Eutrophication is a nutrient enrichment of dams and lakes. Increased eutrophication in dams results in blooms of cyanobacteria. Cyanobacteria are troublesome as they form massive surface scums, impart taste and odour to the water. Some strains of cyanobacteria such as Microcystis aeruginosa are dangerous to humans and animals. They produce toxins that can kill animals drinking the contaminated water and have also been implicated in human illnesses. The study investigated the effectiveness of techniques deployed in controlling cyanobacterial growth in Rietvlei, Roodeplaat and Hartbeespoort Dams. This was done by interpreting data from April 2010 to March 2012. The conditions in the three dams show that Microcystis produced toxins in the summer season and all the variables analysed were favourable for the production of toxins. The methods deployed to rehabilitate the dams do not completely solve the problems of toxins experienced by the dams. / Environmental Sciences / M. Sc. (Environmental Management)

A jararagina, isolada do veneno de Bothrops jararaca, possui efeito pró-inflamatório caracterizado por edema, liberação de citocinas e migração celular. Neste estudo, demonstramos o aumento na expressão gênica da quimiocina IL-8, moléculas de adesão (E-Selectina, V-CAM-1, I-CAM-1), CD-69, Angiopoetina-2 e MMP-10 em HUVECs estimuladas com jararagina. Também investigamos o efeito da jararagina na expressão protéica de moléculas de adesão e da quimiocina IL-8. A molécula de adesão PECAM-1 foi expressa na superfície das HUVECs em todos os tratamentos e intervalos de tempo analisados. Entretanto não observamos aumento da expressão de E-selectina e VCAM-1 após estímulo com a jararagina. A jararagina também não induziu a liberação de IL-8. Nossos resultados sugerem que a jararagina se liga nas células endoteliais, mas o receptor celular envolvido neste efeito ainda não está claro. Este trabalho contribui com a literatura na medida em que elucida a participação de importantes genes dentro do contexto inflamatório desencadeado pela jararagina em células endoteliais. / Jararhagin, from Bothrops jararaca venom, causes a local reaction manifested by edema, cytokine release and inflammatory cells recruitment. In this study we evaluated by real time PCR the expression of 9 genes involved in inflammatory response, triggered by jararhagin in HUVECs. Our results showed a significant increase in the gene expression of chemokine IL-8, adhesion molecules (E-selectin, V-CAM-1, I-CAM-1), CD-69, angiopoietin-2 and MMP-10. We also investigated the effect of jararhagin on expression of adhesion molecules in the surface of HUVECs by flow cytometry. We did not observe increased expression of E-selectin and VCAM-1 molecules on the surface of HUVECs compared with control. Jararhagin did not increase the release of soluble chemokine IL-8 in HUVECs supernatant. Our results suggest that jararhagin binds to endothelial cells, but the cellular receptor involved in this effect remains unclear. This work contributes to the literature highlighting the participation of important genes within the inflammatory context triggered by jararhagin on endothelial cells.

Der pilzliche Erreger der Schwarzen Sigatokakrankheit, Mycosphaerella fijiensis (Mf), befällt fast alle Sorten (cv) der angebauten Banane Musa sp. Dabei sind weder die Reaktionen der Wirtspflanzen gegenüber dem Pathogen noch dessen Pathogenität hinreichend charakterisiert. Eine besondere Bedeutung wurde für pilzliche Sekundärmetaboliten des Pentaketid-Biosyntheseweges als spezifische Toxine innerhalb des Pathosystems postuliert. Die vorliegende Arbeit hat die experimentelle Untersuchung der Wirt-Pathogeninteraktionen zur Zielsetzung und wendet Methoden der Gewebekultur und Chromatographie zur bio-chemischen und physiologischen Charakterisierung des pflanzlichen und pilzlichen Stoff-wechsels an. Bezüglich der pflanzlichen Abwehrmechanismen von resistenten Musa cv konnte eine hypersensitive Reaktion (HR), die Aktivierung des Enzyms PAL und die Anreicherung von post-infektionellen Substanzen zur Hemmung des Pilzwachstums nachgewiesen werden. Im Er-regermetabolismus wurden unter in vitro-Bedingungen die Pentaketide Flaviolin, 2-Hydroxy-juglon, Juglon und 2,4,8-Trihydroxytetralon (-THT) bestimmt. Die Konzentration an 2,4,8-THT konnte sowohl durch die Anwendung des synthetischen Wirkstoffes Tricyclazol ® sowie durch natürliche Aktivatoren aus