Dissertations / Theses on the topic 'Nodularin'

Nodularin (NODLN) is a pentapeptide produced by the filamentous cyanobacterium Nodularia spumigena that is a bloom-forming species in the Baltic Sea. NODLN is an intracellular hepatotoxin, which can have a negative effect on aquatic life including fish. Toxins are released into the water when cells are lysing, e.g. during a decaying bloom. N. spumigena filaments have previously been shown to have a negative effect on perch egg development and perch larval survival. Coastal fish such as perch (Perca fluviatilis) have suffered from recruitment problems in the Baltic Sea the last decades. However, little is known about the impact of toxic cyanobacteria on juvenile perch. In the autumn of 2007, 1+ perch were exposed, during 29 days to either whole live cells (WC) or a crude extract (CE) of broken N. spumigena cells. Chlorophyll a concentrations in the aquaria were 50 µg L -1. Perch were fed chironomidae larvae twice a day. Unexposed perch either fed (CoF) or without food (Co) served as controls. Length and weight of perch were measured at onset and termination of experiment. NODLN content was measured in N. spumigena filaments, crude extract and perch liver samples using liquid chromatography-mass spectrometry (LC-MS). Total lipids (TL) were extracted and quantified from whole-body lyophilised perch excluding livers. No significant differences for length and weight of perch were found between treatments and fed control. NODLN was detected in the crude extract samples, while no NODLN was detected in the perch livers. Moreover TL determination revealed no significant differences between treatments and fed control. Nodularia spumigena did not affect perch in this experiment, probably due to that the critical period of the first year for the perch was exceeded. Therefore, 1+ perch was not as susceptible to the cyanobacterium as eggs, larvae and younger juveniles of fish found in the literature. Perch liver did not contain NODLN, thus either the toxin was detoxicated with no recorded energetic cost or it was not ingested. The variables studied here did not show any effects of NODLN. However, other chemical methods such as enzymatic activity may disclose effects of NODLN.

Cyanobacteria produce a variety of secondary metabolites which possess amongst others antifungal, antibacterial, and antiviral properties. Being primary producers they are also a vital component within the food web. However, certain strains also produce toxic metabolites such as the hepatotoxins microcystin (MC) and nodularin (NOD). Their toxicity in combination with the increasing global occurrence has resulted in a drinking water guideline limit of 1 μg L-1 being issued by the World Health Organisation (WHO). However, these toxins are not only present in water, but can be accumulated by fish and shellfish. Currently, no regulations regarding cyanotoxin contaminated seafood has been established despite similar toxicity to routinely monitored marine toxins such as domoic acid (DA). To facilitate regular monitoring, a high performance liquid chromatography photo diode array (HPLC-PDA) analysis method for the detection of DA was optimised to enable the simultaneous detection of DA and nine cyanotoxins. This method was then utilised to determine cyanotoxin concentration in laboratory cyanobacteria strains. To assess the accumulation and depuration of cyanotoxins in the common blue mussel Mytilus edulis, three feeding trials were performed. During these, mussels were exposed to two cyanobacteria strains, Nodularia spumigena KAC66, Microcystis aeruginosa PCC 7813, both individually and simultaneously. A rapid dose dependent accumulation of cyanotoxins was observed with maximum concentration of 3.4 -17 μg g-1 ww accumulated by M. edulis, which was followed by a much slower depuration observed. During the final feeding trial, with N. spumigena KAC 66 and M. aeruginosa PCC7813, cyanotoxins were still detectable following 27 days of depuration. Mortality in all studies was 7% or less indicating that most mussels were unaffected by the maximum dose of 480 μg L-1 NOD (feeding study 1), 390 μg L-1 MC (feeding study 2), or 130 μg L-1 total cyanotoxins (feeding trial 3), respectively. Mortality in negative control tanks was lower throughout all three feeding trials ( < 1 - 2.6%). Consumption of a typical portion size (20 mussels) would result in ingestion of cyanotoxins at levels significantly higher than the WHO recommended tolerable daily intake (TDI) of 2.4 μg NOD and/or MCs for a 60 kg adult. This value was exceeded not only during the exposure period (maximum levels 270 - 1370 μg cyanotoxins per 20 mussels), but also at the end of the depuration period 39-600 μg cyanotoxins per 20 mussels. These results illustrated that cyanotoxin monitoring of seafood should be considered not only during, but also following bloom events. In an attempt to investigate the cyanotoxin budget of the experimental system, not only mussels, but cyanobacteria cultures, the tank water, and the mussel faeces were also analysed for their cyanotoxin content. Results showed that large quantities of MCs and NOD were unaccounted for during all exposure trials. The combined effect of cyanotoxin metabolism in M. edulis, biotic and/or abiotic degradation, protein binding, and losses during the extraction and analysis were thought to have contributed to the unaccounted cyanotoxin fraction. Mussel flesh was analysed for the presence of glutathione or cysteine conjugates, however, there was no evidence of their occurrence in the samples tested. Due to these discrepancies in the toxin budget of the system, the introduction of correction factors for the analysis of cyanotoxins in M. edulis was suggested in order to protect the general public.

The role and function of hepatotoxins microcystin and nodularin produced by M.aeruginosa PCC 7806 and N. spumigena NSORlO respectively have yet to be elucidated. The mode of transcriptional regulation of these toxins, incorporating DNA binding proteins, was investigated, as an attempt to further understand the key control mechanisms acting on the toxins. The DNA binding proteins that control nitrogen and iron responsive transcription, NtcA and Fur, were identified from M. aeruginosa PCC7806 and N. spumigena NSOR10. Cloning and over-expression in E. coli was followed by mobility shift assays to determine binding characteristics of NtcA and Fur to the promoters, mcyA/D and ndaA/C, those regions that control the toxin encoding gene clusters in M. aeruginosa PCC 7806 and N. spumigena NSOR10, respectively. The results from these studies suggested a role for iron and nitrogen in the transcriptional control of microcystin and nodularin. biosynthesis. As NtcA and Fur classically act to regulate nitrogen and iron dependent genes, a link may be made to the putative function and control of microcystin and nodularin. By identifying the transcription factors NtcA and Fur in these genera, a greater understanding of the link between nutrient levels in the environment and hepatotoxin production in cyanobacteria may be possible.

Reversible phosphorylation of proteins on serine, threonine and tyrosine residues, is now widely accepted to be the principal mechanism for the control of intracellular events in eukaryotic and prokaryotic cells. The nodularins are known to be potent inhibitors of serine/threonine protein phosphatases, PPlc and PP2Ac, with sub-nanomolar inhibition constants. They have been shown to form covalent adducts with the enzymes and are known to be potent hepatotoxins and liver promoters. Nodularin has the general structure: cyclo [(R)-eryphro-beta-methyl-iso-Asp-(S)-X-Adda-(R)-iso- Glu-N-methyldehydrobutyric acid)], where (S)-X is a variable S-amino acid and Adda is the unique beta-amino acid, (2S,3S,8S,9S)-3-amiao-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid. In order to investigate the mode of inhibition and also to probe the active-site binding interactions, we decided to synthesis new analogues of nodularin. Specific inhibitors for either PP1 or PP2A are not presently available, but would be useful biochemical tools in delineating the individual physiological roles of these enzymes. We decided to syntliesise, the potential inhibitor cyclo [betaAla-(2R)-Glu-alpha-OMe-gamma-Pro-(2R)-Asp-alpha-OMe-gamma-(25)-Phe], a stripped-down nodularin macrocycle, and also an analogue which is suitable for synthetic elaboration at the "Adda position". Using solution phase peptide synthesis (LPPS), four such nodularin analogues (both (25)- and (2R)-proline) were synthesised in seventeen steps. The cyclisation between the Phe and Asp residues were carried out using DIPEA under conditions of high dilution. NMR studies (TOCSY, ROESY) have elucidated the three dimensional structures which have been shown to be similar to the natural product, nodularin. A shorter synthesis of these nodularin analogues was developed using solid phase peptide synthesis (SPPS). Two solid phase synthesise of the nodularin macrocycles, cyclo-[betaAla-(2R)- Glu-alpha-OMe-gamma-Pro-(2R)-Asp-beta-(25)-Phe]; one in which Fmoc-(2S)-Phe-betaAla-(2R)-Glu-alpha-OMe-gamma-Pro-(2R)-Asp(alphaO-Wang Resin)-beta-OAllyl is deprotected and then cyclised on the resin prior to cyclisation were found to be successful. Even though the resin-bound synthesis gave low yields for the cyclisation step, compared to the situation in solution, it offered advantages in the construction of the linear isopentapeptide precursor. Initial studies have shown that the nodualrin analogues 130 and 131 are moderate inhibitors (IC50 2.8 mmol) of PP1 when tested using the malachite green system. Studies towards the synthesis of incorporating more suitable Adda functionalities, and the development of a radiolabelled protein phosphatase assay are currently being investigated within the group.

