Muscarinic toxins : characterization of adrenoceptor binding and applicability of membrane anchoring via glycosylphosphatidylinositol tail

Adrenoceptors (ARs), G-protein coupled receptors (GPCRs) at the plasma membrane, respond to endogenous catecholamines noradrenaline and adrenaline. These receptors mediate several important physiological functions being especially important in the cardiovascular system and in the regulation of smooth muscle contraction. Impairments in the function of these receptors can thus lead to severe diseases and disorders such as to cardiovascular diseases and benign prostatic hyperplasia. The Eastern green mamba (Dendroaspis angusticeps) venom has been shown to contain toxins that can antagonize the functions of GPCRs. The most well-known are muscarinic toxins (MTs) targeting muscarinic acetylcholine receptors (mAChRs) with high affinity and selectivity. However, some reports have indicated that these toxins might also act on the α1- and α2-ARs which can be divided into various subtypes; the α1-ARs to α1A-, α1B- and α1D-ARs and α2-ARs to α2A-, α2B- and α2C-ARs. In this thesis, the interaction of four common MTs (MT1, MT3, MT7 and MTα) with the adrenoceptors was characterized. It was also evaluated whether these toxins could be anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) tail. Results of this thesis reveal that muscarinic toxins are targeting several α-adrenoceptor subtypes in addition to their previously identified target receptors, mAChRs. MTα was found to interact with high affinity and selectivity with the α2B-AR whereas MT7 confirmed its selectivity for the M1 mAChR. Unlike MTα and MT7, MT1 and MT3 have a broad range of target receptors among the α-ARs. All the MTs characterized were found to behave as non-competitive antagonists of receptor action. The interaction between MTα and the α2B-AR was studied more closely and it was observed that the second extracellular loop of the receptor functions as a structural entity enabling toxin binding. The binding of MTα to the α2B-AR appears to be rather complex and probably involves dimerized receptor. Anchoring MTs to the plasma membrane did not interfere with their pharmacological profile; all the GPI-anchored toxins created retained their ability to block their target receptors. This thesis shows that muscarinic toxins are able to target several subtypes of α-ARs and mAChRs. These toxins offer thus a possibility to create new subtype specific ligands for the α-AR subtypes. Membrane anchored MTs on the other hand could be used to block α-AR and mAChR actions in disease conditions such as in hypertension and in gastrointestinal and urinary bladder disorders in a cell-specific manner and to study the physiological functions of ARs and mAChRs in vivo in model organisms.

Improving the competitiveness of electrolytic Zinc process by chemical reaction engineering approach

This doctoral thesis describes the development work performed on the leachand purification sections in the electrolytic zinc plant in Kokkola to increase the efficiency in these two stages, and thus the competitiveness of the plant. Since metallic zinc is a typical bulk product, the improvement of the competitiveness of a plant was mostly an issue of decreasing unit costs. The problems in the leaching were low recovery of valuable metals from raw materials, and that the available technology offered complicated and expensive processes to overcome this problem. In the purification, the main problem was consumption of zinc powder - up to four to six times the stoichiometric demand. This reduced the capacity of the plant as this zinc is re-circulated through the electrolysis, which is the absolute bottleneck in a zinc plant. Low selectivity gave low-grade and low-value precipitates for further processing to metallic copper, cadmium, cobalt and nickel. Knowledge of the underlying chemistry was poor and process interruptions causing losses of zinc production were frequent. Studies on leaching comprised the kinetics of ferrite leaching and jarosite precipitation, as well as the stability of jarosite in acidic plant solutions. A breakthrough came with the finding that jarosite could precipitate under conditions where ferrite would leach satisfactorily. Based on this discovery, a one-step process for the treatment of ferrite was developed. In the plant, the new process almost doubled the recovery of zinc from ferrite in the same equipment as the two-step jarosite process was operated in at that time. In a later expansion of the plant, investment savings were substantial compared to other technologies available. In the solution purification, the key finding was that Co, Ni, and Cu formed specific arsenides in the “hot arsenic zinc dust” step. This was utilized for the development of a three-step purification stage based on fluidized bed technology in all three steps, i.e. removal of Cu, Co and Cd. Both precipitation rates and selectivity increased, which strongly decreased the zinc powder consumption through a substantially suppressed hydrogen gas evolution. Better selectivity improved the value of the precipitates: cadmium, which caused environmental problems in the copper smelter, was reduced from 1-3% reported normally down to 0.05 %, and a cobalt cake with 15 % Co was easily produced in laboratory experiments in the cobalt removal. The zinc powder consumption in the plant for a solution containing Cu, Co, Ni and Cd (1000, 25, 30 and 350 mg/l, respectively), was around 1.8 g/l; i.e. only 1.4 times the stoichiometric demand – or, about 60% saving in powder consumption. Two processes for direct leaching of the concentrate under atmospheric conditions were developed, one of which was implemented in the Kokkola zinc plant. Compared to the existing pressure leach technology, savings were obtained mostly in investment. The scientific basis for the most important processes and process improvements is given in the doctoral thesis. This includes mathematical modeling and thermodynamic evaluation of experimental results and hypotheses developed. Five of the processes developed in this research and development program were implemented in the plant and are still operated. Even though these processes were developed with the focus on the plant in Kokkola, they can also be implemented at low cost in most of the zinc plants globally, and have thus a great significance in the development of the electrolytic zinc process in general.

