Itraconazole is a triazole antifungical agent with a broad spectrum of activity belonging to the class of azole, indicated in the treatment of different types of mycotic infections systemic and local. Its mechanism of action is based on the ability to inhibition of synthesis of ergosterol which is a component of vital importance to the cell membrane of the fungi. This drug has only official methodology for raw material, described in British and European Pharmacopeia. In this paper, following the main guidelines for validation, were developed and validated quantitative analytical methods that can be applied both for the raw materials as the finished product containing itraconazole capsules through a high performance liquid chromatography (HPLC) with detection in the ultraviolet (254 nm, C8 reversed-phase column, mobile phase acetonitrile : water (65:35), the temperature of 25 °C) and microbiological assay by agar diffusion, with planning 3x3, employing Candida Albicans ATCC 10231 as microorganism testing in the culture medium antibiotic N° 19 in the region of the ultraviolet spectrophotometry (256 nm, with final solutions in 0.1 mol l-1 hydrochloric acid). All methods showed appropriate linearity, precision, accuracy, robustness and specificity. / O itraconazol é um antifúngico triazólico de amplo espectro de ação pertencente à classe dos azóis, indicado no tratamento de diferentes tipos de infecções micóticas sistêmicas e superficiais. Seu mecanismo de ação baseia-se na capacidade de inibição da síntese do ergosterol que é um componente de vital importância para a membrana das células dos fungos. Este fármaco possui metodologia descrita, somente, para matéria-prima, que se encontram oficializadas nas Farmacopéias Britânica e Européia. Neste trabalho foram desenvolvidos e validados, em conformidade com guias nacionais e internacionais, métodos analíticos quantitativos que podem ser empregados tanto para matéria-prima quanto para forma farmacêutica de cápsulas contendo itraconazol. Utilizaram-se as metodologias: cromatografia a líquido de alta eficiência (CLAE) com detecção na região do ultravioleta (UV) em 254 nm, coluna de fase reversa C8, fase móvel acetonitrila : água (65:35), na temperatura de 25 °C; Ensaio microbiológico por difusão em ágar com planejamento 3x3, empregando Cândida Albicans ATCC 10231 como microrganismo teste em meio de cultura antibiótico n°19 e Espectrofotometria na região do ultravioleta, no comprimento de onda de 256 nm , com soluções finais em ácido clorídrico 0,1 mol.l-1 . Todos os métodos apresentaram linearidade, precisão, exatidão, robustez e especificidade adequados.

Les peptides appartenant à la famille des tachykinines telle que la substance P (SP) sont des acteurs essentiels contribuant à l’hyperalgésie primaire et secondaire. La SP libérée par les neurones afférents primaires, ne provoque pas à elle seule des décharges nociceptives, mais elle potentialise l’effet de divers neurotransmetteurs tel que le glutamate. Pour ces différentes raisons, de nombreuses recherches ont été effectuées avec des antagonistes des récepteurs neurokinines et en particulier des récepteurs NK1. Cependant, malgré des études pré-cliniques prometteuses, les antagonistes du récepteur NK1 n’ont pas montré d’effet significatif chez l’Homme. La biosynthèse des neuropeptides actifs passe par la maturation protéolytique des pro-neuropeptides. La compréhension des mécanismes de la maturation enzymatique des précurseurs des tachykinines, ainsi que l’étude de la stabilité métabolique de la substance P (SP) permettraient d’élucider des stratégies de traitement innovateur en favorisant l’inhibition du processus de maturation ou la production de fragments peptidiques moins actifs ou inactifs. Le premier objectif de cette étude était d’élucider le rôle de la Proproteine convertase 1 (PC1) et de la Proproteine convertase 2 (PC2) dans la maturation de la protachykinine en utilisant des fractions S9 de la moelle épinière des souris du type sauvage (WT), PC1-/+ et PC2-/+. La caractérisation et la quantification des neuropeptides ont été réalisées à l’aide de la chromatographie liquide à haute performance et de la spectrométrie de masse. Les résultats montrent que PC1 et PC2 interviennent dans la maturation de la protachykinine et ces deux enzymes sont essentielles pour la biosynthèse de la Tachykinine58-71, le précurseur de la SP. Une réduction de plus de 50% de la vitesse de formation dans les fractions S9 de la moelle épinière de souris mutantes PC1 et PC2 a été observée. Les résultats obtenus révèlent