Nodularia spumigena is an estuarine cyanobacteria that produces the toxin nodularin. This toxic cyanobacteria is known to have caused death to domestic and wild animals and is recognised as dangerous to human health. N. spumigena causes harmful algal blooms in many parts of the world including Australia. The toxic solutes of N. spumigena are potentially dangerous when contact is made to contaminated water bodies or is ingested by primary consumers. In Australia blooms of N. spumigena are common in the Gippsland Lakes in South-eastern Victoria and cause socio - economic hardships to the local communities. This PhD investigates the toxic effects of N. spumigena and its solutes to a range of aquatic life. A method known as SPME - HPLC showed promise in environmental monitoring of N. spumigena toxins by measuring nodularin from water samples. Other research presented study into the lethal and sublethal effects of on an extract from N. spumigena to aquatic fauna. Resu lts showed the N. spumigena extract was not lethal to many aquatic fauna although zooplankton from the Gippsland Lakes showed mortality at environmental relevant levels. Biochemical studies focusing on animal detoxification and antioxidation enzymes and DNA integrity showed sublethal effects to the N. spumigena extract. Results presented in this thesis show that an extract of N. spumigena elicited detoxification and antioxidation responses in animals tested. Furthermore, the use of the COMET assay showed increased damage to DNA of animals tested. Results also showed that different organs in animals tested responded differently to the aqueous extract, suggesting mode of uptake maybe important in toxicosis. Further, feeding studies with N. spumigena help elucidate mode of uptake using enzyme response biomarkers. The overall results of this research provided an assessment of the toxic affects of N. spumigena on aquatic fauna with special reference to the Gippsland Lakes, Victoria, Australia.

Cyanobacterial secondary metabolites, microcystins (MC) and nodularin (NOD) have become common contaminants in most aquatic ecosystems over recent years presenting a hazard to animal and human health. Unfortunately, these chemically diverse peptide hepatotoxins remain a challenge to most conventional water treatments due to their stable cyclic structures. Over recent years, bioremediation of MC and NOD has become one of the most exciting areas that holds promise for a successful and cost effective solution for water treatment process. The current work presents the biodegradation of MCs and NOD by bacterial isolates from three different bacteria genus Arthrobacter, Brevibacterium and Rhodococcus belonging to Actinobacteria. A total of five isolates representing the three genera have demonstrated an overall metabolism of MC-LR, -LF, -LY, -LW, -RR and NOD in a Biolog MT2 assay. Subsequently, these bacteria were reported to degrade the range of toxins in a separate batch experiment. The bacterial degradation rate of the above cyanobacterial peptides were found to decrease with the multiple subculturing of the bacteria. However, a rapid degradation was discovered when the bacteria were re-exposed to MC or other prokaryotic peptides demonstrating an inducible bacterial biodegradation. Utilising latest molecular biology techniques, the gene responsible for production of MC degrading enzymes was successfully elucidated and its activity was evaluated. Analysis of the degradation products of MC-LR revealed a glutathione conjugate detoxification mechanism involved during the degradation of MC-LR by Rhodococcus sp. (C1). A novel MC degradation pathway was proposed. Further studies were suggested to fully characterise the degradation pathway and to evaluate the MC detoxification mechanism in bacteria.

A contaminação da água para consumo humano por toxinas produzidas por cianobactérias é um problema de saúde pública e das autoridades em todo o mundo. Microcistina-LR (MCLR) é uma cianotoxina heptapeptídica cíclica que inibe as proteínas fosfatases PP1 E PP2A nos hepatócitos. Microcistinas são produzidas por diversos gêneros de cianobactérias e mais de 70 variações estruturais têm sido caracterizadas em florações naturais. Por serem haptenos, as microcistinas são incapazes de induzir uma resposta imune em animais. Conseqüentemente, foi necessário aplicar métodos de conjugação envolvendo a adição de uma proteína carreadora, mcKLH (cationized Keyhole Limpet Hemocyanin). Portanto, o objetivo inicial desta tese foi o de obter anticorpos monoclonal (em camundongos) e policlonal (em coelho) anti- MCLR. Com relação ao anticorpo monoclonal foram obtidos 9 hibridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232), sendo que apenas 5 se mostraram estáveis (k29, k317, k248, k284, k2232). Estes foram selecionados para serem isotipados, expandidos em líquido ascítico, purificados em coluna cromatográfica de proteína-A e titulados. Dentre estes cinco hibridomas secretores de anticorpos, o clone k317 foi o que melhor reconheceu (mais específico) a toxina MCLR. Os anticorpos do sobrenadante de meio de cultura do hibridoma e o fluido ascítico purificado foram identificados pelo ensaio ELISA (Enzyme Linked Immunosorbent Assay) previamente padronizado. Mesmo sensibilizando a placa de ELISA com diferentes antígenos, tais como MCLR-cBSA, MCLR, MCLR, MCRR, MCYR e MCLA, o clone 17 foi o que apresentou melhor linearidade frente às variantes de microcistina. Portanto, o clone 17 (isótipo IgG1) obtido é muito promissor e será usado para detecção de MCLR na água para consumo humano através do desenvolvimento de um kit de ELISA competição. Com relação ao anticorpo policlonal, o antígeno de imunização foi MCLR-mcKLH, enquanto que o antígeno de sensibilização foi MCLR-cBSA para o ensaio de titulação de anticorpos de classe IgG por ELISA indireto. Na seqüencia, foi padronizado um ensaio ELISA competição utilizando somente a toxina MCLR como antígeno de sensibilização. Este método Caseína foi padronizado, validado e comparado com o kit comercial Abraxis®. O kit ELISA competição que utiliza anticorpo policlonal, nomeado como método Caseína, foi avaliado quanto Limite Inferior de Quantificação, Especificidade, Seletividade, influência do metanol no ensaio, Recuperação, Linearidade, Precisão, Exatidão e Robustez. Este método de triagem apresentou excelente resultado quando comparado ao kit comercial Abraxis®, pois foi capaz de detectar tanto variantes de microcistinas como nodularinas no ambiente aquático. O ensaio ELISA competição utilizando anticorpo policlonal anti-MCLR foi submetido à patente pela Agência USP de Inovação (I.N.P.I. 018090046230).
The contamination of drinking water by cyanobacterial toxins is a public health issue and a concern for water authorities throughout the world. Microcystin-LR (MCLR) is a hazardous cyclic heptapeptide cyanotoxin, which inhibits protein phosphatase PP1 and PP2A in hepatocytes. Microcystins are produced by several genera of cyanobacteria and presents more than 70 structural variations characterized in natural blooms. As haptens, microcystins are unable to invoke an immune response in animals. Consequently, the application of conjugation methods with an additional carrier protein, the KLH (Keyhole Limpet Hemocyanin) was necessary. The main objective of this study was to obtain monoclonal (in mice) and polyclonal (in rabbits) antibodies for reacting against MCLR. In what refers to monoclonal antibodies, 9 hybridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232) were obtained; however only 5 were stables (k29, k317, k248, k284, k2232). These were selected to be isotyped, expanded in ascitic fluid, purified by protein-A column chromatography and then, they were titrated. Out of these five antibody-secretor hybridomas, clone k317 was the best to recognize (more specific) the MCLR toxins. Antibodies in hybridoma cell culture supernatant and purified ascites fluid were identified by ELISA assay (Enzyme Linked Immunosorbent Assay) as prior standardized. Even when sensitizing ELISA plate with different antigens, as MCLR-cBSA, MCLR, MCLR, MCRR, MCYR and MCLA, clone 17 presented the best linearity against microcystin variants. Therefore, the obtained clone 17 (isotype IgG1) is a promising clone and shall be used for detecting MCLR in drinking water through the development of a competitive ELISA immunoassay kit. In what refers to the polyclonal antibody, MCLR-mcKLH was used as immunization antigen, while MCLR-cBSA was used as sensitizing antigen for the IgG titration assay by indirect ELISA. In the sequence, a competition ELISA assay was standardized using the MCLR toxin as sensitizing antigen. This Casein method was standardized, validated and compared to the commercial kit Abraxis®. The competition ELISA kit using polyclonal antibody, known as Casein method, was analyzed concerning its Quantification Inferior Limit, Specificity, Selectivity, methanol influence of the assay, Recuperation, Linearity, Precision, Accuracy and Robustness. This screening method reached excellent results if compared to the commercial kit Abraxis®, for being able to detect both the microcystins variants and the nodularins in aquatic environmental. The competition ELISA assay using anti-MCLR polyclonal antibody was submitted to the grant of a patent by USP Innovation Agency (INPI 018090046230).

