Masters of Science / Invasive pneumococcal disease (IPD), which includes potentially fatal conditions such as meningitis, septicaemia and pneumonia poses a threat in children aged <5 years, pneumonia being the leading cause of child mortality worldwide. Even though capsular polysaccharides are the main antigens involved in the immunity to encapsulated bacteria, it was found that in children in that age group, the immune system was unresponsive. Conjugate vaccines however induce immunologic memory and provide long-term protective immunity. Therefore the aim of this project was to develop novel conjugation strategies towards a pneumococcal conjugate vaccines and focuses mainly on the serotypes that are a burden to the African continent. The chemistry involved in developing a conjugate vaccine is of importance beacuse while some polysaccharides contain chemical grouping which can be conveniently utilized for conjugation, many medically important ones require derivatization before they can be coupled to protein. Derivatization of which can be achieved through various strategies, important to note is through hypervalent iodine oxidants. Two hypervalent iodine reagents, O-Methyl substituted-1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (Me-IBX)and modified 1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (mIBX)were successfully synthesized in preparation for the use in polysaccharide, polyribitol phosphate, (PRP) oxidation. The polysaccharide to be oxidised was first size reduced by microfluidisation to allow maximum oxidation. However, the extent to which oxidisation was achieved was not enough to conjugate the polysaccharide to the protein of preference, Bovine Serum Albumin, (BSA).

Although conventional two-dimensional (2D) cell culture is convenient for routine work, researchers are turning to three-dimensional (3D) cell culture for more accurate, physiologically representative information on the way their cells behave and respond to stimuli. Cells can now be routinely cultured in the many commercially available 3D formats. In this study, we developed non-woven scaffolds for 3D cell culture and enhanced cell function. By making use of methods that measure the behaviour of liver cells in the 3D system we were able to demonstrate, compared to standard 2D systems, significantly higher expression of key liver enzymes involved in drug metabolism and albumin production (specifically cytochrome P450). Cell proliferation on the various scaffolds was comparable to that of a commercially available hydrogel 3D cell culture system, AlgimatrixTM. When culturing cells in 3D, the means by which cells are harvested or extracted from the 3D scaffold for downstream applications is more challenging than in 2D. For this reason, many of the 3D scaffolds currently manufactured are either bio-degradable or require the use of salts to dissolve the scaffold which may negatively impact on the cells they contain. By grafting the non-woven scaffolds with the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), we demonstrated that cells growing on the scaffolds are able to be released from the scaffold in a 3D conformation, non-enzymatically, through temperature changes. Selected thermoresponsive non-woven fabrics were also tested in an automated cell culture device for cell proliferation and thermally induced harvesting. One of the applications of a 3D cell culturing system would be in exploration of the many diseases plaguing mankind, in particular malaria which is still responsible for severe disease and mortality, especially in Africa. Most available antimalarials are designed to target the pathogenic blood stages in humans and to address the constant threat of drug resistance. However, to meet the objective of malaria eradication, medicines that block parasite transmission also need to be developed. Molecules that efficiently target the parasite stages in the liver would prevent pathogenesis, symptoms and transmission. Equipped with the knowledge that the infectious sporozoites