, sendo que esta classe de equivalência quando aplicada ao algoritmo de procura dos códigos de treliça ótimos diminui a quantidade de matrizes geradoras a ser investigada. Apresentam-se, vários exemplos de códigos de treliça sobre reticulados quociente nos espaços bi-dimensional, tridimensional e tetra-dimensional com satisfatórios ganhos de codificação e menor energia média das constelações de sinais. / Abstract: This thesis presents some contributions to the coded modulation schemes for the trellis codes based on lattices partitioning. One of the main contributions is the construction of the trellis codes based on novel lattices partitioning and also on chains partitioning. In order to optimize the search for the optimum trellis codes, a search algorithm was proposed. An equivalence class is proposed to exclude the generator matrix of equivalent codes. This equivalence class, when applied to the search algorithm for optimum trellis codes, reduces quite strongly the number of generator matrices to be investigated. Several examples of trellis codes on lattices quotient are shown in bi-dimensional, three-dimensional and tetra-dimensional spaces with satisfactory coding gain and lower average energy of the signal constellations. / Doutorado / Telecomunicações e Telemática / Doutor em Engenharia Elétrica

<p>Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2,4,6,7,8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarity in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here a selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs was reported to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a Kd of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment significantly decreases expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.</p> <p>CBX2 is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable. Here, selections of focused DNA encoded libraries (DELs) were performed for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a Kd of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs <em>in vitro</em>. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation.</p> <p>Targeted covalent inhibitors (TCIs) are rationally designed inhibitors that bind to a target protein and specifically label a non-conserved amino acid on proteins by means of reactive moieties (warheads). TCIs typically function by two steps, in which inhibitors first non-covalently bind to the target protein and then covalent bond formation occurs between the inhibitor- warhead and a proximal nucleophile on protein. Covalent inhibitors or drugs have prolonged target engagement and enhanced pharmacokinetic potency in vivo, compared to non-covalent molecules. Strategies to develop effective warheads of TCIs have been reported for labeling different nucleophilic amino acid residues, of which cysteine and lysine are the most established for covalent labeling. Tyrosine is recently becoming an attractive nucleophile for TCIs as an alternative choice, yet currently developed warheads that label tyrosine do so with modest specificity over other side chains. Here, I report the development of novel Mannich electrophiles and use those electrophiles as covalent warheads on an inhibitor to specifically target tyrosine in protein labeling. To my knowledge, this is first demonstration of the use of Mannich electrophiles in covalent inhibitors. Specifically, I leveraged a previously developed CBX8 chromodomain inhibitor to specifically label a non-conserved tyrosine within CBX8 using cyclic imine derivatives as warheads. This ligand-directed, specific tyrosine conjugation on CBX8 but not on CBX2, significantly improves both the potency and selectivity of inhibition. Biochemical, proteomic, and cellular validation further showed the cyclic imine covalent inhibitors can increase both potency and selectivity to the target protein CBX8 in cells, serving as a robust chemical probe for target function evaluation and modulation. This new type of tyrosine labeling warhead is a useful addition to the toolbox of medicinal chemists for covalent inhibitor development.</p> <p>The following chapters are modified from following publications, with permissions from Sijie Wang, Emily C.Dykhuizen, and Casey J. Krusemark. </p> <p>Wang, S., Denton, K. E., Hobbs, K. F., Weaver, T., McFarlane, J. M., Connelly, K. E., Gignac, M.C., Milosevich, N., Hof, F., Paci, I., Musselman, C. A., Dykhuizen, E.C., Krusemark, C. J. Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor. <em>ACS Chem Biol. </em>2020, 15, 112-131</p> <p>Wang, S., Alpsoy, A., Sood, S., Ordonez-Rubiano, S. C., Dhiman, A., Sun, Y., Krusemark, C. J., Dykhuizen, E. C. A Potent, Selective CBX2 Chromodomain Ligand and its Cellular Activity During Prostate Cancer Neuroendocrine Differentiation. <em>ChemBioChem.</em> 2021, 22, 2335-2344</p> <p>Wang, S., Ordonez-Rubiano, S. C., Dhiman, A., Jiao G., Strohmier B. P., Krusemark, C. J., Dykhuizen, E. C. Polycomb Group proteins in cancer: multifaceted functions and strategies for modulation Modulators. <em>NAR Cancer</em>. 2021, 3, zcab039</p>

