Currently, the only cure for liver failure is orthotopic liver transplantation. However, there are insufficient donor organs available to treat every patient on the transplant list and many die before they are able to receive a liver transplant. The bioartificial liver (BAL) device is a potential extracorporeal treatment strategy utilising hepatocytes or hepatocyte-like cells (HLCs) within a bioreactor to recapitulate normal liver function and therefore ‘bridge’ a patient with liver failure until they receive a transplant. The work in this thesis utilised tissue engineering methods to develop novel approaches to BAL device design through development and characterisation of a polymer membrane scaffold (“PX”) for hollow fibre bioreactor (HFB) culture and a HLC source generated from the transdifferentiation of pancreatic AR42J-B13 (B13) cells. A flat sheet membrane model was used for the development of asymmetrical, hydrophobic polystyrene (PS) phase inversion membranes. Oxygen plasma significantly increased PS membrane surface wettability through addition of oxygen functional groups to create an environment conducive for cell culture. The treated membrane was henceforth referred to as “PX”. The culture medium HepatoZYME+ was investigated for its ability to induce transdifferentiation of B13 cells to HLCs and maintain the hepatic phenotype. Overall, HepatoZYME+-cultured cells experienced viability loss. A diluted version, “50:50”, showed induction of the hepatic markers carbamoylphosphate synthetase-1 (CPS-1) and HNF4α, as well as a change towards a HLC morphology. When using 50:50 as a maintenance medium, transdifferentiated HLCs retained loss of pancreatic amylase and also induction of hepatic markers, with comparable serum albumin secretion to the established Dex + OSM treatment. However, culture viability in 50:50 was still compromised. Therefore, HepatoZYME+ based media were deemed unsuitable for induction and maintenance compared to Dex-based protocols. PX flat sheet membranes were able to support culture of B13 cells and also the human osteosarcoma cell line, MG63, demonstrating improved cell attachment over non-surface treated PS membranes. PX membranes supported transdifferentiation of B13 cells to HLCs, presenting with loss of pancreatic amylase, induction of the hepatic markers transferrin, GS and CPS-1 and serum albumin secretion. Furthermore, PX showed no change in mass or loss of culture surface area over 15 days in culture conditions. Together, the novel membrane material and the media formulation and feeding regime developed have strong potential to be translated to a HFB setting and guide future BAL device design.

Les eaux usées peuvent possiblement être traitées par un système membrane intégré et combinant les procédés de bioréacteur à membrane (BAM) et d’osmose inverse (OI) pour une élimination efficace des micropolluants en vue de la réutilisation des eaux. Cependant, le rejet des concentrats d’OI dans l’environnement pourraient représenter un danger en raison de la toxicité de certains de leurs composés (micropolluants, sels, matières organiques). Une des solutions possibles peut être de recycler le concentrat d’OI vers le BAM. Néanmoins, une étude approfondie s’impose pour une telle configuration car le recyclage mettrait en jeu la recirculation de matière organique non biodégradable, ou de fortes concentrations en sels ou micropolluants, qui pourraient finalement engendrer, directement ou indirectement, un colmatage de la membrane ainsi qu’une modification de l’activité bactérienne dans le BAM. Les effets du recyclage de concentrat d’OI sur les performances de BAM ont été étudiés de deux différentes manières, en distinguant les effets à court-terme (ou court temps de contact) et les effets à long-terme (ou long temps de contact). Les résultats montrent qu’après un temps de contact de 3 heures entre le concentrat et les boues, les concentrations en protéines et polysaccharides dans le surnageant restent inchangées par rapport au début de l’opération. Une analyse HPLC-SEC a permis d’étudier les effets du concentrat d’OI sur la production de matières microbiennes solubles de types protéique. Un pic de concentration en substances protéiques ayant une masse moléculaire de 10 à 100 kDa a été observé dans le surnageant juste après l’addition du concentrat d’OI. Le pouvoir colmatant des boues n’a lui pas été modifié après l’injection du concentrat d’OI. Cette observation ouvre sur la possibilité de développer une opération d’OI comme traitement tertiaire en aval du BAM. La combinaison BAM-OI pourrait donc être une solution envisageable pour traiter le concentrat d’OI. Pour les longs temps de contact, les résultats ont montré que l’impact de l’effluent toxique (concentrat d’OI) sur les boues dépendait du rendement de l’opération d’OI et des caractéristiques du concentrat. Les mêmes tendances ont été observées quelle que soit la composition du concentrat en sels et en matière organique, puisqu’une augmentation de la concentration en protéine a été mise en évidence. L’effet du recyclage du concentrat d’OI a aussi été étudié