Tesi sul tema "Co-Culture systems"

Background: Lymphangioleiomyomatosis (LAM) is a rare progressive neoplastic cystic lung disease that primarily affects women of child bearing age leading to lung destruction, respiratory failure and death. Thought to be a consequence of dysregulated protease expression, cells of unknown origin accumulate in the lung, often forming clusters or nodules of cells with both melanocytic and smooth muscle properties. Some of these cells, known as LAM cells, have bi-allelic mutations in TSC2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures and genotyping analyses suggest that cells without LOH for TSC2 including wild-type fibroblasts are also common within LAM nodules. Hypotheses and aims: We hypothesise that LAM cells recruit wild-type fibroblasts and modify their properties to generate a permissive microenvironment, akin to a tumour stroma including the production and activation of matrix-degrading proteases which contribute to the destruction of the lung parenchyma. This study has therefore deigned in vitro co-culture models with an aim to study the expression patterns and activation of proteases in a LAM lung leading to matrix destruction. Another aim was also to characterise transcriptional differences normal human lung fibroblasts (NHLFs) and LAM-associated fibroblasts (LAFs) and to investigate changes in their gene expression when cultured together with a model LAM cell line, 621-101 angiomyolipoma cells which were derived from a LAM patient and have bi-allelic loss of TSC2. Methods: In vitro 2-dimensional (2D) and 3-dimensionalD (3D) co-culture models were designed and validated using fibroblasts characterised and isolated from 4 LAM lung donors, now termed LAFs, and 621-101 cells. The 3D extracellular matrix (ECM) incorporated the two cell types in a 10:1 ratio embedded in a basement membrane extract (BME) mimicking the lung matrix. An organotypic spheroid model was also developed incorporating both cell types thereby mimicking a LAM nodule. 6 LAM lung and 3 normal lung tissue donors were screened for candidate proteases in LAM pathology using qRT-PCR and identified upregulated proteases which may contribute to a role in LAM pathology. These findings were verified in the 2D and 3D in vitro models as well as ex vivo tissue using a variety of immunostaining techniques, activity assays and ELISA. Lastly, commercially bought NHLF (n=3) and LAF (n=3) were cultured in the presence or absence of 621-101 cells in the 2D Boyden chamber co-culture model. LAF and NHLF RNA was analysed using Affymetrix Human Genome U133 Plus 2.0 Arrays and Genomics Suite and Pathway (Partek). Findings were validated by multiplex assay and immunohistochemistry in 2D and 3D in vitro models and tissue respectively. Inflammatory cell migration and function was examined in co-culture model and LAM tissue. Results: The 3D BME model showed that TSC2-/- 621-101 cells and fibroblasts spontaneously form aggregates and clump together akin to a LAM nodule. The two cell types exhibited strong heterotypic cell-cell adhesive forces and resulted in strictly spherical spheroids. The 3D models designed all showed expression of markers of LAM nodules thereby representing LAM nodules in a dish. Of 30 proteases screened, cathepsin K gene expression was increased almost 15-fold in LAM lung compared to normal tissue and was also found to be elevated in 3D BME model. Cathepsin K in LAM tissue was expressed in the LAM nodules associated with cysts and was expressed exclusively by fibroblasts in the 3D spheroid model. As cathepsin K requires low pH for activity it was determined if LAFs and TSC2-/- cells can acidify the extracellular space. TSC2-/- cells but not LAFs decreased extracellular pH, over 24 hours and pH values < 7 were associated with increased cathepsin K activity in co-cultures. TSC2-/- cells expressed membrane transporters associated with extra-cellular acidification and inhibitors of the sodium bicarbonate co-transporters, carbonic anhydrases and mTOR reduced the pH gradient and decreased CTSK activity in co-cultures. Transcriptomic analysis using the 2D co-culture model showed 148 genes were significantly altered in both NHLF and LAF by 621-101 cells. Soluble factors from 621-101 cells induce pro-inflammatory transcriptional changes in both NHLFs and LAFs and pathway analysis showed enhanced chemokine signalling which highlighted stimulation of mainly the C-X-C motif chemokines and chemokine receptor signalling. The analysis identified 6 C-X-C motif chemokines all possessing a cognate receptor. The gene and protein expression of these chemokines was validated in the in vitro models and in ex vivo LAM lung tissue. Conclusions: The in vitro models are versatile and mimic the LAM lung nodule and environment. A potent matrix degrading protease possibly playing a role in LAM has been identified and using the in vitro models a possible mechanism of activation of CTSK resulting from a synergistic relationship between TSC2-/- cells and LAFs has been demonstrated. Also, soluble factors from the TSC2-/- LAM cell line elicit changes in gene expression in co-cultured fibroblasts. Chemokine signalling is associated with cell migration; elevated chemokine expression may be associated with the recruitment of inflammatory cells to the LAM nodule. The identification of these mechanisms and pathways opens up new avenues for therapeutic interventions in LAM.

This dissertation is a study of the possibilities and processes of constructing strong Chinese brands in the global marketplace. It investigates conceptual and strategic relationships between brands and cultures, focusing specifically on the issue of the unprivileged position of Chinese brands vis-à-vis that of other famous global counterparts. Accordingly, it deploys three illustrative cases from the Chinese context – Jay Chou (a successful Chinese music artist), the 2008 Beijing Olympics opening ceremony, and Shanghai Tang (a global Chinese fashion brand). In so doing, it moves away from the general trend to study the managerial aspects of Western brand building in Chinese contexts, and instead examines how Chinese brands express cultural aspects of their own well-known brand development models in the global marketplace. In short, this study uses a Chinese vantage to examine the emergence of cultural branding (using historical culture and global fashion systems to develop global brands), and its capacity to function as a useful complement to existing models of brand globalisation and global brand culture. The function of the three cases is illustrative and analytic. Collectively, they serve as a lens through which to study Chinese brand development in the global marketplace and examine global brand culture. Each case was fleshed out through various multi-sited ethnographic studies, which consisted of interviewing and observing consumers and managerial workers, the results of which shed light on several important but under-studied aspects of global brand culture. These include Chinese cultural branding in the global context, the cultural approach to branding among various brand actors, and relationships between brands and cultures across branding cultures. Drawing on these examinations, this study not only demonstrates ways in which brands and cultures circulate and construct each other in global brand culture. It also uses these insights to argue for the development of Chinese culture or Chinese-ness into a global brand resource by Chinese brand builders.

Les découvertes récentes sur les communautés microbiennes, ou microbiotes, ont révélé un potentiel biotechnologique considérable dans divers domaines. Ils sont considérés comme essentiels pour accélérer l'innovation dans les systèmes de production alimentaire. Toutefois, les procédés existants ne sont pas adaptés à la culture des microbiotes. La difficulté que représente la culture de microbiotes a notamment pour origine la capacité des microorganismes à interagir par compétition, qui peut conduire à la réduction indésirable de la biodiversité au sein du réacteur de culture. Ce phénomène peut aboutir à l'obtention de communautés qui ne présentent pas les fonctionnalités souhaitées. L'objectif de cette thèse est d'étudier l'influence de la propagation de microbiotes en condition contrôlée sur leur structure et leur fonction. Les travaux de cette thèse ont permis de développer et de déterminer la performance d'un procédé excluant la compétition microbienne pour la culture de communautés bactériennes. La stratégie choisie repose sur le micro-confinement et la ségrégation spatiale des bactéries au sein d'un bouillon de culture structuré en émulsion inverse. Après avoir étudié l'effet de la culture en émulsion inverse sur la croissance de bactéries individuelles, les travaux ont comparé son effet sur la dynamique de communautés propagées selon un régime séquentiel, ou backslopping, avec celui exercé par un système classique non-émulsionné. Les résultats ont montré que l'utilisation d'une émulsion inverse conduit à la génération de nouvelles structures de communautés au cours de la propagation, et que l'utilisation de la culture classique conduit à leur stabilisation. Les comportements différents issus de ces deux systèmes de culture en font des outils complémentaires pour le modelage et la propagation de communautés microbiennes. Enfin, l'effet de la propagation sur la variabilité fonctionnelle de communautés a été étudiée dans un contexte de biopréservation. Le criblage de microbiotes de laits crus propagés a montré qu'ils se différenciaient en termes de robustesse et de reproductibilité de leur activité anti-Listeria, justifiant de tenir compte de la variabilité fonctionnelle des communautés pour leur sélection dans un contexte d'ingénierie de microbiotes
Recent discoveries about microbial communities, or microbiota, have revealed considerable biotechnological potential in a variety of fields. They are considered essential to accelerate innovation in food production systems. However, existing processes are not adapted to the cultivation of microbiota. One major barrier to community propagation is competition between microorganisms, which can lead to an undesirable reduction in biodiversity within the culture reactor. This phenomenon can lead to communities that lack the desired functionality. The objective of this thesis was to study the influence of microbiota propagation, under controlled conditions, on their structure and function. During this work, a process of microbial culture excluding microbial competition for the propagation of bacterial communities was developed. The chosen strategy is based on the micro-confinement and spatial segregation of bacteria within a broth structured as an invert emulsion. The effect of the invert emulsion culture on the growth of individual bacteria was studied, then the effect of this system on the dynamics of communities propagated according to a sequential regime, or backslopping, as well as that exerted by a conventional non-emulsified system was investigated. The results showed that the use of an inverse emulsion leads to the generation of new community structures during propagation, and that the use of the classical culture leads to their stabilization. The different behaviors of these two culture systems make them complementary tools for the modeling and the propagation of microbial communities. Finally, the effect of propagation on the functional variability of communities was studied in a biopreservation context. The screening of propagated raw milk microbiota showed that they differed in terms of robustness and reproducibility of anti-Listeria activity, emphasizing the need to take into account the functional variability of communities when selecting communities of interest for microbiota engineering