que PC1 et PC2 sont impliquées dans la protéolyse de la protachykinine et suggèrent un rôle important de ces enzymes dans la maturation de la protachykinine-1. La protéolyse régule probablement les concentrations extracellulaires de la SP, mais peu d'études ont été menées sur le métabolisme des tachykinines. Dans ce présent travail, nous démontrons que la protéolyse contrôle le niveau de la SP dans la moelle épinière menant à la formation de fragments C-terminaux actifs. La stabilité métabolique de la β-tachykinine58-71 et de la SP était très courte, avec une demi-vie de 5.7 et 3.5 min, respectivement. Plusieurs fragments C-terminaux ont été identifiés, y compris la SP3-11, la SP5-11 et la SP8-11, qui conservent leurs affinités vis-à-vis des récepteurs neurokinines. La stabilité métabolique des fragments C-terminaux était significativement supérieure à celle de la β-Tachykinine58-71 et de la SP. Deux inhibiteurs de Prolyl endopeptidase spécifiques ont été utilisés et ont montré une réduction significative de la vitesse de formation de SP3-11 et de SP5-11. Ainsi, nous avons démontré que le Prolyl endopeptidase est impliqué dans le traitement N-terminal de la SP dans la moelle épinière et dans la formation de la SP3-11 et la SP5-11. Étant donné que la régulation des niveaux endogènes de peptides opioïdes (DynA, Leu-Enk, Met-Enk) et des tachykinines (Tach58-71, SP) dépend fondamentalement de l'activité de PC1 et de celle de PC2, l'analyse des tachykinines et des neuropeptides opioïdes ont été réalisées. Les résultats obtenus révèlent une diminution significative des neuropeptides pro nociceptifs la Tach58-71 (p <0,05), de la SP (p <0,01) et du NKA (P <0,001)), et des neuropeptides opioïdes DynA (p <0,01), de Leu-Enk (p <0,001), de Met-Enk (p <0,001), dans la moelle épinière de souris PC1 - / + et PC2 - / +. Par conséquent, la modulation de l'activité des PCs a un impact important sur les peptides pro-nociceptifs, mais également sur le système opioïde endogène et par conséquent elle affectera significativement les voies modulatrices de la douleur. Ces résultats suggèrent également que la réduction significative des concentrations de peptides pro-nociceptifs peut altérer la réponse du système opioïde endogène. Les analyses des concentrations des peptides opioïdes chez les souris Tac1-/- ont montré spécifiquement que les concentrations en Endomorphine-2 (EM2), en Leu-Enk et en Dyn A sont significativement inférieures que celles obtenues dans la moelle épinière chez les souris WT. Par conséquent, l’absence de la SP a un impact sur les mécanismes endogènes de modulation de la douleur. Mots clés : Tachykinines, substance P, proprotéines convertases, protéolyse, peptides opioïdes, moelle épinière, douleur, chromatographie liquide à haute performance, spectrométrie de masse. / SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons and plays a central role in nociceptive transmission. The SP does not acte alone to cause nociceptive discharges, but it potentiates the effect of various neurotransmitters such as glutamate. For these various reasons, much research has been carried out with antagonists of neurokinin receptors and in particular NK1 receptors. However, despite promising pre-clinical studies, NK1 receptor antagonists have not shown significant effect in Humans. The proteolysis control of endogenous protachykinins has a profound impact on pain perception. Proprotein convertases (PCs) are extensively expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue but the role of PCs remains unclear. The first objective of this study was to decipher the role of PC1 and PC2 in the proteolysis of protachykinins using cellular fractions of spinal cords from wild type (WT), PC1 -/+ and PC2 -/+ mices and mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and β-Tachykinin58-71, an important SP precursor, with over 50 % reduction of the rate of formation in mutant PC1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein. The proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. In the present study, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. The metabolic stability of β-Tachykinin58-71 and SP were very short resulting in half-life of 5.7 and 3.5 min, respectively. Several C-terminal fragments were identified, including SP3-11, SP5-11 and SP8-11, which conserve affinity for the neurokinin receptors. Interestingly, the metabolic stability of C-terminal fragments were significantly superior. Two specific Prolyl endopeptidase inhibitors were used and showed a significant reduction in the rate of formation of SP3-11 and SP5-11 providing strong evidence that Prolyl endopeptidase is involved into N-terminal processing of SP in the spinal cord. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. This study has determined the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1- / + and PC2- /+ mice to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The results revealed a significant decrease of Dyn A (p < 0.01), Leu-Enk (p < 0.001), Met-Enk (p < 0.001), Tach58-71 (p < 0.05), SP (p < 0.01) and NKA (p < 0.001) spinal cord concentrations in both, PC1 -/+ and PC2 -/+ mice. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also showed that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. Our results reveal that Endomorphin-2 (EM2), Leu-Enk and Dyn A were down regulated in Tac1-/- spinal cord tissues that strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These results may have insightful impact on future analgesic drug developments and therapeutic strategies. Key words: Tachykinins, substance P, proprotein convertases, proteolysis, opioid peptides, spinal cord, pain, high performance liquid chromatography, mass spectrometry.

>Magister Scientiae - MSc / Subsistence farmers may contribute significantly to food production, food security, and employment in South Africa. However poor storage practices and contamination with mycotoxins, particularly fumonisins and aflatoxins impacts adversely on production, food safety and food security. Mycotoxins are toxic natural food-borne compounds which frequently contaminate agricultural produce worldwide. They are hazardous to humans and animals and result in significant production losses for farmers. This study focused on former Bantustans in Northern South Africa, namely Vhembe District Municipality (Limpopo) and Gert Sibande District Municipality (Mpumalanga). The aim was to assess mycological and mycotoxin contamination of crops grown by subsistence farmers. A semi-structured questionnaire was administered to randomly thirty-nine households. Data on demographics, storage practices and production during period of 2011 and 2012 cropping seasons were collected. One hundred and fifteen (115) crop samples (maize, beans and peanuts) were collected for analysis. Standard mycological methods and validated mycotoxin analysis methods (HPLC and LC- MS/MS) were used. It was found that maize was the staple food in both provinces, with a significant difference (p = 0.0184) in its production between the two districts; Vhembe produced 0.6 tonnes compared to 2.4 tonnes in Gert Sibande. The majority of the farmers for storage used traditional open wooden cribs (15/20) and steel tanks (5/20) while VDM farmers used sealed store houses 5/19 and 15/19 used polystyrene sacks. Aflatoxin occurrence was low with <1% of GSDM samples contaminated compared to 11% of VDM samples. No significant difference (p > 0.05) was observed in the aflatoxin contamination in VDM samples between the year 2011 and 2012. Samples from VDM households had higher Aspergillus fungal infection (maximum incidence 69%) compared to GSDM (27%) over both seasons. The most frequently isolated Fusarium species in VDM samples was F. verticillioides (92%; 93%), and F. subglutinans (97%; 80%) in GSDM samples over seasons 2011 and 2012, respectively. Highest levels of fumonisins (FB1+ FB2) ranged between 1010 μg/kg and 12168 μg/kg with less than 30% extremely contaminated above the regulated limit in 91% of samples from Limpopo over both seasons (2011 and 2012). Fumonisin levels between the two seasons in VDM showed no significant difference (p>0.05). Only three (less than 5%) from 68% GSDM contaminated maize samples were above the FB1 and FB2 limit. In 2011, there were two highly contaminated maize samples (1762 μg/kg and 4598 μg/kg) with the other samples less than 600 μg/kg, whereas in season two (2012) all samples were below 200 μg/kg, except one highly contaminated sample (26115 μg/kg). None of the beans and peanuts from Mpumalanga was contaminated with mycotoxins above the recommended limit, but from Limpopo 1/5 peanuts was found contaminated with aflatoxin G1 (41 μg/kg). Natural occurrence and contamination of both fumonisin and aflatoxin in stored home-grown maize from VDM was significantly (p < 0.0001) higher than GSDM over both seasons. In general, Limpopo farmers’ experience lower harvests and greater mycotoxin contamination of agricultural produce. This may be attributed in part to poor storage practices and environmental and climatic conditions in that agro-ecological zone.