à différents débits et avec différentes caractéristiques. Les effets sur les performances globales du BAM ainsi que sur son colmatage ont plus particulièrement été investigués. Le taux d’abattement en termes de Demande Chimique en Oxygène (DCO) est, dans tous les cas, supérieur à 93 %, quel que soit le débit de recyclage. Des résultats similaires ont été obtenus en termes de Carbone Organique Dissous. De plus, l’efficacité de la nitrification n’a pas été affectée en présence de concentrat d’OI dans le BAM. L’analyse HPLC-SEC a révélé un pic important de concentration en composés protéiques dans le surnageant, avec des masses moléculaires comprises entre 10 et 100 kDa et entre 100 et 1000 kDa. Par conséquent, une augmentation significative du pouvoir colmatant des boues a été observée et attribuée à la présence de protéines. Par ailleurs, le recyclage du concentrait d’OI n’a pas eu d’effet sur l’élimination de la carbamazépine et du diclofenac dans le BAM. Au contraire, l’élimination du ketoprofene a légèrement baissé, en passant de 94 à 72 %. Enfin, l’effet du recyclage de concentrat d’OI sur la biodégradation a été révélé comme insignifiant, ce qui indique que le recyclage du concentrat d’OI pourrait être une bonne alternative pour réduire les concentrats d’OI et limiter leur rejet dans l’environnement. / Wastewater effluents can be treated by an integrated membrane system combining membrane bioreactors (MBR) and reverse osmosis (RO) for effective removal of micropollutants in the field of high-quality water reuse. However, discharging the RO concentrate waste stream directly into the natural environment could lead to serious problems due to the toxic components contained in the concentrates (micropollutants, salts, organic matter). A possible solution could be the recirculation of RO concentrate waste to the MBR. However, such an operation should be studied in detail since the recirculation of non-biodegradable organic matter or high concentrations of salts and micropollutants could directly or indirectly contribute to MBR membrane fouling and modification of the biodegradation activity. The effects of RO concentrate recirculation on the MBR performances were investigated in two different ways of contact, i.e. short term peak contact and long-term continuous contact at various operating conditions. The results demonstrated that after 3 hours of contact time between the sludge and concentrate, the same values of both protein and polysaccharide concentrations were found in the supernatant, compared to that at the beginning of the reactor. HPLC-SEC analysis was employed to study the effects of RO concentrate on the production of protein-like SMPs. A significant peak of protein-like substances with a molecular size of 10-100 kDa was observed immediately in the supernatant after the addition of RO concentrate. Besides, no significant change was found of the sludge fouling propensity after the injection of RO concentrate into the activated sludge. This finding proposes the opportunities to develop RO process as a tertiary treatment of the membrane bioreactor (MBR), hence, the integrated MBR - RO concept with the RO concentrate recirculation to the MBR might be a solution to treat the concentrate waste stream produced by RO. During the long-term continuous contact, the results demonstrated that the impact of the toxic flow on activated sludge depends on the recovery of the RO step and the characteristics of the concentrate but the same trends were observed whatever the organic matter and salt contents of the concentrates: the concentration of proteins increased. The effects of the reverse osmosis concentrate recirculation, at different flow rates and with different characteristics, to the MBR were investigated. Their impacts on MBR global performances, especially the MBR fouling were evaluated. The removal efficiencies of chemical oxygen demand (COD) at the different flow rates of concentrate were greater than 93%. Similar results for the dissolved organic carbon removal efficiency were found in the MBR. Additionally, the presence of RO concentrate in the MBR did not inhibit the nitrification process. HPLC-SEC analysis employed to study the effects of RO concentrate on the production of protein-like SMPs demonstrated a significant peak of protein-like substances corresponding to 10-100 kDa and 100-1000 kDa molecules in the supernatant. Thus a significant increase of sludge fouling propensity was observed, which could be attributed to the increased quantity of protein-like substances. Furthermore, the recirculation of RO concentrate to the MBR did not significantly affect the removal of carbamazepine and diclofenac in the MBR. Meanwhile, the removal rate of ketoprofen was impacted slightly by the RO concentrate recycling to the MBR (from 94 to 72%). Finally, the effect of the concentrate on sludge activity was studied and no significant effect was observed on biodegradation, indicating that the return of the concentrate to the MBR could be a good alternative for the reduction of concentrate quantities before disposal to the environment.

