Les nanotubes de carbone ou le graphène sont des formes allotropiques du carbone prometteuses pour un large domaine d’applications, mais des modifications de leur surface carbonée sont nécessaires pour leur manipulation et mise en forme. La fonctionnalisation covalente est un des moyens utilisés avec succès dans ce cadre, même si les modifications induites ne sont pas contrôlables. Les travaux réalisés au cours de cette thèse concernent l’utilisation de la réduction chimique des nanotubes et autres nanoformes de carbone, nanocornes, graphène ou nanodisques, pour d’une part obtenir des solutions stables et concentrées dans une gamme de solvants dépendant du type de nanoforme et d’autre part fonctionnaliser de manière covalente les différents types de surface carbonée de ces objets. Dans le cas des nanotubes, des molécules acceptrices ou donneuses d’électrons ont ainsi été greffées permettant la formation d’ensembles donneur-accepteur. D’autre part, le nombre de fonctions chimique greffées a pu être contrôlé, préservant ainsi les propriétés électroniques des nanotubes. / Carbon nanotubes and graphene are promising allotropes of carbon for a large range of applications. Due to their tendency to aggregate, modifications of the carbon surface are required in order to manipulate them. Covalent functionalization is one of the ways commonly used to reach this target even if this route is not controllable. The study presented in this thesis deals with the chemical reduction of carbon nanotubes and other forms of carbon such as nanohorns, graphene, nanocones and nanodisks. Two main results have been demonstrated here. First, adding electrons to these materials renders them soluble at high concentrations in various organic solvents. Second, these reduced materials can be functionalized covalently thanks to the charges excess. Donor-acceptor dyads have thus been prepared by grafting organic dyes such as porphyrin and perylene to the nanotubes. Furthermore the extent of functional groups can be controlled preserving the electronic properties of carbon nanotubes.

The goal of this project was to develop a non-toxic amphiphilic diblock copolymer nanoparticulate drug delivery system that will solubilize paclitaxel (PTX) and retain the drug in plasma. Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) diblock copolymers loaded with PTX were characterized and their physicochemical properties were correlated with their performance as nanoparticulate drug delivery systems. A series of MePEG-b-PCL was synthesized with PCL blocks ranging from 2-104 repeat units and MePEG blocks of 17, 44 or 114 repeat units. All copolymers were water soluble and formed micelles except MePEG₁₁₄-b-PCL₁₀₄, which was water insoluble and formed nanospheres. Investigation of the effects of block length on the physicochemical properties of the nanoparticles was used to select appropriate copolymers for development as PTX nanoparticles. The critical micelle concentration, pyrene partition coefficient and diameter of nanoparticles were found to be dependent on the PCL block length. Copolymers based on a MePEG molecular weight of 750 g/mol were found to have temperature dependent phase behavior. Relationships between the concentration of micellized drug and the compatibility between the drug and core-forming block, as determined by the Flory-Huggins interaction parameter, and PCL block length were developed. Increases in the compatibility between PCL and the drug, as well as longer PCL block lengths resulted in increased drug solubilization. The physicochemical properties and drug delivery performance characteristics of MePEG₁₁₄-b-PCL₁₉ micelles and MePEG₁₁₄-b-PCL₁₀₄ nanospheres were compared. Nanospheres were larger, had a more viscous core, solubilized more PTX and released it slower, compared to micelles. No difference was seen in the hemocompatibility of the nanoparticles as assessed by plasma coagulation time and erythrocyte hemolysis. Micellar PTX had an in vitro plasma distribution similar to free drug. The majority of micellar PTX associated with the lipoprotein deficient plasma fraction (LPDP). In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction. In conclusion, although both micelles and nanospheres were capable of solubilizing PTX and were hemocompatible, PTX nanospheres may offer the advantage of prolonged blood circulation, based on the in vitro plasma distribution data, which showed that nanospheres retained PTX more effectively. / Pharmaceutical Sciences, Faculty of / Graduate

Předmětem této práce bylo popsat agregační vlastnosti kyseliny hyaluronové (HA) a jejích hydrofobně modifikovaných derivátů. Za tímto účelem byly připraveny vodné roztoky kyseliny a dvou derivátů, lišící se stupněm substituce a druhem anorganické soli jako prostředím. Koncentrační řady čisté kyseliny hyaluronové a jejích derivátů byly studovány dvěma metodami, tensiometricky a spektrometricky. Povrchové vlastnosti roztoků byly měřeny s použitím kroužku Du Noüy. Zkoumáno bylo také solubilizační chování ve vodě a v chloridu sodném pomocí dvou sond: Coomassie brilantní modři a sudanu III. Amfifilní deriváty hyaluronanu mohou skutečně tvořit hydrofobní agregáty v roztoku a adsorbovat se na rozhraní vzduch–voda. Tato vlastnost je důležitá v souvislosti s transportními systémy léčiv, protože právě hydrofobní domény agregátů hyaluronanu mohou být použity jako nosiče hydrofobních léčiv. Byly zjištěny kritické agregační koncentrace roztoků derivátů hyaluronanu (tensiometricky a spektrometricky), které odpovídají právě těm koncentracím, při kterých vznikají hydrofobní domény.

