Suspensões oftálmicas de dexametasona e polimixina B foram formuladas variando-se os conservantes e combinando 2 concentrações de polissorbato 20 e hidroxipropilmetilcelulose segundo o projeto fatorial 2n. A estabilidade das preparações foi avaliada através de parâmetros físicos, com ênfase na facilidade de rehomogeneização após período de repouso. Das 16 fórmulas foram selecionadas 8 para avaliação da eficácia antimicrobiana do sistema conservante pelo método de desafio, segundo B.P. 88, frente a Pseudomonas aeruginosa, Pseudomonas cepacia e Sthaphylococcus aureus. O ensaio foi precedido de validação da técnica segundo planejamento estatistico two way e split + plot. Os produtos contendo associação clorhexidina e álcool feniletílico atenderam às exigências do compêndio adotado, em oposição aos sistemas contendo cloreto de benzalcônio e EDTA, além de clorhexidina e EDTA, independente da concentração do tensoativo e agente suspensor. / Ophthalmic suspensions of dexametasone and polymyxin B were formulated varying the preservatives and combining two concentrations of polysorbate 20 and hydroxypropylmethylcellulose according to the factorial project 2n. The stability of the preparations were evaluated through physical parameters, with emphasis on the easing of the rehomogenization after a period of rest. 8 of the 16 formula were selected for an evaluation of the antimicrobial efectiveness of the preservative system through the challenge method, according to B.P. 88, against Pseudomonas aeruginosa, Pseudomonas cepacia and Sthaphylococcus aureus. The test was preceded by a validation of the technique according to statistical planning two way and split + splot. The products containing the association of chorhexidine and phenylethyl alcohol complied to the requirements of the adopted compendium, in opposition to the systems containing benzalkonium chloride and EDTA, besides clorhexidine and EDTA, not depending on the concentration of the surfactant and suspending agents.

Suspensões oftálmicas de dexametasona e polimixina B foram formuladas variando-se os conservantes e combinando 2 concentrações de polissorbato 20 e hidroxipropilmetilcelulose segundo o projeto fatorial 2n. A estabilidade das preparações foi avaliada através de parâmetros físicos, com ênfase na facilidade de rehomogeneização após período de repouso. Das 16 fórmulas foram selecionadas 8 para avaliação da eficácia antimicrobiana do sistema conservante pelo método de desafio, segundo B.P. 88, frente a Pseudomonas aeruginosa, Pseudomonas cepacia e Sthaphylococcus aureus. O ensaio foi precedido de validação da técnica segundo planejamento estatistico two way e split + plot. Os produtos contendo associação clorhexidina e álcool feniletílico atenderam às exigências do compêndio adotado, em oposição aos sistemas contendo cloreto de benzalcônio e EDTA, além de clorhexidina e EDTA, independente da concentração do tensoativo e agente suspensor. / Ophthalmic suspensions of dexametasone and polymyxin B were formulated varying the preservatives and combining two concentrations of polysorbate 20 and hydroxypropylmethylcellulose according to the factorial project 2n. The stability of the preparations were evaluated through physical parameters, with emphasis on the easing of the rehomogenization after a period of rest. 8 of the 16 formula were selected for an evaluation of the antimicrobial efectiveness of the preservative system through the challenge method, according to B.P. 88, against Pseudomonas aeruginosa, Pseudomonas cepacia and Sthaphylococcus aureus. The test was preceded by a validation of the technique according to statistical planning two way and split + splot. The products containing the association of chorhexidine and phenylethyl alcohol complied to the requirements of the adopted compendium, in opposition to the systems containing benzalkonium chloride and EDTA, besides clorhexidine and EDTA, not depending on the concentration of the surfactant and suspending agents.

