Les nanoparticules à base d’acide hyaluronique (AH) sont utilisées pour de nombreuses applications pharmaceutiques. Elles peuvent cibler les tumeurs par interaction avec le CD44,qui est un récepteur biologique surexprimé à la surface de certaines cellules cancéreuses. Dans ce projet nous explorons l’application potentielle de ces nanoparticules dans les domaines cosmétiques, car l’AH est aussi un ingrédient important pour l’hydratation et le renouvellement de la peau. Les copolymères à bloc à base de polypeptides et de polysaccharides ont été synthétisés par une combinaison de polymérisation par ouverture de cycle et de couplage par chimie « click ». Les nanoparticules ont été obtenues par l’auto assemblage de ces copolymères en utilisant un procédé de nano precipitation, dont la taille et la morphologie sont contrôlées par les paramètres expérimentaux. L’interaction entre les nanoparticules d’AH et le CD44 a été quantifiée par la résonance de plasmon de surface(RPS). En comparant avec l’AH libre en solution, les nanoparticules d’AH ont montré une interaction plus efficace avec le CD44, mettant ainsi en évidence un effet de multivalence des nanoparticules. Finalement, la dégradation enzymatique de ces nanoparticules d’AH a été évaluée avec deux types de hyaluronidases, HYAL1 et SPAM-1. La digestion des nanoparticules de l’AH a été significativement ralentie par rapport à l’AH libre. De manière très surprenante, ces nanoparticules de AH ont pu inhiber l’activité de l’enzyme HYAL1 et protéger l’AH libre dans la solution. Enfin, des ligands du récepteur TLR2 de type lipopeptide ont été synthétisés et leurs performances via TLR2 ont été évaluées par RPS. / Nanoparticles based on hyaluronic acid (HA) are widely used in pharmaceutics. They can target the tumor by the interaction with CD44, a biological receptor overexpressed in some cancer cells. In this project, we investigate the potential applications of these nanoparticles in cosmetics, since HA is also an important ingredient for the skin hydration and renewing. Block copolymers based on polypeptides and polysaccharides were synthesized using a combination of ring opening polymerization and “click chemistry”. The nanoparticles were formed by the self-assembly of these block copolymers using a nanoprecipitation process, and their size and morphology were controlled by the experimental conditions. The interaction between nanoparticles and CD44 were measured by surface plasmon resonance(SPR). Compared to free hyaluronic acid chains in solution, the HA-based nanoparticles could interact more efficiently with CD44, thus demonstrating a multivalent effect. The enzymatic degradation of these HA nanoparticles was then evaluated with twohyaluronidases: HYAL1 and SPAM-1. The digestion of the HA nanoparticles was significantly slower than that of free hyaluronic acid. Surprisingly, these HA nanoparticles could even inhibit the activity of the enzyme HYAL1 and protect free HA chains in the solution. Finally, lipopeptide-based ligands of the biological receptor TLR2 were also synthesized and their performances were evaluated by SPR.

