Dendritische Polymere werden im zunehmenden Maße als nicht-virale Vektoren für virus- oder tumor-assoziierte Antigen-Peptide zur Entwicklung neuer immuntherapeutischer Strategien eingesetzt. Diese beruhen auf der Verwendung von dendritischen Zellen (DCs), welche Schlüsselzellen bei der Induktion und Aufrechterhaltung einer T-Zell-basierten Immunantwort darstellen. Im Rahmen dieser Arbeit wurden Nitrilotriessigsäure-funktionalisierte dendritische Glycopolymere (NTA-DG) für den Transport von Antigen-Peptiden in DCs etabliert. Die Ni(II)-NTA-DGs waren durch definierte Komplexierungs- und Freisetzungseigenschaften charakterisiert. So wurde das Antigen-Peptid bei einem pH-Wert unter 6 vom polymeren Träger freigesetzt. Die gebildeten Polyplexe, zwischen Ni(II)-NTA-DG und dem Antigen-Peptid, bewirkten eine Erhöhung der Antigen-Peptid-Aufnahme in immaturen DCs (iDCs). Dieses war nach der Endozytose im frühen endosomalen und lysosomalen Kompartiment von iDCs lokalisiert. Somit kann das Antigen-Peptid am MHC Klasse II-Molekül im lysosomalen Kompartiment ohne sterische Hinderungen durch die Polymeroberfläche binden. Die Polyplexe bewirkten eine Aktivierung der iDCs durch Aufregulation der kostimulatorischen Moleküle CD86 und CD80 sowie der pro-inflammatorischen Zytokine IL-6 und IL-8. Weiterhin wurde die Migrationsfähigkeit und das pro-inflammatorische Potential der Antigen-Peptid enthaltenen maturen DCs (mDCs) aufrechterhalten. Somit stellen Ni(II)-NTA-DGs ein vielversprechendes polymeres Trägersystem für Antigen-Peptide dar.

Thy-1 is a glycosylphosphatidylinositol-anchored protein that is expressed on murine T lymphocytes and is involved in T cell-mediated immune responses. In the presence of costimulatory signals, monoclonal antibody (mAb)-induced signaling through Thy-1 is associated with hallmarks of T cell activation, including IL-2 production and T cell proliferation. Thy-1-induced signaling promotes cytotoxic effector molecule expression, but is unable to trigger delivery of the lethal hit to target cells, suggesting that Thy-1 provides an incomplete T cell receptor (TcR)-like signal. However, the effect of Thy-1 signaling on cytokine production and the development of T helper (Th) cell phenotypes (Th1, Th2, Th17) remains unclear. The purpose of this work was to further our understanding of Thy-1-mediated signal transduction and the role that Thy-1 plays in the development of effector T cell responses. I found that, in the context of costimulatory signals, anti-Thy-1 mAb induced significantly less IL-2 production, CD25 expression and T cell proliferation than anti-TcR? mAb. Several key signaling molecules, including protein tyrosine kinases, zeta chain-associated protein-70 and extracellular signal-regulated kinase were activated with delayed kinetics during Thy-1-mediated T cell activation. The delayed signaling kinetics resulted in the delayed acquisition of cytotoxic effector function and also delayed delivery of the lethal hit to target cells. Interestingly, Thy-1-mediated signaling induced significantly more IL-17 and IL-4 synthesis and less IFN-? synthesis in comparison to TcR-mediated signaling. Moreover, Thy-1-activated CD4+ T cells produced high levels of IL-17 and IL-4 but minimal IFN? when restimulated with anti-Thy-1 mAb or anti-TcR? mAb with or without costimulatory signals. The unique ability of Thy-1 signaling to induce IL-17 production correlated with the expression of the Th17 lineage-specific transcription factor, retinoic orphan receptor gamma t. These observations show that Thy-1 signaling differs from TcR signaling in its ability to induce Th cell cytokines. Taken together, my findings show that Thy-1 signaling can provide the full TcR-like signal required for both the differentiation and triggering of Th cells and cytotoxic T lymphocytes, albeit with delayed kinetics in comparison to TcR signaling. They also suggest that Thy-1 signaling may be important in the development of Th2 and Th17 responses.