Terpenoid transformations over gold catalysts

Nowadays biomass transformation has a great potential for the synthesis of value-added compounds with a wide range of applications. Terpenoids, extracted from biomass, are inexpensive and renewable raw materials which often have a biological activity and are widely used as important organic platform molecules in the development of new medicines as well as in the synthesis of fine chemicals and intermediates. At the same time, special attention is devoted to the application of gold catalysts to fine chemical synthesis due to their outstanding activity and/or selectivity for transformations of complex organic compounds. Conversion of renewable terpenoids in the presence of gold nanoparticles is one of the new and promising directions in the transformation of biomass to valuable chemicals. In the doctoral thesis, different kinds of natural terpenoids, such as α-pinene, myrtenol and carvone were selected as starting materials. Gold catalysts were utilized for the promising routes of these compounds transformation. Investigation of selective α-pinene isomerization to camphene, which is an important step in an industrial process towards the synthesis of camphor as well as other valuable substrates for the pharmaceutical industry, was performed. A high activity of heterogeneous gold catalysts in the Wagner-Meerwein rearrangement was demonstrated for the first time. Gold on alumina carrier was found to reach the α-pinene isomerization conversion up to 99.9% and the selectivity of 60-80%, thus making this catalyst very promising from an industrial viewpoint. A detailed investigation of kinetic regularities including catalyst deactivation during the reaction was performed. The one-pot terpene alcohol amination, which is a promising approach to the synthesis of valuable complex amines having specific physiological properties, was investigated. The general regularities of the one-pot natural myrtenol amination in the presence of gold catalysts as well as a correlation between catalytic activity, catalyst redox treatment and the support nature were obtained. Catalytic activity and product distribution were shown to be strongly dependent on the support properties, namely acidity and basicity. The gold-zirconia (Au/ZrO2) catalyst pretreated under oxidizing atmosphere was observed to be rather active, resulting in the total conversion of myrtenol and the selectivity to the corresponding amine of about 53%. The reaction kinetics was modelled based on the mechanistic considerations with the catalyst deactivation step incorporated in the mechanism. Carvone hydrogenation over a gold catalyst was studied with the general idea of investigating both the activity of gold catalysts in competitive hydrogenation of different functional groups and developing an approach to the synthesis of valuable carvone derivatives. Gold was found to promote stereo- and chemoselective carvone hydrogenation to dihydrocarvone with a predominant formation of the trans-isomer, which generally is a novel synthetic method for an industrially valuable dihydrocarvone. The solvent effect on the catalytic activity as well as on the ratio between trans- and cis-dihydrocarvone was evaluated.