Bacillus amyloliquefaciens FZB42 besitzt einen beeindruckenden Effekt zur Verbesserung des Pflanzenwachstums. Um die Mechanismen, vor allem auf molekularer Ebene, zu verstehen, wurde das komplette Genom von FZB42 in dieser Arbeit sequenziert. Abwesenheit von der weit verbreiteten Phagen-verwandten Genen im Genom von B. subtilis 168, der in enger Verwandtschaft zum FZB42 steht, ist ein besonderes Merkmal. Dagegen enthält das Genom von FZB42 viele DNA-Inseln, in denen unikale Gene in FZB42 als Cluster gefunden wurden. Viele Gene, die möglicherweise zur Pflanzenwachstumsförderung beitragen, wurden in dieser Arbeit identifiziert. B. amyloliquefaciens FZB42 ist natürlich kompetent. Das kompetente Stadium in FZB42 kommt früher als in B. subtilis 168, nämlich während der späten exponentiellen Wachstumsphase. Das FZB42-Genom enthält den kompletten Satz von Genen, die für die Entwicklung der genetischen Kompetenz nötig sind. Ausgenommen von Gene für Quorum-Sensing-System ist die Mehrzahl der Kompetenz-Gene von FZB42 sehr ähnlich zu denen in B. subtilis 168. Das FZB42 Genom birgt ein enormes Potential zur Produktion von sekundären Metaboliten. Genetische Manipulationen wurden durchgeführt, um die Funktionen der trans-AT Domänen und der Modifikationsdomänen in den PKS-Gen-Clustern zu erklären. Mit Ausnahme von fünf Gen-Clustern in B. subtilis 168 (Surfactin, Fengycin, Bacillibactin, Bacillaene und Bacilysin), sind Bacillomycin D, Difficidin, Macrolactin und ein hypothetisches Tripeptid einzigartig im Genom der FZB42. FZB42 kann kein bekanntes ribosomal synthetisiertes Bacteriocin produzieren kann. Gleichzeitig beinhaltet sein Genom ein Gen-Cluster, das wahrscheinlich für die Produktion eines neuartigen Bacteriocins verantwortlich ist. Die eindrucksvolle genetische Kapazität zur Herstellung von antagonistischen sekundären Metaboliten ermöglicht es FZB42, nicht nur erfolgreich neben konkurrierenden Organismen innerhalb seiner natürlichen Umgebung zu überleben, sondern auch Pflanzen gegen pathogene Bakterien und Pilze zu schützen. / Bacillus amyloliquefaciens FZB42 has an impressive effect to improve plant growth. In order to understand the mechanisms, especially at the molecular biological level, the whole genome of FZB42 was sequenced in this work. The absence of extended phage insertions which are typical for the closely related B. subtilis 168 genome is a particular feature. On the other hand, several DNA islands where unique genes in FZB42 were found clustered. Many candidate genes that may contribute to the plant growth promotion were identified in this works. B. amyloliquefaciens FZB42 is naturally competent. FZB42 exhibited its maximal competence earlier than B. subtilis, during late exponential growth. Not surprisingly, the FZB42 genome harbors the complete set of genes necessary for development of genetic competence. The majority of competence genes are highly homologous to their counterparts in B. subtilis 168, excluded from genes for the quorum-sensing system. The FZB42 genome harbors enormous potential for producing secondary metabolites. Genetic manipulation was carried out to investigate the trans-AT domains and some modification domains in the pks gene clusters. With the exception of five gene clusters in B. subtilis 168 (Surfactin, Fengycin, Bacillibactin, Bacillaene and Bacilysin), Bacillomycin D, Difficidin, Macrolactin and a hypothetical tripeptide are unique in the genome of the FZB42. A remarkable feature of the FZB42 genome is that it does not produce any known ribosomally synthesized bacteriocin, whereas a gene cluster probably responsible for production of a new bacteriocin was identified in this work. The impressive genetic capacity to produce antagonistic acting secondary metabolites not only enables FZB42 to cope successfully with competing organisms within its natural environment, but also to protect plants from pathogenic bacteria and fungi.

