The mosquito-borne Usutu virus (USUV) is a zoonotic flavivirus and an emerging pathogen. So far therapeutical options or vaccines are not available in human and veterinary medicine. The bioenergetic profile based on extracellular flux analysis revealed an USUV infection-associated significant increase in basal and stressed glycolysis on Vero and with a tendency for basal glycolysis on the avian cell line TME-R derived from Eurasian blackbirds. On both cell lines this was accompanied by a significant drop in the metabolic potential of glycolysis. Moreover, glycolysis contributed to production of virus progeny, as inhibition of glycolysis with 2-deoxy-D-glucose reduced virus yield on Vero by one log10 step. Additionally, the increase in glycolysis observed on Vero cells after USUV infection was lost after the addition of exogenous type I interferon (IFN) b. To further explore the contribution of the IFN response pathway to the impact of USUV on cellular metabolism, USUV infection was characterized on human A549 respiratory cells with a knockout of the type I IFN receptor, either solely or together with the receptor of type III IFN. Notably, only the double knockout of types I and III IFN receptor increased permissiveness to USUV and supported viral replication together with an alteration of the glycolytic activity, namely an increase in basal glycolysis to an extent that a further increase after injection of metabolic stressors during extracellular flux analysis was not noted. This study provides evidence for glycolysis as a possible target for therapeutic intervention of USUV replication. Moreover, presented data highlight type I and type III IFN system as a determinant for human host cell permissiveness and for the infection-associated impact on glycolysis.

Vor zehn Jahren wurde der (Pro)Renin-Rezeptor [(P)RR] entdeckt und als neuer Bestandteil des Renin-Angiotensin-Systems beschrieben. Neuere Studien ergaben, dass der (P)RR mit der vakuolären H+-ATPase (V-ATPase) assoziiert sein kann, weshalb er auch V-ATPase associated protein 2 (ATP6ap2) genannt wird. In Osteoklasten befinden sich V-ATPase hauptsächlich an der zur Knochenoberfläche gerichteten Plasmamembran und transportieren Protonen in den extrazellulären Raum. Mäuse mit genetischer Deletion verschiedener V-ATPase-Untereinheiten charakterisiert durch einen Anstieg von Knochenmasse (Osteopetrose). In der vorliegenden Arbeit fanden wir heraus, dass (P)RR stark in reifen Osteoklasten in vitro und in vivo exprimiert wird. Mäuse mit genetischer Deletion des (P)RR in Osteoklasten wurden durch einen komplexen Knochen-Phänotyp mit reduzierter Knochendichte charakterisiert. (P)RR-defiziten Osteoklasten wiesen vermehrte Differenzierung und/oder Aktivität in vitro und in vivo auf. Wir postulieren deshalb, dass der (P)RR die in der Plasmamembran lokalisierten V-ATPase nicht direkt reguliert, sondern mit der physiologischen Aktivität der Osteoklasten durch andere Mechanismen interferiert. Macrophagen sind speziell auf die Immunabwehr ausgerichtete Fresszellen (Phagozyten). Phagozytose ist ein wesentlicher Zellprozess der die V-ATPase in Lysosomen braucht um die eingeschlossenen Pathogen zu zerstören. Wir generierten transgene Ratten mit konditionellen knockdown von (P)RR unter Nutzung eines Doxyzyclin-induzierten shRNA-Expressionssystems. Eine effiziente (P)RR-Depletion in Makrophagen wurde durch Behandlung mit Doxyzyclin in vivo im Trinkwasser und in vitro im Kulturmedium erreicht. Die vorliegende Arbeit zeigt, dass die Verschiebung des vesikulären pHs erst ziemlich spät nach (P)RR-Depletion auftritt. Wir fanden heraus, dass (P)RR-Depletion weder Phagozytose noch Endozytose beeinträchtigte, sondern für das Recycling des Transferrin-Rezeptors zur Plasmamembran wichtig ist. / A decade ago, the (pro)renin receptor [(P)RR] was discovered and depicted as a new component of the renin-angiotensin system. However, recent studies have put in evidence that the (P)RR associate with and regulate the vacuolar H+-ATPase (V-ATPase), hence its other name vacuolar H+-ATPase associated protein 2 (ATP6ap2). In osteoclasts, V-ATPases are mainly located at the plasma membrane facing the bone surface and extrude protons into the extracellular space. Mice with genetic deletion of various V-ATPase subunits are characterized by an increase of bone mass (osteopetrosis). In this work, we found that the (P)RR is highly expressed in mature osteoclasts in vitro and in vivo. Mice with genetic deletion of the (P)RR in osteoclasts developed a complex bone phenotype characterized by a reduced bone density. Osteoclasts lacking (P)RR displayed increased differentiation and/or activity in vitro and in vivo. We therefore suggest that the (P)RR does not directly regulate V-ATPases located at the plasma membrane