L‟eutrophisation croissante des écosystèmes aquatiques favorise le développement des cyanobactéries, parmi lesquelles Microcystis est la plus représentée dans les régions tempérées. La capacité de Microcystis à produire une puissante hépatotoxine, la microcystine, est à l‟origine de diverses perturbations écologiques, et de nombreuses nuisances sanitaires. La compréhension des facteurs déterminant la toxicité des efflorescences de Microcystis constitue, de fait, un enjeu majeur des recherches actuelles. Dans ce contexte, l‟objectif premier de ce travail de thèse était d‟étudier la variabilité temporelle et l‟implication potentielle de la toxicité de Microcystis à l‟échelle de son cycle de développement annuel. Pour cela, il était nécessaire de considérer, en particulier, les parties les moins connues du cycle de développement : la phase de survie benthique, et les transitions entre les phases benthique et planctonique, via les processus de recrutement et de sédimentation. Nous avons alors étudié le potentiel toxique des populations de Microcystis grâce à des approches complémentaires menées à différentes échelles spatio-temporelles, en considérant à la fois les gènes impliqués dans la synthèse de microcystines, leur transcription et les concentrations en microcystines. Cette étude s‟est appuyée, en parallèle, sur la caractérisation de la structure génétique des populations de Microcystis dans les compartiments benthique et planctonique. La prise en compte systématique de la phase de vie benthique a tout d‟abord permis d‟améliorer nos connaissances sur cette phase du cycle de développement de Microcystis. Ainsi, Microcystis peut survivre plusieurs années en profondeur dans les sédiments, sans que les populations ne perdent leur potentiel toxique, ou que leur structure génétique soit altérée. En revanche, en surface des sédiments, le potentiel toxique et la structure génétique des populations sont variables, de manière similaire à ce qui peut être observé dans la colonne d‟eau. Enfin, ces travaux ont également mis en évidence l‟influence des phases de transition entre l‟eau et les sédiments dans la variabilité du potentiel toxique et de la structure génétique des populations de Microcystis. Les processus de recrutement benthique et de sédimentation occasionnent, en effet, une sélection génétique, qui, bien que paraissant indépendante du potentiel toxique des génotypes, peut grandement affecter le potentiel toxique des sous-populations benthiques et planctoniques de Microcystis. / The increasing eutrophication of aquatic ecosystems promotes the development of cyanobacteria, among which Microcystis is the most widespread in temperate regions. The ability of this cyanobacterium to produce a potent hepatotoxin, called the microcystin, represent a serious threat for both natural life and human health. Thus, understanding the factors determining the toxicity of Microcystis blooms is a major challenge of actual research. In this context, the main goal of this work was to study the temporal variability and the potential implication of Microcystis toxicity, at the scale of its annual life cycle. For that, it was necessary to consider more particularly, the least known parts of the cycle : the benthic survival phase, and the transition between the benthic and the planktonic phases, through the benthic recruitment and the sedimentation processes. Then, we studied the toxic potential of Microcystis populations through complementary approaches conducted at different spatio-temporal scales, by considering the genes controlling the synthesis of the microcystin, their transcription and the concentrations of microcystin. In parallel, the genetic structure of Microcystis populations was characterized in both benthic and planktonic compartments. By considering systematically the benthic life stage, we were first able to improve our knowledge on this phase of Microcystis development cycle. Thus, Microcystis is able to survive several years in deep sediments, without the population‟s toxic potential or genetic structure being degraded. On the other hand, at the sediment surface, the toxic potential and the genetic structure of the populations vary, in a similar range to what observed in the water column. Furthermore, this work also shed the light on the influence of benthic-pelagic transitions in the variability of the genetic structure and the toxic potential of the populations of Microcystis. Indeed, a genetic selection occurs during the benthic recruitment and the sedimentation processes. Although such a selection does not seem to rely on the toxic potential of the genotypes, it can greatly modify the toxic potential of both benthic and planktonic sub-populations of Microcystis.

