Sabendo-se que o sistema colinérgico muscarínico modula funções cognitivas, propomos estudar as toxinas muscarínicas isoladas do veneno da M. lemniscatus, MT-Mlα e MT-Mlβ, sobre o processo de aprendizado e memória. Ratos Wistar machos foram injetados por via intrahipocampal com MT-Mlα, MT-Mlβ ou solução de Ringer (SRg). Após um período de sete dias de treino no Labirinto Aquático de Morris (LAM), os ratos receberam uma das toxinas ou SRg (dia da inoculação) e 20 min e 24 h após a inoculação foram testados no LAM. A MT-Mlα reduziu o tempo de permanência no quadrante do dia anterior, indicando interferência na evocação da memória. Por outro lado, a MT-Mlβ causou um efeito facilitatório quanto à recuperação da localização da plataforma. A ansiedade foi avaliada no Labirinto em Cruz Elevado, o treino foi feito após 30 min da injeção e o teste 24 h após. O tratamento com a MT-Mlβ mostrou ter um efeito ansiogênico, o que pode ter contribuído para o efeito facilitatório sobre a memória, pois sabe-se que a ansiedade até certo nível, pode favorecer o desempenho cognitivo. / Knowing that the muscarinic cholinergic system modulates cognitive functions, we propose to study the muscarinic toxins isolated from the venom of M. lemniscatus, MT-Mlα and MT-Mlβ, on the process of learning and memory. Male Wistar rats were injected by intrahippocampal pathway with MT-Mlα, MT-Mlβ or Ringer\'s solution (SRG). After a period of seven days training in Morris Water Maze (MWM), rats received a toxin or SRG (Inoculation day) and 20 min and 24 h after inoculation were tested in LAM. The MT-Mlα reduced the time spent in the quadrant of the previous day, indicating interference in the evocation of memory. On the other hand, the MT-Mlβ caused a facilitatory effect in recovering the location of the plataform. Anxiety was assessed in the Elevated Plus Maze, the training was done at 30 min after injection and 24 h after the test. Treatment with MT-Mlβ shown to have an anxiogenic effect, which may have contributed to the facilitatory effect on memory, since it is known that anxiety to a certain level can help cognitive performance.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-16T13:22:52Z No. of bitstreams: 1 Edilaine Alves de Melo.pdf: 641010 bytes, checksum: 2a8032c2676a99782e8fd581d977a950 (MD5) / Made available in DSpace on 2017-03-16T13:22:52Z (GMT). No. of bitstreams: 1 Edilaine Alves de Melo.pdf: 641010 bytes, checksum: 2a8032c2676a99782e8fd581d977a950 (MD5) Previous issue date: 2012-07-27 / The bacterial fruit blotch caused by Acidovorax citrulli is one of the most severe diseases of melon (Cucumis melo), and a major problem in the Northeast, the main melon producing region of Brazil. Strategies for control of bacterial blotch include chemical and physical treatments of seeds and chemical sprays of the plant canopy. Since these treatments are not efficient and resistant melon cultivars do not exist, other strategies have been studied, including biological control. Our objectives were to analyze the efficiency of yeasts in the biocontrol of this disease by protecting seedlings and plants, and by treating melon seeds; and to verify the in vitro activity against the pathogen and the growth promotion of melon plants. None of the 60 yeasts inhibited the growth of the pathogen, but the isolates LMA1 (Rhodotorula aurantiaca), LMS (R. glutinis) and CC-2 (Pichia anomala) stood out as the most effective in protecting seedlings. When tested in plants and seeds, LMA1 and CC-2 maintained effectiveness. In plants, the reductions in disease index (ID) and area under the disease progress curve (AUDPC) compared to the control reach 58.6 and 47.2%, respectively, while seed treatments reduced ID and AUDPC up to 34.3 and 45.5%. These isolates did not promote the growth of melon plants and did not produce killer toxins in vitro. R. aurantiaca (LMA1) and P. anomala (CC-2) were effective in protecting plants and seedlings and for seed treatment of melon. Therefore, the use of these yeasts jointly with other control methods, such as resistant varieties and copper compounds, is important in integrated management of bacterial fruit blotch. / A mancha aquosa causada por Acidovorax citrulli, é uma