>Magister Scientiae - MSc
A novel 3-mercaptopropionic acid capped iridium selenide quantum dots based label free impedimetric immunosensor was successfully constructed. The 3-mercaptopropionic acid capped iridium selenide quantum dots synthesized were studied using HRTEM, revealing the formation of very small sizes, of about 3 nm. The optical Uv-Vis absorption wavelength of the quantum dots is blue-shifted, a phenomenon explained by the effective mass approximation (EMA) for semiconducting materials with sizes below 10 nm. Using cyclic voltammetry it is noted that the quantum dots have interesting electro-catalytical properties. The immunosensor proved to be sensitive towards nodularin, with a very low detection limit of 0.009 ng/mL and is significantly lower than the recent anti-nodularin ELISA kit developed by (Zhou et al., 2011) which has a detection limit of 0.16 ng/mL.Also the dection limit of the immunosensor is below the South African guideline value for microcystin-LR (0-0.8) μg/L (DWAF; 1996). The calibration curve of the 3MPA-GaSe nanocrystal based biosensor was successfully constructed, which exhibited a trend described by Michaelis-Menten, a typical behaviour of enzymatic biosensors. The detection limit of the biosensor is 0.004 nM and is significantly lower than the action limit of 17beta-estradiol, (1.47 x 10-10 M).

Due to heavy nutrient load and adverse climate change the occurrence of toxic cyanobacterial blooms have significantly increased during the last decades. Nodularia spumigena is one of the dominant toxic cyanobacteria which produces massive and inherent blooms in brackish water body, the Baltic Sea, particularly in late summer. Nodularia spp. are known to produce nodularins (NOD) and a range of other bioactive peptides such as spumigins and nodulopeptins, all of which have unclear function. In a recent study, three new nodulopeptins with molecular weight of 899, 901 and 917 were characterised from N. spumigena KAC 66. In the present study, N. spumigena KAC 66 was fractionated by reversed phase flash chromatography and their toxicity was determined by their lethality to Daphnia pulex and D. magna along with inhibition of protein phosphatase 1 assay (PP1). All fractions showed lethality to Daphnids and inhibitory activity against PP1, the toxicity was due to additional compounds as NOD and nodulopeptin 901 were only detected in 7 fractions. Pure NOD was lethal to D. pulex and D. magna LC50= 8.4 μg/mL and 5.0 μg/mL, respectively. The newly characterised nodulopeptin 901 was also tested against D. magna (LC50=>100 μg/mL). NOD and nodulopeptin 901 inhibited PP1 with IC50 0.038 μg/mL and 25 μg/mL, respectively. In common with many studies, the maximum amount of NOD was retained within the cells during the seven week growth experiment. In contrast, as much as ~50% of nodulopeptin 901 was detected in the growth media throughout the duration of experiments. To gain further insight on the effects of environmental stress on growth and production of bioactive metabolites in N. spumigena KAC 66, a range of parameters were investigated which included; temperature, salinity, nitrate and phosphorus. In the present study it was investigated that extreme growth conditions have a considerable effect on biomass and toxin levels by N. spumigena KAC 66. The light intensity ranged from 17.35-17.47 μmol/s/m2, 22°C and 11-20 ‰ of salinity were the optimal growth conditions to obtain maximum biomasses, intra and extracellular peptide contents. At 6.5 mg/L nitrate the maximum growth, as indicated by Chl-a and maximum concentrations of intracellular NOD and nodulopeptin 901 were detected found in week 5 and 4, respectively. Temperature had the greatest effect on peptide production. Whilst growth was similar at 22°C, 25°C and 30°C, increase in temperature had a profound effect on NOD production in that an increase from 22°C to 25°C resulted in a 50% decrease in intracellular NOD levels. At 30°C little or no NOD was detected. In contrast, whilst concentrations of nodulopeptin 901 decreased with increasing temperature, they were still detected at consistent levels suggesting they play an important role. The results from phosphate experiment showed Chl-a, cell biomass and peptide production did not show clear dependency on availability of PO-3 4. This is the first study to evaluate the effects of selected environmental parameters on NOD/nodulopeptin 901 production which ultimately may be helpful to explain the distribution, control of natural blooms and toxin levels of N. spumigena in the Baltic Sea and as well as laboratory based experiments. In an attempt further exploit cyanobacterial diversity, 20 strains were isolated from the Dian Lake and 6 from the Dead Sea. The UPLC-PDA-MS analysis of isolates, Microcystis spp. from Dian Lake, China indicated the presence of several peptides namely MC-LR, cyanopeptolin A and aerucyclamides A-D. These new isolates will be examined for biological activity and chemical characterisation in future studies.

Future climate scenarios in the Baltic Sea project increasing sea surface temperature, as well as increasing precipitation and river runoff resulting in decreased salinity. These changes can severely impact the dynamics and function of brackish water communities, specifically phytoplankton. Phytoplankton are a significant source of organic matter to other trophic levels, and some species can be toxic. Their response to future climate conditions is of great relevance for the health of humans and aquatic ecosystems. The aim of this thesis was to assess the potential for climate-induced changes, such as decreasing salinity, to affect phytoplankton dynamics, physiology and chemical profiles in the Baltic Sea.      Phytoplankton successional patterns in the Baltic Proper consist of a spring bloom where diatoms and dinoflagellates co-occur and a summer bloom dominated by filamentous/colonial cyanobacteria. The consensus is that future warmer conditions will promote filamentous/colonial cyanobacteria blooms. This thesis shows that phytoplankton biomass in the spring bloom was lower in years with milder winters compared with cold winters. This suggests that in terms of annual carbon export to higher trophic levels, loss of biomass from the spring bloom is unlikely to be compensated by summer cyanobacteria. High frequency sampling of phytoplankton performed in this thesis revealed a strong relationship between the dynamics of pico- and filamentous cyanobacteria. Large genetic diversity was found in cyanobacterial populations with high niche differentiation among the same species. At community level, high temperature and low salinity were the main factors shaping the summer cyanobacterial composition. These conditions may promote the predominance of opportunistic filamentous cyanobacteria, e.g. Nodularia spumigena. This species produces various bioactive compounds, including non-ribosomal peptides such as the hepatotoxin nodularin. In this work, N. spumigena subpopulations evolved different physiological strategies, including chemical profiles, to cope with salinity stress. This high phenotypic plasticity ensures survival in future climate conditions. Under salinity stress, some subpopulations displayed shorter filaments as a trade-off. This indicates that the future freshening of the Baltic Sea may promote grazing on filamentous cyanobacteria and modify carbon flows in the ecosystem. In this thesis, Baltic N. spumigena chemotypes and genotypes grouped into two main clusters without influence of geographical origin. Thus, chemical profiling can be used to explore conspecific diversity in closely genetically related N. spumigena subpopulations.      Overall, this thesis has significantly expanded the knowledge on phytoplankton community and population responses to short- and long-term environmental changes, relevant to project the impacts of future climate conditions in the Baltic Sea.