Poor skeletal health results from aging and metabolic diseases such as obesity and diabetes and involves impaired homeostatic balance between marrow osteogenesis and adipogenesis. Tissue engineering provides researchers with the ability to generate improved, highly controlled and tailorable in vitro model systems to better understand mechanisms of homeostasis, disease, and healing and regeneration. Model systems that allow assembly of modules of MSCs, osteoblasts, and adipocytes in a number of configurations to engage in signaling crosstalk offer the potential to study integrative physiological aspects and complex interactions in the face of changes in local and systemic microenvironments. Thus, the overall goal of this dissertation was to examine integrative physiological aspects between MSCs, osteoblasts, and adipocytes that exist within the marrow microenvironment. To investigate the effects of intercellular signaling in different microenvironmental contexts, methods were developed to photolithographically pattern and assemble cell-laden PEG-based hydrogels with high spatial fidelity and tissue-scale thickness for long-term 3D co-culture of multiple cell types. This platform was applied to study effects of crosstalk between MSCs, osteoblasts and adipocytes on markers of differentiation in each cell type. Additionally, responses of MSCs to systemic perturbations in glucose concentration were modulated by mono-, co-, and tri-culture with these cell types in a model of diabetes-induced skeletal disease. Together, these studies provided valuable insight into unique and differential effects of intercellular signaling within the niche environment of MSCs and their terminally differentiated progeny during homeostatic and pathological states, and offer opportunities further study of integrative physiological interactions between mesenchymal lineage cells.

Le diabète mellitus, également désigné comme la maladie du siècle, est une pathologie mortelle qui affecte le système endocrinien. Les mécanismes liés à la rupture de la boucle de rétroaction, qui régule le métabolisme et induit le diabète, ne sont pas entièrement connus. La compréhension des mécanismes d'action de l'insuline est donc essentielle pour le développement de stratégies thérapeutiques efficaces afin du lutter contre cette maladie. Par conséquent, il est impératif de trouver un modèle robuste et fiable, capable de surmonter les limites de la culture cellulaire traditionnelle en 2D et de l'expérimentation animale, pour la recherche sur le diabète. L'objectif de cette thèse est de développer un nouveau modèle de co‐culture foie‐pancréas en utilisant des systèmes microphysiologiques avancés (MPs) afin d’aborder plus efficacement le mécanisme impliqué dans la régulation endocrinienne hépatique et pancréatique. Ce travail met en évidence la capacité des systèmes multi‐organes sur puce qui combinent la compartimentation avancée des cellules en 3D, la microfluidique et la technologie des cellules souches pluripotentes induites (iPSC), pour atteindre une complexité biologique élevée et des fonctions rarement reproduites par une seule de ces technologies d’ingénierie tissulaire
Diabetes mellitus (DM) or the so called disease of the century is a life threatening dysfunction that affects the endocrine system. The mechanisms underlying the break in the feedback loop that regulates the metabolism and the consequent diabetes induction are not fully known. Understanding the mechanisms of insulin action is therefore crucial for the further development of effective therapeutic strategies to combat DM. Accordingly, it is imperative to find a robust and reliable model for diabetes research able to overcome the limitations of traditional 2D in vitro cell culture and animal experimentation. The aim of this thesis is to develop a new liver‐pancreas co‐culture model using advanced microphysiological systems (MPs) to tackle more effectively the mechanism involving the hepatic and pancreatic endocrine regulation. This work highlights the power of multi organ‐on‐chip systems that combines the advanced 3D‐cell compartmentalization, microfluidics and induced pluripotent stem cells (iPSC) technology to achieve a high biological complexity and functions that are rarely reproduced by only one of these tissue engineering technologies

This thesis presents the development and evaluation of two applications for scaffolds in the field of liver tissue engineering. In the first study a poly (D,L lactic acid) (PDLLA) scaffold is used as a three-dimensional template for hepatocyte–hepatic stellate cell (HSC) co-culture. To enhance PDLLA ligand binding capacity scaffolds are surface modified using allylamine plasma deposition and treatment with NaOH. Primary adult rat hepatocytes and HSC are then seeded onto these scaffolds and cultured in static conditions. Scanning electron microscopy (SEM) is used to assess mono-culture and co-culture morphology whilst synthetic and cytochrome P450 function are measured using albumin and testosterone assays. The second study explores the potential for intrahepatic growth factor and extracellular matrix (ECM) delivery from a biodegradable polymer scaffold to promote liver growth and to enhance regeneration. The study is undertaken in rats. The scaffold design and implantation technique are first piloted in a short survival study. Hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF)1, FGF2 and liver derived ECM (L-ECM) are then loaded into poly(lactic-co-glycolic acid) (PLGA) + 5% poly(ethylene glycol) (PEG) scaffolds and implanted into normal and partially hepatectomised liver. Implant morphology is assessed by micro-CT reconstruction. Growth factor bioactivity and release are confirmed by in vitro profiling. Liver growth and volume redistribution are assessed by liver weight analysis. Parenchymal injury and function are quantified by measuring serum aspartate aminotransferase (AST) & bilirubin. 5-bromodeoxyuridine (BrdU) inclusion & MIB-5 immunohistochemistry (IHC) are used to identify hepatocyte and non-parenchymal cell proliferation. Liver-scaffold interaction is characterised by H&E and Masson’s trichrome staining. Non-parenchymal cell migration is assessed by ED-1 and desmin IHC. All histology is then subjected to image analysis.

The aim of this Thesis was to characterise the behaviour and interaction of primary muscle derived cells (MDCs) and motoneurons within a collagen-based 3D in vitro culture system. Cells cultured under uniaxial tension within 3D collagen matrices are known to selforientate along the lines of principle strain. In the case of skeletal muscle cells, this leads to the formation of aligned myotubes, thereby generating cultures which more closely recapitulate the architecture of in vivo muscle. Since maturation of muscle in vivo is dependent on functional innervation, integration of this model with a physiologically correct neural input would further improve both the accuracy and complexity of the in vitro construct. Furthermore, reliable neuromuscular junction formation in 3D culture could have substantial benefits for the study of neuromuscular disease and the testing of novel therapeutic agents. The behaviour of primary rat MDCs within an established collagen-based 3D culture system was optimised and subsequently characterised. A comparison of this model to conventional 2D cell culture techniques was carried out using immunohistochemical and PCR analysis. Investigation of myogenin expression levels over a three week culture period in both 2D and 3D found no significant differences between the two systems, indicating a conserved ability for MDC differentiation in both models. Immunohistochemical data illustrated the alignment of uniaxial myotubes in 3D compared with randomly orientated and branched myotubes in conventional culture, demonstrating the improved biomimicity of myotubes developed in 3D and under directional tension. The presence of motoneurons within the 3D co-culture was found to promote maturation of the MDCs as indicated by levels of macroscopic construct contraction and by quantitative PCR analysis. Co-localisation of pre- and post- synaptic markers in culture indicated the presence of putative synaptic contacts within the model. The model presented in this Thesis represents a step forward in the development of physiologically accurate in vitro models of skeletal muscle, which may help in future investigations of skeletal muscle development, physiology and pathology.

In these studies the ability of a three-dimensional hepatocyte-stellate cell co-culture system to preserve some key aspects of differentiated hepatocyte function in vitro is demonstrated. A poly(DL-lactic Acid) surface allows dynamic and rapid interaction of hepatocytes and stellate cells to form co-culture spheroids in a complex multistage process (shown by time lapse microscopy). After five days the spheroids have developed a substantial extracellular matrix support and hepatic ultra-structure including bile canaliculi, tight junctions, desmosomes and lipid storage. The distribution of the stellate cells in the final structure is related to their motile and aggregating role in spheroid formation, i.e. mainly central and peripheral, and provides a unique and generically applicable insight into the dynamics of multicellular spheroid formation where aggregation is induced by one cell type and imposed on another. The spheroid morphology supports enhanced cell viability relative to hepatocytes in a mono-culture mono-layer. Co-culture spheroids also have superior cytochrome P450 3A and 2B function, and increased inducibility of 2B function, relative to a range of hepatocyte monoculture techniques (HPLC detection of testosterone metabolites). Increased function in co-culture is supported by greater expression of cytochrome P450 3A23,1A2, and 2E1 mRNA relative to monoculture (RT-QPCR). Also, high hepatocyte growth factor mRNA expression in co-culture suggests a post-traumatic, or possibly regenerative, environment. A preliminary study of human hepatocytes co-cultured with rat stellate cells demonstrated prolonged function of cytochrome P450 3A4,2C19 and 2C9. The co-culture spheroids are also shown to maintain a low level of sensitivity to hepatotoxins DDC and amiodarone after seven days in culture. This study shows that stellate cells facilitate spheroid formation, influence spheroid architecture, and are an effective method of preserving some aspects of hepatocyte function in the early stage of culture.

