The effects of habitat complexity and hydraulic conditions on the establishment of benthic stream macroalgae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of crevice size on the establishment of macroalgae in subtropical streams

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression studies in the embryo and in the micropylar endosperm of germinating coffee (Coffea arabica cv. Rubi) seeds

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Spatial and temporal quality of water in the Itupararanga Reservoir, Alto Sorocaba basin (SP), Brazil

Considering the great importance of the Itupararanga Reservoir, Upper Alto Sorocaba basin/SP, this study aimed to report the variations of some parameters of water quality in the spatial and temporal gradients in this multipurpose res- ervoir. The eutrophication of this reservoir was checked using the Carlson Index Modified and the results indicate that the surface water were classified as eutrophic and mesotrophic in wet and dry periods, being characterized the better quality of water in wet period. In the vertical gradient the results showed a stratiphication in all parameters analyzed, except for the electrical conductivity, with good correlation between total phosphorous and chlorophyll-a, indicating that eutrophication of the reservoir changes the conditions of algal growth, mainly in its initial area. Immediate inter- ventions are needed, which must be directed to planning of land use, domestic effluents treatment, taking to an inte- grated management of this important watershed located in the São Paulo State.

Eutrophication and macroalgal blooms in temperate and tropical coastal waters: nutrient enrichment experiments with Ulva spp.

Receiving coastal waters and estuaries are among the most nutrient-enriched environments on earth, and one of the symptoms of the resulting eutrophication is the proliferation of opportunistic, fast-growing marine seaweeds. Here, we used a widespread macroalga often involved in blooms, Ulva spp., to investigate how supply of nitrogen (N) and phosphorus (P), the two main potential growth-limiting nutrients, influence macroalgal growth in temperate and tropical coastal waters ranging from low- to high-nutrient supplies. We carried out N and P enrichment field experiments on Ulva spp. in seven coastal systems, with one of these systems represented by three different subestuaries, for a total of nine sites. We showed that rate of growth of Ulva spp. was directly correlated to annual dissolved inorganic nitrogen (DIN) concentrations, where growth increased with increasing DIN concentration. Internal N pools of macroalgal fronds were also linked to increased DIN supply, and algal growth rates were tightly coupled to these internal N pools. The increases in DIN appeared to be related to greater inputs of wastewater to these coastal waters as indicated by high delta 15N signatures of the algae as DIN increased. N and P enrichment experiments showed that rate of macroalgal growth was controlled by supply of DIN where ambient DIN concentrations were low, and by P where DIN concentrations were higher, regardless of latitude or geographic setting. These results suggest that understanding the basis for macroalgal blooms, and management of these harmful phenomena, will require information as to nutrient sources, and actions to reduce supply of N and P in coastal waters concerned.

A hormonal misunderstanding in Acca sellowiana embryogenesis: levels of zygotic embryogenesis do not match those of somatic embryogenesis

Endogenous levels of IAA, ABA and four types of CKs were analyzed in zygotic and indirect (ISE) and direct somatic embryogenesis of Acca sellowiana. Zygotic and somatic embryos at different developmental stages were sampled for morphological and hormonal analysis. Both embryo types showed substantial asymmetry in hormone levels. Zygotic embryos displayed a conspicuous peak of IAA in early developmental stages. The results outlined the hormonal variations occurring during zygotic and somatic embryogenesis regarding the timing, nature and hormonal status involved in both processes. The short transient pulse of IAA observed on the 3rd day in culture was suggested to be involved with the signaling for the induction of somatic embryogenesis. Fertilized ovule development was associated with increased IAA levels 21-24 days after pollination, followed by a sharp decrease in the cotyledonary stage, both in zygotic and somatic embryos. There was a prominent increase in ABA levels in cultures which generated ISE 24-30 days after pollination, a period that corresponds to the heart and torpedo stages. The levels of total CKs (Z, [9R]Z, iP and [9R]iP) were also always higher in zygotic than in somatic embryogenesis. While zygotic embryogenesis was dominated by the presence of zeatin, the somatic process, contrarily, was characterized by a large variation of the other cytokinin forms and amounts studied. The above results, when taken together, could be related to the previously observed high frequency formation of anomalous somatic embryos formed in A. sellowiana, as well as to their low germination ability.

