Produtos de glicação avançada (AGE) são prevalentes no diabete melito e alteram o metabolismo de lípides e lipoproteínas. Neste estudo, avaliou-se a influência da albumina, isolada do soro de indivíduos controles (C, n =12) e de portadores de diabete melito tipo 1 (DM 1, n=13) e tipo 2 (DM 2, n=11), com controle glicêmico inadequado, sobre a remoção de colesterol de macrófagos, o acúmulo intracelular de lípides, o conteúdo do receptor de HDL, ABCA-1 e a captação seletiva de colesterol esterificado de HDL. Além disso, foi determinada a expressão diferencial de genes em macrófagos tratados com albumina C, DM 1 ou DM 2. A concentração plasmática de albumina glicada foi superior no grupo DM 1 e DM 2 em relação ao C e correlacionou-se positivamente com glicemia, hemoglobina glicada e frutosamina. Albumina sérica foi isolada por cromatografia para separação rápida de proteínas e purificada por extração alcoólica. Albumina DM 1 e DM 2 apresentaram maior conteúdo de carboximetil-lisina e apo A-I quando comparada à albumina C. Macrófagos enriquecidos com LDL acetilada e 14C-colesterol foram tratados com albumina C, DM 1 ou DM 2 e, a seguir, incubados na presença ou ausência de apo A-I, HDL3 ou HDL2 para determinação do efluxo de colesterol. Apesar de removerem maior quantidade de colesterol celular, as albumina DM 1 e DM 2 reduziram o efluxo de colesterol mediado por apo A-I (70% e 45%, respectivamente) e HDL2 (55% e 54%, respectivamente) em comparação à albumina C. Com HDL3, a queda no efluxo de colesterol só foi observada em macrófagos expostos à albumina DM 2 (55%). Macrófagos incubados apenas com albumina C, DM 1 ou DM 2 apresentaram conteúdo lipídico semelhante, evidenciado por coloração com Oil Red O. A adição de apo A-I, HDL3 ou HDL2 reduziu o conteúdo lipídico apenas nas células tratadas com albumina C, mas não com albumina DM 1 ou DM 2. A expressão de ABCA-1 foi diminuída 82% e 25% em macrófagos expostos, respectivamente, à albumina DM 1 e DM 2, em comparação às células tratadas com albumina C. As albuminas DM 1 e DM 2 reduziram a captação de 3H colesteril oleoil éter de HDL por células que superexpressam o receptor SR-BI. Estes resultados também foram obtidos com albumina humana modificada in vitro por glicação avançada. As albuminas isoladas dos pacientes diabéticos aumentaram a expressão de receptores envolvidos na captação de LDL modificadas e de proteínas que modulam vias da homeostase do colesterol. Os resultados deste estudo evidenciaram que a modificação in vivo da albumina por glicação avançada prejudica o transporte reverso de colesterol no diabete melito, por reduzir a expressão de ABCA-1 e a remoção de colesterol de macrófagos, bem como a captação seletiva de colesterol esterificado de HDL pelo SR-BI. Independentemente de variação na concentração e composição de HDL, a glicação da albumina pode contribuir para o acúmulo de lípides em macrófagos e gênese da aterosclerose no diabete melito / Advanced glycation end products are prevalent in diabetes mellitus and alter lipids and lipoprotein metabolism. In this study we analyzed the role of albumin, isolated from control individuals (C, n = 12) and uncontrolled type 1 (DM 1, n = 13) and type 2 (DM 2, n = 11) diabetes mellitus patients on macrophage cholesterol removal, intracellular lipid accumulation, expression of the HDL receptor protein level, ABCA-1and the uptake of esterified cholesterol from HDL. It was also investigated the differential gene expression in macrophages treated with C, DM 1 or DM 2 albumin. Glycated albumin was higher in DM 1 and DM 2 groups as compared to C and was positivetly correlated with glycemia, glycated hemoglobin and fructosamine. Serum albumin was isolated by fast protein liquid chromatography and alchoolic extraction. DM 1 and DM 2 albumin presented a higher amount of carboxymethyllysine and apo A-I as compared to C albumin. In order to determine cholesterol efflux acetylated LDL and 14C-cholesterol enriched J- 774 macrophages were treated with C, DM 1 or DM 2 albumin and then incubated in the absence or presence of apo A-I, HDL3 or HDL2. Although presenting a higher ability to remove cell cholesterol by itself, DM 1 and DM 2 albumin reduced cholesterol efflux mediated by apo A-I (70% e 45%, respectively) and by HDL2 (55% e 54%, respectively) as compared to C albumin. With HDL3 the reduction of the cholesterol efflux was only observed in macrophages treated with DM 2 albumin (55%) in comparison to C albumin. Macrophages incubated with C, DM 1 or DM 2 albumin alone presented similar amount of intracellular lipids as assessed by Oil Red O staining. The addition of apo A-I, HDL3 or HDL2 reduced the lipid content in cells treated with C albumin, but not in those exposed to DM 1 or DM 2 albumin. The expression of ABCA-1 was reduced 82% and 25% in macrophages treated, respectively, with DM 1 or DM 2 albumin, in comparison to C albumin. DM 1 and DM 2 albumin reduced the uptake of 3H colesteril oleoyl ether from HDL by SR-BI overexpressing cells. These findings also were obtained when cells were treated in vitro with human serum albumin submitted to advanced glycation. DM 1 and DM 2 albumin enhanced the expression of receptors involved in the uptake of modified LDL and cholesterol homeostasis. Our findings showed that the advanced glycation of albumin that takes place in diabetes mellitus impairs the reverse cholesterol transport efficiency by reducing the ABCA-1 expression and cholesterol exportation to HDL and also by diminishing the uptake of esterified cholesterol from HDL. Independently of changes in HDL composition and concentration, advanced glycated albumin contributes to cholesterol accumulation in macrophages and atherogenesis in diabetes mellitus