traverse several hepatocytes prior to cell infection, it may be physiologically limiting to culture the exo-erythrocytic stage in vitro in a 2D cell culture system where the hepatocytes are in an unnatural flat conformation, distinctly different to their in vivo counterparts. Moreover, monolayer cell cultures lose their tissue-related functions rapidly, greatly impairing the predictive power of such assays. Thus, the second aim of this thesis was to establish if hepatocytes that have been cultured on 3D non-woven scaffolds improve in vitro sporozoite invasion compared to conventional 2D systems. Sporozoite invasion was detected in the conventional 2D monolayers using a TaqMan® assay but not in the hepatocytes growing in 3D. Future studies beyond the scope of this thesis will include modifications to the 3D scaffold to attempt achieving superior sporozoite invasion in this model system. / Thesis (PhD)--University of Pretoria, 2013. / Biochemistry / unrestricted

Le stress oxydant correspond à un déséquilibre entre les défenses antioxydantes et les espèces pro-oxydantes en faveur de ces derniers. Il joue un rôle central dans de nombreuses pathologies telles que l'obésité, le diabète et les maladies cardiovasculaires. Le diabète est caractérisé par une hyperglycémie chronique, source d'un stress oxydant accru et de dommages oxydatifs tissulaires. Notamment l'hyperglycémie favorise la glycation des protéines aboutissant à la formation de produits avancés de glycation (AGE). Bien que l'action délétère des AGE soit reconnue dans le diabète, leurs rôles au niveau cardiaque et adipeux restent encore assez méconnus. L'objectif de mon travail de thèse a été de déterminer les effets du stress oxydant, induit par l'hyperglycémie et l'albumine glyquée, au niveau des tissus adipeux et cardiaque, mais aussi au niveau de lignées cellulaires adipocytaires et cardiaques. Mes résultats ont montré un impact délétère de l'hyperglycémie tant au niveau cellulaire que tissulaire. De plus, certains dysfonctionnements identifiés au niveau de cœurs ou de tissu adipeux provenant de modèle animaux diabétiques ont pu être reproduits in vitro par l'incubation de lignées cellulaires adipeuses ou cardiaques en présence d'albumine glyquée. Cette étude propose de nouveaux éléments de compréhension sur les dommages de type oxydatif dans le cadre de la pathologie diabétique et ouvre de nouvelle piste d'étude sur le rôle spécifique que pourrait exercer les AGE issus de l'albumine au niveau du tissu adipeux et cardiaque. / Oxidative stress is defined as “an imbalance between oxidants and anti-oxidants in favor of the oxidants”, leading to a disruption of redox signaling and control and/or molecular damage. It plays a central role in many diseases such as obesity, diabetes and cardiovascular diseases. Diabetes is characterized by chronic hyperglycemia, a source of increased oxidative stress and tissue oxidative damage. In particular, hyperglycemia can promote protein glycation leading to the formation of advanced glycation end products (AGEs). Although the deleterious action of AGEs in diabetes is recognized, its impact in the heart and adipose tissues remains relatively unknown. The objective of this thesis was therefore to determine the effects of hyperglycemia-induced oxidative stress together with glycated albumin on the redox balance of adipose and cardiac tissues, and also by in vitro analysis of heart and adipocyte cell lines.This study revealed enhanced oxidative stress and damage induced by hyperglycemia at both cellular and tissue levels. In addition, the oxidative damage identified in heart and adipose tissues isolated from diabetic mice could be reproduced in vitro by the incubation of adipose or cardiac cell lines in the presence of glycated albumin. The current study proposes novel insights into redox imbalance in adipose and heart tissues of diabetic/obese mice and highlights the role of AGEs (especially glycated albumin) as a putative contributor to adipocyte and cardiomyocyte dysfunction.