Sea-based integrated multi-trophic aquaculture (sIMTA) was explored as an amalgam of the processes of polyculture and biofiltration, with the primary objectives of improved aquaculture production efficiency and wastes remediation. The study established a coculture of Atlantic salmon (Salmo salar, L.), Pacific oyster (Crassostrea gigas, Thunberg) and sugar kelp (Saccharina latissima, L.), in Scotland, and explored their production in relation to the background environment, historical aquaculture production modes, regulation and resource-use. The field trials demonstrated the dominance of the ambient environment in regulating trophic linkage of co-cultures. Enhanced growth of the bivalve and macroalgal components, over reference cultures, was only observable when ambient nutrients were limiting or favoured food, i.e. phytoplankton, was scarce. Localised differences within and between sealoch systems were also observed to be of importance. The complex physical processes of particulate waste dispersion and dissolved nutrient diffusion were simulated using established models and field data drivers. This process illustrated the potential benefits of designing the integrated farm to optimise trophic linkages as well as considering the final fate of wastes. The application of sIMTA was explored within the current regulatory regime, illustrating what regulatory gains (statutory and non-statutory) might be possible on account of the process and how the products of integration, as studied, are likely to meet with few regulatory food safety constraints within a UK market. The scarcity of aquaculture resources, partly through inefficient use, was explored. sIMTA is presented as a method which could possibly alleviate some of these resource-use inefficiencies when ambient environment supports, and as such sIMTA is proposed to qualify for priority resource allocations, in the context of greater socio-economic advantage.

A reproducible model of infection of dental tissues by SAG bacteria has been produced which shows attachment patterns of bacteria and the effect on host tissues. This model can be used to further investigate processes involved in endodontic infections, including expression of virulence factors by bacteria and host response from the dental tissues. It may also be used in the future for testing novel antimicrobials for use in treating pulpal disease.

Cystic Fibrosis (CF) is the most common hereditary genetic disorder among Caucasians. Pseudomonas aeruginosa is a major cause of morbidity in cystic fibrosis patients. Chronic infection with P. aeruginosa eventually occurs and is associated with a switch to biofilm formation of the bacteria. The symptoms and pathology of acute and chronic P. aeruginosa infections differ greatly. The first line of defense within the lung is the physical barrier of the lung epithelia. The examination of established biofilm interactions with lung epithelia is difficult. Here, I use the Calgary Biofilm Device co-culture system to conduct the concurrent analysis of established biofilms and planktonic bacteria with A549 lung cells. Comparison of P. aeruginosa biofilm and planktonic bacteria’s effects on A549 lung cells showed that planktonic bacteria caused more A549 cell rounding and death, while biofilm stimulated more IL-8 release by epithelial cells. Biofilm was shown to secrete significantly more Pseudomonal Elastase than planktonic, causing A549 morphological changes and loss of tight junctions. The antimicrobial peptide LL-37 was shown to differentially affect biofilm and planktonic bacteria. LL-37 caused a decrease in twitching of planktonic bacteria and exposure to LL-37 for 48 hours resulted in a decrease in elastase secretion likely due to down-regulated type 2 secretion. When established biofilms were compared with newly adherent biofilms, young biofilms were shown to have characteristics similar to both planktonic bacteria and mature biofilms. From this data we can follow the pattern of bacterial virulence as P. aeruginosa transitions from the planktonic mode of growth to the eventual mature biofilm that is associated with chronic infection. In conclusion, this study provides the foundation for a co-culture system that can be used to study the host-pathogen interactions of mammalian epithelia with established P. aeruginosa biofilms. The future adaptations of this model will better represent the in vivo characteristics of chronic lung infection to delineate ongoing virulence mechanisms of the bacteria causing host cell stimulation and damage.
May 2016

Intimal hyperplasia (IH) is defined as the abnormal migration and proliferation of smooth muscle cells with associated deposition of extracellular matrix in the intimal layer. It is a natural response to endovascular injury induced by procedures such as angioplasty, stent implantation, or atherectomy. Research on the molecular pathways and mediators has led to the discovery of a variety of substances aimed to interrupt or attenuate IH. Heparin, low-molecular-weight heparin, aspirin, corticoids, angiotensin-converting enzyme inhibitors, cyclosporin, vascular endothelial growth factor (VEGF) and other agents have been investigated. However, none of these agents has been used with marked success at the clinical level. Therapeutic angiogenesis studies have demonstrated the potential of heparin binding angiogenic growth factors such as VEGF and basic fibroblast growth factor (bFGF/FGF-2) to treat ischemic heart diseases. Studies on endothelial nitric oxide synthase (eNOS) gene transfer in vivo showed attenuated IH caused by constitutive generation of nitric oxide (NO) via the NOS pathway. FGF-2 increased VEGF mRNA levels in single-cultures of rabbit smooth muscle cells (SMC) and also promoted NO production from endothelial cells (EC). Therefore, we hypothesize that FGF-2 mediates SMC inhibition through the NOS pathway. In order to elucidate the influence of these growth factors, we employed an appropriate SMC-EC co-culture system.Studies on SMC-EC interactions have been established in various in vitro co-culture systems. However, there exist only few co-culture systems in which the structure of a vessel wall is imitated. A direct co-culture model was used in this study to determine the effect of growth factors on the SMC and EC proliferation. In the following study we investigate the effects of VEGF, FGF-2 and FGF-2+VEGF on porcine aorta smooth muscle and endothelial cells. Addition of the higher concentrations (>10 ng/ml) of FGF-2 to the SMC-EC direct co-culture greatly reduced smooth muscle cell numbers and cell cycle S-phase, as judged by propidium iodide DNA analysis using flow cytometry. We also observed that coadministration of FGF-2 with VEGF did not show any difference on SMC proliferation compared to control. These data demonstrate the potent regulatory capabilities of FGF-2 on smooth muscle cell inhibition. Nitric oxide which is generated by the enzyme NOS is hindered by the addition of NOS inhibitor, NG-Methyl-L-arginine acetate (L-NMMA). Utilizing L-NMMA we found that FGF-2 mediated smooth muscle cell inhibition does not follow the NOS pathway. This study is intended to understand the interactions of combination of therapeutic growth factors on vascular cells. The current study is a first step towards an overall goal of setting up an in vivo porcine model for clinical treatment of IH using FGF-2.

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

Hypertension is associated with marked cardiac sympathetic over-activity and end organ hyper-responsiveness. The sympathetic dysfunction is caused by aberrant calcium (Ca²⁺) handling resulting in enhanced neurotransmission. However, it remains unclear whether the sympathetic neuron or the myocytes is the primary driver behind the initiation and maintenance of the autonomic phenotype. The work in this thesis characterises the Ca²⁺ dysfunction and regulation at the membrane level. Further, it employs physiologically coupled sympathetic neurons and ventricular myocytes to determine the cellular driver of cardiac dysautonomia in the pro-hypertensive state. Chapter 1 provides a general overview of the field of autonomic hypertension with a specific focus on the sympathetic control of cardiac excitability. In particular, the role of Ca²⁺ and cyclic nucleotides in the facilitation of neurotransmission are explored. Chapter 2 details the methods used in this thesis. It provides rationale for the approaches taken to record membrane Ca2+ currents, cyclic adenosine monophosphate (cAMP) levels and cAMP-activated protein kinase (PKA) activity, and the development and uses of a co-culture of coupled sympathetic neurons and ventricular myocytes. Chapter 3 describes the successful development of an effective voltage clamp method to isolate whole cell Ca²⁺ currents in sympathetic neurons. It details the issue of space clamp problem when using this technique on peripheral neurons and provides experimental guidance on how to quantify and limit theses issues. Chapter 4 identifies that the pro-hypertensive four-week old neurons from the spontaneously hypertensive rat (SHR) have significantly larger whole cell Ca²⁺ currents when compared to normotensive (Wistar Kyoto-WKY) neurons, that are largely N-type in nature. Restoring the cGMP cyclic nucleotide dysfunction seen in these cells, rescues the ion channel phenotype and bring the Ca²⁺ down to levels seen in the normotensive WKY neuron. Further, it identifies that phosphodiesterase (PDE) 2A inhibition differentially affects the currents in the WKY and SHR, further supporting the notion of PDE2A dominance. Chapter 5 identifies the presence and functional relevance of cGMP cross-talk with the cAMP-PKA pathway in sympathetic neurons. This cross talk is significantly altered in the pro-hypertensive state, via the differential involvement of PDEs. It functionally identifies the presence of PDE3 and PDE2A and provides further evidence that these enzymes could be dysregulated in pro-hypertensive neurons. Chapter 6 describes the use of a co-culture model of ventricular myocytes and sympathetic neurons. Physiological stimulation of the sympathetic neuron with nicotine whilst monitoring cAMP levels in the myocytes confirms that the cellular phenotypes seen in the individual cells are functionally present in the co-culture. Using cross-cultures, it identifies the neuron as the principal driver behind the cardiac sympathetic responses observed in pro-hypertension. The results provide evidence for a dominant role played by the neuron in driving the adrenergic phenotype seen in cardiovascular disease and highlights the potential of using healthy neurons to turn down the gain of neurotransmission, akin to a smart pre-synaptic β-blocker. Chapter 7 forms the concluding discussion that summarises the main findings of this thesis and attempt to place it in a clinical context, and highlights avenues of further research. In particular, the possibility of using a cell therapeutic approach to treat sympathetic hyperactivity.