but rather interferes with osteoclast physiology through other mechanisms. Macrophages are professionalized phagocytes crucial for immune response. Phagocytosis is an essential cellular process, which requires lysosomal V-ATPases for degradation of engulfed pathogens. We generated transgenic rats with a conditional depletion of the (P)RR with the use of a doxycycline-induced shRNA expression system. Efficient (P)RR depletion in macrophages was accomplished by doxycycline treatment in vivo in drinking water and in vitro in culture medium. In this work, we found that the impairment of vesicular pH occurs lately after (P)RR deletion. Also, we found that (P)RR deletion did not impair neither phagocytosis nor endocytosis but rather perturbed the recycling of the transferrin receptor to the plasma membrane.

Kanalrhodpsine (ChRs) sind lichtaktivierbare Kationenkanäle, welche als primäre Fotorezeptoren in Grünalgen dienen. In der Optogenetik werden ChRs verwendet um neuronale Membranen zu depolarisieren und mit Licht Aktionspotentiale auszulösen. Das mit blauem Licht aktivierte Chlamydomonas Kanalrhodopsin 2 (C2) und effiziente Mutanten wie C2 H134R stellen die am häufigsten genutzten depolarisierenden, optogenetischen Werkzeuge dar. Komplementär zu ChRs werden Protonen- und Chloridpumpen aus Archaebakterien zur neuronalen Inhibierung durch lichtinduzierte Hyperpolarisation verwendet. In der vorliegenden Arbeit untersuchten wir die ChR-Chimäre C1V1, ein grünlichtaktiviertes ChR, das sich durch hervorragende Membranlokalisierung und hohe Fotoströme in HEK-Zellen auszeichnet. C1V1 und C1V1-Mutanten mit feinabgestimmten spektralen und kinetischen Eigenschaften ermöglichen die neuronale Aktivierung mit Wellenlängen bis 620 nm sowie die unabhängige Aktivierung zweier Zellpopulationen in Kombination mit C2. Um die strukturelle Basis von Kanalöffnung und Ionentransport in ChRs zu verstehen, wurden gezielt Mutationen in C2 und C1V1 eingeführt. Die Fotoströme der entsprechenden Mutanten wurden auf Kationenselektivität und kinetische Veränderungen untersucht. Während Aminosäuren, die den Kanal an der zytosolischen Seite begrenzen, die Kationenfreisetzung und Einwärtsgleichrichtung der ChRs bestimmen, spielen zentral im Kanal gelegende Aminosäuren ein entscheidende Rolle für Kationenselektivität und -kompetition. Ein enzymkinetisches Modell ermöglichte außerdem die Zerlegung der Fotoströme in Beiträge der verschiedenen, konkurrierenden Kationen. Im letzten Teil der Arbeit wurde pHoenix, ein optogenetisches Werkzeug zur Ansäuerung synaptischer Vesikel, entwickelt. In Neuronen des Hippocampus wurde pHoenix verwendet, um die treibenden Kräfte für die vesikuläre Neurotransmitteraufnahme sowie den Zusammenhang zwischen Vesikelfüllstand und Freisetzungswahrscheinlichkeit zu analysieren. / Channelrhodopsins (ChRs) are light-activated cation channels functioning as primary photoreceptors in green algae. In the emerging field of optogenetics, ChRs are used to depolarize neuronal membranes, thus allowing for light-induced action-potential firing. The blue light-activated Chlamydomonas channelrhodopsin 2 (C2) and high-efficiency mutants such as C2 H134R represent the most commonly used depolarizing optogenetic tools. Complementary to ChRs, green to yellow light-activated proton and chloride pumps originating from archea enable neuronal inhibition by membrane hyperpolarization. In the present work, we developed the chimeric ChR C1V1, a green-light activated ChR with excellent membrane targeting and high photocurrents in HEK cells. Action spectrum and kinetic properties of C1V1 were further fine-tuned by site-directed mutagenesis. The ensemble of C1V1 variants allows for neuronal activation with wavelengths up to 620 nm and can be used in two-color optogenetic experiments in combination with C2 derivatives. In order to understand the structural motifs involved in channel gating and ion transport, conserved residues in C2 and C1V1 were mutated and photocurrents of the respective mutants were analyzed for kinetic characteristics and cation selectivity. In these experiments, residues of the inner gate region were shown to alter cytosolic cation release and inward rectification, whereas central gate residues determine cation competition and selectivity, as well as the equilibrium between the two open channel conformations. Moreover, an enzyme-kinetic model was used to quantitatively dissect ChR photocurrents into the contribution of different competing cations. Finally, we designed pHoenix, an optogenetic tool enabling green-light induced acidification of synaptic vesicles. In hippocampal neurons, pHoenix was used to study both the energetics of vesicular neurotransmitter uptake and the impact of the vesicular contents on synaptic vesicle release.

