Wong Pak Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 86-88). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Dedication --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.ix / List of Appendices --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xx / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Ginseng and Ginsenosides --- p.1 / Chapter 1.2 --- Instrumental Analysis of Ginsenosides --- p.6 / Chapter 1.2.1 --- Thin Layer Chromatography --- p.6 / Chapter 1.2.2 --- Infrared Spectroscopy --- p.7 / Chapter 1.2.3 --- Colorimetry --- p.7 / Chapter 1.2.4 --- Gas Chromatography --- p.7 / Chapter 1.2.5 --- High Performance Liquid Chromatography --- p.8 / Chapter 1.3 --- Objective of the Study --- p.9 / Chapter Chapter 2: --- Experimental --- p.13 / Chapter 2.1 --- History of Electrophoresis and Capillary Electrophoresis --- p.13 / Chapter 2.1.1 --- Electroosmotic Flow (EOF) --- p.14 / Chapter 2.1.2 --- Electrophoretic Migration --- p.18 / Chapter 2.2 --- Reagents and Materials --- p.20 / Chapter 2.2.1 --- Reagents and Glassware --- p.20 / Chapter 2.2.2 --- Instrumentation --- p.20 / Chapter 2.2.3 --- Preparation of Solutions and Wavelength Selection --- p.22 / Chapter 2.2 --- Procedures --- p.23 / Chapter Chapter 3: --- Results and Discussions --- p.24 / Chapter 3.1 --- Initial Selection of the Running Electrolyte --- p.24 / Chapter 3.2 --- Inclusion Additives in the Aqueous Buffer Solution --- p.29 / Chapter 3.2.1 --- Reasons for Addition of Buffer Additives --- p.29 / Chapter 3.2.1.1 --- Cyclodextrin --- p.29 / Chapter 3.3 --- Addition of Surfactants --- p.33 / Chapter 3.3.1 --- Sodium Dodecyl Sulfate (SDS) --- p.35 / Chapter 3.3.2 --- Sodium Cholate --- p.41 / Chapter 3.4 --- Addition of Organic Modifier --- p.43 / Chapter 3.5 --- Effect of pH --- p.46 / Chapter 3.6 --- Effect of the Concentration of the Borate/Phosphate Solution --- p.51 / Chapter 3.7 --- Effect of Capillaries with Different Inner Diameters (I.D.) --- p.54 / Chapter 3.7.1 --- Effect of pH --- p.54 / Chapter 3.7.2 --- Effect of the Buffer Concentration --- p.60 / Chapter 3.7.3 --- Comparison of Migration Time between Capillaries of 50μm and 75μm Inner Diameter --- p.62 / Chapter 3.8 --- Optimization of Other Experimental Parameters --- p.66 / Chapter 3.8.1 --- Applied Voltage --- p.66 / Chapter 3.8.2 --- The Time of Injection --- p.68 / Chapter 3.8.3 --- The Operating Temperature --- p.70 / Chapter 3.9 --- Intra-day and Inter-day Reproducibility --- p.72 / Chapter 3.10 --- Quantitative Analysis of the Ginsenosides --- p.74 / Chapter 3.11 --- Application of the Developed Methodology --- p.78 / Chapter 3.11.1 --- Experimental Procedures --- p.79 / Chapter Chapter 4: --- Conclusion --- p.83 / References --- p.86 / Appendices --- p.89

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 106-113).

Mollicutes (mycoplasmas) are pathogenic in a wide range of mammals (including humans), reptiles, fish, arthropods, and plants. Of the medically important mollicutes, Mycoplasma gallisepticum is of particular relevance to avian agriculture and veterinary science, causing chronic respiratory disease in poultry and turkey. Using two-dimensional electrophoresis based quantitative expression proteomics, the current study investigated the molecular mechanisms behind the phenotypic variability between a M. gallisepticum vaccine strain (6/85) and a competitive, virulent field strain (K5234), two strains which were indistinguishable using commonly accepted genetic methods of identification. Twenty-nine proteins showed a significant variation in abundance (fold change > 1.5, p-value < 0.01). Among others, the levels of putative virulence determinants were increased in the virulent K5234, while the levels of several proteins involved with pyruvate metabolism were decreased. It is hoped that the data generated will further the understanding of M. gallisepticum virulence determinants and mechanisms of infection, and that this may contribute to the optimization of diagnostic methodologies and control strategies.

