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In this work, two projects were pursued. In the first project, I investigated two different subtypes of opioid receptors, which play a key role as target for analgesia. A set of subtype specific fluorescent ligands for μ opioid receptor (MOR) and δ opioid receptor (DOR) was characterised and used to gain insights into the diffusion behaviour of those receptors. It was shown that the novel ligands hold photophysical and pharmacological properties making them suitable for single-molecule microscopy. Applying them to wild-type receptors expressed in living cells revealed that both sub-types possess a heterogeneous diffusion behaviour. Further- more, the fluorescent ligands for the MOR were used to investigate homodomerisation, a highly debated topic. The results reveal that only ≈ 5 % of the receptors are present as homodimers, and thus the majority is monomeric. G-protein coupled receptors (GPCRs) play a major role as drug targets. Accordingly, understanding the activation process is very important. For a long time GPCRs have been believed to be either active or inactive. In recent years several studies have shown, that the reality is more complex, involving more substates. [1, 2, 3, 4] In this work the α 2A AR was chosen to investigate the activation process on a single-molecule level, thus being able to distinguish also rare or short-lived events that are hidden in ensemble mea- surements. With this aim, the receptor was labelled intracellular with two fluorophores using supported membranes. Thus it was possible to acquire movies showing qualita- tively smFRET events. Unfortunately, the functionality of the used construct could not be demonstrated. To recover the functionality the CLIP-tag in the third intracellular loop was replaced successfully with an amber codon. This stop codon was used to insert an unnatural amino acid. Five different mutants were created and tested and the most promising candidate could be identified. First ensemble FRET measurements indicated that the functionality might be recovered but further improvements would be needed. Overall, I could show that single-molecule microscopy is a versatile tool to investigate the behaviour of typical class A GPCRs. I was able to show that MOR are mostly monomeric under physiological expression levels. Furthermore, I could establish intra- cellular labelling with supported membranes and acquire qualitative smFRET events. / In dieser Arbeit wurden zwei Projekte verfolgt. Im ersten Projekt wurden zwei Subtypen der Opioidrezeptoren untersucht, die eine wichtige Rolle für die Wirksamkeit von Analgetika spielen. Ein Set von subtypspezifischen fluoreszierenden Liganden für den MOR und den DOR wurde charakterisiert und eingesetzt, um Einblicke in das Diffuionsverhalten der Rezeptoren zu gewinnen. Es konnte gezeigt werden, dass die neuartigen Liganden sowohl photophysikalische als auch pharmakologische Eigenschaften besitzen, die sie für die Einzelmolekülmikroskopie interessant machen. Versuche mit Opioidrezeptoren, die in lebenden Zellen exprimiert werden, zeigten, dass beide Subtypen heterogenes Diffuionsverhalten aufweisen. Des Weiteren wurden die fluoreszierenden Liganden für den MOR genutzt um Homodimerisierung zu untersuchen, da dies ein kontrovers diskutiertes Thema ist. Die Ergebnisse zeigen, dass lediglich ≈ 5% der Rezeptoren als Homodimere vorliegen und der Großteil monomerisch ist. GPCRs sind besonderem Interesse, weil sie Angriffspunkt vieler Medikamente sind. Deshalb ist es wichtig ihren Aktivierungsmechanismus besser zu verstehen. Lange Zeit wurde angenommen, dass GPCRs entweder aktiv oder inaktiv sind. Neuere Studien zeigten jedoch, dass die Realität komplexer ist und der Prozess Zwischenschritte involviert. [1, 2, 3, 4] In dieser Arbeit wurde der α 2A Adrenorezeptor als prototypischer Klasse A GPCR gewählt, um den Aktivierungsprozess auf Einzelmoleküllevel zu untersuchen. Durch die Betrachtung einzelner Rezeptoren ist es möglich auch seltene oder sehr kurzlebige Ereignisse zu unterscheiden, die in Kollektivmessungen untergehen. Um dies zu erreichen wurde der Rezeptor erfolgreich intrazellulär mit zwei Fluorophoren markiert. Dies gelang durch die Herstellung von „supported membranes", also Zellmembranen die auf einem Objektträger fixiert wurden. Dadurch war es möglich Videos aufzunehmen, die Einzelmolekül-FRET-Ereignisse zeigen. Jedoch gelang es nicht zu zeigen, dass der Rezeptor als Ganzes noch funktional war. Um einen funktionalen Rezeptor zu erhalten, wurde das CLIP-Tag in der dritten intrazellulären Schleife erfolgreich durch ein Stopcodon ersetzt, welches für eine nicht kanonische Aminosäure kodierte. Fünf verschiedene Mutanten wurden kloniert und getestet, wobei der vielversprechendste Mutant identifiziert werden konnte. Erste FRET-Kollektivmessungen deuten darauf hin, dass dieser Mutant funktional sein könnte. Jedoch sind weitere Verbesserungen nötig. Insgesamt konnte ich zeigen, dass Einzelmolekülmikroskopie vielseitige Möglichkeiten bietet um das Verhalten von GPCRs zu untersuchen. Ich konnte nachweisen, dass MOR unter physiologischen Bedingungen hauptsächlich als Monomere vorliegen. Des Weiteren konnte ich Dank supported membranes die Markierung durch Farbstoffe im Intrazellularbereich etablieren und qualitative smFRET Ereignisse aufnehmen.