The aim of this thesis was to determine the solubilizing capacity of microbubbles based on SonoVue®, and phospholipids SonoVue® is made of, by using the UV-VIS spectrophotometry. The concentrations of solubilized substances within these systems was further determined. In particular, the properties of natrium dipalmitoylphosphatidylglycerole and distearoyl¬phosphatidylcholine were investigated. The microbubbles were produced using these phospholipids with the addition of polyethyleneglycol and palmitic acid. The solubilizing capacity was determined using hydrophobic solutes Sudan Red G, Oil Red O, 4-Di-2-Asp and Nile Red in order to obtain a model system of solubilized drugs or other hydrophobic substances. The behavior of solutes in phospholipids and our prepared microbubbles were examined in a moderately polar medium â physiological saline solution (0.15 M NaCl). The vizualization of prepared microbubbles was performed using optical and fluorescence microscopy. 4-Di-2-Asp, as a fluorescence probe, was not suitable for microbubble vizualization. The size of microbubbles that were produced during the experiment was almost the same as the size of microbubbles of commercially made SonoVue®. The concentration of solubilized hydrophobic solutes inside the liposomes of phospholipids ranged from tens to hundreds of micromoles per liter. With increasing concentration of phospholipids the concentration of solubilized solutes also increased. The results of this experiment can be used for further research focused on the solubilization of drugs in microbubbles, and contrast agents which are used in ultrasonography.

This diploma thesis was focused on the system of agarose hydrogels with micellar domains. Polysaccharide agarose was used for the preparation of hydrogel matrices. Septonex was chosen as a surfactant. The release of solubilizate from the agarose hydrogel matrix was studied with a hydrophobic dye Sudan III in order to obtain a model system of hydrophobic solubilized drugs. Sudan III was solubilized via surfactant´s micels. Subsequently, this micellar aqueous solution with the dissolved dye was used to prepare the agarose hydrogel. The system was prepared in the environment of deionized water and physiologic solution. The gradual release of the dye into deionized water and physiologic solution was studied for 22 days using the UV-VIS spectrophotometry method. It was found that after 14 days the concentration of the releasing dyes into the deionized water was decreased. The cause is that after 14 and 22 days there was a slight degradation of gels, which was detected using the rheology method. Using the rheology method it was also found that Septonex does not affect the gel formation process.

The mode of feeding exhibited by different organisms may influence the form or quantity of copper that is bioaccumulated. This hypothesis has been tested by exposing 2 species of sea cucumbers, Pentacta anceps and Stichopus chloronotus, which possess different feeding modes, to varying concentrations of copper. The digestive tract and body wall were dissected and analyzed for copper concentration using atomic absorption spectroscopy. A trend was present, exhibiting a small dose dependent curve. The results did not show a significant difference between species or treatments. This study indicates that feeding mode may influence the amount of copper accumulated but it could not be concluded because of a small sample size. The model used in this study demonstrated that copper was being actively precipitated out of the water and deposited into sediment. Future studies focusing on pollutant uptake may find this useful when evaluating the role of feeding mode or habitat.

