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O objetivo deste estudo foi avaliar a contribuição de linfoproliferação (1 parâmetro) em associação a provas sorológicas de alto desempenho (4 parâmetros), estudo epidemiológico (1) e parasitológico (1) em indivíduos com sorologia convencional inconclusiva para a doença de Chagas. Mostramos que o diagnóstico de doença de Chagas é provável quando 3 ou mais desses parâmetros são positivos em 7 (15/73). A combinação: TESA-blot e linfoproliferação positivos revelou-se útil diante de antecedentes epidemiológicos positivos / This study aims to evaluate the contribution of lymphoproliferation (1 parameter) in association with high performance serological tests (4), epidemiological data (1) and parasitological tests (1) for Chagas disease in patients with inconclusive conventional serological tests. We showed that this diagnosis is probable in individuals presenting > three positive of these 7 parameters (15 of 73 individuals). The combination of TESA-blot, lymphoproliferation was useful when epidemiological data were positive

Neste estudo avaliamos a resposta imune humoral de indivíduos naturalmente expostos à malária em áreas endêmicas no Brasil. Os anticorpos IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE e IgA anti-formas eritrocitárias de Plasmodium falciparum foram determinadas por ELISA. Anticorpos IgG, IgG1, IgG2 de alta avidez e IgG3 de baixa avidez predominaram nos indivíduos sem complicações de malária ou assintomáticos, enquanto anticorpos IgG4, IgE e IgM predominaram nos indivíduos com complicações clínicas por malária. Os resultados mostram que mesmo em regiões com transmissão instável de malária pode ser observado o desenvolvimento de imunidade protetora quando anticorpos apropriados são produzidos / In this study, we have evaluated the humoral immune response of individuals naturally exposed to malaria living in endemic areas of Brazil. We determined IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA antibodies against Plasmodium falciparum blood stages by ELISA. We observed that the level of high avidity IgG, IgG1 and IgG2 and low avidity IgG3 antibodies were higher in asymptomatic individuals or with uncomplicated malaria, while IgG4, IgE and IgM antibodies were higher in individuals with complicated malaria. Taken together the results showed that even in unstable malaria regions it can be observed the development of protective immunity against malaria when appropriate antibodies are produced

The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings. In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.

MSc (Microbiology) / Department of Microbiology / Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds. / NRF

Cardiovascular diseases are a broad group of diseases, such as heart attack and heart failureaffecting the cardiovascular system. The primary cause of cardiovascular diseases isatherosclerosis, and its progression is brought about by oxidative stress and a complex chronicinflammation reaction cascade. Of central importance are proinflammatory cytokines, regulatedby multiple factors, including interleukin (IL) 17A. This project aims to investigate the effectof IL-17A on the inflammatory response of human vascular endothelial cells by quantifyingchemokine C-X-C motif ligand-1 (CXCL1) release when exposed or not to otherproinflammatory mediators such as TNF-𝛼, IL-6 and IL-1β. To investigate this, humanumbilical cord endothelial cells were cultured and then stimulated with IL-17A alone or incombination with other cytokines, namely IL-6/sIL6R, IL-1β, or TNF-𝛼. After an appropriateincubation time following the stimulations, the supernatants of the cells were collected, and theamount of CXCL1 was analysed with ELISA or qPCR, respectively. At a lower concentration(10ng/ml), IL-17A failed to induce a significant level of CXCL1 release from endothelial cells.However, IL-17A + TNF-𝛼 (5ng/ml) greatly enhanced, higher than inductions from individualtreatments combined, level of CXCL1 release from endothelial cells. Furthermore, combiningIL-17A with IL-1β or IL-6 induced non-abundant and abundant upregulation in CXCL1 release,respectively. On transcription level, the amount of CXCL1 mRNA induced by IL-17A alonewas non-significant, but stimulation with TNF-𝛼 and IL-17A + TNF-𝛼 induced significantlyupregulated expression of CXCL1. In conclusion, we found that IL-17A induced synergeticrelease of CXCL1 in human vascular endothelial cells with TNF-𝛼. In addition, the synergisticimpact of IL-17A and TNF-𝛼 in terms of CXCL1 induction in vascular endothelial cells wasevident on a transcriptional level. Our data imply that combined blockage of IL-17A and TNF-𝛼 could have an enhanced therapeutic effect on vascular inflammation.

