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In der vorliegenden Arbeit wurde gezeigt, dass enzymresponsive Peptid-Poly(ethylenglycol)-Konjugate (Peptid-PEG-Konjugate) effizient biotransformiert und proteinresistente Beschichtungen ausbilden können. Die oberflächenspezifische Haftung eines linearen Biokonjugates auf Basis einer literaturbekannten Adhäsionsdomäne für Titandioxid-Oberflächen wurde durch Verlängerung mit einer proteolytisch spaltbaren Erkennungssequenz und einer Suppressionsdomäne temporär unterbunden. Aus einer Serie unterschiedlich modifizierter Biokonjugate wurde eine anionische Suppressionsdomäne als besonders leistungsfähige haftungsunterdrückende Einheit identifiziert. Die Prozessierung des nicht-bindenden Vorläufers mit einer spezifischen Cysteinprotease hervorgehend aus dem Tabakätzvirus (TEV Protease) bewirkte die Abtrennung der eingeführten Modifikation. Durch die Biotransformation wurden die Haftungseigenschaften der polymergebundenen Adhäsionsdomäne zurückgebildet. Das aktivierte Biokonjugat ermöglichte die nicht-kovalente PEGylierung der Metalloxid-Oberfläche. Das Konzept wurde auf divalente Peptid-PEG-Konjugate unter Verwendung verzweigter Adhäsionsdomänen und verlängerter Suppressionsdomänen übertragen. Die proteolytisch aktivierte Dimer-Beschichtung zeigte eine erhöhte Stabilität im Vergleich zum linearen Biokonjugat und demonstrierte vielversprechende Antifouling-Eigenschaften gegenüber der unspezifischen Adsorption eines Modellproteins für Serumproteine des menschlichen Blutes auf Titandioxid-Oberflächen. / The present thesis has shown that enzyme-responsive peptide-poly(ethylene glycol) (peptide-PEG) conjugates can be efficiently biotransformed to create protein-resistant coatings. The surface-specific adsorption of a linear bioconjugate is temporarily suppressed by extending a titanium dioxide adhesion domain known from literature with a proteolytically cleavable recognition site and a suitable interfering domain. From a series of differently modified bioconjugates, an anionic interfering domain was identified as particularly effective to suppress adhesive functions. The enzymatic processing of the non-binding precursor with a specific cysteine protease derived from tobacco etch virus (TEV protease) resulted in the separation of the introduced modification. The adhesive properties of the polymer-bound binding sequence were reproduced by the biotransformation process. The activated bioconjugate allowed the non-covalent PEGylation of the metal oxide surface. The concept was applied to divalent peptide-PEG conjugates using branched adhesion domains and extended interfering domains. The proteolytically activated dimer coating showed increased stability against dilution compared to the linear bioconjugate and demonstrated promising antifouling properties against the non-specific adsorption of a model protein for human blood serum proteins to titanium dioxide surfaces.

