タンデトロン加速器質量分析計業績報告

The presence of many human pharmaceuticals in the aquatic environment is now a worldwide concern and yet little is known of the chronic effects that these bioactive substances may be having on aquatic organisms. This study used mammalian pharmacodynamics to predict the mode of action of the 13-blocker, propranolol, on fish, in order to identify chronic effects in fathead minnows. β-blockers target β1- and β2-adrenergic receptors in humans and hence these receptors were characterised in the fathead minnow. It was found that fish possess β1- and β2-ARs that are structurally very similar to their mammalian counterparts. Further, the distributions of these two β-ARs in various organs of the fathead minnow were similar to those in mammals. Pair-breeding assays were conducted, in which fathead minnows were exposed to various concentrations of propranolol. To discover whether β-ARs had been up or down regulated by propranolol, molecular analysis was conducted on different tissues of the exposed fish using real-time polymerase-chain reactions (RT-PCR). Exposure of fathead minnows to propranolol caused acute toxicity at 10 mg/L. Propranolol caused a statistically significant decrease in reproduction at 1.0 mg/L, dose-related decreases in male weight, condition index and fatpad weight, and a dose-related increase in female GSI. Molecular analysis of βl- and β2-AR expression levels revealed a dose-related decrease in β2-AR expression in fathead liver and heart. LOEC and NOEC values were 0.1 mg/L and 0.01 mg/L, respectively. Propranolol plasma concentrations in fish exposed to water concentrations of 0.1 and 1.0 mg/L were greater than the human therapeutic concentration and hence these data very strongly support the fish plasma model proposed by Huggett et al. (2001).

The effects of three oral dosages (0.4 mg, 0.8 mg, and 1.6 mg) of quercetin on Colon-25 tumors implanted in Balb-c mice were studied. The data in this study show that: (1) certain dosages of quercetin in alcohol solutions, reduces the weight, and size of implanted Colon-25 tumors in Balb-c mice, (2) these same dosages of quercetin all produce a profound neutrophilia combined with a significant lymphopenia at day 20 post-implantation, and (3) there was relatively little evidence of histological changes in the quercetin-treated tumor section which would indicate that the action(s) of quercetin is primarily at the subcellular level probably within the nuclei of the tumor cells.

The bio-manufacturing of recombinant proteins from mammalian cell cultures requires robust processes that can maximize protein yield while ensuring the efficacy of these proteins as human therapeutics. Recognizing that the challenge of improving protein yield and quality can be met through various approaches, this paper presents three strategies currently being developed in our group. A method for rapidly selecting subpopulations of cells with high production characteristics is proposed. This method combines the efficiency of green fluorescent protein/fluorescence-activated cell sorting (GFP/FACS)–based screening with homologous recombination to generate and select high-producing subclones. Next, the development of chemically defined, protein-free media for enhancing monoclonal antibody production is described. Analysis of culture media effects on the genome-wide transcriptional program of the cell is presented as a means to optimize the culture media and identify potential targets for genetic manipulation. Finally, we propose a method for increasing the extent of intracellular sialylation by improving the transport of CMP-sialic acid into the trans-Golgi. This is hypothesized to increase the sialic acid availability, and may enhance the degree of sialylation in the glycoprotein product. / Singapore-MIT Alliance (SMA)

Thesis (M.S.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Cell culture media used in the manufacture of biopharmaceuticals conventionally contain many animal derived components. These components can harbor adventitious agents which can be transmitted through biotherapeutics, employed in the medical treatment of immunocompromised patients. An ADCF (animal derived component free) medium formulation obviates this concern. A rational method for the rapid and efficient screening and optimization of ADCF media while preserving, if not enhancing, cellular growth and protein productivity is needed. CHO (Chinese Hamster Ovary) cells, widely used as a production platform in industry, expressing a recombinant protein, were employed as a model system. Design of Experiment (DOE) and statistical analysis were employed to assess the impact of media formulation on cellular physiology. Metabolic flux, cellular growth, and protein productivity were evaluated as the measures of ADCF media formulation success. Measurements of extracellular metabolites were determined by HPLC and enzymatic methods. Recombinant protein production was measured by HPLC. This research demonstrates the successful screening and optimization of four plant hydrolysate mixtures (2 soy and 2 wheat) as a replacement for animal derived components. / NSERC, ABIN, MABNET