Les émissions anthropiques de dioxyde de carbone (CO2) ont augmenté depuis la révolution industrielle, entraînant des modifications dans les teneurs atmosphériques en CO2 et un accroissement de la pression partielle de CO2 (pCO2) océanique. L’absorption du CO2 par les océans a entraîné une baisse du pH des eaux de surface, correspondant à une augmentation de l’acidité d’environ 30 % par rapport aux valeurs préindustrielles. D’autre part, l’accumulation de CO2 anthropique dans l'atmosphère a également induit un réchauffement des eaux de surface depuis le milieu du 20e siècle. Le devenir des communautés planctoniques face à ces altérations actuelles et futures de leur environnement demeure incertain. On ignore également comment l’acidification et le réchauffement affecteront la production du diméthylsulfure (DMS), un gaz sulfuré d’origine planctonique impliqué dans la régulation du climat. Le but de cette thèse est de déterminer l’impact d’une augmentation de la pCO2 sur le développement des floraisons phytoplanctoniques de l'estuaire maritime du Saint-Laurent (EMSL) et la production de DMS, ainsi que d’évaluer dans quelle mesure le réchauffement des eaux de surface modulera l’effet de l’acidification. Deux grandes expériences ont été menées au cours de la thèse. Une première expérience en microcosmes (~20 L) a été conduite à l’été 2013 afin d’étudier les effets de la pCO2 sur la floraison printanière des diatomées dans L’EMSL, en portant une attention particulière aux processus microbiens régissant la production de DMS. Une seconde expérience de type multifactorielle en mésocosmes (~2600 L) a été conduite à l’automne 2014 afin de déterminer l’impact combiné de l’augmentation de la pCO2 et du réchauffement sur le développement de la floraison automnale de l’EMSL et la production du DMS. Les résultats de l’expérience en microcosmes montrent que les communautés phytoplanctoniques responsables de la floraison printanière dans l’EMSL sont résistantes à des augmentations de la pCO2 supérieures aux valeurs attendues pour 2100. Cette résistance reflète vraisemblablement leur adaptation au milieu estuarien, environnement connu pour ses variations de pCO2 importantes et rapides. Cette première expérience a également mis en évidence une diminution de 15 et de 40 % des concentrations moyennes de DMS chez les communautés respectivement soumises à des pCO2 de 1850 μatm et 2700 μatm par rapport au contrôle (~775 μatm). Des incubations menées en parallèle ont permis de montrer, au moyen de 35S-DMSPd, que l’effet négatif de l’acidification sur le DMS résultait en grande partie d’une diminution de l’efficacité de conversion du DMSP en DMS par les bactéries. La deuxième expérience a également mis en évidence une forte résistance de la diatomée Skeletonema costatum à une large gamme de pH (~8.0–7.2) et de pCO2 (~90–3000 μatm). Lors de cette étude, un réchauffement des eaux de 5 °C a accéléré le développement et le déclin de la floraison, mais n’a pas eu d’effet sur la production primaire intégrée pendant l’expérience. À l’instar de l’expérience en microcosmes, l’augmentation de la pCO2 a provoqué une diminution des concentrations moyennes de DMS de ~66 % dans les mésocosmes les plus acidifiés par rapport aux traitements les moins acidifiés à température in situ (10 °C). L’effet négatif d’une augmentation de la pCO2 sur la production nette de DMS pourrait cependant être annulé par le réchauffement des eaux de surface. En effet, mes résultats révèlent que la production nette de DMS était plus élevée à 15 °C par rapport à 10 °C et ce à toutes les pCO2 testées. Ces résultats inédits suggèrent que le réchauffement attendu des eaux