Os organismos chamados de algas apresentam uma grande diversidade de espécies e ocupam uma grande variedade de nichos ecológicos. Fundamentais para a manutenção das condições que permitem a vida no planeta, inclusive porque constituem a base de cadeias tróficas de ecossistemas aquáticos, as algas vêm sofrendo com o descarte contínuo de resíduos resultantes das mais variadas atividades humanas. Por outro lado, as algas, ao serem expostas aos poluentes, podem indicar a presença dos mesmos em seus habitats por meio de seus biomarcadores. Neste sentido, este trabalho preocupou-se em caracterizar as bases moleculares da toxicidade do hidrocarboneto policíclico aromático (HPA) benzo(a)pireno (BaP), presente no petróleo cru e derivado da combustão parcial de matéria orgânica, sobre a macroalga vermelha Gracilaria birdiae, uma Rhodophyta marinha nativa. Para avaliar a agressividade do poluente, a alga foi exposta a diferentes concentrações do HPA em água do mar, tanto em situações de exposição aguda (24 e 96h) quanto crônica (7 e 15 dias). Após estes períodos de exposição, alguns biomarcadores bioquímicos e fisiológicos da alga tiveram seus comportamentos analisados. Foram eles: taxas de crescimento (TC); duas defesas antioxidantes: os níveis do tripeptídeo de baixo peso molecular glutationa (GSH) e a atividade da enzima superóxido dismutase (SOD); os níveis do dímero de glutationa GSSG; a fotossíntese da macroalga; e seu aspecto de pigmentação geral. Foi possível observar que BaP é tóxico para a macroalga G. birdiae. Aumentos da concentração do HPA nos meios de cultura das algas provocaram menores TC. A partir destes dados de TC foi possível determinar uma curva de inibição do crescimento da alga e a 7 respectiva IC50 de BaP para um período de exposição de 15 dias, com valor de 69 ng do HPA para cada mL de água do mar. Após diferentes exposições a BaP, incluindo em IC50, algas expostas a BaP apresentaram níveis reduzidos do antioxidante GSH, o mesmo efeito que fora observado para GSSG (com exceção da exposição de 96h). O desempenho fotossintético, a atividade de SOD e a pigmentação de G. birdiae mostraram ser sistemas menos sensíveis à presença de BaP, e foram prejudicados em concentrações mais elevadas do poluente, em torno de 10 a 20 µg/mL. Nestas situações, a despigmentação severa da macroalga foi acompanhada de decaimentos expressivos de alguns parâmetros fotossintéticos (i.e., rETR, RQE, Ik, β) da alga. Numa outra abordagem, alguns experimentos foram feitos com o intuito de descobrir se a alga era capaz de eliminar e biorremediar BaP presente na água do mar. Aparentemente, a alga não apresenta tal capacidade; porém, uma cinética mais detalhada se faz necessária para a confirmação desta observação. Concluindo, os resultados do trabalho corroboram outros dados da literatura a respeito da toxicidade de BaP sobre sistemas vivos. Ainda, apesar de a sensibilidade de G. birdiae a BaP não ter sido alta, foi possível apontar alguns sistemas bioquímicos da alga capazes de desempenhar papel como biomarcadores de exposição ao poluente estudado, o que pode auxiliar na tomada de medidas para o manejo e a conservação de áreas impactadas / Algae show a great biodiversity and occupy many different ecological niches. Besides being essential for the maintenance that allow life on Earth, algae are on the basis of aquatic food chains and they suffer with continuous residue discharge that comes from different human activities. However, once algae are exposed to pollutants, biomarkers can indicate its presence on the environment. The aim of this work was on the characterization of benzo(a)pyrene (BaP; a polycyclic aromatic hydrocarbon (PAH) derived from crude oil and from incomplete