Orientadores: Arnaldo Yoshiteru Kuaye, Raquel Monteiro Cordeiro de Azevedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T01:42:08Z (GMT). No. of bitstreams: 1 Soares_CelinaMara_D.pdf: 3203111 bytes, checksum: 0aa846a0bcf7bb5eb0b55f95d9a7f9ff (MD5) Previous issue date: 2005 / Resumo: Amostras ambientais e de alimentos foram coletadas em dois restaurantes institucionais da cidade de Campinas/SP para a investigação das fontes de contaminação por Bacillus cereus e caracterização do perfil enterotoxigênico dos isolados. A pesquisa foi realizada em três etapas: i) análise da contaminação ambiental geral; ii) diagnóstico da contaminação durante processamentos de pratos cárneos e; iii) estudo do comportamento do microrganismo em substrato cárneo mantido a 10 e 30°C. A avaliação da contaminação ambiental geral e durante os processamentos revelou a presença do microrganismo em 80 (74,8%) das 107 amostras de ar ambiente (2,0 a 38,4 UFC/m³) e em 58 (46,4%) das 125 amostras de superfícies de bancadas e de equipamentos analisadas. O microrganismo também foi identificado em amostras de carne processada (10² UFC/g a 2,4x10³ UFC/g) e de condimentos e temperos (1,0x10 a 5,0x10² UFC/g) coletadas durante os processamentos. A produção de enterotoxinas foi investigada através da técnica da reação da polimerase em cadeia (PCR) para os genes hblA, hblD e hblC (codificadores da "hemolisina BL" - HBL) e para os genes nheA, nheB e nheC (codificadores da "enterotoxina não hemolítica" - NHE). Do total de 124 isolados, 117 (94,3%) foram positivos para ao menos um dos genes pesquisados; 18 (14,5%) foram positivos para os três genes codificadores da HBL; 25 (20,2%) foram positivos para os três genes codificadores da NHE; e 7 (5,6%) foram positivos para todos os genes. Um teste imunoenzimático de detecção da NHE (kit BDE-VIA: Bacillus Diarrhoeal Enterotoxin Visual Immunoassay; Tecra) também foi utilizado. Os resultados obtidos com o BDE-VIA revelaram que 89 (71,8%) dos 124 isolados são produtores de NHE. B. cereus apresentou rápido crescimento em substrato cárneo mantido a 30°C, com tempo de geração entre 28,8 e 36,0 min, indicando que a carne exposta a temperaturas abusivas é propícia ao desenvolvimento do microrganismo. A 10°C, o tempo de geração oscilou entre 10,16 e 28,38 h. A presença de cepas potencialmente enterotoxigênicas observadas em amostras ambientais atestam a importância do ambiente como fonte de contaminação pelo microrganismo. Os resultados da pesquisa permitem concluir que, tanto o controle do microrganismo no ambiente de serviços de alimentação como o adequado processamento dos alimentos, são fundamentais para a redução do risco de doenças de origem alimentar associadas a B. cereus / Abstract: Environmental and food samples were collected in two food services in the city of Campinas/SP for investigation of Bacillus cereus contamination sources and characterization of strains toxin profiles. The research has been made in three steps: i) analysis of general environmental contamination; ii) diagnosis of contamination during processing cooked meat; and iii) study of microorganism behavior in meat substrate stored at inappropriate temperatures. The results indicated the presence of microorganism in 80 (74.8%) of the 107 environmental air samples (2.0 to 38.4 CFU/m³) and in 58 (46.4%) of the 125 samples of equipments and benches surfaces. The microorganism also was identified in cooked meat samples (10² CFU/g to 2.4x10³ CFU/g) and in spices samples (1.0x10 CFU/g to 5.0x10² CFU/g), collected during the processing. The potential of enterotoxin production was investigated using polymerase chain reaction (PCR) methods for genes hblA, hblD e hblC (encoding hemolysin BL) and for genes nheA, nheB and nheC (encoding non-hemolytic enterotoxin - NHE). From all the 124 strains, 117 (94.3%) were positive for at least one gene; 16 (12.9%) were positive for the three HBL encoding genes; 25 (20.2%) were positive for the three NHE encoding genes; and 7 (5.6%) were positive for all genes. The Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDE-VIA; Tecra) also was used for NHE detection. The results obtained with BDE-VIA revealed that 89 (71.8%) from the 124 strains are NHE producers. B. cereus cells showed fast growth in meat substrate stored at 30°C with generation time between 28.8 and 36.0 min, indicating that meat stored at inadequate temperatures is propitious to the microorganism development. At 10°C, the generation time varied between 10.16 and 28.38 h. The presence of potentially enterotoxigenic strains in the environmental samples confirm the importance of environment as a source of microorganism contamination. The results allow to conclude that either the control of microorganism in food services environment either the proper food processing are fundamental to reduce the risks of foodborne diseases associated to B. cereus / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos

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.