La mortalité du diabète de type 2 est liée à ses complications cardiovasculaires (CVD). L'identification de nouveaux biomarqueurs associés à la dysfonction endothéliale, permettrait d'en améliorer le dépistage, la prévention. Au cours du diabète de type 2, l'hyperglycémie est associée à un fort stress oxydant. Nous nous sommes proposés ici d'évaluer l'impact de la glycoxydation sur la principale protéine circulante, l'albumine, et d'identifier si les modifications glycoxydatives de l'albumine dans le diabète avait un rôle dans la phyiopathologie des CVD du diabète de type 2. Nous avons pu mettre en évidence des modifications structurales et fonctionnelles importantes de l'albumine au cours du diabète de type 2 avec la formation entre autres de produits avancés de glycation (AGEs). Ces modifications glycoxydatives sont associées à des effets cellulaires pro-oxydant et pro-inflammatoire via une augmentation de l'expression du Récepteur aux AGEs (RAGE). Ces observations suggèrent que les formes glycoxydées d'albumine présentent un rôle central dans les mécanismes menant à la dysfonction endothélilale. Il reste néanmoins à évaluer l'intérêt du dosage des formes modifiées de l'albumine dans le dépistage des CVD au cours d'une étude clinique prospective de grande ampleur. Le développement d'une méthode de dosage rapide et reproductible des fractions d'albumine modifiées, comme celui de l'IMA (albumine modifiée par l'ischémie), en faciliterait la mise en œuvre. / Type 2 diabetes is dramatically associated with an enhanced cardiovascular complication risk. The identification of novel biomarkers associated with endothelial dysfunction remains highly warranted to improve diabetes screening and prevention. Oxidative stress and protein modifications are frequently observed in numerous disease states. Albumin, the major circulating protein in blood, can undergo increased glycoxidation in diabetes. Objectives of my thesis were to clarify the impact of glycoxidative modification of albumin on its structure and its functions and to determine whether such impairments may be encountered in albumin purified from diabetics. The occurrence of oxidative modifications was found to be enhanced in in vitro glycoxidized HAS and albumin purified from diabetics, after determination of their free thiol group content, relative electrophoretic migration, carbonyl content, and antioxidant activities. In addition, glycoxidized albumin exhibited impaired pharmaceutic molecule binding capacities. Cells treated with glycoxidized albumin exhibited a proinflammatory state attested by an overgeneration of intracellular reactive oxygen species, enhancements in RAGE expression, and an accumulation of carbonylated proteins.Methods to detect IMA (ischemia modified albumin) were developed and applied to diabetics patients. Relationships have been established between specific pathological parameters (cardiovascular disorders, hyperglycemia…) with an enhanced glycoxidative modification of albumin in diabetics. We thus propose that impaired albumin structure and function in relation in the enhanced oxidant stress observed in diabetics might be involved in the increased mortality risk of these patients.

In this study the heat-induced gelation of two (readily available) proteins, which contain disulphide bonds, has been investigated over a range of protein concentrations in the presence and absence of the presence of the reductant, dithiothreitol at neutral pH. The proteins selected in this study were: β-Lactoglobulin and bovine serum albumin. These proteins have different number of disulphide bonds and possess different protein secondary structures. The influences of the reductant and protein concentration on their heat-induced gelation were explored to see whether the proteins were able to form protein hydrogels and that the mechanical properties of the resulting protein hydrogels were controllable. The tilting test tube method revealed that both proteins formed macroscopic hydrogels, at protein concentrations above the critical gelation concentration and that the critical gelation concentration was constantly lower in the presence of the reductant. Micro-DSC revealed that both proteins had completely denatured upon heating and that the denaturation temperature and enthalpy were significantly lower in the presence of the reductant. IR spectroscopy revealed that both proteins undergo major secondary structure transitions that resulted in the formation of fibers that are rich in β-sheet structure upon heating and that the protein lost some secondary structure before any heating and gained more β-sheet structure in the presence of the reductant. Both proteins had partially denatured before any heating in the presence of the reductant and that β-LG underwent aggregation that was accompanied by the loss of native β-sheet structure and the formation of intermolecular β-sheet structure, while that BSA underwent aggregation that was accompanied by the loss of native α-helix structure and the formation of intermolecular β-sheet structure. Cryo-TEM revealed that both proteins formed fibers (10 nm in diameter) that exist as single entities at low protein concentrations and become entangled into macroscopic networks, at protein concentrations above the critical gelation concentration and that more fibers and denser macroscopic networks were formed in the presence of the reductant. Oscillatory rheology revealed that both proteins formed macroscopic networks exhibit viscoelastic behaviour and that their elastic modulus had increased in the presence of the reductant and with increasing protein concentration.