The widespread acceptance of topical formulations intended for local and/or regional activity has prompted renewed interest in developing a model to determine the bioavailability of drugs in order to establish bioequivalence as a means of evaluating formulation performance of multisource products and also for use during formulation development. Current in vivo techniques such as blister suction and skin biopsy amongst others used to determine the bioavailability and/or bioequivalence of topical formulations are either too invasive to generate appropriate concentration-time profiles or require large numbers of study subjects thereby making the study expensive and time-consuming. Moreover, there are currently no sampling techniques that can demonstrate dermal bioavailability and/or bioequivalence of topical formulations intended for local and/or regional activity. Dermal microdialysis is a relatively new application of microdialysis that permits continuous monitoring of endogenous and/or exogenous solutes in the interstitial fluid. The technique is involves the implantation of semi-permeable membranes which are perfused with an isotonic medium at extremely slow flow rates and collection of microlitre sample volumes containing diffused drugs. Tape stripping, a relatively older technique, has been extensively used in comparative bioavailability studies of various topical formulations. However, due to shortcomings arising from reproducibility and inter-subject variation amongst others, the published FDA guidance outlining the initial protocol was subsequently withdrawn. The incorporation of transepidermal water loss with tape stripping has garnered renewed interest and has been used for the determination of drug bioavailability from a number of topical formulations. Hence the primary objective of this research is to develop and evaluate microdialysis sampling and tape stripping techniques, including the incorporation of the determination of transepidermal water loss, to assess the dermal bioavailability of ketoprofen from topical gel formulations and to develop models for bioequivalence assessment. A rapid UPLC-MS/MS method with requisite sensitivity for the analysis of samples generated from dermal microdialysis was developed and validated which accommodated the microlitre sample volumes collected. An HPLC-UV method was developed and validated for the analysis of samples generated from the in vitro microdialysis and in vivo tape stripping studies. The work presented herein contributes to a growing body of scientific knowledge seeking to develop a model for the determination of bioequivalence of pharmaceutically equivalent topical formulations intended for local and/or regional activity in human subjects.

Orientador: Carol Hollingworth Collins / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T07:24:16Z (GMT). No. of bitstreams: 1 Silva_CarlaGrazieliAzevedoda_D.pdf: 4489958 bytes, checksum: f1c0299f6db7beed410c437dcdfc5886 (MD5) Previous issue date: 2013 / Resumo: Este trabalho apresenta o desenvolvimento de fases estacionárias (FE) para cromatografia líquida de alta eficiência em fase reversa (CLAE-FR) e cromatografia com fluido supercrítico (CFS) a partir da imobilização térmica de poli(metiloctadecilsiloxano) (PMODS) sobre suportes de sílicas metalizadas com zircônio e titânio. O processo de imobilização térmica do PMODS foi otimizado aplicando-se planejamento composto central. O polímero, os suportes e as fases estacionárias foram caracterizados por testes físicoquímicos e cromatográficos. As fases estacionárias Si(PMODS), Si-Zr(PMODS) e Si-Ti(PMODS) apresentaram os melhores resultados quando foi aplicada a temperatura de 120 °C por 16 horas. As melhores FE foram submetidas ao processo de capeamento. Estas FE mostraram eficiências entre 82.000 e 90.000 pratos m e boas separações com picos simétricos para compostos apolares e picos com simetria adequada, segundo parâmetros estabelecidos pela Farmacopéia Americana (United States Pharmacopeia), para compostos polares, avaliados pelos testes de Engelhardt, Tanaka, Neue, SRM 870 e pelo modelo dos parâmetros de solvatação utilizando CFS. A presença do óxido metálico no suporte resultou em FE com maior estabilidade química em condições drásticas de fase móvel (pH 1,7 e pH 10 a 50 °C), quando comparada com fases similares baseadas em sílica nua. A reação de capeamento melhorou a estabilidade química