by Yu Wing Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-126). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.vi / 摘要 --- p.viii / Table of Contents --- p.x / List of Tables --- p.xvi / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter CHAPTER 2 --- Literature Review --- p.3 / Chapter 2.1 --- Toxoplasma gondii --- p.3 / Chapter 2.1.1 --- Morphology and Life Cycle of T. gondii --- p.3 / Chapter 2.1.2 --- Routes of Transmission --- p.7 / Chapter 2.2 --- Toxoplasmosis --- p.8 / Chapter 2.2.1 --- Influences and Symptoms --- p.8 / Chapter 2.2.2 --- Treatment of Toxoplasmosis --- p.10 / Chapter 2.2.2.1 --- Antitoxoplasma Drugs --- p.10 / Chapter 2.2.2.2 --- Toxoplasma Vaccines --- p.12 / Chapter 2.3 --- Major T. gondii Surface Antigen - P30 --- p.16 / Chapter 2.4 --- Plants as Bioreactors --- p.19 / Chapter 2.4.1 --- Advantages of Plant Bioreactors --- p.19 / Chapter 2.4.2 --- Plant-based Vaccines --- p.20 / Chapter 2.4.2.1 --- VP2 Capsid Protein of Mink Enteritis Virus --- p.21 / Chapter 2.4.2.2 --- Hepatitis B Surface Antigen --- p.21 / Chapter 2.4.2.3 --- Norwalk Virus Capsid Protein --- p.22 / Chapter 2.5 --- Tobacco Expression System --- p.23 / Chapter 2.5.1 --- Transformation Methods --- p.23 / Chapter 2.5.1.1 --- Agrobacterium-mediated Transformation --- p.23 / Chapter 2.5.1.2 --- Direct DNA Uptake --- p.24 / Chapter 2.6 --- Phaseolin and Its Regulatory Sequences --- p.26 / Chapter CHAPTER 3 --- Expression of P30 in Transgenic Tobacco --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Materials and Methods --- p.29 / Chapter 3.2.1 --- Chemicals --- p.29 / Chapter 3.2.2 --- Oligos: Primers and Adapters --- p.29 / Chapter 3.2.3 --- Plant Materials --- p.31 / Chapter 3.2.4 --- Bacterial Strains --- p.31 / Chapter 3.2.5 --- Construction of Chimeric Genes --- p.31 / Chapter 3.2.5.1 --- Modification of pET-ASP30ΔPI --- p.32 / Chapter 3.2.5.2 --- Cloning of P30 into Vectors with Different Promoters --- p.38 / Chapter 3.2.5.2.1 --- Cloning ofP30 into Vector with CaMV 35S Promoter --- p.38 / Chapter 3.2.5.2.2 --- Cloning of P30 into Vector with Maize Ubiquitin 1 Promoter --- p.38 / Chapter 3.2.5.2.3 --- Cloning of P30 into Vector with Phaseolin Promoter --- p.38 / Chapter 3.2.5.2.4 --- Cloning of P30 into Vector with Phaseolin Promoter and Phaseolin SP --- p.39 / Chapter 3.2.5.3 --- Cloning of P30 into Agrobacterium Binary Vector pBI121 --- p.44 / Chapter 3.2.6 --- Transformation of Agrobacterium by Electroporation --- p.49 / Chapter 3.2.7 --- "Transformation, Selection and Regeneration of Tobacco " --- p.50 / Chapter 3.2.8 --- GUS Assay --- p.51 / Chapter 3.2.9 --- Synthesis of Single-stranded DIG-labeled DNA Probe --- p.51 / Chapter 3.2.10 --- Extraction of Genomic DNA from Leaves --- p.52 / Chapter 3.2.11 --- PCR of Genomic DNA with P30 Specific Primers --- p.53 / Chapter 3.2.12 --- Southern Blot Analysis of Genomic DNA --- p.53 / Chapter 3.2.13 --- Extraction of Total RNA from Leaves or Developing Seeds --- p.54 / Chapter 3.2.14 --- Reverse Transcription-Polymerase Chain Reaction of Total RNA --- p.55 / Chapter 3.2.15 --- Sequencing of RT-PCR Product --- p.56 / Chapter 3.2.16 --- Northern Blot Analysis of Total RNA --- p.56 / Chapter 3.2.17 --- Extraction of Total Protein from Leaves or Mature Seeds --- p.57 / Chapter 3.2.18 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.58 / Chapter 3.2.19 --- Purification of 6xHis-tagged Proteins --- p.58 / Chapter 3.2.20 --- Western Blot Analysis of Total Protein --- p.59 / Chapter 3.2.21 --- In vitro Transcription and Translation --- p.60 / Chapter 3.2.21.1 --- Construction of Transcription Vector Containing Chimeric P30 Gene --- p.60 / Chapter 3.2.21.2 --- In vitro Transcription --- p.60 / Chapter 3.2.21.3 --- In vitro Translation --- p.60 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- Construction of Chimeric P30 Genes --- p.65 / Chapter 3.3.2 --- "Tobacco Transformation, Selection and Regeneration " --- p.65 / Chapter 3.3.3 --- Detection of GUS Activity --- p.67 / Chapter 3.3.4 --- Detection of P30 Gene in Transgenic Plants --- p.69 / Chapter 3.3.4.1 --- PCR of Genomic DNA --- p.69 / Chapter 3.3.4.2 --- Southern Blot Analysis --- p.72 / Chapter 3.3.5 --- Detection of P30 Transcript in Transgenic Plants --- p.75 / Chapter 3.3.5.1 --- RT-PCR --- p.75 / Chapter 3.3.5.2 --- Sequencing of RT-PCR Product --- p.79 / Chapter 3.3.5.3 --- Northern Blot Analysis --- p.79 / Chapter 3.3.6 --- Detection of P30 Protein in Transgenic Plants --- p.83 / Chapter 3.3.6.1 --- Western Blot Analysis of Total Protein and Ni-NTA Purified Proteins --- p.83 / Chapter 3.3.7 --- In vitro Transcription and Translation --- p.92 / Chapter 3.3.7.1 --- In vitro Transcription --- p.92 / Chapter 3.3.7.2 --- In vitro Translation --- p.92 / Chapter CHAPTER 4 --- Discussion --- p.97 / Chapter 4.1 --- General Conclusion --- p.97 / Chapter 4.2 --- Further Speculations and Investigations --- p.100 / Chapter 4.2.1 --- Other Protein Detection Procedures --- p.100 / Chapter 4.2.2 --- In vitro Transcription and Translation --- p.100 / Chapter 4.2.3 --- Gene Silencing at Transcription and/or Post-transcription Levels --- p.101 / Chapter 4.2.4 --- Gene Silencing at Translation and/or Post-translation Levels --- p.102 / Chapter (A) --- AUG Context Sequence --- p.102 / Chapter (B) --- Codon Usage --- p.103 / Chapter (C) --- N-end Rule --- p.107 / Chapter (D) --- Phaseolin Sorting Signal --- p.107 / Chapter CHAPTER 5 --- Future Perspectives --- p.109 / Chapter 5.1 --- Codon Modification of the P30 Gene --- p.110 / Chapter 5.2 --- Fusion of the P30 Gene with the LRP Gene --- p.117 / Chapter CHAPTER 6 --- Conclusion --- p.118 / References --- p.119

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-06-06T13:43:29Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_vania_trevelin.pdf: 930781 bytes, checksum: 87425d2c5f79b86f6c423cc28ab11eb3 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-06T14:30:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_vania_trevelin.pdf: 930781 bytes, checksum: 87425d2c5f79b86f6c423cc28ab11eb3 (MD5) / Made available in DSpace on 2017-06-06T14:30:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_vania_trevelin.pdf: 930781 bytes, checksum: 87425d2c5f79b86f6c423cc28ab11eb3 (MD5) Previous issue date: 2014-10-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A floricultura traz muitos benefícios sociais e econômicos, já que é possível produzir em pequenas áreas, criando trabalho e desta forma mantendo o homem no campo. O Brasil, por apresentar climas e solos bastante diversificados, é um país que possibilita o cultivo das mais variadas espécies de flores e plantas ornamentais, tanto de origem nativa ou exótica, como de clima temperado ou tropical. A Gypsophila paniculata é uma espécie com grande mercado entre as flores de corte, podendo ser propagada comercialmente através de métodos vegetativos, como enraizamento de estacas. Outra planta ornamental de destaque é a Dracaena sanderiana, conhecida como Bambu-da-sorte, sendo atualmente uma das plantas de vaso mais populares em todo o mundo. Os arranjos formados foram popularizados segundo a tradição chinesa do Feng Shui, sendo oferecidos como presentes com intuito de trazer sorte, energizar ambientes e dissipar fluxos negativos. A tecnologia da cultura de tecidos pode auxiliar na propagação destas espécies, visando à produção de mudas com elevada qualidade genética e sanitária, substituindo assim, os métodos de propagação por sementes ou estacas. Tanto as técnicas convencionais de micropropagação, como o uso de biorreatores podem ser utilizadas para a propagação destas espécies ornamentais, viabilizando aumentar a oferta de mudas para o mercado. Os estudos foram conduzidos em experimentos que visaram à multiplicação de G. paniculata, em meios MS semissólido e líquido em biorreatores com sistema de imersão temporária (STI) e o estabelecimento e indução inicial de brotações de D. sanderiana, testando diversos explantes e reguladores de crescimento, bem como avaliar o estresse oxidativo, ocorrido in vitro na fase de estabelecimento. Neste contexto, o trabalho teve como objetivos: a) realizar uma análise comparativa entre a multiplicação de