<p>Parkinson’s disease is a neurodegenerative disorder characterized by a loss of dopaminergic neurons, where mitochondrial dysfunction and neuroinflammation are implicated in this process. However, the exact mechanisms of mitochondrial dysfunction, oxidative stress and neuroinflammation leading to the onset and development of Parkinson’s disease are not well understood. There is a lack of tools necessary to dissect these mechanisms, therefore we engineered genetically encoded fluorescent biosensors to monitor redox status and an inflammatory signal peptide with high spatiotemporal resolution. To measure intracellular redox dynamics, we developed red-shifted redox sensors and demonstrated their application in dual compartment imaging to study cross compartmental redox dynamics in live cells. To monitor extracellular inflammatory events, we developed a family of spectrally diverse genetically encoded fluorescent biosensors for the inflammatory mediator peptide, bradykinin. At the organismal level, we characterized the locomotor effects of mitochondrial toxicant-induced dopaminergic disruption in a zebrafish animal model and evaluated a behavioral assay as a method to screen for dopaminergic dysfunction. Pairing our intracellular redox sensors and our extracellular bradykinin sensors in a Parkinson’s disease animal model, such as a zebrafish toxicant-induced model will prove useful for dissecting the role of mitochondrial dysfunction and inflammation in Parkinson’s disease. </p>

Escherichia coli enteroagregativa (EAEC), destaca-se como um importante patógeno emergente causador de diarréia persistente em países em desenvolvimento e de diarréia aguda em países desenvolvidos. A grande heterogeneidade dos fatores de virulência caracteriza esta categoria, porém não foi estabelecido um marcador genético comum a todas as amostras de EAEC. O padrão de adesão agregativa (AA) em células HEp-2 e HeLa é a forma de caracterização e diagnóstico mais precisos desta categoria. Uma das toxinas envolvidas na patogênese é Plasmid-encoded toxin (Pet) pertencente à classe das proteínas autotransportadoras com características de uma serino protease denominada SPATEs. Iniciou-se este estudo com a determinação do padrão de adesão de 164 amostras EAEC, previamente caracterizadas como sonda pCVD432 ou onda AA positiva. Assim, 141 (86%) amostras, que apresentaram padrão de adesão agregativo, foram caracterizadas como EAEC. Face aos resultados obtidos, confirmou-se a baixa especificidade da sonda AA. A pesquisa do gene pet, por meio de ensaio de PCR, resultou na positividade de 12 (8,5%) amostras. Prosseguiu-se esse estudo com a padronização da reação de immuno-dot. Utilizando-se 300 &#181;L do sobrenadante bacteriano, soro policlonal anti-Pet e o conjugado nas diluições 1/50 e 1/2.500, respectivamente, resultados bastante reprodutíveis foram obtidos. O método foi mais sensível que a detecção do gene por PCR. Por esse ensaio, detectou-se a toxina Pet em 16 (11,3%) das 141 amostras EAEC. Nenhuma das amostras controle negativo foi reconhecida pelo soro anti-Pet, assim como as amostras de E. coli produtoras das mais diversas toxinas. Apesar da baixa prevalência de amostras de EAEC produtoras da toxina Pet, neste estudo padronizou-se um método rápido, sensível, específico e de baixo custo para pesquisa desta toxina mostrando o potencial diagnóstico deste ensaio para uso em inquéritos epidemiológicos, o que poderá permitir determinar o papel da Pet no desenvolvimento de diarréia aquosa. / Enteroaggregative Escherichia coli (EAggEC) is an emerging diarrheal pathogen, whose pathogenesis is thought to comprise colonization of the intestinal mucosa with the release of secretogenic toxins. One of the toxin involved is the plasmid-encoded toxin (Pet), which is secreted by the autotransporter mechanism and belongs to a growing class of Enterobacteriaceae autotransporter proteins. Since the characteristic aggregative adherence pattern of EAggEC is associated with the presence of a large plasmid called pCVD432, DNA probes and PCR primers derived from this plasmid have been recommended as a screening method for EAggEC in the clinicai laboratory. In this study 164 E. coli isolates positive for the pCVD432 probe were tested for adherence to HEp-2 cells in which 141 isolates showed aggregative pattern, 12 isolates from them amplify a 1037-bp DNA fragment corresponding to pet gene by PCR. Using this samples we standardized an immuno-dot assay for EAggEC detection through Pet toxin as target antigen. 300 &#181;l of bacterial supernatant were applied in a PVDF membrane, and using a rabbit polyclonal sera anti-Pet the expression of the toxin by immuno-dot was in the same isolates in which the gene was detected. Besides no negative controls reacted with Pet antisera, in which we included 40 isolates with no virulence markers for diarrheagenic E. coli and E. coli expressing toxins other than Pet. This method proves to be rapid, sensitive, specific and low cost, demonstrating this potential as diagnosis for Pet expression and its association with diarrhea.