das FE e diminuiu o número de silanóis residuais. As FE Si-Zr(PMODS), Si-Ti(PMODS), Si-Zr(PMODS)ec e Si- Ti(PMODS)ec apresentam potencialidade na separação de fármacos psicoativos, filtros ultravioleta (UV), xantinas e hidrocarbonetos policíclicos aromáticos (HPA), utilizando CLAE e CFS. / Abstract: This work presents the development of stationary phases (SP) for high performance liquid chromatography in the reversed phase (RP-HPLC) and for supercritical fluid chromatography (SFC) prepared by the sorption and thermal immobilization of poly(methyloctadecylsiloxane) (PMODS) onto silica and metalized silica supports modified with zirconium and titanium. The immobilization process was optimized using central composite design for determination of the best conditions of time and temperature. The polymer, supports and stationary phases were characterized with physico-chemical and chromatographic tests. The stationary phases Si(PMODS), Si-Zr(PMODS) and Si-Ti(PMODS) presented the best results when a temperature of 120 °C for 16 hours was applied. The best SP were submitted to endcapping processes. These SP showed efficiencies between 82,000 and 90,000 plates m and good separations with symmetric peaks for apolar and polar compounds evaluated by the Engelhardt, Tanaka, Neue and SRM 870 tests and the solvation parameter model using supercritical fluid chromatography (SFC). The presence of metallic oxide on the supports resulted in stationary phases with better chemical stability under drastic conditions of mobile phase (pH 1.7 and pH 10 at 50 °C), when compared to similar stationary phases based on bare silica. The endcapping reaction improved the chemical stability. The SP Si-Zr(PMODS), Si- Ti(PMODS), Si-Zr(PMODS)ec e Si-Ti(PMODS)ec presented potentialities for the separation different pharmaceutical compounds used as psychotropic drugs, ultraviolet (UV) filters, xanthines, and polycyclic aromatic hydrocarbons (PAH), using separations in HPLC and SFC. / Doutorado / Quimica Analitica / Doutor em Ciências

Orientador: Isabel Cristina Sales Fontes Jardim / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T08:24:02Z (GMT). No. of bitstreams: 1 Oshita_Daniele_D.pdf: 4645263 bytes, checksum: 6628dba7b8449d1cf1f86afa3dcae9e2 (MD5) Previous issue date: 2013 / Resumo: Este trabalho envolve o desenvolvimento, a otimização e a validação de um método analítico para determinação de multirresíduos de agrotóxicos em amostras de morango, por cromatografia líquida acoplada à espectrometria de massas sequencial (LC-MS/MS). No preparo de amostra utilizou o método QuEChERS (quick, easy, cheap, effective, rugged and safe), que foi testado nas três versões, Original, AOAC Official Method e European Committee for Standardization (CEN) Standard Method EN 15662, além da versão CEN 15662 modificada. Também foram otimizados os solventes de extração, massas do agente secante e, na etapa de clean-up por extração em fase sólida dispersiva (d-SPE), o sorvente comercial PSA (primary secondary amine), alguns preparados no laboratório à base de polímeros de siloxano, como octadecil, octil, amino, fenil, e a mistura PSA e octadecil. As avaliações dos métodos foram baseadas, principalmente, nos valores de recuperação e nos estudos sobre o uso de diferentes sorventes, outros parâmetros que estimam a eficiência do clean-up também foram utilizados, como aspecto físico do extrato final, quantidade de coextratos da matriz, obtida por medidas gravimétricas, e efeito matriz. O método desenvolvido foi validado por meio dos parâmetros analíticos de seletividade, limite de detecção (LOD), limite de quantificação (LOQ), linearidade, exatidão e precisão, conforme o guia Sanco para análises de resíduos de agrotóxicos em alimentos e, posteriormente, amostras comerciais de morango da região de Campinas foram analisadas. O método validado por LC-MS/MS apresentou-se seletivo, preciso, exato e atingiu concentrações abaixo dos respectivos limites máximos de resíduos (LMR) para determinação de agrotóxicos em morango. Este método foi transferido para cromatografia líquida de ultra eficiência (UHPLC), que mostrou redução no tempo de análise, na vazão da fase móvel (FM) e no volume de injeção de amostra e da FM, e similaridade na detectabilidade dos analitos / Abstract: This work involves the development, optimization and validation of an analytical method for multiresidue determination of pesticides in