The design of versatile biohybrid nanosized materials has revealed itself as a promising avenue towards biomedical applications in today´s life sciences. In this regard the combination of components of synthetic and natural origin facilitates an applicability which is supposed to be far beyond the sum of their single components. These biohybrid structures (BHS) can be built by a huge variety of building blocks including solid or soft nanoparticles, peptides/proteins, polynucleotides or low molecular weight drugs. Along with the latter the attachment of biologically active entities or imaging moieties, e. g. enzymes, fluorescence markers or targeting motifs display thereby a key step towards the development of carrier systems for drug delivery purposes. Among the soft nanoparticles especially dendritic polymers such as perfectly branched dendrimers or hyperbranched polymers are considered as ideal building blocks, since they allow an easy tailoring of crucial properties such as solubility, biocompatibility or bioactivity by means of surface functionalization. Especially in the field of targeted drug delivery the crucial role of sizes and size distributions of carriers has been highlighted recently, since it critically influences important factors such as circulation time or biodistribution within the body. The ability of avidin to form high molecular weight associates with biotinylated macromolecules as well as its inherent properties makes it a suitable candidate for passive and active targeting in combination with biotinylated (bio-)polymers. Furthermore, along with the covalent attachment of bioactive moieties, non-covalent attachment is a frequently used approach, because it is assumed to only require stoichiometric mixing. In context of the latter molecular recognition processes such as the avidin-biotin, β-cyclodextrin-adamantane or Ni(II)-NTA-histidine-tag interactions have shown to be fruitful strategies for the attachment of bioactive entities. The overall aim of this work was to fabricate BHS based on dendritic glycopolymers with varied sizes in the nano- and micrometer range as models for biomedical applications e. g. carriers for drug delivery. Therefore the molecular recognition of avidin with biotin derivatives and β-cyclodextrin with adamantane derivatives was utilized in order to tailor final sizes, functionality or catalytic activity of those BHS.

Interleukin-10 ist ein Paradebeispiel eines immunhemmenden Zytokins. Es konnte nachgewiesen werden, dass eine Reihe von Tumoren Interleukin-10 produziert, um einer Antitumor-Immunantwort zu entgehen. Viele Studien haben sich mit dem Einfluss von Interleukin-10 auf die antigenpräsentierenden Fähigkeiten der Dendritischen Zellen beschäftigt. Es gibt eindeutige Hinweise, dass der Effekt von tumorproduziertem Interleukin-10 nicht nur in einer hemmenden Wirkung auf die Ausreifung Dendritischer Zellen besteht, sondern dass Interleukin-10 zu einer Reduktion der Anzahl an Dendritischen Zellen führen kann. Ziel dieser Arbeit ist es daher, den Mechanismus für eine solche depletierende Wirkung auf die Dendritischen Zellen zu analysieren. Hierzu wurden die Effekte von Interleukin-10 auf die frühe Differenzierung von Dendritischen Zellen aus Monozyten untersucht. Die Zugabe von Interleukin-10 zu einem Differenzierungscocktail aus Interleukin-4 und Granulozyten/Makrophagen-Kolonie-stimulierendem-Faktor führt zu einer nachhaltigen Hemmung des Differenzierungsprozesses von Monozyten zu Dendritischen Zellen. Bereits 48h nach Beginn der Zellkultur konnte mit Hilfe von cDNA-Microarray-Analysen gezeigt werden, dass Interleukin-10 nicht nur einen Differenzierungs-hemmenden Effekt ausübt, sondern auch die Entstehung aberranter Zellphänotypen bewirkt. In weiteren Experimenten konnte gezeigt werden, dass die Effekte des Interleukin-10 in der frühen Differenzierungsphase weitgehend irreversibel sind. Zusammenfassend können die Ergebnisse zur Erklärung beitragen, wie es bei Patienten mit Tumoren unter dem Einfluss von Interleukin-10 zu einer Reduktion der absoluten Zahl Dendritischer Zellen kommen kann.