de surface pourrait contrer en partie l’effet négatif de l’acidification sur la production nette de DMS dans l’EMSL et possiblement dans l’océan mondial. / Anthropogenic carbon dioxide (CO2) emissions have increased since the industrial revolution, leading to modifications in atmospheric CO2 content and an increase in oceanic CO2 partial pressures (pCO2). The uptake of CO2 by the oceans has resulted in a lowering of surface water pH, corresponding to an increase in the acidity of the oceans by ~30 % compared with pre-industrial times. Furthermore, climate change resulting from the accumulation of anthropogenic CO2 in the atmosphere is responsible for the observed warming of sea surface temperatures since the mid 20th century. The fate of planktonic communities in the face of these changes in the marine environment over the next century remains uncertain. Even less understood are the possible interactions of acidification and warming on the production of dimethylsulfide (DMS), a sulfur-containing gas produced by planktonic communities and involved in climate regulation. The aim of this thesis is to determine the impact of heightened pCO2 on the development of the phytoplanktonic blooms in the Lower St. Lawrence Estuary (LSLE), and their production of DMS, as well as to evaluate how concomitant warming could modulate the effects of acidification. Two intricate experiments were carried out during this study. First, a microcosm experiment (~20 L) was conducted in the summer of 2013 to assess the effects of pCO2 on the development of the LSLE spring diatom bloom, paying special attention to the microbial processes governing the production of DMS. Second, a multifactorial mesocosm experiment (~2600 L) was carried out in the fall of 2014 to investigate the combined effects of pCO2 and temperature on the development of the fall bloom in the LSLE and the production of DMS. Results from our microcosm experiment show that the blooming phytoplankton community of the LSLE during spring is resistant to pCO2 increases superior to the expected values for 2100. This resistance likely reflects its adaptation to the estuarine setting, an environment known for rapid and intense fluctuations of pCO2. This first experiment has also highlighted a reduction of the average concentrations of DMS by 15 and 40 % in planktonic assemblages respectively subjected to pCO2 of ~1850 μatm and ~2700 μatm compared to the control (~775 μatm). Parallel incubations have shown, using 35S-DMSPd, that the negative effect of acidification on DMS mostly stemmed from a decrease in the conversion efficiency of DMSP to DMS by bacteria. The second experiment has also highlighted a strong resistance of the diatom Skeletonema costatum to a wide range of pH (~8.0–7.2), and corresponding pCO2 (~90–3000 μatm). In this study, a warming of 5 °C accelerated the development and decline of the bloom, but did not affect the integrated primary production over the duration of the experiment. As in the first experiment, heightened pCO2 resulted in a decrease of average concentrations of DMS of ~66 % in the most acidified mesocosms compared to the least acidified mesocosms at in situ temperature (10 °C). However, the negative effect of an increase in pCO2 on the net production of DMS could be mitigated by a warming of surface waters. Indeed, my results reveal that the net production of DMS was higher at 15 °C compared to 10 °C over the whole pCO2 gradient in our mesocosm study. These novel results suggest that warming of surface waters could mitigate, at least partly, the negative effect of acidification on DMS net production in the LSLE and perhaps in the world’s oceans.