The preservative concentration difference in treated wood was investigated to understand the component distribution; a study of different chemical extractions of treated wood waste was carried out and certain reagents were realized to be feasible to the preservative component removal. During fixation, the preservative components redistributed between earlywood and latewood and concentration gradients at depths also developed. Different solvent extractions of CCA treated wood were tested and ion exchange, chelation and metal dissolving were all mechanisms for component extraction. The transition of Cr(III) to Cr(VI) by oxidizing reagents (NaClO and H2O2) can make possible the direct reuse of extracted chemicals as a preservative. Different reaction factors in the oxidant extractions were compared and higher pHs significantly improved the oxidizing capability of the reagents and CCA component removal. Fresh and aged CCA treated wood generally responsed similarly to the oxidant extractions. However, arsenic in aged wood was more difficult to be removed by NaClO, while, H2O2 was more efficient to extract CCA components from aged wood than fresh wood. Monoethanolamine (Mea) efficiently extracted copper (above 90%) from ACQ treated wood and the formation of stable neutral Cu(Mea)2 in sufficient Mea solution is the main mechanism for Mea extraction. Little wood structure degradation occurred during the process. Mea (10%~15%) extraction was fast and the effect of temperature was insignificant. Cu diffusion in the longitudinal direction was the most significant compared to other wood directions. To further promote Mea extraction, repeated extraction (batch-based and column-based) was performed and proved to be more efficient, feasible and economical than one-time extraction. Column-based continuous Mea extraction showed both high Cu removal (up to 99%) and Cu accumulation in the extract. After the preservative treated wood waste is decontaminated significantly, the extract solution can be reused by directly mixing with the preservative treating solution, which is the most straightforward procedure for the recycling of chemicals removed from the preservative treated wood.

The preservative concentration difference in treated wood was investigated to understand the component distribution; a study of different chemical extractions of treated wood waste was carried out and certain reagents were realized to be feasible to the preservative component removal. During fixation, the preservative components redistributed between earlywood and latewood and concentration gradients at depths also developed. Different solvent extractions of CCA treated wood were tested and ion exchange, chelation and metal dissolving were all mechanisms for component extraction. The transition of Cr(III) to Cr(VI) by oxidizing reagents (NaClO and H2O2) can make possible the direct reuse of extracted chemicals as a preservative. Different reaction factors in the oxidant extractions were compared and higher pHs significantly improved the oxidizing capability of the reagents and CCA component removal. Fresh and aged CCA treated wood generally responsed similarly to the oxidant extractions. However, arsenic in aged wood was more difficult to be removed by NaClO, while, H2O2 was more efficient to extract CCA components from aged wood than fresh wood. Monoethanolamine (Mea) efficiently extracted copper (above 90%) from ACQ treated wood and the formation of stable neutral Cu(Mea)2 in sufficient Mea solution is the main mechanism for Mea extraction. Little wood structure degradation occurred during the process. Mea (10%~15%) extraction was fast and the effect of temperature was insignificant. Cu diffusion in the longitudinal direction was the most significant compared to other wood directions. To further promote Mea extraction, repeated extraction (batch-based and column-based) was performed and proved to be more efficient, feasible and economical than one-time extraction. Column-based continuous Mea extraction showed both high Cu removal (up to 99%) and Cu accumulation in the extract. After the preservative treated wood waste is decontaminated significantly, the extract solution can be reused by directly mixing with the preservative treating solution, which is the most straightforward procedure for the recycling of chemicals removed from the preservative treated wood.

Les microalgues sont bien connues pour produire de nombreuses molécules bioactives qui sont de plus en plus utilisées dans les industries pharmaceutiques et agroalimentaires. Mon projet de thèse avait pour but de mettre en évidence des activités antimicrobiennes d’origine naturelle valorisables en cosmétique notamment en tant que conservateurs et antiacnéens. Des milieux de culture en phase stationnaire de croissance d’une collection de 113 microalgues cultivées au sein du laboratoire ont été prélevés. La présence d’activités antibactérienne et / ou antifongique a été évaluée sur différents microorganismes modèles. Ce criblage m’a permis d’isoler 5 microalgues sécrétant des molécules inhibant la croissance de bactéries appartenant aux genres Salmonella, Staphylococcus et Propionibacterium. La suite de mon travail a porté sur la microalgue référencée S555 qui montre une activité d’inhibiton totale de la croissance de 3 bactéries Gram + : S. aureus, S. epidermis et P. acnes, ces 2 dernières espèces étant impliquées dans l’acné. Cette microalgue a alors été mise en culture en pilote pré-industriel afin de confirmer ces inhibitions. Un élargissement du spectre d’action suggère que ces activités sont spécifiques des bactéries Gram positives. De plus, le(s) composé(s) actif(s) de S555 ne présente(nt) aucune cytotoxicité ou potentiel irritant sur des fibroblastes en culture, les rendant potentiellement utilisables en dermocosmétique. Enfin, un fractionnement du milieu de culture et de la biomasse de cette microalgue a été mis en oeuvre pour séparer le(s) composé(s) actif(s). Leur caractérisation est actuellement en cours. / Microalgae are well-known to produce many bioactive molecules which are used more and more in both pharmaceutical and agroalimentary companies. The aim of my PhD project was to discover antimicrobial activitiesof natural origin which may be used in cosmetic in particular as preservatives and antiacne. Culture media in stationary phase of growth of a 113 microalgae collection, cultivated in the laboratory, were harvested. The presence of antibacterial and / or antifungal activities was evaluated on different microorganisms. This screening allowed to isolate 5 microalgae secreting molecules inhibiting the growth of bacteria belonging to Salmonella, Staphylococcus and Propionibacterium genera. Then my work concerned the S555 microalgae which show a total inhibiton activity of the growth on 3 Gram + bacteria : S. aureus, S. epidermis and P. acnes, these 2 last species being involved in the acne disease. This microalgae was then cultivated in industrial condition in order to confirm these inhibitions. A widening of the action spectrum suggests that these activities are specific to Gram + bacteria. Moreover, the S555 active compound(s) present no cytotoxicity or irritability potential on fibroblasts in vitro,making them potentially usable in dermocosmetic. Lastly, a fractionation of the culture medium and biomass of S555 microalgae were performed to separate the active compound(s). Their characterization is currently in progress.