Cadmium and Chromium Toxicity to Pseudokirchneriella subcapitata and Microcystis aeruginosa

The toxicity of cadmium and chromium to Pseudokirchneriella subcapitata and Microcystis aeruginosa was evaluated through algal growth rate during 96h exposure bioassays. Free metal ion concentrations were obtained using MINEQL(+) 4.61 and used for IC50 determination. Metal accumulations by the microorganisms were determined and they were found to be dependent on the concentration of Cd2+ and Cr6+. IC50 for P. subcapitata were 0.60 mu mol L-1 free Cd2+ and 20 mu mol L-1 free Cr6+, while the IC50 values for M. aeruginosa were 0.01 mu mol L-1 Cd2+ and 11.07 mu mol L-1 Cr6+. P. subcapitata accumulated higher metal concentrations (0.001 - 0.05 mu mol Cd mg(-1) dry wt. and 0.001 - 0.04 mu mol Cr mg(-1) dry wt) than the cyanobacteria (0.001 - 0.01 mu mol Cd mg(-1) dry wt and 0.001 - 0.02 mu mol Cr mg(-1) dry wt). Cadmium was more toxic than chromium to both the microorganisms.

Use of solvents and environmental friendly materials for applications in Green Chemistry

The present Thesis studies three alternative solvent groups as sustainable replacement of traditional organic solvents. Some aspects of fluorinated solvents, supercritical fluids and ionic liquids, have been analysed with a critical approach and their effective “greenness” has been evaluated from the points of view of the synthesis, the properties and the applications. In particular, the attention has been put on the environmental and human health issues, evaluating the eco-toxicity, the toxicity and the persistence, to underline that applicability and sustainability are subjects with equal importance. The “green” features of fluorous solvents and supercritical fluids are almost well-established; in particular supercritical carbon dioxide (scCO2) is probably the “greenest” solvent among the alternative solvent systems developed in the last years, enabling to combine numerous advantages both from the point of view of industrial/technological applications and eco-compatibility. In the Thesis the analysis of these two classes of alternative solvents has been mainly focused on their applicability, rather than the evaluation of their environmental impact. Specifically they have been evaluated as alternative media for non-aqueous biocatalysis. For this purpose, the hydrophobic ion pairing (HIP), which allows solubilising enzymes in apolar solvents by an ion pairing between the protein and a surfactant, has been investigated as effective enzymatic derivatisation technique to improve the catalytic activity under homogeneous conditions in non conventional media. The results showed that the complex enzyme-surfactant was much more active both in fluorous solvents and in supercritical carbon dioxide than the native form of the enzyme. Ionic liquids, especially imidazolium salts, have been proposed some years ago as “fully green” alternative solvents; however this epithet does not take into account several “brown” aspects such as their synthesis from petro-chemical starting materials, their considerable eco-toxicity, toxicity and resistance to biodegradation, and the difficulty of clearly outline applications in which ionic liquids are really more advantageous than traditional solvents. For all of these reasons in this Thesis a critical analysis of ionic liquids has been focused on three main topics: i) alternative synthesis by introducing structural moieties which could reduce the toxicity of the most known liquid salts, and by using starting materials from renewable resources; ii) on the evaluation of their environmental impact through eco-toxicological tests (Daphnia magna and Vibrio fischeri acute toxicity tests, and algal growth inhibition), toxicity tests (MTT test, AChE inhibition and LDH release tests) and fate and rate of aerobic biodegradation in soil and water; iii) and on the demonstration of their effectiveness as reaction media in organo-catalysis and as extractive solvents in the recovery of vegetable oil from terrestrial and aquatic biomass. The results about eco-toxicity tests with Daphnia magna, Vibrio fischeri and algae, and toxicity assay using cultured cell lines, clearly indicate that the difference in toxicity between alkyl and oxygenated cations relies in differences of polarity, according to the general trend of decreasing toxicity by decreasing the lipophilicity. Independently by the biological approach in fact, all the results are in agreement, showing a lower toxicity for compounds with oxygenated lateral chains than for those having purely alkyl lateral chains. These findings indicate that an appropriate choice of cation and anion structures is important not only to design the IL with improved and suitable chemico-physical properties but also to obtain safer and eco-friendly ILs. Moreover there is a clear indication that the composition of the abiotic environment has to be taken into account when the toxicity of ILs in various biological test systems is analysed, because, for example, the data reported in the Thesis indicate a significant influence of salinity variations on algal toxicity. Aerobic biodegradation of four imidazolium ionic liquids, two alkylated and two oxygenated, in soil was evaluated for the first time. Alkyl ionic liquids were shown to be biodegradable over the 6 months test period, and in contrast no significant mineralisation was observed with oxygenated derivatives. A different result was observed in the aerobic biodegradation of alkylated and oxygenated pyridinium ionic liquids in water because all the ionic liquids were almost completely degraded after 10 days, independently by the number of oxygen in the lateral chain of the cation. The synthesis of new ionic liquids by using renewable feedstock as starting materials, has been developed through the synthesis of furan-based ion pairs from furfural. The new ammonium salts were synthesised in very good yields, good purity of the products and wide versatility, combining low melting points with high decomposition temperatures and reduced viscosities. Regarding the possible applications as surfactants and biocides, furan-based salts could be a valuable alternative to benzyltributylammonium salts and benzalkonium chloride that are produced from non-renewable resources. A new procedure for the allylation of ketones and aldehydes with tetraallyltin in ionic liquids was developed. The reaction afforded high yields both in sulfonate-containing ILs and in ILs without sulfonate upon addition of a small amount of sulfonic acid. The checked reaction resulted in peculiar chemoselectivity favouring aliphatic substrates towards aromatic ketones and good stereoselectivity in the allylation of levoglucosenone. Finally ILs-based systems could be easily and successfully recycled, making the described procedure environmentally benign. The potential role of switchable polarity solvents as a green technology for the extraction of vegetable oil from terrestrial and aquatic biomass has been investigated. The extraction efficiency of terrestrial biomass rich in triacylglycerols, as soy bean flakes and sunflower seeds, was comparable to those of traditional organic solvents, being the yield of vegetable oils recovery very similar. Switchable polarity solvents as been also exploited for the first time in the extraction of hydrocarbons from the microalga Botryococcus braunii, demonstrating the efficiency of the process for the extraction of both dried microalgal biomass and directly of the aqueous growth medium. The switchable polarity solvents exhibited better extraction efficiency than conventional solvents, both with dried and liquid samples. This is an important issue considering that the harvest and the dewatering of algal biomass have a large impact on overall costs and energy balance.

