by Chan Wing-yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 93-95). / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- "Clinical Significance of Fe, Cu and Zn" --- p.2 / Chapter 1.3 --- Alternative Methods of Analysis --- p.5 / Chapter 1.4 --- Principles of Energy Dispersive X-ray Fluorescence Spectrometry --- p.11 / Chapter 1.5 --- Research Plan --- p.20 / Chapter CHAPTER 2 --- EXPERIMENTAL --- p.22 / Chapter 2.1 --- Energy Dispersive X-ray Fluorescence Analysis --- p.22 / Chapter 2.1.1 --- Apparatus --- p.22 / Chapter 2.1.2 --- Reagents --- p.25 / Chapter 2.1.3 --- Procedure --- p.28 / Chapter 2.2 --- Atomic Absorption Spectrometric Analysis --- p.32 / Chapter 2.2.1 --- Apparatus --- p.32 / Chapter 2.2.2 --- Reagents --- p.32 / Chapter 2.2.3 --- Procedure --- p.34 / Chapter CHAPTER 3 --- RESULTS AND DISCUSSION --- p.38 / Chapter 3.1 --- Optimisation of Excitation Conditions --- p.38 / Chapter 3.1.1 --- Effect of Filter --- p.38 / Chapter 3.1.2 --- Effect of Tube Voltage --- p.43 / Chapter 3.1.3 --- Effect of Tube Current --- p.44 / Chapter 3.2 --- Optimisation of Preconcentration Procedure --- p.46 / Chapter 3.2.1 --- Effect of Sample Area and Collimator Size --- p.46 / Chapter 3.2.2 --- Effect of pH --- p.51 / Chapter 3.2.3 --- Effect of Ligand Concentration --- p.54 / Chapter 3.2.4 --- Effect of Mixing Time --- p.57 / Chapter 3.2.5 --- Effect of Standing Time --- p.59 / Chapter 3.2.6 --- Study of Sample Homogeneity --- p.61 / Chapter 3.3 --- Optimisation for Deproteination --- p.63 / Chapter 3.3.1 --- Effect of Different Protein Precipitants --- p.63 / Chapter 3.3.2 --- Effect of Trichloroacetic Acid Concentration --- p.65 / Chapter 3.3.3 --- Effect of Hydrochloric Acid Concentration --- p.67 / Chapter 3.3.4 --- Effect of Temperature --- p.69 / Chapter 3.3.5 --- Effect of Incubation Time --- p.71 / Chapter 3.4 --- Study of Blanks --- p.74 / Chapter 3.5 --- Construction of Calibration Curves --- p.77 / Chapter 3.6 --- Determination of Detection Limit and Sensitivity --- p.84 / Chapter 3.7 --- Accuracy and Reproducibility Tests --- p.86 / Chapter 3.8 --- Parallel Check --- p.89 / Chapter CHAPTER 4 --- CONCLUSION --- p.92 / REFERENCES --- p.93

Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2009 / Obesity is a complex and chronic disease that is associated with many complications including type 2 diabetes (T2D). However, the mechanism leading to these events is unclear. To determine the role played by obesity in the development of T2D, it was purposed to examine serum protein profiles of diet-induced obese Wistar rats. These protein(s) will be used as potential biomarkers for early detection, diagnosis, as target for drug discovery and hopefully treatment of obesity and obesity-induced disorders. An animal model of obesity-induced T2D was established by feeding male Wistar rats a diet with a high fat content for 44 weeks. Weight changes and food intake were monitored weekly during the diet phase. Fasting blood glucose levels were measured while blood was collected for serumpreparation every second week for the first eight weeks; using these parameters, a model for the identification of diet-induced obese rats was established. Serum protein profiles were then compared between the two groups using 2D-PAGE analysis coupled to MALDI-MS at termination of the study. Several proteins showed differences in their expression when compared between the low fat (LF) and high fat (HF) groups. However, only proteins that showed expression that was either two fold low or high between the two groups were considered to be differentially expressed. High fat fed rats weighed significantly heavier starting at the fourth week on diet and throughout the study, their glucose homeostasis and serum protein expression was altered. Three protein spots were identified as dysregulated in the HF group, where Apolipoprotein-AIV was found to have been up-regulated whereas C-reactive protein and Fetuin-A were down-regulated. These proteins might help in the understanding of the mechanism(s)that underlie the pathogenesis of obesity and its related disorders. However, further studies are required to determine the relationship between these proteins, obesity development and its comorbidities.

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published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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In recent years the study of serum glycoproteins has advanced rapidly. Much of this interest in serum protein-carbohydrate complexes has been inspired by findings that many physiological and pathological conditions are associated with elevated serum protein bound carbohydrates. Paper electrophoFesis has been used in many studies of the serum glycoproteins of the human and experimental animals. Reports on the electrophoretic distribution of serum glycoproteins in most animals, however, are lacking. [...]

It is widely accepted that the initial event that significantly influences biocompatibility is the nearly instantaneous adsorption of proteins from biological fluids onto the biomaterial surface. For blood-contacting devices, the complex layer of adsorbed plasma proteins is generally unfavourable and leads to major complications, including thrombus formation, inflammatory tissue responses, and microbial infections. Furthermore, protein interaction with passive films on metallic biomaterial surfaces may contribute to enhanced in vivo corrosion. To gain a better understanding of this phenomenon, the present thesis investigated the fundamental aspects of the interaction of the serum protein fibrinogen with a medical-grade stainless steel 316LVM surface using electrochemical and IR spectroscopy techniques. Aspects of this interaction included the thermodynamics and kinetics of fibrinogen adsorption, the effect of fibrinogen adsorption on the corrosion behavior of 316LVM stainless steel, and the conformational changes of fibrinogen upon its adsorption onto the stainless steel surface. / It was shown that fibrinogen readily adsorbs onto the 316LVM stainless steel surface. Increases in the bulk protein concentration resulted in a corresponding increase of the surface coverage, a dependence that was described by the Langmuir isotherm. Large, negative values of the calculated Gibbs energy of adsorption indicated a highly spontaneous and strong adsorption of fibrinogen onto the 316LVM stainless steel at all investigated temperatures. Although the adsorption process was shown to be endothermic under the applied experimental conditions, the primary driving force for the adsorption process was found to be the positive entropy gain that arises from structural loss and/or rearrangement of the protein upon adsorption, as well as dehydration of the protein and stainless steel surface during the adsorption process. Kinetic measurements indicated that fibrinogen adsorption occurs rapidly. / It was determined that for short contact times (1 hour), the addition of fibrinogen to the electrolyte enhanced the corrosion rate of the 316LVM stainless steel at the open circuit potential. For longer contact times (24 hours), an increase in the polarization resistance values was obtained, indicating an enhanced corrosion resistance of the material. It was postulated that the protein was not capable of complexing the well-stabilized passive film, and instead remained adsorbed to form a protective barrier to diffusion of oxygen-containing species from the electrolyte to the stainless steel surface. / The secondary structure of the surface-adsorbed fibrinogen molecules was investigated by modeling the experimental PM-IRRAS spectra. It was shown that the protein lost a certain extent of its secondary structure upon adsorption to the steel surface. Fibrinogen molecules adsorbed from more dilute solutions were also shown to possess a lower alpha-helical content than those adsorbed from more concentrated solutions, suggesting they laid on the stainless steel surface in a more linear configuration.