Natural Killer (NK) cells can recognize and lyse a large variety of tumor cells and have been of interest as a potential cancer treatment option. Our group has developed a particle-based NK cell expansion method that utilizes plasma membrane particles (PM-particles) derived from K562 cells genetically engineered to express membrane bound IL21 and 41BBL(K562-mbIL21-41BBL), two proteins that stimulate growth and activity of NK cells. This method selectively expands highly cytotoxic NK cells > 400-fold in 14 days of culture. Currently NK cells are expanded in vitro using Fetal Bovine Serum (FBS) as a serum-supplement to promote cell growth. While effective, the use of animal products is not preferred in cell cultures grown for clinical purposes. This project tested Human Platelet Lysates (HPL) as a potential replacement for FBS in NK cell culture. NK cells were expanded using PM21-particle based expansion method with either FBS or HPL as supplements. Their growth characteristics, phenotype and functionality were assessed and compared. Results of this study determined that HPL is a viable option to replace FBS in NK cell culture for clinical applications, as there was no significant difference between the two serum supplements.

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor b1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

No description available.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Grape and wine phenols consist of flavanols which is the building blocks for tannins. These building blocks are called monomers which consist of catechins, epicatechins, epigallocatechins and epicatechin-gallate. Tannin is important in wine as it contributes to bitterness, mouth feel (astringency) and maturation potential of the wine. Futhermore it has a health benefit as an antioxidant. Anthocyanins are responsible for the colour of red wine. The anthocyanins combine with tannins to form stable polymeric pigments. Due to the importance of tannins and anthocyanins in wine, it is imperitative that different winemaking techniques are used to extract as much of these components as possible and that the analysis is done quickly and accurately. The aim of this study was to evaluate different winemaking techniques and their extraction of tannins and anthocyanins into the wine. Too much tannin extraction can have a negative effect on the sensory quality of the wine. Therefore a second aim was to evaluate the mouth feel properties of a Shiraz wine. A third aim was to compare the two tannin precipitation methods in terms of time efficiency, repeatability and the ease of practice. To investigate the amount of tannin concentration extracted by different winemaking techniques, two cultivars (Cabernet Sauvignon and Shiraz) were used. These treatments included the addition of an enzyme during fermentation [E], cold maceration [CM], post maceration [PM] and the combination of cold and post maceration [CM+PM]. The grapes were harvested in two different climatic areas during the 2008 and 2009 vintages. The two climatic areas were classified according to the Winkler scale as a III (Morgenster) and a IV (Plaisir de Merle). The grapes were harvested at two different ripeness levels in order to evaluate the effect of the different winemaking processes on the extraction of tannins and anthocyanins. One harvest was before (LB) and the other after (HB) the commercial harvest. The results of this study showed significant differences in the phenolic composition of the wines. It was found that the warmer area showed higher tannin concentrations than the cooler area for both cultivars. In the 2008 Cabernet Sauvignon the CM extracted higher concentrations of tannin from the cooler area at both ripeness levels. In the warmer area, CM extracted the highest tannin concentration HB, but the CM+PM extracted the highest tannin concentration from Cabernet Sauvignon at the LB and CM at the HB of the warmer area. In 2009 the PM extracted the highest concentration of tannin at the lower ripeness level, while the E treatment extracted the highest concentration from the warmer area. In the cooler area the CM+PM extracted the highest concentration of tannin at a lower ripeness level, while there were no siginicant differences between the different treatments at the higher ripeness level. The highest anthocyanin concentration was found in the cooler area. The CM treatment was found to have no effect on anthocyanin extraction. Different methods are available to quantify the tannin concentration in wine. Two of the most popular tannin analytical methods are the bovine serum albumin (BSA) and the methyl cellulose precipitable tannin (MCP) methods. The BSA method is a very complex method which uses at least 3 times more reagents than the MCP method. The MCP method only analyzes tannins, while the BSA method analyzes tannins, monomeric pigments (MP), small polymeric pigments (SPP) and large polymeric pigments (LPP). In this study a good correlation was found between the two tannin precipitation methods (R2 – 0.88). There is controversy regarding the variability of these methods. Some scientists found that the