Actuellement, il existe deux types de peintures anti-salissures marines sur le marché : - Les Self-Polishing Coatings (SPC), revêtements auto-polissants à base de liants polymères hydrolysables, efficaces par relargage de biocides dans le milieu marin mais toxiques pour l’environnement ; - Les Fouling Release Coatings (FRC), revêtements hydrophobes à base de silicone, sans biocides, qui limitent la force d’adhésion des salissures mais ne sont pas efficaces en mode statique.L’objectif de cette étude est d’élaborer des peintures anti-salissures marines hybrides FRC/SPC. Pour ce faire, des polymères diblocs à la fois hydrolysables et présentant une faible énergie de surface ont été synthétisés et caractérisés puis utilisés en tant que liants dans la formulation de peintures anti-salissures marines.Le procédé de polymérisation RAFT a été employé afin de synthétiser des polymères avec une architecture et une masse molaire contrôlée. Deux approches ont été abordées :- Des polymères ont été synthétisés à base de monomères de faible énergie de surface et hydrolysables (le méthacrylate de (heptaméthyl-trisiloxy)diméthylsilyle et le méthacrylate de bis(triméthylsilyloxy)méthylsilyle) avec un co-monomère méthacrylate de méthyle ou méthacrylate de butyle. Il a notamment pu être montré que les copolymères de structure dibloc présentent une énergie de surface plus faible que les copolymères statistiques.- Des polymères ont été synthétisés à partir de monomères hydrolysables tel que le méthacrylate de tert-butyldiméthylsilyle et de monomères de faible énergie de surface tel que le méthacrylate de poly(diméthyl-siloxane).Les propriétés d’érosion (type SPC) et d’énergie de surface (type FRC) ont été étudiées pour les liants seuls et les peintures formulées, avant et pendant leur immersion en eau de mer artificielle. L’efficacité anti-salissures marines des peintures formulées a été évaluée lors de leur immersion in-situ en Mer Méditerranée. / Two main types of antifouling coatings are present on the market: - The Self-Polishing Coatings (SPC): they are based on hydrolysable polymer binders and their antifouling efficiency relies on the release of biocides in the marine surrounding. Unfortunately, they are toxic for the marine environment- The Fouling Release Coatings (FRC): these hydrophobic silicone-based coatings limit the adhesion strength of fouling organisms and are biocide-free. Nevertheless, they are not efficient during idle periods.The aim of this study is to develop FRC/SPC hybrid antifouling coatings. Thus, diblock polymers both hydrolysable and with a low surface energy have been synthesized and characterized, then used as binders for the formulation of antifouling coatings.Polymerization by the RAFT process has been used to synthesize well-architectured polymers with controlled molecular weights. Two different approaches have been considered:- Polymers have been synthesized from low surface energy and hydrolysable monomers ((heptamethyl-trisiloxy)dimethylsilyl methacrylate and bis(trimethylsilyloxy)methylsilyl methacrylate) and a co-monomer of methyl or butyl methacrylate. It has been demonstrated that the diblock copolymers exhibit a lower surface energy than the statistical copolymers.- Polymers have been synthesized from hydrolysable monomers (tert-butyldimethylsilyl methacrylate) and low surface energy monomers (poly(dimethylsiloxane) methacrylate).The erosion properties (SPC-type) and the hydrophobicity (FRC-type) have been studied for both the binders and the formulated coatings, before and during their immersion in artificial seawater. The antifouling efficiency of the formulated coatings has been evaluated during their in-situ immersion in the Mediterranean Sea.

L’objectif de ces travaux est de synthétiser des copolymères diblocs et triblocs à base d'unités monomères méthacrylate de tert-butyldiméthylsilyle et diméthylsiloxane. Le choix de ces unités monomères repose sur l'élaboration de films polymères hydrolysables dans le milieu marin et de faible énergie libre de surface, respectivement. Ces polymères ont été caractérisés puis utilisés comme liants dans la formulation de peintures anti-salissures marines SPC/FRC hybrides. Les performances des revêtements obtenus ont alors été comparées aux deux types de revêtements anti-salissures marines disponibles sur le marché : - les revêtements auto-polissants (Self-polishing copolymer, SPC), à base de liants polymères hydrolysables, efficaces par relargage de biocides dans le milieu marin et par érosion, mais toxiques pour l’environnement ; - les revêtements Fouling Release" (FRC), hydrophobes à base de silicone, et non toxiques, qui limitent la force d’adhésion des salissures mais sans efficacité en mode statique.Le procédé de polymérisation RAFT a été employé afin de synthétiser des polymères avec des architectures, des compositions et des masses molaires contrôlées. Des macro-agents de transfert de chaîne à base de poly(diméthylsiloxane)s ont été préalablement synthétisés à partir de poly(diméthylsiloxane)s mono et di-hydroxylés, de masses molaires 1000 , 5000 et 10000 g.mol-1. Trois séries de copolymères ont été préparées avec des masses molaires allant de 12000 à 60000 g.mol-1 et des teneurs en unités diméthylsiloxanes