Escherichia coli enteroagregativa (EAEC), destaca-se como um importante patógeno emergente causador de diarréia persistente em países em desenvolvimento e de diarréia aguda em países desenvolvidos. A grande heterogeneidade dos fatores de virulência caracteriza esta categoria, porém não foi estabelecido um marcador genético comum a todas as amostras de EAEC. O padrão de adesão agregativa (AA) em células HEp-2 e HeLa é a forma de caracterização e diagnóstico mais precisos desta categoria. Uma das toxinas envolvidas na patogênese é Plasmid-encoded toxin (Pet) pertencente à classe das proteínas autotransportadoras com características de uma serino protease denominada SPATEs. Iniciou-se este estudo com a determinação do padrão de adesão de 164 amostras EAEC, previamente caracterizadas como sonda pCVD432 ou onda AA positiva. Assim, 141 (86%) amostras, que apresentaram padrão de adesão agregativo, foram caracterizadas como EAEC. Face aos resultados obtidos, confirmou-se a baixa especificidade da sonda AA. A pesquisa do gene pet, por meio de ensaio de PCR, resultou na positividade de 12 (8,5%) amostras. Prosseguiu-se esse estudo com a padronização da reação de immuno-dot. Utilizando-se 300 &#181;L do sobrenadante bacteriano, soro policlonal anti-Pet e o conjugado nas diluições 1/50 e 1/2.500, respectivamente, resultados bastante reprodutíveis foram obtidos. O método foi mais sensível que a detecção do gene por PCR. Por esse ensaio, detectou-se a toxina Pet em 16 (11,3%) das 141 amostras EAEC. Nenhuma das amostras controle negativo foi reconhecida pelo soro anti-Pet, assim como as amostras de E. coli produtoras das mais diversas toxinas. Apesar da baixa prevalência de amostras de EAEC produtoras da toxina Pet, neste estudo padronizou-se um método rápido, sensível, específico e de baixo custo para pesquisa desta toxina mostrando o potencial diagnóstico deste ensaio para uso em inquéritos epidemiológicos, o que poderá permitir determinar o papel da Pet no desenvolvimento de diarréia aquosa. / Enteroaggregative Escherichia coli (EAggEC) is an emerging diarrheal pathogen, whose pathogenesis is thought to comprise colonization of the intestinal mucosa with the release of secretogenic toxins. One of the toxin involved is the plasmid-encoded toxin (Pet), which is secreted by the autotransporter mechanism and belongs to a growing class of Enterobacteriaceae autotransporter proteins. Since the characteristic aggregative adherence pattern of EAggEC is associated with the presence of a large plasmid called pCVD432, DNA probes and PCR primers derived from this plasmid have been recommended as a screening method for EAggEC in the clinicai laboratory. In this study 164 E. coli isolates positive for the pCVD432 probe were tested for adherence to HEp-2 cells in which 141 isolates showed aggregative pattern, 12 isolates from them amplify a 1037-bp DNA fragment corresponding to pet gene by PCR. Using this samples we standardized an immuno-dot assay for EAggEC detection through Pet toxin as target antigen. 300 &#181;l of bacterial supernatant were applied in a PVDF membrane, and using a rabbit polyclonal sera anti-Pet the expression of the toxin by immuno-dot was in the same isolates in which the gene was detected. Besides no negative controls reacted with Pet antisera, in which we included 40 isolates with no virulence markers for diarrheagenic E. coli and E. coli expressing toxins other than Pet. This method proves to be rapid, sensitive, specific and low cost, demonstrating this potential as diagnosis for Pet expression and its association with diarrhea.