Phytoplankton Response to Environmental Variables and Organic Pollutants. Laboratory Cultures and Numerical Simulations Experiments

Herbicides are becoming emergent contaminants in Italian surface, coastal and ground waters, due to their intensive use in agriculture. In marine environments herbicides have adverse effects on non-target organisms, as primary producers, resulting in oxygen depletion and decreased primary productivity. Alterations of species composition in algal communities can also occur due to the different sensitivity among the species. In the present thesis the effects of herbicides, widely used in the Northern Adriatic Sea, on different algal species were studied. The main goal of this work was to study the influence of temperature on algal growth in the presence of the triazinic herbicide terbuthylazine (TBA), and the cellular responses adopted to counteract the toxic effects of the pollutant (Chapter 1 and 2). The development of simulation models to be applied in environmental management are needed to organize and track information in a way that would not be possible otherwise and simulate an ecological prospective. The data collected from laboratory experiments were used to simulate algal responses to the TBA exposure at increasing temperature conditions (Chapter 3). Part of the thesis was conducted in foreign countries. The work presented in Chapter 4 was focused on the effect of high light on growth, toxicity and mixotrophy of the ichtyotoxic species Prymnesium parvum. In addition, a mesocosm experiment was conducted in order to study the synergic effect of the pollutant emamectin benzoate with other anthropogenic stressors, such as oil pollution and induced phytoplankton blooms (Chapter 5).

Charakterisierung einer pflanzlichen Variante des Cytoskelettproteins gamma-Tubulin durch Strukturmodellierung, Überexpression und Analyse der Interaktionspartner