Os biossurfatantes (BS) são produtos de origem microbiana com propriedades tensoativas e emulsificantes. Estes compostos são candidatos para substituir os surfatantes sintéticos em aplicações industriais devido à sua menor toxicidade, maior biodegradabilidade, maior diversidade química e maior eficiência e efetividade em condições físicas extremas de salinidade, pressão e temperatura. O uso comercial e industrial dos BS ainda não é sustentável devido a seu alto custo de produção relacionado principalmente ao baixo rendimento. A utilização de substratos de baixo custo e ferramentas estatísticas para melhorar o rendimento de produção dos BS são duas das principais estratégias para tratar este problema. O objetivo do trabalho foi estudar a produção e recuperação do BS produzido por B. luteolum, visando o melhoramento na sua produção através do uso do planejamento fatorial e caracterizar a estrutura química do BS. A partir dos resultados, determinou-se que a adsorção em resina foi mais efetiva para a recuperação do BS comparada com a precipitação ácida. A produção do BS foi melhorada através de um planejamento fatorial 23 usando como fatores as concentrações da fonte de carbono (vaselina), a fonte de nitrogênio (nitrato de amônio) e a água do mar artificial e como resposta a tensão superficial da solução 0,1% de BS. A maior produção de BS foi obtida com 4% de fonte de carbono, 2% de fonte de nitrogênio e 20% de água do mar artificial gerando tensão superficial de 27 mNm-1. O BS foi caracterizado como uma mistura de lipopeptídeos com ácidos graxos cujo comprimento da cadeia variou entre 10-18 unidades de carbono e um conteúdo de proteína total de 5%. Três estruturas químicas foram sugeridas para os compostos ativos: dois prolina-lipídeos com os ácidos graxos C16:0 e C18:0 respectivamente e um lipopeptídeo com uma sequência peptídica Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) ligada a uma cadeia de ácido graxo C16:0. Não observou-se atividade antimicrobiana contra as cepas de S. aureus, E. coli, S. enteritidis, L. monocytogenes e S. mutans nas faixas de concentrações de BS testadas. O uso de vaselina como substrato para a produção do BS sugere que a bactéria e o BS podem ser explorados para aplicações como a biorremediação e a recuperação melhorada de petróleo (EOR). / Biosurfactants (BS) are microbial-derived molecules showing tensoactive and emulsification properties. These compounds are candidates to replace synthetic surfactants for industrial applications due to their less toxicity, greater biodegradation capacity, greater chemical diversity and greater efficiency and effectiveness under extreme physical conditions of salinity, pressure and temperature. Commercial and industrial use of BS is not sustainable due their high production cost mainly related to low production yields. The use of low cost substrates and statistical tools to enhance the production yield of biosurfactants are two of the main strategies to deal with that problem. The objective of this work was to study the production and recovery of the BS produced by B. luteolum, aiming to enhance its production through a factorial experimental design, and to characterize the chemical structure of the BS. It was found that resin adsorption was more effective than acid precipitation to recover the BS. The production of BS was enhanced through a factorial experimental design 23 using the concentrations of the carbon source (mineral oil), the nitrogen source (ammonium nitrate) and artificial seawater as the factors and the surface tension of a solution 0,1% of BS as the response. The value of factors that enhanced the production of BS were 4% of carbon source, 2% of nitrogen source and 20% of artificial sea water showing a surface tension of 27mNm-1. The BS was characterized as a mix of lipopeptides with fatty acid chains varying between 10-18 carbon units and a total protein content of 5%. Three chemical structures were proposed for the active compounds: two proline-lipids with the fatty acid chains C16:0 e C18:0 respectively and a lipopeptide with a peptide sequence Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) linked to a fatty acid chain C16:0. BS did not show antimicrobial activity against S. aureus, E. coli, S. enteritidis, L. monocytogenes and S. mutans at concentration range tested. The use of mineral oil as a substrate for the production of the BS suggests that the bacteria and the BS can be explore for applications as bioremediation and enhanced oil recovery (EOR).

Biosurfactants are naturally occurring surface active compounds with unique properties such as biodegradability, low toxicity and tolerance to extreme conditions. These unique properties promote their use as alternatives to traditional petrochemical and oleochemical surfactants, as they satisfy requirements for environmentally friendly manufacturing processes. However, the cost of biosurfactants is still significantly higher than chemical surfactants which hinders their large-scale commercialisation. This work presents an investigation into the production of surfactin, a lipopeptide biosurfactant, exploiting its foamability characteristics for the design and implementation of a recirculating continuous foam fractionation column operated in parallel with a bioreactor. Surfactin is a powerful amphiphilic compound produced by Bacillus subtilis. It is a plant-elicitor with antimicrobial properties offering a huge potential in the food and agricultural industries. However, surfactin has extreme foamability even at low concentrations. This foamability can lead to production problems such as large volumes of uncontrolled overflowing foam with significant product and biomass losses. Here, it is demonstrated that this overflow can be controlled, or eliminated, by integrating a foam fractionation system to the bioreactor in a recirculating loop. A dual production and separation process was engineered and enabled reaching high surfactin productivity in a controlled manner. After elucidating the surface properties of surfactin-rich broth, a foam fractionation column was designed for bench-scale production. Operation of the recirculating column in parallel with the bioreactor enabled air flow to be independently controlled for each unit. Surfactin solutions of various concentrations were tested to relate foamability to concentration over a range of bubble sizes. The sintered glass pore size was revealed to be the main factor influencing the enrichment, with a positive correlation with increasing pore size. Characterisation of the fermentation production rate enabled fractionation column air flow rate to be controlled to ensure sufficient foam surface area for product adsorption. The airflow rate was identified as the main factor impacting on the surfactin recovery rate. This characterisation enabled broth feed flow rate to be controlled to balance the removal rate with the production rate. Two processes were created coupling the newly designed fractionation column with the bioreactor operated either in aerated or non-aerated conditions. Under aerated settings, controlled surfactin production was successfully achieved at a productivity of 0.0019 g L-1 h-1 whilst simultaneously recovering 91% of the product at a maximum enrichment of 79 and 116 through the column and overflow routes, respectively. Under non-aerated settings, overflowing foam was fully avoided and 90% of the product was recovered solely through the fractionation column at an enrichment ratio of 40 under non-optimised settings. Additionally, up to 14% (g/g) increase in surfactin production was observed with the coupling of the fractionation column demonstrating a further benefit as a bioprocess intensifying device for surfactin production. This work provides a benchmark for a robust system for surfactin production, substantially improving the productivity at bench scale, potentially leading the way to more productive and less costly industrial processes for surface active compounds in a wide range of industrials fields.