das doenças mais severas do meloeiro (Cucumis melo) e um dos principais problemas para o Nordeste, a principal região produtora de melão do Brasil. Estratégias para o controle da mancha aquosa incluem tratamentos químicos e físicos das sementes e químico da parte aérea da planta. Uma vez que esses tratamentos não são eficientes e cultivares resistentes de meloeiro inexistem, outras estratégias têm sido investigadas, dentre elas o controle biológico. Os objetivos deste trabalho foram analisar a eficiência de leveduras no biocontrole dessa doença pela proteção de plântulas e plantas e pelo tratamento de sementes de meloeiro, além de verificar a atividade in vitro contra o patógeno e a promoção do crescimento de plantas de meloeiro. Nenhuma das 60 leveduras testadas inibiu o crescimento do patógeno, porém os isolados LMA1 (Rhodotorula aurantiaca), LMS (R. glutinis) e CC-2 (Pichia anomala) destacaram-se como os mais eficientes na proteção de plântulas. Quando testadas em plantas e sementes, LMA1 e CC-2 mantiveram a eficácia. Em plantas, as reduções de índice de doença (ID) e área abaixo da curva de progresso da doença (AACPD) em relação à testemunha foram de até 58,6 e 47,2%, respectivamente, enquanto que o tratamento de sementes reduziu o ID e AACPD em até 34,3 e 45,5%. Esses isolados não promoveram o crescimento do meloeiro e não produziram toxinas killer in vitro. R. aurantiaca (LMA1) e P. anomala (CC-2) foram eficazes na proteção de plântulas e plantas e no tratamento de sementes de meloeiro. Portanto, a utilização dessas leveduras junto a outros métodos de controle, tais como cultivares resistentes e utilização de compostos cúpricos, será importante no manejo integrado da mancha aquosa.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-06Bitstream added on 2014-06-13T19:40:32Z : No. of bitstreams: 1 gomes_pc_dr_rcla.pdf: 875503 bytes, checksum: 13ea84ba63273890b35fe9374e841f94 (MD5) / As aranhas do gênero Phoneutria (Aranae, Ctenidae) são comumente conhecidas como aranha armadeira ou aranha da banana, devido à posição de ataque/defesa que elas assumem contra uma presa/competidor e devido sua alta incidência nas plantações de bananas. Estas aranhas são solitárias, errantes (não constroem teias), muito agressivas e responsáveis por muitos casos graves de acidentes por envenenamento e de morte registrados. Estas aranhas são amplamente distribuídas nas regiões temperadas da América do Sul, com várias espécies já descritas. A espécie Phoneutria nigriventer é mais comum nas regiões centroeste e sudeste do Brasil. Um grande número de neurotoxinas tem sido purificado de venenos de aranhas do gênero Phoneutria. No entanto, as neurotoxinas não – protéicas de baixa massa molecular precisam ser isoladas e caracterizadas estrutural e funcionalmente. Essas toxinas são de potencial interesse na neuroquímica como ferramentas para investigações do sistema nervoso. O objetivo do presente estudo foi a caracterização estrutural e funcional da Nigriventrina, uma nova neurotoxina não-protéica de baixa massa molecular, isolada da fração hidrofílica do veneno da aranha Phoneutria nigriventer por (RPHPLC) sob gradiente de acetonitrila. A elucidação estrutural foi realizada com HRESIMS, ESI-MS, ESI-MS/MS e espectroscopia de Ressonância Magnética Nuclear 1D e 2D. A toxina apresentou massa molecular de 422Da (C12H14N4O13) e foi caracterizada como hidrazil-dioxopiperidina. A caracterização biológica foi realizada pela aplicação icv da Nigriventrina em cérebro de rato, seguido pelo acompanhamento da expressão da proteína Fos e dupla marcação imunoistoquímica. Todos os neurônios duplamente marcados foram associados ao receptor ionotrópico de glutamato NMDA (N-metil-D-aspartato), subtipo NMDA-NR1. A Nigriventrina apresentou afinidade... / The spiders of the genus Phoneutria (Aranae, Ctenidae) are commonly known as “armed spider” or “banana spider”, because of the attack–defense position that they assume against a prey/competitor and their high incidence in banana plantations. These spiders are solitary, wandering (no web), very aggressive, responsible for many cases of severe envenomation and recorded mortality. These spiders are widely distributed in the warm regions of South