Intracellular calcium is a major determinant of a wide variety of cell functions and thus of organ function. In order to get a clear picture of the intracellular calcium status it is preferable to assess the content of the various intracellular calcium pools as well as the characteristics of the transmembrane calcium movements, Le., the magnitude of the transmembrane Ca2+ flux upon stimulation and the rate of the subsequent return to baseline levels. The first aim of this study was to establish and evaluate the methods in the laboratory. The methods investigated include atomic absorption spectrometry, graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry for the determination of the total cell calcium content, fluorescence spectrophotometry for the determinations of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The methods eventually identified as feasible included fluorescence spectrophotometry for the determination of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The newly developed fluorescent calcium indicator, fura-PE3, was presently shown to be the most reliable fluorescent indicator for the intracellular free Ca2+ determinations. The best method for the calcium localisation by transmission electron microscopy was an adaptation of the antimonate precipitation technique. The following objectives were set in order to contribute to the knowledge in chronic renal failure; examination of the intracellular free Ca2+ content in the neutrophils of end stage renal failure patients on maintenance haemodialysis treatment, as the result of renal failure, dialysis treatment and medication combined; examination of the characteristics of the transmembrane Ca2+ movements; investigation of the intracellular calcium distribution in the neutrophils; exploration of a possible link between the alterations in intracellular calcium status and factors known to influence the calcium status, including the lipid composition of the membrane, the oxidative status as reflected by anti-oxidant vitamin levels, as well as the levels of parathyroid hormone, and ionised serum calcium. This study involved 14 chronic renal failure patients on maintenance haemodialysis. An increase in intracellular free Ca2+, the magnitude of the transmembrane Ca2+ flux upon fMLP stimulation and an increase in the rate of the subsequent decrease in intracellular free calcium were found. In separating the patients into those receiving rHuEPO and those not receiving rHuEPO, it was seen that the significance in the increase in intracellular free Ca2+ could be ascribed to the values obtained in those patients receiving rHuEPO - despite the fact that they were the only patients receiving calcium channel blockers. No overt indications of oxidative stress could be detected by anti-oxidant vitamin levels. Nevertheless, a decrease in the content of specific membrane fatty acids occurred, supporting the previous suggestions of the presence of a mild chronic inflammatory condition in the chronic renal failure patient on maintenance haemodialysis treatment. These results suggest that factors other than those associated with uraemia, such as rHuEPO administration, might result in an increase in intracellular free Ca2+ in cells of CRF/MHT patients. The magnitude of the rHuEPD-induced increase in intracellular free Ca2+ and the effects of the various calcium channel blockers need urgent further investigation as ineffective counteraction of the rHuEPO effect, as indicated by the relative ineffectivity of Norvasc, may have serious side-effects. / Dissertation (MSc)--University of Pretoria, 2000. / Physiology / unrestricted

Cells transduce signals from the external environment to the inside through phosphatidylinositol-3,4,5-phosphate (PIP3), a major signaling lipid on the plasma membrane. PIP3 is generated by the action of a family of lipid kinases called Class I phosphoinositide 3-kinases (PI3Ks) and controls an array of essential cellular functions including growth, proliferation, survival, metabolism and cytoskeletal architecture. PI3Ks are large heterodimeric complexes composed of a catalytic p110 subunit and a regulatory subunit. Crucial to healthy PIP3 production is the interpretation of diverse activating inputs arising from signaling proteins on the membrane by these subunits. A member of the PI3K family, PI3Kγ is a master regulator of immune functions with therapeutic implications in cancer immunity and inflammatory disease. PI3Kγ is distinct from other well studied PI3Ks due to the presence of unique regulatory mechanisms that control its ability to integrate signals from G-protein coupled receptors, small GTPases, immunoglobulin receptors and toll-like receptors. However, unlike the other well characterized PI3Ks, there are significant gaps in understanding of the molecular details of these mechanisms and how regulatory processes are translated into functions elicited by PI3Kγ in its unique milieu within the immune system. To understand PI3Kγ regulation, I utilized a synergy of cutting-edge approaches including protein biochemistry, X-ray crystallography, cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry to elucidate the unique regulatory features within its catalytic and regulatory subunits and how these features are disrupted in disease. These studies significantly advanced our understanding of how this enzyme functions and provided novel avenues for potentially targeting the enzyme better in therapy. This dissertation will consist of an introduction chapter summarizing PI3Kγ regulation and its role in disease, followed by three data chapters investigating previously uncharacterized regulatory mechanisms that control its function and how these mechanisms are implicated in disease. These data chapters are followed by a final chapter describing conclusions and future directions. In summary, the work presented in this thesis provides novel insights into the unique regulatory features in the catalytic and regulatory subunits of PI3Kγ that mediate its stimulation by upstream activating partners and the mechanisms by which these features are disrupted in disease. Further, these studies have facilitated the effective characterization of potent molecules that can specifically target PI3Kγ in disease. Altogether, the findings of this dissertation constitute a major advancement in our understanding of PI3K regulation. / Graduate