Tesis por compendio / [ES] El pepino dulce (Solanum muricatum) y el tomate de árbol (S. betaceum) pertenecen al grupo de cultivos de la familia Solanaceae. Estos dos cultivos son originarios de América del Sur y actualmente se cultivan en varios países con climas tropicales, subtropicales y mediterráneos. Han sido infrautilizados durante mucho tiempo y han cobrado relevancia solo en los últimos años debido a su alta calidad nutricional. El pepino dulce exhibe niveles significativos de potasio, vitamina C y carotenoides y se informa que presenta propiedades antioxidantes, antidiabéticas, antiinflamatorias y antitumorales. Sus frutos se pueden consumir tanto como postre o en ensaladas. El tomate de árbol también destaca por su alto contenido en compuestos bioactivos como carotenoides, antocianinas, flavonoides y vitaminas. Varios productos como jugos, mermeladas, salsas y productos farmacéuticos son elaborados a partir de sus frutos. Debido a que estos cultivos se han introducido en nuevas regiones, donde pueden estar expuestos a estreses bióticos y abióticos que pueden amenazar su producción, y dado que el pepino dulce se ve especialmente afectado por la escasez de agua, fue necesario realizar un estudio para determinar la respuesta de siete cultivares de pepino dulce a parámetros fisiológicos y bioquímicos al estrés por sequía. Este trabajo puede ayudar a desarrollar programas de selección y mejoramiento que permitan generar nuevas variedades más tolerantes a la sequía. Por otro lado, en los países de clima mediterráneo, el pepino dulce se cultiva como cultivo protegido, aplicando las mismas técnicas agrícolas que otras solanáceas como el tomate y el pimiento. Estos sistemas agrícolas también brindan condiciones óptimas para el desarrollo de enfermedades como Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), virus del mosaico del pepino (PepMV) y virus del mosaico del tomate (ToMV), que potencialmente podrían causar grandes daños a los cultivos de pepino dulce. Por tal motivo, se realizó un estudio para evaluar la respuesta de una colección de pepino dulce y sus parientes silvestres contra estas cuatro enfermedades, y encontrar fuentes de resistencia/tolerancia a estos patógenos. Aunque el tomate de árbol es un cultivo frutal importante debido a su valor nutricional y efectos beneficiosos para la salud, actualmente no hay información genómica y transcriptómica disponible públicamente. Por lo tanto, fue fundamental secuenciar el transcriptoma de dos cultivares de tomate de árbol con frutos morados (A21) y frutos anaranjados (A23). Estos dos cultivares han sido ampliamente utilizados y cultivados comercialmente en países de la región andina como Ecuador y Colombia. La obtención del primer transcriptoma de tomate de árbol ha permitido realizar un estudio comparativo entre el tomate de árbol y sus especies cercanas, tomate y patata, identificar genes implicados en la ruta de biosíntesis de carotenoides y desarrollar marcadores de polimorfismo de nucleótido único (SNP). En general, esta Tesis Doctoral aporta información relevante sobre la respuesta del pepino a diversos estreses ambientales, que puede ser utilizada para el desarrollo de nuevas variedades de pepino resistentes a múltiples estreses. Mientras que en tomate de árbol, el desarrollo de herramientas genómicas acelerará los programas de mejoramiento. / [CA] El cogombre dolç (Solanum muricatum) i tomata d'arbre (S. betaceum) pertanyen al grup de cultius de la família Solanaceae. Aquests dos cultius són originaris d'Amèrica del Sud i actualment es cultiven en diversos països amb climes tropicals, subtropicals i mediterranis. Han sigut infrautilitzats durant molt de temps i han cobrat rellevància només en els últims anys a causa de la seua alta qualitat nutricional. El cogombre dolç exhibeix nivells significatius de potassi, vitamina C i carotenoides i s'informa que presenta propietats antioxidants, antidiabètiques, antiinflamatòries i antitumorals. Els seus fruits es poden consumir tant com postres o en ensalades. La tomaca d'arbre també destaca pel seu alt contingut en compostos bioactivos com carotenoides, antocianinas, flavonoides i vitamines. Dels seus fruits s'elaboren diversos productes com a sucs, melmelades, salses i productes farmacèutics. Pel fet que aquests cultius s'han introduït en noves regions on poden estar exposats a estressos biòtics i abiòtics que poden