This thesis, which is a continuation of the previous Bachelor thesis, is dedicated to the study of polyelectrolyte complexes between hyaluronan (HA) and bovine serum albumin (BSA). Interactions between HA and BSA and a formation of complexes were studied by SEC-MALLS method, where a molar mass, a root mean square (rms) radius, a hydronamic radius and an intrinsic viscosity of particles of the system were defined. Furthermore the interactions were studied by calorimetric measurement ITC, where thermodynamic character of complex formation was determined. Additionally, dynamic and electrophoretic light scattering methods (DLS and ELS) were applied, where hydrodynamic radius and a value of the zeta potential were defined. Also the effect of higher temperature on the character and complex formation was examined by SEC-MALLS and ITC measurements. The interactions were confirmed at any of the used media, but with different efficiency. It was also proved that complexes become smaller in their radii and viscosity with growing BSA concentration. Furthermore it was proved, that the high ionic strength hinders surface charges of HA and BSA molecules and minimizes their mutual interactions. At the higher ionic strength the radii of the complexes, their intrinsic viscosity and zeta potential are increased. The higher temperature has only minimal effect on the formation of the complexes.

Stellung und Aufgaben Internationaler Rohstofforganisationen sind häufig ambivalent. Schwächen hat insbesondere das Scheitern des Internationalen Zinnrates 1987 aufgezeigt. Der Beitrag erläutert deren Ursachen und verknüpft die Vorschriften der Rohstoffabkommen mit dem Welthandels- und dem allgemeinen Völkerrecht.

This dissertation is a composite of some of the research that I have conducted during the course of my PhD study. The larger goal of this dissertation is to renew the interests among the scientific community for an otherwise under-appreciated technique called Isothermal Titration Calorimetry. The resurgence of calorimetry in the biophysical community and the shift to investigations of more complex biological systems signal a real need for more sophisticated analysis techniques. This dissertation expounds on new ITC analysis methods that we have developed as well as results from the study of thermodynamic properties of higher order DNA structures. In 1978, Peter Privalov described the first use of microcalorimetry to obtain the thermodynamic properties for removing calcium from parvalbumin III protein. Fast forward 36 years: modern day electronics, highly efficient thermally conductive and chemically inert materials, in conjunction with sensitive thermal detectors, has transformed the original calorimeter into a device capable of measuring heat changes as small as 0.05 nanowatts, which is equivalent to capturing heat from an incandescent light bulb a kilometer away. However, analytical methods have not kept pace with this technology. Commercial ITC instruments are typically supplied with software that only includes a number of simple interaction models. As a result, the lack of analysis tools for more complex models has become a limiting factor for many researchers. We have recently developed new ITC fitting algorithms that we have incorporated into a userriendly program (CHASM©) for the analysis of complex ITC equilibria. In a little over a year, CHASM© has been downloaded by over 370 unique users. Several chapters in this dissertation demonstrate this software’s power and versatility in the thermodynamic investigations of two model systems in both aqueous and non-aqueous media. In chapter VI, we assembled a model NHE-III1 : a novel structure of Gquadruplex in a double stranded form and studied its structural complexity and binding interactions with a classical G-quaduplex interactive ligand known as TMPyP4. In chapter VII, we reported the thermodynamic properties of a novel PAH system in which weak dispersion forces are solely responsible for formation of the supramolecular complexes.