While treating domestic wastewater, algae can be grown and harvested for biofuel production. Water is a vital resource and it is imperative to conserve and reuse as much as possible. Several pilot and lab scale experiments were conducted to further research into a full scale wastewater treatment and biofuel production facility. This thesis will include these topics: nitrogen and phosphorus concentrations of clarified wastewater pond feed, nitrogen and phosphorus transformations by algae, and the potential of algae cell disruption technologies to increase nutrient solubilization. The pilot scale experiments were conducted using nine 33 m2, 0.3 m deep multi-culture raceway algae ponds being continuously mixed. These ponds were setup in triplicates and fed municipal wastewater in the form of primary clarifier effluent. The three experiments conducted using these pilot scale ponds were: grab and 24-hour composite influent comparison, nitrogen and phosphorus of various hydraulic residence times (HRTs), and nitrogen and phosphorus nutrient depletion with water recycle. The grab and 24-hour composite comparison was conducted from July 30 to December 3, 2014 and compared the dissolved reactive phosphorus (DRP) and total ammonia nitrogen (TAN) concentrations of the two sample types. Equations were made to convert from grab to 24-hour concentrations. The sample types for both DRP and TAN were on average within 10% and essentially the same. A comparison of data from ponds operating at different HRTs was collected from October 30 and November 6, 2013 and October 29 and November 5, 2014. There were linear correlations between different HRTs and both soluble nitrogen and DRP. Equations were made to calculate the expected removal of ponds using the HRT. The nutrient depletion with water recycle experiment was conducted during October 15-29, 2015. Soluble nitrogen removal was linear with a rate of 1.5 mg-N/L-day and required 14 days to drop below 5 mg-N/L in the ponds. DRP removal was also linear with a rate of 0.18 mg-P/L-day. The algae cell disruption solubilization experiments were conducted using homogenization, sonication, autoclaving, and boiling pretreatment technologies. Algae harvested from the pilot ponds was anaerobically digested and then aerobically digested in an attempt to reuse nutrients for continued growth. It was found that there was no significant difference between the pretreated and non-pretreated digested samples. Keywords: algae, raceway pond, nutrient transformation, anaerobic digestion, aerobic digestion, assimilation, volatilization, nitrogen, phosphorus, pretreatment of algae, nutrient resolubilization, nutrient solubilization

Morphine is an opioid agonist, which can exert cardioprotective effects under certain conditions. Lipid rafts are considered important platforms for membrane organization of signaling proteins and, therefore, these structures could play a role in the effects of morphine, which acts through the opioid receptors. The aim of this thesis was to investigate the distribution of the main components of the opioid receptor and Gi/o-mediated signaling pathway in lipid rafts isolated from rat myocardium, which was affected by various doses of morphine. Because we used different isolation techniques with different solubilization agents (Triton X-100, CHAPS, cholate and sodium carbonate) for preparation of lipid rafts, it was of interest to characterize more closely these preparations. Another aim of this study was to investigate how different methods of isolating these structures affect activity of the key target enzyme of the opioid signaling pathway, i.e. adenylyl cyclase. The presence of signaling molecules of the Gi/o/AC pathway of the opioid system in membrane rafts was confirmed and the distribution of selected proteins was dependent on the type of extractant. We also observed the effect of morphine on the localization of proteins in lipid rafts. Different extractants provided different degree of...