Cyanobacteria can form nuisance proliferations and produce large concentrations of toxins that pose a health hazard. This thesis investigates cyanotoxin production by New Zealand benthic cyanobacteria. Cyanobacteria were sampled from lakes, reservoirs, streams, and rivers. Thirty-five strains were isolated into culture and screened for genes involved in the biosynthesis of common cyanotoxins. Positive results were confirmed and cyanotoxin concentrations quantified using analytical chemistry techniques. Genes involved in anatoxin a/homoanatoxin a biosynthesis were detected in nine out of ten Phormidium cf. uncinatum strains isolated from a single mat. Anatoxin a was confirmed in these strains by LC–MS/MS at concentrations from 0.3 to 6.4 mg kg⁻¹. One strain also produced homoanatoxin-a. Anatoxin-a variation between strains may explain the wide range in anatoxin a concentrations previously observed in New Zealand. The sxtA gene involved in saxitoxin biosynthesis was identified in Scytonema cf. crispum strains. Saxitoxin was confirmed in strains and environmental samples by Jellett PSP Rapid Test and HPLC–FD. Gonyautoxins, neosaxitoxin, and decarbamoyl derivatives were also detected. This study is the first identification of these compounds in Scytonema and in New Zealand cyanobacterial strains. These strains were isolated from recreational and pre-treatment drinking water reservoirs, highlighting the risk benthic cyanobacteria pose to human and animal health. Experiments were undertaken using cultures of Phormidium and Scytonema to determine how growth influences cyanotoxin production. The effects of iron and copper stress on P. autumnale were also investigated. High iron concentrations disrupted attachment mechanisms. Iron and copper had a significant effect on growth, without significantly affecting anatoxin a production. However, the maximum anatoxin a quota was consistently observed during early exponential growth. Scytonema cf. crispum produced higher saxitoxin quota throughout exponential growth than during the stationary phase. Both the Phormidium and Scytonema growth experiments indicate that high toxin quota can be expected early in benthic mat development, making early detection of these proliferations important.

During summer, the Baltic Sea is subjected to the world’s largest cyanobacterial blooms. These blooms are linked to eutrophication and raise many questions concerning their effects on the ecosystem. To understand their impacts on the food web dynamics, it is essential to assess growth responses of grazers to these cyanobacteria. In the northern Baltic proper, copepods are the most important herbivores providing an essential link between the primary producers and higher trophic levels. In this Thesis, Papers I & II evaluate methods to estimate copepod growth in response to feeding conditions in situ. The most conspicuous diazotrophic filamentous cyanobacterium in the Baltic Sea is Nodularia spumigena, a producer of nodularin which is highly toxic to vertebrates, yet its ecological role is largely unknown. In Paper III, reciprocal interactions between cyanobacteria, sympatric algae and copepods are studied. The results suggest that nodularin is likely involved in allelopathic interactions, but it is not an inducible defense against grazers. Furthermore, the results of Papers IV & V, indicate that natural assemblages of N. spumigena and Anabaena spp. may support copepod reproduction and that total diazotrophic filamentous cyanobacteria appear to provide a beneficial feeding environment for the feeding stages of copepod nauplii, most probably by stimulating the microbial communities that nauplii feed upon. Since cyanobacterial blooms are projected to increase due to global climate change, the combined effects of toxic cyanobacteria, ocean acidification and global warming predicted for year 2100 are further investigated on copepods in Paper IV. Taken together, these studies indicate that filamentous diazotrophic cyanobacteria contribute to sustaining secondary productivity and have potential implications of management practices with respect to combating eutrophication, global climate change and sustaining fish feeding conditions.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Nodulation is a symbiosis between plants and bacteria called rhizobia which leads to the formation of a lateral organ called a nodule where nitrogen fixation occurs. Nodulation can be divided into two developmental programmes, rhizobial infection, and nodule organogenesis, both of which are required for nitrogen fixation. In my PhD studies, I focused on the study of rhizobial infection using the Medicago truncatula-Sinorhizobium meliloti model system. NIN is an important transcription factor in the nodulation signalling pathway that functions both in rhizobial infection and nodule organogenesis. Gene expression profiling of nin during rhizobial infection has enabled me to identify its potential downstream targets including several NIN-dependent infection-related genes and, surprisingly, genes involved in mycorrhization that are apparently repressed by NIN. The identification of these genes has provided insight into how NIN functions in rhizobial infection and revealed potential cross-talk between nodulation and mycorrhization pathways. In the course of this work I discovered two infection mutants. One of the mutants, cbs1, was cloned in collaboration with a colleague in the lab. It encodes a Cystathionine Beta-Synthase domain containing protein with a potential role in reactive oxygen species homeostasis. The other mutant, which I named knocks but can’t enter (kce), is blocked at an early stage of infection. I used conventional mapping and next-generation sequencing technologies to genetically map kce to a known rhizobial infection gene LIN. The kce mutant developed nodules with central rather than peripheral vascular bundles resembling nodules from the more ancient Frankia-actinorhizal symbiosis. Using kce and other infection mutants I demonstrate that this abnormal nodule architecture results from a failure of infection to reach the nodule cortex. Based on this finding, I predict that Nod factor activated signalling in the nodule cortex plays a role in determining nodule structure.

La déficience en phosphore est l’un des facteurs limitant la production de légumineuses à graines, dont le haricot (Phaseolus vulgaris). La diversité génétique de cette espèce en Amérique latine a permis d'identifier avec le CIAT, des lignées recombinantes du croisement de DOR364 et BAT477 à fort pouvoir fixateur d'azote (FSN) et à forte efficacité d'utilisation de phosphore (EUP). Les résultats obtenus au cours de ce travail nous ont permis de répondre à notre objectif principal de thèse qui consistait à vérifier la pertinence des critères d'adaptation de la fixation symbiotique de l'azote chez le haricot à la disponibilité de P que nous avons déterminé. Nous avons démontré, d'une part, en milieu hydroaéroponique, que les racines nodulées du haricot, excrètent dans leur rhizosphère une quantité de H+ qui est corrélée à la perméabilité nodulaire. Ce qui laisse penser qu'une part d'H+ excrétée par les racines nodulées est liée à la fixation symbiotique de N2. D'autre part, nous avons démontré que sous déficience en P les lignées tolérantes acidifient plus leur rhizosphère que les sensibles. En multisites les facteurs les plus déterminants de la hiérarchisation des sites sont N-total et P-total. Deux groupes de lignées se distinguent, les tolérantes versus les sensibles à la faible fertilité des sols en P. La lignée locale se distingue des groupes précédents. Elle est capable de croître dans différents sols, notamment les contraignants avec une grande capacité à noduler, par conséquent, à compenser la déficience en N. Nous avons également pu démontrer que les biomasses nodulaire et aérienne sont corrélées au P Olsen du sol
Phosphorus deficiency is one of the limiting factor for grain legume production like bean (Phaseolus vulgaris). The results obtained during this work enabled us to confirm our main aim of this work which is checking the relevance of the criteria of adaptation of the symbiotic nitrogen fixation of bean to P availability. We showed, in controlled conditions that the nodulated roots of bean, release in their rhizosphere a quantity of H+ which is correlated with the nodul permeability. What lets think that a share of H+ released is related to the symbiotic N2 fixing. In addition, we showed that under P deficiency the tolerant lines acidify more their rhizosphere that the sensitive ones. Into multisite trial the most determining factors of the hierarchisation of the sites are total-N and total-P. Two groups of lines are distinguished: tolerant versus sensitive to P deficiency. The local farmer line is distinguished from the studied lines. It is able to grow in various soil fertility, in particular the constraining soils. ones with a great capacity with noduler, consequently, to compensate for deficiency in NR. We also could show that shoot and nodule biomass are correlated with the Olsen-P Olsen of the soil

Phenotypic characterizations demonstrated that diversity among 19 naturalized North American and 5 commercial Kura clover ( Trifolium ambiguum M.B.) rhizobial strains was limited. Growth chamber and field evaluations indicated the superiority of North American isolates, increasing foliage accumulation by 30% when compared to commercial inoculant strains. Nitrogen fertilization, however, produced greater accumulations in all evaluations. Genetic diversity among 128 isolates from the lower Caucasus was significant. Nodulation specificity of rhizobia from the lower Caucasus was demonstrated to be more complex than what was reported in the literature, as plants of different ploidy levels and even of different species were, in some cases, nodulated by the same isolate. Specificity of a given rhizobial strain varied depending on the isolate. This study identified naturalized North American rhizobial isolates that are more efficient than currently used commercial strains and increased the genetic diversity of Kura clover rhizobia currently available.

