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Tolerance of Phaeodactylum tricornutum to cadmium and hydrogen peroxide
Get PDFSiden starten på moderne jordbruk, industrialisering og urbanisering har omfanget av forurensning fra tungmetaller eskalert. Den økte mengden forurensning fra tungmetaller har blitt en stor bekymring, spesielt på grunn av deres toksisitet og persistens i miljøet.
Målet med denne studien var å undersøke effekten av kadmium og hydrogenperoksid på Phaeodactylum tricornutum. Å forstå effektene kan gi verdifull informasjon når man vurderer bruken av P. tricornutum i biomonitorering og “phycoremediation”.
Generelt var det ingen fysiologisk effekt fra 0.1 og 0.5 mg/L Cd2+ på P. tricornutum. Imidlertid ble det observert en signifikant effekt for konsentrasjoner på 2.5 og 12.5 mg/L Cd2+ med redusert vekst, cellelevedyktighet og nøytralt lipidinnhold. Det var også en reduksjon av flere fotosyntetiske parametere. Det var en økning i opptak av Cd2+ og forholdet mellom opptak av Cd2+ og eksponeringskonsentrasjonene av Cd2+ var lineært. Det var endret opptak av forskjellige andre elementer som respons på 2.5 og 12.5 mg/L Cd2+. Genekspresjonsprofilering viste at det var en oppregulering av gener relatert til transport og avgiftning av kadmium i P. tricornutum. Det var også oppregulering av gener relatert til generelt stress, fotosyntese og lipidmetabolisme. Totalt sett viste P. tricornutum høy toleranse for kadmium og et potensial for bruk i biomonitorering og “phycoremediation”.
P. tricornutum viste følsomhet for hydrogenperoksid (H2O2). Eksponering med konsentrasjoner på 0.03 mM H2O2 hadde ingen effekt på veksten. Konsentrasjoner på 0.1, 0.3 og 0.5 mM H2O2 ga redusert vekst. Eksponering for 0.03 og 0.1 mM H2O2 hadde liten eller ingen effekt på fotosyntetiske parametere. For eksponering for 0.3 og 0.5 mM H2O2 kunne ingen verdier for fotosyntetiske parametere oppnås, bortsett fra ikke-fotokjemisk quenching med verdier rundt null. Fra disse resultatene ser det ut til at terskelen for toleranse av P. tricornutum overfor H2O2 ligger et sted mellom 0.1 og 0.3 mM.Since the start of modern agriculture, industrialization and urbanization, the extent of pollution from heavy metals has escalated. The increased level of pollution from heavy metals has become of great concern, particularly because of their toxicity and persistence in the environment.
The goal of this study was to investigate the effects of cadmium and hydrogen peroxide on the diatom Phaeodactylum tricornutum. Understanding the effects could provide valuable information when considering the use of P. tricornutum in biomonitoring and phycoremediation.
In general, there was no physiological effect of 0.1 and 0.5 mg/L Cd2+ on P. tricornutum. However, a significant effect was observed for concentrations of 2.5 and 12.5 mg/L Cd2+ with reduced growth, cell viability and neutral lipid content. There was also a reduction of several photosynthetic parameters. There was an increase in Cd2+ uptake and the relationship between the uptake of Cd2+ and the exposure concentrations of Cd2+ was linear. There was altered uptake of various other elements in response to 2.5 and 12.5 mg/L Cd2+. Gene expression profiling showed that there was upregulation of genes related to transport and detoxification of cadmium in P. tricornutum. There was also upregulation of genes related to general stress, photosynthesis and lipid metabolism. Overall, P. tricornutum showed a high tolerance to cadmium and a potential for use in biomonitoring and phycoremediation.