interzellulärem Blattgewebe resistenter Musa cv stark gesteigert werden. Mf-Rohextrakte und ausgewählte pilzliche Substanzen wurden auf ihre Dosis-Wirkungsbeziehung gegenüber verschiedenen Musa cv untersucht. Unter in vivo-Bedingungen Mf-inokulierter Bananenpflanzen führte die Anwendung des Wirkstoffes zu einer als "zerstörerische HR" bezeichneten Nekrotisierung des Blattes in anfälligen und resistenten Sorten. Diese Ergebnisse belegen die Bedeutung des 2,4,8-THT für die Ausprägung nekrotischer Blatt-symptome, die in Abhängigkeit von der Konzentration zum jeweiligen Zeitpunkt Musa cv-spezifische Wirkungen hervorruft: In der resistenten Sorte führte die Steigerung von 2,4,8-THT zur HR und der Elizitierung postinfektioneller Abwehrreaktionen, die zur Inkompatibilität zwischen Pathogen und Wirtspflanze führen; in der anfälligen Sorte erreicht 2,4,8-THT erst nach Ausbildung einer kompatiblen Interaktion mit biotropher Ernährung toxische Dosen und wirkt als Virulenzfaktor in der nekrotrophen Phase der Pathogenese. / The fungal pathogen causing black Sigatoka disease, Mycosphaerella fijiensis (Mf), attacks almost all varieties (cv) of cultivated bananas and plantains Musa sp. However, neither the reactions of host plant in relation to the pathogen nor its pathogenicity has been characterized in detail. A special significance has been attributed to fungal secondary metabolites of pentaketid pathway as host-specific toxins within the pathosystem. The present study refers to the experimental investigation of host-pathogen interactions using tissue culture as well as chromatographic methods in order to characterize biochemical and physiological metabolisms of plant and fungus. With reference to plant defense mechanisms of resistant Musa cv, hypersensitive reaction (HR), activation of phenylalanine-ammonia lyase and accumulation of postinfectional substances which blocked fungal growth have been demonstrated. Using in vitro conditions, the pentaketide metabolites flaviolin, 2-hydroxyjuglone, juglone and 2,4,8-trihydroxytetralone (2,4,8-tht) had been determined. 2,4,8-tht concentration was significantly increased through the application of the synthetic compound tricyclazole ® and through natural activators extracted from resistant Musa cv intercellular space of leaf tissue. Dose-effect relationship between crude extracts and selected fungal substances applied to different Musa cv were investigated. Using in vivo conditions, the application of tricyclazole ® to host plants inoculated with Mf resulted in extensive necrosis of susceptible and resistant Musa cv leaves, characterized as "devastating HR". These results proof the importance of 2,4,8-tht for the development of necrotic leaf symptoms that causes host-specific reactions depending on their concentration at different moments of pathogenesis: The resistant Musa variety increases 2,4,8-tht causing HR and elicitation of postinfectional defense reactions that lead to incompatibility between pathogen and host plant; in the susceptible variety, 2,4,8-tht attains toxic doses only after establishment of a compatible interaction including biotrophic nutrition of the pathogen, acting as a virulence factor during the necrotrophic phase of pathogenesis.

O para-cresol (4-metilfenol) e as guanidinas são compostos envolvidos em uma série de processos bioquímicos e fisiológicos. Em condições normais esses compostos são eliminados do organismo pelos rins. Entretanto, em pacientes com doença renal crônica (DRC), esses compostos acumulam nos fluidos biológicos e tecidos, e são de difícil remoção, gerando alta taxa de mortalidade em pacientes humanos hemodialisados. Cromatografia gasosa (GC) e cromatografia líquida de alta eficiência (HPLC) são as técnicas analíticas mais utilizadas para a separação e quantificação de p-cresol e guanidinas. A derivatização pré ou pós-coluna geralmente é empregada para aumentar a volatilidade, absorbância ou fluorescência desses compostos, permitindo a detecção e quantificação. A ninidrina é um dos reagentes mais utilizados para a derivatização das guandinas pois apresenta alta sensibilidade, solubilidade em água e o procedimento aplicado para a derivatização é simples, permitindo a automação do sistema de preparo de amostras quando necessário. Embora haja vários estudos sobre a concentração e os efeitos desses compostos no sangue de pacientes humanos com DRC, a concentração de p-cresol e das guanidinas