The objective of my PhD study was to develop and characterize new methods and sensors based on fluorescence resonance energy transfer (FRET) for bioanalysis. Chapter 3 describes the use of FRET between donor fluorophores and acceptor labeled murine macrophage cells. FRET microscopy was used to determine whether the donor molecules truly permeate through the cell membrane or only adsorb to the cell surface. This method was found to be partially successful since the donor red tail fluorescence overlapped with the sensitized acceptor fluorescence and led to false reading of FRET. We found that is easier to monitor delivery of acceptor molecules into donor-labeled cells. Using donor labeled cells it was possible to determine whether the acceptor molecules were actually delivered into cells. However, a relatively high acceptor concentration in the hundreds of micromolar level was needed to obtain measurable FRET signals in the 3-D cellular system. The results underscored the need to reduce the dimensionality of FRET systems in order to increase the FRET efficiency between donor and acceptor molecules. Chapter 4 describes the development of FRET sensing lipobeads labeled with donors and their use to evaluate the interactions of acceptor molecules with the phospholipid membrane of FRET sensing lipobeads. The change in the dimensionality of the system in which FRET occurs, improved the sensitivity of our measurements by 3-folds compared to FRET measurements in solution. We concluded that a molecular recognition component had to be added to the sensing particles to further increase their selectivity and sensitivity. Chapter 5 describes the development of FRET trap sensing beads and their use for screening nonfluorescent carbohydrates and glycoproteins. The FRET sensing technique was based on binding between dextran molecules labeled with Texas Red (Dextran-TR) and polystyrene microparticles labeled with Fluorescein tagged Concanavalin A (FITC-ConA). It was found that carbohydrates and glycoproteins inhibit the binding between dextran-TR and FITC-ConA labeled particles. The inhibition effect was concentration dependent thus enabled screening carbohydrates and glycoproteins based on their inhibition potency. The dissertation critically evaluates the performance of FRET microscopy and FRET based sensors in delivery and screening applications.

In summer of 2012 archaeological excavations were conducted at the Iberville Housing Projects in New Orleans, Louisiana. The excavations were conducted in order to gather archaeological data pertaining to the site’s history as part of New Orleans’ notorious vice district, Storyville. During excavation a cache of 765 turquoise glass seed beads was uncovered along the east wall of Test Unit #1. The cache, found at a depth of around 83 cm below the ground surface, suggests, in conjunction with other artifacts found at this level, that the beads were deposited at the site between 1810 and 1830. This cache of seed beads is unique at the site both in its context and in the quantity of beads that were found. The presence of the bead cache suggests that there may have been an active trading economy at the site, as beads similar to those found at the Iberville site are important elements in informal economies of the eighteenth century. This paper discusses the possibility that an alternative or informal reciprocal, non-cash based economy was in operation on the periphery of New Orleans in the early nineteenth century.

The present research investigates the interaction between Sri Lanka and its closest neighbouring region, South India, during the Early and Middle Historic periods. This connection has often been studied based on the textual evidence available on either side with little regard for the material evidence. Therefore, previous studies have fallen short of providing an objective understanding of the interactions. Furthermore, the focus of previous studies has been large-scale, state-mandated interaction. In contrast, this study has adopted a novel approach through the perspective of personal adornment and has been able to trace far closer contact between the two regions than official interaction suggests. To understand interpersonal interactions between the two regions, objects of personal adornment from seven different sites in Sri Lanka and South India were analysed. The patterns that emerge from assemblages of objects of adornment, including beads and other non-bead adornments were observed. The rationale behind this analysis was that body beautification expresses the individual and social identities of people across time and space. Consequently, it was anticipated that, by observing artefacts that are expressions of the identities and preferences of the general populace, this would shed light on interpersonal contact between the two regions. The patterns visible from the analysis of assemblages illustrate strong similarities between the two regions, during the Early and Middle Historic Periods. This is likely to have been the result of exchanging goods, ideas and technological knowledge. This study has also revealed that amidst shared cultural traits, each region developed preferences distinct from each other. The ethnographic study carried out provides further evidence of interactions between the two regions, which is missing in the texts and the archaeological record. These interactions probably reflect those which existed in the past. The integrated evidence used in this study clearly indicates longstanding, continuous personal-level interactions, between Sri Lanka and South India, which were hitherto unknown.