two methods show a good correlation with HPLC, while others found that there was no such correlation between the precipitation methods and the HPLC. The MCP method had a practical advantage as it could be performed in half the time required for the BSA method. This has a significant impact in scenarios where a high sample throughput is required although it only measures total tannin. The phenolic composition and mouth feel of the wine was strongly influenced by the climatic area. In the warmer area the effect of tannin concentration on mouth feel was much less than in the cooler area. The wine made of riper grapes, was more grippy, bitter and numbing than the wines made from greener grapes. The E treatment was especially associated with a dry, grippy sensation. / AFRIKAANSE OPSOMMING: Druif en wyn fenole bestaan uit flavanole wat weer die boublokke is van tanniene. Hierdie boublokke, wat bekend staan as monomere, betsaan uit katesjiene, epikatesjiene, epigallokatesjiene an epikatesjien-gallaat. Tanniene is belangrik in wyn aangesien dit bydra tot bitterheid, mondgevoel (vrankheid) asook die verouderingspotensiaal van wyn. As antioksidante hou dit ook gesondheidsvoordele in. Antosianiene dra by tot die kleur van rooiwyn. Antosianiene kombineer met tanniene om meer stabiele polimeriese pigmente te vorm. As gevolg van die belangrikheid van tanniene en antosianiene is dit van uiterse belang dat verskillende wynmaak tegnieke gebruik word om ekstraksie in die wyn te bevoordeel en dat die analitiese metode so vinnig en akkuraat as moontlik gedoen word. Die eerste doel van hierdie studie was om die ekstraksie van tanniene en antosianiene deur middel van verskillende wynmaak tegnieke te evalueer. Te veel tanniene in die wyn kan negatiewe sensoriese kwaliteit tot gevolg het. Daarom is die tweede doel om die sensoriese kwaliteit van Shiraz wyn te evalueer. Die derde doel van hierdie studie was die twee tannien presipitasie metodes met mekaar te vergelyk in terme van die moeilikheidsgraad van die metode, tyd doeltreffendheid en herhaalbaarheid. Verskillende wynmaak tegnieke (ensiem byvoegings [E], koue maserasie [CM], verlengde dopkontak [PM] en ‘n kombinasie van koue maserasie en verlengde dopkontak [CM+PM]) is vergelyk ten opsigte van tannien en antiosianien ekstraksie. In 2008 en 2009 is twee kultivars (Cabernet Sauvignon en Shiraz) in twee verskillende klimatologiese areas gepars. Hierdie areas is geklassifiseer in die Winklerskaal as ‘n IV (Plaisir de Merle) en ‘n III (Morgenster). Om die effek van die verskillende wynmaak tegnieke op die ekstraksie van antosianiene en tanniene te vergelyk, is hierdie twee kultivars by twee verskillende rypheidsgrade geoes. Die eerste oes was net voor kommersiële oes (LB) en die tweede oes het net na kommersiële oes (HB) plaasgevind. Die 2009 Shiraz wyn is organolepties beoordeel om die effek van die verskillende wynmaak tegnieke op die wyn se mondgevoel te vergelyk. Die resultate van hierdie studie toon beduidende verskille in die fenoliese samestelling van die wyne. Dit is gevind dat die warmer area hoër tannien konsentrasies het as die koeler area. In 2008 het die CM+PM die meeste tanniene uit die Cabernet Sauvignon geëkstraheer by LB en die CM by HB in die warmer area. Die CM het in die koeler area meer tanniene geëkstraheer by beide die LB en HB rypheidsgrade. In 2009 het PM die meeste tanniene geëkstraheer by LB terwyl E die meeste tanniene geëkstraheer in die warmer area. In die koeler area het CM+PM die meeste tanniene geëkstraheer, terwyl geen van die behandelings ‘n effek gehad het by HB. Die meeste antosianien konsentrasie was in die koeler area gevind as in die warmer area. In beide 2008 (LB en HB) en 2009 (LB) het CM die meeste antosianiene geëkstraheer, terwyl geen behandeling ‘n effek gehad het by HB. Twee van die mees populêre tannien analitiese metodes is die BSA (bovine serum albumien) en die MCP (metielsellulose presipitasie) metodes. Die BSA metode is ‘n baie meer ingewikkelde metode waarvoor drie keer meer reagense gebruik word as vir die MCP metode. Maar waar die MCP net tanniene ontleed, ontleed die BSA metode tanniene, monomere (MP), klein polimeriese pigmente (SPP) en groot polimeriese pigmente (LPP). Dit help indien daar gekyk wil word na die evolusie van polimeriese pigmente. In hierdie studie is bevind dat daar ‘n redelike korrelasie (R2 – 0.88) tussen die BSA en MCP metode bestaan. Die herhaalbaarheid van die metodes het redelike kontroversie veroorsaak, waar sommige navorsers bevind het dat die BSA metode nie so herhaalbaar is soos eers bevind is nie. Die MCP metode het ’n praktiese voordeel aangesien dit in die helfde van die tyd van die BSA metode uitgevoer kan word. Dit het ‘n groot impak indien ‘n groot hoeveelheid monsters ontleed moet word. Die fenoliese samestelling en mondgevoel word sterk beïnvloed deur die klimatologiese area. In die warmer area was die effek van tannien konsentrasie op mondgevoel kleiner as in die koeler area. Die wyn van ryper druiwe het meer harder, verdowingseffek en bitter nasmaak gehad as by die wyn van groener druiwe. Die ensiem behandeling was meer geassossieerd met droë mond gevoel.