no sangue de cães com DRC e os efeitos associados a tais compostos foram pouco estudados. Assim, são necessários estudos similares aos realizados em humanos, para o desenvolvimento de novas estratégias de tratamento veterinário para cães. Para isso, é importante o desenvolvimento de uma metodologia analítica para a quantificação desses compostos no sangue de cães. Nesse trabalho desenvolveu-se, validou-se e aplicou-se um método para análise de p-cresol e guanidinas em plasma de cães urêmicos com detecção por fluorescência. A reação de derivatização das guanidinas com a ninidrina foi revisada, caracterizada por espectrometria de massas (MS) e otimizada utilizando planejamento fatorial. Foi analisado um total de 63 amostras de plasma de cães incluindo grupo controle e urêmico. A concentração média de guanidina, metilguanidina e p-cresol livre determinada foi maior no grupo urêmico do que no grupo controle, a exemplo do que ocorre em humanos, sugerindo um comportamento metabólico similar desses compostos em cães. O método desenvolvido possibilitou as análises, sendo relativamente simples, evitando um preparo de amostras complexo. Por sua vez, a otimização por planejamento fatorial da reação de derivatizacão com ninidrina resultou em ganhos na área dos picos cromatográficos que chegaram a 1000%. / Para-cresol (4-methylphenol) and guanidino compounds are involved in a series of biochemical and physiological cycles. Under ordinary conditions, these compounds are excreted from the body by healthy kidneys. However, in uremic patients, the concentration of these compounds is highly increased in biological fluids and tissues and is of hard removal, increasing the mortality rate in dialysis patients. Gas chromatography (GC) and high performance liquid chromatography (HPLC) are the most used techniques for separation and quantification of p-cresol and guanidino compounds. For the guanidines determination, derivatizing reactions are usually employed to enhance the absorbance, fluorescence or volatility of these compounds, allowing the quantification. Ninhydrin is one of the most used reagents for derivatization because it shows high sensitivity and presents water solubility. The procedure employed for the derivatization reaction is simple, allowing the automation of sample preparation system, when it is required. There are few studies quantifying the concentration of guanidino compounds and p-cresol in blood of uremic dogs, therefore, a better understanding of the role of these compounds in the metabolism of dogs are needed. For this reason, it is important to develop an analytical method for the quantification of these compounds in dog\'s blood. The development of a method for simultaneous determination of these compounds, which is not reported in the literature, can improve the analytical productivity, decreasing the time spent with sample preparation and analysis. Here, a method based in reversed-phase HPLC for simultaneous quantification of guanidino compounds and free p-cresol in plasma of uremic dogs with fluorescence detection was developed, validated and applied. For this purpose, the ninhydrin derivatization reaction was reviewed, characterized by mass spectrometry (MS) and optimized using central composite design (CCD). A total of 63 samples of dog plasma, including healthy and uremic groups, were analyzed. The mean concentration of guanidine, methylguanidine and free p-cresol determined by the validated method, for the uremic group, were higher than the healthy group, alike usual for humans, suggesting a similar metabolic behavior of these compounds in dogs. The obtained method is relatively simple avoiding complex sample preparation and the experimentally designed optimization of the ninhydrin reaction by CCD yielded a gain of area as high as 1000% for the chromatographic peaks.