O Monometoxi-polietilenoglicol succinimidil ácido propiônico (mPEG-SPA 5 e 20 kDa) foi analisado como adjuvante e inibidor da atividade neurotóxica da toxina tetânica (TxT) adsorvida ou não em Al(OH)3, à qual o polímero foi conjugado. Avaliou-se a toxicidade das amostras por DL50, demonstrando que a atividade neurotóxica da TxT foi inibida. A via subcutânea foi mais efetiva na indução de resposta à TxT tratada pelo mPEG-SPA e o efeito adjuvante do Al(OH)3 se deu pela intramuscular. Trinta cavalos foram submetidos a esquema de imunização seletiva, dividindo-se os dezoito escolhidos em grupos para imunização com TxT conjugada ao mPEG-SPA 5.000 e 5.000(2X) e TxT adsorvida ou não. Os soros dos cavalos foram analisados por ToBI Teste, que avaliou a evolução da resposta imune. Os soros também foram analisados por imunodifusão, eletroforese e immunoblotting, tendo este indicado uma provável superioridade antigênica da TxT Fluida relativamente aos adjuvantes. A conjugação mPEG-SPA provou ser efetiva na produção do soro antitetânico terapêutico para uso humano. / Monometoxypolyethylene glycol succinimidyl propionic acid (SPA-mPEG 5 and 20 kDa) was analyzed as adjuvant and inhibitor of tetanus toxin neurotoxic activity (TxT) adsorbed or not by Al(OH)3, to which the polymer was conjugated. The samples toxicity was evaluated by DL50, disclosing that TxT neurotoxic activity was inhibited. The subcutaneous inoculation was more effective in induction of response to TxT treated with SPA-mPEG and the adjuvant effect of Al(OH)3 was evidenced by the intramuscular. Thirty horses were submitted to a selective scheme of immunization and eighteen were divided in groups to be immunized with TxT conjugated to SPA-mPEG 5,000 and 5,000(2X) and TxT adsorbed or not. The horses sera were analysed by ToBI Test, which evaluated the immune response development. The sera were also analysed through immunodifusion, electrophoresis and immunoblotting and the last one indicates a probable antigenic superiority of TxT fluid relatively to the adjuvants. The SPA-mPEG conjugation proved to be effective for anti-tetanus human therapeutic serum production.

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.

O veneno produzido pela aranha Phoneutria nigriventer causa priapismo. O pré-tratamento de camundongos com inibidores da Óxido nítrico-sintase, antes de se injetar a toxina, inibe este priapismo. A toxina causa ativação do gene c-fos no núcleo paraventricular do hipotálamo (NPV). Este estudo tem como objetivo ampliar os conhecimentos relacionados ao mecanismo da ereção peniana causada pela toxina Tx2-6. A toxina foi injetada no NPV e os resultados não mostraram o envolvimento direto do mesmo com o priapismo. Procuramos então avaliar se há aumento na produção de óxido nítrico (NO) quando o tecido é submetido à TX2-6 e o resultado foi negativo. Foram feitos também experimentos com animais que sofreram ablação cirúrgica bilateral dos nervos cavernosos. Estes, quando injetados com a toxina, tiveram ereção peniana, porém não apresentaram exposição total do pênis, possivelmente devido à fibrose causada pela denervação. Estes resultados nos levam a crer que a ereção causada pela toxina Tx2-6 pode não ser mediada pela produção de NO e não depende da inervação peniana. / The venom produced by Phoneutria nigriventer spider causes priapism. Mice pretreatment with Nitric oxide-synthase inhibitors, before injecting the toxin, inhibits the priapism. The toxin causes c-fos gene activation in paraventricular nucleus of hypothalamus (PVN). This study aims to widen some knowledge concerning penile erection mechanism caused by Tx2-6 toxin. The toxin was injected in the PVN and the results didnt show its direct relation with the priapism. We analyzed if there is any increase of nitric oxide (NO) production when the tissue is submitted to TX2-6 and the result was negative. Experiments were carried out on mice which suffered bilateral surgical ablation of the cavernosum nerves. When these animals were injected with the Tx2-6 toxin, there was penile erection. However, the erection of the denervation group didnt present total penis exposition possibly due to the fibrosis caused by the denervation. These results reveal that erection caused by Tx2-6 toxin might not be mediated by NO production and it does not depend on penile nerves.