combustion of organic matter) toxicity against the red macroalga Gracilaria birdiae, a marine brazillian Rhodophyta. BaP toxicity in marine water was investigated under different exposure concentrations after acute (24 and 96h) and chronic (7 and 15 days) conditions. At the end of the exposures time, some of the algae`s biochemical and physiological biomarkers were analyzed: growth rate (GR); two antioxidant defenses: the low molecular weight peptide glutathione (GSH) and superoxide dismutase (SOD) enzyme activity; glutathione disulfide (GSSG) levels; the macroalga photosynthetic capacity; and the general colour aspect. Increased BaP concentrations led to a decrease of GR values. From GR data it was possible to obtain an inhibition growth curve and the respective BaP IC50 for a 15-day exposure time period with value of 69 ng/mL. After different BaP exposure conditions, including IC50, exposed algae presented decreased GSH levels, the same effects observed for GSSG (except for 96h exposure). The photosynthetic yield, SOD activity and colour aspect of G. birdiae appeared to be more resistant systems against BaP, and were affected only in higher BaP concentrations (10 to 20 µg/mL). In such situations, G. birdiae lost its red colour, which was accompanied by considerable photosynthetic parameters (i.e., rETR, RQE, Ik, β) decay. In a different approach, some experiments were done in order to discover any BaP clean-up and bioremediation played by G. birdiae. Preliminary results suggests that the alga has low remediation efficiency, although more investigations need to be done to confirm this. In summary, the results presented here show similar tendencies with literature data in respect of BaP toxicity against living organisms. Even though G. birdiae presented reasonable resistance to BaP, it was possible to identify some of the alga`s biochemical systems as BaP exposure biomarkers. This situation can be useful for impacted areas assessment and management.

Les objectifs de cette thèse sont de déterminer les mécanismes cellulaires et moléculaires mis en jeu lors de la co-exposition de cellules hépatiques à l'éthanol, un toxique alimentaire, et au benzo[a]pyrène (B[a]P), un important contaminant de l’environnement émis lors de combustions incomplètes. L’exposition d’hépatocytes primaires de rat pendant 8h favorise leur collaboration via l’induction d’une déplétion membranaire en cholestérol par le B[a]P, ce qui facilite l’action de l’éthanol à déstabiliser les lysosomes via la phospholipase C-1 pour entraîner la mort par apoptose. Lors d’une exposition répétée sur 96h dans les cellules WIF-B9, celles-ci provoquent une mort précoce par nécrose suivie d’une apoptose tardive via leurs métabolismes. Leur toxicité impliquerait un remodelage membranaire et un stress oxydant avec la production d’espèces réactives de l’oxygène et la variation de l’homéostasie du fer. / The aim of this work is to determine cellular and molecular mechanisms implicated in the co-exposure to ethanol, a dietary toxic substance, and benzo[a]pyrene (B[a]P), a major environmental contaminant, found during incomplete combustions. Primary rat hepatocytes exposed during 8h showed a cooperation effect between the two molecules through the depletion of membrane cholesterol by B[a]P, which promote ethanol action to destabilize lysosomes through phospholipase C-1 and facilitate apoptosis cell death. After repeated exposure during 96h of WIF-B9 cells, these two molecules provoke an early cell death by necrosis and a late apoptosis through their metabolisms. Their toxic effects implicate membrane remodeling and oxidative stress with reactive oxygen species production and modifications in iron pool.