INTRODUÇÃO: O aracnídeo Phoneutria nigriventer, também conhecido por aranha-armadeira, possui um veneno complexo, contendo vários peptídeos que ativam canais iônicos nas células. Dentre estes, só dois neuropeptídeos, Tx2-5 e Tx2-6, destacam-se por relaxar o músculo liso trabecular do corpo cavernoso, induzindo ereção peniana em camundongos e ratos. Este efeito tem sido associado à produção de oxido nítrico pela ativação de óxido nítrico sintases. No entanto, faltam estudos mais amplos para determinar o papel de Tx2-6 na indução da ereção. OBJETIVOS: Identificar os genes diferencialmente expressos no tecido erétil de camundongos após indução da ereção pela Tx2-6 utilizando microarranjos de oligonucleotídeos. Validação dos resultados obtidos nos microarranjos por PCR quantitativa e imuno-histoquímica. MATERIAIS E MÉTODOS: Camundongos machos e adultos da linhagem Swiss foram divididos em dois grupos: controle (n=10), inoculados pela via intracavernosa com 20 l de solução salina; e tratado (n=10), os quais receberam 0,006gg/animal do peptídeo Tx2-6 diluído em 20 l de salina pela via intracavernosa. Uma hora após o início da ereção no grupo tratado todos os animais foram sacrificados e retirou-se o pênis. Este último foi dividido em dois fragmentos, uma parte do material foi congelada em nitrogênio líquido e mantida a 80°C até a extração do RNA para os experimentos de microarranjos e PCR quantitativa; outra parte foi utilizada para avaliação imuno-histoquímica. RESULTADOS: No grupo tratado a ereção foi observada 30-45 minutos após aplicação de Tx2-6 e mantida durante 120 minutos. Os camundongos de grupo controle não apresentaram nenhum indício de ereção. Nos experimentos de microarranjos, onde foram analisados 34.000 genes representando o genoma total do camundongo, identificou-se 3.803 (12,3%) genes com expressão diferencial de pelo menos ±1,5 vez entre os grupos (1.823 genes superexpressos e 1.980 genes subexpressos no grupo tratado comparado ao controle). Os genes ednrb, sparc, fn1, sstr2, pdgfr foram selecionados para validação dos microarranjos por PCR quantitativa e confirmaram a superexpressão em relação aos controles. As proteínas Fn1, Sstr2 e Pdgfr resultaram aumentadas no grupo tratado após avaliação imuno-histoquímica. CONCLUSÕES: A inoculação de Tx2-6 pela via intracavernosa alterou o perfil de expressão gênica no tecido erétil de camundongos. O número de genes superexpressos foi similar ao de genes subexpressos. Serão necessários outros estudos para entender melhor as vias moleculares que Tx2-6 afeta na indução da ereção peniana. / INTRODUCTION: The Phoneutria nigriventer arachnid, also known as armed-spider, has a complex venom, composed by several peptides that affect cellular ionic channels. Among these, only two neuropeptides, Tx2-5 and Tx2-6 induce penile erection in mice and rats and this effect has been associated with the production of nitric oxide by the activation of nitric oxide synthases. Moreover, there is a scarcity of studies focusing on the role of Tx2-6 in the induction of erection. OBJECTIVES: To identify the differently expressed genes in the erectile tissue of mice after erection induction by Tx2-6 using oligonucleotide microarrays. To validate microarray results by quantitative PCR and immunohistochemistry. MATERIALS AND METHODS: Swiss adult male mice were divided in two groups: control (n=10) were injected intracavernously with 20 gl of saline solution; and treated (n=10) were injected intracavernously with 0.006gg/mouse of the Tx2-6 peptide diluted in 20 gl of saline solution. After checking the penile erection in the treated group, all mice were sacrificed one hour after the beginning of erection for the removal of the penis. Penile organ was divided into two fragments, one piece was immediately frozen in liquid-nitrogen and stored at -80°C until RNA extraction to make the microarray and quantitative PCR experiments; the other was reserved for immunohistochemistry analysis. RESULTS: In the treated group, erection was noticed 30-45 minutes after Tx2-6 inoculation and lasted for 120 minutes. Control mice did not present any sign of erection. Considering as differentially expressed genes with a ±1.5 fold expression difference, of the 34,000 genes on the microarray we identified 3,803 (12.3%) genes differentially expressed between the groups (1,823 genes up-regulated and 1,980 genes down-regulated in the treated group compared to controls). The ednrb, sparc, fn1, sstr2, pdgfr genes were selected for validation of microarray results by using quantitative PCR and confirmed the up-regulation when compared to controls. After immunohistochemistry analysis the Fn1, Sstr2 and Pdgfr proteins were found increased in the treated group. CONCLUSIONS: The intracavernous inoculation of Tx2-6 modified the gene expression profile of erectile tissue of mice. The number of upregulated and down-regulated genes was similar. Further studies are needed to understand the molecular pathways that Tx2-6 affect to induce penile erection.