Um novo ciclo de tratamento térmico denominado como têmpera e partição vem sendo desenvolvido em aços com elevados teores de silício, como rota para obtenção de estruturas com frações consideráveis de austenia retida. Essa rota de tratamento m térmico consiste em realizar uma têmpera temperaturas intermediárias entre Ms e Mf, seguido de um reaquecimento com manutenção em patamares isotérmicos por certos intervalos de tempo, objetivando estabilizar a austenita remanescente através da partição do carbono a partir da martensita supersaturada. No presente trabalho, duas ligas de ferros fundidos nodulares convencionais, com diferentes teores de silício e manganês, foram submetidas a ciclos de têmpera e partição. As amostras foram austenitizadas a 900°C por duas horas. Uma das ligas foi temperada em óleo a 160°C e a outra a 170°C por 2 minutos. Imediatamente após a têmpera as amostras foram reaquecidas em temperaturas entre 300 e 450°C por intervalos de tempo que variaram entre 2 e 180 minutos. A caracterização microestrutural foi realizada através de microscopia eletrônica de varredura (MEV) e difração de raios x. A caracterização mecânica foi feita através de ensaios de energia absorvida ao impacto, dureza HRC e ensaios de tração. A caracterização microestrutural evidenciou que os ciclos de têmpera e partição são viáveis na obtenção de frações consideráveis de austenita retida nos ferros fundidos nodulares. A caracterização mecânica evidenciou que foi possível obter boas combinações de energia absorvida ao impacto, resistência à tração e alongamento. Em todas as condições testadas é possível perceber uma janela de processo bem definida caracterizada por valores crescentes das propriedades mecânicas nos primeiros minutos do ciclo de partição e que decrescem após certo intervalo de tempo. O conjunto de propriedades mecânicas obtidas através dessa rota de tratamentos térmicos indica que os ferros fundidos nodulares submetidos ao ciclo de têmpera e partição podem se constituir como alternativa tecnológica para aplicações comerciais nas quais os ferros fundidos nodulares austemperados já são materiais consolidados.
A new heat treatment cycle known as quenching and partitioning has been developed in commercial steel alloys containing silicon as a way to obtain structures with controlled fractions of retained austenite. This heat treatment cycle consists in performing a quenching in temperatures between Ms and Mf, followed by a reheating with isothermal holding by different time intervals. The aim of this cycle is to achieve the austenite stabilization by diffusion of carbon from the supersaturated plates of martensite. In this work, two conventional ductile cast iron alloys, with two different contents of silicon and manganese were heat-treated in quenching and partitioning cycle. The samples were austenitized at 900°C for two hours, followed by quenching in oil at 160° C and 170° C for two minutes. Immediately after quenching, the samples were reheated at temperatures between 300 and 450°C for time intervals between 2 and 180 minutes. The microstructural characterization was performed using electronic microscopy (SEM) and x-ray diffraction. The mechanical characterization was performed using impact tests, hardness and tensile strength tests. The microstructural characterization showed that the cycles of quenching and partitioning are viable to obtain considerable fractions of retained austenite in nodular cast by this heat treatment route. The mechanical characterization showed that it was possible to obtain good combinations of energy absorbed in the impact, tensile strength and elongation. In all tested conditions was possible to perceive a well-defined process window characterized by increasing values of mechanical properties in the first minutes of the partitioning step, and decrease after certain time intervals. The set of mechanical properties obtained by this route of heat treatments indicates that nodular cast iron subjected to tempering and partitioning cycle can be constituted as an alternative technology for commercial applications in which austempered ductile irons are already consolidated materials.

Plusieurs especes de legumineuses tropicales appartenant au genre aeschynomene portent des nodules fixateurs d'azote a la fois sur leurs racines et sur leur tige. Ces especes peuvent etre classees en trois groupes d'inoculation croisee. L'infection de la tige d'a. Afraspera a lieu au niveau de sites de nodulation predetermines selon un processus d'infection directement intracellulaire qui ne fait intervenir ni poils absorbants, ni cordons d'infection. La plupart des rhizobia isoles des nodules caulinaires sont capables de fixer l'azote moleculaire en cultures pures. L'etude taxonomique de ces rhizobia a permis de les classer dans le genre bradyrhizobium. Sur le plan des applications agronomiques, les potentialites de fixation d'azote considerables de ces plantes permettent leur utilisation comme engrais vert en riziculture irriguee. Les rendements en riz ont pu etre doubles dans des essais en microparcelles. De plus, la fixation d'azote par les nodules caulinaires n'est pas inhibee par l'azote combine du sol

The production of ductile cast iron and austempered ductile cast iron - ADI- usually involves an inoculation 0.03-0.06% Magnesium and the presence of Chromium, Nickel, Copper and Molybdenum. The addition of Niobium is not common. Reports in theliterature show that Niobium leads to improved mechanical properties of the ductile cast iron and recent findings indicate that the 0.5% Niobium increases its strength and impact strength, but decreases its hardness in relation to a ductile iron without Niobium. The process of production of cast iron with Niobium is difficult, according to the literature, due to the presence of carbon at higher levels, which are typical of this alloy. This study presents a methodology for the production of cast iron with Niobium and analyzes the effects of the addition of 0.23%, 0.47%, 0.67% and 0.85% Niobium to a ductile cast iron in their microstructures, tensile strength, yield strength and elongation, impact resistance, wear and fatigue properties. The study showed that the volume fraction of perlite increases and the number of spheroidal graphite decreases as the content of Niobium is increased, leading to an increase in tensile strength and hardness of these materials. These is also an increase in the tensile ductility, impact resistance and elongation for an addition of 0.23% Niobium. For austempered ductile cast iron with niobium, the results indicate a preponderance of the influence of the matrix compared to the influence of the addition of Niobium.
A produção de ferros fundidos nodulares e de ferros fundidos nodulares austemperados ADI- geralmente envolvem uma inoculação e nodulização de 0,03-0,06% de Magnésio e a presença de Cromo, Níquel, Cobre e Molibdênio. A adição de Nióbio não é comum. Os relatos na literatura mostram que o Nióbio leva a melhores propriedades mecânicas do ferro fundido nodular e, resultados recentes indicam que 0,5% de Nióbio aumenta a resistência ao impacto, mas diminui sua dureza, em relação a um ferro fundido nodular comum. O processo de produção de ferros fundidos com Nióbio é dificultado, segundo dados da literatura, devido à presença de Carbono em teores mais elevados, que são típicos dessa liga. Este estudo apresenta uma metodologia de produção de ferros fundidos com Nióbio e analisa os efeitos da adição de 0,23%, 0,47%, 0,67% e 0,85% de Nióbio a um ferro fundido nodular, nas suas microestruturas, resistência à tração, limite de escoamento e alongamento, resistência ao impacto, propriedades de desgaste e fadiga. O estudo mostrou que a fração de volume de perlita aumenta e o número de esferóides de grafita parece diminuir à medida que o teor de Nióbio é aumentado, conduzindo a um aumento na resistência à tração e dureza destes materiais, associado a valores importantes de alongamento, impacto e fadiga. A resistência ao desgaste também aumenta discretamente à medida que se aumenta o teor de Nióbio. Para o ferro fundido nodular austemperado com Nióbio, os resultados indicam uma preponderância da influência da matriz em relação à adição de Nióbio para quase todas as propriedades estudadas.