Nesse estudo, utilizamos a oleoresina de capsaicina, extraído da pimenta Malagueta (Capsicum frutensens) e da pimenta Dedo-de-moça (Capsicum baccatum), para a preservação de amostras de madeira contra o ataque do fungo Paecilomyces variotti. Os preservantes naturais foram aplicados em corpos de prova de madeiras do gênero Pinus sp. e Hymenae sp. (Jatobá) com as dimensões 5,0 x 3,0 x 1,0 (cm). A seguir, esses corpos de prova foram expostos ao fungo para o acompanhamento do seu desenvolvimento. As análises mostraram que o preservante natural retardou o crescimento do fungo, sendo a oleoresina de capsaicina extraído da pimenta Malagueta a mais eficiente se comparada à oleoresina extraída da pimenta Dedo-de-moça e ao óleo de linhaça. A partir da medida de ângulo de contato observou-se que o preservante de oleoresina da pimenta Malagueta proporcionava uma maior molhabilidade para as duas espécies de madeiras. A técnica de FTIR-ATR indicou que os preservantes não modificaram a estrutura das madeiras e a análise de raios X revelou que o desenvolvimento do fungo provocou uma perda de estabilidade e periodicidade nas estruturas das madeiras. Através do teste de proporção utilizado para a análise do desenvolvimento do fungo, comprovou-se estatisticamente que o seu crescimento foi menor para as amostras com os preservantes das pimentas. Pelo MEV foi possível visualizar as estrutura de hifas do fungo sobre a madeira. E a perda de massa para ambas as espécies de madeiras foram menores quando foram utilizados os preservantes, sendo o Pinus a espécie que sofreu maior degradação. / In this study, were used the oleoresin capsaicin, extracted from Capsicum frutensens and Capsicum baccatum, for the preservation of wood samples against the attack of the Paecilomyces variotti fungus. The natural preservatives were applied to Pinus sp. and Hymenaea sp. (Jatobá) specimens with the dimensions 5.0 x 3.0 x 1.0 (cm). Subsequently, these specimens were exposed to fungus and their development was monitored. . Analyses showed that the natural preservative slowed the growth of the fungus. Action of the oleoresin capsaicin extracted from Chilli pepper is the most efficient when compared to pepper oleoresin extracted from Capsicum frutensens and Capsicum baccatum and also with the linseed oil. The contact angle measured showed that the preservative of Oleoresin Chilli Pepper offered a higher wettability for both wood species. FTIR-ATR technique indicated that the preservatives did not change wood structure and X-ray analysis revealed that the development of the fungus caused a loss of stability and periodicity in the wood structures. At proportion test to analyze the development of the fungus, it was shown statistically that their growth was lower for the samples with preservatives peppers. It was possible to visualize the hyphae structure by scanning electronic microscopy technique. Mass loss of both wood specie was lower when preservative was used, and Pine species was more degraded.