The gas-phase ion chemistry of hydroxyl- and amino-substituted polycyclic aromatic hydrocarbons (PAHs) and their protonated counterparts were studied using mass spectrometry. Ions were generated using an electron ionization (EI) source and the unimolecular chemistry of metastable ions was studied by performing mass-analysed ion kinetic energy spectrometry (MIKES) experiments with a magnetic sector tandem mass spectrometer. Collision-induced dissociation (CID) experiments were used in conjunction with MIKES experiments to determine ion structure. The ten molecules studied were: 1-naphthol, 2-naphthol, 1-naphthylamine, 2-naphthylamine, 1-aminoanthracene, 2-aminoanthracene, 1-phenanthrol, 9-phenanthrol, 1-hydroxypyrene and 1-aminopyrene. Since it is believed that larger PAHs, on the order of more than 50 carbon atoms, populate the interstellar medium, the goal of this study was to attempt to extrapolate the results from smaller systems to larger ones. The trends found include: hydroxy-substituted PAH radical cations lose carbon monoxide spontaneously and amino-substituted PAH radical cations lose HCN. Mechanisms for both processes are proposed, and it appears from the present results that this process should extrapolate to larger PAHs. Another trend found was that all the remaining fragment ions were always a closed ring. Protonated amino-substituted PAHs were generated by electrospray ionization using a quadruple time-of-flight mass spectrometer. By protonating 1-naphthol and 2-naphthol using methane in the high-pressure EI source, it was found that they lost exclusively H2O. As for 2-naphthylamine, 1-aminoanthracene and 2-aminoanthracene, it was found that 2-naphthylamine lost NH3 and a hydrogen atom, NH3being the dominant channel. However, as the ion size 3 increases, the hydrogen-loss channel became the dominant channel. This means that larger PAHs will likely lose exclusively a hydrogen atom to reform the parent radical cation.

Mes recherches s’inscrivent dans une problématique de remédiation des sols pollués par deshydrocarbures aromatiques polycycliques (HAP) avec comme modèle de polluant organiquepersistant le benzo[a]pyrène (BaP). La persistance de ce type de molécules dans les sols estprincipalement due à deux paramètres limitants : leur faible biodisponibilité en raison de leur faible solubilité aqueuse et la difficulté pour les microorganismes d’amorcer l’oxydation de molécules aussi stables. Notre approche a consisté en l’utilisation de polysaccharides à base d’amidon comme matrice permettant de stimuler la solubilisation du BaP et comme support à la réaction de Fenton (Fe²⁺ + H2O2 → Fe³⁺ + OH- + OH⁺) en tant que producteur d’un puissant oxydant : le radical hydroxyl OH⁺. Une étude théorique de modélisation moléculaire a permis de mettre en évidence dans lamolécule d’amidon des sites de fixation préférentiels aussi bien pour le BaP que pour le cation Fe ²⁺. Par la suite, l’étude expérimentale a eu pour objectif la synthèse chimique d’amidon modifié utilisantdeux types d’agent alkylant, des époxydes et des anhydrides d’acides carboxyliques. Après la caractérisation chimique de ces molécules par spectroscopie RMN et FTIR, les études de relation structure activité (par fluorescence, MEB et MEB-EDS) entre les amidons modifiés, le BaP et le cation Fe ²⁺ ont permis le criblage d’une vingtaine de molécules d’amidons modifiés et la sélection des amidons modifiés les plus solubles et les plus efficaces dans la solubilisation du BaP. Par exemple, la molécules d’amidon (P17) bialkylé par un époxyde à trois carbones et un anhydride à onze carbones présente une solubilité aqueuse de 4,41 g/l (amidon natif 0,4 g/l) et une capacité à stimuler la solubilité du BaP d’un facteur 20. La dernière étape de mon travail de thèse aborde, in vitro, les applications potentielles des amidons modifiés pour la dégradation du BaP. Les essais réalisés avec la réaction de Fenton nous ont suggéré une hypothèse originale dans laquelle il semblerait que la matrice polysaccharique produirait des radicaux carbohydrates possédant un temps de demi-vie largement supérieur comparé à celui de l’hydroxyl radical leur conférant une meilleure capacité à atteindre et à oxyder le BaP. Les premiers essais couplant l’oxydation chimique et les potentialités de dégradation par des champignons saprotrophes doivent être encore optimisés afin d’utiliser tout le potentiel de la biodiversité des champignons. / My researchs concern