Die vorliegende Dissertation beschäftigt sich mit der Optimierung und der Untersuchung der Materialeigenschaften des thermoelektrischen Materials Ag1-xPb18Sb1+yTe20 (englisch Lead-Antimony-Silver-Telluride: LAST). Bei LAST handelt es sich um Bleitellurid mit geringen Anteilen von Silber und Antimon, welche teilweise gelöst den Gitterplatz von Blei substituieren (Einbau in PbTe-Matrix) bzw. Fremdphasen auf m- und nm-Skala bilden. Seine hohe thermoelektrische Güte wird dabei hauptsächlich der geringen thermischen Gitterleitfähigkeit zugeschrieben, die in ersten Veröffentlichungen mit dem Auftreten nanoskaliger, Silber- und Antimonreicher Einschlüsse und deren Funktion als Phononenstreuer erklärt wurde. Das durch Schmelzsynthese hergestellte Bulkmaterial wurde im Rahmen der Arbeit durch Gefügeabbildung und Elementanalytik untersucht. In Kooperation mit den Projektpartnern sollte daraus eine Korrelation von Struktur- und Funktionseigenschaften abgeleitet, sowie eine reproduzierbare Syntheseroute entwickelt werden. Die elektronenmikroskopische Abbildung der Mikrostruktur erfolgte dabei auf zwei Größenskalen. Auf der µm-Skala wurde die Oberfläche des Bulkmaterials auf Homogenität und Zusammensetzung sowie Anteil des Fremdphasenbestands untersucht. Trotz des sehr komplexen Phasenbestandes aufgrund des quaternären Phasendiagramms und der Vielzahl relevanter Syntheseparameter konnte ein Zusammenhang zwischen Zusammensetzung (Regulierung des Silber- und Antimonanteils bzw. dessen Verhältnis), Temperbedingungen und thermoelektrischen Eigenschaften hergestellt werden. Mithilfe des detektierten Phasenbestandes konnte die Existenz einer Mischungslücke im quasibinären Phasendiagramm 2PbTe-AgSbTe2 nachgewiesen werden. Dabei bilden die Zusammensetzungen zwei der ermittelten Fremdphasen die Phasengrenzen. Die beobachtete spinodale Entmischung erzeugte eine extrem hohe Grenzflächendichte und kann somit ebenfalls einen Beitrag zur Senkung der Wärmeleitfähigkeit liefern. Für die Analyse der Mikrostruktur auf nm-Skala wurden aus der LAST-Matrix mithilfe der fokussierten Ionenstrahltechnik elektronentransparente Schnitte gefertigt. Abhängig von Temperbedingungen und dem Verhältnis von Silber und Antimon wurden auch hier fremdphasige Einschlüsse entdeckt. Dabei konnte ein optimaler Temperbereich von 500 bis 550 °C (bezogen auf einen hohen Gütewert) mit dem Auftreten dieser Einschlüsse korreliert werden. Eine allgemeine, direkte Zuordnung des Vorhandenseins von Nanostrukturen zu guten oder schlechten thermoelektrischen Eigenschaften konnte im Allgemeinen jedoch nicht nachgewiesen werden. Vielmehr wurden deutliche Hinweise gefunden, dass auch die Anordnung von Punktdefekten (Blei-Substitution durch Silber und Antimon) und ggf. Agglomerate aus Punktdefekten in der LAST-Matrix eine Rolle bei der Senkung der Wärmeleitfähigkeit spielen. Im hochaktuellen Entwicklungsgebiet selbstorganisierender Nanostrukturen mit Auswirkungen auf thermoelektrische Eigenschaften wurden substantielle Fortschritte bei der Entwicklung geeigneter, LAST-basierter Thermoelektrika für mittlere Einsatztemperaturen erzielt. Die gewonnenen Erkenntnisse dieser Arbeit zeigen Optionen zur Erzeugung hocheffizienter thermoelektrischer Bauelemente auf, wie unter anderem die bestätigte Stabilität bis zu relativ hohen Einsatztemperaturen (> 500 °C) zeigt.