Wie alle Eukaryoten besitzen auch höhere Pflanzen ein mikrotubuläres Cytoskelett. Einige Funktionen dieses Cytoskeletts sind relativ stark konserviert, andere dagegen scheinen sehr pflanzenspezifisch zu sein. Dies betrifft insbesondere charakteristische mikrotubuläre Netzwerke, die bei der Neubildung und der Verstärkung der Zellwände wichtige Rollen übernehmen. Wie der Aufbau dieser Netzwerke kontrolliert wird, ist bisher relativ unklar. Typische Mikrotubuli organisierende Zentren (MTOC), insbesondere Centrosomen oder Spindelpolkörper, sind bei höheren Pflanzen nicht beobachtet worden. Von pilzlichen und tierischen Organismen weiß man, dass gamma-Tubulin (gTUB) mit seinen assoziierten Proteinen in den MTOC bei der Nukleation von Mikrotubuli eine Schlüsselfunktion hat. Dieses Mitglied der Tubulin-Superfamilie wird aber auch in Pflanzen gefunden, dessen genaue Funktion bisher unbekannt ist. Zu Beginn der Arbeit wurden mittels in silico Berechnungen Strukturmodelle des pflanzlichen gTUBs aus Nicotiana tabacum erarbeitet, da die Struktur, die zu einem Verständnis der pflanzlichen Wachstumsregulation beitragen könnte, bisher unbekannt ist. Auf Grundlage der bioinformatischen Daten konnte für weitere Studien eine notwendige gTUB-Deletionsmutante entwickelt werden. Für Röntgendiffraktionsstudien und gTUB-Interaktionspartneranalysen war die Verfügbarkeit verhältnismäßig großer Proteinmengen notwendig. Die Expression der gTUB-Volllängensequenz in gelöster und aktiver Form stellte einen immanent wichtigen Zwischenschritt dar. Das Escherichia coli T7/lacO-Expressionssystem lieferte, trotz vielversprechender Erfolge in der Vergangenheit, kein gelöstes rekombinantes gTUB. So wurden zwar verhältnismäßig hohe Expressionsraten erzielt, aber das rekombinante gTUB lag quantitativ als Inclusion bodies vor. Eine Variationen der Expressionsparameter sowie umfangreiche Versuche mittels verschiedenster Konstrukte sowie potentiell die Löslichkeit erhöhenden Tags gTUB in gelöster Form in E. coli zu exprimieren blieben erfolglos. Eine Denaturierung der Inclusion bodies und Rückfaltung wurde aufgrund der wohl bei der Tubulinfaltung notwendigen komplexeren Chaperone sowie thermodynamischer Überlegungen ausgeschlossen. Die höher evolvierte Chaperonausstattung war ein Hauptgrund für die Verwendung der eukaryotischen Hefe-Expressionssysteme K. lactis und des S. cerevisiae-Stammes FGY217 zur gTUB-Expression. So konnten nach der Selektion nur transgene Hefe-Zellen dokumentiert werden, die die gTUB-Expressionskassette nachweislich an der vorgesehenen Zielposition in ihrem Genom integrierten, aber keine dokumentierbare Expression zeigten. Die wahrscheinlichste Begründung hierfür ist, dass ein erhöhter intrazellulärer gTUB-Titer mit dem Zellwachstum und der Zellteilung dieser eukaryotischen Organismen interferierte und durch Rückkopplungen die rekombinante gTUB-CDS aus N. tabacum ausgeschaltet wurde. Der Versuch einer transienten gTUB-Überexpression in differenzierten Blattgeweben höherer Pflanzen war eine logische Konsequenz aus den vorherigen Ergebnissen und lieferte, wenn auch nicht die für eine Proteinkristallisation notwendigen Mengen, gelöstes gTUB. Bestrebungen einer stabilen Transfektion von A. thaliana oder BY-2-Zellkulturen mit einer gTUB-CDS lieferten keine transgenen Organismen, was starke Interferenzen der rekombinanten gTUB-CDS in den Zellen vermuten lies. Transfektionsversuche mit nur GFP tragenden Konstrukten ergaben hingegen eine hohe Anzahl an transgenen Organismen, die auch verhältnismäßig starke Expressionsraten zeigten. Die erzielten Proteinmengen bei der transienten gTUB-Überexpression in N. benthamiana Blattgeweben, in Co-Expression mit dem Posttransriptional Gene Silencing-Suppressorprotein p19, waren für einen Pull-Down sowie eine massenspektroskopische Analyse der Interaktionspartner ausreichend und ergaben Befunde. Eine abschließende Auswertung des erarbeiteten massenspektroskopischen Datensatzes wird jedoch erst dann möglich sein, wenn das Tabak-Proteom vollständig sequenziert ist. Die Erweiterung der bestehenden pflanzlichen Vergleichsdatenbanken um das bisher bekannte Tabak-Proteom vervielfachte die Anzahl der in dieser Studie identifizierten gTUB-Interaktionspartner. Interaktionen mit dem TCP1-Chaperon untermauern die Hypothese der zur Faltung pflanzlichen gTUBs notwendigen Chaperone. Beobachtete gTUB-Degradationsmuster in Verbindung mit Interaktionen des 26S-Proteasoms deuten auf eine Gegenregulationen bei erhöhtem gTUB-Titer auf Proteinebene hin. Da Blattgewebe selbst nur noch über eine sehr geringe und inhomogene Teilungsaktivität verfügen ist diese Regulation hoch spannend. Auch konnte durch Co-Expression des PTGS-Suppressorproteins p19 gezeigt werden, dass bei der gTUB-Expression eine Regulation auf RNA-Ebene erfolgt.