O urânio (U) é um elemento tóxico radioativo encontrado na natureza, normalmente presente na água e nos alimentos e acumula-se preferencialmente em ossos. Nestes, a medula óssea constitui o alvo com o maior risco radiobiológico. Foram utilizados 60 ratos wistar recém desmamados, com vinte e dois dias de vida. Destes, trinta e cinco foram tratados com ração suplementada de 50ppm (parte por milhão) de Nitrato de Uranila e vinte e cinco foram mantidos como controle. Os animais tratados foram separados em seis grupos com cinco animais cada e os grupos controle com três animais. Foi feita a eutanásia dos 5 animais de cada grupo alimentado com urânio e 3 animais de cada grupo de controle com intervalo de tempo de 3 e 4 dias para avaliar alterações histopatológicas, hematológicas, na densidade mineral óssea e medir o teor de urânio acumulado em ossos, em função do tempo, utilizando a técnica de registro de traços de fissão SSNTD (Solid State Nuclear Track Detector). Nas avaliações histopatológicas foi observada congestão, fibrose e necrose hepática, degeneração vacuolar e desarranjo cordonal dos hepatócitos. Essas alterações iniciaram-se em animais alimentados durante três dias com ração contendo U e se intensificaram nos animais tratados durante onze dias, sugerindo que tenha ocorrido combinação de efeitos toxicológicos e radiobiológicos. Foi observada degeneração vacuolar, cilindros hialinos, fibrose e necrose nos rins dos animais alimentados com ração suplementada de U, a partir de quatorze dias de alimentação, decorrentes da nefrotoxicidade do Nitrato de Uranila. Foi observado que não ocorre alteração da densidade mineral óssea no curto prazo; porém, os animais tratados durante 21 e 28 dias, ou seja, expostos ao U por período mais longo, tiveram a densidade mineral óssea diminuída. Ocorreu substancial acúmulo de urânio nos ossos, onde foi observado 1,139 ± 0,057 ppm em ossos e 0,705 +- 0,092 ppm em dentes. Os animais dos grupos controle apresentaram teor de urânio praticamente constante no decorrer do estudo. Não foi observada alteração do teor de urânio em ração comercial. / Uranium (U) is a radioactive toxic element found in the environment, naturally present in water and food, with preference for accumulation in bone. In the latter, marrow is the target with the highest radiobiological risk. It was carried out a study with sixty Wistar rats, twenty two days old, starting at the post weaning period. From this total, thirty five animals fed with chow containing Uranyl Nitrate at a concentration of 50 ppm (parts per million) were selected as the treated group, while the remaining twenty five were the control group. Treated animals were divided into six groups with five animals each plus six control groups with three animals each. Five animals of the treated group and three of the control group were sacrificed at intervals of four days to observe histopathologic, hematologic, and bone mineral density (BMD) alterations, as well as to measure the uranium content in bone as function of time, using the Solid State Nuclear Track Detector technique. It was observed congestion, vacuolar degeneration, hepatocytes misalignment, fibrosis and necrosis in liver. These alterations were initiated in treated animals fed for three days with diets containing U and intensified in the animals treated for eleven days, suggesting the occurrence of an intertwining between radiobiological and toxicological effects. It was also observed vacuolar degeneration, hyaline cylinders, fibrosis and necrosis in the kidneys of the treated animals, all initiated after fourteen days of treatment, and these effects were attributed to the nephrotoxic character of the Uranyl Nitrate. It was found out that the BMD was not altered in the short range term of treatment, that is, treatments of twenty-one and twenty-eight days, but appreciably reduced in the long range term. There was substantial accumulation of uranium in bones and teeth, where it was measured concentrations of 1.139 ± 0.057 ppm and 0.705 ± 0.092 ppm, respectively. The uranium concentration in the bones of animals of the control group were low and approximately constant.