Neste estudo foi acessada a diversidade taxonômica e potencial biológico de actinobactérias isoladas de três biomas brasileiros com atividade antagônica contra Sclerotinia sclerotiorum. No total, 354 isolados foram obtidos da Coleção de Microrganismos de Importância Agrícola e Ambiental (CMAA) da Embrapa - Meio Ambiente. A atividade antagônica contra S. sclerotiorum foi avaliada por meio de ensaio in vitro. Foram selecionados 55 isolados, que apresentaram alguma porcentagem de inibição do crescimento de S. sclerotiorum. A identificação e análise da diversidade dos isolados, usando o gene 16S rRNA, demonstrou que 49 isolados são pertences ao genêro Streptomyces, quatro ao genêro Micromonospora, um a Kocuria e um a Actinomadura. Todas as actinobactérias apresentaram compatibilidade com bactérias benéficas do solo, exceto o isolado Caat P8 35. Três isolados, 1AS2a, Caat P5 55 e Caat P8 79, inibiram tanto o crescimento micelial quanto a germinação de escleródios de S. sclerotiorum. Os isolados Bc V1 06 e 3AS4 apresentaram hidrólise dos meios de fósforo orgânico e inorgânico. Com a ideia de conhecer o possível mecanismo de ação para solubilização de fósforo, o extrato aquoso do isolado 3AS4 foi submetido à análise de HPLC e o perfil cromatográfico obtido foi similar ácido glucônico. A avaliação desse isolado em casa de vegetação evidenciou diferenças na altura das plantas de soja, após seis semanas, quando inoculadas com a 3AS4. A relação Parte-aérea/Raiz foi significativamente maior quando adicionado o fósforo junto com a actinobactéria. Plantas sem adição de fósforo e com o isolado 3AS4 evidenciaram desenvolvimento similar na Parte-aérea/Raiz quando comparadas com plantas inoculadas com 40 kg ha-1 de PR e SPT. A avaliação da atividade enzimática evidenciou nove isolados capazes de hidrolisar quitina e laminarina como único substrato, sendo que três deles, 1AS2a, Caat P5 55 e Caat P8 79, foram avaliados quantitativamente, para a atividade enzimática quitinase (β 1-4 -Nacetilglucosaminidase) os resultados foram 0.03, 0.22 e 0.16 UI respectivamente, e a atividade glucanase (β 1-3-glucanase) foi 0.23, 0.25 e 0.26 UI respectivamente. Extratos orgânicos das actinobacterias obtidos com diclorometano (26) e acetato de etila (15) demostraram inibição do crescimento do fungo, somente o isolado 1AS2a apresentou inibição na concentração de 165 μg.mL-1. Estudos espectrométricos revelaram a prescença de um composto da família das bafilomicinas como principal composto ativo. A anotação do genoma do isolado 1AS2a revelou a presença de 33 operons gênicos envolvidos em vias biossínteticas para produção de compostos antimicrobianos, nos quais um deles, apresentou 100% de similaridade para operon de sintese de bafilomicina. Estudos taxonômicos mostraram que as características filogenéticas, morfológicas e químicas do isolado 1AS2c são consistentes com o gênero Streptomyces. Entretanto, algumas diferenças no perfil taxonômico, Hibridização DNA:DNA e análise de Multilocus confirmou o isolado 1AS2cT como linhagem tipo para uma nova espécie de Streptomyces, para qual o nome Streptomyces rhizosphaericola sp. nov. Com os resultados obtidos podemos afirmar: 1. Biomas brasileros possuem uma grande diversidade taxonômica e funcional 2. Os isolados de Actinobacteria posuem capacidade antagonica contra o fungo S. sclerotiorum 3. O isolado 3AS4 posui a capacidade de solubilização de fósforo e estimulação de crescimento vegetal do em condições de casa de vegetação 4. A efetiva produção de compostos antifúngicos do isolado 1AS2a, revelados diante o uso combiando de ferramentas espectroscopicas e bioinformaticas. Considerando todo isso, existe um enorme potencial de biocontrole em isolados de Actinobactérias de diferentes biomas do Brasil que podem ser uma nova opcão para uso na agricultura. / In this study, the taxonomic diversity and biological potential of actinobacteria isolated from different Brazilian biomes with antagonistic activity against Sclerotinia sclerotiorum were accessed. In total, 354 isolates were obtained from the Embrapa - Environment Collection of Microorganisms of Agricultural and Environmental Importance (CMAA). The antagonistic activity against S. sclerotiorum was evaluated by in vitro assay. In total, 55 isolates were obtained, all isolates showed some percentage of visible growth inhibition against S. sclerotiorum. The identification and analysis of the diversity of the isolates using the 16S rRNA gene showed that 49 isolates belong to the Streptomyces genus, 4 isolates belonging to the genus Micromonospora, 1 