Les changements environnementaux globaux (CEG) menacent les écosystèmes marins et les populations humaines qui en dépendent. Une recherche scientifique croissante tente d’évaluer les impacts des changements environnementaux sur les écosystèmes et les services écosystémiques, notamment pour guider les politiques publiques. Focalisée sur les systèmes socio-écologiques (SSE) des récifs coralliens, cette thèse analyse les approches proposées dans la littérature et conçoit de nouvelles méthodologies, évaluations et indicateurs pour guider la science et l’action publique. Nous montrons qu’une stratégie de recherche régionale doit prendre en compte la complexité et produire de meilleures projections des impacts des CEG sur les récifs coralliens et les services associés. Nous cartographions des indicateurs à l’échelle globale pour évaluer où la dépendance des sociétés aux récifs coralliens sera affectée par les menaces globales dues à un niveau de CO2 élevé. Nous analysons comment la science répond aux impacts des CEG sur les récifs coralliens et nous identifions des pistes pour la recherche. Enfin, nous opérationnalisons une facette de la vulnérabilité, la capacité d’adaptation écologique, pour servir d’outil pour évaluer l’effectivité des actions locales dans un contexte de CEG. Ce manuscrit contribue à des avancées théoriques et méthodologiques sur l’évaluation des impacts, de la vulnérabilité et de l’adaptation aux CEG. Il développe des approches interdisciplinaires pour l’étude des SSE et des services écosystémiques, ciblant les récifs coralliens comme étude de cas. Enfin, il analyse l’émergence d’un champ scientifique sur les solutions aux GEC pour les récifs coralliens. / Global environmental change (GEC) in the ocean threatens marine ecosystems and the people who depend on them. A growing scientific effort is attempting to evaluate the impacts of environmental changes on ecosystems and ecosystem services and guide policy-making to respond to this global issue. Focusing on social-ecological systems of coral reefs, this thesis critically reviews the approaches put forward in the literature to understand gaps and to design new methodologies, assessments, and indicators to guide science and policy. Our findings show that a regionally targeted strategy of research should address complexity and provide more realistic projections about the impacts of GEC on coral reefs ecosystems and ecosystem services. We map global-scale indicators to understand where human dependence on coral reef ecosystems will be affected by globally-driven threats expected in a high-CO2 world. We then analyze how science is responding to the challenge posed by GEC on coral reefs and to identify gaps in research.Finally, we attempt to operationalize an overlooked component of vulnerability assessments, ecological adaptive capacity, to serve as a tool to help assess where local actions can be effective in the context of climate change. This manuscript contributes to theoretical and methodological advances to evaluate impacts, vulnerability and adaptation to GEC. It develops interdisciplinary approaches for the study of social-ecological systems and ecosystem services, targeting coral reefs as a case study. Finally, it synthesizes critically the emergence of a scientific field on solutions to GEC for coral reef social-ecological systems.

Coccolithophores are unicellular microscopic algae (Haptophyta) surrounded by calcium carbonate plates that are produced during their life cycle. These species, whose contemporary contributor is Emiliania huxleyi, are mainly found in the sub-polar and temperate oceans, where they produce huge blooms visible from space. Coccolithophores are sensitive to ocean acidification that results from the ongoing accumulation of anthropogenic carbon dioxide (CO2) in the atmosphere. The response of these organisms to global change appears to be related to the reduction of their ability to produce calcium carbonate at the cellular level. At the community levels, one anticipates changes in the carbon fluxes associated to their blooms as calcification is reduced. However, the consequences of such environmental changes on this species are speculative and require improvements in the description of the mechanisms controlling the organic and inorganic carbon production and export.<p><p>The first aspect of this work was to study the response of these organisms to artificially modified CO2 concentrations representative of the conditions occurring in the past (glacial) and those expected by the end of the century (2100). Two different levels were examined: the continuous monospecific cultures (chemostats) allowed us to work at the cellular level while the mesocosms gave light to the mechanisms taking place in an isolated fraction of the natural community. The second aspect of this work consisted of field studies carried out during four cruises (2002, 2003, 2004 and 2006) in the northern Bay of Biscay, where the occurrence of E. huxleyi blooms were observed in late spring and early summer. We describe the vertical profiles of biogeochemical variables (nutrients, chlorophyll-a, dissolved inorganic chemistry, particulate carbon, transparent exopolymer particles (TEP)) and study processes such as primary production, calcification and bacterial production. The properties of these blooms are compared with those reported in the literature and enriched with original measurements such as the abundance and concentration of TEP that could play an important role in carbon export to the deep ocean, modifying the properties of the settling ballasted aggregates.