G. paniculata in vitro pelo sistema convencional e por biorreatores com sistema de imersão temporária; b) definir um protocolo eficiente para o estabelecimento de D. sanderiana e analisar a atividade das enzimas antioxidantes da mesma, durante o processo de estabelecimento in vitro. Quanto aos resultados de micropropagação e multiplicação de G. paniculata, o sistema de biorreator de imersão temporária foi mais eficiente, demonstrando maior proliferação de brotações e maior número de folhas. Para D. sanderiana os resultados obtidos mostraram que o procedimento de desinfestação mais promissor para a espécie foi com hipoclorito de sódio a 2% por 15 minutos e lavagens de 10 minutos com Cetazima 250 mgL-1, com adição de PPM (Plant Preservative Mixture) ao meio de cultura. Dentre os meios testados, o meio MS semissólido suplementado com 15 mgL-1de 2ip e 0,5 mgL-1 AIA e posterior diminuição para 2mgL-1de 2ip, foram os que apresentaram os melhores resultados quanto ao estabelecimento e indução de gemas. Quanto às análises enzimáticas, após 20 dias in vitro, os explantes de D. sanderiana já apresentaram uma adaptação ao ambiente, independente de serem expostos a luz ou ao escuro. / Floriculture brings many social and economic benefits, since it is possible to produce in small areas, fostering employment and thus keeping the man in the country. Brazil, by presenting very diverse climates and soils, is a nation that enables the growth of a wide variety of flowers and ornamental plants, both from native and exotic origins, and from temperate and tropical climates. The Gypsophila paniculata is a species that provides a large market among the cut flowers and can be commercially disseminated by vegetative methods such as rooting. Another outstanding ornamental plant is the Dracaena sanderiana, known as lucky bamboo, being currently one of the most popular pot plants worldwide, whose arrangements were popularized according to the Chinese tradition of Feng Shui, being offered as a gift with the intention of bringing luck, energizing environments and dispel negative flows. The tissue culture technology can aid spreading these species, aiming at producing seedlings with high genetic quality and health, thus taking the place of the methods of propagation by seeds or cuttings. Both conventional micropropagation techniques and the use of bioreactors can be used for the propagation of these ornamental species, enabling to increase the supply of seedlings to market. These studies were conducted through experiments which aimed at the reproducing of G. paniculata in semisolid and liquid media in bioreactors with a temporary immersion system (TIS) and the establishment and initial shoot induction of D. sanderiana, testing several explants and growth regulators, as well as at evaluating oxidative stress in vitro, occurred in the establishment phase. In this context, the study aimed at: a) conducting a comparative analysis between the multiplication of Gypsophila paniculata in vitro, both by conventional system and through bioreactors with temporary immersion system; b) defining an efficient protocol for in vitro establishment of Dracaena sanderiana and analyzing the activity of its antioxidant enzymes during the establishment process. Regarding the results of G.paniculata’s micropropagation and multiplication, the temporary immersion bioreactor system was more efficient, demonstrating greater proliferation of shoots and a greater number of leaves. Concerning D. sanderiana, results showed that the most promising decontamination procedure for the species was with sodium hypochlorite 2% for 15 minutes and 10 minute washes with Cetazima 250 mgL-1, with the addition of PPM to the cultural environment. Among the media tested, MS semisolid environment supplied with 15 mgL-1 2ip and 0.5 mgL-1 AIA and subsequent decrease to 2mgL-12ip presented the best results regarding the bud establishment and formation. In the enzymatic analysis, after 20 days in vitro, D.sanderiana explants already presented an adaptation to the environment, whether it be exposed to light or dark.