Systemic lupus erythematosus (SLE) is an autoimmune, inflammatory disease characterized by autoantibody production and an activated type I interferon system. Cardiovascular disease (CVD) is as a major cause of morbidity and mortality. The aim of this thesis was to identify genetic risk factors for CVD in SLE. The role of T cells in regulation of the interferon-α (IFNα) production by plasmacytoid dendritic cells (pDCs) was also investigated.    In paper I, a thicker intima, thinner media and increased intima/media ratio was found in young premenopausal women with SLE compared to healthy controls indicating increased cardiovascular risk. As traditional ultrasound assessment of the common carotid intima-media thickness (CCA-IMT) in SLE has given conflicting results separate measurement of the intima and media can be a useful tool to identify SLE patients at increased risk of CVD.    In paper II, an association was demonstrated in SLE between a STAT4 risk allele and ischemic cerebrovascular disease and presence of anti-phospholipid antibodies (aPL). The association remained after adjustment for traditional CVD risk factors. A possible mechanism for this association is that the risk allele leads to increased production of aPL, which promotes thromboembolism.    In paper III, a genetic locus in IRF8 was identified to be associated to coronary heart disease (CHD) in SLE. The association remained after adjustment of other CHD risk factors.  Patients with the IRF8 risk variant had increased CCA-IMT, more carotid plaques and reduced frequency of circulating B cells. Weaker binding of nuclear protein to the risk allele was demonstrated, suggesting a regulatory function of the IRF8 risk variant.    In paper IV, activated T cells were found to strongly enhance the IFNα production by pDC stimulated with RNA-containing immune complexes via GM-CSF and IL-3. Activated SLE T cells enhanced the IFNα production to the same extent as T cells from healthy controls. This finding together with previous observations in SLE of increased levels of GM-CSF and IL-3 suggests that T cells contribute to the activated type I interferon system in SLE.    In conclusion, this thesis demonstrates that genetic predisposition is important for CVD in SLE and describes a new role for T cells in the pathogenesis of SLE.

The work in this thesis concerns mechanisms of excitability of neurons. Specifically, it deals with how neurons respond to input, and how their response is controlled by ion channels and other active components of the neuron. I have studied excitability in two systems of the nervous system, the hippocampus which is responsible for memory and spatial navigation, and the peripheral C–fibre which is responsible for sensing and conducting sensory information to the spinal cord. Within the work, I have studied the role of excitability mechanisms in normal function and in pathological conditions. For hippocampus the normal function includes changes in excitability linked to learning and memory. However, it also is intimately linked to pathological increases in excitability observed in epilepsy. In C–fibres, excitability controls sensitivity to responses to stimuli. When this response becomes enhanced, this can lead to pain. I have used computational modelling as a tool for studying hyperexcitability in neurons in the central nervous system in order to address mechanisms of epileptogenesis. Epilepsy is a brain disorder in which a subject has repeated seizures (convulsions) over time. Seizures are characterized by increased and highly synchronized neural activity. Therefore, mechanisms that regulate synchronized neural activity are crucial for the understanding of epileptogenesis. Such mechanisms must differentiate between synchronized and semi synchronized synaptic input. The candidate I propose for such a mechanism is the fast outward current generated by the A-type potassium channel (KA). Additionally, I have studied the propagation of action potentials in peripheral axons, denoted C–fibres. These C–fibres mediate information about harmful peripheral stimuli from limbs and organs to the central nervous system and are thereby linked to pathological pain. If a C–fibre is activated repeatedly, the excitability is altered and the mechanisms for this alteration are unknown. By computational modelling, I have proposed mechanisms which can explain this alteration in excitability. In summary, in my work I have studied roles of particular ion channels in excitability related to functions in the nervous system. Using computational modelling, I have been able to relate specific properties of ion channels to functions of the nervous system such as sensing and learning, and in particular studied the implications of mechanisms of excitability changes in diseases. / <p>QC 20102423</p>