Solid phosphoric acid is a catalyst used for the upgrading of light olefins into fuels. To delve into the mechanism of olefin dimerisation over the catalyst, the oligomerisation of 1- hexene was investigated over a wide range of operating conditions. The reaction progression of 1-hexene dimerisation over solid phosphoric acid was interpreted by means of kinetic experiments for both a linear hexene (1-hexene) and a branched hexene (2,3-dimethylbutene). The reaction rate for both reagents was described by using an elementary kinetic model. From the experimental data it was shown that the rate of dimerisation of branched hexenes was faster than the rate observed for linear hexene dimerisation. To correlate the two sets of kinetic data, the reaction network was expanded to incorporate skeletal isomerisation of 1-hexene with dimerisation only taking place by the co-dimerisation of linear and branched hexenes and the dimerisation of branched hexenes. The fit of the kinetic equation demonstrated that the reaction rate of 1-hexene is essentially controlled by the rate of skeletal isomerisation. Due to the large activation energy for skeletal isomerisation, low reaction temperatures favoured the co-dimerisation of linear and branched hexenes whereas at higher temperatures, the reaction rate was dominated by the dimerisation of branched hexenes. The product distribution indicated that, because of the fast rates of both cracking and secondary dimerisation (dimerisation of cracked products), the product distribution instantaneously reached a pseudo equilibrium after the dimerisation of hexenes. Therefore the carbon distribution was found to depend only on the reaction temperature, not on the residence time in the reactor. Solid phosphoric acid is a supported liquid phosphoric acid where the condensed state of the acid, e.g. ortho phosphoric acid (H3PO4) and pyro phosphoric acid (H4P2O7), is dependent on the quantity of water present in the reaction mixture. With a decrease in water content, the distribution of acid shifts and the ortho phosphoric acid becomes more condensed (H4P2O7, H5P3O9 etc.), i.e. high water content → low acid strength, low water content → high acid strength. The experiments completed at various degrees of catalyst hydration and free acid loading showed that the rate of reaction over solid phosphoric acid was dependent on the acid strength of the catalyst. The effect of acid strength on the reaction rate was integrated into the rate constants by means of an exponential dependency on acid strength. It was also shown that both the product distribution and the degree of branching remained unaffected by acid strength. The constant product indicates that the rate of cracking is limited by the rate of oligomerisation of hexenes, irrespective of the acid strength of the catalyst. Since the product from the dimerisation of 1-hexene could be used as fuel, the quality of the desired fuel would therefore depend solely on the reaction temperature, not on the hydration of the catalyst. The work performed in this thesis has been published in two peer-review articles: 1. Schwarzer R.B., Du Toit E. and Nicol W. (2008) Kinetic model for the dimerisation of 1-hexene over a solid phosphoric acid catalyst, Applied Catalysis A: General, 340, 119-124. 2. Schwarzer R.B., Du Toit E. and Nicol W. (2009) Solid phosphoric acid catalysts: The effect of free acid composition on selectivity and activity for 1-hexene dimerisation, Applied Catalysis A: General, 369, 83-89. / Thesis (PhD(Eng))--University of Pretoria, 2012. / Chemical Engineering / unrestricted