Thesis (MPhil)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Stellenbosch University was approached to assist with developing a techno-financial model for an urban freshwater aquaculture system in Philippi, Cape Town. Rapidly growing urban areas are predominantly becoming concentrated zones for malnutrition and poverty which require attention. Having enough food to eat does not mean that a family is food secure, the problem is usually associated with the lack of access to nutritious food. Fish is seen as an extremely healthy food which has the potential to effectively support food security and alleviate malnutrition. Aquaculture is identified as a largely underdeveloped sector in South Africa. It is currently undergoing rapid transition, being promoted by government as an industry that has potential to develop and create jobs, provide food security and grow the South African economy. Aquaponics- a method to integrate aquaculture with growing crops in a symbiotic system is a highly resource efficient closed-integrated food producing technology which has the potential to benefit from South African biosecurity regulations and climate-geographic characteristics. It is viewed as an effective food production alternative to deal with the challenges of declining high quality freshwater resources and available arable land. Training and capacity building is important for the development of aquaponic technology. This study explores and identifies the advantages aquaponic technology development would have in South Africa. The study has reviewed and assessed the fundamental principles for aquaculture production and management required for aquaponic systems development and management. A practical case study identifies the daily challenges and design parameters of aquaponic systems. The study is concluded with a techno-financial project proposal which shows how aquaponic systems can be planned.
AFRIKAANSE OPSOMMING: Universiteit Stellenbosch was genader om 'n tegno-finansiele model to ontwikkel vir 'n stedelike akwakultuur plaas in Philippi, Kaapstad. The tempo waarteen die stedelike areas groei ontwikkel kommerwekkende uitdagings soos wanvoeding en armoede. In hierdie studie is vis geindentifiseer as 'n uiters voedsame aanvulling in die dieet van Suid Afrikaners. Akwakultuur is grootliks agter in terme van ontwikkeling. Dit word beskou as 'n sektor wat groot potensiaal inhou vir Suid Afrika se eknomiese groei, werkskepping en voedselsekuriteit. Akwaponika is die hersirkulerende integrasie van akwakultuur en hidroponika. Akwaponika hou groot voordele in terme van Suid Afrika se biosekuriteit regulasies and geografiese eienskappe en is 'n effektiewe manier om gebruikte akwakultuur te suiwer. Opleiding en beplanning word gesien as 'n fundamentele benadering tot suskesvolle akwaponika ontwikkeling. Hierdie studie bestudeer die Suid Afrikaanse omgewing en potensiaal vir akwaponika ontwikkeling. Die fundamentele beginsels van akwakultuur en hidroponika bestuur en produksie is saamgesit wat beskou word as die aanbevele manier om akwaponika te bestuur. 'n Praktiese gevallestudie toon die daaglikse uitdagings aan en gee raad oor daaglikse bestuur van akwaponika stelsels. Die studie word afgesluit met 'n tegno-finansiele model wat wys hoe om 'n akwaponika sisteem te beplan.

Bacterial biofilms are involved in most device-associated infections. This thesis has developed an in-situ method to treat biofilm and a coculture for testing new antimicrobial biomaterials. We used surface immobilisation to promote Alpha-amylase and silver ion’s disruption of S. aureus biofilms. The treatment also helped coating the surface with silver particles to prevent recurrence. We also formed a coculture of S. aureus and pre-osteoblastic cells and showed that bacteria preferentially attached to the osteoblasts and became more resistant to antibiotic treatment. Overall, this thesis has laid down the foundation for the development and study of new anti-biofilm biomaterials.

Although electronic cigarettes (ECs) have been widely promoted as safer alternatives to tobacco cigarettes, limited scientific data is currently available on their possible health effects. The current study aims to investigate the potential effects of ECs using a novel in-house designed cigarette/EC aerosol delivery system and physiologically relevant 2D and 3D in-vitro human airway models. Submerged cultures of BEAS 2B and CALU 3 bronchial epithelial cells were used to investigate the effects of nicotine and its oxidative metabolite cotinine. Results demonstrated that neither nicotine nor cotinine had any significant impact on the bronchial epithelial cell viability or IL-6/IL-8 pro-inflammatory mediators’ production. Further, treatment of submerged cultures of a number of airways related cell types to extracts of commercially available ECs of different nicotine strength, flavourings and brands showed that while the differing nicotine strengths had no impact on the cell viability, flavourings significantly influenced cell viability, with strawberry and cherry flavoured ECs demonstrating the highest cytotoxicity. Moreover, same flavours from different brands produced different effects on cells. Finally, a co-culture human airways model consisting of CALU 3 bronchial epithelial cells and MRC-5 pulmonary fibroblasts cultured at air-liquid-interface were treated to whole cigarette smoke (WCS) and EC vapour (ECV) at different exposure times (7 m, 1 h, 2 h, 3 h, 4.5 h and 6 h) as per the ISO:3308 standard smoking regime using an in-house designed bespoke, automated aerosol delivery system. Results demonstrated that while WCS caused a significant reduction in cell viability post 7 min exposure, ECV produced cytotoxic effects only at exposure times ≥ 3 h. A significant increase in oxidative stress and IL-6/IL-8 production was observed post 3 h ECV treatment, both of which are hallmark characteristics of airway inflammatory conditions like chronic obstructive pulmonary disorder. Overall, results from the current study suggest that ECs have the potential to cause substantial airways damage, and as yet, cannot be regarded as safe alternatives to tobacco cigarettes. Importantly, a standardised testing method is urgently required in order to elucidate on the long-term health effects of ECs.

Stress promotes the use of methamphetamine and other recreational substances and is often implicated in relapse to stimulant use. Thus, it is of critical importance to examine the consequences of the co-occurance of stress and methamphetamine use. Activity of the glutamatergic N-methyl D-aspartate (NMDA) receptor system appears to be involved in the neurotoxic effects of both chronic stress and methamphetamine exposure. The current studies investigated the hypothesis that chronic pre-exposure to the stress hormone corticosterone (CORT) results in an increase of NMDA receptor activity and that this will potentiate the neurotoxic effects of methamphetamine (METH). Co-cultures of the ventral tegmental area, nucleus accumbens, and medial prefrontal cortex were pre-exposed to CORT (1 μM) for 5 days prior to co-exposure to METH (100 μM) for 24 hours to investigate the combined effects on neurotoxicity and protein density of NMDA receptor subunits. The combination of CORT and METH resulted in significant neurotoxicity within the medial prefrontal cortex compared to either CORT or METH alone. The CORT+METH-induced toxicity was attenuated by co-exposure to the NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV; 50 μM) during the 24 hour CORT and METH co-exposure. Although CORT alone did not significantly alter the density of the NR1 and NR2B subunits of the NMDA receptor, METH exposure for 24 hours resulted in a significant loss of the polyamine sensitive NR2B subunit. Co-exposure to CORT and METH also resulted in decreased extracellular glutamate while not significantly altering extracellular dopamine. These results suggest an enhancement of NMDA receptor systems or downstream effectors in areas of the mesolimbic reward pathway following chronic pre-exposure to CORT, which leads to enhanced neuronal vulnerability to future excitotoxic insults. This may be of critical importance as use of psychostimulants such as METH and other drugs of abuse may produce excitotoxic events in these areas, thus further compromising neuronal viability.