A prevalência de diabetes mellitus (DM) está aumentando constantemente em todo o mundo a uma taxa alarmante. Devido às suas complicações que causam uma maior morbidade, invalidez e mortalidade, o DM representa um enorme problema para a saúde pública. Com isso, ações, tanto em diagnóstico como em tratamento, são necessárias para desacelerar a tendência atual e prevenir o desenvolvimento das complicações do DM. Recentemente, a hemoglobina glicada (HbA1c) foi introduzida nos critérios diagnósticos de DM. Os resultados de HbA1c são igualmente apropriados para o diagnóstico, apesar de não necessariamente detectarem DM nos mesmos indivíduos detectados pelos critérios de glicemia. No entanto, os níveis de HbA1c podem ser influenciados por qualquer condição que altere a vida útil dos eritrócitos e metabolismo da hemoglobina, independentemente da glicemia, resultando em erro diagnóstico neste grupo da população com estas condições. Além disso, estudos epidemiológicos revelaram que a glicemia pós-prandial tem um maior risco de causar complicações cardiovasculares em relação à hiperglicemia persistente e pode ser acessada com precisão usando a albumina glicada (AG) e não a HbA1c. Nesse contexto, estudos recentes têm evidenciado que a AG pode ser um marcador para diagnóstico do DM e também ser utilizado como um marcador alternativo à HbA1c para o controle glicêmico. No entanto, esses estudos, foram realizados apenas na população asiática e podem não ser aplicáveis a outros grupos étnicos. Por isso, mais investigações para a validação do desempenho diagnóstico da AG na predição do DM em diferentes grupos etnicos são necessárias. Neste estudo avaliamos o desempenho da AG no diagnóstico do DM em 242 indivíduos brasileiros com idade média de 54,4 anos (+ 13,0). Baseando-se nos valores de glicose plasmática durante o teste oral de tolerância à glicose (TOTG), o DM foi detectado em 31,8%. AG ≥16,8% apresentou acurácia similar para a detecção de DM conforme definido por HbA1c >6,5%. O uso da razão glicemia de 2h pós-sobrecarga de 75g de glicose (2hPG) e AG (2hPG/AG) melhora a sensibilidade, reduz o número de diagnósticos incorretos por AG ou HbA1c >6,5% e possui um acurácia comparável ao TOTG, indicando que o uso de uma estratégia aplicando a razão da glicemia pós-prandial (GPP) real e AG (GPP/AG) pode ser mais conveniente para pacientes e aumentar o desempenho diagnóstico do teste. Estudos para validar esta estratégia são necessários. / The prevalence of diabetes mellitus (DM) is constantly increasing worldwide at an alarming rate. Due to its complications that cause greater morbidity, disability and mortality, DM represents a heavy burden on public health. Therefore, actions in both, diagnosis and treatment, are necessary to slow down the current tendency and avoid the development of DM complications. Recently, the glycated hemoglobin test (HbA1c) was introduced in the diagnostic criteria for DM. The HbA1c results are equally appropriate for diagnostic testing, though not necessarily detect DM in the same subjects detected by plasma glucose criteria. However, blood HbA1c levels may be influenced by any condition that changes the lifespan of erythrocytes and hemoglobin metabolism regardless of glycemia, resulting in the misdiagnosis of this population group. In addition, epidemiological studies have shown that postprandial glycemia has a higher risk of causing cardiovascular complications than chronic hyperglycemia and can be accurately assessed using the glycated albumin (GA) test rather than HbA1c. In this context, recent studies have shown that GA may be a marker for the diagnosis of DM and also be used as an alternative marker to HbA1c on many occasions. However, these studies have been conducted only in the Asian population and may not be applicable to other ethnic groups. Therefore, further investigations to validate the diagnostic performance of GA in the prediction of DM in different ethnic groups are necessary. In this study, we evaluated the GA performance in the diagnosis of DM in 242 Brazilian individuals with a mean age of 54.4 years (+ 13.0). Based on plasma glucose values during oral glucose tolerance test (OGTT), DM was detected in 31.8%. AG ≥16.8% presented similar accuracy for detecting DM as defined by a HbA1c >6.5%. The use of the 2-h plasma glucose after a 75-g OGTT and GA (2hPG/GA) ratio improves sensitivity, reduces the number of incorrect diagnoses by GA or HbA1c >6.5% and has an accuracy comparable to OGTT, indicating that the use of approach applying the postprandial glucose (PPG) and GA (PPG/GA) may be more convenient for patients and increase the diagnostic performance of the test. Studies to validate this approach are needed.

