Enterohemorrhagic E. coli (EHEC) infection is a leading cause of acute kidney failure in otherwise healthy children, and a leading cause of foodborne illness with an outsized economic impact from outbreaks. EHEC secrete two Shiga-like toxins (Stx1 and Stx2) which are AB5 holotoxins that inhibit protein synthesis in cells expressing the toxin receptor Gb3. Infection with EHEC typically begins with a diarrheal prodrome that can progress in 5-15% of cases to hemolytic uremic syndrome (HUS), a clinical diagnosis characterized by thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy. Historically, strains of EHEC expressing Stx2 have been associated with more severe disease. We hypothesized that tissue injury due to the toxins leads to the release of damage-associated molecular patterns (DAMPs), which act through inflammatory receptors to promote the endothelial dysfunction that drives this disease alongside the inciting Shiga toxins. Here we demonstrate that two well-characterized DAMPs, extracellular histones and HMGB1, are produced in two different mouse models when Stx2 is present; one model represents challenge with the toxin alone, and the second model introduces toxin through secretion with a lysogenized bacterium, C. rodentium, mimicking EHEC colonization. We investigate whether Stx1, Stx2, or histones affect the endothelial expression of well-characterized members of the protein C pathway, namely the endothelial protein C receptor (ECPR), protease-activated receptor 1 (PAR1), and thrombomodulin (TM), on human aortic (HAEC) and human renal glomerular endothelial cells (HRGEC). We show that Stx and/or histones reduce endothelial expression of these anti-coagulant molecules and histones dramatically increase expression of pro-thrombotic PAR-1. These changes lead to physiologically important decreases in activated protein C (APC), a critical anti-coagulant and cytoprotective molecule. Finally, we demonstrate that histones exacerbate thrombin's barrier-disruptive effects on the endothelium, and prevent APC's protective effects. These data provide novel mechanistic insight into the endothelial dysfunction that characterizes HUS and also provide a new perspective on systemic consequences of the bacterial Shiga toxins that might drive organ injury in susceptible patients.

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Microcosms were used in this research to assess the environmental risk associated with two types of stressors: genetically-engineered microorganisms (GEMs) and toxic chemicals. Approaches used to evaluate the potential environmental impact of these two stressors are fundamentally different, and arise mainly from the fact that the GEMs are living and capable of replication. Risk assessment for genetically-engineered Erwinia carotovora strain L-864 included investigations of its persistence and effect on structure and function of aquatic microcosms. Densities of genetically-engineered and wild type E. carotovora declined at the same rate in water or in sediment, falling in 32 days below the level of detection by viable counts. Selective media, antibiotic resistance, and most probable number analysis were used to enumerate genetically-engineered E. carotovora in environmental samples. This technique was able to detect as few as 1 to 10 target cells/10 g soil. In thermally-perturbed aquatic microcosms, genetically-engineered E. carotovora persisted significantly longer than in unperturbed microcosms, suggesting the vulnerability of stressed ecosystems to colonization by GEMs. Competition study showed that the genetically-engineered E. carotovora did not displace the wild type strain. Effects of genetically-engineered and wild type strain on indigenous bacteria belonging to specific functional groups important in nutrient cycling were similar: inoculation of either strain caused a temporary increase in densities of total and proteolytic bacteria, while it did not affect amylolytic and pectolytic bacteria. Treatment with engineered bacteria did not change biomass values of the receiving community, but caused a transitory increase in its metabolic activity. The inability of genetically-engineered E. carotovora to persist, displace resident species, and affect metabolic activity of a community indicates a low risk of adverse ecological effects in aquatic systems. Microcosms were also used to assess environmental risk for toxic chemicals. A study was conducted to assess the fate of sediment-associated copper and to investigate its effects on the structure and function of the aquatic community. Most of the added copper was bound to sediment particles. In microcosms containing 100 µg Cu/kg sediment, chlorophyll a content and respiration significantly decreased compared to the control. Addition of 1000 µg Cu/kg sediment caused a decrease in production, respiration, respiration/biomass ratio, ATP, and chlorophyll a. The last study compared responses of Simocephalus exspinosus (Daphniidae) to copper during a single-species test to responses of S. exspinosus populations in a microcosm test. Responses of S. exspinosus were similar in both test systems: there was an increased production of young at 30-46 µg Cu/l, while the organisms did not survive exposure to concentrations > 100 µg Cu/l. In these studies, microcosms showed a potential to predict fate and effects of chemical and biological contaminants released into the biosphere. / Ph. D.