P. tricornutum showed sensitivity to hydrogen peroxide (H2O2). Exposure with concentrations of 0.03 mM H2O2 had no effect on growth. Concentrations of 0.1, 0.3 and 0.5 mM H2O2 resulted in reduced growth. Exposure to 0.03 and 0.1 mM H2O2 had little to no effect on photosynthetic parameters. For exposure to 0.3 and 0.5 mM H2O2 no values for photosynthetic parameters could be obtained, except for non-photochemical quenching with values close to zero. From these results it appears that the threshold for tolerance of P. tricornutum to H2O2 lies somewhere between 0.1 and 0.3 mM
Testing the sensitivity of Phaeodactylum tricornutum to environmental conditions and pollutants
Get PDFKiselalger har betydelig økologisk betydning som primærprodusenter da de er ansvarlige for mer enn 20% av global karbonfiksering og er viktige komponenter i den marine næringskjeden. Deres unike egenskaper gjør dem til attraktive kandidater for ulike bioteknologiske formål. Derfor er det essensielt å forstå hvordan kiselalger reagerer på miljøforandringer og å finne optimale vekstforhold som kan gi bedre utbytte i industrien. Denne avhandlingen undersøker responsen til kiselalgen Phaeodactylum tricornutum på økte konsentrasjoner av NaCl og MgCl2, og på per- og polyfluoralkylstoffer (PFASer).
Saliniteten i sjøvann varierer betydelig i kystnære strøk, og kiselalger må tilpasse seg disse variasjonene. I den første delen av avhandlingen undersøkes virkningene av forhøyede nivåer av NaCl og MgCl2 på vekst, celledød, fotosyntetisk effektivitet og kapasitet, samt genuttrykk. Veksten avtok med økende saltkonsentrasjoner, med signifikant vekstreduksjon mellom 0,428 og 0,856 M for eksponeringer i 96 timer og én måned. Celledød inntraff for konsentrasjoner over 1,712 M innen 24 timer, og ble påvist ved hjelp av flowcytometri og propidium-jodidfarging. Ingen forskjeller i vekst ble observert mellom korttids- og langtidseksponering, noe som indikerer fravær av veksttilpasning etter langtidseksponering. Puls-amplitude-modulasjons-fluorometri viste økt fotosyntetisk effektivitet og kapasitet for begge salter. Målinger av osmolalitet indikerte flere partikler og høyere salinitet av NaCl-løsninger sammenlignet med MgCl2. Genuttrykk ble analysert ved revers transkripsjon-kvantitativ polymerasekjedereaksjon (RT-qPCR) og RNA-sekvensering. Funnene viste næringsmangel i den ueksponerte kontrollen og antydet at endringer i ionetransport ble indusert for å motvirke salinitetstress. Samlet sett viser resultatene at NaCl er mer potent enn MgCl2 ved samme kloridkonsentrasjon, noe som antyder at Na+ kan ha en større rolle i toksisiteten enn Cl-.
De siste årene har effekten av PFASer på mennesker, samt regulering av stoffene, fått mye oppmerksomhet. Til tross for at sjøvann antas å være det endelige oppsamlingsstedet for PFASer, har få studier undersøkt virkningene på primærprodusenter nederst i den marine næringskjeden. Den andre delen av denne avhandlingen undersøker effekten av de to vanligste påviste PFASene på vekst av P. tricornutum. Relativ fluorescens ble målt med plateleser og ble brukt som en indikator på vekst. EC50 for 96 timers inhibering av relativ fluorescens ble funnet ved ca. 160 mg/l for perfluoroktansyre (PFOA) og 80 mg/l perfluoroktansulfonat (PFOS). Dette antyder biomagnifisering i den marine næringskjeden, som til slutt kan påvirke mennesker. Videre forskning er nødvendig for å forstå de biologiske mekanismene bak denne toleransen og den toksiske virkemåten til PFASer i kiselalger og i P. tricornutum.Diatoms hold significant ecological importance as primary producers responsible for more than 20% of global carbon fixation and they are essential components of the marine food chain. Their unique traits make them potential candidates for various biotechnological applications. Understanding how diatoms respond to changing environments and identifying optimal conditions for growth and productivity is of great value. This thesis examines the responses of the diatom Phaeodactylum tricornutum to increasing concentrations of NaCl and MgCl2, and to per- and polyfluoroalkyl substances (PFASs).
Seawater salinity fluctuates substantially in the habitats of P. tricornutum, and the diatom must adapt to tolerate these changes. The first part of the thesis investigates the effects of elevated levels of NaCl and MgCl2 on growth, cell death, photosynthetic efficiency and capacity, and gene expression. Growth declined with increasing salt concentrations, with significant decreases between 0.428 and 0.856 M for 96 hour and long-term exposure. The 24 hour cell death was found for concentrations above 1.712 M using flow cytometry and propidium iodide staining. Short-term and long-term effects on growth were compared, indicating no adaption. Pulse-amplitude-modulation (PAM) fluorometry revealed increased photosynthetic efficiency and capacity for both salts. Measurements of osmolalities indicated more particles and higher salinity of the NaCl solutions compared to MgCl2. Gene expression was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing, revealing nutrient depletion in the non-exposed control and changes in ion flux as a mechanism to counteract salinity stress. Overall results showed that NaCl is more potent than MgCl2 at the same chloride concentration, suggesting that Na+ may contribute more to toxicity than Cl-.