In real life, immediately after joint replacement, the artificial joint is actually bathed in blood (and clotted blood) instead of synovial fluid. Blood contains large molecules and cells of size ~ 5 to 20 μm suspended in plasma and considered to be a non-Newtonian (pseudoplastic) fluid with density of 1060 Kg/m³ and viscosity ~ 0.01 Pas at shear rates of 3000 s⁻¹ (as obtained in this work). The effect of these properties on friction and lubrication is not fully understood and, so far to our knowledge, hardly any studies have been carried out regarding friction of metal-on-metal bearings with various clearances in the presence of lubricants such as blood or a fluid containing macromolecules such as hyaluronic acid (HA) which is a major component of synovial fluid increasing its viscosity and lubricating properties. In this work, therefore, we have investigated the frictional behaviour of a group of Smith and Nephew Birmingham Hip Resurfacing implants with a nominal diameter of 50mm and diametral clearances in the range ~ 80 μm to 300 μm, in the presence of blood (clotted and whole blood), a combination of bovine serum (BS) with hyaluronic acid (HA) and carboxymethyl cellulose (CMC, as gelling agent) adjusted to a range of viscosities (~0.001-0.2 Pas), and bovine serum with CMC adjusted to a similar range of viscosities. These results suggested that reduced clearance bearings have the potential to generate high friction especially in the presence of blood which is indeed the in vivo lubricant in the early weeks after implantation. Friction factors in higher clearance bearings were found to be lower than those of the lower clearance bearings using blood as the lubricant. Similar trends, i.e. increase in friction factor with reduction in diametral clearance, were found to be also the case using a combination of BS+CMC or BS+HA+CMC as lubricants having viscosities in the range 0.1-0.2 and 0.03-0.14 Pas, respectively. On the other hand, all the lubricants with lower viscosities in the range 0.001-0.0013 and 0.001-0.013 Pas for both BS+CMC and BS+HA+CMC, respectively, showed the opposite effect, i.e. caused an increase in friction factor with increase in diametral clearance. Another six large diameter (50mm nominal) BHR deflected prostheses with various clearances (~ 50-280μm after cup deflection) were friction tested in vitro in the presence of blood and clotted blood to study the effect of cup deflection on friction. It was found that the biological lubricants caused higher friction factors at the lower diametral clearances for blood and clotted blood as clearance decreased from 280μm to 50μm (after deflection). The result of this investigation has suggested strongly that the optimum clearance for the 50 mm diameter MOM BHR implants to be ≥150μm and <235μm when blood lubricant used, so as to avoid high frictions (i.e. avoid friction factors >0.2) and be able to accommodate a mixed lubrication mode and hence lower the risk of micro- or even macro-motion specially immediately after hip implantation. These suggested optimum clearances will also allow for low friction (i.e. friction factors of <0.2-0.07) and reasonable lubrication (dominantly mixed regime) for the likely cup deflection occurring as a result of press-fit fixation.

