Submitted in fulfilment of the requirements of the Degree of Master of Technology: Chemistry, Durban University of Technology, 2012. / The present work involves the interaction studies of chiral compounds with the Human Serum Albumin (HSA) protein using computational and experimental methods. The HSA protein has multiple binding sites that forms the basis for its exceptional ability to interact with many organic and inorganic molecules, which makes this protein an important regulator of intercellular fluxes and the pharmacokinetic behaviour of many drugs. This study was undertaken to evaluate the related pharmacokinetic and enantioselective binding parameters of the racemic catechin enantiomers with the HSA. Accordingly, this work involved a method development for the chiral separation of a racemic compound, by capillary electrophoresis-electrokinetic chromatography (CE-EKC) with a highly sulphated beta-cyclodextrin (HS--CD) as a chiral selector. The experimental work was supported by two molecular docking studies. The first included the mimicking of the host-guest interactions between a chiral selector and an enantiomeric compound. The second study included the estimation of the pseudo enantioselective (ES) binding of catechin to HSA. Overall, it was found that CE-EKC is the preferred method for the(±)-catechin binding to HSA protein evaluation. Moreover, the technique used in this work is not restricted to HSA or polyphenols, but can also be applied to other proteins and ligands that possess chirality. Furthermore, the molecular docking approaches also proved to be very useful for the evaluation of chiral recognition systems and for elucidation of the ligand-protein interactions.

OBJECTIVE: The main aim of this thesis was to investigate the role of nutrition during "early" HIV-infection in African women. METHODS: Data reported in this investigation formed part of two cross-sectional studies, the THUSA and Mangaung studies. The Mangaung study investigated women and, therefore, the sub-sample of the THUSA study was chosen accordingly. The data of the two studies were kept and analysed separately. The investigation consisted of 1040 women from the THUSA study, aged between 15 and 90 years of which 120 (11.5%) were HIV infected. The Mangaung study comprised of 488 women aged between 25 and 44 years of which 248 (51%) women were infected. Demographic data, anthropometric measurements, health outcome variables and habitual nutrient intakes by a quantified food frequency questionnaire were used. The SPSS statistical package (version 14.0; SPSS Inc., Chicago, Illinois, 2005) was used to analyse data. Descriptive statistics were done expressing variables as means, medians, standard deviations (SD), standard errors (SE) and confidence intervals (CI). An analysis of variance (ANOVA) was done to test for significance between the HIV-infected and non-infected groups in both studies. Partial correlations were done in the infected and non-infected groups to determine associations between dietary / nutrient intake, anthropometry and the biological health variables. In the THUSA study we controlled for age, education level, degree of urbanization and alcohol intake and in the Mangaung study for age, education level and alcohol intake. Nutrient intakes of both infected and non-infected women above and below median values as well as in the first and fourth quartile of total cholesterol (TC) and albumin distribution were compared to assess the role of nutrients in the observed decreases in TC and albumin of HIV-infected women. RESULTS AND DISCUSSION: The dietary intakes of the HIV-infected women in both the studies did not differ significantly from the non-infected women. Total serum cholesterol, albumin, fibrinogen and blood pressure were significantly lower in the HIV-infected women in both the THUSA and Mangaung studies. The non-infected THUSA women with lower serum cholesterol levels (than the median) had significantly lower intakes of percentage energy from fat (25.2 versus 26.4%, p ≤0.027), percentage energy from total protein (11.6 versus 12.1%, p≤0.000), animal protein (25.6 versus 27.7g, p≤0.005), and significantly higher intakes of plant protein (32.2 versus 29.4g, p≤0.002) and fibre (16.9 versus 15.89 p≤0.029). There were no significant differences observed in the nutrient intakes in the infected women with serum cholesterol levels above and below the median. In the Mangaung study no significant nutrient intake differences were observed in both of the HIV-infected and non-infected women with lower and higher than the median TC levels. In the THUSA study, higher intakes of fat (percentage energy) were close to significant (27.3 versus 24.5%, p≤0.053) in the infected women with higher (than the median) albumin levels. In the non-infected group with higher albumin levels, significant differences were observed in percentage energy from fat (26.6 versus 24.9%; p≤0.001) protein (12.2 versus 11.6%; p≤0.001) and carbohydrate (62.8 versus 65.2%; p≤0.000). Higher intakes of saturated fat (SATFAT) (17.7 versus 16.1g, p≤0.008), monounsaturated fats (MUFAT) (19.3 versus 17.4g, p≤0.004) as well as higher intakes of animal protein (28.5 versus 24.4g, p≤0.000) were observed in the group with higher than the median levels of serum albumin. In the Mangaung study the HIV-infected women (with higher than the median serum albumin levels), had significantly higher intakes of energy (13 275 versus 11 622 kJ, p≤0.022), polyunsaturated fatty acids (32.3 versus 17.3g, p≤0.036), dietary cholesterol (412.9 versus 344.5mg, p≤0.043) and plant protein (42.3 versus 35.3g, p≤0.008). No differences were observed in the non-infected women. The further analyses, comparing the dietary intakes in both studies of infected and non-infected women with TC and albumin levels in the first and fourth quartiles, showed that in the THUSA study, non-infected women with lower TC levels had significantly lower intakes of protein (% of total energy), total fat (% of total energy) and vitamin B12 and significantly higher intakes of total energy (TE), plant protein, total carbohydrate, % TE from carbohydrate, dietary fibre, added sugar and thiamine. In the infected women saturated fatty acids (SATFAT), calcium and the fat ratio (polyunsaturated/saturated ratio) differed significantly between women with TC levels in the first and the fourth quartile. A significant higher intake of riboflavin was seen in the non-infected women from Mangaung with TC levels in the fourth quartile, while significant higher intakes of energy, total protein, animal protein, total fat, SATFAT, MUFAT, total carbohydrate, phosphorus, chromium and iodine was seen in the infected women with TC levels in the fourth quartile. These results suggest that a more "westernized" diet with higher intakes of energy, and animal derived foods (SATFAT and calcium) could have protected against the detrimental decreases in TC observed in HIV infection. Significant differences were observed in the intakes in the non-infected THUSA women who had serum albumin in the first and fourth quartiles. lntakes in percentage energy from protein and fat, animal protein, total fat, SATFAT, MUFAT, calcium, zinc, vitamin C and fat ratio, were significantly lower in the women with albumin levels in the first quartile. Significantly higher carbohydrate intakes were observed in the women who had serum albumin levels in the first quartile. In the Mangaung study, significant differences were seen in the intakes between infected women who had serum albumin levels in the first and fourth quartiles. lntakes of total energy, protein, fat, MUFAT, SATFAT, carbohydrate, magnesium, zinc, chromium, biotin, pantothenic acid and iodine were significantly lower in the infected women with serum albumin levels in the first quartile. In the non-infected women significantly lower intakes of calcium were observed in the group who had serum albumin levels in the first quartile compared to those who had serum albumin levels in the fourth quartile. These results also suggest that a more "westernized” diet was associated with higher albumin levels in HIV-infected women. CONCLUSION: It is well known that nutrition has an integral part to play in the care of people living with HIV/AIDS (PLWHA). Maintaining proper nutrition, weight and immune function is thought to delay disease progression, prolong the asymptomatic phase and improve survival. These analyses suggest that the "prudent" diet generally regarded as an optimal diet for prevention of non-communicable diseases, may not be the optimal diet for PLWHA. The overall analyses therefore suggest that a more "westernized" diet, higher in fat and protein could be more beneficial to asymptomatic HIV-infected women compared to that of a more "prudent" diet. As these studies were not primarily designed to investigate HIV and nutrition, the role of a higher energy, fat and animal protein intake ("western" diet) in asymptomatic HIV warrants urgent investigation. / Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