remediation of polluted soil by polycyclic aromatic hydrocarbons (PAH) with the benzo[a]pyrene (BaP) as a model of persistent organic pollutant. The persistence of these molecules into soil is mainly due to both limiting parameters: their weak biodisponibility due to their low aqueous solubility and the difficulty for microorganisms of starting the oxidation of such stable molecules. Our approach consisted in the use of polysaccharides like starch matrix for stimulating the solubilization of BaP and allowing on such support the Fenton reaction (Fe²⁺+H2O2 → Fe ³⁺+ OH -+ OH ⁺) as a producer of high oxidant i.e. the hydroxyl radicals OH ⁺. A theoretical computer modelling study conducted on potato starch permits to identify the preferential sites for BaP and iron complexation. Thereafter, the experimental study aimed the chemical modification of starch using two types of alkylated agents i.e. epoxides and anhydrides of carboxylic acids. After the chemical characterization of these molecules by NMR and FTIR spectroscopy, the structure activity relationship between the modified starches, BaP and Fe2+ (as studied by fluorescence, SEM and SEM-EDS) allowed the screening of modified starches and the selection of the most soluble starches and the most effective one in BaP solubilization. For example, the molecule of starch (P17) bi-alkyl substituted by an epoxy group with three carbons and an anhydride with eleven carbons has an aqueous solubility of 4,41 g/L (native starch 0,4 g/L) and a capacity to stimulate the BaP solubility by a 20-factor. The last step of this work approaches, in vitro, the potential application of the modified starch for BaP degradation. The tests carried out with Fenton reaction suggested an original hypothesis based on the production of carbohydrate radicals having a higher half-life time compared with that of the hydroxyl radical itself, conferring them a better capacity to reach and to oxidize BaP. The first tests coupling chemical oxidation and the potentialities of fungal degradation must be still optimized in order to use all the potential biodiversity of fungi. / Cercetările acestei teze se înscriu în cadrul unei probleme de remediere a solurilor poluate cu hidrocarburi aromatice policiclice (HAP),având ca model de poluare benzo [a] pirenul (BaP). Persistenţa acestor moleculelor, de acest tip, în soluri este dată în principal de doi parametri limitanţi: biosponibilitatea lor scăzută, datorită solubilităţii apoase scăzute, şi dificultatea, pentru microorganisme de a oxida aceste molecule stabile. Abordarea nostră constă în utilizarea polizaharidelor, având ca suport amidonul: ca matrice permanentă de stimulare a solubilităţii BaPului şi ca suport în reacţia Fenton (Fe²⁺+H2O2 → Fe ³⁺+ OH -+ OH ⁺) producătoare de un oxidant foarte puternic precum radicalul hidroxil OH ⁺. Un studiu teoretic de modelare moleculară a permis punerea în evidenţă în molecula de amidon a unor site-uri de fixare preferenţiale atât pentru BaP cât şi pentru cationul Fe²⁺. Apoi, un studiu experimantal a avut ca obiectiv sinteza chimică a amidonului modificat utilizând două tipuri de agenţi de alchilare: epoxizii şi anhidridele acizilor carboxilici. După caracterizarea chimică a acestor molecule prin spectroscopie RMN şi FTIR, studiile de relaţie structură-activitate (prin fluorescenţă, MEB şi MEB-EDS) între amidonurile modificate, BaP şi cationul Fe²⁺ au permis verificarea a douăzeci de molecule de amidon modificate şi selecţionarea amidonurilor modificate cele mai solubile şi mai eficace în solubilizarea BaPului. De exemplu, molecula de amidon (P17) bialchilată printr-un epoxid cu trei atomi de carbon şi o anhidridă cu unusprezece carboni, prezintă o solubilitate apoasă de 4,41 g/L (faţă de amidonul nativ 0,4 g/L) şi o capacitate de stimulare a solubilităţii BaP de un factor 20. Ultima etapă a acestei teze de doctorat abordează, in vitro, aplicaţiile potenţiale ale amidonurilor modificate pentru degradarea BaPului. Încercările realizate cu reacţia Fenton ne sugerează o ipoteză originală în care se pare că matricea polizaharidică produce radicali carbohidraţi posedând un timp de viaţă superior, comparativ cu cel al radicalului hidroxil, conferind o mai bună capacitate de captare şi oxidare a BaPului. Primele încercări cuplând oxidarea chimică şi potenţialitatea de degradare prin fungi saprotrofi mai trebuie încă optimizată pentru utilizarea întregului potenţial de biodiversitate a fungilor.