Orientador: Dimas Tadeu Covas / Banca: José Orlando Bordin / Banca: Carmino Antônio de Souza / Banca: Orlando Castro e Silva Júnior / Banca: Aparecida Maria Fontes / Resumo: Alguns trabalhos realizados recentemente relatam que as células-tronco mesenquimais (CTM) podem ser induzidas à aquisição de marcadores hepatocíticos pelo transplante em modelos animais de dano hepático, ou pelo cultivo in vitro com fatores de crescimento e citocinas. O presente trabalho teve por objetivo avaliar o comportamento das CTM frente à indução da diferenciação hepatocítica. As CTM foram isoladas da medula óssea de quatro doadores saudáveis, caracterizadas e submetidas ao protocolo de indução à diferenciação hepatocítica in vitro e in vivo. As células induzidas in vitro apresentaram mudanças na sua morfologia, mostrando a morfologia semelhante à do hepatócito, porém, o perfil imunofenotípico não foi modificado. As células induzidas também não apresentaram o aumento dos transcritos de albumina, citoqueratina 18 e citoqueratina 19 quando analisadas por RT-PCR em tempo real, e não alteraram a expressão de albumina, citoqueratina 18 e alfafetoproteína como demonstrado por imunofluorescência. Quando analisadas in vivo, as CTM demonstraram o potencial migratório para o tecido hepático danificado de camundongos imunodeficientes. Em conjunto, os resultados sugerem que as CTM da medula óssea não são capazes de se diferenciar em hepatócitos quando estimuladas in vitro pela metodologia utilizado neste trabalho, mas são capazes de migrar para o tecido hepático danificado in vivo, o que sugere o seu papel no reparo do fígado. A contribuição para o reparo pode estar associada com o efeito parácrino dessas células. / Abstract: Some recently works have been reported that mesenchymal stem cells (MSC) can be induced to the acquisition of hepatocytic markers for the transplant in animal models of liver damage, or for the in vitro culture with growth factors and cytokines. The present work aim is to evaluate the behavior of the MSC in front of the induction of the hepatocytic differentiation. The MSC was isolated from the bone morrow of 4 normal donators, characterized and submitted to the protocol of in vitro and in vivo induction of hepatocytic differentiation. The in vitro induced cells showed morphology changes acquiring hepatocytes-like morphology. However, the immunophenotypic profile of those cells was not modified. The induced cells did not present increase of the albumin, cytokeratin 18 and cytokeratin 19 transcripts, when analyzed by real time RTPCR. The expression of albumin, cytokeratin 18 and alpha foetoprotein was also not modified as demonstrated by immunofluorescence. In vivo, the MSC have demonstrated the migratory potential for the damaged liver of immunodeficient mice. Together, the results suggest that the bone morrow MSC are not capable of in vitro hepatocytic differentiating according to the approach in this work, but are capable to homming into damaged hepatic tissue in vivo. This migration capacity suggests their role in the repair mechanisms. / Doutor

The T cell receptor (TCR) activation induced signaling cascade is a major driver of T cell effector responses such as cytokine production and actin cytoskeletal rearrangement. Characterizing chemical modulators of this pathway has the benefits of both revealing basic science knowledge about these signaling processes and providing foundation for development of novel therapeutics. Glycerol Monolaurate (GML) is a naturally occurring fatty acid monoester that is found as a monoglyceride in human breast milk and coconut oil. It is widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a wide range of bacteria, fungi, and enveloped viruses. Because of this, GML has been developed as a preventative for menstrual associated Toxic Shock Syndrome, and is being tested to prevent HIV transmission and superficial skin infections. Interestingly, GML suppresses mitogen induced lymphocyte proliferation and inositol triphosphate production, suggesting that GML has immunomodulatory functions. This thesis mechanistically examined how GML affects human primary T cells. Chapter III describes how GML potently altered order and disorder dynamics in the plasma membrane that resulted in reduced membrane-localized clustering of the proteins LAT, PLC-γ, and AKT, events integral for proper TCR signal propagation. Altered membrane signaling events induced selective inhibition of TCR-induced signaling events. Specifically GML reduced the phosphorylation of the regulatory P85 subunit of PI3K, and AKT and abrogated calcium influx. Functionally, GML treatment potently reduced TCR-induced production of the cytokines IL-2, IFN-γ, TNF-α, and IL-10. Chapter V shows that GML causes the mis-localization of the ARPC3 subunit of the Arp2/3 complex that leads to the formation of abnormal filopodia structures, and reduced cellular adhesion. Chapter V shows that human serum albumin binds directly to GML on the 12 carbon acyl chain. This interaction reverses GML induced suppression of TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane, AKT phosphorylation, and cytokine production. These findings establish GML as a T cell suppressive agent in addition to an antimicrobial agent. This observation reveals the potential role of naturally occurring GML in human breast milk in the formation of microbiota and immune tolerance in the infant gastrointestinal tract. It also allows for optimization of the current applications of GML in various commercial products and therapeutic strategies. Finally this information provides the rationale to investigate GML in new remedial avenues as a topical agent to treat excessive inflammation in the skin, and vaginal and gut mucosal regions.