Securing water in the right quantities at the right quality for the right price is a major issue around the world. Membranes are making an increasingly important contribution to meeting this need; however their performance is limited by fouling. This thesis reports on an investigation into the fouling of systems related to water treatment using the Direct Observation Through the Membrane (DOTM). The investigation focused on the measurement of critical flux and observation of particle behaviour under a variety of conditions and for a number of different particles. The range of meanings attributed to critical flux in the literature was analysed and several proposals made for the improved use of the concept. In particular, critical flux determination techniques were classified by whether they measure resistance changes or particle deposition; leading to the definition of Critical Resistance and Critical Deposition Fluxes. In this thesis the deposition definition is used exclusively. The effect of Reynolds number and spacer orientation on critical flux was correlated for spacer-filled channels. The heterogeneous deposition patterns observed with regions of heavy deposition next to areas of little or no deposition. This pattern was related to the local hydrodynamics of spacer cells (a few mm2 in size). The correlations developed for critical flux in spacer-filled channels were adjusted for submicron particle size and incorporated into a SpiralWound Module (SWM) leaf model and then used to simulate the fouling of SWM leaves under a range of operating conditions and operating policies. The Mass Balance technique of critical flux determination was also briefly assessed. The applicability of critical flux criteria to SWM arrays was discussed. Fouling, particle behaviour and critical flux were also investigated in air-sparged systems. The post-cleaning water flux was found to be enhanced when the membrane is fouled in the presence of bubbles. The rate of flux decline was reduced by bubbles. Critical flux increased with air flowrate, and decreased with increased liquid flowrate and concentration. Bubbles caused particles to periodically deposit on the membrane. Particles were observed to stream past the membrane under the influence of back-diffusive forces. Video clips of particulate fouling are provided.

The purpose of this project is to evaluate the potential of a bioreactor system to degrade DDT based upon co-metabolism of indigenous bacteria. The study was performed with soil samples spiked with four different concentrations of DDT. The prepared sludge was circulated at a steady rate of revolution per minute in bioreactors with added M8 solution, cabbage leaf extract and molasses. The experiment was carried out for 7 days and chemical analysis and toxicity testing was accomplished at the beginning and the end of the experiment. The chemical analysis was essential to support the conclusions of the ecotoxicology tests. Ecotoxicology test was performed for the assessment of the toxicity (in terms of bioavailable measures) of the sludge samples, and was carried out with the Ostracodtoxkit sediment toxicity test, with the freshwater benthic crustacean test species Heterocypris incongruens. As part of the project the potential of the bioremediation method phytoremediation have been studied. Brassica Juncea seeds have been cultivated in the soil spiked with four different concentrations of DDT for one month, under stable circumstances. Growth of the plants was measured at the end of the experiment, and a chemical analysis was carried out. A thorough literature review was carried out for both the bioreactor and the phytoremediation experiments in order to obtain information about methods and theoretical background. The ecotoxicology tests and the chemical analysis showed increased p,p’- DDT concentrations in the bioreactors I. and II. at the end of the 7 day experiment, the reasons of which are not known, and require further studies. / -

Tissue engineering strategies that combine porous biomaterial scaffolds with cells capable of osteogenesis or bioactive proteins have shown promise as effective bone graft substitutes. Attempts to culture bone tissue-engineering constructs thicker than 1mm in vitro often result in a shell of viable cells and mineralized matrix surrounding a necrotic core. To address this limitation, we developed a perfusion bioreactor system that improves mass transport throughout large cell-seeded constructs. Additionally, we established and validated 3-D computational methods to model flow and shear stresses within the microporosity of perfused constructs. Micro-CT scanning and analysis techniques were used to non-destructively monitor mineral development over time in culture. CFD modeling of axial perfusion through cylindrical scaffolds with a regular microarchitecture revealed a uniform flow field distributed throughout the scaffold. Perfusion resulted in a 140-fold increase in mineral deposition at the interior of 3 mm thick polymer scaffolds seeded with rat bone marrow