allant de 3% à 57%.Les propriétés de prise en eau, d'érosion (type SPC) et de mouillabilité (type FRC) ont été étudiées pour les liants seuls et les revêtements formulés avec et sans biocides. L'évolution de l'hydrophobie de surface des revêtements pendant leur immersion en eau de mer artificielle a été suivie. L'efficacité anti-adhésion bactérienne d'une série de copolymères, sous forme de vernis et de revêtements formulés, a été étudiée vis-à-vis de deux souches de bactéries marines. Enfin, l’efficacité anti-salissure marine des vernis et des revêtements formulés avec et sans biocides a été évaluée lors d'une immersion in-situ en Mer Méditerranée pendant 16 mois au maximum. / The aim of this study is to synthesize diblock and triblock copolymers based on tert-butyldimethylsilyl methacrylate and dimethylsiloxane monomer units. These monomer units have been selected to elaborate polymer films both hydrolysable in the marine environment and with a low surface energy. These copolymers have been fully characterized and have been formulated to develop FRC/SPC hybrid antifouling coatings. The performances of these new coatings have been compared to the two main types of antifouling coatings on the market:- the Self-Polishing coatings (SPC), based on hydrolysable polymer binders with an efficiency relied on the release of biocides in the marine environment and the erosion of the coating. Unfortunately, these coatings toxic for the marine environment;- the Fouling Release Coatings (FRC), based on hydrophobic and non-toxic silicone-based coatings which limit the adhesion strength of fouling organisms. Nevertheless, they are not efficient during idle periods. Block copolymers with controlled architecture, chemical composition and molar masses have been synthesized via the RAFT process from poly(dimethylsiloxane)-based chain transfer agents. These macro-chain transfer agents have been previously prepared from mono- and di-hydroxylated poly(dimethylsiloxane)s with molar masses of 1,000, 5,000 and 10,000 g.mol-1. Three series of copolymer have been synthesized with molar masses ranging from 12,000 to 60,000 g.mol-1 and a mass content of dimethylsiloxane units ranging from 3% to 57%. The water uptake, the erosion properties (SPC type) and the hydrophobicity (FRC-type) have been studied for both the binders and the coatings formulated with and without biocides. The evolution of the hydrophobic properties of the coatings' surface has been investigated during their immersion in artificial seawater. The anti-adhesion properties of one series of copolymers have been investigated toward two marine bacterial strains. Then, the antifouling efficiency of the binders and the coatings formulated with and without biocides has been evaluated during their in-situ immersion in the Mediterranean Sea for 16 months at a maximum.

The marine surface-associated bacterium Pseudoaltermonas tunicata, produces a range of compounds that inhibit fouling organisms, including invertebrate larvae, bacteria, algal spores and fungi. In addition to these antifouling compounds P. tunicata cells produce both a yellow and a purple pigment. The aim of this study was to further characterise the antifouling activities, their regulation and relationship with pigmentation, and the ecological significance of P. tunicata and related organisms. It was discovered that the anti-algal compound was extracellular, heat sensitive, polar and between 3 and 10 kDa in size. The anti-fungal compound was found to be the yellow pigment and active against a wide range of fungal and yeast isolates. Chemical analysis suggests that this compound consists of a carbon ring bound to a fatty-acid side chain. Genetic analysis supports the chemical data for the active compound as a mutant in a gene encoding for a long-chain fatty-acid CoA ligase was deficient for anti-fungal activity. To address the regulation of antifouling compounds and their relationship to pigmentation transposon mutagenesis of P. tunicata was performed. Mutants lacking the yellow pigment displayed a reduced ability to inhibit fouling organisms. Further analysis of these mutants identified genes involved with the synthesis and regulation of synthesis of pigment and antifouling compounds. One of these mutants was disrupted in a gene (wmpR) with similarity to the transcriptional regulators ToxR from Vibrio cholerae and CadC from Escherichia coli. Analysis of global protein expression using two-dimensional gel electrophoresis showed that WmpR is essential for the expression of at least fifteen proteins important for the synthesis of fouling inhibitors. The ecological significance of antifouling bacteria was addressed by assessing the antifouling capabilities of a collection of bacteria isolated from different marine surfaces. Overall, isolates from living surfaces displayed more antifouling traits then strains isolated from non-living surfaces. Five dark-pigmented strains originating from the alga Ulva lactuca were further studied. Phylogenetic and phenotypic analysis revealed that they were all members of the genus Pseudoalteromonas and were closely related to P. tunicata. Two strains represented a novel species within the genus and were taxonomically defined as P. ulvae sp. nov.