amenaçar la seua producció, atés que el cogombre es veu especialment afectat per l'escassetat d'aigua, va ser necessari realitzar un estudi per a determinar la resposta de set cultivars de cogombre dolç a paràmetres fisiològics i bioquímicos a l'estrés per sequera. Aquest treball pot ajudar a desenvolupar programes de selecció i millorament que permeten generar noves varietats més tolerants a la sequera. D'altra banda, als països de clima mediterrani, el cogombre dolç es cultiva com a cultiu protegit, aplicant les mateixes tècniques agrícoles que unes altres solanáceas com la tomaca i el pimentó. Aquests sistemes agrícoles també brinden condicions òptimes per al desenvolupament de malalties com Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), virus del mosaic del cogombre (PepMV) i virus del mosaic de la tomaca (ToMV), que potencialment podrien causar grans danys als cultius de cogombre dolç. Per tal motiu, es va realitzar un estudi per a avaluar la resposta d'una col·lecció de cogombre dolç i els seus parents silvestres contra aquestes quatre malalties, i trobar fonts de resistència/tolerància a aquests patògens. Encara que la tomaca d'arbre és un cultiu fruiter important a causa del seu valor nutricional i efectes beneficiosos per a la salut, actualment no hi ha informació genòmica i transcriptómica disponible públicament. Per tant, va ser fonamental seqüenciar el transcriptoma de dues cultivars de tomaca d'arbre amb fruits morats (A21) i fruits ataronjats (A23). Aquestes dues cultivars han sigut àmpliament utilitzats i cultivats comercialment en països de la regió andina com l'Equador i Colòmbia. L'obtenció del primer transcriptoma de tomaca d'arbre ha permés realitzar un estudi comparatiu entre la tomaca d'arbre i les seues espècies pròximes, tomaca i creïlla, identificar gens implicats en la ruta de biosíntesi de carotenoides i desenvolupar marcadors de polimorfisme de nucleòtid únic (SNP). En general, aquesta Tesi Doctoral aporta informació rellevant sobre la resposta del cogombre a diversos estressos ambientals, que pot ser utilitzada per al desenvolupament de noves varietats de cogombre resistents a múltiples estressos. Mentre que en tomaca d'arbre, el desenvolupament d'eines genòmiques accelerarà els programes de millorament. / [EN] Pepino (Solanum muricatum) and tree tomato (S. betaceum) belong to the group of crops of the Solanaceae family. These two crops are native to South America and currently are grown in various countries with tropical, subtropical and Mediterranean climates. They have been underutilized for a long time and have become relevant only in recent years due to their high nutritional quality. Pepino exhibit significant levels of potassium, vitamin C and carotenoids and it is reported to present antioxidant, antidiabetic, anti-inflammatory and antitumor properties. Its fruits can be consumed both as a dessert or in salads. Tree tomato also highlights high content of bioactive compounds such as carotenoids, anthocyanins, flavonoids and vitamins. Severals products such as juices, jams, sauces and pharmaceutical products are made from its fruits. Due to these crops have been introduced into new regions, where they may be exposed to biotic and abiotic stresses that can threaten their production, and since pepino is specially affected by water scarcity, a study was needed to determine the response of seven pepino cultivars to physiological and biochemical parameters to drought stress. This work can help develop selection and improvement programs that allow the generation of new varieties that are more tolerant to drought. On the other hand, in countries with a Mediterranean climate, pepino is grown as a protected crop, applying the same agricultural techniques as other solanaceous plants such as tomato and pepper. These agricultural systems also provide optimal conditions for the development of diseases such as Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), pepino mosaic virus (PepMV) and tomato mosaic virus (ToMV), which could potentially cause great damage to pepino crops. For this reason, a study was performed to evaluate the response of a collection of pepino and their wild relatives against these four diseases, and find sources of resistance/tolerance to those pathogens. Although tree tomato is an important fruit crop due to its nutritional value and beneficial health effects, there is currently