stromal cells. The total detected mineral volume tripled as the construct length was increased from 3 to 9 mm. Increasing scaffold length to 9 mm did not affect the mineral volume fraction (MVF) within the full volume of each construct. Mineral volume, spatial distribution, density, particle size and particle number were then quantified on cell-seeded constructs in 5 different culture environments. The effect of time varying flow conditions was compared with continuous perfusion as well as two different control cell culture methods in an attempt to enhance mineralized matrix within the constructs. Intermittent elevated perfusion and dynamic culture in an orbital rocker plate produced the greatest amount of mineral within 9 mm long constructs compared to low continuous flow and high continuous flow cases. Together, these studies indicate that dynamic culture conditions enhance construct development with regards to cell viability, mineralized matrix deposition, growth rate, and distribution. Furthermore, these techniques provide a rational approach to selecting perfusion culture conditions that optimize the amount and distribution of mineralized matrix production. Finally, the established perfusion bioreactor, in combination with micro-CT analysis, provides a foundation for evaluating new scaffolds and cell types that may be useful for the development of effective bone graft substitutes.

The effect of steady shear stress on somatic embryos were investigated in a flow chamber and evaluated at different time intervals using microscopy technique. The development of meristematic cell clusters, i.e. the immature embryos, into a polarized somatic embryo, and the effect on the localization of the suspensor cells that form during development of the immature embryos, were studied as a function of shear stresses. With the distribution and growth rate of the meristematic and suspensor cells, the effect of stress on the embryo development was established. Furthermore, the effect of shear stress on the cells at molecular level, the reaction of integrin-like proteins, the production of reactive oxygen species and the pore size of the cell walls involved in the shear stress responses, were investigated with molecular techniques. In general, shear stress inhibits meristematic cells growth. Meristematic cells grow fastest at shear rate of 86 s-1 among all the tested shear stress conditions. By combining the results of meristematic cells growth and suspensor cells formation, it suggests that there is a critical shear rate between 86 and 140 s-1, at which no suspensor cells form. The unidirectional flow with different shear stresses helps the polarized growth and the unidirectional alignment of suspensor cells. Reactive oxygen species and integrin-like protein are detected in the stressed cells as cellular responses to shear stresses. By monitoring the pore size and uptake time of cells to macromolecules with solute-exclusive experiments, it suggests that the stressed cells expedite the response to plasmolyzing components that are used to induce maturation treatment thus affect the response to maturation stimuli.

Phylogenetic hybridization and phenotypic fingerprinting were applied to the analysis of bacterial communities in wastewater treatment systems. These approaches were aimed at (i) developing monitoring tools able to foresee operational problems, and (ii) providing the rationale to optimize the operation of bioreactors. The work presented is intended to first describe the community found in two reactors treating pulp and paper mill effluent, and second evaluate the possibilities of these techniques with respect to the development of new monitoring tools. / Phylogenetic membrane hybridization showed that the bacterial communities were dominated by Alpha and Beta Proteobacteria, a structure probably linked to the low F:M ratio. Other important factors determining the community structure were the proportion of COD in the high molecular weight fraction, the sludge age, phosphate addition, and the concentration of specific compounds (alcohols, phenols, volatile fatty acids) in the influent. The community structure partly determined the sludge characteristics demonstrating its potential value in the assessment of reactor performance. The results obtained by phylogenetic membrane hybridization suggest that the probes used in a monitoring tool would not need to be targeted to the species level to provide relevant information. However, they also suggest that the technique is more sensitive to changes in population density as opposed to changes in bacterial metabolism. / Phenotypic fingerprinting measured a smaller difference between the communities of the two reactors studied than what was measured by phylogenetic membrane hybridization. However, differences in heterotrophic activities observed between the two communities were linked to differences in influent composition.

Thèse (M.Ress.Renouv.)-- Université du Québec à Chicoutimi, 2002. / Document électronique également accessible en format PDF. CaQCU