Les plantes bénéficient d’une plasticité développementale assurée par des zones méristématiques actives tout au long de leur cycle de vie afin de s’adapter aux contraintes environnementales. Les parties aériennes et les racines sont chacune soumises à des stimuli environnementaux indépendants qui doivent être intégrés au niveau de la plante entière afin d’assurer une coordination du développement de ces différents organes, impliquant des échanges de signaux à longue distance, dits systémiques. Chez les légumineuses, le système racinaire est non seulement capable de se ramifier en racines latérales, mais est aussi capable de former de nouveaux organes, les nodules fixateurs d’azote atmosphérique, grâce à une symbiose avec des bactéries, appelées rhizobia, lorsque le sol est pauvre en azote minéral. La formation et le maintien de ces nodules sont cependant couteux en énergie et doivent donc être strictement régulés par la plante via des signalisations hormonales afin d’inhiber ou de favoriser la nodulation selon ses besoins. Des hormones peptidiques de la famille CLE (« Clavata3 (CLV3)/Embryo Surrounding Region ») et CEP (« C-terminally Encoded Peptide »), produites dans les racines, ont notamment été identifiées comme jouant respectivement un rôle systémique négatif ou positif sur la nodulation, de manière dépendante des récepteurs SUNN (« Super Numeric Nodules ») et CRA2 (« Compact Root Architecture 2 ») dans les parties aériennes, respectivement. La problématique de cette thèse était donc de comprendre comment des peptides de signalisation régulent à la fois localement et systémiquement la nodulation. A cette fin, deux objectifs ont été définis : 1) identifier des mécanismes moléculaires associés aux voies de signalisation systémiques CLE/SUNN et CEP/CRA2 régulant la nodulation ; 2) caractériser de nouveaux peptides CLE régulant la nodulation. Dans un premier temps, j’ai participé à démontrer que les voies systémiques CLE/SUNN et CEP/CRA2 agissent indépendamment pour réguler de manière antagoniste la nodulation. Dans un second temps, des acteurs racinaires en aval de la voie CLE/SUNN ont été identifiés, révélant un lien avec les récepteurs des molécules signales des rhizobia, les NFs (« Nod Factors »), ainsi qu’avec des protéines à « F-box » nommées TML1 (« Too Much Love 1 ») et TML2, et un rôle négatif de ces protéines TML sur la nodulation a pu être caractérisé. Enfin, un acteur commun agissant en aval des voies CLE/SUNN et CEP/CRA2 a été identifié : le miR2111. Ce microARN produit dans les parties aériennes est régulé de manière opposée par les deux voies systémiques, et clive les transcrits des gènes TMLs dans les racines, contrôlant ainsi le nombre de nodules formés en fonction de la disponibilité en azote et de la préexistence de nodules. Le deuxième objectif de thèse a permis de révéler l’existence d’un peptide CLE dont l’expression est induite dans l’épiderme racinaire en conditions symbiotiques, CLE37. Ce gène code pour un peptide TDIF (« Tracheary Element Differentiation Inhibitory Factor »), et des approches par ARN interférence et de mutagénèse ont révélé que ce gène est requis pour moduler la croissance racinaire en réponse aux rhizobia, inhibant la croissance des racines principales et augmentant le diamètre racinaire. Ainsi, le recrutement de ce peptide de signalisation pourrait permettre d’accommoder le développement racinaire pour soutenir les besoins de la symbiose fixatrice d’azote
As sessile organisms, plants benefit of a high developmental plasticity through the maintenance of active meristems during their whole life cycle to adapt to environmental constraints. Shoots and roots are subjected to independent environmental stimuli that must be integrated at the whole plant level to coordinate growth and development of these different organs, implying the existence of endogenous systemic (long-distance) signals. Legume plants besides forming lateral roots, are also able to form new root organs, the nitrogen-fixing nodules, thanks to a symbiotic interaction with bacteria called rhizobia when mineral nitrogen availability is limited in the soil. However, formation and maintenance of nodules is energetically costly and must be tightly controlled by the host plant through hormonal signalling pathways in order to inhibit or to promote nodulation depending on its needs. Peptide hormones from the CLE (Clavata3 (CLV3)/Embryo Surrounding Region) and CEP (C-terminally Encoded Peptide) families, produced in roots, were notably identified as regulating systemically nodulation, respectively negatively and positively, depending on the SUNN (Super Numeric Nodules) and CRA2 (Compact Root Architecture 2) receptors in shoots, respectively. The problematic of this Ph.D. thesis was therefore to investigate how signalling peptides regulate nodulation locally and systemically. To this end, two objectives were defined: 1) to identify molecular mechanisms linked to the CLE/SUNN and CEP/CRA2 systemic pathways; 2) to characterize new CLE peptides regulating nodulation. First, I participated in demonstrating that the CLE/SUNN and CEP/CRA2 systemic pathways act independently to regulate antagonistically nodulation. Then, molecular effectors acting in roots downstream of the CLE/SUNN pathway were identified, revealing a link with NFs (Nod Factors) receptors and with F-box proteins called TML1 (Too Much Love 1) and TML2, and both TML proteins were shown to regulate negatively nodulation. Finally, a shared downstream actor of the CLE/SUNN and CEP/CRA2 pathway was identified: the miR2111. This microRNA, produced in shoots, is regulated antagonistically by the two systemic pathways, and cleaves TML transcripts in roots, therefore controlling nodule number depending on nitrogen and rhizobial cues. The second Ph.D. thesis objective allowed highlighting a CLE peptide which expression is induced in the root epidermis by symbiotic conditions: CLE37. This gene encodes a TDIF (Tracheary Element Differentiation Inhibitory Factor) peptide, and RNA silencing as well as mutagenesis approaches revealed that it is required to modulate root development in response to rhizobia, inhibiting primary root growth and enhancing stele root diameter. The recruitment of the CLE37 signalling peptide could thus allow accommodating root development to support symbiotic nitrogen fixation needs

The Rhizobium-legume symbiosis is an opportunistic association between two symbiotic partners that results in the formation of the root nodule. The process depends on the expression of a number of plant and bacterial genes that are considered critical for the establishment and maintainance of the symbiotic state. The merits of a mutational approach to the analysis of symbiosis have been recognized for several years and transposon Tn5 mutagenesis of Rhizobium has led to the identification of several symbiotic genes. This study describes the use of Tn5 mutagenesis for the isolation of symbiotically defective mutants of Bradyrhizobium japonicum. Two classes, auxotrophic and cell surface-altered mutants defective in nodule formation, have been characterized. In B. japonicum USDA 122, histidine auxotrophs that are defective in nodulation have been studied. The mutagenized DNA region has been cloned and the wild-type DNA region isolated by hybridization and complementation. In B. japonicum 61A76, Tn5-induced cell surface-altered mutants have been isolated by selecting for bacteriophage resistance. Several parameters have been used to demonstrate alterations in cell surface components. It has been shown that the Tn5 insertion is not the primary cause of the mutation in two of the characterized mutants. Complementation tests have led to the isolation of a wild-type DNA-containing cosmid, pPS23A, that overcomes the symbiotic defect in one of the mutants. Analysis of the cell surface showed a partial restoration of surface components in the complemented mutant.

Five herbicides commonly used for suppression of weed growth in white clover seed crops were tested for toxicity against white clover (Trifolium repens), Rhizobium trifolii and the nitrogen fixing symbiosis formed between these two organisms. Trials were carried out on R.trifolii on solid and in liquid media to determine if growth of this bacterium was affected by the presence of the 5 herbicides. Paraquat and MCPB substantially inhibited bacterial growth on solid medium. Bentazone, fusilade and kerb caused very small zones of growth inhibition of R.trifolii on solid agar at high concentrations. None of the herbicides tested affected growth of R.trifolii in liquid culture. In vitro studies of herbicide toxicity toward white clover were carried out to identify interactions of herbicide activity with rhizobial inoculation and supplied nitrogen, and to attempt to identify the targets of herbicide activity. Nodules grown under in vitro conditions were excised and used for ultrastructural examination. Herbicides were applied to plants grown in vitro at two different stages of plant growth. White clover proved to be very sensitive to all herbicides at the early seedling stage. Three week old plants were more tolerant. Pot experiments in a glasshouse environment indicated the relevance of in vitro experiments of herbicide toxicity against plants and gave information on the effect of soil water levels on herbicide activity. Paraquat was extremely toxic to white clover both in vitro and in pot experiments. Nodulation is affected to some extent directly by this herbicide although dessication of foliage probably has some role in halting activity of the nitrogenise enzyme. MCPB caused severe deformation of root tissue both in vitro and in pot experiments. It must be either contaminated with the active form of this herbicide, MCPA, or is being broken down to the active form by bacterial or chemical action. Bentazone did not damage white clover or nodule activity in a consistent way in vitro. However this herbicide did have a deleterious effect on both plant weight and nodulation when applied to white clover grown in soil, particularly under conditions of low soil moisture. Fusilade showed a direct effect on the activity of nitrogenase in vitro. Fusilade also acted more severely against plants of higher nutritional status, and appeared to affect nodule activity directly rather than affecting nodules via damage to other plant parts. Kerb was very toxic to seedling white clover in vitro although older plants were not as susceptible and were stimulated by high concentration of kerb. In pots white clover was slightly inhibited by kerb at recommended concentration while 10 x this concentration did not cause any inhibition of nodulation or plant growth. Differences in results between in vitro and pot studies of toxicity of these herbicides to white clover appear to be due to the different application methods used. In vitro herbicides were applied to the whole plant while in pot experiments herbicides were foliarly applied, hence more uptake by roots would be expected. Pot experiments indicated that changes in nodulation generally reflected changes in plant growth rather than an independent activity of the herbicide on nodulation.