<p></p><p>The antifouling paint Fabi 3959 is painted on the hulls of vessels to avoid fouling caused by marine organisms attached to surfaces. The paint is registered for use on pleasure boats and other vessels weighing over 200 kg which are mainly running on the Swedish west coast (www.kemi.se).</p><p>Fabi 3959 contains copper as its active component, which is highly toxic to marine organisms and thus classified as a biocide.</p><p>Fabi antifouling paint was tested under laboratory conditions on the red macro alga <em>Ceramium tenuicorne</em>, in natural brackish water taken from the Baltic Sea. The <em>Ceramium</em> growth inhibition-test was performed using cloned algae exposed to leakage water with and without sediment. The samples containing only water held concentrations in the range of 0.11% of volume-18% of volume per liter, while the samples using sediment held doses measuring between 0.11% of volume-36% of volume leakage water per liter.</p><p>The study showed a growth inhibiting effect on the <em>Ceramium</em> in both water and sediment samples down to the lowest concentration used in the test. There was a difference between the water series and the sediment series in the EC₅₀ values of the leakage water. The mean EC₅₀value was almost 10 times lower within the sediment series compared to the water series (0.114±0.10 and 1.024±0.75, respectively). This indicates that the sediment series are more toxic to <em>Ceramium</em> than the water series. However, if the mean values of EC₅₀ are expressed as copper-concentration, there is no clear difference between the two series (0.59 ± 0.13µg/l for the sediment series and 0.62 ± 0.12 µg/l for the water series). Apparently, the test did not indicate that the sediment was absorbing the copper. Instead it cannot be excluded that another substance involved could have a growth inhibiting impact on <em>Ceramium</em>.</p><p> </p> / <p> </p><p>Båtbottenfärgen Fabi 3959 målas på fartygsskrov för att undvika påväxt av marina organismer. Färgen är registrerad att användas på fritidsbåtar och andra fartyg med en egenvikt på över 200 kg och med huvudsaklig fart på Västkusten (www.kemi.se). Den aktiva komponenten i Fabi 3959 är koppar, vilket är mycket giftigt för marina organismer och därför klassificeras den som en biocid.</p><p>Fabi båtbottenfärg testades i laborativ miljö, på den röda makroalgen <em>Ceramium tenuicorne</em> i naturligt brackvatten från Östersjön. Ett tillväxthämningstest utfördes på <em>Ceramium</em>-kloner vilka exponerades för lakvatten i bägare med och utan sediment. Proverna endast innehållande vatten bestod av koncentrationer i intervallen 0,11-18 volym% per liter medan proverna med sedimentvatten hade koncentrationer på 0,11-36 volym% per liter.</p><p>Studien visade på en signifikant tillväxthämningseffekt på <em>Ceramium</em> i både vatten och sediment, ner till den lägsta använda koncentrationen. Det förelåg en tydlig skillnad mellan vattenserierna och sedimentserierna med avseende på resultaten av EC₅₀-värdena på lakvattnet; EC₅₀-värdena av den toxiska nivån för lakvattnet visade sig ligga tio gånger lägre i sedimentserierna än i vattenserierna (0,114 ± 0,10 i sediment och 1,024 ± 0,75 i vatten). Medelvärdena av EC₅₀för koppar visar inte någon påtaglig skillnad mellan serierna (0,59 ± 0,13µg/l för sediment 0,62 ± 0,12 µg/l för vatten). Testet indikerade därmed inte att sedimentet absorberade koppar. Istället föreligger en möjlighet att en annan substans orsakade tillväxthämning på<em> Ceramium.</em></p><p> </p>

<p>Antifouling paints are applied on vessels to prevent growth of fouling organisms such hasbarnacles. Presently, there are a number of different paints available on the Swedish marketwith different strategies and active substances. The paints might work by either continuouslyreleasing biocides or physically by peeling off or provide an easily cleansed surface whereorganisms cannot attach. The physically working paints do not need to register an activesubstance since its purpose is not to affect living organisms by a chemical or biological modeof action. In this study, two commercially available paints, the copper-based Fabi 3959(International Paint Ltd) and physically eroding, biocide-free