Weed control in young coffee plantations through post emergence herbicide application onto total area

This study was carried out to investigate the efficiency of several herbicides under field conditions, by post-emergence application onto the entire area, their effect on the control of weeds in young coffee plantations and commercial coffee and bean intercropping system, as well as on both crops. Seedlings of Coffea arabica cv. Red Catuaí with four to six leaf pairs were transplanted to the field and treated according to conventional agronomic practices. A bean and coffee intercropping system was established by sowing three lines of beans in the coffee inter-rows. At the time the herbicides were sprayed, the coffee plants had six to ten leaf pairs; the bean plants, three leaflets; and the weeds were at an early development stage. Fluazifop-p-butyl and clethodim were selective for coffee plants and controlled only Brachiaria plantaginea and Digitaria horizontalis efficiently. Broad-leaved weeds (Amaranthus retroflexus, Bidens pilosa, Coronopus didymus, Emilia sonchifolia, Galinsoga parviflora, Ipomoea grandifolia, Lepidium virginicum, and Raphanus raphanistrum) were controlled with high efficiency by sole applications of fomesafen, flazasulfuron, and oxyfluorfen, except B. pilosa, C. didymus, and R. raphanistrum for oxyfluorfen. Sequential applications in seven-day intervals of fomesafen + fluazifop-p-butyl, or clethodim, and two commercial mixtures of fomesafen + fluazifop-p-butyl simultaneously controlled both types of weed. Cyperus rotundus was only controlled by flazasulfuron. Except for fluazifop-p-butyl and clethodim, all herbicide treatments caused only slight injuries on younger coffee leaves. However, further plant growth was not impaired and coffee plant height and stem diameter were therefore similar in the treatments, as evaluated four months later. Fomesafen, fluazifop-p-butyl, and clethodim, at sole or sequential application, and the commercial mixtures of fomesafen + fluazifop-p-butyl were also highly selective for bean crop; thus at doses recommended for bean crop, these herbicides may be applied to control weeds in coffee and bean intercropping systems by spraying the entire area.

Rice seedling and plant development as affected by increasing rates of penoxsulam under controlled environments

Rice is a major staple in many countries. Weed control is one of the factors limiting higher rice yield. ALS (acetolactate synthase)-inhibiting herbicides are desirable weed control herbicides because of their high efficacy, low toxicity to mammalians, and low rates used. An important herbicide characteristic is high selectivity to the crop, since it facilitates fast crop establishment and greater crop advantage over the weeds. The objectives of this work were to study the effects of increasing rates of the ALS-inhibiting herbicide penoxsulam on seed integrity and germination, and seedling and plant development of rice cv. BRS Pelota under controlled laboratory and greenhouse conditions. The results showed that penoxsulam affected rice germination and seedling and plant growth at rates above 54 g a.i. ha-1, and that penoxsulam is safe for rice seedling development at the currently recommended rates.

Selective inhibition of human tankyrases

Tankyrases belong to the Diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme superfamily, also known as poly(ADP-ribose) polymerases (PARPs). They catalyze a covalent post-translational modification reaction where they transfer ADP-ribose units from NAD+ to target proteins. Tankyrases are involved in many cellular processes and their roles in telomere homeostasis, Wnt signaling and in several diseases including cancers have made them interesting drug targets. In this thesis project, selective inhibition of human tankyrases was studied. A homogeneous fluorescence-based assay was developed to screen the compound libraries. The assay is inexpensive, operationally easy, and performs well according to the statistical analysis. Assay suitability was confirmed by screening a natural product library. Flavone was identified as the most potent inhibitor in the library and this motivated us to screen a larger flavonoid library. Results showed that flavones were indeed the best inhibitor of tankyrases among flavonoids. To further study the structure-activity relationship, a small library of flavones containing single substitution was screened and potency measurements allowed us to generate structure-activity relationship. Compounds containing substitutions at 4´-position were more potent in comparison to other substitutions, and importantly, hydrophobic groups improved isoenzyme selectivity as well as the potency. A flavone derivative containing a hydrophobic isopropyl group (compound 22), displayed 6 nM potency against TNKS1, excellent isoenzyme selectivity and Wnt signaling inhibition. Protein interactions with compounds were studied by solving complex crystal structures of the compounds with TNKS2 catalytic domain. A novel tankyrase inhibitor (IWR-1) was also crystallized in complex with TNKS2 catalytic domain. The crystal structure of TNKS2 in complex with IWR-1 showed that the compound binds to adenosine site and it was the first known ARTD inhibitor of this kind. To date, there is no structural information available about the substrate binding with any of the ARTD family members; therefore NAD+ was soaked with TNKS2 catalytic domain crystals. However, analysis of crystal structure showed that NAD+ was hydrolyzed to nicotinamide. Also, a co-crystal structure of NAD+ mimic compound, EB-47, was solved which was used to deduce some insights about the substrate interactions with the enzyme. Like EB-47, other ARTD1 inhibitors were also shown to inhibit tankyrases. It indicated that selectivity of the ARTD1 inhibitors should be considered as some of the effects in cells could come from tankyrase inhibition. In conclusion, the study provides novel information on tankyrase inhibition and presents new insight into the selectivity and potency of compounds.