No / When subjected to positive ion electrospray ionisation (ESI+) mass spectrometry (MS), indoles with a 3-alkyl substituent show a propensity to form novel [2M-H](+) 'covalently bound dimers'. This process, which appears to be initiated in the nebuliser of the instrument, is mechanistically interesting, analytically useful and potentially significant in organic synthesis. A selection of 2- and 3-substituted indoles have been synthesised and analysed by ESI-MS. The formation of the 'homo' and 'hetero' dimers of these compounds has been investigated using ESI+ mode. The mechanism of formation of the observed 'dimeric' species has been probed by synthesising authentic samples of the dimeric compounds. 'Dimeric' species corresponding to [2M-H](+) have been observed for all 3-substituted indoles studied, but not for indoles substituted in just the 2-position. By infusing equimolar mixtures of labelled and unlabelled indoles through the instrument, the expected approximately statistical mixture of homo- and heterodimeric species has been observed. Further experiments have established that this novel dimerisation occurs in the droplets formed in the nebuliser of the instrument. It has been shown that 3-substituted indoles form [2M-H](+) dimers in high abundance in the spray obtained from the nebiliser of an ESI+ instrument. The mechanism for the dimerisation does not involve the known 2M dimeric species that is readily formed in the solution-phase chemistry of indoles.

SAS-6 is the structural core of the forming centriole - a cylindrical protein complex, which is an essential component of the centrosome. Oligomerisation of SAS-6 is crucial for successful centriole duplication and is achieved through two dimerisation domains in the SAS-6 protein; a long C-terminal coiled-coil domain and a globular N-terminal dimerisation domain. As core components of the centrosome, centrioles help facilitate various cellular functions. They are involved in the anchoring of flagella and cilia to the membrane and in coordinating the spindle apparatus during chromosome segregation. A deeper insight into the molecular mechanisms at play in the centriole duplication process would have implications on our understanding of fundamental cell division processes and a number of related diseases. Here the involvement of an unstudied loop region in the C. elegans SAS-6 N-terminal domain dimerisation is described. Combining structural biology, biophysical and computational techniques, the molecular interactions of this loop were explored, contributing to the oligomerisation of SAS-6 at the N-terminal dimer interface. Furthermore, the screening and testing of small molecule inhibitors of the SAS-6 N-terminal domain dimerisation is described, targeting a hydrophobic pocket in the domain. Two candidate compounds are presented as a result of the screens and next steps towards structure based compound design are suggested, based on computational analysis. The search for inhibitory compounds includes a set-up of an in-house virtual screening pipeline, as well as in vitro screening efforts and a new crystallographic structure of the H. sapiens SAS-6 N-terminal domain. By investigating the making and breaking of the SAS-6 N-terminal domain dimerisation, light is shed on so far neglected details of this essential protein-protein interaction and advancements towards a SAS-6 oligomerisation inhibitor described, which could ultimately be used for new approaches in cell cycle research and might open up new avenues for medical research by binding a disease relevant target.

The complementary value of vibrational spectroscopy and mass spectrometry in obtaining structural information on a range of tricyclic indoles with various ring patterns has been investigated, focusing particularly on whether these heterocycles with a functional group containing oxygen in the third ring should be described as ketoindoles or hydroxindolenines. Parallels between certain fragmentations of ionised indoles and electrophilic substitution in solution have been identified. A mechanistically interesting and analytically useful interesting dimerisation, leading to the formation of [2M-H]+ ions, has been discovered in the positive ion electrospray mass spectra of 3-alkylindoles. This dimerisation, which occurs in the nebuliser of the instrument, offers a potential new route to bisindoles under milder conditions than those employed in classical solution chemistry. Facile formation of C=N bonds by condensation of C=O and H2N has been shown to provide a means of preparing protonated imines and protonated quinoxalines from mixtures of the requisite (di)carbonyl compounds and (di)amines, thus further illustrating how organic synthesis is possible in the droplets in the nebuliser of the instrument. Possible metal catalysed coupling reaction routes to bisindoles have been explored. Acyl transfer reactions from nitrogen to carbon have been investigated in 1-acyl-2-methylindoles and orthogonally protected sulphonamides. These processes have been shown to be intermolecular and intramolecular, respectively. The latter rearrangement, which may be prevented when necessary by choosing the nitrophenylsulphonamide protecting group, offers a route to acyl, carboalkoxy and carboaryloxy aromatic compounds, some of which are difficult to prepare.