America, and several species have been described. The Phoneutria nigriventer are the most common in central and south-eastern regions of Brazil. A large number of neurotoxins have been purified from the venoms of Phoneutria spiders. However, the non-proteic low molecular mass neurotoxins must be isolated and structural and functionally characterized. These toxins have an interesting potential in neurochemistry as tools for investigations of nervous system. The objective of the present study was the structural and functional characterization of “Nigriventrine”, a novel non-proteic low molecular mass neurotoxin, isolated from the hydrophilic fraction of Phoneutria nigriventer spider venom by (RP-HPLC) under gradient of acetonitrile. The structural elucidation was carried out with HRESIMS, ESI-MS, ESI-MS/MS and Nuclear Magnetic Resonance 1D and 2D spectroscopy. The toxin presented molecular mass of 422Da (C12H14N4O13) and was characterized as hydroxyl-hydrazyl-dioxopiperidine. The biological characterization was performed by the i.c.v application of nigriventrine in rat brain, followed by the monitoring of the expression of Fos protein and doublelabeling immunohistochemistry. All doubly labeled neurons were associated to Nmethyl- D- aspartate/subtype of ionotropic glutamate receptor (NMDA-NR1). The nigriventrine presented affinity mainly to cortical regions, which are linked to perceptions and the voluntary muscles control of the animals... (Complete abstract click electronic access below)

Os venenos das serpentes são compostos protéicos complexos com extensas atividades biológicas. Estas moléculas abrangem até 95% do peso seco do veneno, e compreendem enzimas, toxinas não enzimáticas e proteínas atóxicas. Dentre as complicações fisiológicas, a Insuficiência Renal Aguda - IRA é bem comum nos acidentes crotálicos, onde as concentrações renais do veneno se apresentam até 50% maiores do que na concentração plasmática. A fração tóxica responsável por essa complicação é a crotoxina. O veneno das serpentes, quando irradiado com raios gama de 60Co, tem a sua toxicidade diminuída, entretanto, são mantidas as suas propriedades imunológicas. Nossa hipótese é que a utilização de veneno irradiado em substituição ao nativo pode reduzir a taxa de lesões renais em animais de grande porte durante a produção de soros antiofídicos, propiciando melhor qualidade de vida e bem estar desses animais. No presente estudo realizou-se o isolamento da crotoxina por técnicas cromatográficas, a partir do veneno da serpente Crotalus durissus terrificus. Parte da toxina isolada foi reservada e denominada crotoxina nativa (CTXN) enquanto que outra parte foi irradiada com 2 kGy de raios gama oriundos de uma fonte de 60Co e denominada crotoxina irradiada (CTXI). Diferentes grupos de camundongos Balb/c receberam a toxina nativa ou irradiada e tiveram seus rins removidos em diferentes tempos pós-injeção da crotoxina. Realizou-se a nefrectomia dos animais e foi feito o preparo dos cortes histológicos em Hematoxilina-Eosina (HE) e cortes de imunohistoquímica com anticorpo policlonal de coelho anticrotoxina. As alterações histopatológicas encontradas nas amostras foram glomerulonefrite (GN), congestão capilar (CC) e necrose tubular aguda (NTA). Verificou-se que a GN teve maior ocorrência no grupo CTXN que no grupo CTXI. A NTA foi dominante no grupo CTXN em relação ao grupo CTXI. A CC foi predominante no grupo CTXI que no grupo CTXN. Entretanto esta diferença não é estatisticamente significante segundo o teste t Student de amostras independentes. O tratamento imunohistoquímico revelou que a concentração de antígeno marcado com o anticorpo é decrescente segundo os intervalos de tempo de eutanásia dos animais do grupo inoculado com CTXI, pois a crotoxina quando irradiada tem a sua excreção renal facilitada. Conclui-se que as alterações histopatológicas GN e a NTA foram significativamente menos frequentes em rins de animais inoculados com CTXI. Diferentemente da crotoxina nativa, a crotoxina irradiada é detectada pelo anticorpo anticrotoxina apenas nos tempos intermediários pós-inoculação e em baixa concentração, indicando que a radiação gama promoveu mudanças na forma de ligação da toxina ao