Made available in DSpace on 2017-07-21T20:00:01Z (GMT). No. of bitstreams: 1 Murilo Delgobo.pdf: 2288840 bytes, checksum: d9ce3a086e9ee92b92ac76ac76264e04 (MD5) Previous issue date: 2014-02-21 / Brown-Spider’s venom (Loxosceles sp.) is presented as a complex mixture of toxins, able to induce skin necrosis with gravitational spreading, intense inflammatory response, edema induction and increase in vascular permeability in vivo. Recently, the biotechnological potential of the toxins was explored by its use in clinical test, in the study of inflammatory response, as a research tool in cell biology, as a biopesticide and in immunotherapy, through the production of antiserum. In this context, immunotherapy is broadly spread through the production of vaccines, available for the treatment of deleterious reactions developed in accidents. In the present work, we investigated if immunological tolerance induction to dermonecrotic recombinant toxin (LiRecDT1) and its mutated form (LiRecDT1 H12A), through its oral administration, could modulate inflammatory and deleterious responses triggered by dermonecrotic toxin. For this purpose, an oral tolerance protocol was designed, consisting in the administration of 10 μg of LiRecDT1 and LiRecDT1 H12A three times in a week, for three weeks. Adult Swiss mice were further immunized, and oral tolerance induction was observed by reduction in serum levels of IgG antibody anti-toxin when compared with control group. It was observed that mice tolerant to LiRecDT1 H12A present a reduction in paw edema, caused by the injection of 6 μg of dermonecrotic toxin in plantar surface hind paw. Mice tolerized with LiRecDT1 and challenged with 50 μg of dermonecrotic toxin, exhibited higher survival, when compared to control group. This effect was not observed in mice tolerized to LiRecDT1 H12A. The present findings suggested that oral tolerance induction to LiRecDT1 H12A was able to alleviate inflammatory responses triggered by dermonecrotic toxin in paw edema and oral tolerance to LiRecDT1 increase survivability in challenge. The results shown that LiRecDT1 and LiRecDT1 H12A can be explored as a tool in the induction and study of oral tolerance phenomena. The generation of T regulatory cells (Tregs) and following involvement of immunosuppressive cytokines might take part in the modulation of immune response. / O veneno de aranha-marrom (Loxosceles sp.) apresenta-se como uma mistura complexa de toxina, capazes de causar necrose com espalhamento local, intensa resposta inflamatória, indução de edema e aumento da permeabilidade vascular in vivo. Recentemente, o potencial biotecnológico das toxinas foi explorado através de seu uso em análises clínicas, no estudo da resposta inflamatória, como ferramenta de pesquisa na biologia celular, como biopesticidas e na imunoterapia, através da produção de anti-soro. No presente trabalho, foi investigado se a indução de tolerância imunológica à toxina dermonecrótica recombinante (LiRecDT1) e sua forma mutada (LiRecDT1 H12A), através de sua administração oral, poderia modular as respostas inflamatórias e deletérias causadas pela toxina dermonecrótica. Para tal, foi desenvolvido um protocolo para indução de tolerância oral, consistindo na administração de 10 μg de LiRecDT1 e LiRecDT1 H12A três vezes por semana, durante três semanas. Camundongos Swiss fêmeas adultas foram posteriormente imunizadas, e a indução de tolerância foi confirmada pela diminuição nos níveis de anticorpos IgG anti-toxina em relação ao grupo controle, resultado que aponta a obtenção de sucesso na indução de tolerância imunológica. Observou-se que animais tolerantes a LiRecDT1 H12A apresentaram uma diminuição no edema desenvolvida na pata, causado pela aplicação de 6 μg de LiRecDT1 na superfície plantar traseira. Animais tolerizados com LiRecDT1 e desafiados com 50 μg intraperitoneal de toxina dermonecrótica apresentaram maior índice de sobrevivência, quando comparados ao grupo controle. Esse efeito não foi observado em animais tolerizados com LiRecDT1 H12A. Os dados obtidos no presente trabalho sugerem que a indução de tolerância oral à LiRecDT1 H12A é capaz de atenuar o desenvolvimento da resposta inflamatória no edema de pata, enquanto a tolerância oral a LiRecDT1 foi capaz de aumentar a sobrevivência em animais desafiados com LiRecDT1. Os resultados demonstram que as toxinas LiRecDT1 e LiRecDT1 H12A podem ser exploradas como ferramenta na indução e estudo da tolerância oral. A geração de células T regulatórias (Tregs) e subsequente participação de citocinas imunossupressoras devem estar envolvidas na modulação da resposta imune.