Capillary electrophoresis chip has gradually ripe along with the development of MEMS technology. However, such these electrophoresis chips was design closed-channel form whose process including the micro-channel forming and chip bonding and so on, so the cost is higher. In addition, if these chips use repeated will cause some pollution problems such as obstruction or difficult to clean in the closed-channel. Therefore, to fabricate a non-closed microfluidic chip system will resolve the issues above listed. In this study has successful developed a convenient and low-cost thread microfluidic system, the thin polyester thread is instead of the traditional closed separation channel. And to avoid the cross contaminations that the separation channel can free replace a new electrophoresis separation channel by the roller equipment. Thread microfluidic systems can take advantage of capillary action to move, as a disposable chromatography flow channel, and the electric field is applied to this system for electrophoresis separation and electrochemical detection in the backend. This research develop an novel process technology, the hot embossing technology shape from concave embossing and metal coating procedure in PMMA, the salient pillow-electrode structure has be produced in PMMA board, the salient electrode structure set up the polyester fiber thread, the polyester fiber thread is as the electrophoresis separation channel, and electrochemical detect samples in back-end. In this study take plasma treatment to improve wettability and surface roughness of the polyester fiber thread, in order to improve the operational effectiveness of the thread microfluidic systems. The cyclic voltammetry measure potassium ferricyanide samples and the results showed that the performance of thread microfluidic system significantly increase after the plasma treatment, the measuring current value is 10 times greater than without the plasma treatment, and the estimated detection limit of potassium ferricyanide is around 6.25 £gM in the plasma treatment one. In addition, the thread microfluidic devices with plasma treatment has successful separation and detection the mixing samples of 0.3 mM chlorine, bromine and iodine ions, and the signal of the S/N ratio is 6 times higher than the without plasma treatment one, and the number of theoretical plates of electrophoresis separation also enhance to 28% in the plasma treatment one. In addition, in order to further enhance the detection sensitivity of the thread microfluidic systems, the study design and fabricate the concave-shaped three-dimensional electrode structure, and to achieve greater contact area between thread and the electrode. Thread microfluidic system with plasma treatment detect in concave-shaped three-dimensional electrode and flat electrode respectively, the results showed that concave-shaped electrodes in the potassium ferricyanide detection limit that measured current value is 10 times greater than flat electrode, and a mixture of dopamine and catechol sample are electrophoresis separated that concave-shaped 3D electrode whose measured S/N ratio is 5 times higher than flat electrode, and the number of theoretical plates is 1.5 times higher than the flat electrode. This study develops a novel thread microfluidic system that will provide a simple process and low-cost, and can solve the shortcomings of conventional closed-channel electrophoresis chips. In future, in this study develop the thread microfluidic system architecture will have contribute for fast electrophoresis separation and detection.

Capillary electrophoresis (CE) is an excellent analytical separation method with promising features such as small sample volumes (µL to pL), fast analysis times (s), high selectivity and efficiency, and excellent compatibility with biological samples. However, the inability of conventional CE detectors to sense biologically active compounds that are optically and electrochemically inactive limits their use for biosensing and drug screening. We have developed a highly stable electrophysiological detection platform consisting of ion channel (IC) reconstituted in synthetic bilayer membrane also known as black lipid membranes (BLM) suspended across a functionalized microaperture to be coupled to a high resolution capillary separation channel. Low energy surface modifiers were used to drastically improve the electrical, mechanical, and temporal stability of BLMs. Glass microapertures modified using tridecafluoro 1, 1, 2, 2-tetrahydrodimethylchlorosilane facilitated the rapid formation of highly stable BLMs due to the amphiphobic property (H₂O/oil repellency). Furthermore, a combination of chemically modified aperture surfaces and chemical cross-linking within the lipid membrane were used to dramatically improve BLM stability. Partial cross-linking within the bilayer maintained fluidity which allowed reconstitution of ion channel proteins while maintaining the stability of BLM-IC platform. The stable BLM-IC across glass pipette aperture was coupled to microchip electrophoresis and was shown to withstand field strength (>250 V/cm) from separation channel. Additionally, planar microapertures fabricated in SU8 were used for the formation of stable BLM-IC platform towards the construction of an integrated device. The chemical properties of the SU8 supported the formation and cross-linking within polymerizable lipid or lipid bilayer doped with polymerizable methacrylate monomers. Additionally, we expressed ion channel coupled receptor fusion protein in HEK 293 cells towards the development of ion channel sensors for wide range of ligand detection in BLM sensor platforms. The pharmacology of IC functionalized with muscarinic acetyl choline (M2-K) receptor using cell based assay by patch clamp electrophysiology showed activation by acetylcholine and inhibition by atropine. Thus this platform holds a great promise as the next-generation integrated analysis system for rapid screening of biologically active compounds (eg. glucagon) in complex matrix such as whole blood and urine for the diagnosis and management of chronic disease such as diabetes.

Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.

Orientadora: Marileda Bonafim Carvalho / Banca: Julieta Rodini Engracia Moraes / Banca: Aguemi Kohayagawa / Resumo: Para este estudo foram avaliados 20 cães adultos sadios (10 machos e 10 fêmeas) e 24 cães adultos (16 machos e 8 fêmeas) com insuficiência renal crônica (IRC), com o objetivo de estudar a proteinúria. Foram empregadas técnicas para determinação quantitativa de proteína da urina e para a separação de suas frações por meio de SDS-PAGE. As quantidades de proteínas presentes na urina de cães sadios foram pequenas, mas o estudo dos perfis eletroforéticos mostrou haver diferenças entre machos e fêmeas. Apesar da ampla variação das concentrações de proteínas entre os animais, o grupo dos portadores de IRC apresentou proteinúria intensa. Os resultados dos portadores de IRC foram agrupados de acordo com o sexo ou em função do tipo de lesão renal predominante, para comparação dos eletroforetogramas. As análises revelaram que os perfis eletroforéticos seguem um padrão para os machos e outro pra as fêmeas. Ainda, o padrão o perfil eletroforético observado quando há predomínio de lesões glomerulares difere do observado quando há predomínio de lesões túbulo-intersticiais. / Abstract: This trial was conducted in 20 adult health dogs (10 male and 10 female) and in 24 adult dogs (16 male and 8 female) in chronic renal failure (CRF), in order to study urinary protein loss. Determination of total urinary proteins and their separation with SDS-PAGE were employed. The results show that CRF dogs had higher proteinuria when compared to controls and the amount of detected protein had large difference among them. In the CRF there are different patterns of protein bands occurrence and distribution according to the patient sex and predominant renal lesion. The difference between protein bands occurrence of male and female already exists in normal dogs. / Mestre

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2001-01-16Bitstream added on 2014-06-13T19:15:42Z : No. of bitstreams: 1 toledo_egh_me_jabo.pdf: 1480955 bytes, checksum: 264147d52e4fdcb0c91216e8ebe11eca (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Para este estudo foram avaliados 20 cães adultos sadios (10 machos e 10 fêmeas) e 24 cães adultos (16 machos e 8 fêmeas) com insuficiência renal crônica (IRC), com o objetivo de estudar a proteinúria. Foram empregadas técnicas para determinação quantitativa de proteína da urina e para a separação de suas frações por meio de SDS-PAGE. As quantidades de proteínas presentes na urina de cães sadios foram pequenas, mas o estudo dos perfis eletroforéticos mostrou haver diferenças entre machos e fêmeas. Apesar da ampla variação das concentrações de proteínas entre os animais, o grupo dos portadores de IRC apresentou proteinúria intensa. Os resultados dos portadores de IRC foram agrupados de acordo com o sexo ou em função do tipo de lesão renal predominante, para comparação dos eletroforetogramas. As análises revelaram que os perfis eletroforéticos seguem um padrão para os machos e outro pra as fêmeas. Ainda, o padrão o perfil eletroforético observado quando há predomínio de lesões glomerulares difere do observado quando há predomínio de lesões túbulo-intersticiais. / This trial was conducted in 20 adult health dogs (10 male and 10 female) and in 24 adult dogs (16 male and 8 female) in chronic renal failure (CRF), in order to study urinary protein loss. Determination of total urinary proteins and their separation with SDS-PAGE were employed. The results show that CRF dogs had higher proteinuria when compared to controls and the amount of detected protein had large difference among them. In the CRF there are different patterns of protein bands occurrence and distribution according to the patient sex and predominant renal lesion. The difference between protein bands occurrence of male and female already exists in normal dogs.