no publicly available genomic and transcriptomic information. Therefore, it was essential to sequence the transcriptome of two tree tomato cultivars with purple fruits (A21) and orange fruits (A23). These two cultivars have been widely used and cultivated commercially in countries of the Andean region such as Ecuador and Colombia. Obtaining the first tree tomato transcriptome has made it possible to perform a comparative study between tree tomato and its close species, tomato and potato, identify genes involved in the carotenoid biosynthesis pathway, and develop single nucleotide polymorphism (SNP) markers. In general, this Doctoral Thesis provides relevant information on the response of pepino to various environmental stresses, which can be used for the development of new varieties of pepino resistant to multiple stresses. While in tree tomato, the development of genomic tools will accelerating up breeding programs. / Pacheco Toabanda, JE. (2022). Development of Biotechnological Tools for the Genetic Improvement of Pepino (Solanum Muricatum) and Tree Tomato (S.betaceum) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189205 / Compendio

Que o envenenamento pela serpente Bothrops jararaca causa distúrbios hemorrágicos sistêmicos, com alteração da coagulação e fibrinólise sangüíneas, é notório. Contudo, pouco se sabe sobre a ação in vivo desse veneno sobre as plaquetas. Em estudos recentes, demonstrou-se que esse veneno causa trombocitopenia, distúrbios da agregação e diminuição do número de corpos densos plaquetários, que, dessarte, sugeriam a ativação das plaquetas circulantes. Com o escopo de comprovar esta hipótese e melhor caracterizar as ações in vivo desse veneno sobre as plaquetas, serviu-se de um modelo experimental que empregava coelhos para o envenenamento pela B. jararaca. No grupo experimental, os animais foram injetados i.v. com o veneno da B. jararaca (60 µg/kg) e no grupo controle com salina. Previamente à administração de salina ou veneno, os coelhos tiveram suas plaquetas marcadas ex vivo com NHS-biotina. Para a avaliação das alterações plaquetárias, amostras de sangue foram coletadas seqüencialmente, em intervalos de tempo que variaram de 1 a 144 horas após a administração do veneno ou salina. Durante o envenenamento, houve trombocitopenia, hipofibrinogenemia, elevação dos níveis plasmáticos do fator de von Willebrand, diminuição da função plaquetária no sangue total induzida pela botrocetina e pelo colágeno e diminuição da secreção de ATP. Não obstante, os níveis plasmáticos de fator plaquetário 4, um marcador específico da ativação plaquetária in vivo, e os níveis intraplaquetários de serotonina se mantiveram constantes. Pela citometria de fluxo, observou-se um decréscimo significativo da expressão do epítopo da GPIIb-IIIa reconhecido pelo anticorpo monoclonal P2, porém isso não foi observado ao utilizar-se anticorpos policlonais. A expressão de fibrinogênio ou dos produtos de degradação do fibrinogênio/fibrina (PDF) na membrana plaquetária também não sofreu alteração significativa ao longo do tempo. Houve, todavia, elevações significativas da P-selectina plaquetária, um receptor cuja expressão é indicativa de ativação plaquetária, e do epítopo induzido por ligantes (LIBS1) da GPIIIa. A porcentagem de plaquetas reticuladas na circulação, assim como os tempos de sobrevivência plaquetária, não foram estatisticamente diferentes entre os dois grupos. As análises histológicas e imuno-histoquímicas dos órgãos dos coelhos mostraram que as plaquetas circulantes são retidas entre redes de fibrina nos capilares pulmonares. Os resultados obtidos sugerem que a trombina engendrada pelos componentes pró-coagulantes deste veneno desempenha uma função essencial na patogenia dos distúrbios da coagulação e plaquetários observados neste modelo de envenenamento. O aumento da expressão de P-selectina no grupo experimental comprovou a hipótese inicial de que as plaquetas dos coelhos envenenados são verdadeiramente ativadas na circulação. Os dados ora apresentados demonstram definitivamente que a diminuição do fibrinogênio ou o aumento dos PDF não são a causa fundamental da disfunção plaquetária observada no envenenamento botrópico e que outro(s) composto(s) parece(m) estar envolvido(s) com estes distúrbios plaquetários. / In spite of being well established that Bothrops jararaca snake venom causes blood coagulation and fibrinolysis