Tall fescue (Festuca arundinacea Schreb.) is often infected by the endophytic fungus, Acremonium coenophialum (Morgan-Jones and Gams) The fungus produces ergo peptide alkaloids, especially ergovaline. Consumption of endophyte-infected (ED fescue forage by cattle decreases serum prolactin and average daily weight gains, which results in economic losses for producers. Anecdotal reports suggest cattle with fescue toxicosis may not respond to vaccination. Hyperprolactinemia decreases cell-mediated and humoral immune responses in mice. Therefore, steers grazing EI or endophyte-free (EF) fescue forages were vaccinated to assess humoral immune responses. Steers grazing EI fescue exhibited classical clinical signs of fescue toxicosis, and mounted humoral immune responses to vaccination, despite hyperprolactinemia. Lymphocyte proliferation responses to mitogens in mice fed EI diets were similar to mice fed EF diets. Production of interferon gamma and interleukin-4 was higher by splenocytes from mice fed EI diets, whereas interleukin-2 tended to be lower. Fescue toxicosis may stimulate T helper cell 2 subset of CD4⁺ T cells. The T_H2 subset may augment humoral immune responses to vaccination. / Ph. D.

Pyrenophora tritici-repentis (Ptr) is the necrotrophic fungus responsible for tan spot of wheat (Triticum aestivum). Ptr causes disease on susceptible wheat cultivars through the production and secretion of host-selective toxins (HSTs). HSTs are compounds that are only known to be produced by fungi and considered to be primary determinants of pathogenicity. Infiltration of these toxins into sensitive wheat elicits the same symptoms as the pathogen, which simplifies investigations of host- pathogen interactions due to exclusion of the pathogen. These characteristics make HSTs ideal molecules to dissect molecular plant-microbe interactions. Known HSTs of Ptr include Ptr ToxA (ToxA), Ptr ToxB (ToxB) and Ptr ToxC (ToxC). ToxA is the most characterized toxin of Ptr, as well as the first proteinaceous HST identified. The proposed mode-of-action for ToxA includes internalization into sensitive wheat mesophyll cells, localization to the chloroplast, photosystem perturbations and elicitation of high amounts of reactive oxygen species (ROS), all of which lead to necrosis. However, it is still unknown how ToxA is transported to the chloroplast. To identify additional interacting components involved in ToxA symptom development, genes were silenced in tobacco plants (Nicotiana benthamiana) using the tobacco rattle virus (TRV) virus-induced gene-silencing (VIGS) system. Four genes were identified that potentially could play a role in ToxA-induced cell death: a 40S ribosomal subunit, peroxisomal glycolate oxidase (GOX), a thiamine biosynthetic enzyme (Thi1), and the R-gene mediator, Sgt1. Ptr exhibits a complex race structure determined by the HST(s) produced and the symptom(s) elicited on sensitive wheat cultivars. Currently, there are eight characterized races and other HSTs and races have been proposed. Isolate SO3 was discovered in southern Oregon and elicits ToxA-like symptoms on a wheat differential set, yet lacks the ToxA gene. The transcriptome of SO3 was sequenced, assembled, and aligned to a ToxA-producing isolate, Pt-1C-BFP, which will aid in the identification of the protein(s) that may be responsible for these ToxA-like symptoms. SO3 contains a set of 497 sequences that were not found in the ToxA-producing isolate Pt-1C-BFP (BFP). These sequences should be further investigated to identify those that encode small secreted proteins (SSPs) and could potentially serve as HSTs and pathogenicity factors of SO3. / Graduation date: 2013