Title from PDF of title page (University of Missouri--Columbia, viewed on February 24, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Gary Stacey. Vita. Includes bibliographical references.

La symbiose qui s'etablit entre les legumineuses et les rhizobia conduit au developpement d'un nouvel organe racinaire, la nodosite, dont la morphogenese necessite l'expression de genes vegetaux specifiques. Le gene enod40 s'exprime dans les cellules corticales en division des l'initiation du primordium nodulaire et code pour un arn a petites orf. Ce travail de these contribue a l'analyse de l'action du gene enod40 lors de la nodulation. Nous avons montre qu'en situation de carence azotee et en absence de rhizobia, la surexpression d'enod40 induisait la dedifferenciation et la division des cellules du cortex racinaire. De plus, les plantes transgeniques m. Truncatula surexprimant enod40 ont presente, lors de leur interaction avec les bacteries rhizobia, une acceleration de leur cinetique de nodulation, et lors de leur interaction avec le champignon glomus mosseae, un nombre accru de mycorhizes. L'analyse de deux lignees transgeniques presentant de la cosuppression a montre une correlation entre la non-expression d'enod40 et une reduction du nombre de nodosites par plante ainsi qu'une perturbation de leur developpement. Nous avons aussi cherche a determiner la forme moleculaire du produit du gene et a analyser les mecanismes moleculaires mis en jeu lors de son action. Par microbombardement et par fusions traductionnelles, nous avons montre que l'arn enod40 possedait au moins 2 regions biologiquement actives. L'activite de la region 5 de l'arn enod40 serait liee a la traduction d'une petite orf de 13 acides aminees, tandis que l'activite de la region 3 serait liee a la traduction d'une autre orf de 27 acides aminees chez m. Truncatula. Enod40 participerait donc au controle de la formation et de la differenciation des organes symbiotiques au sein du cortex racinaire. Il ferait partie d'une nouvelle classe de regulateurs de croissance chez les plantes. La caracterisation d'un tel gene ouvre de nouvelles perspectives sur la signalisation chez les vegetaux.

Nous avons réalisé la synthèse de précurseurs d'analogues sulfures de facteurs NOD, messagers chimiques intervenant lors du phénomène de nodulation rencontré dans l'association symbiotique entre certaines plantes légumineuses et des bactéries de la famille rhizobium. Cette association permet à la plante de fixer l'azote atmosphérique préalablement métabolisé par les bactéries. Ces analogues devraient permettre de mieux comprendre les différents processus intervenant au cours de la symbiose plante/ bactérie ainsi qu'étudier l'hydrolyse des facteurs NOD par des enzymes, les chitinases. Nous avons choisi de préparer des analogues fonctionnalisés pour lersquels seule la liaison O-glycosidique entre deux unités serait remplacée par une liaison thioglycosidique. Grâce à l'utilisation de donneurs de glycosyle sous la forme de trichloroacétimidate, nous avons réalisé la synthèse d'un thiotrisaccharide ainsi que celle d'un thiotétrasaccharide, précurseur d'analogues de facteurs NOD, dont les unités réductrices et non réductrices sont potentiellement différenciées en vue de leur fonctionnalisation. Par ailleurs, le transfert d'une unité de glucosamine sur un thiodisaccharide, à l'aide de la bétâ-1,4-galactosyltranférase, a conduit à la préparation d'un thiotrisaccharide. Cette dernière étape a permis d'envisager la synthèse d'un thiotétrasaccharide par voie chimio-enzymatique. Ces précurseurs devraient conduire à de nouveaux analogues sulfures de facteurs NOD sur lesquels des tests biologiques pourraient être réalisés afin d'évaluer l'activité de nodulation ainsi que la résistance aux chitinases

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Existem dois níveis de bauxitas distintos no platô Miltônia-3 localizado na Província Bauxitífera de Paragominas-PA. Estes níveis estão separados por um horizonte de laterita ferruginosa (LF) pseudopsolítica a concrecionária que marca um hiato entre dois ciclos distintos de formação do perfil bauxítico atual. As bauxitas do nível superior (2° ciclo de formação) possuem características nodulares a concrecionárias e as do nível basal (1° ciclo de formação) composto por uma bauxita concrecionária (BC) mais íntegra fisicamente somado a outro nível de bauxita mais friável com porções argilosas para a sua base (BCBA). Percebeuse tratar da Bauxita Nodular Concrecionária (BNC) localizada no nível superior, de horizonte enriquecido em gibbsita, com baixos teores de sílica reativa e ferro, teores esses muito semelhantes àqueles encontrados no horizonte do principal minério de bauxita (BC) do perfil. Em observações de campo, nas frentes de lavra e nos testemunhos de sondagem constatou-se que esta BNC é uma gradação do horizonte de Bauxita Nodular (BN) acima. Essa gradação é observada pelo aumento do tamanho dos nódulos bauxíticos, onde os seus pseudopisólitos Fe-gibbsíticos intercrescem por coalescência, diminuindo os teores de ferro e sílica disseminados marcados pela mudança de coloração, passando de amarelo-lilás, até atingir uma coloração vermelho-alaranjado a ocre, à medida que se avança na profundidade. Nota-se também marcante diminuição até o completo desaparecimento dos pseudopisólitos Al-ferruginosos, além da diminuição do volume de argila gibsítica-caulinítica neste nível. Com base nos estudos como petrografia macroscópica e microscópica, MEV/EDS, DRX e análises químicas, além de Análise de Componentes Principal (ACP) e estatística descritiva, foram desenvolvidas duas propostas de modelos de evolução sobre a gênese do nível superior de bauxitas nodulares deste depósito laterítico-bauxítico, considerando: Modelo (1) - Origem a partir da degradação das bauxitas originais (1º Ciclo), relacionadas a um 2º Ciclo de Lateritização que consiste na preexistência da bauxita matura (BC), sobreposto pela LF, que foi recoberto por “Argila de Belterra”. Este novo nível nodular imposto (BN), ocorre pelo processo de coalescência onde houve a junção da fase aluminosa residual, resultante da migração do Fe e Si em solução para fora deste nível e pela migração do Al dos níveis vizinhos acima do capeamento (CAP) e abaixo deste de LF e BC, formando e concentrando em larga escala a gibbsita preferencialmente e secundariamente a caulinita. Com a contínua evolução deste nível de BN, observa-se um amadurecimento da porção basal deste nível, formando a BNC cujos nódulos estão intercrescidos, se conectando localmente, consumindo os níveis vizinhos acima da BN e os níveis abaixo de LF e BC, até o total consumo destes; Modelo (2) - Sua origem a partir de um 2º Ciclo de Lateritização, porém a partir de uma deposição sedimentar posterior sobre o perfil laterítico do 1º Ciclo. Com a exposição de uma rocha fonte como um plúton granitóide (Granito Cantão, Japiim, Jonasa, Ourém e Ney Peixoto do Neoproterozóico), gnaisse (embasamento cristalino arqueano) ou sedimentos siliciclásticos (Formações Itapecuru e Ipixuna do Cretáceo Superior), cuja degradação intempérica possibilitou a geração de sedimentos de natureza argilosa preferencialmente caulinítica durante o Paleógeno até o início do Oligoceno?. Houve a migração de Fe, Si, Ca, Na, etc. para fora deste nível, preservando e concentrando in situ o Al e O, além do Si residual. O processo de coalescência permitiu a junção da fase aluminosa residual, concentrando preferencialmente gibbsita e secundariamente caulinita, fechando o primeiro ciclo de formação de bauxita. Em seguida houve um soerguimento regional, seguido por processos erosivos que possibilitaram a exposição deste perfil bauxítico anteriormente formado, sob clima sazonal, com abundância de água meteórica e intensa insolação se intercalando, onde se desenvolveu a LF, de ocorrência regional marcando um hiato entre os ciclos de formação destas bauxitas. Nova movimentação regional de rebaixamento, que possibilitou a deposição de sedimentos de origem siliciclástica, que serviram de rocha fonte para um novo ciclo de formação de bauxita durante o Mioceno Superior. Podem ser as mesmas rochas cuja degradação física e química forneceram os sedimentos para o 1° ciclo de formação de bauxitas. Repetindo o processo de coalescência da fase aluminosa residual, com o desenvolvimento em larga escala preferencialmente da gibbsita e secundariamente caulinita, fechando o segundo ciclo de formação de BN e BNC.
There are two distinct levels of bauxites on the Miltônia-3 plateau located at the Bauxite Province of Paragominas-PA. These levels are separated by a pseudopsolitic to concretionary ferruginous laterite (FL) horizon, marking a hiatus between two distinct cycles of the current bauxite profile formation. The bauxites of the upper level (2nd cycle of formation) have nodular to concrete characteristics whereas those of the basal level (1st cycle of formation) are composed by a more physically complete concrete bauxite (CB) added by another level of a more friable bauxite with clayey portions for its base (concrete bauxite with clayey bauxite - CBCB). It was noticeable the CNB located at the upper level of gibbsite-enriched horizon with low reactive silica and iron contents, which are very similar to those found on the horizon of the main bauxite ore (CB) of the profile. In field observations, on the survey fronts and in the drill holes it was found that this CNB is a gradation of the above Nodular Bauxite (NB) horizon. This gradation is observed by the increase in the size of the bauxite nodules, where their Fe-gibbsite pseudopsolites grows up by coalescence, decreasing the diffused iron and silica contents marked by the change in color from lilac-yellow to a red-orange color, to ocher, in higher depths. It is also noticeable a decrease until the complete disappearance of the Al-ferruginous pseudopsolites, in addition to the decrease of the volume of gibsytic-kaolinite clay at this level. Based on this study using macroscopic and microscopic petrography, SEM/EDS, XRD and chemical analysis, as well as Principal Component Analysis (PCA) and descriptive statistics, two evolution model proposals were developed on the genesis of the upper level of nodular bauxites of this lateritic-bauxite deposit, considering: Model (1) - Origin from the degradation of the original bauxites (1st Cycle), related to a 2nd Lateritization Cycle which consists of the preexistence of mature bauxite (CB), overlapped by FL, which was covered by "Belterra Clay". This new nodular level (NB) occurs through the coalescence process whereby the residual aluminous phase junction occurred, resulting from the migration of Fe and Si in solution out of this level and by the migration of the neighboring levels above the clayey overburden (CAP) and below that of FL and CB, forming and concentrating large scale gibbsitepreferably and secondarily to kaolinite. With the continuous evolution of this level of NB, a maturation of the basal portion of this level is observed, forming the CNB whose nodules are interincreased, connecting locally, consuming neighboring levels above NB and levels below FL and CB, up to the total consumption of these; Model (2) - Its origin from a 2nd Lateritization Cycle, however from a later sedimentary deposition on the lateritic profile of the 1st Cycle. With the exposure of a source rock as a granitoid pluton (Cantão, Japiim, Jonasa, Ourém and Ney Peixoto of Neoproterozoic granites), gneiss (Archaean crystalline basement) or siliciclastic sediments (Itapecuru and Ipixuna Formations of the Upper Cretaceous), whose weathering degradation made it possible the generation of sediments of clayey nature preferentially kaolinite during the Paleogene until the beginning of the Oligocene. Migration of Fe, Si, Ca, Na, etc. occurred outside this level, preserving and concentrating the Al and O in situ, in addition to the residual Si. The process of coalescence allowed for the addition of the residual aluminous phase, preferentially concentrating the gibbsite and secondarily kaolinite, closing the first cycle of bauxite formation. Thereafter, there was a regional upwelling, followed by erosive processes that allowed for the exposure of this previously formed bauxite profile, under a seasonal climate, with an abundance of meteoric water and intense intercalated insolation, where the FL developed, of regional occurrence marking a hiatus between the formation cycles of these bauxites. New regional retraction movement, which allowed for the deposition of sediments of siliciclastic origin, which served as source rock for a new bauxite formation cycle during the Upper Miocene. They may be the same rocks from which physical and chemical degradation provided sediments for the 1st cycle of bauxite formation. Repeating the coalescence process of the residual aluminous phase, with the large scale development preferably of the gibbsite and secondarily kaolinite, closing the second cycle of NB and CNB formation.