The natural hip joint in healthy people has a very low friction with very little (or no) wear. It works as a dynamically loaded bearing and is subjected to about 1-2 million cycles of loading per year. The applied load is the body weight which is tripled when walking and even higher during other activities such as running and jumping. Unfortunately these joints are not always healthy due to various causes such as fractures or disease leading to severe pain which necessitates joint replacement. Currently, the orthopaedic industries are working towards developing an ideal artificial hip joint with low wear, low friction, good lubrication, better fixation/stability and biocompatibility. Many different designs and materials have been investigated with some promising new implants which can be used depending on patients' individual need (large or small joint), activity and age. In this work, two types of artificial hip joints were tested for friction and lubrication studies: Metal-on-Metal (MoM) Biomet hip resurfacing ReCaps with large diameters (>35-60 mm) and different diametral clearances (~ 60-350 µm), and Zirconia Toughened Alumina (ZTA) heads against carbon-fibre-reinforced poly-ether-ether ketone (CFR PEEK) cups with different diameters (>35-60 mm) and diametral clearances (60-1860 µm). Seven serum-based lubricants with different viscosities were used with and without carboxy methyl cellulose (CMC) additions as gelling agent to increase viscosity depending on the CMC content. The maximum load applied was 2000 N for the stance phase with a minimum load of 100 N for the swing phase. A Pro-Sim friction hip simulator was used to investigate the frictional torque generated between the articulating surfaces so as the friction factor can be calculated. Stribeck analysis was then employed to assess the mode of lubrication. For the metal-on-metal hip resurfacing joints, the friction factors were in the range 0.03-0.151 and those for the ZTA ceramic heads versus CFR PEEK cups were in the range 0.006-0.32. Stribeck analyses showed mainly mixed lubrication for both MoM and ZTA ceramic-on-CFR PEEK joints. The experimental results were in agreement with most of the theoretical calculations suggesting mixed lubricating regimes at low viscosities and moving on to fluid film lubrication at higher viscosities. Joints with larger-diameters, lower clearances and lower surface roughness exhibited a higher lambda ratio suggesting improved lubrication. Viscosity flow curves for the serum-based lubricants having viscosity ≤ 0.00524 Pas showed non-linear relationship between viscosity and shear rate indicating non-Newtonian flow with pseudoplastic or shear-thinning characteristic, i.e. viscosity decreased as shear rate increased up to shear rates of ~ 1000 s⁻¹. However, at shear rates greater than 1000 s⁻¹ Newtonian flow became dominant with almost constant viscosity, i.e. a linear relationship between shear stress and shear rate. On the other hand, viscosity flow curves for the lubricants with viscosity ≥ 0.0128 Pas showed non-Newtonian behaviour up to a shear rate of 3000 s⁻¹ with shear-thinning characteristic.

This diploma thesis is focused on coagulation of humic substances (HS) and BSA (Bovine Serum Albumin) protein which was chosen as a representative of proteins contained in AOM (Algal Organic Matter). Additionally, possible interactions between these compounds were also investigated. It was found that the optimal dosage of coagulant is much higher for HS compared to BSA. The best removal of both HS and BSA was reached in slightly acidic pH range and it is attributed mainly to charge neutralization and adsorption mechanisms. The maximum removal rate was 70 % for humic substances and 80 % for BSA. The results show that BSA has a positive effect on coagulation of HS (resulting in a lower coagulant demand) and vice versa while BSA was removed more efficiently than HS. The existence of interactions between BSA and humic substantces during coagulation was demonstrated in certain pH ranges and it can occur even without the presence of coagulant. These interactions are highly dependent on pH that determines charge properties (and hence reactivity) of organic matters. Finally, the comparison of BSA and cyanobacterial proteins shows that their behavior during coagulation is similar. Consequently, BSA can be used as a model compound representing AOM proteins, especially their high molecular weight fraction....

This thesis is focussed on the investigation of matrices for matrix-assisted diffusion-ordered spectroscopy (MAD). Diffusion-ordered spectroscopy (DOSY) is a family of experiments where the resonances in the chemical shift dimension are further dispersed in an extra dimension according to diffusion coefficient. A typical DOSY spectrum shows one single diffusion coefficient for all the resonances coming from one single species. However, If two or more resonances overlap, the diffusion resolution of the DOSY spectrum is compromised and a spurious diffusion coefficient results, intermediate between the species. In case of signal overlap, the use of more advanced processing methods aids to separate two analytes that differ by at least 30% in diffusion coefficient. In practice, many mixtures contain species of similar diffusion coefficients whose resonances overlap in the chemical shift dimension. The addition of co-solutes can modify the chemical environment (matrix), with which different analytes interact to different extents, and enhance the diffusion resolution of DOSY. However, the addition of co-solutes can risk the benefits of DOSY by increasing the probability of signal overlap. Signal overlap in MAD is avoided by using a 1H NMR-invisible surfactant such as sodium perfluorooctanoate (NaPFO), which has replaced each proton by a fluorine atom. PFO micelles are a tunable matrix which allows the separation of analytes via coulombic interactions by adjusting the pH. Differences in diffusion coefficient in NaPFO solution can be analysed using a modified Lindman's law to model the diffusion coefficient as a function of pH. The model rationalises the binding constants of analytes to PFO micelles with good accuracy, subject to the spectral data quality. Another alternative to resolve diffusion coefficients using the invisible MAD approach is by means of a commercially available alkyl surfactant like cetyltrimethylammonium bromide (CTAB). CTAB in high ionic strength solution forms worm-like micelles whose resonances can be filtered out from the final DOSY spectrum. CTAB worm-like micelles have short transverse relaxation times compared to all of the analytes in the mixture. If a transverse relaxation filter is positioned at the beginning of a standard DOSY pulse sequence, as in PROJECT-Oneshot, the strong CTAB signals vanish and leave behind only the analyte resonances and hence avoid signal overlap. Finally, the use of bovine serum albumin (BSA) as a potential invisible matrix, using a similar approach to CTAB worm-like micelles is investigated, using a relaxation-weighted DOSY pulse sequence to suppress most of the BSA background signal (at a cost in analyte signal to noise ratio). An alternative to suppress most of the BSA background and preserve most of the analyte signal is by means of mild transverse relaxation filtration and spectral editing to obtain an edited DOSY spectrum that shows only the analyte signals. Nonetheless, it is a shame that useful MAD results can only be obtained under a narrow set of conditions: i) different mole ratios BSA: analyte to aid diffusion resolution, ii) mild T2 filtration to improve analyte signal to noise ratio and iii) spectral editing to remove residual BSA background.