isolated to the genus Kocuria and 1 isolated to the genus Actinomadura. All the actinobacteria showed compatibility with the soil beneficial bacteria evaluated except the isolate Caat P8 35. Three isolates, 1AS2a, Caat P555 and Caat P8 79 inhibited both mycelia and S. sclerotiorum sclerotia. The isolates Bc V1 06 and 3AS4 showed hydrolysis using organic and inorganic phosphorus media. In order to know the possible mechanism of action for solubilization of phosphorus, the aqueous extract of the 3AS4 isolate was submitted to HPLC analysis, the chromatographic profile obtained was similar to gluconic acid. Greenhouse evaluation showed differences in height of soybean plants after 6 weeks when inoculated with 3AS4. The Shoot/Root ratio was significantly higher when the phosphorus was added along with the actinobacteria. Plants with no phosphorus addition and with 3AS4 showed similar development in the Shoot/Root when compared to inoculated plants with 40 kg.ha-1 of PR and SPT. The evaluation of the enzymatic activity evidenced nine isolates efficient of hydrolyzing chitin and laminarin as the only carbon substrate, three of them; 1AS2a, Caat P5 55 e Caat P8 79 isolates (β-1-4-N-acetylglucosaminidase) was 0.03, 0.22 and 0.16 IU respectively, however, glucanase activity (β-1-3-glucanase) was 0.23, 0.25 and 0.26 IU respectively. Organic extracts of the actinobacteria obtained with dichloromethane (26) and ethyl acetate (15) demonstrated inhibition of fungus growth, only the 1AS2a isolate presented inhibition at the concentration of 165 μg.mL-1. Spectrometric studies have revealed the presence of a compound of the bafilomycins family as the main active compound. The annotation of the genome of the 1AS2a isolate revealed the presence of 33 gene operons involved in biosynthetic pathways for the production of antimicrobial compounds, in which one of them presented 100% similarity to the bafilomycin synthesis cluster. Taxonomic studies have shown that the phylogenetic, morphological and chemical characteristics of the 1AS2c isolate are consistent with the genus Streptomyces. However, some differences in the taxonomic profile, DDH and Multilocus analysis confirmed the isolated 1AS2cT as a type strain for Streptomyces, for which the name Streptomyces rhizosphaericola sp. Nov. With the results obtained we can corroborate: 1. Brazilian biomes possess a great taxonomic and functional diversity 2. Detection of the antagonism of Actinobacteria isolates against S. sclerotiorum fungus 3. Phosphorus solubilization and plant growth stimulation of 3AS4 isolate under conditions of greenhouse 4. The effective antifungal compounds production of the 1AS2a isolate, revealed in the combining use of spectroscopic and bioinformatics tools. Considering all this, there is an enormous potential for biocontrol in isolates of Actinobacteria from different Brazilian biomes that may be a new option for use in agriculture.

Os manguezais são ecossistemas encontrados na transição entre os ambientes terrestre e marinho apresentando uma biodiversidade funcional única e conseqüentemente flora e fauna específica. Por se tratar de um ambiente inóspito e pouco explorado é de grande importância o estudo desta comunidade a fim de se obter isolado com potencial biotecnológico. Sabe-se que tecidos vegetais são habitados por microrganismos denominados endofíticos, cuja interação com a planta hospedeira pode conferir características vantajosas ao mesmo. Este trabalho teve como objetivo estudar a comunidade bacteriana endofítica cultivável de manguezais, além de determinar a produção enzimática e a utilização de isolados na promoção de crescimento vegetal. Para tanto, foram coletadas amostras de ramo das espécies vegetais Rhizophora mangle, Avicenia nitida, Laguncularia racemosa para o isolamento de bactérias endofíticas. Os locais e épocas amostrados foram manguezais do litoral paulista em Bertioga (local com e sem impacto ambiental) e em Cananéia (local considerado preservado) durante o verão e inverno de 2007 e 2008. Foi obtido grande número de isolados em todos os locais e épocas avaliados, porém a análise estatística não apresentou diferença significativa entre as variáveis avaliadas, com exceção da planta L. rancemosa no período do verão em Bertioga impactado a qual apresentou baixa freqüência. Dentre os isolados obtidos selecionou-se para estocagem aproximadamente 1000 isolados os quais foram submetidos a testes enzimáticos in vitro a fim de realizar uma triagem inicial. Destes, 75% apresentaram atividade para pelo menos uma das enzimas avaliadas. Selecionou-se