O benzo[a]pireno é um contaminante da classe dos HPAs (Hidrocarbonetos Policíclicos Aromáticos) oriundo do processo pirolítico a partir da combustão incompleta da matéria orgânica. Possui potencial carcinogênico, mutagênico e baixa degradação no meio ambiente, gerando preocupações ambientais. O presente estudo visa desenvolver uma metodologia de biorremediação eficiente para o contaminante benzo[a]pireno em solo inerte, previamente contaminado com uma concentração conhecida. Para a biorremediação foi avaliado o potencial da utilização de um consórcio microbiano com a bactéria Pseudomonas aeruginosa, e os fungos Candida albicans, Aspergillus flavus e Fusarium sp. Para comprovação da redução do contaminante foi realizada uma quantificação inicial e final das amostras via cromatografia gasosa acoplada à espectrometria de massas (GC-MS). As análises foram realizadas a partir da correlação das áreas dos picos de padrões com as áreas obtidas das amostras extraídas. O tempo de incubação dos microrganismos foi de cinquenta dias, com monitoramento do crescimento microbiano a cada sete dias. As amostras foram divididas em três grupos, caracterizando a triplicata de todas as análises. Para cada grupo foi monitorado o crescimento dos microrganismos na presença e na ausência do contaminante. Para cada via foi pesado 55 g de solo, adicionado a solução contendo 6,2 mg L-1 do contaminante benzo[a]pireno (menos a via do branco) e adicionado 800μL do consórcio microbiano. As amostras ficaram em estufa na temperatura de 35°C durante o período de incubação. A quantificação inicial anterior ao processo de incubação dos microrganismos, foi obtida uma média de 3,74 mg kg-1, representando a adsorção do contaminante no solo. Para o monitoramento microbiano, foram observadas pelas curvas do crescimento dos microrganismos representativas diferenças, nas amostras do branco, que não continham B[a]P obtiveram um leve crescimento, tempo de vida curto, em torno de sete a quatorze dias, e um decrescimento brusco e repentino, enquanto as amostras com o contaminante B[a]P mostraram um número maior de Unidades Formadoras de Colônias (UFC) por grama de amostra, tempo de vida maior e um decrescimento normal. Esta diferença no comportamento dos microrganismos indicou a utilização o carbono orgânico do contaminante como fonte de energia, demonstrando a possibilidade da efetivação do processo de biorremediação. Após esta etapa, realizou-se a quantificação final do solo, foi obtida uma média de 1,29 mg kg-1. Avaliando ambas concentrações, antes e após o processo, constatou-se uma degradação do contaminante em 65,51%±0,95. Com estes resultados, comprovou-se a eficiência da metodologia proposta neste trabalho para a biorremediação do benzo[a]pireno. / Benzo[a]pyrene is a contaminant class of PAHs (Polycyclic Aromatic Hydrocarbons) arising from the pyrolytic process of incomplete combustion of organic matter. Are carcinogenic potential , mutagenic and low degradation in the environment, causing environmental concerns. This study aims to develop a methodology for efficient bioremediation of contaminant benzo[a]pyrene in soil inert previously contaminated with a known concentration. For bioremediation potential was evaluated using a microbial consortium with Pseudomonas aeruginosa bacteria and Candida albicans , Aspergillus flavus and Fusarium sp fungi. To prove the reduction of contaminant was performed to quantify the initial and final samples via gas chromatography- mass spectrometry (GC - MS). The analyzes were performed from the correlation of peak areas of standards with the areas obtained from extracted samples. The incubation of microorganisms was fifty days with monitoring of microbial growth every seven days. The samples were divided into three groups, featuring all of triplicate analyzes. For each group was monitored growth of micro-organisms in the presence and absence of the contaminant. For each route was weighed 55 g of soil added to a solution containing 6,2 mg L-1 of the contaminant benzo[a] pyrene ( the path of least white) and added to 800μL of a microbial consortium, which consists of a mixture of each species. The samples were left in an oven at 35°C. The