Un changement rapide et d’ampleur vers des pratiques agricoles qui contribuent à la protection de l’environnement et la santé humaine est nécessaire. Dans de nombreux cas, ces pratiques alternatives existent, mais elles ne sont pas mises en œuvre du fait de contraintes au niveau de la parcelle, de l’exploitation, du territoire, de la filière et/ou à une échelle globale. Dans ma thèse, j’ai développé une méthodologie d’accompagnement du changement de pratiques prenant en compte les déterminants du choix des pratiques aux différentes échelles. J’ai appliqué cette méthodologie sur un cas d’étude précis : la gestion des bioagresseurs telluriques, en particulier des nématodes à galles, en maraîchage sous abris provençal. Les nématodes à galles causent des dommages importants sur les cultures maraîchères au niveau provençal (40% des exploitations touchées) et mondial. Leur gestion actuelle repose essentiellement sur l’usage de nématicides non sélectifs causant des dommages en matière environnementale et de santé humaine. Tout d’abord, j’ai réalisé une analyse sociotechnique montrant que le système agri-alimentaire maraîcher provençal était majoritairement verrouillé autour de l’utilisation des techniques « de désinfection radicale des sols », excluant ainsi la mise en œuvre de techniques alternatives agroécologiques. Ce verrouillage était constitué d’un ensemble de freins interconnectés qui ont entravé le changement de pratiques et auxquels prenait part une diversité de parties prenantes au niveau provençal et au-delà : les agriculteurs, l’amont et l’aval de la filière (consommateurs inclus), la R&D et les acteurs des politiques publiques. Suite à cette analyse, j’ai étudié des innovations couplées existantes facilitant la mise en œuvre d’une protection agroécologique des cultures dans les systèmes légumiers français. Cette « traque aux innovations » nous a permis d’identifier 5 types d’innovations couplées, et pour chaque type les combinaisons de leviers sociotechniques mobilisés et leurs conditions de mise en œuvre. En parallèle, j’ai mis au point un jeu sérieux me permettant de partager efficacement le résultat de l’analyse sociotechnique avec les parties prenantes du problème. Ce jeu sérieux m’a également permis de faciliter la gestion des connaissances et la créativité chez les parties prenantes et de favoriser leur collaboration, afin d’initier la conception de solutions innovantes adaptées au problème traité. Enfin, j’ai mobilisé les travaux précédents (analyse, traque et jeu) lors de 4 ateliers de coconception avec les parties prenantes. J’ai créé et mobilisé une diversité de dispositifs d’accompagnement dans ces ateliers. Ils ont permis de concevoir des solutions de plus en plus élaborées pour faciliter le changement de pratiques. Au total, nous avons collectivement conçu 50 solutions innovantes dont 41 innovations couplées, ouvrant ainsi l’espace des solutions possibles. Nous avons également évalué une partie des innovations couplées. En discussion, je pointe les pistes d’action et de recherche prometteuses pour faciliter l’implémentation de pratiques agroécologiques de gestion des bioagresseurs telluriques en maraîchage provençal sous abris. Je discute les évolutions possibles du dispositif méthodologique que j’ai développé au cours de ma thèse. Je propose ainsi d'améliorer son efficacité et de compléter le processus de conception en précisant les conditions de mise en œuvre des innovations conçues, en les évaluant et en les ancrant dans le système agri-alimentaire territorial. Enfin, je montre que ce travail contribue à établir des bases théoriques et méthodologiques à l’accompagnement du changement de pratiques par la reconception multi-échelle de systèmes agricoles. Les parties A « Problématique » et C « Discussion » de ma thèse sont rédigées en français. La partie B est constituée de trois articles et d’un chapitre de thèse rédigés en anglais
A rapid and far-reaching change towards farming practices that contribute to the protection of the environment and the human health is needed. In many cases, these alternative practices exist but are not implemented due to interconnected barriers at the plot, farm, territory, value chain and/or global level. In my thesis, I developed a methodology taking into account the determinants of the farming practice choices at the different levels to support the change in farming practices. I applied this methodology to a specific case study: the management of soil-borne pests and diseases, mainly root-knot nematodes, in sheltered vegetable farming systems in Provence (France). The impact of root-knot nematodes on vegetable crops is significant both in Provence (40% of farms affected) and worldwide. Their management is essentially based on the use of non-selective nematicides that are damaging for the human health and the environment.First, I carried out a sociotechnical analysis showing that most of the Provençal agri-food system was locked around the use of "radical soil disinfection" techniques, thus excluding the implementation of alternative agroecological techniques. This lock-in arose from interconnected barriers to the change in practices, involving a diversity of stakeholders at the Provençal level and beyond it: farmers, upstream and downstream of the sector (including consumers), R&D and public policy actors. Following this analysis, I studied existing coupled innovations that foster the implementation of agroecological crop protection in French vegetable systems. This “tracking of innovations” led us to identify 5 types of coupled innovations, and for each of them, the combinations of sociotechnical levers mobilized and the way they were implemented. Meanwhile, I developed a serious game enabling the effective sharing of the sociotechnical analysis results to the stakeholders of the studied problem. This serious game also enabled to facilitate stakeholders’ knowledge management and creativity and the collaboration between them, for initiating the design of innovative solutions tailored for the problem under study. Finally, I mobilized the previous works (analysis, tracking, serious game) in 4 co-design workshops conducted with the stakeholders. I created and implemented several methods in these workshops to design increasingly elaborate solutions that favor change in practices. As a result, we collectively designed 50 coupled innovations including 41 coupled innovations, thus opening up the space of possible solutions. We evaluated part of the complex coupled innovations.In the discussion, I point out the promising avenues of action and research to facilitate the implementation of agroecological practices for the management of soil-borne pests and diseases in Provençal sheltered vegetable farming systems. I discuss the possible evolution of the methodology I developed during this thesis, in order to improve its efficiency and complete the design process. I make proposals to specify the conditions of the implementation of the innovations designed, evaluate them and anchor them in the territorial agri-food system. Finally, I show that this work contributes to establishing theoretical and methodological bases to multi-level redesign of agricultural systems for accompanying changes in farming practices.Parts A "Problem" and C "Discussion" of my thesis are written in French. Part B consists of three articles and one chapter of the thesis written in English

Engagerat ledarskap är grunden till att skapa en god kvalitetskultur och för attlyckas krävs medarbetarskapets delaktighet. Forskare menar att stödet förnärvarande ledare i vården har fram tills idag varit tämligen outvecklat. Syftetmed denna studie var att förstå framgångsfaktorer för hur kvalitetskulturen isvensk sjukvård kan kopplas mot ledarnas möjlighet att främja ett gottmedarbetarskap. Detta utifrån en förklarande sekventiell mixad metod med tvåkvantitativa mätningar som slutligen resulterade i en kvalitativ intervju. Enmätning av kvalitetskulturen vid svenska sjukhus utfördes utifrån ett tidigareframtaget mätinstrument för att mäta kvalitetskultur. Mätinstrumentetpresenterades genom 13 beteendepar som främjar respektive hindrar enkvalitetskultur. Denna mätning visade att det idag råder en generellt godkvalitetskultur vid svenska sjukhus. Genom denna mätning kunde enregressionsanalys utföras som kopplades samman mot sjukhusens resultat iNationell patientenkät. Ett statistiskt signifikant beteende kunde uppmätassom enligt denna mätning skapar nöjdare patienter ju mer medarbetarnaupplever att detta beteende förekommer i deras organisation. Beteendet ärnär vi har ett problem tar vi reda på grundorsaken innan vi beslutar om enlösning. Detta beteende togs med till två framgångsrika sjukhus för att djupareförstå hur dessa arbetar med medarbetarskapet i just detta beteende. Utifrånworkshop med dessa två sjukhus är slutsatsen att ledare behöver ha erfaritden kvalitetskultur och det medarbetarskap de ska bära för att främja ettmedarbetarskap som kopplats samman med kvalitetskulturen. I jakten på attnå framgång är en stark kvalitetskultur eftersträvansvärd men utifrånworkshoparna ser författarna ingen möjlig snabb lösning för att nå dit.Resultatet visar att dagens ledare i vården behöver stöd i form av mentor ellerreflektion kring ledarskap själv eller i grupp för att utvecklas. I konstruktiv andaär det med förbättringskunskap i grunden som allas delaktighet i arbetet medständiga förbättringar bedrivs. Detta kräver allas reflektion, ärlighet, mod,öppenhet och förtroende där allt grundar sig i att alla vill varandras väl!
Committed leadership is the foundation for creating a good quality culture andto succeed, the participation of employees is required. Researchers argue thatsupport for the current leaders in healthcare has until now been ratherundeveloped. The purpose of this study was to understand how the qualityculture in Swedish healthcare can be linked to the leaders' ability to promotegood co-workership. This study was based on an explanatory sequentialmixed method with two quantitative measurements that ultimately resulted in aqualitative interview. A measurement of the quality culture at Swedishhospitals was taken based on a previously developed instrument formeasuring quality culture . The measuring instrument comprises 13behavioral pairs that promote or hinder a quality culture. This measure showsthat there is generally a good quality culture at Swedish hospitals at present.Through this measurement, a regression analysis was done which links to thehospital's results in the National Patient Survey. A statistically significantbehavior was observed, and according to this measurement, is likely to createmore satisfied patients as more professions feel that this behavior occurs intheir organization. The behaviour in question can be described as: when wehave a problem, we find out the root cause before we decide on a solution.This behavior was brought into two successful hospitals in order to understandmore deeply how they work with the co-workership in this particular behavior.Based on the workshop with these two hospitals, we conclude that leadersneed to have experienced the quality culture and the co-workership they areassumed to carry in order to promote an employee culture that is linked to thequality culture. In pursuit of success, a strong quality culture is desirable, butbased on the workshops, the authors see no possible quick solution to reachit. The result shows that today's leaders in healthcare need support in the formof a mentor or time for reflection on leadership on their own or in groups inorder to develop. In constructive approach, it is with improvement knowledgethat everyone's involvement in the work of continuous improvement isconducted. This requires reflection, honesty, courage, openness and trustfrom everyone involved and intentions rooted in the wellness and prosperity ofall.

2019-06-27

Although nano-sized metal colloids are used in industrial and medicinal applications, little is known about the potential liver toxicity of these materials after occupational or intentional exposures. To begin to resolve some outstanding hepatotoxicity concerns, the inflammatory response of hepatocytes after exposure to metal colloids was assessed. Four ~30-nm-sized metal colloids, including silver (nano-Ag), copper (nano-Cu) and nickel (nano-Ni) were examined in an effort to understand the induced cytokine expression in a murine liver cell line (AML12). Here we also utilized another system, co-cultures of hepatocytes, Kupffer’s cells, and lymphocytes isolated from C57BL6 mice. Cells were exposed to the materials over dose-response (0.1mg/L to 1000mg/L) and time-dependent (4 h, 48 h, and 1-week) studies. Cytotoxicity was measured via metabolism of resazurin and validated via MTT assay and cell counts. Inflammatory response was determined by cytokine profiles (TNF-a and IL-6), as well as by mRNA and protein expression of heat shock protein (Hsp70). Results from cells exposed to nano-Ag to doses of up to 100mg/L exhibited no significant changes in cytotoxicity, IL-6, or TNF-a production, or Hsp70 expression. Both nano-Cu and nano-Ni exposed cells exhibited decreased metabolism, increased Hsp70 induction, and increased inflammatory responses (IL-6 and TNF-a). Dynamic light scattering and electron microscopy were used to characterize particle size and surface charge. All three metal colloidal systems demonstrated different particle size distributions, agglomerated sizes, and surface zeta potentials. Furthermore, each metal colloid system elicited different inflammatory biomarker responses and stress protein expression.