La chimioembolisation est une thérapie loco-régionale qui consiste à injecter, au moyend’un microcathéter, un principe actif et un agent d’occlusion vasculaire de manière la plussélective possible dans les artères nourricières du processus pathologique. Les microsphèresde chimioembolisation sont des microsphères calibrées et chargeables en principe actif,développées ces dernières années afin d’optimiser la chimioembolisation et permettre unelibération ciblée et contrôlée du principe actif au sein du territoire pathologique. L’utilisationde ces microsphères n’a encore jamais été appliquée à la chimioembolisation du poumon. Ellepourrait être intéressante dans le traitement des tumeurs pulmonaires malignes en permettantune imprégnation de la tumeur par un anticancéreux, tout en évitant une toxicité systémiquede ce dernier et dans le traitement des hémoptysies massives en évitant les récidives, dues àune recanalisation des vaisseaux après embolisation, par l’utilisation d’un inhibiteur duremodelage vasculaire.Notre travail a consisté à évaluer les performances de libération de l’irinotécan et dusirolimus à partir des microsphères de chimioembolisation au niveau systémique et au niveautissulaire, sur des modèles de chimioembolisation pulmonaire chez la brebis. Nos résultats ontmontré que les microsphères de chimioembolisation ne permettaient pas une délivrancetissulaire prolongée de l’irinotécan pour espérer obtenir une imprégnation efficace d’un lobepulmonaire. Les microsphères chargées en sirolimus semblent permettre une libérationcontrôlée du principe actif et paraissent intéressantes pour prévenir la recanalisation.Les microsphères de chimioembolisation doivent être améliorées pour permettre unelibération prolongée du principe actif. Des études complémentaires notamment en termesd’efficacité (modèle tumoral) doivent être réalisées pour montrer l’intérêt d’utiliser lachimioembolisation pulmonaire par microsphères en pratique clinique. / Chemoembolization is a loco-regional therapy, which consists of delivering selectively and directly to the pathologic area, by means of catheters through the vasculature, a drug and an embolic agent. The purpose is to achieve nutrient and oxygen starvation of the tumor, to minimize chemotherapy wash-out with prolonged contact with tumor tissue and therefore to increase the local drug concentration and reduce systemic toxicity. Drug eluting beads are a new generation of calibrated embolization beads, which behave as a drug delivery system. They have been developed in order to optimize chemoembolization and to control precisely the release and the dose of drug into the treatment site. Drug eluting beads have never been used for lung chemoembolization. It may be interesting to evaluate them in the treatment of lung tumors in order to impregnate the tumor with an anticancer drug while avoiding systemic toxicity of this drug and in the treatment of massive hemoptysis to avoid recurrences, induced by a recanalization of vessels after embolization, by using an inhibitor on vascular remodeling. Our purpose was to evaluate the release performances of irinotecan and sirolimus from drug eluting beads, in systemic circulation and in lung tissue, in sheep lung chemoembolization models. Our results showed that drug eluting beads did not allow a sufficient sustained delivery of irinotecan to expect to obtain an effective impregnation of a pulmonary lobe. Sirolimus eluting beads seem to allow a drug controlled release and appear interesting to prevent recanalization. Drug eluting beads have to be improved in order to allow sustained and controlled release of the drug. Complementary studies especially efficacy studies have to be investigated for showing the interest to use lung chemoembolization with drug eluting beads in clinical practice.

Integrins are transmembrane heterodimeric proteins that link extracellular matrix (ECM) to cytoskeleton and have been shown to function as mechanotransducers in non-muscle cells. Synthetic integrin-binding peptide triggers Ca2+ mobilization and contraction in vascular smooth muscle cells (VSMCs) from rat afferent arteriole, indicating that interactions between ECM and integrins modulate vascular tone. RGD, an integrin binding peptide, triggered contraction in cultured VSMCs as observed by Electric Cell-Substrate Impedance Sensing technique. To examine whether integrins transduce extracellular mechanical stress into intracellular Ca2+ signaling events in VSMCs, unidirectional mechanical force was applied to freshly isolated renal VSMCs through paramagnetic beads coated with fibronectin (FN, natural ligand of α5β1 integrin in VSMCs). Pulling of fibronectin-coated beads with electromagnet triggered Ca2+ sparks, followed by global Ca2+ mobilization. Paramagnetic beads coated with low-density lipoprotein (LDL), whose receptors are not linked to cytoskeleton, were minimally effective in triggering Ca2+ sparks and global Ca2+ mobilization. Pre-incubation with ryanodine, cytochalasin-D, or colchicine substantially reduced the occurrence of Ca2+ sparks triggered by fibronectin-coated beads. Binding of VSMCs with antibodies specific to the extracellular domains of alpha5 and beta1 integrins triggered Ca2+ sparks simulating the effects of fibronectin-coated beads. Anti-β2- integrin antibody served as the negative control. Traction force microscopy studies showed that only the force transduced via integrins could potentially trigger cytoskeletal remodeling in cultured VSMCs. Atomic force microscopy revealed a significant increase in surface roughness in VSMCs when treated with RGD peptide though there was no difference in the maximum deflection of the force curves. Pre-incubation of microperfused afferent arterioles with ryanodine or integrin specific binding peptide inhibited pressure-induced myogenic constriction. In conclusion, integrins transduce mechanical force into intracellular Ca2+ signaling events in renal VSMCs. Integrin-mediated mechanotransduction is probably involved in myogenic response of afferent arterioles. Thus, integrins can potentially act as sensors for myogenic response phenomenon and affect the autoregulatory mechanism in the vasculature.