Zugl.: Berlin, Humboldt-Univ., Diss., 2008

Os biossurfatantes (BS) são produtos de origem microbiana com propriedades tensoativas e emulsificantes. Estes compostos são candidatos para substituir os surfatantes sintéticos em aplicações industriais devido à sua menor toxicidade, maior biodegradabilidade, maior diversidade química e maior eficiência e efetividade em condições físicas extremas de salinidade, pressão e temperatura. O uso comercial e industrial dos BS ainda não é sustentável devido a seu alto custo de produção relacionado principalmente ao baixo rendimento. A utilização de substratos de baixo custo e ferramentas estatísticas para melhorar o rendimento de produção dos BS são duas das principais estratégias para tratar este problema. O objetivo do trabalho foi estudar a produção e recuperação do BS produzido por B. luteolum, visando o melhoramento na sua produção através do uso do planejamento fatorial e caracterizar a estrutura química do BS. A partir dos resultados, determinou-se que a adsorção em resina foi mais efetiva para a recuperação do BS comparada com a precipitação ácida. A produção do BS foi melhorada através de um planejamento fatorial 23 usando como fatores as concentrações da fonte de carbono (vaselina), a fonte de nitrogênio (nitrato de amônio) e a água do mar artificial e como resposta a tensão superficial da solução 0,1% de BS. A maior produção de BS foi obtida com 4% de fonte de carbono, 2% de fonte de nitrogênio e 20% de água do mar artificial gerando tensão superficial de 27 mNm-1. O BS foi caracterizado como uma mistura de lipopeptídeos com ácidos graxos cujo comprimento da cadeia variou entre 10-18 unidades de carbono e um conteúdo de proteína total de 5%. Três estruturas químicas foram sugeridas para os compostos ativos: dois prolina-lipídeos com os ácidos graxos C16:0 e C18:0 respectivamente e um lipopeptídeo com uma sequência peptídica Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) ligada a uma cadeia de ácido graxo C16:0. Não observou-se atividade antimicrobiana contra as cepas de S. aureus, E. coli, S. enteritidis, L. monocytogenes e S. mutans nas faixas de concentrações de BS testadas. O uso de vaselina como substrato para a produção do BS sugere que a bactéria e o BS podem ser explorados para aplicações como a biorremediação e a recuperação melhorada de petróleo (EOR). / Biosurfactants (BS) are microbial-derived molecules showing tensoactive and emulsification properties. These compounds are candidates to replace synthetic surfactants for industrial applications due to their less toxicity, greater biodegradation capacity, greater chemical diversity and greater efficiency and effectiveness under extreme physical conditions of salinity, pressure and temperature. Commercial and industrial use of BS is not sustainable due their high production cost mainly related to low production yields. The use of low cost substrates and statistical tools to enhance the production yield of biosurfactants are two of the main strategies to deal with that problem. The objective of this work was to study the production and recovery of the BS produced by B. luteolum, aiming to enhance its production through a factorial experimental design, and to characterize the chemical structure of the BS. It was found that resin adsorption was more effective than acid precipitation to recover the BS. The production of BS was enhanced through a factorial experimental design 23 using the concentrations of the carbon source (mineral oil), the nitrogen source (ammonium nitrate) and artificial seawater as the factors and the surface tension of a solution 0,1% of BS as the response. The value of factors that enhanced the production of BS were 4% of carbon source, 2% of nitrogen source and 20% of artificial sea water showing a surface tension of 27mNm-1. The BS was characterized as a mix of lipopeptides with fatty acid chains varying between 10-18 carbon units and a total protein content of 5%. Three chemical structures were proposed for the active compounds: two proline-lipids with the fatty acid chains C16:0 e C18:0 respectively and a lipopeptide with a peptide sequence Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) linked to a fatty acid chain C16:0. BS did not show antimicrobial activity against S. aureus, E. coli, S. enteritidis, L. monocytogenes and S. mutans at concentration range tested. The use of mineral oil as a substrate for the production of the BS suggests that the bacteria and the BS can be explore for applications as bioremediation and enhanced oil recovery (EOR).