tecido renal, facilitando sua eliminação e diminuindo, portanto, as chances de lesão. Esses resultados reforçam as evidências de que o uso de veneno irradiado no processo de imunização garante uma melhor qualidade de vida e bem estar aos animais soroprodutores. / Snake venoms are complex mixtures of proteins and peptides with a wide spectrum of physiological targets such as the blood coagulation and cardiovascular systems and the motor end plate among others. Acute renal failure is a common complication in accidents with the South American rattlesnake. The toxin involved in this pathology is crotoxin, a major component of the venom in terms of concentration and toxicity. Snake venoms, when irradiated with 60Co gamma rays present a significant decrease in toxicity while the immunogenic properties of its components are preserved. The use of irradiated rattlesnake venom is an attractive alternative for antisera production since it might reduce the appearance of renal lesions improving the welfare and lifespan of animals employed for antivenom production. In the present work, we have isolated crotoxin from crude venom of C. d. terrificus and irradiates part of it with 2 kGy of gamma rays from 60Co and compared the effects of native (CTXN) and irradiated (CTXI) crotoxin on the mice renal function. We conducted nephrectomy of animals and preparation of histological sections with hematoxylin-eosin (HE) and immunohistochemistry with polyclonal rabbit anticrotoxin for analysis in optical microscopy. Histopatological changes found in the samples were glomerulonephritis (GN), capillary congestion (CC) acute kidney tubular necrosis (NTA). Immunohistochemistry indicates that the concentration of bound antibody labelled antigen decreases in a time dependent manner. The GN ante NTA were significantly less frequent in kidneys of animals inoculated with irradiated toxin. The capillary congestion was evidenced in all animals treated with irradiated or native crotoxin. Concluding, ionizing radiation appears to mitigate the nephrotoxic effects of crotoxin and may represent an alternative for antivenin production, improving welfare for seroproductor animal.

Made available in DSpace on 2016-04-28T20:16:30Z (GMT). No. of bitstreams: 1 2009 - Vanessa de Magalhaes Ferreira.pdf: 3832895 bytes, checksum: 54d34bf9e767ef211ef5b82de690ac7b (MD5) Previous issue date: 2009-03-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / At Guaiba island (Mangaratiba municipality, south coast of Rio de Janeiro) is located the Association of Marine Maricultores of Mangaratiba s farm. The region has excellent conditions for growth of molluscs bivalves: mild water temperatures, mesotrophic and with good sanitary conditions (under bacteriological aspects). The crop is the main shellfish mussel Perna perna, which feeds mainly phytoplanktonic organisms (microalgae). However, the phytoplankton community may be present harmful microalgae such as dinoflagellates Dinophysis spp. that can produce diarrhoetic toxins. The main diarrhoetic phycotoxin produced by these microalgae is okadaic acid (OA), previously detected in the region and associated with D. acuminata. This phycotoxin is implicated in the syndrome Diarrhoetic Shellfish Poisoning, which affects humans, showing the acute effect gastrointestinal symptoms (vomiting, abdominal pain and diarrhea) and chronic promotion of carcinogenesis in the stomach and intestine. Regarding the animal sanity OA affects the immune system of mussels making them susceptible to pathogens and pollutants. In addition is genotoxic and can lead to loss of genetic biodiversity of P. perna. This effect can generate mutations that compromise the future generations of shellfish can lead to disastrous consequences for the mitiliculture. Dinophysis as planktonic organism, has a limited ability to swim. However, under conditions of stability of the water column the dinoflagellate may attach to certain depths, which may become an aggravating factor in the contamination of shellfish if production of OA. This distribution on the microscale is from the interaction between biotic and abiotic factors. Thus the present work to identify and quantify the Dinophysis species present at