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The acidification has been a known problem in Sweden for several decades. Sulphurous compounds, spread from the British Isles and the European continent led to a decrease in the pH-value of the rain that fell over Sweden. Since the acidification was discovered in the 1960s, active measures against the sulphurous deposition have been undertaken. The sulphurous deposition has decreased by 90 %, and the problem was for some time considered under control, until recently when a new era of the acidification may have started. Due to the increased demand of renewable energy, and Sweden’s potential to use biomass instead of fossil fuels, whole tree harvesting has been more utilized. Studies indicate that the forest soils are depleted in base cations in a faster rate when whole tree harvesting is performed compared to regular stem harvesting. Mass balance calculations and simulations indicate that an increased bio uptake of base cations due to whole tree harvesting leads to an increased biological acidification. However, although many studies agree that the impact of the whole tree harvest on the base cation supply of the soils is significant, long running Swedish experiments indicate that the difference between whole tree harvesting and regular stem harvesting diminishes over time. After a 40 year period, the difference in base cation supply between whole tree harvested soils and stem harvested soils are small. The reason for this could be different processes that reallocate base cations from different pools, which are not usually studied. The aim has been to investigate and evaluate different chemical extraction methods (Aqua Regia, HCl, EDTA, BaCl2, NH4OAc and water) capability to extract the base cations calcium, potassium, magnesium and sodium from four different Swedish forest soils and what this means for our understanding of how much base cations a soil contains. The extractions indicated that there is a statistical significant difference between the methods ability to extract base cations. Generally Aqua Regia was the most potent method, followed by HCl, EDTA, BaCl2, NH4OAc and water in decreasing order of effectiveness to extract the base cations. Linear correlations were found between EDTA, BaCl2 and NH4OAc. The internationally widely used method NH4OAc was considered to be at risk of underestimating the amount of base cations in the soil. / Försurningsproblematiken har länge varit ett känt problem i Sverige. Svavelhaltiga föroreningar som spreds från de brittiska öarna och den europeiska kontinenten ledde till att pH-värdet i regnet som föll över Sverige sjönk. Sedan upptäckten på 60-talet har aktiva åtgärder vidtagits mot utsläppen vilket har lett till en minskning av de försurande föroreningarna med 90 %. På grund av den stora utsläppsreduktionen som skett ansågs försurningsproblematiken vara under kontroll, tills nyligen då en ny etapp av för- surningen kan ha påbörjats. På grund av den ökande efterfrågan av förnyelsebar energi, i kombination med Sveriges stora skogstillgångar, har helträdsskörd av träd blivit alltmer nyttjad. Studier visar att markens baskatjonförråd utarmas i större utsträckning av helträdsskörd, då även grenar, rötter och toppar tas om hand jämfört med vanlig stamskörd då endast stammen tas med från skogen. Massbalanssimuleringar antyder att ett ökat bioupptag av baskatjoner på grund av helträdsskörd leder till en ökad biologisk försurning. Trots att många studier är överens om helträdsskördens inverkan på markens innehåll av baskatjoner visar lång- liggande försök i Sverige att skillnaderna mellan uttag av hela träd och stamved minskar med tiden. Efter en period på 40 år återstår endast små skillnader mellan avverknings- metoderna. Orsakerna till varför mätningarna och massbalansberäkningarna och simuleringarna inte stämmer överens kan vara många, t.ex. att det finns processer som kan omfördela baskatjoner från de som vanligtvis studeras. Syftet har varit att undersöka och utvärdera olika kemiska extraktionsmetoders (Aqua Regia, HCl, EDTA, BaCl2, NH4OAc och vatten) förmåga att extrahera baskatjonerna kalcium, kalium, magnesium och natrium från fyra olika skogsjordar i Sverige och vad resultaten betyder för vår uppfattning av mängden baskatjoner i marken. Extraktionerna visade att en statistiskt signifikant skillnad fanns mellan metodernas förmåga att extrahera de olika baskatjonerna. Generellt var Aqua Regia den metod som extraherade den största mängden baskatjoner, HCl, EDTA, BaCl2, NH4OAc och vatten följde i fallande ordning efter förmåga att extrahera baskatjonerna. Linjära korrelationer mellan EDTA, BaCl2 och NH4OAc upptäcktes. Den internationellt ofta använda metodiken för att extrahera baskatjoner, NH4OAc, ansågs riskera att underskatta mängden baskatjoner i marken.