Healthy articular joints exhibit remarkable lubrication due in large part to the complex rheological and tribological behavior of the synovial fluid (SF) that lubricates the joints. Current approaches that seek to elucidate such remarkable lubrication usually focus on the roles of high molecular weight SF components such as lubricin and hyaluronic acid but frequently overlook the role of serum albumin (SA), although it represents 90% of the protein content of SF. In this thesis, we used the Surface Forces Apparatus to investigate in detail the structural and tribological response of SA thin films when sheared between model surfaces and subjected to a large range of shearing parameters. Our data indicate that, under shear, SA films reproduce closely the shear response previously reported for SF, i.e., film thickening and formation of numerous long-lived aggregates accompanied by low friction and efficient surface protection against damage. More specifically, our detailed investigation of shear parameters reveals that (i) strong anchoring of SA to surfaces promotes the formation of large rod-like shaped aggregates that enable rolling friction and keep surfaces far apart, preventing damage, (ii) aggregation mechanism is irreversible, which makes aggregates long-lived (though mobile) in the contact, and (iii) aggregate formation only occur when SA was sheared above a ‘critical’ amplitude Ac and a critical shear velocity Vc. Collectively, our results provide experimental evidence of the role of globular proteins, such as SA, in lubrication and establish a correlation between shearing parameters, formation and stability of aggregates, low friction and wear protection. Although our findings are based on experiments involving rigid, nonporous surfaces hence can hardly be generalized to compliant and porous cartilage surfaces, they are applicable to other rigid tribosystems such as artificial joints and will certainly advance our understanding of joint implants’ lubrication in SF mediated by protein aggregation, with implications for future design of artificial joints and therapeutic interventions.