Syftet är att undersöka hur ett antal gymnasielärare som undervisar i svenska ser på datortillgång och datoranvändning, samt vilka krav de ställer på elevernas datoranvändning i kursen Svenska A. Undersökningen genomfördes via en webbenkät, vilken distribuerades under mars 2009. Resultatet av webbenkäten ställs mot beskrivningar av förutsättningar för skrivande och aktuella styrdokument. Tillgången på lärardatorer är otillräcklig och tillgången på elevdatorer är ännu mera otillräcklig. Datormedierad kommunikation används för att effektivisera undervisningen och för att nå eleverna med information. En majoritet av lärarna i undersökningen har inte erbjudits kompetensfortbildning inom området datormedierad kommunikation av sin arbetsgivare. I kursen Svenska A erbjuds eleverna varierande datortillgång, allt från varje lektion till en gång per kurs. Lärarnas krav på elevernas datorkompetens i kursen varierar mellan ”inga krav” och omfattande krav. I diskussionen belyser jag hur ny teknik assimileras av skola och undervisning och sätter in mina resultat i en tidskontext. / The purpose of my investigation is to study the view of supply of computers and the use of computers among a num¬ber of senior high school teachers, teaching Swedish. I also investigate their demands on pupils and their use of computers in the course “Svenska A”. My web inquiry was distributed during March 2009. The results of the inquiry have been compared with descriptions of requirements for writing and with former and present curricula. The supply of computers for teachers is insufficient and the supply of computers for pupils is even more so. Communication mediated by computer is being used to give information to pupils and to make teaching more efficient. A majority of the teachers have not been offered further training by their employers within the field of communication mediated by computer. In the course “Svenska A” pupils are being offered to use computers in varying degrees, from once per course till every lesson. The teachers’ claims on the pupils’ competence in their use of computers vary from “no demands” to wide demands. In the discussion I shed light on how new technology is being assimilated by schools and education. I also insert my results in a context of time.

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Estate planning is the arrangement and management of an estate owner’s estate to the effect that the estate owner and his beneficiaries can enjoy maximum benefit from his worldly possessions during his lifetime and after his death. Unfortunately, for estate owners and their beneficiaries, a deceased estate has to pay an executor’s fee, estate duty and capital gains tax on the demise of the estate owner, which means the amount the estate owner intended his heirs to receive, might be substantially decreased. For decades trusts have been used for estate planning purposes. The decision of the estate owner to utilise a trust for estate planning purposes involves the disposal of growth assets from the estate owner’s estate to the trust. This ensures that the value of a growth asset is pegged in the estate owner’s estate and the asset continues to grow in the trust. The asset is disposed of by way of a loan account in favour of the estate owner and the parties agree that the outstanding amount is payable on demand. In his will, the estate owner then bequeaths the outstanding amount back to the trust. However, Paragraph 12(5) of the Eighth Schedule to the Income Tax Act 58 of 1962 stated that capital gains tax will be levied in cases where a debt is reduced or discharged by a creditor for no consideration or for an amount which is less than the outstanding amount. In ABC Family Trust ITC 1793 the estate owner transfer assets to the trust on a loan account and in her will, bequeathed the exact outstanding amount back to the trust. It was argued on behalf of the trust that the bequest constituted a set-off and not a discharge of the debt. However, the court stated that the set-off took place because of the “operation of law” which is specifically included in the definition of a “disposal” for capital gains tax purposes. The court applied Paragraph 12(5) and found that the trust is liable to pay capital gains tax on the full outstanding amount. In XXX Trust ITC 1835 the estate owner also transferred asset to a trust, but in her will she bequeathed the residue of her estate, and not the exact outstanding amount, to the trust. In this case the court placed emphasise on the intention of the estate owner and not on the possible application of Paragraph 12(5). The court found that it was not the intention of the estate owner to discharge or reduce the debt for no consideration. Subsequently, it was found that the trust is not liable for capital gains tax. Since these two cases Paragraph 12(5) has been deleted and Paragraph 12A inserted in the Eight Schedule to the Income Tax Act 58 of 1962. The focus of this mini-dissertation is to determine which estate planning tools were available to estate owners to prevent a capital gains liability under Paragraph 12(5). The capital gains tax effect that Paragraph 12A might have on estate planning is also discussed. / LLM (Estate Law), North-West University, Potchefstroom Campus, 2014