disturbances in patients, scant information about blood platelet disorders during envenomation is available. In recent investigations, thrombocytopenia, platelet aggregation disturbances and decreased numbers of platelet dense bodies were observed following venom administration, suggesting that circulating platelets had been activated. In order to prove this hypothesis and to gain a better characterization of the in vivo role of this venom on platelets, an experimental model of B. jararaca envenomation was utilized. Rabbits were injected i.v. either with B. jararaca venom (60 µg/kg) (experimental group) or saline (control group). Previously to saline or venom administration, rabbit platelets were labeled ex vivo with NHS-biotin. To evaluate platelet disturbances, blood samples were collected consecutively, at time intervals that varied from 1 to 144 hours after venom or saline administration. During envenomation, there were thrombocytopenia, hypofibrinogenemia, elevation of von Willebrand factor plasma levels, reduced botrocetin- and collagen-induced platelet aggregation in whole blood, and decreased ATP secretion. However, plasma levels of platelet factor 4, a specific marker of in vivo platelet activation, and intraplatelet serotonin levels remained constant. By flow cytometry, a significant decrease on the expression of GPIIb-IIIa epitope recognized by P2 monoclonal antibody was observed; however, this was not observed when polyclonal antibodies were employed. Fibrinogen or fibrin(ogen) degradation product (FDP) expression on platelet surface showed no significant alteration. Nonetheless, significant elevations of platelet P-selectin, a receptor whose expression is indicative of platelet activation, and of ligand-induced binding sites (LIBS1) of GPIIIa were noted. The percentage of circulating reticulated platelets, as well as platelet survival times, were not statistically different between the two groups. Histopathological and immunohistochemical analyses of rabbit organs demonstrated that circulating platelets were sequestered among fibrin deposits in pulmonary capillaries. These results suggest that thrombin generated by procoagulating components of B. jararaca venom has an essential role in the pathogenesis of platelet and coagulation disorders in this experimental model. Increased expression of P-selectin in the experimental group proves the initial hypothesis that platelets of envenomed rabbits are indeed activated in the circulation. The data presented herein demonstrate definitively that decreased fibrinogen or increased FDP levels are not the primary cause of the platelet dysfunction observed in bothropic envenomation, but other substances seem to be responsible for it.

Introdução: Apesar dos avanços nas técnicas de diagnóstico e tratamento, o câncer de próstata avançado ainda é uma condição letal. Terapêuticas mais eficazes são necessárias para reduzir as taxas de morbi-mortalidade associadas à doença. A Proteína-78 regulada pela glicose (GRP78), uma proteína de choque térmico envolvida na apresentação de antígenos, foi recentemente descrita como sendo um possível marcador molecular para o câncer de próstata. Ainda mais, a resposta imune a essa proteína mostrou correlação com o desenvolvimento de doença hormônio-independente e com pior sobrevida para a doença. Objetivos: Neste estudo, avaliou-se a hipótese de que a GRP78 poderia ser usada como marcador molecular em câncer de próstata no desenvolvimento de um sistema de receptor-ligante, através do uso da tecnologia de apresentação de fagos. Casuística e métodos: Inicialmente, foram clonados dois peptídeos que apresentam afinidade à proteína regulada pela GRP78 (os peptídeos WIFPWIQL e WDLAWMFRLPVG) no vetor fUSE5, criando-se fagos com capacidade teórica de ligação à mesma proteína. Posteriormente foi testada a capacidade de ligação desses fagos à GRP78 na membrana de células prostáticas malignas em solução, em xeno-tumores in vivo e em metástases ósseas de câncer de próstata humano. Resultados: Demonstrou-se que ambos os fagos se ligam especificamente à GRP78 in vitro, em comparação à proteínas com seqüência semelhante (proteínas de choque térmico 70 e 90) e não semelhante (albumina sérica bovina). Em seguida, mostrou-se que esses fagos se ligam com afinidade pelo menos 30 vezes maior à células de câncer de próstata que o fago controle, e que os fagos são