quantification process prior to initial incubation of microorganisms was obtained an average of 3,74 mg kg-1, representing the adsorption of contaminants in the soil. For monitoring microbial curve was observed for the growth of microorganisms that the samples of the blank, containing no B[a]P had a slight growth short lifetime , about seven to fourteen days and decrease rate, while samples with contaminant B[a]P showed greater formation of Colony Forming Units (CFU) per gram of sample, lifetime , and a greater decrease normally. This difference in behavior indicated microorganisms using organic carbon as a dopant source of power, demonstrating the possibility of the realization of the bioremediation process . After this step, the measurement was carried out final soil was obtained an average of 1.29 mg kg-1. Evaluating concentrations both before and after the process , it was found a degradation of contaminant in 65.51 ± 0.95 % . With these results proved the efficiency of the methodology proposed in this work for the bioremediation of benzo[a]pyrene.

L’obésité et les maladies métaboliques associées, telles que les stéatopathies hépatiques (ou NAFLD, pour non-alcoholic fatty liver disease), sont en constante augmentation dans la population mondiale. La stéatose hépatique, correspondant au premier stade de la NAFLD et caractérisée par une simple accumulation de lipides dans le foie est considérée comme bénigne. Cependant, cette pathologie est susceptible d'évoluer en stéatohépatite (ou NASH, pour non-alcoholic steatohepatitis), bien plus grave puisqu’elle est additionnée d’une nécrose et d’une inflammation. L’origine de cette évolution est multifactorielle : l’alcool ou les médicaments peuvent en être à l’origine, mais de plus en plus d’études font part du rôle probable que pourraient avoir les contaminants de l’environnement. Ainsi dans cette étude, nous nous sommes intéressés à l’impact de la co-exposition à l’éthanol et au benzo[a]pyrène (B[a]P), un xénobiotique de la famille des hydrocarbures aromatiques polycycliques, sur la progression de la NAFLD vers la NASH. Pour cela, nous avons utilisé la lignée cellulaire hépatique humaine HepaRG. Nous avons établi, sur cette lignée, un modèle de NAFLD en incubant les cellules avec un mélange d’acides gras pendant 14 jours, nous permettant de réaliser des investigations chroniques sur les effets du B[a]P et de l’éthanol. Au terme de ces traitements, nous avons constaté que l’alcool potentialisait la cytotoxicité du B[a]P uniquement sur les cellules HepaRG stéatosées. Cette cytotoxicité était également accompagnée d’une production de cytokines pro-inflammatoires, témoignant d’un état pouvant être apparenté à une NASH. Nous avons également mis en évidence une modification du profil de métabolisation du B[a]P, accompagnée d’une sur production de ROS, un dysfonctionnement global de la chaîne respiratoire mitochondriale et une induction de la voie apoptotique. Enfin, ces observations étaient absentes ou minorées lorsque l’exposition était individuelle et lorsque les cellules n’étaient pas stéatosées. Ainsi, ces résultats suggèrent l’importance de la prise en compte d’une exposition multifactorielle à des xénobiotiques dans la progression de la NAFLD, mais également du fait que la présence de NAFLD pourrait à elle seule aggraver la toxicité de xénobiotiques. / Obesity and associated metabolic diseases such as non-alcoholic fatty liver diseases (NAFLD) are steadily increasing in the global population. Fatty liver (also called steatosis) which is the first stage of NAFLD described by a retention of lipids in the liver is considered to be benign. However, this pathology is likely to evolve into non-alcoholic steatohepatitis (NASH), much more severe since it is supplemented with necrosis and inflammation. This evolution is multifactorial in origin: alcohol or drugs may be involved, but more and more studies have mentioned the likely role of environmental contaminants. Thus, in this study we were interested in the impact of an alcohol/benzo[a]pyrene (B[a]P, a xenobiotic of the family of polycyclic aromatic hydrocarbons) co-exposure on the progression of NAFLD towards NASH. To this end, we used the human