One-dimensional nanostructures have offered unique advantages in many fields. Protein based nanotubes, in particular, are desirable for biomedical applications due to their ease of functionlization and intrinsic biocompatibility. Template-assisted methods are widely used to fabricate cylindrical nanostructures like carbon nanotubes, metal nanowires, polymer nanorods, etc. In the fabrication of protein nanostructures, the layer by layer (LbL) technique has long been applied to deposit protein multilayers on planar and spherical substrates. The success in each area led to the conclusion that the combination of these two techniques will potentially bring us the capability of fabricating protein nanotubes in a more controllable fashion. In this work, protein nanotubes have been successfully deposited inside nanoscopic pores by sequential filtration of bovine serum albumin (BSA) solution at pH 3.8 and pH 7.0 through the channels in the anodic aluminum oxide (AAO) template. The morphologies of the obtained nanostructures have been examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Also, a simple analysis from UV/Vis spectroscopy has shown that the solutions used in our experiment will not significantly damage the bioactivity of BSA. Our future work will focus on strengthening the mechanical stability of the protein nanotubes and controlling their morphology more precisely.

Membrane chromatography is a promising process for the isolation, purification, and recovery of proteins, enzymes, and nuclear acids. Comparing with traditional beads column chromatography, membrane chromatography can faster, easier and cheaper to mass-produce. And also, it is easy to set up and scale up. In this thesis, we are trying to study the performance of membrane chromatography, and the mixture of HSA and chicken egg white is used as an example. We are investigating the purification of Human serum albumin (HSA) from chicken egg white in terms of precondition, dilution, purification method, product recovery, product purity and product cost. HSA, is a very important clinical protein. In order to obtain low cost, high efficiency and less risk HSA, recombinant DNA technology is used. Many kinds of host organism have been used to produce recombinant HSA (rHSA). In this thesis, a kind of ion-exchange membrane (Mustang Q membrane capsule) chromatography was used. The membrane capsule is disposable because it is designed for use in pharmaceutical production. For this project, a cleaning method was used which made the membrane capsule reusable. Washing with 4 mL 1 M NaCl and 4 mL NaOH was sufficient for this purpose. Since the egg white protein solution was very viscous, it needs to be diluted before loaded on FPLC. Dilute experiment was done to find the best dilution level. In this thesis, we found that 5 times dilution was best not only for high efficiency but also for FPLC operation. After getting the basic conditions, some purification experiments were done to find the optimal operation condition to purify HSA form chicken egg white protein solution by changing buffer pH, salt concentration in elution buffer and gradient used to elute proteins. The best purification condition for loading buffer is Tris-HCl buffer A (4.75g/L, pH 9.5) and the elution buffer is Tris-HCl buffer A + 0.2M NaCl. The purity of HSA recovered was 93% on the Mustang Q membrane capsule at 1 ml/min when the mixture of HSA and chicken egg white was diluted 10 times. And the yield was 85%. The impurity is probably ovoglobulin as suggested by the result of SDS-PAGE, whose molecular weight is close to 40kd. To characterize the separation capability of the Mustang Q membrane capsules, equilibrium adsorption and breakthrough curve studies were made using bovine serum albumin (BSA). 1mg/mL BSA solution was used to get the breakthrough curve with different flow rate ranging from 1 to 4 ml/min. With a flow rate is 1 ml/min, breakthrough curve were obtained with different concentrations of BSA ranging from 1 to 16 mg/mL. The dynamic binding capacity was found to be from 9.1 to 119.1 mg/mL. The equilibrium adsorption isotherm showed Langmuir isotherm behavior with dissociation constant and a maximum adsorption capability. According to the result of isotherm adsorption, a multi-plate mathematical model was used to get the theoretical breakthrough curve. By fitting the theoretical breakthrough curve to the experimental breakthrough curve, constants in the multi-plate model were obtained and were used to estimate the axial dispersion coefficient of the membrane capsule. The estimated axial dispersion coefficient of 2.45*10-6 cm2/s is very small which means that the axial ispersion is not significant. The adsorption process is therefore controlled by radial radius dispersion or film dispersion.

Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007.

No description available.