Notre système immunitaire détecte les microorganismes via des molécules absentes de l’hôte appelées MAMPs. Mais étant donné que les MAMPs sont exprimés par tous les microorganismes indépendamment de leur potentiel pathogène, ce mécanisme n’explique pas comment le système immunitaire distingue les microorganismes pathogènes des non-pathogènes. De récents travaux ont mis en évidence un mécanisme de détection de l’activité des facteurs de virulence bactériens. En utilisant la drosophile, notre laboratoire a précédemment démontré que l’activation de la Rho GTPase Rac2 par la toxine CNF1 d’Escherichia coli induisait une réponse immunitaire innée conservée au cours de l’évolution chez le mammifère et similaire à l’immunité induite par les effecteurs chez la plante. Par la suite, nous avons évalué l’importance de cette réponse immunitaire au cours de la bactériémie chez la souris et démontré le rôle central de la cytokine IL-1β dans l’élimination des bactéries en réponse à la détection de CNF1. Des expériences in vitro nous ont permis d’identifier les mécanismes moléculaires mis en jeu et l’inflammasome responsable de l’activation de la caspase-1 et du clivage de l’IL-1β. De manière intéressante, CNF1 est toujours co-exprimée avec la toxine hémolysine-α (HlyA) dans les souches pathogènes d’E. coli. En outre, nous avons découvert que l’HlyA bloquait l’élimination des bactéries induite par CNF1 au cours de la bactériémie et inhibait la sécrétion de l’IL-1β. Ici, nous avons rapporté le premier exemple d’immunité induite par une toxine (CNF1) et contrecarrée par une autre (HlyA). / Our immune system detects microorganisms via molecules absent from the host called MAMPs. Since MAMPs are shared by all microorganisms regardless of their pathogenic potential, this mechanism does not explain how the immune system distinguishes between pathogenic and non pathogenic microorganisms. The detection of the activities of pathogen-encoded virulence factors has emerged as a new paradigm of pathogen recognition. Using Drosophila we previously demonstrated that the Escherichia coli CNF1 toxin-induced activation of the Rho GTPase Rac2 is sufficient to initiate a defense signal evolutionarily conserved from flies to mammals and similar to Effector-Triggered Immunity in plants. We further addressed the importance of this innate immune mechanism during bacteremia in mice and demonstrated the central role of the IL-1β cytokine in the clearance of bacteria in response to the detection of CNF1. In vitro experiments allowed us to identify the involved molecular mechanisms and the inflammasome responsible of caspase-1 activation and IL-1β maturation. Interestingly, CNF1 is always co-expressed with α-hemolysin toxin in pathogenic E. coli. In addition, we found that HlyA blocked the elimination of bacteria induced by CNF1 during bacteremia and inhibited the secretion of IL-1β. Here, we have reported the first example of immunity induced by a toxin (CNF1) and counteracted by another (HlyA).