Venomous animals have evolved a vast array of peptide toxins for prey capture and defense. Nature has evolved the venoms into a huge library of active molecules with high selectivity and affinity, which could be explored as therapeutics or serve as a template for drug design. The individual components of venom i.e. toxins are used in ion channel and receptor studies, drug discovery, and formulation of insecticides. ‘Venominformatics is a systematic bioinformatics approach in which classified, consolidated and cleaned venom data are stored into repositories and integrated with advanced bioinformatics tools and computational biology for the analysis of structure and function of toxins.’ Conus peptides (conopeptides), the main components of Conus venom, represent a unique arsenal of neuropharmacologically active molecules that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes and neuronal receptors. Ziconotide (ω-conotoxin MVIIa from Conus magus (Magician's cone snail)), is proven as an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain (U.S. Food and Drug Administration. Center for Drug Evaluation and Research) attesting to the pharmaceutical importance of Conus peptides. From the point of view of protein sequence and structure analysis, conopeptides can serve as attractive systems for the studies in sequence comparison, pattern extraction, structure–function correlations, protein–protein interactions and evolutionary analysis. Despite their importance and extensive experimental investigations on them, they have been hardly explored through in silico methods. The present thesis is perhaps the first attempt at deploying a multi-pronged bioinformatics approaches for studies in the burgeoning field of conopeptides. In the process of sequence-structure-function studies of conopeptides, we have created several sequence patterns of different conopeptide families and these have been accepted for inclusion in international databases such as PROSITE, the first pattern database to have been developed (http://www.expasy.org/prosite) and INTERPRO (http://www.ebi.ac.uk/interpro). More importantly, we have carried out extensive literature survey on the peptides for which we have defined the patterns to create PROSITE compatible documentation files (PDOC6004, PDOC60025 and PDOC60027). We have also created a series of sequence patterns and associated documentation filesof pharmaceutically promising peptides from plants and venomous animals (including O-conotoxin and P-conotoxin superfamily members) with knottin scaffold. Knottins provide appealing scaffolds for protein engineering and drug design due to their small size, high structural stability, strong sequence tolerance and easy access to chemical synthesis. The sequence patterns and associated documentation files created by us should be useful in protein family classification and functional annotation. Even though patterns might be useful at the family level, they may not always be adequate at the superfamily level due to hypervariability of mature toxins. In order to overcome this problem, we have demonstrated the applicationos of multi-class support vector machines (MC-SVMs) for the successful in silico classification of the mature conotoxins into their superfamilies. TheI- and J-conotoxin-superfamily members were analyzed in greater detail. On the basis of in silico analysis, we have divided the 28 entries previously grouped as I-conotoxin superfamily in UniProtKB/Swiss-Prot (release 49.0) into I1 and I2 superfamilies inview of their having two different types of signal peptides and exhibiting distinct functions. A comparative study of the theoretically modeled structure of ViTx from Conus virgo, a typical member of I2-conotoxin superfamily, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels. Putative complexes created by us of very recently characterized J-superfamily conotoxin p11-4a with Kv1.6 suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. This finding provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profile against subtypes of the potassium channels for these conopeptides. Furthermore, the present thesis reports the application of order-statistic filters and hydrophobicity profiles for predicting the location of membrane-spanning helices. The Proposed method is in particular effective for the class of helical membrane proteins, namely the therapeutically important voltage-gated ion channels, which are natural targets of several conotoxins. Our suggested ab initio approach is comparatively better than other spatial filters, confirming to the efficacy of including the concept of order or ranking information for prediction of TM helicdes. Such approaches should be of value for improved prediction performance including in large-scale applications. In addition, anlaysis has been carried out of the role of context in the relationship between form and function for the true PDB hits of some nonCys-rich PROSITE