Guaiba island and verify whether the formation of density along the water column over a year. Were collected monthly, in high tide, over a vertical profile of 8 meters deep. The microalgae were collected with Van-Dorn bottle and fixed with formalde?do for further work on microscopy. We measured temperature, salinity, Secchi depth. Climatological data of rainfall, intensity and direction of winds were released by Vale. We identified 6 species of Dinophysis: D. acuminata, D. caudata, D. fortii, D. ovum, D. tripos and D. rotundata. The Cluster analysis revealed D. acuminata was dominant (Fitopac v1.6, p <0001), besides being the most frequent and abundant throughout the year. Only the species during summer showed higher cell density (average of 297 c?lulas.l-1, a low relative abundance according literature) in the stratum of 0-4 meters deep, than in the stratum > 4 meters (160 c?lulas.l-1), significantly different from the rest of the year (ANOVA, p <0.0001). D. fortii and D. rotundata were codominant in summer and autumn/winter, respectively. The remaining species occurred as incidental. D. ovum only occurred in summer and this work is the first report of this species in Brazilian waters. Redundancy analysis showed that the hydrological and climatological parameters analyzed only salinity and wind (selected by the Monte Carlo test) explained 11% of the variance of the data distribution of Dinophysis. Although no analysis has been performed for the presence of okadaic acid that all identified species are reported as potential producers of toxins diarrhea. Thus it is necessary for an evaluation of risk for diarrhoetic toxins aimed at animal health and safety to public health in the region. / Na ilha Gua?ba (munic?pio de Mangaratiba, litoral sul do Rio de Janeiro) localiza-se a fazenda marinha da Associa??o de Maricultores de Mangaratiba. O principal molusco cultivado ? o mexilh?o Perna perna, que se alimenta principalmente de organismos fitoplanct?nicos (microalgas). No entanto, na comunidade fitoplanct?nica podem estar presentes microalgas nocivas, como os dinoflagelados Dinophysis spp. que podem produzir toxinas diarr?icas. A principal ficotoxina diarr?ica produzida por essas microalgas ? o ?cido okadaico (AO), j? anteriormente detectado na regi?o e associado ? D. acuminata. Tal ficotoxina encontra-se implicada na s?ndrome Envenenamento Diarr?ico por Moluscos, que acomete humanos, apresentando como efeito agudo sintomatologia gastrintestinal (v?mito, dor abdominal e diarr?ia) e cr?nico promo??o de carcinog?nese no est?mago e intestino. Em rela??o ? sanidade animal o AO afeta o sistema imune dos mexilh?es tornando-os suscept?veis ? pat?genos e poluentes. Al?m disso, ? genot?xico e pode levar ? perda de biodiversidade gen?tica de P. perna. Dinophysis, como todo organismo planct?nico, apresenta uma restrita capacidade de nata??o. No entanto, sob condi??es de estabilidade da coluna d ?gua o dinoflagelado pode agregar-se em determinadas profundidades, o que pode vir a se tornar um agravante na contamina??o de moluscos se houver produ??o de AO. Tal distribui??o em microescala resulta da intera??o entre fatores bi?ticos e abi?ticos. Dessa forma o presente trabalho buscou identificar e quantificar as esp?cies de Dinophysis presentes na ilha Gua?ba, bem como verificar se houve a forma??o de adensamentos ao longo da coluna d ?gua ao longo de um ano. Foram realizadas coletas mensais, ao longo de um perfil vertical de 8 metros de profundidade, sempre na preamar. As microalgas foram coletadas com garrafa Van- Dorn e fixadas com formalde?do para posterior realiza??o de microscopia em campo claro e contraste de fase. Foram mensuradas: temperatura, salinidade, profundidade Secchi. Dados climatol?gicos de pluviosidade, intensidade e dire??o dos ventos foram cedidos pela Cia. Vale. Foram identificadas 6 esp?cies de Dinophysis: D. acuminata, D. caudata, D. fortii, D. cf ovum, D. tripos e D. rotundata. A an?lise de classifica??o revelou que D. acuminata foi dominante (Fitopac v1.6, p<0,001), al?m de ser a esp?cie mais frequente e abundante ao longo de todo o ano. Apenas durante o ver?o a esp?cie apresentou maior densidade celular (m?dia de 297 c?lulas.l-1, uma abund?ncia