Optimization and validation of the solid-liquid extraction technique for determination of picloram in soils by high performance liquid chromatography

The objective of this study was to optimize and validate the solid-liquid extraction (ESL) technique for determination of picloram residues in soil samples. At the optimization stage, the optimal conditions for extraction of soil samples were determined using univariate analysis. Ratio soil/solution extraction, type and time of agitation, ionic strength and pH of extraction solution were evaluated. Based on the optimized parameters, the following method of extraction and analysis of picloram was developed: weigh 2.00 g of soil dried and sieved through a sieve mesh of 2.0 mm pore, add 20.0 mL of KCl concentration of 0.5 mol L-1, shake the bottle in the vortex for 10 seconds to form suspension and adjust to pH 7.00, with alkaline KOH 0.1 mol L-1. Homogenate the system in a shaker system for 60 minutes and then let it stand for 10 minutes. The bottles are centrifuged for 10 minutes at 3,500 rpm. After the settlement of the soil particles and cleaning of the supernatant extract, an aliquot is withdrawn and analyzed by high performance liquid chromatography. The optimized method was validated by determining the selectivity, linearity, detection and quantification limits, precision and accuracy. The ESL methodology was efficient for analysis of residues of the pesticides studied, with percentages of recovery above 90%. The limits of detection and quantification were 20.0 and 66.0 mg kg-1 soil for the PVA, and 40.0 and 132.0 mg kg-1 soil for the VLA. The coefficients of variation (CV) were equal to 2.32 and 2.69 for PVA and TH soils, respectively. The methodology resulted in low organic solvent consumption and cleaner extracts, as well as no purification steps for chromatographic analysis were required. The parameters evaluated in the validation process indicated that the ESL methodology is efficient for the extraction of picloram residues in soils, with low limits of detection and quantification.

Mefenpyr-diethyl action on fenoxaprop-p-ethyl detoxification in wheat varieties

Safeners protect crops against herbicide injury. The aim of this study was to examine the differential susceptibility of five wheat (Triticum aestivum) varieties to the herbicide fenoxaprop-p-ethyl, as well as the performance of mefenpyr-diethyl on minimizing herbicide injury and on lipid contents. Varieties BRS 49, CD 104, CEP 24, IAPAR 78 and Rubi were sprayed with fenoxaprop-p-ethyl (69 g ha¹), fenoxaprop-p-ethyl + mefenpyr-diethyl (69 g + 18.75 g ha-1), or mefenpyr-diethyl (18.75 g ha-1). Plants were evaluated visually for injury at 7 and 14 days after treatment (DAT). Glutathione S-transferase (GST) activity was assayed in aerial parts at 7 DAT, and lipid content was measured at 14 DAT. Varieties CEP 24, IAPAR 78 and Rubi were more tolerant to fenoxaprop-p-ethyl than BRS 49, and CD 104 rapidly recovering from the slight phytotoxicity symptoms produced by the herbicide. Mefenpyr-diethyl prevented crop injury associated with the herbicide. GST activity did not correlate directly with fenoxaprop-p-ethyl detoxification. However, lipid content was related to the susceptibility of wheat to fenoxaprop-p-ethyl treatment.