Des produits efficaces pour stimuler le système immunitaire auraient de très nombreuses applications dans le domaine de la santé publique : lutte contre la résistance aux antibiotiques, épidémies, protection des personnels de secours et des populations en cas de crise sanitaire ou d’attentat biologique. Aujourd’hui, les rares produits immunostimulants efficaces sont coûteux, dangereux, et donc réservés au traitement de pathologies graves ciblées comme les cancers, aplasies ou infections virales chroniques. La petite protéine G Rac1 fut identifiée comme une potentielle cible thérapeutique. Activée par de nombreux pathogènes, elle contrôle l’inflammation et la mise en place des défenses de l’hôte. CNF1 est une toxine bactérienne activant fortement Rac1, ce qui conduit à sa dégradation protéasomale. Le maintien d’un pool de Rac1 activé permettrait d’augmenter les défenses immunitaires. En utilisant CNF1 comme un outil permettant de diminuer celui-ci, un test cellulaire fut mis au point pour cribler 17 680 composés. A l’aide de différents paramètres de tri, un ensemble de molécules fut identifié comme capable de prévenir la dégradation de Rac1 médiée par CNF1.De manière inattendue, la plupart des composés semblent posséder un effet anti-inflammatoire, en baissant la sécrétion de cytokines de cellules stimulées. Le potentiel thérapeutique et en recherche de tels composés doit être évalué. En parallèle, deux autres composés montrent une protection anti-toxines large spectre (CNF1, toxine diphtérique, toxine de Shiga, toxine B de C. difficile). De même, leur mécanisme d’action, encore inconnu, pourrait permettre le traitement d’infections variées. / Immune system boosters could have many applications in public health: fighting antibiotics resistance, epidemics, protection of health care staff and populations during health crisis or biological warfare. Currently, the rare efficient immune stimulants are costly and dangerous; hence, they are earmarked to severe and targeted pathologies such as cancers, aplasia, or chronic viral infections. The small G protein Rac1 was identified as a potential therapeutic target. Once activated by various pathogens, it controls inflammation and host defenses establishment. CNF1 is a bacterial toxin that strongly activating Rac1, leading to its proteasomal degradation. Maintaining an activated Rac1 pool could enhance immune defenses. By using CNF1 as a tool to reduce it, a cellular bioassay was developed and optimized to screen 17 680 compounds. Through various filters, a group of molecules was identified to prevent CNF1-mediated Rac1 depletion. Unexpectedly, most of them seems to possess anti-inflammatory properties, down-regulating cytokines production from stimulated cells. The therapeutic potential of such compounds must be now evaluated. In parallel, two other molecules show a broad-spectrum anti-toxins protection (CNF1, diphtheria toxin, Shiga toxin, toxin B from C. difficile). Their unknown mode of action may allow the treatment of various infections.

<p>Potreba za potpunijom informacijom o važnosti kontaminacije useva Alternaria<br />toksinima su nedavno dobili na značaju. Imajući u vidu toksičnost metabolita koje<br />produkuju pojedine vrste gljive iz roda Alternaria u sistemu od njive do trpeze uticalo<br />je na to da se u sklopu istraživanja u oblasti mikotoksikologije registruje porast<br />interesovanja za Alternaria toksine i gljive koje ih produkuju. S obzirom na činjenicu<br />da sa na&scaron;ih prostora nema dostupnih podataka o prirodnoj kontaminaciji p&scaron;enice sa<br />Alternaria toksinima, a o pona&scaron;anju Alternaria toksina tokom procesa prerade p&scaron;enice nema dostupnih podataka u naučnoj literaturi, proizi&scaron;li su ciljevi ove doktorske disertacije. Osnovni cilj planiranih istraživanja u ovoj doktorskoj disertaciji je bio sagledavanje uticaja konvencionalno primenjenih mehaničkih (či&scaron;ćenje i suvo<br />mlevenje) i hidrotermičkih (ekstrudiranje) tehnolo&scaron;kih postupaka na redukciju<br />prisutnih Alternaria toksina u p&scaron;enici. Primenom postupaka či&scaron;ćenja p&scaron;enice<br />uobičajenih u praksi rada silosne i mlinske čistione postiže se značajna redukcija<br />prisustva svih ispitivanih Alternaria toksina u zrnenoj masi.U postupku mlinskeprerade p&scaron;enice, čak