labelled Mille Light (HempelFärg AB) were compared to Hard Racing superior, containing copper and the forbiddensubstance Tributyltin. Fabi International is only allowed to be used on the Swedish west coastdue to 6% added as active substance while the biocide-free Mille Light is eligible for eastcoast usage. The toxic effect from respective paint was investigated by assembling a growthinhibition test with the red macro alga Ceramium tenuicorne. The results show that all thestudied paints had a negative effect on growth and therefore leaked substances inconcentrations high enough to be harmful to the alga. The toxic response differed with theeffect on growth being in the following order, Hard racing superior>Fabi >Mille Light.Implications regarding the current legalization involving biocide-free labelled antifoulingpaints are discussed.</p>

<p>Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. </p><p>The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge <i>Geodia barretti</i> and its ability to inhibit larval settlement of the barnacle <i>Balanus improvisus</i> was determined. With an EC₅₀ value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from <i>Geodia barretti</i>, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT_1-7 receptors, whereas barettin possessed specific affinity to 5-HT_2A, 5-HT_2C and 5-HT₄, while 8,9-dihydrobarettin interacted with 5-HT₄. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC₅₀ value of 34 nM, displayed the highest settlement inhibition.</p><p>The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus <i>Agelas</i>. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified.</p><p>In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges <i>Geodia barretti</i> and <i>Agelas</i> spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.</p>

Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge Geodia barretti and its ability to inhibit larval settlement of the barnacle Balanus improvisus was determined. With an EC50 value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from Geodia barretti, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT1-7 receptors, whereas barettin possessed specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted with 5-HT4. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC50 value of 34 nM, displayed the highest settlement inhibition. The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus Agelas. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified. In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges Geodia barretti and Agelas spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.

In order to prevent algae, barnacles and sea poppies from growing on boat bottoms, the hull is painted with antifouling paint each year. Most antifouling paint contains environmentally hazardous, chemically active substances, such as copper. The environmentally hazardous substances then leak into the environment and accumulate in the harbor's bottom sediment. During resuspension through boat traffic and wave action, these substances spread further into the ocean where they may affect other organisms such as fish and algae negatively. In order to detect copper leakage from the boat industry and to increase knowledge about the fate of copper in the sediments on the Swedish west coast our study examined ten ports and marinas along the Swedish west coast and analyzed its copper content in the bottom sediment. In addition, ten bathing areas were investigated as control group. Sediment samples were taken and analyzed in the laboratory for copper content per dry weight of bottom sediment.  Laboratory analyses were conducted using the atomic absorption spectrometer (AAS). Results were analyzed for statistical significance using the IBS SPSS Statistics 24 software. In addition, the measured copper concentration from the various sites were compared with Norwegian copper thresholds in marine waters, as Sweden has no guideline value for the marine environment. The result shows a significant difference (p&lt;0.05) between ports / marinas and bathing areas. Furthermore, a connection between copper concentration in the sediments in marinas and number of berths per marina was found.