internalizados por essas células. Posteriormente, mostrou-se que os fagos rastrearam xeno-tumores prostáticos quando injetados in vivo num modelo animal de câncer de próstata. Finalmente, mostrou-se que os fagos ligam-se especificamente à GRP78 expressa em metástases ósseas de adenocarcinoma prostático humano. Conclusões: Os fagos criados apresentam capacidade de ligação específica à GRP78 in vitro, em células em suspensão e in vivo. A estratégia e o sistema de receptor-ligante definidos no presente estudo podem ter implicacões relevantes no desenvolvimento de terapias dirigidas para o tratamento do câncer de próstata. / Introduction: Despite the advances in diagnosis and treatment, advanced prostate cancer remains a lethal condition. Improved methods of therapy are needed to reduce the morbidity and mortality rates associated with this disease. The Glucose-regulated protein-78 (GRP78), a stress-responsive heat-shock protein involved in antigen presentation, was recently described as a possible molecular marker for prostate cancer. Moreover, immune response against this protein was shown to have correlation with the development of androgen-independent prostate cancer and shorter overall survival. Objectives: We hipothesized that GRP78 could be used as a molecular marker for prostate cancer in the development of a receptor-ligand system, by using phage display technology. Patients and methods: We initially cloned two GRP78-targeting peptides (WIFPWIQL and WDLAWMFRLPVG) into a fUSE5-based phage. We then tested binding capacity of the phage to GRP78 in vitro, to GRP78 expressed in intact prostate cancer cell membranes, to a prostate cancer xenograft and to human bone metastases. Results: We showed that both phage created bound specifically to GRP78 in vitro, in comparison to related (Heat-shock proteins 70 and 90) and unrelated control proteins (bovine serum albumin). Next, we showed that these phage bound at least 30 times more to prostate cancer cells than the control phage, and were also internalized into these cells. Both GRP78-binding phage showed a strong homing in vivo to a human prostate cancer xenograft in a mouse model. Finally, we showed that both phage bound specifically to GRP78 expressed in human prostate cancer bone metastases. Conclusions: Both phage are capable of binding specifically to GRP78 in vitro, in the context of intact prostate cancer cells and in vivo. The strategy and the ligand-receptor system we have defined in this study may have relevant implications in the development of targeted therapies for the treatment of prostate cancer.

We now report an in depth analysis of the successful in vitro enzymatic synthesis of PHB utilizing the three-enzyme system from the bacteria Cupriavidus necator. Using HPLC methodology developed in this laboratory, and by adding each enzyme in a step-wise manner, we follow each individual stage in the three-enzyme route for PHB synthesis and delineate all stoichiometric relationships. We report the construction of the first metabolic model developed specifically for analyzing in vitro enzymatic PHB synthesis. We developed a hands-on student laboratory for culturing, producing, isolating, and purifying the bacterial biopolyesters PHB. We now report the first structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using MS, MS/MS, and homo- and hetero-nuclear NMR analyses.We describe HPLC methodology to separate the isomers of several iso-CoA-containing compounds and report the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl-transfer reactions. We describe a simple regioselective synthesis of iso-CoA from CoA. We also demonstrate a plausible mechanism, which accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. Herein we report that phenylaminoethyl selenide compounds protect DNA from peroxynitrite-mediated single-strand breaks. The mechanism of protection against peroxynitrite mediated DNA damage was investigated by HPLC. The chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. The unique chemistry of the reaction between peroxynitrite and HOMePAES was investigated using HPLC and HPLC/MS. We report the development of novel CDB derivatives, which are selective COX-II inhibitors. A series of compounds were assayed with an in vitro colorimetric inhibitor screening and with a whole blood ELISA screening and the results indicate that MST is a selective inhibitor of COX-II.