relativa considerada baixa pela literatura) no estrato de 0-4 metros de profundidade, do que no estrato > 4 metros (160 c?lulas.l-1), significativamente diferente do resto do ano (ANOVA, p<0.0001). D. fortii e D. rotundata foram co-dominantes no ver?o e outono/inverno, respectivamente. As demais esp?cies ocorreram como acess?rias. D. ovum apenas ocorreu no ver?o e o presente trabalho ? o primeiro relato da esp?cie em ?guas brasileiras. An?lise de redund?ncia revelou que dos par?metros hidrol?gicos e climatol?gicos analisados apenas salinidade e vento (selecionadas pelo Teste de Monte Carlo) explicaram 11% da vari?ncia dos dados de distribui??o de Dinophysis. Embora n?o tenha sido realizada an?lise para a presen?a do ?cido okadaico salienta-se que todas as esp?cies identificadas s?o relatadas como potenciais produtoras de toxinas diarr?icas. Dessa forma faz-se necess?rio a realiza??o de uma avalia??o de risco em rela??o ?s toxinas diarr?icas com vistas ? sanidade animal e ? seguran?a para a sa?de p?blica.

Shiga producing E. coli (STEC) O157 is a zoonotic pathogen. In humans STEC O157 causes bloody diarrhoea and potentially fatal renal failure. Cattle are the major reservoir, where bacteria are limited to the intestinal tract and do not cause clinical signs of disease. Previous studies indicate that shiga toxins produced by STEC O157, suppress STEC-specific cellular immune responses in vivo. This study aimed to initially examine the humoral immune response in cattle following natural challenge and the effects of a toxoid vaccination on this humoral STEC specific-immune response. We determined a statistically significant suppression in Tir specific IgA in STEC O157 positive cattle compared to O157 negative cattle but not in super shedding cattle. Following toxoid vaccination we determined a significant increase in flagellin specific IgG1 antibody levels in toxoid vaccinated animals despite lower numbers of positive faecal samples compared to placebo vaccinated controls. These results suggest that shiga toxins produced by STEC O157 are actively suppressing the STEC specific immune response in natural colonisation. To clarify this suppression further calves were orally challenged with STEC O157 (either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain) and their STEC specific immune responses monitored. STEC specific systemic antibody responses were variable and weak in some cases. STEC specific local antibody responses were only significantly increased following challenge with the PT21/28 Stx2a+Stx2c+ challenge. Transcripts for genes associated with immune responses, and in particular B cell activation, at the terminal rectum were analysed by reverse transcriptase quantitative PCR. Suppression of IL2RA transcripts was observed in calves challenged with PT21/28 Stx2a+Stx2c+ compared to control calves but not with the other two STEC O157 strains tested. This study also aimed to determine the effects of cattle colonisation with STEC O157 on the immune response to a non-bacterial T-cell dependent antigen, ovalbumin (OVA). Cattle were orally challenged with either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain or unchallenged. Calves were subcutaneously immunised with OVA five days post challenge, on two separate occasions with a two week interval. Lymphocytes from lymph nodes local to the immunisation site demonstrated significantly increased OVA-specific proliferation and OVA-specific activation of CD4+ and CD8+ cells in calves that were challenged with the PT21/28 Stx2c+ strain (but not with the other two challenge strains), compared to unchallenged controls. These results indicate that colonisation with STEC O157 can alter local adaptive immune responses to non-bacterial antigens in a strain dependent manner, unexpectedly enhancing the immune response rather than suppressing it. Circulating T cell responses were unaffected. In conclusion this study provides some further evidence of adaption of the host immune response by STEC O157, which is strain dependent, and variable. It seems unlikely from the data in this study that STEC O157 colonisation is having a major impact on the responses of cattle to other vaccines or infections in the field.