hepatic cell line called HepaRG. In this cell line, we established an in vitro model of NAFLD by incubating the cells with a mixture of fatty acids for 14 days, whereby we would carry out some chronic investigations about the effects of B[a]P and ethanol. At the end of these treatments, we found that alcohol potentiated B[a]P cytotoxicity on steatotic HepaRG cells. This cytotoxicity was also combined with pro-inflammatory cytokine production, suggesting the presence of a NASH condition. We also demonstrated a change in the metabolic profile of B[a]P, in addition to a ROS production, a global dysfunction of the mitochondrial respiratory chain and an induction of apoptosis. Finally, these observations were absent or decreased when the exposure was individual and when the cells were not steatotic. Thus, these results suggest the importance to consider multifactorial exposure to xenobiotics in the progression of NAFLD as well as the fact that the presence of NAFLD alone could exacerbate xenobiotic toxicity.

The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.

The effect of native hyaluronan addition on CMC of excluded surfactants (Tween 20, BETADET THC 2, SDS, CTAT a CTAB) in physiological solution (0,15 M NaCl) has been investigated by fluoresence spectroscopy with Pyrene and Nile red as probes. The greatest influence on CMC has been observed in systems with cationic surfactant. System of cetyltrimethylamonnium bromid has been closely studied. Solubilization experiments with a hydrophobic dye Sudan red has been realized and aggregation number of this system has been determinated via fluorescence quenching with cetylpyridinium chloride as quencher and Pyrene as fluorescence probe. Addition of hyaluronan (native or hydrophobicaly modified) reduces aggregation number of system.

This diploma thesis is focused on determination of aggregation behavior of selected phospholipids (lecithin; 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) in water and on the effect of native hyaluronan addition of various molecular weights and concentrations on this behavior. The behavior has been investigated with fluorescence spectroscopy using pyrene and perylene as fluorescence probes being able to penetrate into hydrophobic cavities of formed aggregates. Critical aggregation concentration and the concentration at which lecithin begins to aggregate have been determined. Regarding 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, it was possible to determine only the beginning of aggregation value. The values of this parameter for lecithin and for 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine correspond in the order. It has been investigated, that the addition of native hyaluronan has only in some systems slight effect on the aggregate behavior of selected phospholipids.

In this work a new simplified procedure for isolation and determination of PAH in smoked meat products was developed. This procedure is using ASE as selective extraction technique. Its principle is addition of silicagel activated during 18 hours at 140 °C directly to the extraction cell in ratio 10 g to 1 g of homogenized sample. In the next step the sample is extracted by n-hexane at 100 °C and 10 MPa for three 10 minutes static extraction cycles. The flush volume is 60 % and the purge time 120 sec. During the extraction silicagel adsorb koextracted lipids and thus the obtained extract is suitable for immediate HPLC-FLD analysis. On the contrary, the classical extraction procedures with nonpolar solvent require difficult purification of extract for removal of unwanted lipids. Mostly applied techniques here are GPC and SPE. The new procedure was validated for determination of B[a]P as well as for determination of 4 PAH (B[a]A, CHR, B[b]F, B[a]P) in smoked sausages. It meets all requirements to be set by Regulation (EC) No 333/2007, respective by new Regulation (EC) No 836/2011. After the method validation, the content of 4 PAH was investigated in twelve samples of various smoked sausages manufactured in Slovakia. Two analyzed products showed high PAH levels exceeding maximum legislation limits set for B[a]P as well as for sum of 4 PAH by Regulation (EC) No 835/2011.