Les peptides pénétrants (CPP) se caractérisent par deux propriétés : ils pénètrent dans l'espace intracellulaire et favorisent l'internalisation de cargaisons moléculaires auxquelles ils sont associés. Si les CPP sont très utilisés comme vecteurs en recherche fondamentale, la méconnaissance des mécanismes de pénétration et de leurs distributions intracellulaires limite leur utilisation thérapeutique. Il est admis que les CPP et leurs cargaisons sont internalisés par transport actif (endocytose) et par transport passif (translocation directe). J'ai étudié la translocation directe à l'échelle moléculaire en utilisant des membranes modèles. Les CPP usuels sont internalisés et permettent l'accumulation de cargaisons dans des vésicules unilamellaires. J'ai alors démontré que la translocation directe se déroule via la formation de complexes neutres et hydrophobes CPP-phospholipides.La pénétration intracellulaire des CPP est le plus souvent étudiée par microscopie confocale. J'ai démontré que des fortes concentrations locales de CPP induit une auto-inhibition de leur fluorophore. Cet artefact a conduit à des erreurs d'interprétation dans la littérature quant à la localisation des CPP. Un protocole permettant de révéler la fluorescence éteinte a été proposé et conduit à réévaluer la localisation subcellulaire des CPP ainsi que l'importance relative des mécanismes d'internalisation.Ces résultats ont permis de développer rationnellement de nouveaux vecteurs pénétrants : les oligoarginines acylées par des chaînes grasses dont des insaturations sont de stéréochimie cis. Leur internalisation passive particulièrement importante conduit à la libération de la cargaison dans le cytosol. / Cell penetrating peptides (CPPs) are short cationic sequences capable of shuttling bioactive cargoes into eukaryotic cells. If CPPs are common delivery tools in basic research, their therapeutic use is currently limited because their internalization mechanisms and intracellular distributions remain to be elucidated. In living cells there is evidence for both endocytosis of the CPPs and for “direct translocation”, an energy-independent uptake pathway. I analyzed the direct translocation phenomenon at the molecular level with model membranes. CPPs are internalized into large unilamellar vesicles and trigger the internalization of various cargoes. I then demonstrated that direct translocation occurs through membranes via the formation of a neutral and hydrophobic CPP-anionic phospholipids complex. CPPs internalization is mostly analyzed by confocal microscopy. I demonstrated that fluorescence self-quenching occurs if fluorescently labeled CPPs are locally too concentrated. This severe artifact led to misinterpretation of the subcellular distribution of CPPs. I developed a reliable procedure to avoid this artifact and ranked subcellular regions of living cells depending on their CPP concentration. As a result, I was able to rationalize the subcellular distribution of CPPs and to deduce their penetration mechanisms. The studies that I performed provided valuable information that I used to design a new family of delivery vectors: minimalist oligoarginines peptides acylated by unsaturated fatty acids (cis unsaturations). The direct translocation of these lipopeptides is particularly important yielding to an efficient delivery of a cargo inside the cytosol of living cells.

There has been increasing concern regarding the emergence of multi-drug resistant pathogens. The resistance develops when pathogens, especially bacteria, are frequently exposed to conventional antibiotics, as they are heavily used in both human and livestock. This is due to the high target specificity of conventional antibiotics, which places pathogens in high selective pressures and eventually results in drug resistant by mutations. To address this issue, global actions and cooperation are needed. At the same time, new technologies and strategies need to be developed. Host defense peptides (HDPs) are widely found in the innate immune system. They show both direct antimicrobial properties and immunomodulatory activities. The multifaceted functions of HPDs make them less likely to promote antimicrobial resistance. Thus, they are promising as new therapeutics to treat multi-drug resistant infections. In fact, several drug candidates derived from HDPs have entered the clinical trial, but none of them got into the clinic. This is due to several challenges associated with HDPs, such as low in vivo stability, high cost of manufacturing, and toxicity to mammalian cells. In this dissertation, we explored the ability of a new type of unnatural scaffolds (γ-AApeptides) to mimic the functions of HDPs, including both broad spectrum antimicrobial properties and immunomodulatory activities. Furthermore, the efforts to identify simpler and more drug like γ-AApeptide based antimicrobial agents were also discussed. The findings in this dissertation may lead to the development of potential drug candidates to treat multi-drug resistant infections.

Immunological and ultrastructural changes in mononuclear cells exposed to Mycobacterium avium serovar-specific glycopeptidolipid (GPL) and the chemically derived R-lipid (lipopeptide fragment) were examined.

No description available.