i u slučaju prerade p&scaron;enice u kojoj je sadržaj pojedinih Alternaria toksina ispod granice detekcije u frakcijama mlevenja p&scaron;enice u koje dospevaju periferni delovi p&scaron;eničnog zrna, odnosno kasnijim prolazi&scaron;tima krupljenja i mlevanja, kao i sporednim proizvodima meljave, može se očekivati povi&scaron;en sadržaj Alternaria toksina. Optimalni efekti procesa ektrudiranja u slučaju kori&scaron;ćenja jednopužnog ekstrudera ostvaruju se u slučaju visoke vlage polaznog materijala (w=24%) velike brzine protoka materijala (q=25 kg/h) i srednje brzine obrtanja puža ekstrudera (v=390 obrtaja/min) pri čemu se postiže redukcija TeA=60-65 %, AOH=90 % i AME=95 %. Dodatni cilj je bio da se sagleda učestalost pojave Alternaria toksina, i stekne preliminarni uvid u pojavu i opseg koncentracije Alternaria toksina u p&scaron;enici sa teritorije Vojvodine sa ciljem da se dobiju preliminarni podaci o uticaju proizvodne<br />godine, lokaliteta i najkarakterističnijih klimatskih uslova na pojavu Alternaria toksina<br />u p&scaron;enici, kao i da se sagleda uticaj uslova introdukovane stimulacije i inhibicije<br />kontaminacije p&scaron;enice sa Alternaria spp. na sadržaj Alternaria toksina u p&scaron;enici. Analiza učestalosti incidencije Alternaria toksina u p&scaron;enici sa područja Vojvodine ukazuju na evidentnu pojavu značajnog broja partija p&scaron;enice kontaminirane sa TeA, praćenu sporadičnom pojavom partija kontaminiranih sa AOH i AME. Kako prosečne, tako i maksimalne koncetracije TeA u p&scaron;enici iz Vojvodine proizvedene u trogodi&scaron;njem periodu od 2011. do 2013. godine bile su vi&scaron;e od zvanično objavljenih vi&scaron;egodi&scaron;njih podataka od strane Evropske agencije za bezbednost hrane (EFSA). Utvr&ntilde;ene koncentracije AME i AOH u p&scaron;enici iz Vojvodine su daleko niže od koncentracije TeA kao najče&scaron;ćeg kontaminenta iz grupe Alternaria toksina. Preliminarno ima indicija da se najvi&scaron;e, ekstremne koncentracije ispitivanih Alternaria toksina u p&scaron;enici iz pojedinih proizvodnih godina i iz najugroženijih regiona mogu povezati sa vi&scaron;im prosečnim dnevnim temperaturama sa jedne i povećanom sumom padavina u maju i junu sa druge strane u odnosu na dugogodi&scaron;nji prosek (1981-2010). Inokulacija p&scaron;enica sa Alternaria tenuissima dovodi po povećanja koncentracije TeA u zrnenoj masi u odnosu na netretiranu p&scaron;enicu, dok tretman iste p&scaron;enice fungicidom utiče na smanjenje prisutva ovog Alternaria toksina &scaron;to ukazuje da infekcija prodire u zrno u većem obimu kod inokulisanih uzoraka p&scaron;enice, dok je u netretiranim i za&scaron;tićenim manje izražena. Koncetracija Alternaria toksina u plevi p&scaron;enice je značajno vi&scaron;a u odnosu na zrnenu masu ovr&scaron;ene p&scaron;enice &scaron;to ukazuje na izvesnu za&scaron;titnu funkciju pleve od prodiranja Alternaria toksina u unutra&scaron;nji deo endosperma zrna p&scaron;enice. Krajnji cilj istraživanja je bio da se na osnovu dobijenih podataka o efikasnosti primenjenih tehnolo&scaron;kih postupaka u pogledu redukcije Alternaria toksina u p&scaron;enici i podataka u učestalosti i uzrocima pojave Alternaria toksina u p&scaron;enici da okvirna procena mogućnosti smanjenja rizika od pojave visokih udela Alternaria toksina u proizvodima prerade p&scaron;enice na trži&scaron;tu. U lancu proizvodnje, prometa, skladi&scaron;tenja i prerade p&scaron;enice kao proizvodi sa niskim rizikom u pogledu pojave i koncentracije Alternaria toksina mogu se istaći bela bra&scaron;na i hidrotermički obra&ntilde;eni proizvodi, kao &scaron;to su naprimer ekstrudirani proizvodi od p&scaron;enice. Kao proizvodi visokog rizika u pogledu pojave i koncentracije Alternaria toksina ističu se pleva dobijena tokom žetve p&scaron;enice, nečistoće dobijene u procesu silosnog postupka či&scaron;ćenja p&scaron;enice, tamne frakcije bra&scaron;na dobijene u postupku mlevenja p&scaron;enice i sporedni prozvodi mlevenja p&scaron;enice &ndash; mekinje i osevci.