Doctor Scientiae / The blood-brain barrier (BBB) is a restrictive interface located between the blood circulation and the central nervous system (CNS), regulating the homeostatic environment of the neuronal milieu, by controlling the permeability of the cerebrovasculature. Currently, we cannot fully comprehend the regulatory features and the complexity of BBB morphology to allow for intervention clinically. The thesis consists of four publications. The methodology paper proposes a novel experimental design to visualize the morphological architecture of immortalized mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells (BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in preparation for visualization of the paracellular (PC) spaces between adjacent BECs, employing high-resolution electron microscopy (HREM), with vested interest in the morphological profile of the developing BEC. The second publication addresses and reports on the nanosized detail of BEC monolayer morphology utilizing high-resolution scanning electron microscopy (HR-SEM) and published the first descriptions of the extrusion of a basement membrane from developing in vitro BECs. Moreover, we categorized and discussed two types of nanotubule (NT) development specific for the establishment of the BEC monolayers. NTs can occur via nanovesicle extrusion onto the BEC membrane surfaces, which fuse, forming tunneling NTs (TUNTs) between adjacent BECs. Furthermore, cytoplasmic extensions of BEC membrane leading edges give rise to tethering NT (TENTs), which result in overlapping regions across the PC spaces, resulting in PC occlusion. BEC NT communication is illuminated in a third publication utilizing immunofluorescence microscopy, which reports on the molecular, cytoskeletal elements governing NT formation. This study shows, for the first time, f-actin and α-tubulin cytoskeletal proteins extending between the soma of the cells and NT cytoskeletal structures within an in vitro BBB model. Thereafter, the effects depolymerizing agents, Cytochalasin D and Nocodazole, were investigated on f-actin and α-tubulin cytoskeletal protein generation,functionality of NT morphology, cell division and permeability. For the first time, we show that f-actin possesses an additional function, key to tight junction, plaque protein organization. Moreover, it facilitates TENT formation, essential for cytoplasmic projection across PC spaces. Conversely, α-tubulin facilitates known functions: (i) transportation, (ii) cytokinesis, (iii) cellular division, and (iv) possesses a novel function as the molecular cytoskeletal backbone of TENTs, which facilitates BBB impermeability. A critical review evaluates past literature, in light of the current findings emanating from this study. The review critiques the concept of BEC cilia, which have been reported in the literature, comprised of tubulin and actin, but at low-resolution. In the light of our novel observations, nowhere in transmission electron microscopy do we observe cilia on the BECs, we postulate that NTs have been misnamed and mischaracterized as cilia. The thesis endeavors to elucidate the complexity of BEC nanostructures by examining the emerging role of the nanoscopic landscape of BBB development and the changing nature of BEC morphology, NT formation and associated cytoarchitectural underpinnings governing NT morphology. The research study attempts to, with a view to create new avenues for treating brain pathology, revolutionize our interpretation of barrier-genesis on a nanoscale.

The cellular mechanisms by which nervous systems evolve to match evolutionary changes occurring in the rest of the body remain largely unexplored. In a distal visual neuropil of a previously unexamined ancient dipteran family, Stratiomyidae, homologues of all of the periodic neurons known already from more recent Diptera can be recognized, occupying the same locations within the unit structure. This points to extreme developmental stasis for more than 200 million years, conserving both cell identity and position. The arborizations that some neurons make also have remained conservative, but others show marked differences between families in both size and branching patterns. At the electron-microscopical level, extensive differences in synaptic connectivity are found, some sufficient to radically redefine the systems roles of particular neurons. The findings bear out an earlier prediction that changes in the connectivity matrix linking conserved neurons may have been a major factor in implementing evolutionary change in the nervous system.