L'insuffisance rénale chronique (IRC) est une affection caractérisée par une perte progressive de la fonction rénale. L’IRC est associée à l'accumulation de diverses toxines urémiques. Les toxines urémiques ou solutés de rétention de l'urémie sont des composés qui s'accumulent chez les patients atteints d'IRC en raison d'un défaut de clairance rénale et qui exercent des effets biologiques délétères. Les hémodialyses éliminent mal les toxines urémiques liées aux protéines (PBUT), en raison de leur liaison aux protéines plasmatiques, en particulier la sérumalbumine humaine. En conséquence, les toxines urémiques liées aux protéines s'accumulent chez les patients atteints d'IRC et leur concentration ne peut que difficilement être diminuée chez les patients atteints d'insuffisance rénale terminale (IRT). Mes travaux sont principalement centrés sur les toxines urémiques, en particulier les toxines urémiques liées aux protéines, comme l’indoxyl-sulfate (IS), l'acide phénylacétique (PAA) et le p-crésyl-glucuronide (p-CG); et la zinc-alpha2-glycoprotéine (ZAG) qui est une « middle molécule ». Nous avons étudié le rôle de l'IS dans le développement de la résistance à l'insuline et d'autres troubles métaboliques associés à l'IRC, ainsi que ses effets sur l'inflammation et le stress oxydant. Nous avons exploré les propriétés de liaison du PAA et du p-CG à la sérumalbumine, qui est la plus abondante protéine dans le plasma humain. Enfin, nous avons essayé de développer une nouvelle stratégie d'élimination des PBUT, à l’aide de déplaceurs/compétiteurs chimique. / Chronic kidney disease (CKD) is a condition characterized by progressive loss of kidney function. CKD is associated with the accumulation of various uremic toxins. Uremic toxins or uremic retention solutes are compounds that accumulate in patients with CKD due to impaired renal clearance and exert deleterious biological effects. Protein-bound uremic toxins (PBUT) is poorly removed by hemodialysis because of its binding to plasma proteins, particularly human serum albumin. As a result, protein-bound uremic toxins accumulate in patients with CKD and their concentration can hardly be reduced in patients with end-stage renal disease (ESRD). My work focuses mainly on uremic toxins, particularly protein-bound uremic toxins such as indoxyl-sulfate (IS), phenylacetic acid (PAA) and p-cresyl-glucuronide (p-CG); and zinc-alpha2-glycoprotein (ZAG) which is a "middle molecule". We investigated the role of IS in the development of insulin resistance and other metabolic disorders associated with CKD, as well as its effects on inflammation and oxidative stress. We have investigated the binding properties of PAA and p-CG to serum albumin, which is the most abundant protein in human plasma. Finally, we tried to develop a new strategy to eliminate PBUTs, using chemical displacers / competitors.

L’Insuffisance Rénale Chronique (IRC) résulte de la destruction progressive et irréversible des reins. Elle est associée à une rétention de toxines urémiques à l’origine des nombreuses complications de la maladie rénale chronique (cardiovasculaires, osseuses ou métaboliques). Nos travaux se sont focalisés sur l’impact de la dysfonction rénale et de l’urémie sur la fonction barrière de l’intestin et la motilité intestinale. Deux modèles d’IRC ont été implémentés : un modèle animal, chez la souris, par néphrectomie chimique (régime alimentaire enrichi en adénine) et un modèle cellulaire d’urémie en incubant des cellules coliques Caco-2 avec 10% de plasma de patients hémodialysés (HD). Le transit, la motilité, la perméabilité intestinale et la régulation des protéines des jonctions serrées ont été explorés. Les animaux urémiques présentent un transit gastro-intestinal ralenti et une perméabilité intestinale augmentée associés à une dérégulation de l’expression et de l’abondance des protéines des jonctions serrées dans le côlon (surexpression de la Claudine 1). La perméabilité de la monocouche cellulaire de Caco-2 incubées avec du plasma HD est significativement augmentée et est associée à une augmentation de l’expression et de l’abondance de la Claudine-1. En IRC, la motilité digestive et la fonction barrière de l’intestin sont significativement altérées. Ces dysfonctions pourraient contribuer à la production intestinale et l’absorption des toxines urémiques accélérant ainsi la progression du syndrome urémique et installant un véritable « cercle vicieux ». / Chronic Kidney Disease (CKD) result from a progressive kidney dysfunction. CKD is associated with an increase in the concentration of uremic toxins inducing CKD-associated metabolic alterations. Our work focused on the impact of renal dysfunction on gut permeability and gut motility. In vivo, CKD was induced in mice by chemical nephrectomy (adenine-enriched diet); In vitro, Caco-2 cells were incubated for 24h with 10% (v/v) of uremic plasma to mimic the uremic environment. Gastrointestinal transit time, gut motility, intestinal permeability and expression of tight junction proteins were explored. In vivo, kidney failure was associated with an impaired gastrointestinal transit and an increased intestinal permeability associated with a dysregulation of tight junction proteins (mainly claudine-1 overexpression). The Caco-2 monolayer permeability was significantly increased in cell monolayers incubated with uremic plasma. Claudine-1 expression and abundance was increased. In CKD, gut motility and gut permeability (e.g. « leaky » gut) are significantly impaired. Generally speaking, these gut dysfunctions could promote the production and the absorption of uremic toxins contributing to the uremic syndrom.