This diploma thesis deals with preparation and subsequent characterization of albumin (BSA) and mixed hyaluronan-albumin (HA+BSA) hydrogels. The experimental characterization was based on the rheological measurements of the influence of ionic strength, pH and molar mass of HA on the gelation process and the properties of formed gels, as well as the time stability of these properties. Following object of the study was swelling of hydrogels in water and buffers with different pH value. Characterization was extended by thermal analysis, morphology description using scanning electron microscopy (SEM) and study of transport properties of hydrogels for selected model substance. Due to increasing ionic strength, the gel point was shifted towards higher temperature values and the resulting gels exhibited higher values of complex moduli. In case when pH was lower than isoelectric point of BSA, attractive electrostatic interactions between albumin and hyaluronan took place and the faster gelation occurred, compared to the case when pH was higher than isoelectric point of BSA. On the contrary, values of complex moduli increased with increasing pH. The gel samples prepared in the presence of low molecular weight HA reached higher values of complex moduli, compared to samples with the high molecular weight HA. Rheological properties of BSA gels were stable over time, whereas the mixed HA+BSA were characterized by significant decrease of complex moduli during the first three days of storage. Due to heating up to 100 °C, more than 80 % of the weight of studied samples was lost, as a result of water evaporation. Further heating of hydrogels, up to 250 °C, did not cause other changes associated with weight loss, in contrast with crystalline powder BSA, in which another weight loss at 220 °C was observed, as a result of the beginning degradation. Swelling studies, as well as SEM images, indicated higher crosslink density of BSA gels, compared to HA+BSA samples, which exhibited higher values of swelling ratio. The lowest values of equilibrium swelling ratio were observed at pH 4, which is close to the isoelectric point of albumin. Calculated diffusion coefficients relative to diffusivity of methylene blue in water ranged between the order of 10E-2 for diffusion into physiological solution and the order of 10E-3 for the diffusion into water. Ongoing transport of model dye from gel to solution was significantly slower, compared to its diffusion in water, as a result of gel porosity and electrostatic immobilization of positively charged molecules of methylene blue on the negatively charged BSA and HA chains.

The structural and mechanical properties of a surface often play an integral part in the determination of the cell adhesion strength and design parameters for creating a biodegradable electrospun scaffold. Nanofibers composed of the globular proteins bovine serum albumin (BSA) and fibronectin were produced by electrospinning with the electrospun protein scaffold serving as an extracellular matrix to which adhesion interaction will exist with cells via cell surface integrin. This interaction is vital in regulation cell differentiation, growth and migration and cell adhesion. We will demonstrate the ability to manipulate ligand-receptor interaction, the properties of the electrospun fibers, control and the formation of focal adhesions sites in cells cultured on the fibers with the ultimate goal of developing a biomimetric scaffold to investigate how cell adhesion molecules modulate cell behavior in a 3-dimentional culture.