In response to a need for a natural antimicrobial to replace sodium benzoate, cinnamic acid was chosen. Due to cinnamic acid's solubility issues, α-cyclodextrin was used as a host molecule to form an inclusion complex with the cinnamic acid molecule. The cinnamic acid: α-cyclodextrin inclusion complex was then characterized using phase solubility analysis, proton nuclear magnetic resonance (H-NMR), and solid inclusion. Phase solubility analysis verified the maximum amount of cinnamic acid that α-cyclodextrin was able to host. H-NMR was used to determine the complex association constant, determine the chemical shifts of available protons, and yield a stoichiometry for the complex. The solid inclusion complex allowed for a physical formation of the complex, yielding further information in support of the complex stoichiometry. Microbiological tests were also performed to quantify the antimicrobial abilities of the complex, the guest, and the host against the yeast Saccharomyces cerevisiae and mold Paecilomyces variotii. Results indicated that approximately 990.29 ppm in aqueous solution was the maximum amount of cinnamic acid in the complex. The 2:1 stoichiometry yields an association constant of 21.7 M-1. Results also indicated that the cinnamic acid readily conformed to fit within the α-cyclodextrin host molecule, which remained a rigid structure. An 8.9% weight to weight of cinnamic acid was calculated for the solid inclusion again reinforcing a 2:1 stoichiometry. Microbiological studies showed little to no inhibition power by the complex at varying concentrations against S. cerevisiae and P. variotii. Free cinnamic acid showed greater antimicrobial activity compared with free α-cyclodextrin and the complex. / Master of Science in Life Sciences

As a response of the need for a natural antimicrobial to replace sodium benzoate's use as a preservative in beverages, twenty eight compounds known to have antimicrobial activity were evaluated to quantify their solubility. Twenty three of the compounds evaluated are components of plant essential oils and the remaining five compounds are alkyl esters of para-hydroxybenzoic acid. The test compounds were evaluated for aqueous solubility as well as their solubility in an acid-based beverage mixture. The compounds were found to be practically insoluble (< 100 mg/L), very slightly soluble (100 mg/L – 1,000 mg/L) or slightly soluble (1,000 mg/L to 10,000 mg/L). o-Methoxycinnamaldehyde, trans,trans-2,4-decadienal, cinnamic acid, and citronellol were complexed with α- and β-cyclodextrin and evaluated through phase solubility analyses. The complexes formed showed improved aqueous solubility for all compounds. Complexation with α-CD resulted in an increase of aqueous solubility of o-methoxycinnamaldehyde by 10-fold, trans,trans-2,4-decadienal by 3.2-fold, cinnamic acid by 6.3-fold, and citronellol by 8-fold. In addition, complexation with β-CD resulted in an increase of aqueous solubility of o-methoxycinnamaldehyde by 1.6-fold, trans,trans-2,4-decadienal by 3.1-fold, cinnamic acid by 1.7-fold, and citronellol by 1.6- fold. The storage stability of the α-CD complexes of o-methoxycinnamaldehyde trans,trans-2,4-decadienal and citronellol were evaluated for 7 days in an acid-based beverage solution by SPME GC-MS. The complexes exhibited varying levels of degradation throughout the duration of the study all. The concentration of o-methoxycinnamaldehyde detected by SPME GC-MS decreased by 61.7%. Similarly, the concentration of trans,trans-2,4-decadienal and that of citronellol decreased by 62.7% and 43% respectively. Additionally, a comparison by UV/Vis of the storage stability of the complexes stored in glass and PET containers was performed. The storage stability comparison proved that absorption into the PET polymer membrane did not occur. / Master of Science

Interest in the use of mass timber in building and construction is growing worldwide, this is due to the structural integrity and reduced environmental footprint of timber-based structures. Concerns associated with the biological and environmental degradation of mass timber necessitate the development of adequate protection strategies to ensure the durability of these products. Preservative treatment is a proven technique that increases the durability and performance of wood in-service and can also be applied to large-sized timber panels such as cross-laminated timber (CLT). Therefore, this study focused on investigating the feasibility of treating prefabricated 3- and 5-layer CLT panels with Copper-azole type C (CA-C) and micronized copper azole (MCA) preservatives. Further, we studied the effects of panel layup and thickness on the preservative impregnation in CLT. Based on the experimental results, we found adequate preservative penetration and retention in the treated 3- and 5-layer CLT panels, particularly in CA-C treated panels. Also, the lengthwise layup shows better treatment results in both CA-C and MCA-treated panels. In addition to the preservative-treatment of CLT panels, this dissertation covers the development of lignin-reinforced polyurethane adhesive (PUR) for bonding glue-laminated timber (Glulam). Herein, the glulam were fabricated and bonded using lignin-reinforced PUR at different wt% (1, 2, and 3) and tested for shear strength, wood failure and delamination. The lignin-treated PUR samples showed improved adhesion properties via high shear strength and reduced delamination compared to the control specimens. Thus, the lignin-reinforced PUR adhesive shows great potential as a bio-based and environment-friendly wood adhesive for producing glulam used in structural applications.