Anticorpos recombinantes vêm sendo utilizados como ferramenta diagnóstica por serem produzidos com baixo custo e em larga escala. Partiu-se de um gene sintético que codifica um fragmento de anticorpo (scFv) específico contra a toxina termoestável, com otimização de códons para expressão em Escherichia coli. Esse gene foi amplificado no vetor de clonagem e subclonado em vetor de expressão pET28a. Células E. coli BL21(DE3) foram transformadas com o plasmídeo recombinante e induzidas em meio de expressão. O fragmento de anticorpo obtido estava contido na fração insolúvel, portanto foi submetido a purificado por cromatografia de afinidade ao níquel na presença de ureia, seguido de renaturação. A molécula se apresentou funcional e sem reatividade com inespecífica por ensaios de imunofluorescência e ELISA. Além disso, mostrou-se estável quando armazenada a 4ºC, sendo assim uma ferramenta promissora para ser utilizada no diagnóstico de ETEC para detecção da toxina ST. / Recombinant antibodies have been used as diagnostic tools since they can be produced at low cost and on a large scale. A synthetic gene encoding an antibody fragment (scFv) specific for the heat-stable toxin (ST) with optimized codon for Escherichia coli expression was employed. This gene was amplified in the cloning vector and subcloned into pET28a expression vector. E. coli BL21(DE3) cells were transformed with the recombinant plasmid and induced. Large amounts of antibody fragment were found in the insoluble fraction. Thus it is submitted to nickel-affinity chromatography in urea presence, followed by refolding step. By immunofluorescence assay and ELISA, the obtained antibody showed to be functional with no cross-reaction to the negative controls. Furthermore, it was stable when stored at 4 °C, therefore a promising tool for ETEC diagnosis detecting the ST toxin.

The release of harmful chemicals into the Earth system is a threat to the survival of human societies and ecosystems. There are different types of harmful chemicals, some possess characteristics that are more harmful than others. Chemicals causing local effects have a short-term impact on the Earth system, whereas others are persistent in nature. The second becomes a global pollution issue because these substances accumulate, resulting in cascades through the Earth system. The chemical pollution issue is a scale challenge, meaning that the pollution issue occurs within and across levels and scales. Chemical pollution is not treated as a scale challenge in the decision-making process today. In the literature it is stated that chemical pollution is usually studied by focusing on single institutions, which hinders the possibility to investigate the linkages between the institutions affecting the scale dynamics. In this study the scale and cross-scale perspective was used to identify institutions that are affecting the work for a toxin free environment on a national and municipality level in Sweden. Governance Architecture was used as an analytical tool to investigate the key hindering and facilitating governance mechanisms to achieve sustainable chemical management in Sweden from a cross-scale perspective. The analytical tool was based on the Earth System Governance theory and scale dynamics. Sweden was used as a case to apply this tool. Two methods were used to gather empirics: literature analysis and in-depth interviews. The findings reveal an overrepresentation of the hindering factors being a result of a scale or a policy gap issue. These gaps are created by the lack of interdisciplinarity in the decision-making process, and the current legislation and politics which influences the implementation of the strategies on the different institutional levels. To overcome these hinders the current strategies and legislation needs to be in collaboration. This study contributes to empiric development within policy studies on how the chemical pollution issue needs to be conceptually formulated to achieve a toxin free environment within and cross-scales. It also contributes to method development by addressing the gap of including several institutions in cross-scale studies.