In recent years, the adverse effects of PFASs on humans and the regulation of the contaminants have received much attention. Despite seawater being hypothesized as the final sink for PFASs, few studies have examined the impact on primary producers at the base of the marine food chain. The second part of this thesis investigates the effects of the two most commonly detected PFASs on growth of P. tricornutum. Relative fluorescence was measured using a plate reader and used as an indicator for growth. The 96 hour EC50 for inhibition of relative fluorescence was found at approximately 160 mg/L for perfluorooctanoic acid (PFOA) and 80 mg/L for perfluorooctane sulfonate (PFOS). Tolerance to these high concentrations suggests magnification up the marine food chain, ultimately affecting humans. Further research is necessary to understand the biological mechanisms behind this tolerance and the toxic mode of action of PFASs in diatoms
Phaeodactylum tricornutum tolerance to cadmium and copper
Get PDFIndustrialiseringen har ført til økt utslipp av tungmetaller i både akvatiske og terrestriske miljøer,
og dette kan ha stor påvirkning på levende organismer, inkludert marine mikroalger. Til tross for
mikrogalgers fundamentale roller i marine økosystemer, er kunnskapen vår om deres sensitivitet
og toleranse for miljøfaktorer og forurensinger, inkludert tungmetaller, begrenset.
Målet for denne masteroppgaven var å undersøke effektene av kadmium og kobber eksponering i
den marine kiselalgen Phaeodactylum tricornutum. Dette er verdifullt for å kunne forstå hvordan
økene nivåer av tungmetaller i miljøet vil påvirke mikroalgesamfunn og for å kunne evaluere
potensialet for å kunne bruke P. tricornutum innen bioremedieringsteknologi. Ingen signifikante
fysiologiske påvirkninger ble observert for eksponeringsnivåer på 0.1 og 0.5 mg/L Cd2+, mens
behandling med 2.5 og 12.5 mg/L Cd2+ resulterte i redusert vekst, redusert fotosyntetisk aktivitet,
økt celledød, reduserte nivåer av nøytrale lipider, og endringer i elementinnhold. Kadmiumopptak
i cellene ble vist å øke lineært med økende nivåer av kadmium i vekstmediet.
Genekspresjonsanalyse viste oppregulering av flere gener som er antatt å være relatert til kadmium
transport og detoksifisering i P. tricornutum. Cellene viste lavere toleranse for kobbereksponering,
sammenlignet med kadmium, med signifikant reduksjon av vekst og fotosyntetisk aktivitet som
respons på kobbernivåer under 1 mg/L. Sammen indikerer disse funnene at P. tricornutum kan
tolerere høye nivåer av kadmium eksponering og viser potensiale for bruk innen
bioremedieringsteknologi.The industrialization has led to an increase in heavy metal pollution both in aquatic and terrestrial
environments and this can have major impacts on living organisms, including marine microalgae.
Despite the fundamental role of microalgae in marine ecosystems, our knowledge of their
sensitivity and tolerance to environmental factors and pollutants, including heavy metals, is limited.
The goal of this thesis was to investigate the effects of cadmium and copper exposure in the marine
diatom Phaeodactylum tricornutum. This is important to understand how rising levels of heavy
metals in the environment will influence microalgal communities and for evaluating the potential
of using P. tricornutum in bioremediation technologies. No significant physiological responses
were observed at exposure levels of 0.1 and 0.5 mg/L Cd2+, while treatment with 2.5 and 12.5 mg/L
Cd2+ resulted in reduced growth, reduced photosynthetic activity, increased cell death, reduced
neutral lipid content, and alterations in cellular content of elements. The cadmium uptake by the
cells was demonstrated to increase linearly with increasing levels of cadmium in the growth
medium. Gene expression profiling showed upregulation of several genes believed to be associated
with transport and detoxification of cadmium in P. tricornutum. The P. tricornutum cells showed
lower tolerance to copper exposure compared to cadmium, with significant reduction in growth
and photosynthetic activity in response to copper levels below 1 mg/L. Altogether, the results
indicate that P. tricornutum can tolerate high levels of cadmium exposure and show potential for
applications within the bioremediation technology
Genetic, molecular and functional studies of RAC GTPases and the WAVE-like regulatory protein complex in Arabidopsis thaliana.