碩士
國立中興大學
生命科學系所
103
Biofuel production is using renewable biomass as substrate to produce fuels such as hydrogen,ethanol and butanol. Lignocellulose is the most abundant renewable feedstock to produce biofuel. Lignocellulose is mainly composed of cellulose, hemicelluloses and lignin. However, glucose-based is surrounded by hemicellulose, lignin and pectin, which make it hard to be decomposed. This study was aimed to establish a functional bacterial consortia as a lignocellulose digestion system for biofuel production through serial repeated batch culture. The major bacterial composition of batch culture were termite gut isolated Bacillus pumilus MGB 5 and Clostridium acetobutylicum ATCC 824. This system does not require sterilization, aeration and shake.On an eight day incubation period the system could produce 275 ml /L H2, ethanol 1250 mg/L, butanol 71 mg/L, acetate acid 2275 mg/L, butyrate acid 661 mg/L and left 64% cellulose and hemicellulose from mango powder biomass. The major bacteria in these system are Enterobacteriaceae, Enterococcus, Clostridium, Enterobacter, Bacillus. In the experiment , we found out if we use 3% mango powder as carbon source which can produce higher butanol than using 1% glucose as carbon source.This simple system is very potential to replace the traditional system which use glucose as carbon source to produce hydrogen and butanol.

碩士
長庚大學
生化與生醫工程研究所
101
In recent years , cellulosic ethanol is the one of renewable energy that can replace tranditional petrochemical fuel . Agricultural wastes contains large amounts of cellulose , hemicellulose , lignin and other components , cellulose and hemicelluloses can transform to glucose and xylose by hydrolyzing . Furthermore , it can produce bioethanol . The initial flask experiments , it shows that the ethanol yield is 3.18 g / L , the conversion rate was 62.4% when the rotention speed is 75 rpm , the ratio of Z. mobilis and P. stipitis is 1: 3 , and separated-immobilization of fermentation conditions . Then , we use rice straw as substrate , to discuss aeration rate, stirring speed and other factors , expect to get the best ethanol yield and metabolic rate . The experiment will be simultaneous saccharification and co-fermentation (SSCF) in reactor to investigate the high substrate concentrations and repeated batch . At present , the initial concentrate in addition to the 10 g / L rice straw , we also discuss 20 g / L and 30 g / L . The results show that the initial substrate concentration is higher , the lower the rate of ethanol conversion . And in the experiment ,we found that the substrate concentration of 30 g / L, the bottom of the aerobic zone caused by congestion due to rice straw powder , this problem will be used repeated batch culture to improve .In hydrolyzate discuss, we add 10 g/L rice straw as raw material which the medium after over-liming , the ethanol yield can be achieved 814 ppm. After then, the repeated-bacth fermentation medium

碩士
長庚大學
化工與材料工程學系
99
Swelling and disruption of Ca-alginate immobilization carrier by aeration and agitation are inevitable in the co-culture system that cause low production rate. In this study, we improved the carrier materials and develop a suitable gel matrix for the long-term immobilization culture of strains and established the optimal operating conditions for high-efficient ethanol production using a modified bioreactor. Our results showed that Ba-alginate (10% w/v) carrier had better enzyme activity than Ca-alginate carrier in the flask experiments using Carboxymethylcellulose (CMC). Under the optimal operation conditions of Ba-alginate 4%、aeration rate 1 vvm、agitation rate 150 rpm、2 day pre-culture, our modified co-culture cell bioreactor had ethanol production concentration of 778 ppm/10g-CMC at 8 hr with the theoretical maximum yield of 15.6%. Among the different pretreatment substrates of rice straw, the ethanol concentration was 600 ppm at 7 hr with the theoretical maximum yield of 24% and 1254 ppm at 29 hr with the theoretical maximum yield of 16.7% for 10 g/L and 30 g/L rice straw substrates, respectively. In a repeat-batch culture conditions with the total pretreatment rice straw concentration of 20 g/L, the total ethanol production can reached 1002 ppm at 34 hr, the yield for three batches were about 24%, 12%, 18%, respectively.

碩士
國立臺灣大學
藥學研究所
95
Nitric oxide (NO●) plays double-edged roles in human, not only important in physiological functions but also involved in many pathological pathways. Particularly, the overproduction of NO● generated by inducible nitric oxide synthase (iNOS) is associated with many neuronal disorders such as Parkinson’s disease, Alzheimer’s disease, and cerebral ischemic neuronal damage, etc. Arginine deiminase (rADI), expressed in some microorganisms but not in mammalian cells, can catalyze L-arginine (L-arg) to L-citrulline (L-cit). ADI has been reported the inhibitory activity of iNOS-mediated NO●production. A co-culture of BV2 (a murine microglial cell line) and SHSY5Y (a human neuroblastoma cell line) was established to understand the role of rADI in iNOS-mediated neurotoxicity. The co-culture was treated with the combination of 2 μg/mL of lipopolysaccharide (LPS) and 1 ng/mL of interferon-γ (IFN-γ) for 2 days, and successfully induced iNOS-mediated neuronal death. In the co-culture system, 1 mU/mL of purified recombinant ADI (rADI) was administrated for 2 and 3 days into the co-culture with LPS/IFN-γ treatment. The cell viability and NO● production were measured by MTT assay and Griess method, respectively. In addition, the specific neuronal viability and functionality were analyzed by neuron-specific immunostaining and 3H-dopamine uptake assay, respectively. The results showed that rADI treatment significantly preserved the cell viability (89.2±2.2 % of the initial cells) and decreased the NO● production (19.5±5.5 μM) on day 2, compared with the cells with LPS/IFN-γ treatment only (21.1±4.1 % and 67.0±1.3 μM, respectively) by MTT assay and Griess assay. Furthermore, the results of immunostaining showed that rADI treatment substantially preserved the neurons, and the dopamine uptake function was also maintained on day 2 (from 35.7±11.4% to 103.0±12.6 % of the initial cells) by rADI treatment. In addition, the neuroprotection of 3-day rADI treatment was observed better than 2-day treatment on day 4 by MTT assay (76.8±11.7 and 42.7±2.0 % of the initial cells, respectively), and dopamine uptake assay (75.1±10.8 and 34.5±10.5 % of the initial cells, respectively). To understand the possible neuroprotection mechanism of rADI treatment, replenishment of L-arg into the co-culture system with rADI treatment was conducted. Besides, the treatments of 1400W (a selective iNOS inhibitor) and vinyl-L-NIO (a selective neuronal NOS (nNOS) inhibitor) in the co-culture system were also performed. The results showed that the replenishment of L-arg abolished the neuroprotective and NO● suppressive effect of rADI in the co-culture system. In addition, the treatment 1400W preserved the cell viability (EC50=2.3 μM) and inhibited NO● production (IC50=5.7 μM), but vinyl-L-NIO did not. The results indicated that the neuroprotective mechanism of rADI is via L-arg depletion which inhibits the NO● production produced by iNOS, but not nNOS. According to the accumulative results, the conclusions are that rADI can protect the neurons from LPS/IFN-γ induced neurotoxicity in the co-culture system. rADI not only preserves the neuron viability but also maintains the functionality. The protection mechanism of rADI may be via depletion of L-arg and subsequently inhibits the iNOS mediated NO● production. To evaluate the therapeutic role of rADI in iNOS mediated neuronal disorders, further investigations in the neuroprotection of rADI in primary cell and ischemic mice model are needed in the future.

碩士
國防醫學院
生物及解剖學研究所
93
The type and amount of melanin is packaged into melanosomes in melanocyte then transferred to the neighboring 36 keratinocytes to modulate skin color. However, the actual processes involved in this transfer have not been defined clearly. Various methods have been used before to study the melanosomes transfer, for examples: (1) by using scanning electron microscopy to observe the melanosomes transfer；(2) by using protease inhibitor to verified melanosome transfer mechanism in vivo with histological examination；(3) labeling melanocytes with fluorescing dye (succinimidyl ester of carboxyfluorescein diacetate (CFDA)) or gold dextrin then co-cultured with keratinocytes to implicate melanosome transfer activity. The methods mention above are all complicated and time consuming. Therefore, establish a simple, rapid, direct and reliable method to examine specific transfer of melanosme from melanocyte to keratinocytes is very important for screening inhibitory compounds from Chinese herb to modulate skin color.In this study, a co-culture system of melanocytes and keratinocytes was established and the melanosome transfer activity was examined by triple labeling of immunofluorescence. The number of keratinocytes with melanosome after co-culture of keratinocytes and melanocytes with serine protease inhibitor AEBSF for 72 hours or with MSH for 24 hours was analysed by flow cytometry and confocal microscopy. The data indicated that the co-culture model system combined with triple labeling of immunofluorescence to observe the melanosomes transfer activity from melanocytes to keratinocytes was feasible.

碩士
國立成功大學
微生物暨免疫學研究所
95
Trichomonas vaginalis, a protozoan parasite of the urogenital -vaginal tract, is the causative agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in human. In male, the trichomoniais is usually asymptomatic, although it may cause irritating urethritis or prostatitis. In female, trichominiasis is associated with a wide spectrum of clinical signs ranging from a relatively asymptomatic state to severe vaginitis with a foul-smelling vaginal discharge. T. vaginalis may act as a potential catalyst in the acquisition of secondary infection such as that caused by human papillomavirus, the organism responsible for the pathogenesis of cervical cancer. The adherent clump of this protozoan will destruct the epithelial cell and induce pathogenesis by contact-dependent cytotoxicty. A co-culture system of T. vaginalis and human cervical epithelium cancer cell line (Z172 cell) has been established in this study. Both of the protozoan and host cell grow well in the same culture condition and atmosphere. When Z172 cell exposure under T. vaginalis attack, the morphology of the host cells become round shape, shrinkage, detach, and part of the cells are died. The single Z172 cell is easier attacked by T. vaginalis then colonial cells. The more co-culture time and the more adhesion rate were observed in this study. After 12 hours’ co-culture, the adhesion between protozoan and cell become the most significantly than other time points observation. Does the pathological changes of the Z172 cell are derived by the physical or chemical after the adhesion of T. vaginalis are needed to more studies in the future.