Mouse monoclonal antibodies directed against Pichinde virus (PCV) were produced to evaluate their application as vehicles for the delivery of antiviral drugs or toxins to virus-infected cells. Four monoclonal antibodies, PC4.9A6, PC4.9D3, PC4.7C2, and PC4.8D3, were of the IgG2a subisotype and reacted with acetone-fixed and live PCV-infected Vero-76 cells. In vivo stained splenic macrophages derived from PCV-infected hamsters that had been injected with fluorescein-labeled PC4.9A6 (FITC9A6) demonstrated a 400% increase in total fluorescence over similarly treated, non-infected cells when analyzed by flow cytometry. This is an indication that FITC-9A6 does have some ability to specifically target PCV infected cells in vivo. Radioimmunoprecipitation of viral proteins showed that all the antibodies precipitated two different PCV proteins, one of 64,000 daltons and another of 38,000 daltons. These proteins are, respectively, PCV ix nucleoprotein (NP) and a breakdown product of NP that is present in PCV infected cells. An immunofluorescent assay (IFA) for PCV was developed. This IFA was used for antiviral drug assays against PCV. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to fixed, virus infected cells at 24 h after infection and counting the fluorescent cells. The 50% effective dose (EDso) for ribavirin against PCV using this IFA was 6.0 IJ. g/ml. The EDso of ribavirin using inhibition of marginal PCV cytopathogenic effect after 12 days was 6.0 IJ. g/ml and using plaque reduction after 5 days is 2.5 IJ. g/ml, indicating that this IFA was of comparable sensitivity. An immunotoxin (IT) was produced by the conjugation of gelonin to PC4.9A6. This IT was tested in vivo in PCV-infected MHA hamsters. It was not active against the disease at the dosage tested and by the intraperitoneal (i.p.) treatment route employed in this study. The positive control, ribavirin, administered i.p. for 14 days at a dosage of 40 mg/ ml significantly increased the number of survivors. Three of 5 IT toxicity control animals developed some humoral response that inhibited PC4.9A6 binding to infected cells. They did not show any humoral response to the gelonin moiety of the IT.

Resorcinarenes can be functionalized at their upper and lower rims. In this work, the upper rim of a resorcinarene was functionalized with glutamic acids and the lower rim was functionalized with either methyl or undecyl alkyl groups. The cavitands were characterized by nuclear magnetic resonance (NMR), mass spectrometry (MS), UV-vis spectroscopy, dynamic light scattering (DLS) and electron microscopy. The binding of resorcinarene with amine guests was studied in DMSO by UV-vis titration. The obtained binding constants (K values) were in the range of 12,000-136000 M-1. The resorcinarenes were shown to form aggregates in a variety of solvents. The aggregates were spherical as confirmed by DLS, SEM and TEM experiments. Dynamic light scattering (DLS) experiments revealed the size of the aggregates could be controlled by cavitand concentration, pH, and temperature. The resorcinarene with undecyl alkyl group were adsorbed onto 55% cross-linked styrene-divinylbenzene resin to prepare a new stationary phases for ion chromatography (IC) columns. The new column packing material was applied in determination of uremic toxins and water contaminants. The new IC column afforded separation of the five uremic toxins : guanidinoacetic acid, guanidine, methylguanidine, creatinine, and guanidinobenzoic acid in 30 minutes. Detection and quantification of uremic toxins helps diagnose kidney problems and start patient care. Gradient elutions at ambient temperature with methanesulfonic acid (MSA) as eluent resulted in detection levels in water from 10 to 47 ppb and in synthetic urine from 28 to 180 ppb. Trace levels of creatinine (1 ppt) were detected in the urine of a healthy individual using the columns. The new IC stationary phase separated cationic pharmaceuticals including a group of guanidine compounds in surface water. Detection limits in the range of 5 - 32 µg L-1 were achieved using integrated pulsed amperometric detection (IPAD) for guanidine (G), methylguanidine (MG), 1,1-dimethylbiguanidine (DMG), agmatine (AGM), guanidinobenzoic acid (GBA) and cimetidine (CIM). Suppressed conductivity (CD) and UV-vis detection resulted in limits of detection similar to IPAD, in the range of 1.7 - 66 µg L-1, but were not able to detect all of the analytes. Three water sources, river, lake, and marsh, were analyzed and despite matrix effects, sensitivity for guanidine compounds was in the 100 µg L-1 range and apparent recoveries were 80-96 %. The peak area precision was 0.01 - 2.89% for IPAD, CD and UV-vis detection.