The emergence of microneedle (MN) technologies offers a route for a pain free, straightforward and efficient way of transdermal drug delivery, but technological barriers still exist which pose significant challenges for manufacture of MN systems with high volume outputs at low cost. The main aim of this research was to develop new ways for MN manufacture primarily using micro-injection moulding processes with high performance engineering thermoplastics. During the moulding process these polymeric melts will be subjected to extreme stress and temperature gradients and detailed material characterisation combined with in-line monitoring is desirable to optimise the moulding parameters and will help in achieving sharp microneedles with acceptable quality. Hence high shear rheology of these selected materials was performed at wall shear rates carried out in excess of 107 s-1 over a range of temperatures to predict the flow behaviour of polymer melts at such high shear strain rates. This information was fed into injection moulding simulation software tools (Moldflow) to assist the MN production process design. The optimal design was then used to produce a full 3D solid model of the injection mould and mould insert. Furthermore various design of experiments were conducted considering input parameters such as injection pressure, injection speed, melt temperature, filling time and mould cavity temperature. Response variables including product quality and data acquired from the cavity pressure and temperature transducers were used to optimise the manufacturing process. The moulded MNs were geometrically assessed using a range of characterisation techniques such as atomic force microscopy, confocal microscopy and scanning electron microscopy. An attempt to make hollow MNs was performed and encountered many challenges like partial cavity filling and part ejection during processing. Studies were carried out to understand the problem and identified the major problem was in tool design and improvements to the moulding tool design were recommended. Plasma treatment and mechanical abrasion were employed to increase the surface energy of the moulded polymer surfaces with the aim of enhancing protein adsorption. Sample surface structures before and after treatment were studied using AFM and surface energies have been obtained using contact angle measurement and calculated using Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness resulting in better adsorption and release of BSA. To assist design-optimisation and to assess performance, a greater understanding of MN penetration behaviour is required. Contact stiffness, failure strength and creep behaviour were measured during compression tests of MN against a steel surface, and in-vitro penetration of MNs into porcine skin. The MN penetration process into porcine skin was imaged using optical coherence tomography. Finally, a finite element model of skin was established to understand the effect of tip geometry on penetration. The output of findings from this research will provide proof of concept level development and understanding of mechanisms of MN penetration and failure, facilitating design improvements for micro-injection moulded polymeric MNs.

The Caco-2 cell assay is widely used for in vitro permeability measurements. However, a draw back with the assay that this study will focus on improving, is compound adsorption to the plastic material. Lipophilic compounds such as Cyclosporin A and Peptide J, that will be used in this study, tend to bind to the plastic material in the assay. This can result in poor recovery and misleading permeability predictions. Bovine serum albumin (BSA) is an alternative used today to prevent this but is not always successful.    The aim of this study is therefore to improve the Caco-2 permeability assay by adding phospholipid covered silica beads (PLB) to the basolateral chamber. The role of the PLB is to bind the compound of interest and decrease the amount of compound bound to the plastic material and thus better predict the permeability of the compound of interest.   The PLB was produced using phosphatidylcholine and silica beads. Caco-2 cells were seeded and maintained for 21-29 days ahead of the experiment. PLB concentration of 20, 60 and 100 mg/ml were prepared. Samples were analyzed with HPLC-MSMS. The results showed that with increasing PLB concentration we had a significantly decrease in non-specific plastic binding resulting in reliable permeability predictions, concluding that the hypothesis was correct.

No description available.