Investigations detailed in this thesis constitue a part of continuing programme of research work undertaken in this laboratory on vitamin binding proteins. Avidin from the chicken egg white, streptavidin &om the bacterium Streptromyces avidin and biotin binding proteins (BBP-I and BBP-11) from chicken egg yolk constitute a family of proteins that bind the vitamin biotin with extremely high affinities. The yolk BBPs are involved in the deposition of the vitamin in the developing oocyte in chicks whereas an antimicrobial function has been attributkl to avidin.. The fact that all these proteins bind the vitamin in the same manner, unlike biotin-dependent enzymes, indicates that the structural features involved in ligand binding could be similar, if not identical in these proteins. To delineate the basis of putative structural similarity among these proteins, studies were carried out using antibodies as the immunological probes. Avidin, a homotetremer glycoprotein, with a subunit Mr of 17,000 has been purified to homogeneity from chicken egg white using a novel procedure involving ammonium sulphate fractionation, ethanol precipitation and S-Sepharose column chromatography. Despite their lesser abundance in chicken egg yolk associated with a large amount of interfering lipids during the purification, both BBP-I (monomer and shown to be precursor for BBP-11) and BBP-I1 (tetramer) have been purified to homogeneity by employing a common method using butanol extraction to remove the lipids, DEAE-Sephacel column chromatography, biotin-AH-Sepharose affinity chromatography and fast performance liquid chrometography (FPLC) system. The purity of all these proteins was confirmed by SDS-PAGE analysis.

The reactions of the early and intermediate stages of protein glycation were studied using bovine serum albumin and hen egg white lysozyme as model proteins, and D-glucose and D-fructose as model reducing sugars. The reactions were studied under low moisture and limiting sugar conditions in the presence and absence of oxygen in a closed system. Several analytical methods, including the fluorescamine assay, total Maillard fluorescence and solubility measurement, size exclusion chromatography, and electrospray ionization mass spectrometry were used to follow the glycation reaction. The effect of glycation on the structure of proteins was also studied by electrospray ionization mass spectrometric peptide mapping. The results showed that under low moisture conditions, the initial rate of glucation was 5 to 6 times higher than the initial rate of fructation, both in the presence and in the absence of oxygen. The presence of oxygen in the reaction system induced selectivity of the glycation of the protein amino groups during the initial stages of the reaction, due to competition between glycation and glycoxidation. The reaction, however, occurred at a higher rate in the presence of oxygen than in its absence, especially during the latter stages of the reaction. The more reactive glycoxidation products, formed during the initial stages of the reaction also participated in the glycation reaction and increased the involvement of arginine residues in the glycation reaction. The glycation reaction also resulted in a heterogeneous mixture of protein glycoforms with varying degrees and extent of glycation. The heterogeneity of the glycated species was further complicated by the involvement of glycoxidation products in the glycation reaction, resulting in a distribution of different classes of glycoconjugates. Limited glycation improved protein solubility and increased solvent accessibility into the interior of the protein, thereby, increasing protein digestibility. Electrospr