Use of herbicides on turfgrass

In crop production, weeds must be controlled so as not to adversely affect crop yield and crop quality. Thus, a low level of weeds infesting a field, in most instances, is not a problem. Except in sod or seed production, turfgrass does not have a yield component. The value of turfgrass is its inherent aesthetic quality and usability. Aesthetic quality is the beauty and value that turfgrass adds to a managed landscape. Usability can be the durability of a sport field, trueness of golf putting green roll, or reduction in soil loss from water runoff or wind. Any weed presence in turfgrass can decrease the aesthetic quality and usability of turfgrass. Utilizing herbicides is the only way to completely control weeds in a turfgrass stand. While it is possible to reduce weed populations using cultural or mechanical management practices, it is impossible to completely eliminate weeds as can be accomplished with herbicides. This manuscript will review the major herbicides used in turfgrass in the United States with respect to their modes of action, herbicide family, and primary use in turfgrass.

Growth inhibitors in turfgrass

Well-maintained lawns are comfortable and safe places for leisure activities and sports practice, and they also bring environmental benefits; for example, they reduce soil exposure to erosion and releases atmospheric CO2, thus reducing the greenhouse effect. However, regardless of the purpose of use or the choice of the plant species to form the lawn, the highest costs involve cutting that is needed to keep the turfgrass at its appropriate height. Successive lawn cutting operations are necessary basically because of the vegetative and reproductive growth of turfgrass which, in Brazil, occurs mainly from October to March. Expenditures with successive mechanical cuttings have fostered the search of alternative procedures to keep lawn plants at appropriate height, such as the use of plant growth inhibitors, an increasingly interesting procedure. Since the use of this technology in Brazil is still at its early stage, the aim of this literature review is to examine aspects associated with lawn management by using growth inhibitors. Another alternative is to increase the knowledge of the classification and rational application of the different compounds currently available in the market.

Sorption of fomesafen in Brazilian soils

The study of the dynamics of a herbicide in the soil focus on the interactions with environmental components to obtain agronomic efficiency, ensuring selectivity to the culture and risk reduction of environmental impact. This study evaluated the sorption process of fomesafen in the Brazilian soils Ultisol, Cambisol, and Organosol. Besides soil, washed sand was used as an inert material for determination of the sorption ratio of fomesafen in the soil. The bioassay method was applied, using Sorghum vulgare plants as bio-indicator of herbicide presence. Plant poisoning evaluation and harvest for dry matter determination were carried out 21 days after sorghum sowing. To calculate C50, the nonlinear log-logistic model was applied and sorption ratios of the herbicide were obtained in different soils. The decreasing sorption ratio of formesafen in the soils was: Organosol > Ultisol > Cambisol. It was concluded that the contents of organic matter and clay in the soils were the attributes that most influenced fomesafen sorption.

Directed-spray application of paraquat and diuron in physic nut plants

There is little information about the selectivity of herbicides in physic nut (Jatropha curcas) in Brazil. Therefore, this study aimed to evaluate the selectivity of different doses and mixtures of paraquat and diuron in direted-spray applications in physic nut plants in greenhouse conditions. The study used a randomized block design, with five replicates. The treatments were: paraquat (200 and 600 g ha-1), diuron (1,000 and 2,000 g ha-1), paraquat + diuron (200 + 1,000 g ha-1), paraquat + diuron (200 + 2,000 g ha-1), paraquat + diuron (600 + 1,000 g ha-1), paraquat + diuron (600 + 2,000 g ha-1) and a control (no application). Directed-spray application was performed at 70 days after sowing by the lower third of the plants. The treatments of diuron and paraquat + diuron mixtures affected the growth and photosynthetic activity of physic nut plants, injuries being more pronounced at doses of diuron of 2,000 g ha‑1, while the isolated application of paraquat at doses of 200 and 600 g ha-1 showed good selectivity potential for physic nut plants.