The urinary bladder is a unique organ in that its normal function is storage and release of urine, and vasculature in its wall exhibits specialized features designed to accommodate changes in pressure with emptying and filling. Although we have previously described the fine details of the microvasculature of the urinary bladder of the rabbit and dog, information on the fine details of the microvasculature of the mouse bladder were deemed to be of value because of the increasing use of this species in developing genetic models for studying human disorders. The present study shows that many of the special features of the microvasculature of the mouse urinary bladder are similar to those described in the rabbit and dog, including vessel coiling, abundant collateral circulation, arterial sphincters, and a dense mucosal capillary plexus.

The urinary bladder is a unique organ in that its normal function is storage and release of urine, and vasculature in its wall exhibits specialized features designed to accommodate changes in pressure with emptying and filling. Although we have previously described the fine details of the microvasculature of the urinary bladder of the rabbit and dog, information on the fine details of the microvasculature of the mouse bladder were deemed to be of value because of the increasing use of this species in developing genetic models for studying human disorders. The present study shows that many of the special features of the microvasculature of the mouse urinary bladder are similar to those described in the rabbit and dog, including vessel coiling, abundant collateral circulation, arterial sphincters, and a dense mucosal capillary plexus.

The urinary bladder is an unusual organ in that its normal function includes filling and emptying with alternating changes in internal pressure. Although fluctuations in blood flow to the bladder wall are known to accompany these changes, detailed descriptions of the bladder microvasculature are sparse. The present study uses vascular corrosion casting and scanning electron microscopy to describe the three-dimensional anatomy of the microvasculature of the urinary bladder of the dog. Specialized features of that microvasculature, including collateral circulation, vessel folding, vessel orientation, the presence of valves and sphincters, and mucosal capillary density, that may enhance and control blood flow during normal bladder function, are described and discussed.

The cast products Mg–Sn based alloys are promising candidates for automobile industries, since they provide a cheap yet thermally stable alternative to existing alloys. One drawback of the Mg–Sn based alloys is their insufficient hardness. The hardenability can be improved by engineering the microstructure through additions of Zn to the base alloy and selective aging conditions. Therefore, detailed knowledge about the microstructural characteristics and the role of Zn to promote precipitation hardening is essential for age hardenable Mg-based alloys. In this work, microstructural investigation of the Mg–1.4Sn–1.3Zn–0.1Mn (at.%) precipitation system was performed using TEM. The chemical composition of the precipitates was analyzed using EDS. APT was employed to obtain precise chemical information on the distribution of Zn in the microstructure. It was found from microstructural studies that different precipitates with varying sizes and phases were present; lath-shaped precipitates of the Mg2Sn phase have an incoherent interface with the matrix, unlike the lath-shaped MgZn2 precipitates. Furthermore, nano-sized precipitates dispersed in the microstructure with short-lath morphology can either be enriched with Sn or Zn. On the other hand, APT analysis revealed the strong repulsion between Sn and Zn atoms in a portion of the analysis volume. However, larger reconstruction volume required to identify the role of Zn is still limited to the optimization of specimen preparation.

Carbon nanotube forests are vertically grown tubular formations of graphene. Samples were grown with an injection chemical vapor deposition method on substrates of silicon with various deposited layers and bare fused silica. The morphology of the forest is characterized by the height, density, and presence of defects. Total diffuse reflectance and transmittance measurements were taken in the 2-16 �m spectral range and correlated to the forest’s specific morphology. From these correlations, the conditions necessary to maximize the absorption of the forest were found and exploited to cater sample growth for specific substrates to make ideal absorbers. From the transmittance data, the absorption coefficient is found via Beer-Lambert’s Law and also correlated to sample morphology, giving us an indication of the height of the forest needed for ideal absorption. Two models were used to attempt to reproduce the experimental absorption coefficient: an effective medium theory using a Maxwell Garnett approximation and by treating the carbon nanotube forest as an effective cylindrical waveguide with walls of graphite. Each model leads to a set of fitting parameters providing a better physical understanding of the forests. It was found that the effective medium theory gave results loosely corroborated with electron microscopy, but had trouble fitting the experimental data, and the index of refraction it provides does not behave like a unified medium. The waveguide model fits the data well, but it requires more experimental evidence to be more conclusive. The theoretical models need more work, but fabrication of ideal absorbers has been achieved on various substrates providing framework for their usage in radiometry and spectroscopy.