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

Functional characterization of transcription factor USF

USF, Upstream Stimulatory Factor, is a family of ubiquitous transcription factors that contain highly conserved basic helix-loop-helix leucine zipper DNA binding domains and recognize the core DNA sequence CACGTG. In human and mouse, two members of the USF family, USF1 and USF2, encoded by two different genes, contribute to the USF activity. In order to gain insights into the mechanisms by which USFs function as transcriptional activators, different approaches were used to map the domains of USF2 responsible for nuclear localization and transcriptional activation. Two stretches of amino acids, one in the basic region of the DNA binding domain, the other in a highly conserved N-terminal region, were found to direct nuclear localization independently of one another. Two distinct activation domains were also identified. The first one, located in the conserved N-terminal region that overlaps the C-terminal nuclear localization signal, functioned only in the presence of an initiator element in the promoter of the reporter. The second, in a nonconserved region, activated transcription in the absence of an initiator element or when fused to a heterologous DNA binding domain. These results suggest that USF2 functions in different promoter contexts by selectively utilizing different activation domains.^ The deletion analysis of USF2 also identified two dominant negative mutants of USF, one lacking the activation domain, the other lacking the basic domain. The latter proved useful for testing the direct involvement of USFs in the transcriptional activation mediated by the viral protein IE62.^ To investigate the biological function of USFs, foci and colony formation assays were used to study the growth regulation by USFs. It was found that USFs had a strong antagonistic effect on cellular transformation mediated by the bHLH/LZ protein Myc. This effect required the DNA binding activity of either USF 1 or USF2. Moreover, USF2, but not USF1 or other mutants of USFs, was also found to have strong inhibitory effect on the cellular transformation by E1a and on the growth of HeLa cells. These results demonstrate that USFs could potentially regulate growth through two mechanisms, one by antagonizing the function of Myc in cellular transformation, the other by mediating a more general growth inhibitory effect. ^

Performance enhancement of large industrial wastewater stabilization ponds

A 6-month-long, bench-scale simulation of an industrial wastewater stabilization pond (WSP) system was conducted to evaluate responses to several potential performance-enhancing treatments. The industrial WSP system consists of an anaerobic primary (1ry) WSP treating high-strength wastewater, followed by facultative secondary (2ry) and aerobic tertiary (3ry) WSPs in series treating lower-strength wastewater. The 1ry WSP was simulated with four glass aquaria which were fed with wastewater from the actual WSP system. The treatments examined were phosphorus supplementation (PHOS), phosphorus supplementation with pH control (PHOS+ALK), and phosphorus supplementation with pH control and effluent recycle (PHOS+ALK+RCY). The supplementary phosphorus treatment alone did not yield any significant change versus the CONTROL 1ry model pond. The average carbon to phosphorus ratio of the feed wastewater received from the WSP system was already 100:0.019 (i.e., 2,100 mg/l: 0.4 mg/l). The pH-control treatments (PHOS+ALK and PHOS+ALK+RCY) produced significant results, with 9 to 12 percent more total organic carbon (TOC) removal, 43 percent more volatile organic acid (VOA) generation, 78 percent more 2-ethoxyethanol and 14 percent more bis(2-chloroethyl)ether removal, and from 100- to 10,000-fold increases in bacterial enzyme activity and heterotrophic bacterial numbers. Recycling a 10-percent portion of the effluent yielded less variability for certain physicochemical parameters in the PHOS+ALK+RCY 1ry model pond, but overall there was no statistically-detectable improvement in performance versus no recycle. The 2ry and 3ry WSPs were also simulated in the laboratory to monitor the effect and fate of increased phosphorus loadings, as might occur if supplemental phosphorus were added to the 1ry WSP. Noticeable increases in algal growth were observed at feed phosphorus concentrations of 0.5 mg/l; however, there were no significant changes in the monitored physicochemical parameters. The effluent phosphorus concentrations from both the 2ry and 3ry model ponds did increase notably when feed phosphorus concentrations were increased from 0.5 to 1.0 mg/l. ^