The preservative challenge test is a regulatory requirement specified in various pharmacopoeias to determine the efficacy of preservatives. However, such testing is a labour-intensive repetitive task and often requires days before results can be generated. Microbial biosensors have the potential to provide a rapid and automated alternative to the traditional viable counting currently in use. However, the selection of appropriate promoters is essential. The bioluminescent reporter strains used in the current study comprise the Photorhabdus luminescence lux CDABE reporter genes under the control of five individual constitutive Escherichia coli promoters: outer lipoprotein (lpp); twin arginine translocase (tatA); lysine decarboxylase (ldc); lysyl t-RNA (lysS); and ribosomal protein (spc). The promoter plus lux CDABE constructs were cloned, ligated into the plasmid vector pBR322 and transformed into E. coli ATCC 8739. The bioluminescence intensity in the decreasing order of constitutive promoter was lpp > spc> tatA> ldc > lysS. The five biosensor strains tested successfully in PET assays and demonstrated accuracy with a minimum detection limit of 103 CFU/ml, a detection range of 6 orders magnitude, and yielded equivalent results to methods currently recommended by the pharmacopoeias. The bioluminescent biosensors were used to monitor the efficacy of preservatives; sorbic acid at concentrations of 0.031% to 0.2% at pH 5.0, and benzalkonium chloride at concentrations of 0.0062% to 0.00039% alone and in combination with 0.03% EDTA. The 99.9% percentage of bioluminescence reduction of tatA-lux, ldc-lux, lysS-lux, and spc-lux was statistically equivalent to the 3 log10 CFU/ml reduction as required by the Pharmacopeias’. Strong significant correlations between bioluminescence and the methods recommended by the pharmacopoeias were obtained when the biosensor strains were challenged with preservatives, for all except lpp-lux E. coli. The bioluminescence expressed by the lpp-lux biosensor was significantly lower during long-term stationary phase than it was for any of the other biosensors and was also significantly lower than for any of the other biosensors in the presence of preservatives. Since the plasmid copy number and viable counts for lpp-lux did not change under these conditions, it suggests that perhaps lpp-lux was down regulated under stress conditions. There were no statistically significant differences between the results of the bioluminescence assays and the results of the viable count and ATP chemiluminescence assay. Virtual foot printing (using Regulon DB database) demonstrated that two crp binding sites overlapping the -10 regions are located on the negative strand of the lysS promoter sequences and that one crp binding site is located in lpp. The biosensor strains ldc-lux exhibited levels of bioluminescence per cell significantly lower than spc in the presence of preservatives whilst there was a significant increase in bioluminescence per cell by tatA-lux under alkaline conditions (pH 8.9) during long-term stationary phase. Amongst the five biosensor strains tested in the current work, it was determined that the spc-lux strain would be the most attractive candidate for further work, since the bioluminescence expressed per cell was significantly greater, by 10-1000 times, than that expressed by the other four promoters when challenged with the preservatives tested with excellent significant correlations between bioluminescence expression and viable counts in the PET assays with the various preservatives in this study (R2: 8.79-1.00). The bioluminescent biosensor strains showed no statistical differences from the control strains (wildtype E.coli ATCC 8739 and E.coli carrying a promoterless [pBR322.lux] for adneylate energy charge (AEC), plasmid copy number (PCN) bioluminescence or viable counts over 28 days. The emission of bioluminescence by the four bioreporter strains across 28 days is reflected by the stability of PCN with correlations of 0.78-0.90, except for lpp-lux with R2: 0.59. The following promoter elements were found likely to assist greater expression of bioluminescence: an A+T level of approximately 50% between the -40 and -60 regions (the UP element); a G+C level of approximately 50% within the -10 and +1 regions; the extended -10 region and -10 region of consensus sequence RpoD (σ70/D).