Estate planning is the arrangement and management of an estate owner’s estate to the effect that the estate owner and his beneficiaries can enjoy maximum benefit from his worldly possessions during his lifetime and after his death. Unfortunately, for estate owners and their beneficiaries, a deceased estate has to pay an executor’s fee, estate duty and capital gains tax on the demise of the estate owner, which means the amount the estate owner intended his heirs to receive, might be substantially decreased. For decades trusts have been used for estate planning purposes. The decision of the estate owner to utilise a trust for estate planning purposes involves the disposal of growth assets from the estate owner’s estate to the trust. This ensures that the value of a growth asset is pegged in the estate owner’s estate and the asset continues to grow in the trust. The asset is disposed of by way of a loan account in favour of the estate owner and the parties agree that the outstanding amount is payable on demand. In his will, the estate owner then bequeaths the outstanding amount back to the trust. However, Paragraph 12(5) of the Eighth Schedule to the Income Tax Act 58 of 1962 stated that capital gains tax will be levied in cases where a debt is reduced or discharged by a creditor for no consideration or for an amount which is less than the outstanding amount. In ABC Family Trust ITC 1793 the estate owner transfer assets to the trust on a loan account and in her will, bequeathed the exact outstanding amount back to the trust. It was argued on behalf of the trust that the bequest constituted a set-off and not a discharge of the debt. However, the court stated that the set-off took place because of the “operation of law” which is specifically included in the definition of a “disposal” for capital gains tax purposes. The court applied Paragraph 12(5) and found that the trust is liable to pay capital gains tax on the full outstanding amount. In XXX Trust ITC 1835 the estate owner also transferred asset to a trust, but in her will she bequeathed the residue of her estate, and not the exact outstanding amount, to the trust. In this case the court placed emphasise on the intention of the estate owner and not on the possible application of Paragraph 12(5). The court found that it was not the intention of the estate owner to discharge or reduce the debt for no consideration. Subsequently, it was found that the trust is not liable for capital gains tax. Since these two cases Paragraph 12(5) has been deleted and Paragraph 12A inserted in the Eight Schedule to the Income Tax Act 58 of 1962. The focus of this mini-dissertation is to determine which estate planning tools were available to estate owners to prevent a capital gains liability under Paragraph 12(5). The capital gains tax effect that Paragraph 12A might have on estate planning is also discussed. / LLM (Estate Law), North-West University, Potchefstroom Campus, 2014

Targeting DNA has the advantage over proteins for cancer remediation because of the fewer copies of the ligands required for the desired therapeutic effect. Traditionally, covalent DNA binders like alkylating agents have been used to induce genetic instability through the formation of DNA lesions and strand breaks, leading to cellular apoptosis. The primary drawback of this treatment is the non-specific binding that affects both cancerous and non-cancerous cells. G-quadruplexes are the DNA secondary structures that are present in abundance near the promoter regions of the oncogenes and are involved in the regulation of their activities. A ligand-mediated stabilization of G-quadruplexes in the promoter regions and down-regulation of the associated oncogenes have been validated. In contrast to alkylating agents, G-quadruplex ligands induce genetic stabilization through non-covalent interactions. They can be designed to interact specifically with G-quadruplex DNA over duplex DNA, which reduce side effects arising from the off-targeting. G-quadruplex ligands invariably have the large planar aromatic moiety to interact with G-quadruplexes through π- π stacking interactions. For determining the size effect of the aromatic moiety on stabilization of G-quadruplexes, a series of ligands were synthesized by conjugating nucleobases or 1,10-phenanthroline with an aminoglycoside, neomycin. The resulting conjugates increased the binding affinity synergistically and enabled us to study the effect of the stacking moiety required for G-quadruplex stabilization. Nucleobase-neomycin conjugates did not show stabilization stabilize of human telomeric G-quadruplex. 1,10-Phenanthroline-neomycin conjugate (7b) on the other hand binds to human telomeric G-quadruplex with a Ka of (8.92.4)×108 M-1 and inhibits telomerase activity at 1.56 µM probably through G-quadruplex stabilization. Moving forward, we further enlarged the aromatic moiety by tethering two 1,10-phenantholine molecules together through a five-atom linker. The resulting molecule (2-Clip-phen) was conjugated with various amino-containing side chains. 2-Clip-phen derivatives showed at least 30 times weaker binding to duplex DNA over G-quadruplex DNA. In addition, compounds showed a preference for the antiparallel G-quadruplex conformation over parallel and hybrid G-quadruplex conformations, as shown in the CD spectroscopy studies. Ligands 11 and 13 induced the formation of an antiparallel G-quadruplex from random coils and stabilize it to 60 oC (Tm) in a salt-free condition. Mass spectrometry study showed the formation of a two-tetrad G-quadruplex with the 2-Clip-phen ligand. Docking study showed that the ligand interacts most favorably with antiparallel G-quadruplex conformation, which is supported further by the larger thermal stabilization effect on antiparallel G-quadruplex compared with other G-quadruplex conformations. Our study suggests that 2-Clip-phen can be used as a scaffold for designing G-quadruplex binding ligands that preferentially bind to antiparallel G-quadruplexes, which has never been reported before.

Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects. Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function. Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.