Particles and macromolecular components, including biopolymers (protein and carbohydrate), are viewed as the main foulants in the complex feed submerged membrane filtration systems such as membrane bioreactor (MBR). This work focused on two aspects of fouling in complex fluids: 1- Assessing fouling propensity and mechanisms for various model solutions. 2- Using of two specific solutions modelling biomass found in MBR for a better understanding of the fouling mechanisms in submerged MBR processes. Filtrations were carried out with 0.22 ??m PVDF hollow fibre membrane. Alginate was used as a model for polysaccharide, bovine serum albumin (BSA) as a model for protein, (un)washed yeast and bentonite were representing suspended solid contents. According to the data obtained during this study the fouling propensity of each model solution was classified as follow in a decreasing order: Alginate &gt unwashed yeast &gt washed yeast &gt BSA &gt bentonite for one-component solutions; and Alginate-washed yeast &gt Alginate-BSA &gt Alginate-bentonite &gt Alginate-unwashed yeast for two-component solutions. Introducing the alginate increased the reversible fouling (except BSA). Passive adsorption had a significant effect on fouling of alginate even before the beginning of the filtration. Washed yeast and a mixture of washed yeast + BSA were then used as model solutions to simulate the activated sludge found in MBR. The concentration of washed yeast and BSA used in this study were calculated in order for the characterisations of the two model solution to match (in terms of biopolymer contents) those of MBR biomasses reported in the literature. By rinsing, backwashing and chemical cleaning of the membrane, three fouling layers of upper, intermediate and lower were defined respectively. Results obtained from the analysis of the biopolymers found in the cleaning solutions allow a better understanding of the fouling mechanisms occurring for the two model solutions used in this study: for washed yeast, the lower layer and for washed yeast + BSA , the upper and intermediate layers were found to have relatively high biopolymeric composition. This was explained by higher concentration of solids on the membrane surface and by higher biopolymer interactions when washed yeast was mixed with BSA.

<p>In this thesis, the structure and stability of globular proteins adsorbed onto nanometer-sized hydrophilic silica particles were investigated using differential scanning calorimetry (DSC), hydrogen/deuterium exchange (HDX), and mass spectrometry (MS). The adsorption process itself was characterized with fluorescence and absorption spectroscopy and surface plasmon resonance (SPR). The combination of these methods offered a unique insight into adsorption-induced changes within proteins related to their adsorption characteristics. DSC contributed with thermodynamic information on the overall structural stability within the protein population. HDX in combination with MS contributed information on the structure and stability of adsorbed proteins with focus on changes within the secondary structure elements. In order to increase the structural resolution in this part of the investigation, proteolysis was performed prior to the MS analyzing step. Knowledge on the protein adsorption process was utilized in a practical approach called ligand fishing. In this approach, SPR was used to monitor the chip-based affinity purification of a protein with MS used for protein identification.</p><p>Adsorption isotherms revealed that electrostatic interactions play an important role in the adsorption of proteins to hydrophilic surfaces. DSC investigation revealed that the thermal stability of proteins reduces with increasing electrostatic attraction between the protein and the surface and that this effect diminishes at higher surface coverage. The mass-increase due to exchange between protein hydrogen atoms and deuterium atoms in solution was investigated as a function of time. This gave insight into adsorption-induced changes in the structural stability of proteins. By combining DSC and HDX-MS, it was possible to differentiate between adsorption-induced changes in the secondary and tertiary structure. Additionally, if limited proteolysis was performed, the investigations gave insight into the orientation and protein segment specific changes in the stability of proteins adsorbed to silica surfaces. The adsorption of proteins to silica particles also provided the basis for a new experimental design that allows handling of minute amounts of proteins in a ligand fishing application, as used in the field of functional proteomics.</p>