</p> / <p><span style="font-size:11px;">The need for more complete information about the importance of contamination of crops with Alternaria toxins recently gained in importance. The toxicity of metabolites produced by certain species from Alternaria genus in the whole supply chain resulted in the area of mycotoxicological research in increased interest for Alternaria toxins and fungi producing them. Having in mind that there are no available data about natural contamination of wheat with Alternaria toxins in the region of Serbia, and not any data is available in academic literature about the influence of processing procedures on Alternaria toxins in wheat, the aims of this PhD thesis have been defined. The basic aim of this dissertation was to investigate the influence of conventional mechanical (cleaning and dry milling) and hydrothermal (extrusion) technological processes on reduction of Alternaria toxins present in wheat. The application of wheat cleaning processes common in wheat warehouses and mills significant reduction of concentration of all investigated Alternaria toxins is achieved. In wheat milling process, even for wheat with concentration of Alternaria toxins under the limit of detection, in fraction containing peripheral kernel parts, including last break and milling flows and by products, increased concentration of Alternaria toxins can be expected. Optimal effects of extrusion process with application of single screw extruder are achieved in the case of high raw material moisture (w=24%), high capacity (q=25 kg/h) and medium speed of extruder screw rotation (v=390 rpm) when reduction of TeA=60-65%, AOH=90% and AME=95% is achieved. Additional aim was to obtain data about the frequency of Alternaria toxins including occurrence and ranges of concentration of Alternaria toxins in wheat for the region of Vojvodina with the aim to obtain preliminary data about the influence of production season, locality and the most characteristic climatic conditions on occurrence of Alternaria toxins in wheat, as well as to perceive the influence of introduced </span>stimulation and inhibition of wheat contamination with Alternaria spp. on concentration of Alternaria toxins in wheat. Analysis of frequency of occurrence of Alternaria toxins in wheat from the region of Vojvodina points out at evident incidence of significant number of wheat lots contaminated with TeA accompanied with sporadic occurrence of lots contaminated with AOH and AME. Both, average and maximal concentrations of TeA in wheat produced in Vojvodina in the period from 2011 to 2013 were above the official multiannual data published by European Food Safety Agency (EFSA). Concentrations of AME and AOH in wheat from Vojvodina were far under the concentrations of TeA as the most frequent contaminant from the group of Alternaria toxins. Preliminary there are indications that the highest concentrations of analysed Alternaria toxins in wheat from certain production seasons and the most affected production regions can be related with higher average daily temperatures at one, and increased sum of precipitation at the other hand in comparison to multiannual average (1981-2010). Inoculation of wheat with Alternaria tenuissima resulted in increased concentration of TeA in comparison to non-treated wheat, while the treatment of the same wheat with fungicide influences at decrease of the same Alternaria toxins, pointing out that the infection penetrates to higher extent into inoculated wheat while in non-treated and protected wheat this process is less expressed. Concentration of Alternaria toxins in wheat chaff is much higher in comparison to harvested wheat pointing out at certain protective function of chaff in the process of penetration of Alternaria toxins into inner kernel parts. Final aim of the research was to provide the general assessment of possibilities for reduction of risk of occurrence of high Alternaria toxins concentrations in wheat products at the market based on obtained data about efficiency of applied technological processes for reduction of Alternaria toxins in wheat products and obtained data on frequency of occurrence of Alternaria toxins. In the wheat supply chain white flours and hidrothermically processed like extruded wheat products can be emphasized as the products with low risk regarding Alternaria toxins. As the high risk products regarding Alternaria toxins chaff obtained during harvesting, impurities obtained in wheat cleaning processes, dark flours obtained in wheat milling process and wheat milling by-products, shorts and bran, can be emphasized.</p>