Olfactory ensheathing cells (OECs) are the chief glial population of the mammalian olfactory nervous system and are thought to be responsible for the successful directional growth of new olfactory axons throughout the life of adult mammals. Due to this unique property, OECs have been targeted as a potential cellular transplantation therapy for spinal cord injury. In order to effectively isolate OECs for intraspinal transplantation, more knowledge must be gained on their phenotypic properties. We investigated the neurochemical features of OECs in a variety of mammalian species (including hamsters, rabbits, monkeys, mice, and pigs) using three biomarkers: glial fibrillary acidic protein (GFAP), S100β, and α-smooth muscle actin (αSMA). In addition, we tested the ability of a few biomatrices to sustain and promote OEC growth and survival in vitro. The rationale for using biomatrices is to provide a supportive environment for glial and axonal growth in the spinal lesion. Here, we found that mucosal and bulbar OECs from all five of the aforementioned mammalian species express S100β. Expression of GFAP, however, was not consistent across the five species. Both mucosal and bulbar OECs of monkeys express αSMA; only bulbar OECs of hamsters and only mucosal OECs of rabbits express αSMA as well. Though αSMA immunostaining was not detected in the OECs of adult mice, in adult mutant mice lacking αSMA expression, OECs displayed perturbed ultrastructural morphology. None of the biomatrices used (methacrylated glycol chitosan, arginine-glycine-aspartic acid – grafted methacrylated glycol chitosan, and agarose) were able to promote OEC proliferation. Isolated strips of rodent olfactory lamina propria (the deep connective tissue layer in the olfactory mucosa containing primary sensory axons and OECs) showed sustained growth when cultured for 10 days. In sum, these findings highlight the following points: the efficacy of S100β and αSMA as biomarkers for mammalian OECs in vivo; the potential for isolated strips of lamina propria to provide a natural, supportive environment for OECs during intraspinal transplantation; the failure of methacrylated glycol chitosan and its derivatives, as well as agarose, to promote OEC proliferation. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-07-27 15:29:47.642

No description available.

Recently a whole cell based biosensor has been developed in our laboratory that consists of a monolayer of human umbilical vein endothelial cells (HUVECs) on the asymmetric cellulose triacetate (CTA) membrane of an ion selective electrode (ISE). When a confluent cell monolayer is formed across the membrane, response from the sensor is inhibited due to inhibited ion transport across the membrane. When the cell based biosensor is exposed to permeability modifying agents, the permeability across the cell monolayer is altered facilitating more ion transport and as a result the response from the sensor increases. This sensor response can be related to the concentration of these agents. One objective of this research was to investigate the ability of the sensor to detect a physiological toxin, alpha toxin from Staphylococcus aureus. Studies demonstrated that the biosensor can detect 0.1ng/ml alpha toxin. Considering the fact that the concentration of this toxin is 100-250 ng/ml in whole blood in humans, this biosensor has the ability to act as the diagnostic tool for staphylococcal diseases. Another part of this research was to investigate the ability of the biosensor to measure angiogenesis by measuring the changes in permeability induced by cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and tumor necrosis factor andamp;aacute; (TNF- andamp;aacute;) individually and in combination. The sensor response was then compared with the common in vitro assays for angiogenesis. The study demonstrated that at the concentrations studied the sensor response in the presence of cytokines was much higher than that observed for other angiogenesis assays, thereby bolstering the potential of the biosensor to act as a quick screening tool for angiogenesis. Furthermore, epithelial cell based sensor responses to the same cytokines were compared with the responses from endothelial cell based sensor and the mechanisms behind the increased sensor response were elucidated. Finally, to investigate the ability of the sensor to screen cancer, the biosensor was exposed to the serum from healthy individuals and cancer patients. The results showed that the sensor can distinguish between healthy individuals and cancer patients and the results correlate with the stages of cancer.

Perturbations of the complex intrinsic and extrinsic factors that contribute to cellular differentiation can have many consequences ranging from dedifferentiation to cell death. The overall objective of my research is to investigate the factors that contribute to the maintenance of mature midbrain neurons and glia. In order to address this objective, I first carried out a detailed immunocytochemical analysis to demonstrate that histone deacetylase inhibitor (HDACI) treatment of differentiated midbrain neurons in culture results in an overall destabilization of neuronal phenotype, which leads to caspase-independent cell death. GFAP positive astrocytes are refractory to the effects of HDACI treatment, suggesting that inhibition of HDACs has differential effects on neurons and glia. HDACI treatment alone was not sufficient to induce neuronal dedifferentiation as evidenced by RT-PCR analysis of stem/progenitor markers, and recovery experiments. Finally, I demonstrate that cortical neurons do not undergo cell death in response to HDACI treatment, suggesting that there may be microenvironmental factors that promote the susceptibility of midbrain neurons to the neurotoxic effects of HDACI. In the second part of this thesis I determined the molecular mechanism that was at least partly responsible for the effects of HDACI treatment on midbrain neurons. Gene expression profiling of HDACI treated midbrain cultures revealed a strong down-regulation of immune related factors. This observation is supported by the loss of microglia in HDACI treated midbrain cultures. I also provide evidence that Toll-like receptor (TLR) signaling, likely through the activation of Interleukin-6 (IL-6) expression, mediates HDAC-dependent neuronal survival. These data provide new evidence that the neuroimmune system is an extrinsic regulator for the homeostasis and survival of neurons.