patterns. We have found specific examples of true hits of some PROSITE patterns displaying structural plasticity by assuming significantly different local conformation, depending upon the context. The work was carried out as a part of the research interest in our group in studying structural and other features of protein sequence patterns. The Contributions of the candidate to venominormatics include, creation of protein sequence patterns and information highlighting the importance of the patterns as gleaned from the lteratures for family classification: profile HMM and MC-SVMs for conotoxin superfamily classification; in silico characterization of I1 and I2 conotoxin superfamilies; studies of interaction with Kv1 channels of typical members of I2 and 3 conotoxin superfamilies and development of improved methods for detecting membrane-spanning helices. Chapter I starts with a brief account of venominformatics; bioinformatics for venoms and toxins. Chapter 2 presents a regular expression based classification of Conus peptides. Chapter 3 revisits the 28 entries previously grouped as I-conotoxin superfamily in UniProt Swiss-Prot knowledgebase (release 49.0) having four disulfide bonds with Cys arrangement C-C-CC-CC-C-C and they inhibit or modify ion channels of nerve cells. Chapter 4 describes pseudo-amino acid composition and MC-SVMs approach for conotoxin superfamily classification. Chapter 5 describes in silico detection of binding mode with Kv1.6 channel of J-superfamily conotoxin p114a from bermivorouos cone snail, Conus planorbis. Chapter 6 presents a comparative sequence-structure-function analysis of naturally occurring Cys-rich peptides having the Knottin or inhibitor cystine knot(ICK) scaffold, from different plants and venomous animals based on information available in the knottin database(http://knottin.cbs.cnrs.fr/). Chapter 7 describes the application of order-statistic filters and hydrophobicity profiles for detecting membrane-spanning helices. Chapter 8 describes the role of context in the relationship between form and function for the true PDB hits of some non Cys-rich PROSITE patterns. Chapter 9 summaries the important findings of the present studies on naturally occurring bioactive Cys-rich peptides with emphasis on Conus peptides and their interactions with respective target such as voltage-gated ion channels.

Phan Me is a midland-mountainous commune of Phu Luong district, Thai Nguyen province, Vietnam where the agricultural activities are predominant occupation. The investigation on the use of plant protection products in here showed that chemical pesticides have been commonly used in agricultural activities. Although some local people have a good attitude in using plant protection products, the status of using chemical pesticides that were not recommended (like Vofatox) has been quite popular due to the old cultivation customs. The local people have not applied any measures for wastewater treatment, or plant protection product package treatment. Besides, the use of plant protection products in higher concentration than recommendation for vegetables or crops caused the residues of toxics in agricultural products as well as in cultivation soil. The accumulation of toxic residues in the environment is a serious matter of concern because of their significant impact on the environment, agricultural ecosystem and human health. / Phấn Mễ là một xã trung du miền núi của huyện Phú Lương, tỉnh Thái Nguyên, Việt Nam với hoạt động sản xuất nông nghiệp là chủ yếu. Qua thực tế điều tra tình hình sử dụng thuốc bảo vệ thực vật (BVTV) tại đây cho thấy hầu hết các loại hình sản xuất nông nghiệp đều sử dụng thuốc BVTV. Một số người dân đã có ý thức trong việc sử dụng thuốc BVTV tuy nhiên do tập quán canh tác từ lâu đời nên việc sử dụng các loại thuốc bị khuyến cáo, chẳng hạn như Vofatox, vẫn còn khá phổ biến. Người dân chưa tiến hành các biện pháp để xử lý nước thải, bao bì, vỏ chai thuốc BVTV sau khi sử dụng. Bên canh đó, họ còn sử dụng thuốc BVTV không đúng liều lượng cho một số loại cây rau để lại dư lượng thuốc trên nông sản cũng như đất canh tác. Sự tích lũy dư lượng của những chất độc hại này trong môi trường là vấn đề đáng lo ngại do sự ảnh hưởng nghiêm trọng của chúng đối với môi trường sống, hệ sinh thái nông nghiệp và sức khỏe con người.