碩士
國立成功大學
微生物及免疫學研究所
95
Trichomonas vaginalis, a protozoan parasite of the urogenital -vaginal tract, is the causative agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in human. In male, the trichomoniais is usually asymptomatic, although it may cause irritating urethritis or prostatitis. In female, trichominiasis is associated with a wide spectrum of clinical signs ranging from a relatively asymptomatic state to severe vaginitis with a foul-smelling vaginal discharge. T. vaginalis may act as a potential catalyst in the acquisition of secondary infection such as that caused by human papillomavirus, the organism responsible for the pathogenesis of cervical cancer. The adherent clump of this protozoan will destruct the epithelial cell and induce pathogenesis by contact-dependent cytotoxicty. A co-culture system of T. vaginalis and human cervical epithelium cancer cell line (Z172 cell) has been established in this study. Both of the protozoan and host cell grow well in the same culture condition and atmosphere. When Z172 cell exposure under T. vaginalis attack, the morphology of the host cells become round shape, shrinkage, detach, and part of the cells are died. The single Z172 cell is easier attacked by T. vaginalis then colonial cells. The more co-culture time and the more adhesion rate were observed in this study. After 12 hours’ co-culture, the adhesion between protozoan and cell become the most significantly than other time points observation. Does the pathological changes of the Z172 cell are derived by the physical or chemical after the adhesion of T. vaginalis are needed to more studies in the future.

碩士
國立臺灣大學
醫學工程學研究所
101
In this study, we used mesenchymal stem cells (MSCs) 3A6, cancer cells SW620 and fibroblasts Hs68. First, we demonstrated that 3A6 is similar with MSCs by flow cytomerty analysis and differentiation assay. In addition, antigen markers were used to mark cancer stem cells. Chitosan is a natural, biodegradable, biocompatible, non-toxic and U.S. Food and Drug Administration (FDA) approved polysaccharide. In this study, chitosan was used as the coating substrates. When mesenchymal stem cells, cancer cells and fibroblasts were cultured on chitosan substrates, all cells became suspended and aggregated into spheroids. We used the antigens of transmembrane glycoprotein CD44 on the cell membrane to mark cells in order to compare the influence of different materials, and determine the causes of any discrepancies. Furthermore, we cultured MSCs, cancer cells, and fibroblasts on chitosan substrates in direct co-culture systems to explore the interactions between the three types of cells. We labeled these cells by the CellTrackerTM and used the inverted fluorescence microscope to observe the morphology and distribution of cells on substrates. In addition, we used confocal fluorescence microscope to check the arrangement of 3D spheroids. Besides, we used the antigens of transmembrane glycoprotein CD44 to mark cells in order to observe the effect of different substrates on the co-culture systems. Finally, we used the MTT assay, immunofluorescence staining Ki-67 and LIVE/DEADR Viability/Cytotoxicity Assay Kit to prove and discuss the status of cells on different positions of cell spheroids.

碩士
國立臺灣大學
口腔生物科學研究所
101
Periodontal disease is a chronic inflammatory reaction of periodontal tissues that potentially leads to tooth loss. It can affect daily activity, such as chewing and speech. Unfortunately, there is no effective therapy for periodontal disease. Use of tissue engineering technologies and stem cell therapy on Periodontal bone regeneration is a very promising treatment. Adipose-derived stem cell has been proved to its multipotency and have significant outcome in many disorders. Amniotic membrane is anti-inflammation, anti-angiogenic, low immunogenicity, and enriched with growth factor. We create a co-culture system with adipose-derived stem cell and amniotic membrane in periodontal osseous defect of rat model. The result suggest that the co-culture system could enhance periodontal bone regeneration. We believed this study could provide the base of the co-culture system as stem cell therapy and amniotic membrane transplantation in clinical periodontology.