The aim of this diploma thesis was to reveal the taxonomic position and phylogenetic relationships of the dominant planktonic algae in two acid metal-rich lakes (Hromnice Lake and Plešné Lake, Czech Republic) and to compare these isolates with other closely related strains with the focus on the tolerance to various toxic metals (Cr, Al, Cu, Mn, Zn, Hg). The phylogenetic analyses showed that both strains belong to species Coccomyxa simplex. It was the first evidence that specifically this species is capable to become the dominant phytoplankton alga in the extreme environment of acid lakes with increased supply of phosphorus. Based on 18S rDNA analysis, four independent phylogenetic lineages were revealed within the genus Coccomyxa with three of them containing isolates from acid freshwaters. Furthermore, new strains of the recently described species Coccomyxa polymorpha were found growing in various chemical solutions. The toxicity test revealed that Coccomyxa simplex strains isolated from Hromnice and Plešné lakes did not show any peculiar resistance to increased metal concentrations. A significantly strain-specific response was recorded in case of aluminum, however, it was not related to the concentration of this metal in the original habitat. The ability to thrive in extreme habitats is probably...

- 4 - ABSTRACT During the revision of epiphytic species of the Lecanora subfusca group in the Czech Republic, nine taxa have been recorded. L. cinereofusca and L. exspersa are reported for the first time from the country. L. circumborealis has been excluded from the list of Czech lichens. L. cinereofusca and L. horiza could be considered as extinct. L. rugosella and L. subrugosa are regarded as extreme morpholocial forms from L. chlarotera and L. argentata. These morphotypes corelate with ecological conditions: substrate (nutrient enriched bark) and habitat (eutrophisation effect). In case of L. subrugosa, this speculation was confirmed by molecular data (ITS rDNA sequences). Several new secondary metabolites have been discovered during the detailed research of chemical lichen compounds. These substances belonging to terpenoids are taxonomically important and very helpful for distinguishing single species. The abundance of L. allophana and L. chlarotera has decreased during last decades. Contrarily, L. pulicaris expanded slightly. The main reasons of changes in distribution are air pollution and acidification of substrates as the impact of acid rains. The rate of substrate acidification has been shown on example of L. pulicaris. In the past, this lichen predominated on acid-barked porophytes. Nowadays, it...

Estudaram-se neste trabalho os efeitos da deposição atmosférica sobre o balanço de elementos químicos, analisando-se as entradas e saídas dos elementos através do ciclo hidrológico durante quatro anos (setembro de 1991 a agosto de 1995) na Floresta Atlântica, situada nas vizinhanças do pólo industrial de Cubatão, São Paulo. Por meio de amostragens quinzenais e de determinação da concentração iônica, avaliaram-se, em três florestas diferentemente atingidas pela impacto da poluição aérea – Pilões-menos poluída, área de referência; Moji-fortemente poluída e Paranapiacabamoderadamente poluída – os seguintes aspectos: entrada de elementos pela água de chuva; transferência pela água que atravessa a cobertura vegetal e pela solução do solo a 10cm, 60cm e 100cm de profundidade; saída e neutralização pela água de nascentes e de pequenos córregos, bem como as alterações químicas no solo induzidas pela deposição atmosférica. Na área mais poluída (Moji), as concentrações iônicas médias encontradas na precipitação que atinge a floresta e na precipitação que atinge o solo foram, respectivamente: 5 e 10 mg.L-1 para o sulfato-S, 0,5 e 0,7 mg.L-1 para o nitrato, 0,63 e 1,14 mg.L-1 para o fluoreto, 2,2 e 2,8 mg.L-1 para o amônio-N, 0,69 e 2,15 mg.L-1 para o magnésio, 3,1 e 7,7 mg.L-1 para o cálcio. Na floresta de referência, menos poluída (Pilões), as concentrações iônicas foram em média, 1/3 a 1/9 inferior a esses valores, refletindo claramente as diferenças nas cargas poluidoras que atingem cada floresta. Verificou-se que a composição química da solução do solo muda completamente com a infiltração da água. As concentrações de nitrato aumentaram de 3 a 22 vezes com a infiltração da água nos primeiros 10cm de solo do Pilões e Moji, respectivamente, valores estes