strawberry samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sample preparation used the QuEChERS (quick, easy, cheap, effective, rugged and safe) method, which was tested in three versions, Original, AOAC Official Method and European Committee for Standardization (CEN) Standard Method EN 15662, and also CEN 15662 modified version. The factores optimized were extraction solvents, amount of drying agent and in the clean-up step by dispersive solid phase extraction (d-SPE), the commercial sorbent PSA (primary secondary amine), several prepared in the laboratory based on siloxane polymers, such as octadecyl, octyl, amine, phenyl, and the mixture PSA and octadecyl. The evaluation of the methods was based mainly on the recovery values and for the study of different sorbents, other parameters that estimate the efficiency of the clean-up were also used such as the physical aspect of the final extract, the amount of interference matrix obtained using gravimetric measurements, and the matrix effect. The developed method was validated by the analytical parameters of selectivity, limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy and precision, as described in the Sanco guide for analysis of pesticide residues in foods. After, commercial strawberry samples from the Campinas region were analyzed. The validated method by LC-MS/MS was selective, precise, accurate and reached levels below the respective maximum residue limits (MRLs) for the determination of pesticides in strawberries. This method was transferred to ultra high performance liquid chromatography (UHPLC), which showed a reduction in analysis time, the mobile phase (MP) flow rate and the injection volume of the sample and MP, and similarity in the detectability of the analytes / Doutorado / Quimica Analitica / Doutora em Ciências

Un nouveau système réactif de type phénolique plus respectueux de l’environnement, c’est-à-dire sans phénol, résorcinol ou formaldéhyde a été caractérisé lors de ces travaux de thèse. Ce système implique la copolymérisation du phloroglucinol et du téréphthalaldéhyde (TPA) en solution aqueuse très diluée. Dans un premier temps, une recherche bibliographique a permis de sélectionner les techniques expérimentales pertinentes pour suivre l’avancement de ces systèmes évolutifs complexes en solution. La combinaison d’analyses chromatographiques et spectroscopiques a été nécessaire pour la caractérisation de la partie soluble du système phloroglucinol-TPA. Un mécanisme de polymérisation conduisant à des espèces linéaires a été proposé pour des temps courts de réaction. L’augmentation de la fraction insoluble lors de la réaction dans les analyses nécessitant une étape de solubilisation met en évidence un second mécanisme de réaction des oligomères entre eux. Une méthode de suivi de l’état du système par mesures rhéologiques a été également développée pour le contrôle de production. Dans un deuxième temps, ce système a été utilisé pour la conception d’un adhésif Phloroglucinol-TPA-Latex. Celui-ci a été étudié à l’état liquide lors du ‘mûrissement’ et à l’état solide après réticulation sous la forme de films libres pour différentes situations modèles. La stabilité de l’adhésif lors du ‘mûrissement’ a été vérifiée même si des réactions de condensation au sein du système Phloroglucinol-TPA se poursuivent. Après réticulation, la morphologie à deux phases des films observée est spécifique à la nature de la phase latex. Il a été conclu que le nouveau système phloroglucinol-TPA a un comportement similaire à celui du système résorcinol-formaldéhyde en termes de mécanismes de réaction et lors de son utilisation en présence de latex. Celui-ci a donc la capacité de remplacer le système initial dans l’élaboration d’un nouvel adhésif plus respectueux de l’environnement. / A new phenolic-type reactive system more environment friendly, namely phenol-, resorcinol- or formaldehyde-free, was characterized during this Ph.D. This system consists in the copolymerization of Phloroglucinol and Terephthalaldehyde (TPA) in highly diluted water solution conditions. First, a literature study allowed a selection of relevant experimental technics in order to follow the advancement of these complex systems in solution. The combination of chromatographic and spectroscopic analyses is required to characterize the soluble part of the Phloroglucinol-TPA based system. A mechanism of reaction to synthesize