The complementary value of vibrational spectroscopy and mass spectrometry in obtaining structural information on a range of tricyclic indoles with various ring patterns has been investigated, focusing particularly on whether these heterocycles with a functional group containing oxygen in the third ring should be described as ketoindoles or hydroxindolenines. Parallels between certain fragmentations of ionised indoles and electrophilic substitution in solution have been identified. A mechanistically interesting and analytically useful interesting dimerisation, leading to the formation of [2M-H]+ ions, has been discovered in the positive ion electrospray mass spectra of 3-alkylindoles. This dimerisation, which occurs in the nebuliser of the instrument, offers a potential new route to bisindoles under milder conditions than those employed in classical solution chemistry. Facile formation of C=N bonds by condensation of C=O and H2N has been shown to provide a means of preparing protonated imines and protonated quinoxalines from mixtures of the requisite (di)carbonyl compounds and (di)amines, thus further illustrating how organic synthesis is possible in the droplets in the nebuliser of the instrument. Possible metal catalysed coupling reaction routes to bisindoles have been explored. Acyl transfer reactions from nitrogen to carbon have been investigated in 1-acyl-2-methylindoles and orthogonally protected sulphonamides. These processes have been shown to be intermolecular and intramolecular, respectively. The latter rearrangement, which may be prevented when necessary by choosing the nitrophenylsulphonamide protecting group, offers a route to acyl, carboalkoxy and carboaryloxy aromatic compounds, some of which are difficult to prepare.

Human mitochondrial ATP synthase is an enzyme containing 18 unlike subunits located in the inner mitochondrial membrane (IMM), where the catalytic F1 domain extends into the mitochondrial matrix and the FO domain, which contains the c8-ring rotor, the a-subunit and the supernumerary subunits, is anchored in the IMM. All the subunits, apart from the a- and A6L-subunits, are encoded in the nucleus and require transport into the mitochondria before being assembled. The a- and A6L-subunits are encoded on the mitochondrial genome. The respiratory complexes generate the proton motive force (PMF), which ATP synthase uses to generate ATP from ADP and Pi. Rotation of the α- and β-subunits with the central stalk γ-, δ- and ε-subunits is prevented by coupling the F1 domain to the FO domain via the peripheral stalk (the OSCP-, F6-, d- and b-subunits). ATP hydrolysis is prevented by the natural inhibitor of the enzyme, IF1, binding to the F1 domain. In addition to the aand, b-subunits, the FO domain contains the c8-ring and six supernumerary subunits not involved in the catalytic activity of ATP synthase. The roles of five of these subunits in the assembly of ATP synthase, the e-, f-, g-, DAPIT- and 6.8 kDa proteolipid-subunits, were investigated by suppressing or disrupting their expression individually. The e-subunit is the first of the supernumerary subunits to assemble, then the g-subunit followed by the f-, 6.8 kDa proteolipid- and DAPIT-subunits. All five supernumerary subunits investigated were required to facilitate the dimerisation and oligomerisation of ATP synthase. The e-, f- and g-subunits were found to be important for maintaining mitochondrial respiratory capacity.

<p>Fibroblast growth factors (FGFs) constitute a family (currently FGF-1 to FGF-23) of polypeptides that are essential in embryonal development and adult physiology, in animals from nematodes to humans. FGFs bind to four receptor tyrosine kinases, denoted FGFR-1 to FGFR-4. For proper function, the FGFs and their receptors depend on specific polysaccharide co-receptors, denoted heparan sulfate (HS). This thesis describes HS regulation of FGFR-1 signal transduction using blood vessel endothelial cells as a model.</p><p>We have determined HS structural features, necessary for FGF-2 induced FGFR-1 activation, using chemically modified heparin, which is structurally related to HS. Modified heparin, lacking sulfation at the 6-O position was inhibitory for FGFR-1 kinase activation and FGF-2 induced angiogenesis. Inhibition of blood vessel formation using modified heparin could be useful in treatment of diseases characterized by excess blood vessel formation. The critical role of HS sulfation for proper growth factor function was further underscored using an embryonal stem (ES) cell model. ES cells lacking expression of two isoforms of N-deacetyl N-sulfotransferase, NDST-1 and –2, failed to undergo embryonal development and to establish a vascular system. Exogenous heparin could not support development, but HS delivered from other ES cells allowed formation of primitive vessels and subsequent sprouting angiogenesis.</p><p>We have, furthermore, shown that the mechanism whereby HS supports FGF receptor activation is qualitative, as well as quantitative. Kinase activity could be induced by FGF-2 in the absence of HS, but this allowed only selected phosphorylation. In the presence of HS, the kinase activity was stabilized, allowing a broader spectrum of phosphorylation of sites on the FGF receptor itself as well as on cytoplasmic substrates. Finally, using selected microarrays, we have examined the potential regulation of enzymes in the HS biosynthesis pathway and of different proteoglycans to which HS is attached. Overall, we found no evidence for dramatic regulation on the transcriptional level, but could identify specific upregulation of HS proteoglycan syndecan-2, during blood vessel formation in vitro.</p><p>In conclusion, our studies demonstrate selective and complex regulation of HS synthesis and structure, essential in guiding growth factor function during health and disease.</p>