bastante expressivos. Em menor proporção, isso também foi observado para o sulfato e cloreto. As mudanças nas concentrações dos elementos durante a passagem da água pelo ecossistema são explicadas pelas reações químicas que ocorrem nos diferentes compartimentos. Assim, nas florestas mais poluídas (Moji e Paranapiacaba) elas são caracterizadas pela retenção praticamente total do amônio, e parcial do sulfato, nas camadas superficiais do solo, pela liberação do alumínio dos minerais do solo e pela lixiviação extremamente alta de nitrato em conseqüência do processo de nitrificação da matéria orgânica. A deposição atmosférica nas florestas investigadas é bastante elevada e variável. Entram anualmente no solo, pela precipitação: de 122 a 255 kg.ha-1 de enxofre na forma de sulfato; de 7 a 70 kg.ha-1 de nitrogênio, principalmente na forma de amônio; e de 4 a 28 kg.ha-1 de fluoreto. Com a entrada de amônio e de nitrogênio orgânico no sistema, reações de nitrificação ocorrem na camada superficial do solo, principalmente das áreas mais atingidas pela poluição. Na floresta mais poluída (Moji), a saída de nitrato pela água do solo a 100cm de profundidade chega anualmente a 271 kg de N por hectare, enquanto a saída de enxofre alcança o valor de 386 kg de S por hectare, anualmente. A acidificação do solo em decorrência da entrada e transferência de nitrogênio e enxofre provoca a liberação de alumínio dos minerais do solo e a lixiviação de formas iônicas (mais de 240 kg de Al por ha anualmente). A transferência de íons alumínio para o lençol freático e para águas de nascentes e córregos acarreta um problema ecológico sério, pois esses íons consomem a alcalinidade e a água fica sujeita a acidificação. / The effects of atmospheric deposition upon element balance (input and output) and turnover through the hydrological cycle have been investigated from September 1991 to August 1995 (four years), in the vicinity of the industrial complex of Cubatão, State of São Paulo, Brazil. The aim was to assess atmospheric deposition and fluxes through precipitation, soil water at 10cm, 60cm and 100cm depths, and surface water as well as to investigate possible soil changes induced by atmospheric deposition. Three sites were chosen which differed significantly with respect to pollution impact: Pilões (reference site, less polluted), Moji (most polluted) and Paranapiacaba (mildly polluted). Annual averages of ionic concentrations in precipitation found in open field and below the tree canopy amounted to 5 and 10 mg.L-1, respectively, for sulfate-S, 0.5 and 0.7 mg.L-1 for nitrate, 0.63 and 1.14 mg.L-1 for fluoride, 2.2 and 2.8 mg.L-1 for ammonium-N, 0.69 and 2.15 mg.L-1for Mg, and 3.1 and 7.7 mg.L-1 for Ca at the most polluted site (Moji). The relatively "clean" reference site (Pilões) attained 1/3 to 1/9 of these averages, thus clearly reflecting the difference in air pollution load. Chemical composition in the liquid phase is completely changed when precipitation infiltrates the soil profile. Nitrate concentration increases by the factor 3 to 22. A clear increase is also found for sulfate and chloride. Concentration changes during ecosystem passage of seepage are interpreted in relation to chemical reactions taking place in different compartments. They are characterized by an almost complete retention of ammonium and some retention of sulfate in the upper soil layers, and at the most polluted site by mobilization of Al from soil minerals and very high leaching of nitrate as a consequence of nitrification of organic matter. The forests under investigation are characterized by a very high input from the atmosphere. Between 122 and 255 kg S per hectare are annually carried into soil by precipitation in the form of sulfate, 7 to 70 kg of nitrogen mainly in the form of ammonium, 4 to 28 kg of fluoride. Input of ammonium and organic bound nitrogen is followed by nitrification in the topsoil. At the most polluted site (Moji), nitrate output with seepage amounts to 271 kg N per ha and year, sulfate output to more than 386 kg S. Soil acidification associated with turnover of sulfur and nitrogen is followed by the release of aluminum from soil minerals, and leaching of ionic forms of Al (up to 240 kg Al per hectare annually). Transfer of aluminum ions to groundwater and surface water can have serious ecological effects. Alkalinity is consumed, and the water is subject to acidification.