então isolados com atividade enzimática para no mínimo três ou mais enzimas para testes mais específicos chegando a um número de 115 isolados. A identificação dos isolados foi realizada por seqüênciamento parcial do gene 16S rDNA. Dentre os isolados identificados, os gêneros mais freqüentes foram: Alcaligenes, Bacillus, Brevundimonas, Chryseobacterium, Curtobacterium, Enterobacter, Erythrobacter, Exiguobacterium, Novosphingobium, Ochrobactrum, Pantoea, Pseudomonas, Staphylococcus, Stenotrophomonas, Xanthomonas entre outros. Os resultados demonstraram que dos isolados avaliados: 69% são produtores de fosfatase, 69% de protease, 60% de endoglicanase, 58% de lípase, 43% de amilase e 21% de esterase. Além destes resultados, dos 115 isolados avaliados, 35% apresentou a capacidade de sintetizar AIA (ácido indol acético) e 45% a capacidade de fixar nitrogênio atmosférico. Os isolados com os melhores resultados quanto a fixação de nitrogênio e síntese de AIA foram inoculados em plântulas de cana-de-açúcar e monjoleiro. Os experimentos foram desenvolvidos em casa de vegetação e em viveiro respectivamente. As bactérias selecionadas não promoveram o crescimento de cana-de-açúcar, sendo inclusive observado um crescimento superior da testemunha em relação aos tratamentos com inoculação bacteriana. Já com monjoleiro foi observado que o isolado de Enterobacter sp. apresentou aumento na massa da matéria fresca e seca da parte aérea e da raiz. Assim, os dados apresentados demonstraram potencial de aplicação das bactérias endofíticas isoladas dos manguezais brasileiro, tanto na busca de novos compostos, como enzimas, para aplicação industrial, quanto no desenvolvimento de inoculantes visando a promoção de crescimento de espécies vegetais utilizadas na agricultura e reflorestamento / The mangrove is an ecosystem of transition between the terrestrial and marine environments, featuring a unique functional biodiversity, providing consequently a specific flora and fauna. Due to its inhospible characteristics, few study explore its very importance the study of this community to get isolated with biotechnological potencial. It is known that the plant tissues are inhabited by microorganisms known as endophytes and this interaction may confer advantageous for both. The aiming of this work was the evaluation of the potencial of the mangrove endophytic bacteria to enzymatic production as well their potencial to plant growth promotion. The bacterial endophytic isolades were obtained from branches from Rhizophora mangle, Avicenia nitida and Laguncularia rancemosa mangrove species. The vegetal samples were colleted in the mangroves at Bertioga (with and without environmental impact) and Cananeia (a preserved area) during two different time, summer a winter on 2007 and 2008. We obtained large numbers of isolates in all areas and times evaluated, but the statistical analysis showed no significant difference between the variables, except the plant L. rancemosa in the summer, Bertioga impacted area, that presented low frequency of isolation. Arbitrarily, 1000 were stored and all those evaluated to enzymatic production. Of these, 75% presented activity for at least one of the evaluated enzymes. It was selected 115 isolates that showed activity for at least three or more enzymes. The molecular identification of these isolates were performed by partial sequencing of the 16S rDNA. The predominant identified genera were: Alcaligenes, Bacillus, Brevundimonas, Chryseobacterium, Curtobacterium, Enterobacter, Erwinia, Erythrobacter, Exiguobacterium, Novosphingobium, Ochrobactrum, Pantoea, Pseudomonas, Staphylococcus, Stenotrophomonas, Xanthomonas and others. Among the evaluated isolates: 69% produce phosphatase, 69% produce protease, 60% produce endoglucanase, 58% produce lipase, 43% produce amylase and 21% produce esterase. Even, 35% and 45% are able to produce IAA and nitrogen fixation respectively. Isolates that showed good characteristics to IAA and nitrogen fixation were inoculated with sugar cane and monjoleiro (Acacia polyphylla) seedlings. All experiments were conducted in greenhouse. None selected bacteria promoted the sugar cane growther, being the development of control treatment better than bacterial inoculation. It was observed an increase in their fresh and dried biomass. Thus, the results show the huge potential of endophytic bacteria from mangrove to the discovery news compounds, as enzymes, to industrial application and to the development of new inoculants aiming the plant growth promotion of agriculture and reforestation