linear oligomers was proposed for short reaction times. The increase of the non-soluble part during the reaction in the analyses made after a solubilization step leaded to the conclusion of another type of mechanism where the oligomers are involved. A monitoring method to follow the state of the system by rheological measurement was also developed for the production process. Secondly, this system was used in the preparation of an adhesive based on Phloroglucinol, TPA and Latex. This adhesive was studied in a liquid state during it ‘maturation’ and in a solid state after curing resulting in films from several model systems. The stability of the system during maturation was checked even if condensation reactions of the Phloroglucinol-TPA still took place. After curing, a two-phase morphology of the films was observed which was found to be specific to the nature of the latex phase. It was concluded that the new Phloroglucinol-TPA system has a similar behavior to the one of Resorcinol-Formaldehyde in terms of mechanism and use in presence of latex. It showed the capability to replace the initial system in the preparation of a new adhesive more environment friendly.

No description available.

Wheat bran is a promising material containing a wide range of useful components, including proteins. In addition, it is produced in significant volumes. Currently, wheat bran is used for the production of energy by combustion and for feed purposes. Gradually, new methods of valorization of this material are being sought. One of the possibilities of using wheat bran is the isolation of proteins, hydrolysis, and separation of selected amino acids. This diploma thesis deals with this issue, it is focused on the recovery of arginine and leucine from a protein isolate. Proteins were extracted from wheat bran by changing the pH. Thanks to the subsequent lyophilization a protein isolate was gained. Prior to hydrolysis of the resulting isolate, a stability test of arginine and leucine amino acid standards was first performed, to which various hydrolysis methods were applied. Acid hydrolysis using a mineralizer, which was applied to the protein isolate, was proved to be the most effective. This was followed by the derivatization of the hydrolysates with OPA and analysis of the resulting hydrolysates by high-performance liquid chromatography with UV-VIS detection. Then, suitable adsorption and desorption conditions were optimized. It was found that the time dependence does not affect the amount of adsorbed material on the sorbent. Therefore, an application time of 15 minutes was chosen. While optimizing the amount of used standard, it was found that the optimal weight was 0.25 g of sorbent. The selected conditions were applied to the protein hydrolyzate. Two fractions were obtained by the separation of selected amino acids due to the change in the pH of the citrate buffer. After the application of this procedure, 0.26 g of arginine and 0.82 g of leucine were obtained from one kilogram after evaporation. From evaporation two, 1.01 g of arginine and 0.25 g of leucine were obtained after evaporation.

This diploma thesis focuses on the determination of sulfonamide antibiotics especially the possibility of elimination of these substances from the aquatic ecosystem. Nowadays, environmental contamination of the pharmaceuticals and their residues is a serious concern. Main sources of this contamination are wastewater treatment plants (WWTPs), where these compounds are not effectively removed by contemporary conventional technology. For this reason, new methods are being developed and tested that could eliminate the number of contaminants entering the environment in this way. There is a possibility to use the potential of the enzymatic system of wood-decay fungi, especially white rot fungi. Six representatives of sulfonamide antibiotics were selected and isolated from the aquatic matrix via solid phase extraction. The final identification and quantification method was high performance liquid chromatography with mass spectrometric detection. Monitoring of the concentration level of selected sulfonamide antibiotics at the inflow and effluent at the Brno-Modřice WWTP was carried out weekly. Moreover, the effectiveness of elimination of selected antibiotics from the aquatic ecosystem by the use of Trametes versicolor wood-decay fungi cultured on a suitable carrier was verified.