Em levantamentos realizados no acervo do Centro de Arqueologia de São Paulo, nas coleções relativas aos sítios arqueológicos localizados na região do triângulo histórico de São Paulo e adjacências, foram identificadas 29 contas de vidro e de material orgânico em três contextos do século XIX: a Praça das Artes, o Solar da Marquesa de Santos e a Casa n.°1. Através da análise das técnicas de produção, são levantadas informações sobre cronologia e origem desses artefatos. Em uma pespectiva contextual global, essas miçangas dialogam com outras de contextos africanos ou da diáspora africana, tal como o Cais do Valongo, no Rio de Janeiro e Kindoki, no Congo. São Paulo no século XIX era uma cidade com forte presença da população africana. Eles estavam nas ruas, praças, pontes, chafarizes, mercados e igrejas, com seus batuques e capoeiras, formando verdadeiros territórios negros através de materialidades, identidades e agências. As contas e miçangas delimitavam hierarquias sociais internas a esses grupos e participavam da construção da paisagem negra da cidade de São Paulo. / The Archaeological Center of São Paulo have collections related to archaeological sites located in the region at the São Paulo downtown and its surroundings. Between them, 29 glass and organic beads were identified in three 19th century contexts: the Praça das Artes, the Solar da Marquesa de Santos and the Casa n.1. Through the analysis of the production techniques, information about the chronology and origin of these artifacts is collected. From a global contextual perspective, these beads dialogue with others from African or African diaspora contexts, such as the Cais do Valongo in Rio de Janeiro and Kindoki in the Congo. São Paulo in the 19th century was a city with strong presence of African population. They were in the streets, squares, bridges, fountains, markets and churches, with their batuques and capoeiras, shaped the territories black through materialities, identities and agencies. The beads and miçangas delimited social hierarchies internal to these groups and participated in the construction of the black landscape of the city of São Paulo.

PHAs are a class of biopolymers consisting of (R)-3-hydroxy-fatty acids and are produced by the majority of eubacteria and some archaeal bacteria as carbon storage material. In general, PHA is synthesised when a carbon source is available in excess while another essential nutrient is limited. The key enzyme of PHA biosynthesis, the PHA synthase, catalyses the polymerisation of the substrate (R)-3-hydroxyacyl-CoA to PHA accompanied by the release of coenzyme A. PHA is stored intracellularly as inclusions, the so-called PHA granules. When the external carbon source becomes exhausted, bacteria can metabolise these carbon inclusions by degradation of the polymer. PHA granules are water-insoluble, spherical inclusions of approximately 50-500 nm in diameter which consist of a hydrophobic polyester core surrounded by a phospholipid layer with embedded and attached proteins. One could consider isolated PHA granules as bio-beads due to their structure and size. In this study we tested if the PHA synthase can be used as an anchor molecule in order to display proteins of interest at the PHA granule surface. Furthermore, these modified PHA granules were analysed for their potential applicability as bio-beads in biotechnological procedures. The concept of using the PHA synthase as granule-anchoring molecule for display of proteins of interest was established by the functional display of the ß- galactosidase at PHA granules. This “proof of concept” was followed by the display of biotechnologically more interesting proteins. The IgG binding domain of protein A as well as streptavidin, which is known for its biotin binding ability, were fused to the PHA synthase, respectively, and therefore localised at the PHA granule surfaces during PHA granule assembly, resulting in functional bio-protein A -beads and bio-streptavidin-beads. Moreover, their applicability in biotechnological assays was demonstrated. Recently, we fused the green fluorescent protein (GFP) to the PHA synthase and demonstrated that the PHA granule assembly does not start randomly distributed in the cytoplasm but occurred localised at or near the cell poles. To further investigate if the localisation of the PHA granule formation process is due to polar positional information inherent to the PHA synthase, different mutated versions of the PHA synthase of Cupriavidus necator were created and analysed for a potential alteration in localisation. Furthermore, the phasin protein PhaP1 of C. necator was fused to HcRed, a far-red fluorescent protein, and localisation studies were accomplished when the fusion protein was expressed under different conditions in Escherichia coli.

<p>I have statistically worked up a material from 228 graves from the late Iron Age in the area of Mälardalen.</p><p>In my material I have gathered the incidence of combs, knifes, beads, weapons whetstones and torshammarsrings.</p><p>I have found that beads are overrepresented in graves of women and whetstones in graves of men. I only found weapons in graves from male.</p><p>I found no indication from my statistic hypothesis (Z-test) that a grave that contains more than three beads should define the grave of a woman. A grave that contains a lot of beads, more than 20, consider I as a female gender.</p><p>Combs, knifes and torshammarsring are considered as gender neutral.</p>

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types. Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection. Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate. This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.