Get PDFSmall GTP-binding proteins are molecular switches that serve as important regulators of numerous cellular processes. In animal and plant cells, the Rho family of small GTPases participate in e.g. organisation of the actin cytoskeleton, production of reactive oxygen species through the NADPH oxidase complex, regulation of gene expression. The three most extensively studied subgroups of the Rho GTPase family are Cdc42, Rho and Rac. One of the mechanisms by which animal Rac and Cdc42 GTPases regulate actin filament organisation is through activation of the ARP2/3 complex, a multimeric protein complex which induces branching and nucleation/elongation/polymerisation of actin filaments. Activation of the ARP2/3 complex by Rac and Cdc42 is mediated through the proteins WAVE and WASP, respectively.
In a search for Ras-like GTPases in Arabidopsis, we identified a family of genes with similarity to Rac GTPases. Screens of cDNA and genomic libraries resulted in the finding of 11 genes named ARACs/AtRACs. Genes encoding Rho, Cdc42 or Ras homologues were not identified. Expression analysis of AtRAC1 to AtRAC5 indicated that AtRAC1, AtRAC3, AtRAC4 and AtRAC5 are expressed in all parts of the plant, whereas AtRAC2 is preferentially expressed in root, hypocotyl and stem.
The AtRAC gene family can be divided into two main groups based on sequence similarity, gene structure and post-translational modification. AtRAC group II genes contain an additional exon, caused by the insertion of an intron which disrupts the C-terminal geranylgeranylation motif. Instead, group II AtRACs contain a putative motif for palmitoylation. Phylogenetic analyses indicated that the division of plant RACs into group I and group II occurred before the split of monocotyledonous and dicotyledonous plants. Analyses of the genes neighbouring AtRAC genes revealed that several of the plant RAC genes have been created through duplications.
The restricted/tissue-specific expression pattern of AtRAC2 led us to do a more detailed expression analysis of this gene. A 1.3 kb fragment of the upstream (regulatory) sequence of AtRAC2 directed expression of GUS or GFP to developing primary xylem in root, hypocotyl, leaves and stem. In root tips, the onset GUS staining or GFP fluorescence regulated by the AtRAC2 promoter slighty preceded the appearance of secondary cell walls. In stems, GUS staining coincided with thickening of xylem cell walls. Transgenic plants expressing constitutively active AtRAC2 displayed defects in the polar growth of leaf epidermal cells, indicating that AtRAC2 may be able to regulate the actin cytoskeleton. Surprisingly, an AtRAC2 T-DNA insertion mutant did not show any observable phenotypes. GFP fusion proteins of wild type and constitutively active AtRAC2 were both localised to the plasma membrane. The data suggest that AtRAC2 is involved in development of xylem vessels, likely through regulation of the actin cytoskeleton or NADPH oxidase.
The role of RAC GTPases in regulation of the actin cytoskeleton in plants is well documented. However, although the ARP2/3 complex had been identified in plants/Arabidopsis, the mechanisms regulating this complex were unknown. Through database searches, we identified three Arabidopsis genes, AtBRK1, AtNAP and AtPIR, which encoded proteins with similarity to subunits of a protein complex shown to regulate the activity of WAVE1 in mammalian cells. T-DNA inactivation mutants of AtNAP and AtPIR displayed morphological defects on epidermal cells undergoing polar expansion, such as trichomes and leaf pavement cells. The phenotypes were similar to those observed for ARP2/3 complex mutants, suggesting that AtNAP and AtPIR act in the same pathway as the ARP2/3 complex in plants. The actin cytoskeleton in atnap and atpir mutants was less branched than in wild type plants; instead, actin filaments aggregated in thick actin bundles.
Finally, we have recently discovered a small gene family encoding putative WAVE homologues. In mammalian cells, Rac activates WAVE1 through binding to PIR121 or Sra1 (the mammalian homologues of AtPIR). The discovery of a putative WAVE regulatory complex as well as putative WAVE homologues in Arabidopsis suggests that plant RAC GTPases regulate organisation of the actin cytoskeleton during polar growth at least partly through the ARP2/3 complex, using an evolutionarily conserved mechanism.dr.scient.dr.scient
Genetic, molecular and functional studies of RAC GTPases and the WAVE-like regulatory protein complex in Arabidopsis thaliana.