I characterize the behavior of microcantilever arrays which utilize the in-plane photonic transduction that I've previously developed and evaluate the performance of the microcantilever arrays in simple sensing scenarios with integrated microfluidics. First the thermal responses of microcantilevers with a variety of patterns of deposited gold films are compared. Using a scanning electron microscope, I observe the deflection thermal sensitivities of 300 µm long microcantilevers to be -170.82 nm/K for a full gold coating and -1.93 nm/K for no gold coating. Using the photonic transduction method I measure a thermal sensitivity of -1.46 nm/K for a microcantilever array with no gold. A microcantilever array integrated with microfluidics is exposed to a solution of bovine serum albumin (BSA) followed by solutions of various pH's. In all cases I observe a previously unreported transient deflection response. We find that the transient response is due to temporary nonuniform concentration distributions. In response to nonspecific binding of BSA, I observe a transient surface stress of -0.23 mN/m that agrees well with the -0.225 mN/m predicted by simulations. We hypothesize that the deflection response to pH changes is due to stress generated by conformational changes of bound BSA.The deflection response of an integrated microcantilever array to different types of flow and different flow rates is observed. Simulations of the deflection response match well with experimental results but disagree at higher flow rates. For flow rates greater than 200 µL/min, the limitation of the differential signal's dynamic range becomes apparent. We then investigate flow driven by an on-chip reciprocating reservoir pump. We demonstrate that it is possible to use the reciprocating pump to achieve high flow rates while making deflection measurements in-between reservoir actuations. Investigations of the microcantilever array noise show that flicker noise dominates below 10 Hz, while above 10 Hz, readout noise dominates. A minimum deflection noise density of 15 pW/√Hz is achieved. To improve the signal-to-noise ratio I develop algorithms for a digital lock-in amplifier with a digital phase-lock loop. In simulation the lock-in amplifier is able to improve the SNR by up to a factor of 6000, and self-lock to a noisy carrier signal without an external reference signal.

No description available.

No description available.

No / Biosimilar pharmaceuticals are complex biological molecules that have similar physicochemical properties to the originator therapeutic protein. They are produced by complex multi-stage processes and are not truly equivalent. Therefore, for a biosimilar to be approved for market it is important to demonstrate that the biological product is highly similar to a reference product. This includes its primary and higher order structures and its aggregation behaviour. Representative lots of both the proposed biosimilar and the reference product are analysed to understand the lot-to-lot variability of both drug substances in the manufacturing processes. Whilst it is not easy to characterise every variation of a protein structure at present additional analytical technologies need to be utilised to ensure the safety and efficacy of any potential biosimilar product. We have explored the use of Taylor dispersion analysis (TDA) to analyse such batch to batch variations in the model protein, bovine serum albumin (BSA) and compared the results to that obtained by conventional dynamic light scattering analysis (DLS). Inter and intra batch differences were evident in all grades of BSA analysed. However, the reproducibility of the TDA measurements, enabled the stability and reversibility of BSA aggregates to be more readily monitored. This demonstrates that Taylor dispersion analysis is a very sensitive technique to study higher order protein states and aggregation. The results, here, also indicate a correlation between protein purity and the physical behaviour of the samples after heat shocking. Here, the protein with the highest quoted purity resulted in a reduced increase in the measured hydrodynamic radius after heat stressing, indicating that less unfolding/aggregation had occurred. Whilst DLS was also able to observe the presence of aggregates, its bias towards larger aggregates indicated a much larger increase in hydrodynamic radii and is less sensitive to small changes in hydrodynamic radii. TDA was also able to identify low levels of larger aggregates that were not observed by DLS. Therefore, given the potential for immunogenicity effects that may result from such aggregates it is suggested that TDA may be suitable in the evaluating detailed batch to batch variability and process induced physical changes of biopharmaceuticals and biosimilars.

Alzheimer's disease (AD) was first described more than 100 years ago and is today the most common cause of dementia. It is one of the progressive neurodegenerative diseases that affect 47 million people around the world between the ages of 60 and 90. One of the contributing factors to AD is extracellular amyloid – β (Aβ) plaques that form as a result of protein aggregation. These Aβ proteins are neurotoxic, leading to degeneration of brain neurons and loss of cognitive abilities. Because AD largely affects society, researchers are constantly working to find a cure, which currently does not exist. The purpose of this study was to use Drosophila melanogaster as a living organism model for the expression of two types of Aβ proteins related to AD, Arctic (Glu22Gly) and TandemAβ, and to study the survival of these AD flies when Bovine serum albumin (BSA) was added to the fly food. The hypothesis was that BSA would be effective in slowing down and/or preventing formation of toxic Aβ-aggregates. The focus was therefore to investigate whether the AD flies would live longer if they were allowed to eat Bovine serum albumin and whether the soluble/insoluble Aβ levels in these flies would decrease in comparison to the control AD flies that were not allowed to eat BSA. The effect of BSA on toxicity was evaluated using survival assay on male flies and the levels of soluble/insoluble Aβ were evaluated using Meso Scale Discovery (MSD) on female flies. In both experiments, the following six groups of flies were examined: myow1118 ± BSA; myoArctic ± BSA; myoTandemAβ ± BSA. Conclusions from the studies are that the survival of AD flies could not be extended by adding 0.61 mM BSA to the food, rather the data showed a weak but significant toxic effect in the presence of BSA in the AD flies. However, MSD data showed a reduction of insoluble Aβ aggregates and an equilibrium shift from insoluble Aβ aggregates to soluble Aβ aggregates in the presence of BSA in the AD flies. Equilibrium shifts were particularly detectable in Myo-TandemAβ flies fed with BSA. In Myo-Arctic flies fed with BSA only reduction of insoluble Aβ could be detected. This shows that it is not the amount of Aβ aggregates that is decisive for toxicity, but rather the presence of specific aggregates that have toxic properties. If BSA shows good results in further studies, it could be used in the future to improve AD symptoms in patients.

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.