The outcome of the innate immune response to biomaterials mainly determines whether the material will be incorporated in the body to fulfill its desired function or, when it gets encapsulated, will be rejected in the worst case. Macrophages are key players in this process, and their polarization state with either pro- (M1), anti-inflammatory (M2), or intermediate characteristics is crucial for deciding on the biomaterial’s fate. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates the proper healing and subsequent regeneration. Therefore, biomaterial-based polarization may aid in driving macrophages in the desired direction. However, the in vivo process is highly complex, and a mono-culture of macrophages in vitro displays only one part of the cellular system, but, to this date, there is a lack of established co-cultures to assess the immune response to biomaterials. Thus, this thesis aimed to establish a functional co-culture system of human macrophages and human mesenchymal stromal cells (hMSCs) to improve the assessment of the immune response to biomaterials in vitro. Together with macrophages, hMSCs are involved in tissue regeneration and inflammatory reactions and can modulate the immune response. In particular, endogenously derived hMSCs considerably contribute to the successful engrafting of biomaterials. This thesis focused on poly(ε-caprolactone) (PCL) fiber-based scaffolds produced by the technique of melt electrowriting (MEW) as biomaterial constructs. Via this fabrication technique, uniform, precisely ordered scaffolds varying in geometry and pore size have been created in-house. To determine the impact of scaffold geometries and pore sizes on macrophages, mono-cultures incubated on scaffolds were conducted. As a pre-requisite to achieve a functional co-culture system on scaffolds, setups for direct and indirect systems in 2D have initially been established. These setups were analyzed for the capability of cell-cell communication. In parallel, a co-culture medium suitable for both cell types was defined, prior to the establishment of a step-by-step procedure for the co-cultivation of human macrophages and hMSCs on fiber-based scaffolds. Regarding the scaffold morphologies tested within this thesis to improve M2-like polarization, box-shaped scaffolds outperformed triangular-, round- or disordered-shaped ones. Upon further investigation of scaffolds with box-shaped pores and precise inter-fiber spacing from 100 µm down to only 40 µm, decreasing pore sizes facilitated primary human macrophage elongation accompanied by their differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 µm. To the best of my knowledge, this was the first time that the elongation of human macrophages in a 3D environment has been correlated to their M2-like polarization. Thus, these results may set the stage for the design, the assessment, and the selection of new biomaterials, which can positively affect the tissue regeneration. The cell communication of both cell types, detected via mitochondria exchange in direct and indirect co-cultures systems, took place in both directions, i.e., from hMSCs to macrophages and vice versa. Thereby, in direct co-culture, tunneling nanotubes enabled the transfer from one cell type to the respective other, while in indirect co-culture, a non-directional transfer through extracellular vesicles (EVs) released into the medium seemed likely. Moreover, the phagocytic activity of macrophages after 2D co-cultivation and hence immunomodulation by hMSCs increased with the highest phagocytic rate after 48 h being most pronounced in direct co-cultivation. As the commonly used serum supplements for macrophages and hMSCs, i.e., human serum (hS) and fetal calf serum (FCS), respectively, failed to support the respective other cell type during prolonged cultivation, these sera were replaced by human platelet lysate (hPL), which has been proven to be the optimal supplement for the co-cultivation of human macrophages with hMSCs within this thesis. Thereby, the phenotype of both cell types, the distribution of both cell populations, the phagocytic activity of macrophages, and the gene expression profiles were maintained and comparable to the respective standard mono-culture conditions. This was even true when hPL was applied without the anticoagulant heparin in all cultures with macrophages, and therefore, heparin was omitted for further experiments comprising hPL and macrophages. Accordingly, a step-by-step operating procedure for the co-cultivation on fiber-based scaffolds has been established comprising the setup for 3D cultivation as well as the description of methods for the analysis of phenotypical and molecular changes upon contact with the biomaterial. The evaluation of the macrophage response depending on the cultivation with or without hMSCs and either on scaffolds or on plastic surfaces has been successfully achieved and confirmed the functionality of the suggested procedures. In conclusion, the functional co-culture system of human macrophages and hMSCs established here can now be employed to assess biomaterials in terms of the immune response in a more in vivo-related way. Moreover, specifically designed scaffolds used within the present thesis showed auspicious design criteria positively influencing the macrophage polarization towards the anti-inflammatory, pro-healing type and might be adaptable to other biomaterials in future approaches. Hence, follow-up experiments should focus on the evaluation of the co-culture outcome on promising scaffolds, and the suggested operating procedures should be adjusted to further kinds of biomaterials, such as cements or hydrogels
Der Verlauf der angeborene Immunantwort auf Biomaterialien bestimmt maßgebend, ob das Material vom Körper angenommen wird, um so seine gewünschte Funktion zu erfüllen, oder ob es zur Einkapselung und im schlimmsten Fall zur Abstoßung kommt. Makrophagen spielen in diesem Prozess eine Schlüsselrolle, und ihr Polarisationszustand, entweder pro (M1), antiinflammatorisch (M2) oder ein dazwischenliegender Subtyp, ist dabei von entscheidender Bedeutung. Während ein vorübergehender proinflammatorischer Anfangszustand hilfreich ist, verschlechtert eine anhaltende Entzündung eine zeitnahe Heilung und die anschließende Regeneration. Daher könnte eine durch Biomaterialien beeinflusste Polarisation hilfreich sein, um die Makrophagen in die gewünschte Richtung zu lenken. Die in vivo Reaktion ist jedoch äußerst komplex und die Kultivierung von Makrophagen in vitro stellt nur einen Teil des Prozesses dar. An etablierten Co-Kultursystem zur Untersuchung der immunmodulierenden Eigenschaften von Biomaterialien mangelt es jedoch. Daher war es Ziel dieser Arbeit ein funktionelles Co-Kultursystem von humanen Makrophagen und humanen mesenchymalen Stromazellen (hMSCs) zu etablieren um die in vitro Bewertung der Immunantwort nach Kontakt mit Biomaterialien zu verbessern. Von Interesse sind hMSCs hierbei, da sie zusammen mit Makrophagen an der Geweberegeneration und an Entzündungsreaktionen beteiligt sind. Zudem weisen MSCs immunmodulierende Eigenschaften in Hinblick auf Makrophagen auf und sind aktiv am Verlauf der Fremdkörperreaktion nach der Transplantation von Biomaterial beteiligt. Im Rahmen dieser Arbeit wurden Poly(ε-caprolactone) (PCL)-Scaffolds auf Faserbasis als Biomaterialkonstrukte verwendet, welche mit der Technik des Melt Electrowriting (MEW) hergestellt wurden. Mit dieser Technik kann sowohl die Form der Scaffolds als auch die Porengröße variiert werden. Um Unterschiede der Scaffoldgeometrien und Porengrößen in Hinblick auf die Makrophagenreaktion zu untersuchen, wurden zunächst Versuche mit Makrophagen-Monokulturen durchgeführt. Zur Etablierung eines funktionellen Co-Kultursystem, wurde zu Beginn ein Aufbau für ein direktes und indirektes System in 2D erstellt. Dieser Aufbau wurde anschließend auf die Möglichkeit der Zell-Zell-Kommunikation darin analysiert. Weiterhin wurde ein, für beide Zelltypen, geeignetes Kulturmedium definiert, gefolgt von der Etablierung eines Protokoll für die Co-Kultivierung beider Zelltypen auf faserbasierten Scaffolds. Im Bezug zu dieser Arbeit wurden Scaffolds mit unterschiedlicher Geometrie mittels der Technik des Melt Electrowriting hergestellt um die Veränderung der Makrophagenpolarisation zu untersuchen. Dabei zeigte sich eine verstärkte M2-Polarisation auf Scaffolds mit einer kastenförmigen Morphologie, verglichen mit dreieckigen, runden oder ungeordnet-strukturierten Scaffolds. Die weitere Untersuchung von Scaffolds mit kastenförmigen Poren und präzisen Faserabständen von 100 µm bis zu 40 µm zeigte das kleinere Porengrößen die Elongation primärer menschlicher Makrophagen förderten. Begleitet wurde die verstärkte Elongation mit einer gesteigerten Polarisation in Richtung des M2 Typs. Dieser Effekt war nach Kultivierung von Makrophagen auf Scaffolds mit 40 µm Poren am stärksten ausgeprägt. Im Rahmen dieser Arbeit konnte damit erstmals eine länglichen Morphologie humaner Makrophagen mit einer Polarisierung in den M2 Typ korreliert werden. Diese Ergebnisse könnten daher für das Design neuer Biomaterialien, welche sich positiv auf die Geweberegeneration auswirken sollen, von Bedeutung sein. Die Zellkommunikation beider Zelltypen, welche über Mitochondrienaustausch im direkten und indirekten Co-Kultur-System nachgewiesen wurde, fand sowohl ausgehend von Makrophagen als auch von hMSCs statt. Dabei ermöglichten „Tunneling Nanotubes“ in der direkten Co-Kultur den Transfer von Mitochondrien von einem Zelltyp zum jeweils anderen, während in der indirekter Co-Kultur ein ungerichteter Transfer durch in das Medium freigesetzte extrazelluläre Vesikel (EVs) stattfand. Darüber hinaus wurde die phagozytotische Aktivität von Makrophagen nach Co-Kultivierung untersucht, um die immunmodulatorischen Eigenschaften von hMSCs nachzuweisen, wobei die höchste phagozytotische Aktivität nach 48 stündiger Co-Kultivierung festgestellt wurde. Da die üblicherweise verwendeten Serumzusätze für Makrophagen (humanes Serum (hS)) und hMSCs (fötales Kälberserum (FCS)) bei längerer Kultivierung den jeweils anderen Zelltyp nicht unterstützen konnten, wurden diese Seren durch humanes Thrombozytenlysat (hPL) ersetzt. Dieses erwies sich im Rahmen dieser Arbeit als optimale Ergänzung für die gemeinsame Kultivierung beider Zelltypen in der Co-Kultur. Dabei wurden der Phänotyp und die Populationsverteilung beider Zelltypen, sowie die phagozytotische Aktivität und die Veränderung des Genexpressionsprofils von Makrophagen untersucht und mit den jeweiligen Standard-Monokulturbedingungen verglichen. Des Weiteren konnte gezeigt werde, dass eine Zugabe von Heparin in Zellkulturen mit Makrophagen und hPL nicht nötig ist. Daher wurde auf den Zusatz von Heparin für alle weitere Experimente, die hPL und Makrophagen umfassten, verzichtet. Im letzten Teil der Arbeit wurde ein Protokoll für die Co-Kultivierung auf MEW Scaffolds erstellt. Neben der Etablierung eines Setups für die 3D-Kultivierung wurden sowohl Protokolle zur Bewertung phänotypischer als auch molekularer Veränderungen entwickelt. Durch Feststellung von Unterschieden in der Makrophagenreaktion in Abhängigkeit zu der Kultivierung mit / ohne hMSCs und entweder auf Scaffolds oder Plastik-Kulturschalen konnte die Funktionalität der Protokolle nachgewiesen werden. Mit dem in dieser Arbeit etabliertem funktionellen Co-Kultursystem von humanen Makrophagen und hMSCs können zukünftig Biomaterialien mit einem stärkeren in vivo -Bezug in Hinblick auf die Immunantwort bewertet werden. Darüber hinaus deuten Ergebnisse auf speziell konstruierte MEW-Scaffolds ein vielversprechendes Designkriterium für neu entwickelte Biomaterialien an, wobei die Polarisation der Makrophagen in Richtung des entzündungshemmenden, heilungsfördernden Typens durch eine gesteuerte Morphologieänderung beeinflusst werden kann. An diese Arbeit anschließende Experimente sollten sich auf die Untersuchung vielversprechender Scaffolds mittels Co-Kultivierung sowie auf die Anpassung der etablierten Protokolle an andere Biomaterialgruppen, wie beispielsweiße für Zemente oder Hydrogele, konzentrieren

碩士
長庚大學
生化與生醫工程研究所
102
In this study, we developed a novel bioreactor that simultaneously enhanced the saccharification and fermentation process for bioethanol production. Initially, A. niger and T. reesei were co-immobilized on PU (polyurethane) carrier, and co-cultured in our bioreactor by using carboxymethylcellulose (CMC) as substrate, then Z. mobilis alginate beads were added into this bioreactor together to overcome the problem of simultaneous saccharification and bioethanol fermentation. By using pentose or hexose as subsdtrate, the effects of immobilization speed and inoculum ratio of Z. mobilis and P. stipitis on alcohol production in suspension cultivation were studied. The immobilization results showed that alcohol concentration was 2200 ppm when two strains co-immobilized, while alcohol concentration was 2485 ppm when two strains separately-immobilized. Z. mobilis and P. stipitis were competitive when they were co-immobilized in the same beads. The optimum speed results showed that higher speed had less beneficial effects on ethanol production when using Z. mobilis mono-cultivation fermentation. The higher speed had positive effects on ethanol production when using P.stipitis mono-cultivation fermentation .The inoculum ratio results showed that co-cultivation of Z:P=1:1 was better than mono-cultivation of Z:P=1:0. Our results also showed that the yield of the bioethanol could reach 737 ppm at 36 hours, when PU soaked with 10 g/L CMC in this co-culture system. The further results showed that the maximum ethanol concentration increased to 937 ppm when the CMC concentration was increased from 10 to 15 g/L.

碩士
長庚大學
化工與材料工程研究所
95
Environmental pollution and agricultural wastes have spread all over the world in recent years so we, in order to do effective use of the offal. The purpose for study potapo starch to regard as substrate material and then utilile Aspergillus awamori and Rhizopus Japonicus of fungers to co-culture.It will make starch to come into being glucose after saccharifiction.Afterward, we use efficiency better saccharomycete- Saccharomyces cerevisiae IR-2 to research .Add S. cerevisiae IR-2 into the fungous co-suspension system in the best saccharification time (most reduce sugar), and let the system to become three strains of microorganisms co-suspension system.To find the suitable condition for culture the three strains of microorganisms system to go on different proportion and the amount of spores to addtion. At the same time,the system can avoid co-suspension during the saccharification process that accumulate the large amount reduce sugar make the damage of feedback inhibition,the saccharification and ferment processes can go on at the same moment. In the study,the best proportion to add A. awamor and R. japonicus is 7.5: 2.5,the large amount reduce sugar in the 28th hour and we get 2.152 mg/ml reduce sugar ,so we will go on next experiment that we find out the best addtion amount is 109 spores/ml to receive the concentration of best addtion by the experimental result. Follow-up the experiment , in which the best concentration for addtion is 0.1 x108 cell/ml on 12hr hour. It can get 0.57% of ethanol concentration. Transfer rotational speed for 100 rpm on 36th hour can up to 1.25%