Au cours de cette thèse, nous avons été amenés à développer des méthodes analytiques afin de contrôler la qualité de protéines thérapeutiques en tant que produits finis ou d'évaluer le potentiel de certaines protéines à être des candidats médicaments. Deux protéines ont été étudiées : l'albumine de sérum humain (HSA) et l'antithrombine (AT). Ces protéines diffèrent par leur structure, leur glycosylation et leurs activités biologiques. Une méthode d'électrophorèse capillaire de zone (CZE) a permis de séparer neuf formes dans des préparations commerciales d'HSA plasmatique, dont des formes native, cystéinylée, glyquées et tronquées. Une autre méthode CZE a permis de séparer plus de huit formes dans des préparations commerciales d'AT plasmatique, dont des formes native, latente et hétérodimérique. Les deux méthodes CZE développées ont permis de mettre en évidence des différences significatives quant à la composition de préparations commerciales d'HSA ou d'AT produites par cinq fournisseurs différents. Des méthodes d'électrophorèse capillaire couplée à la spectrométrie de masse (CE-MS), avec un analyseur quadripôle-temps de vol (Q-TOF) et une ionisation électrospray (ESI), ont également été développées. Ces méthodes CE-MS ont permis d'identifier non seulement de nombreuses glycoformes et formes apparentées de l'AT, mais également des formes dimériques. Une méthode CE-MS en conditions non-dénaturantes a permis de montrer que la forme native et la forme latente de l'AT (deux conformères) présentaient un spectre de masse spécifique, permettant de les différencier sans ambiguïté. Afin d'évaluer l'affinité de variants d'AT pour l'héparine, une méthode d'électrophorèse capillaire d'affinité (ACE) a été développée. Cette méthode ACE a permis d'analyser les variants d'AT directement à partir des surnageants de culture cellulaire dans lesquels ils sont produits. Ainsi, il a été montré que certains variants présentaient une affinité très élevée pour l'héparine, en accord avec les mutations réalisées. Enfin, dans le cadre des études d'affinité entre l'AT et l'héparine, nous avons exploré une méthode nouvelle basée sur l'analyse de la dispersion de Taylor (TDA). / During this Ph.D., we developed analytical methods in order to check the quality of therapeutic proteins as finished products or to assess the potential of some proteins to be drug candidates. Two proteins were studied : human serum albumin (HSA) and antithrombin (AT). These proteins are very different regarding their structure, glycosylation, and biological functions. A capillary zone electrophoresis (CZE) method enabled to separate nine forms in commercial preparations of HSA issued from human plasma, among which native, cysteinylated, glycated, and truncated forms. Another CZE method allowed separating more than eight forms in commercial preparations of AT issued from human plasma, among which native, latent, and heterodimeric forms. Both CZE methods enabled to highlight significant differences in the composition of marketed preparations produced by five competitive pharmaceutical companies. Capillary electrophoresis-mass spectrometry (CE-MS) methods, using a quadrupole-time of flight (Q-TOF) analyzer and electrospray ionization (ESI), were also developed. These CE-MS methods enabled to identify not only a high number of glycoforms and related forms of AT, but also dimeric forms. A CE-MS method developed in non-denaturing conditions showed that native and latent forms of AT (two conformers) exhibited a specific mass spectrum, allowing to unambiguously distinguish them. With the aim to assess the binding affinity of AT variants toward heparin, we developed an affinity capillary electrophoresis (ACE) method. This ACE method enabled to analyze AT variants directly from the cell culture supernatants used to produce them. It has been shown that some AT variants had a very high affinity for heparin, in good agreement with the performed mutations. Finally, a new method based on Taylor Dispersion Analysis (TDA) was investigated to study the affinity between AT issued from plasma and heparin.

Living conditions of every organism are influenced by various factors at this time. Some of them have positive effect on organism, some negative. Basic condition for surviving is the ability to resist and adapt to changing metabolic and living conditions. Every single stress effect can lead to changes in metabolism but organisms have ability to develope sufficient mechanisms for stress response. Some of them are similar for all living organisms (enzyme production, endogenous primary stress metabolites) some of them are specific for certain organism or stress type. Cell stress response can be observed on different levels (proteomic, genomic, metabolomic). In proper conditions it can be used indrustrially. In this work, influences of various stress factors were studied. These factors were applied on selected organisms – carotenogenic yeast and plant materials. Yeast stress response was induced by osmotic and oxidation stress factors. Changes on proteomic level and in production of selected secondary metabolites were observed. Proteome was analyzed by 1D and 2D electrophoresis with subsequent analysis of proteins by mass spectrometry. Yeast strain Rhodotorula glutinis CCY 20-2-26 showed the best adaptation to stress factors, which was moreover accompanied by overproduction of carotenoids. This finding can be premise for next industrial production of carotenoids. In plant samples predominantly enzymes and metabolites involved in antioxidant response were studied.