No full textSmall GTP-binding proteins are molecular switches that serve as important regulators of numerous cellular processes. In animal and plant cells, the Rho family of small GTPases participate in e.g. organisation of the actin cytoskeleton, production of reactive oxygen species through the NADPH oxidase complex, regulation of gene expression. The three most extensively studied subgroups of the Rho GTPase family are Cdc42, Rho and Rac. One of the mechanisms by which animal Rac and Cdc42 GTPases regulate actin filament organisation is through activation of the ARP2/3 complex, a multimeric protein complex which induces branching and nucleation/elongation/polymerisation of actin filaments. Activation of the ARP2/3 complex by Rac and Cdc42 is mediated through the proteins WAVE and WASP, respectively. In a search for Ras-like GTPases in Arabidopsis, we identified a family of genes with similarity to Rac GTPases. Screens of cDNA and genomic libraries resulted in the finding of 11 genes named ARACs/AtRACs. Genes encoding Rho, Cdc42 or Ras homologues were not identified. Expression analysis of AtRAC1 to AtRAC5 indicated that AtRAC1, AtRAC3, AtRAC4 and AtRAC5 are expressed in all parts of the plant, whereas AtRAC2 is preferentially expressed in root, hypocotyl and stem. The AtRAC gene family can be divided into two main groups based on sequence similarity, gene structure and post-translational modification. AtRAC group II genes contain an additional exon, caused by the insertion of an intron which disrupts the C-terminal geranylgeranylation motif. Instead, group II AtRACs contain a putative motif for palmitoylation. Phylogenetic analyses indicated that the division of plant RACs into group I and group II occurred before the split of monocotyledonous and dicotyledonous plants. Analyses of the genes neighbouring AtRAC genes revealed that several of the plant RAC genes have been created through duplications. The restricted/tissue-specific expression pattern of AtRAC2 led us to do a more detailed expression analysis of this gene. A 1.3 kb fragment of the upstream (regulatory) sequence of AtRAC2 directed expression of GUS or GFP to developing primary xylem in root, hypocotyl, leaves and stem. In root tips, the onset GUS staining or GFP fluorescence regulated by the AtRAC2 promoter slighty preceded the appearance of secondary cell walls. In stems, GUS staining coincided with thickening of xylem cell walls. Transgenic plants expressing constitutively active AtRAC2 displayed defects in the polar growth of leaf epidermal cells, indicating that AtRAC2 may be able to regulate the actin cytoskeleton. Surprisingly, an AtRAC2 T-DNA insertion mutant did not show any observable phenotypes. GFP fusion proteins of wild type and constitutively active AtRAC2 were both localised to the plasma membrane. The data suggest that AtRAC2 is involved in development of xylem vessels, likely through regulation of the actin cytoskeleton or NADPH oxidase. The role of RAC GTPases in regulation of the actin cytoskeleton in plants is well documented. However, although the ARP2/3 complex had been identified in plants/Arabidopsis, the mechanisms regulating this complex were unknown. Through database searches, we identified three Arabidopsis genes, AtBRK1, AtNAP and AtPIR, which encoded proteins with similarity to subunits of a protein complex shown to regulate the activity of WAVE1 in mammalian cells. T-DNA inactivation mutants of AtNAP and AtPIR displayed morphological defects on epidermal cells undergoing polar expansion, such as trichomes and leaf pavement cells. The phenotypes were similar to those observed for ARP2/3 complex mutants, suggesting that AtNAP and AtPIR act in the same pathway as the ARP2/3 complex in plants. The actin cytoskeleton in atnap and atpir mutants was less branched than in wild type plants; instead, actin filaments aggregated in thick actin bundles. Finally, we have recently discovered a small gene family encoding putative WAVE homologues. In mammalian cells, Rac activates WAVE1 through binding to PIR121 or Sra1 (the mammalian homologues of AtPIR). The discovery of a putative WAVE regulatory complex as well as putative WAVE homologues in Arabidopsis suggests that plant RAC GTPases regulate organisation of the actin cytoskeleton during polar growth at least partly through the ARP2/3 complex, using an evolutionarily conserved mechanism
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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