OBJECTIVE: The main aim of this thesis was to investigate the role of nutrition during "early" HIV-infection in African women. METHODS: Data reported in this investigation formed part of two cross-sectional studies, the THUSA and Mangaung studies. The Mangaung study investigated women and, therefore, the sub-sample of the THUSA study was chosen accordingly. The data of the two studies were kept and analysed separately. The investigation consisted of 1040 women from the THUSA study, aged between 15 and 90 years of which 120 (11.5%) were HIV infected. The Mangaung study comprised of 488 women aged between 25 and 44 years of which 248 (51%) women were infected. Demographic data, anthropometric measurements, health outcome variables and habitual nutrient intakes by a quantified food frequency questionnaire were used. The SPSS statistical package (version 14.0; SPSS Inc., Chicago, Illinois, 2005) was used to analyse data. Descriptive statistics were done expressing variables as means, medians, standard deviations (SD), standard errors (SE) and confidence intervals (CI). An analysis of variance (ANOVA) was done to test for significance between the HIV-infected and non-infected groups in both studies. Partial correlations were done in the infected and non-infected groups to determine associations between dietary / nutrient intake, anthropometry and the biological health variables. In the THUSA study we controlled for age, education level, degree of urbanization and alcohol intake and in the Mangaung study for age, education level and alcohol intake. Nutrient intakes of both infected and non-infected women above and below median values as well as in the first and fourth quartile of total cholesterol (TC) and albumin distribution were compared to assess the role of nutrients in the observed decreases in TC and albumin of HIV-infected women. RESULTS AND DISCUSSION: The dietary intakes of the HIV-infected women in both the studies did not differ significantly from the non-infected women. Total serum cholesterol, albumin, fibrinogen and blood pressure were significantly lower in the HIV-infected women in both the THUSA and Mangaung studies. The non-infected THUSA women with lower serum cholesterol levels (than the median) had significantly lower intakes of percentage energy from fat (25.2 versus 26.4%, p ≤0.027), percentage energy from total protein (11.6 versus 12.1%, p≤0.000), animal protein (25.6 versus 27.7g, p≤0.005), and significantly higher intakes of plant protein (32.2 versus 29.4g, p≤0.002) and fibre (16.9 versus 15.89 p≤0.029). There were no significant differences observed in the nutrient intakes in the infected women with serum cholesterol levels above and below the median. In the Mangaung study no significant nutrient intake differences were observed in both of the HIV-infected and non-infected women with lower and higher than the median TC levels. In the THUSA study, higher intakes of fat (percentage energy) were close to significant (27.3 versus 24.5%, p≤0.053) in the infected women with higher (than the median) albumin levels. In the non-infected group with higher albumin levels, significant differences were observed in percentage energy from fat (26.6 versus 24.9%; p≤0.001) protein (12.2 versus 11.6%; p≤0.001) and carbohydrate (62.8 versus 65.2%; p≤0.000). Higher intakes of saturated fat (SATFAT) (17.7 versus 16.1g, p≤0.008), monounsaturated fats (MUFAT) (19.3 versus 17.4g, p≤0.004) as well as higher intakes of animal protein (28.5 versus 24.4g, p≤0.000) were observed in the group with higher than the median levels of serum albumin. In the Mangaung study the HIV-infected women (with higher than the median serum albumin levels), had significantly higher intakes of energy (13 275 versus 11 622 kJ, p≤0.022), polyunsaturated fatty acids (32.3 versus 17.3g, p≤0.036), dietary cholesterol (412.9 versus 344.5mg, p≤0.043) and plant protein (42.3 versus 35.3g, p≤0.008). No differences were observed in the non-infected women. The further analyses, comparing the dietary intakes in both studies of infected and non-infected women with TC and albumin levels in the first and fourth quartiles, showed that in the THUSA study, non-infected women with lower TC levels had significantly lower intakes of protein (% of total energy), total fat (% of total energy) and vitamin B12 and significantly higher intakes of total energy (TE), plant protein, total carbohydrate, % TE from carbohydrate, dietary fibre, added sugar and thiamine. In the infected women saturated fatty acids (SATFAT), calcium and the fat ratio (polyunsaturated/saturated ratio) differed significantly between women with TC levels in the first and the fourth quartile. A significant higher intake of riboflavin was seen in the non-infected women from Mangaung with TC levels in the fourth quartile, while significant higher intakes of energy, total protein, animal protein, total fat, SATFAT, MUFAT, total carbohydrate, phosphorus, chromium and iodine was seen in the infected women with TC levels in the fourth quartile. These results suggest that a more "westernized" diet with higher intakes of energy, and animal derived foods (SATFAT and calcium) could have protected against the detrimental decreases in TC observed in HIV infection. Significant differences were observed in the intakes in the non-infected THUSA women who had serum albumin in the first and fourth quartiles. lntakes in percentage energy from protein and fat, animal protein, total fat, SATFAT, MUFAT, calcium, zinc, vitamin C and fat ratio, were significantly lower in the women with albumin levels in the first quartile. Significantly higher carbohydrate intakes were observed in the women who had serum albumin levels in the first quartile. In the Mangaung study, significant differences were seen in the intakes between infected women who had serum albumin levels in the first and fourth quartiles. lntakes of total energy, protein, fat, MUFAT, SATFAT, carbohydrate, magnesium, zinc, chromium, biotin, pantothenic acid and iodine were significantly lower in the infected women with serum albumin levels in the first quartile. In the non-infected women significantly lower intakes of calcium were observed in the group who had serum albumin levels in the first quartile compared to those who had serum albumin levels in the fourth quartile. These results also suggest that a more "westernized” diet was associated with higher albumin levels in HIV-infected women. CONCLUSION: It is well known that nutrition has an integral part to play in the care of people living with HIV/AIDS (PLWHA). Maintaining proper nutrition, weight and immune function is thought to delay disease progression, prolong the asymptomatic phase and improve survival. These analyses suggest that the "prudent" diet generally regarded as an optimal diet for prevention of non-communicable diseases, may not be the optimal diet for PLWHA. The overall analyses therefore suggest that a more "westernized" diet, higher in fat and protein could be more beneficial to asymptomatic HIV-infected women compared to that of a more "prudent" diet. As these studies were not primarily designed to investigate HIV and nutrition, the role of a higher energy, fat and animal protein intake ("western" diet) in asymptomatic HIV warrants urgent investigation. / Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007.