Chemical control of Pileamicrophylla in cattleya seedlings (Cattleyatenebrosa x Cattleyaleopoldy)

Species of the orchidaceae family are grown for marketing flowers and among them the genus Cattleya stands out. However, due to its slow growth, the substrate is subjected to weed infestation. Therefore, this study aims to evaluate the selectivity and efficiency of herbicides in controlling Pilea microphylla in Cattleya orchid seedlings (Cattleya tenebrosa x Cattleya leopoldy). We used a completely randomized design with four replications. The evaluated herbicides were oxyfluorfen (0, 120, 240 and 480 g ha-1), flumioxazin (0, 12.5, 25 and 40 g ha-1), nicosulfuron (0, 20, 40 and 80 g ha-1) mesotrione (0, 96, 144 and 192 g ha-1), clethodim (0, 60, 84 and 108 g ha-1) and metsulfuron-methyl (0, 1.2, 1.8 and 2.4 g ha-1). At post-emergence applying time, the Cattleya plants had three bulb sand were 10 cm tall, while P. microphylla, where 5 cm tall. Nicosulfuron, mesotrione and clethodim herbicides did not control P. microphylla, while oxyfluorfen and flumioxazin showed over 90% efficiency level sin controlling P. microphyllafrom 14 days after application (DAA). As to metsulfuron-methyl, it showed efficiency superior to 90% from the control dose of 1.8 g ha-1 at 28 DAA. All herbicides were selective plants of Cattleya, however, only oxyfluorfen, flumioxazin and metsulfuron-methyl were effective in controlling P. microphylla.

How glyphosate may affect transgenic soybean in different soil and phosphorus levels

The technology that employs genetic modifications brought a significant increase in the utilization of glyphosate. Transgenic soybean has been suffering injury, even though it possesses a resistance mechanism to glyphosate. Currently, there are only a few studies on the dynamics of glyphosate in transgenic soybean planted in soils with different textures interacting with phosphorus concentrations. This study focused on assessing the effects of glyphosate in transgenic soybean plants on different types of soil and at different phosphorus levels. The experimental design was completely randomized, in factorial design: 2 x 6 x 3, that being 2 soil types, 6 doses of glyphosate and 3 levels of phosphorus, and four replications. Plants were cultivated for thirty days in pots with two types of soil, one being clayey (Red-Yellow Latosol) and the other sandy (Quartzarenic Neosol). They received one, two, and three times the maintenance dose of fertilization of phosphorus, corresponding to: 170, 250 and 330 kg of P2O5 ha-1 to QN, and 380, 460 and 540 kg P2O5 ha-1 to RYL, respectively. Glyphosate was applied at six different doses: 0, 1,200, 2,400, 12,000, 60,000 and 120,000 g ha-1 of active ingredient. Plant height, a and b chlorophyll, and shoot were lower for the plants that received lower doses of glyphosate, regardless of the type of soil. Greater availability of phosphorus and lower amount of glyphosate used in Quartzarenic Neosol soil provided for less phytointoxication symptoms in transgenic soybean.

Application of Sulfentrazone in Stages of Germination of IAC 90 Cassava Cuttings in Clay Soils and Sandy

Weeds interfere dramatically in the productive potential of cassava; however, information regarding herbicides that are selective to crops is still scarce. Thus, the aim in this study was to assess the initial growth of IAC 90 cassava plants after the application of sulfentrazone at different stages of germination of cassava in clayey and sandy soils. Three experiments were simultaneously deployed: the first experiment consisted in the application of sulfentrazone in the non-germinated stage of cassava cuttings; the second one in the stage of germinated cassavas cuttings (0.9 cm shoots); and the third one in applications in the stage of cassava cuttings with buds emerging (6.5 cm shoots and emerging from the soil). For each experiment the experimental design in randomized blocks was used in the 2 x 5 factorial arrangement with four replications. The factors were composed of two soils (sandy and clayey) and five doses of sulfentrazone (0, 250, 500, 750 and 1,000 g ha-1). It was found that depending on the herbicide dose, development stage of the buds of cassava cuttings and the type of soil, damage can occur in the initial development of the IAC 90 cassava plants. The greatest potential of sulfentrazone selectivity has occurred in applications in the non-germinated cassava cuttings stage and in doses lower than 500 g ha-1 in the clayey soil.