Afin de mieux comprendre le mécanisme de reconnaissance des facteurs de nodulation par les légumineuses, événement déclencheur du processus de fixation de l’azote, une série d’analogues des facteurs de nodulation associés à la luzerne et à la vesce a été préparée. Ces analogues sont préparés en utilisant une méthode chimioenzymatique, qui consiste à produire le squelette oligosaccharidique par des cellules E. Coli recombinantes et à venir fixer la chaîne lipidique par voie chimique. Dans un premier temps, des analogues non hydrolysables par les amidases du sol ont été synthétisés en remplaçant la fonction amide par une fonction amine. Les produits présentent une activité en nodulation, laissant penser qu'il ne se produit pas un clivage entre la chaîne lipidique et le sucre lors du processus de reconnaissance. Cependant, la perte d'activité observée suggère que la fonction amide interagit d'une façon ou d'une autre avec un éventuel récepteur. Dans un deuxième temps, nous nous sommes orientés vers une méthode plus systématique pour l'étude du rôle de la structure de la chaîne, en utilisant les réactions à multi-composants de type Ugi ou Passerini. Celles-ci feraient intervenir l'isonitrile du squelette oligosaccharidique, un aldéhyde, un acide et, dans le cas de la réaction d'Ugi, une amine. En faisant varier la nature de ces trois derniers composés, on devrait pouvoir arriver rapidement à un grand échantillonnage d’analogues. Les essais de couplage réalisés sur la glucosamine se sont révélés encourageants laissant prévoir la possible extrapolation au tétramère
The process of nitrogen fixation by leguminous plants is initiated by the exchange of signal compounds: flavonoids secreted by the plant and nodulation factors (Nod factors) secreted by the bacterium. Nod factors consist in a short chitin oligosaccharidic backbone (typically tetra or pentameric) that is N-acylated at the non-reducing end by a fatty acid. Ln order to understand the role of the structural elements of the bacterial molecule (the nodulation factor) that are involved in the nodulation induction, we have prepared analogs able to trigger the organogenesis in the plant. The focus is on the symbiotic relationship between alfalfa or vetch and their specific rhizobia. The tetrameric backbone was produced by the appropriate E. Coli recombinant cells. The first type of analogs are lipo-chitooligosaccharides in which the fatty-acid is fixed on the sugar via an amine. The sulfated compounds were tested on alfalfa and proved to be still active in nodulation induction, suggesting that there is no cleavage of the fatty-acid during the recognition process. However a decrease of activity seems to prove the influence of the amide group in the recognition process. In a second time, we considered the synthesis of various analogs with modified lipid chains by a method using multi-component reactions such as Passerini and Ugi reactions. Preliminary experiments with glucosamine derivatives are very promising and extrapolation to the tetrameric compounds are in progress

The microsegregation of Mn, Cu, Cr, Mo, Ni and Si has been measured in cast ductile iron and in ductile iron which has been quenched when partially solidified. Effective segregation coefficients have been determined for each of the elements, and used to calculate the segregation on the basis of the Scheil equation. The calculated values agree reasonably well with the values of the solute concentration as a function of the solid fraction measured in quenched samples. The microstructure of the solid phases during the solidification of ductile iron has been observed. Solidification of eutectic ductile iron begins with the independent nucleation of austenite and graphite in the melt. Later the graphite nodules are enveloped by austenite, and further solidification takes place by the thickening of the austenite layers enveloping the graphite. Isolated pockets of interdendritic melt are the last material to solidify. On the basis of the measured segregation of the different alloying elements, the mechanisms by which the segregation affects the microstructure are considered, and an explanation for the effect of segregation on the hardenability of ductile iron is proposed. A mathematical model of the solidification of eutectic ductile iron is formulated which includes heat flow, nucleation and growth of graphite nodules, and the segregation of Si. The model uses equilibrium temperatures given by the ternary Fe-C-Si equilibrium diagram. Using the mathematical model, cooling curves, nodule count and nodular size distribution are determined as a function of position in the casting sample. The results are compared to measured temperatures, nodule count and nodule size in rod castings of 12.5, 20 and 43mm radius. There is good agreement between the calculated and measured values for the 43mm radius rod, and not quite good agreement for the rods of smaller radii. The changes in solidification predicted by the model when some solidification parameters are varied are consistent with experimental observations with the same variation in the parameters.
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