La sécrétion de vésicules membranaires (VMs) constitue un processus physiologique important qui a particulièrement été étudié chez les Bactéries et les Eucaryotes. La récente découverte de la production de VMs chez les Archées souligne cependant que ce phénomène est universel et suggère que le dernier ancêtre commun aux trois domaines, LUCA (Last Universal Common Ancestor), produisait certainement des VMs. Les VMs des Archées n’ayant pour le moment été étudiées que chez certaines Crénarchées (ex : G/ Sulfolobus), nous avons entrepris de caractériser les VMs produites par un groupe d’Euryarchées hyperthermophiles anaérobies, les Thermococcales. Dans la première partie de cette étude, nous avons examiné le mécanisme de production ainsi que la composition en lipides et en protéines des VMs de trois espèces de Thermococcales: Thermococcus kodakaraensis, Thermococcus gammatolerans et Thermocococus sp. 5-4. Nous avons observé que les VMs sont sécrétées par un processus de bourgeonnement à partir de l’enveloppe cellulaire similaire à la formation des ectosomes par les cellules eucaryotes. De plus, les VMs sont fréquemment libérées en groupes, formant de grosses protubérances ou des filaments ressemblant aux nanopodes récemment décrits chez les Bactéries. Des différences de structure et de composition protéique sont observées entre les VMs des trois souches étudiées. Cependant, les VMs et les membranes cellulaires d’une même souche ont des compositions protéique et lipidique très proches, confirmant que les VMs sont produites à partir des membranes des cellules. Les VMs et les membranes cellulaires des trois souches comportent notamment un récepteur de peptides de la famille OppA (Oligopeptide-binding protein A) et des homologues de cette protéine ont été identifiés dans les VMs de certaines souches de Sulfolobus.Les VMs sécrétées par les Thermococcales sont associées à de l’ADN et cette association les protègent contre la thermodégradation. Nous montrons dans notre étude que les cellules de T. kodakaraensis transformées avec le plasmide navette plC70 relâchent des VMs comportant ce plasmide. De façon intéressante, ces VMs peuvent être utilisées pour transférer pLC70 à des cellules dénuées de plasmides, suggérant que les VMs pourraient être impliquées dans le transfert d’ADN entre cellules à haute température.Dans la seconde partie de cette étude, nous nous sommes particulièrement intéressés à la souche Thermococcus nautilus, une Thermococcale produisant des VMs associées de manière sélective à deux plasmides contenus dans la cellule. L’un d’eux correspond notamment à un génome viral défectueux de la lignée d’adenovirus PRD1. Ceci indique que les VMs peuvent être un moyen de transport pour des génomes viraux et suggère que la production de VMs par des cellules ancestrales pourraient avoir joué un rôle dans l’apparition des virus.En plus d’être impliquées dans le transport de plasmides/virus, les VMs produites par T. nautilus exercent un effet toxique sur certaines souches de Thermococcales, probablement dû au convoyage de toxines. Même si ces « thermococcines » nécessitent d’être caractérisées, il s’agit de la première mise en évidence d’une activité toxique liée aux VMs chez les Thermococcales. / Secretion of membrane vesicles (MVs) is an important physiological process that has been extensively studied in Bacteria and Eukarya. The recent discovery that Archaea produce MVs shows that this process is universal and suggests that the Last Universal Common Ancestor, LUCA, certainly produced MVs. As these archaeal MVs have been only studied in some Crenarchaeota (ex: G/ Sulfolobus), we started characterizing MVs produced by Thermococcales, a group of hyperthermophilic anaerobic Euryarchaeota.In the first part of this study we examined the mechanism of production as well as the protein and lipid composition of MVs produced by three strains of Thermococcales: Thermococcus kodakaraensis, Thermococcus gammatolerans and Thermocococus sp. 5-4. We observed that MVs are released by a budding process from the cell envelope that is similar to ectosome formation in eukaryotic cells. Moreover, clusters of MVs often form filamentous structures and protuberances on cell surfaces, resembling recently described bacterial nanopods. Differences in structure are observable between MVs of the three species, as well as in their protein composition. However, MVs and cell membranes from the same species have a quite similar protein and lipid composition, confirming that MVs are produced from cell membranes. A major protein present in cell membranes and MVs from the three strains is the oligopeptide-binding proteins (OppA), which has homologues in MVs from Sulfolobus species. Thermococcales MVs harbor DNA and protect this DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harboring this plasmid. Interestingly, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature. In the second part of this study, we were specially interested in the strain Thermococcus nautilus, a Thermococcale that produces MVs selectively enriched in two plasmids from the cell. Notably, one of them corresponds to the genome of a defective virus from PRD1-adenovirus lineage. This indicates that MVs can be used as vehicles for the transport of viral genomes and suggests that production of MVs by ancestral cells could have played a role in the origin of viruses.In addition to be involved in transport of plasmids/viruses, MVs from T. nautilus display a toxic effect on some strains of Thermococcales, maybe due to the delivery of toxins. Even if these “thermococcins” remain to be characterized, this is the first time that a toxic activity associated with MVs has been shown in Thermococcales.