Microcystis são cianobactérias que apresentam formações de florações e produção de microcistina, uma hepatotoxina que causa envenenamentos e promove tumores hepáticos. Os objetivos deste trabalho foram estudar o efeito combinado da radiação gama com outros agentes físicos em células de Microcystis panniformis e investigar a atenuação da microcistina através da ação de feixes de nêutrons e elétrons e radiação gama. Observou-se que os tratamentos combinados aumentaram a inviabilidade celular e que houve atenuação da microcistina após a sua exposição aos três tipos de radiações ionizantes. Com a combinação de agentes físicos é possível reduzir as doses necessárias para o controle populacional de Microcystis panniformis e a exposição à feixes de elétrons é a radiação indicada para maior atenuação da microcistina. / Microcystis are cyanobacteria exhibiting blooms formation and production of microcystin, a hepathotoxin responsible for poisoning and consequent development of hepathic tumors. The goals of this work are the study of the combined effect between gamma radiation with other exogenous physical agents in cells of Microcystis panniformis, as well to investigate the attenuation of microcystin by neutron and electron beams and gamma radiation. It was found out that combined treatments, as radiation with another physical agent, caused lowering of the cellular viability and that had attenuation of microcystin after your exposition at three kind of ionizing radiation. The results showed that with the association of physical agents it is possible to reduce the doses needed for the population control of Microcystis panniformis and that exposition of electron beams have proved to be the most indicated radiation type for attenuation of microcystin.

O crescimento desordenado das cidades tem trazido à tona problemas de saneamento e degradação dos recursos naturais, entre eles a água. O despejo de efluentes domésticos e industriais têm ocasionado a eutrofização dos mananciais, culminando com a proliferação dos fitoplânctons. Esta proliferação tem chegado ao ponto, em certos momentos, de acarretar episódios de floração de algas. Entre os organismos fitoplanctônicos que se desenvolvem no ambiente, estão as cianobactérias, com vários gêneros capazes de produzir diversas cianotoxinas, tais como as microcistinas, anatoxinas, cilindrospermopsinas (CY), saxitoxinas (PSPs), entre outras. Com o aumento da freqüência dos episódios de floração de algas, a probabilidade da ocorrência destas toxinas também aumenta. Como conseqüência disto e devido às exigências legais, os corpos dágua devem ser monitorados para garantir a qualidade da água para consumo humano. Com vistas ao monitoramento da presença das cianotoxinas CY e PSPs, foram realizadas investigações em alguns corpos dágua do Estado de São Paulo. Das investigações realizadas, a neosaxitoxina foi identificada pela primeira vez no Reservatório Billings e os congêneres, saxitoxina, goniautoxina 2, goniautoxina 3, foram identificados de forma inédita em amostras de água deste reservatório. Com relação à CY, foi desenvolvido um método analítico, parcialmente validado. Entretanto, esta cianotoxina não foi localizada nas amostras de água ou cianobactérias das águas superficiais dos corpos dágua estudados. Este estudo mostra a importância do monitoramento da qualidade das águas dos mananciais quanto à presença de cianotoxinas, especialmente daqueles corpos dágua com a finalidade do consumo humano. / Cities growth usually occur in an unorganized manner. This tendence can generate a variety of sanitary problems, including the degradation of natural resources, such as water bodies. As a consequence, domestic and industrial efluents cause eutrofication of water reservoir, increasing the natural level of phytoplancton, what may form algal bloom. Among the phytoplanktonic organisms that grow in this modified environment it is found the cyanobacteria. Some of them can produce different types of cyanotoxins such as microcystin, anatoxin, cylindrospermopsin (CY) and saxitoxin (PSPs). The probability of production of these cyanotoxins increase according to frequent occurrence of algal blooms episodes. Consequently, water bodies monitoring becomes important to assure water quality. The aim of this project was to develop a specific method to identify the presence of cyanotoxins CY and to investigate PSPs in water bodies in São Paulo State. The results confirmed the presence of neosaxitoxin (NEO), a toxin of PSPs family. It was the first time that Neo was indentified in Billings Reservoir along with other PSPs types: saxitoxin, gonyautoxin 2, gonyautoxin 3. Although the study also included CY monitoring, CY was not identified in the tested samples. The present study confirmed the importance of continuous searching and monitoring of water bodies to grant quality to water used for human consumption.