Bacillus thuringiensis (Bt) é uma bactéria entomopatogênica encontrada em várias partes do mundo e tem a capacidade de produzir cristais durante a fase estacionária do crescimento. Esses cristais são toxinas compostas por proteínas ativas contra uma ampla variedade de fases imaturas de insetos. As toxinas do Bt têm sido usadas como bioinseticidas por décadas no controle de insetos das ordens Coleoptera, Lepidoptera e Diptera, e algumas toxinas já foram selecionadas como efetivas contra ácaros, nematoides e insetos sugadores de floema da ordem Hemiptera, como os afídeos. Recentemente, foi comprovada a patogenicidade de três isolados de Bt a outro inseto dessa ordem, o psilídeo Diaphorina citri. Esse psilídeo é vetor das bactérias Candidatus Liberibacter spp., agentes causais da principal doença dos citros: o Huanglongbing ou HLB. A descoberta da capacidade endofítica de Bt em plantas de diferentes espécies vegetais abriu perspectivas para novos estudos usando o Bt para controle de insetos sugadores, como a D. citri. O objetivo deste trabalho foi avaliar o efeito patogênico de cada toxina Cry ou Cyt presente nestes três isolados através de isolados de Bt recombinantes expressando as toxinas individualmente. Foram montados bioensaios para confirmar a taxa de mortalidade causada por isolados de Bt contra ninfas de D. citri. Os bioensaios foram feitos usando cinco plantas de laranja doce (Citrus sinensis (L.) Osbeck) e dez ninfas de 3º ínstar de D. citri por planta, além dos controles (sem Bt). A mortalidade das ninfas foi avaliada diariamente durante cinco dias. Durante a avaliação, as ninfas mortas foram coletadas para o isolamento do Bt além da detecção do gene da toxina por PCR. No final do experimento, as folhas jovens onde as ninfas se alimentavam também foram avaliadas quanto à presença do Bt. Nossos resultados confirmaram que os isolados de Bt previamente identificados como patogênicos a D. citri causaram elevada mortalidade (em média 68-93%) em ninfas de 3º ínstar. Dentre os isolados recombinantes testados, um deles apresentou grande destaque, causando a mortalidade de 68-81% das ninfas em 48 horas e 83-93% em 120 horas. Esse isolado possui grande potencial de uso no controle biológico de D. citri através da produção de um bioinseticida ou produção de plantas de citros transgênicas. / Bacillus thuringiensis (Bt) is an entomopathogenic bacterium found in several locations around the world and has the ability to produce crystals during the stationary phase of growth. These crystals are composed of protein toxins that are active against a wide variety of insect larvae. The Bt toxins have been used for decades as biopesticides to control insects of the orders Coleoptera, Lepidoptera, and Diptera. Some toxins have been selected as effective for mites, nematodes and phloem-sucking insects of the order Hemipera, such as aphids. The pathogenicity of strains of Bt to Diaphorina citri, an hemipteran known as Asian citrus psyllid, has been recently demonstrated. This psyllid is the vector of the bacteria Candidatus Liberibacter spp., the causal agents of the main disease of citrus: Huanglongbing or HLB. The discovery of the ability of Bt strains to endophytically colonize plants of different species has opened new perspectives for studies. The aim of this work was to evaluate the pathogenic effect of each Cry or Cyt toxin present in these three isolates through the use of recombinant Bt isolates expressing each toxin individually. A series of bioassays were done in order to confirm the mortality caused by Bt strains against nymphs of D. citri. Bioassays were performed in five seedlings of Citrus sinensis (L.) Osbeck (five replicates/ bioassay) and using ten D. citri 3rd instar nymphs to each assay plant. A suspension containing Bt spores was inoculated via drench in each seedling, and five seedlings were maintained as negative control (without Bt). Mortality was assessed daily for five days. During the bioassays, dead nymphs were collected for further Bt isolation and detection by PCR. At the end of the experiment, young leaves where nymphs feed were also collected to Bt isolation and detection. Our results confirmed that the previously identified Bt isolates pathogenic to D. citri cause high mortality to 3rd instar nymphs. Among the ten recombinant isolates tested, one of them stood out, causing 68-81 % and 83-93% mortality after 48 and 120 hours of inoculation, respectively. The isolation of Bt from dead nymphs and young leaves, and PCR performed with specific primers confirmed the presence of the Bt isolates in psyllids and plants, and their involvement in psyllid mortality. This recombinant Bt with the higher mortality rate found in our results has the potential to be used as a bioinsecticide to control D. citri and the toxin gene can be used to genetic engineering of citrus.