Coupling homogeneous catalysts to dendrimers with rigid nuclei is one possible strategy to recycle these compounds in an industrial environment. During this study attempts were made to attach the well-defined ruthenium carbene complex 2 to the alicyclic compound 1A. To achieve this goal attempts were made to functionalise 1A with anime groups to facilitate the attachment of phosphine groups. The catalyst 2 would then be attached through a phosphine exchange reaction. Oximes were used as precursors in attempts to prepare cage amines from 1A. For this purpose, the dioxime 28 was prepared from 1A. Reduction of 28 was unsuccessful. Molecular modelling showed that the lobes of the LUMOs of the oxime carbon atoms of 28 do not protrude from the total electron density of this molecule. This observation indicates that 28 would probably not react with nucleophiles, such as the hydride ion. Molecular modelling was used to probe the unreactive nature of oxime 28. The probe revealed that the imide ring deactivates the oxime groups in this compound. Based on these results, attempts were made to change the carbon framework of 1A to eliminate unwanted interaction between the carbonyl groups. Clemmensen reduction of 1A did not yield the expected ketol 40, but gave a mixture of 62 and 63. Extended reaction times yielded 63 only. Reduction of 1A with zinc and acetic acid produced a mixture of 62 and 40 that could not be separated. Consequently, the applicability of 62 as a possible dendrimer nucleus was investigated. Compound 62 was obtained by oxidisation of the diol 63 with sodium periodate. Attempts to synthesise the dioxime 69 from 62 failed and only the mono oxime 71 was obtained. Reduction of 71 with lithium aluminium hydride was unsuccessful. Molecular modelling revealed that the oxime carbon atom does not have a LUMO and that the carbonyl carbon atom would probably be unreactive towards nucleophiles. Several other attempts were made at reducing the oxime 71 to an amine. None of these attempts met with any success. The reason for the unreactive nature of 71 is less clear than in the case of 28. Failure to produce a cage amine from 1A or derivatives of 1A meant failure in functionalising the cage system with phosphine groups and coupling the Grubbs-I catalyst. It seems that cage compounds based on 1A are generally not suitable as starting materials for amine-functionalised dendrimer nuclei. / Thesis (M.Sc. (Chemistry))--North-West University, Potchefstroom Campus, 2011.

Coupling homogeneous catalysts to dendrimers with rigid nuclei is one possible strategy to recycle these compounds in an industrial environment. During this study attempts were made to attach the well-defined ruthenium carbene complex 2 to the alicyclic compound 1A. To achieve this goal attempts were made to functionalise 1A with anime groups to facilitate the attachment of phosphine groups. The catalyst 2 would then be attached through a phosphine exchange reaction. Oximes were used as precursors in attempts to prepare cage amines from 1A. For this purpose, the dioxime 28 was prepared from 1A. Reduction of 28 was unsuccessful. Molecular modelling showed that the lobes of the LUMOs of the oxime carbon atoms of 28 do not protrude from the total electron density of this molecule. This observation indicates that 28 would probably not react with nucleophiles, such as the hydride ion. Molecular modelling was used to probe the unreactive nature of oxime 28. The probe revealed that the imide ring deactivates the oxime groups in this compound. Based on these results, attempts were made to change the carbon framework of 1A to eliminate unwanted interaction between the carbonyl groups. Clemmensen reduction of 1A did not yield the expected ketol 40, but gave a mixture of 62 and 63. Extended reaction times yielded 63 only. Reduction of 1A with zinc and acetic acid produced a mixture of 62 and 40 that could not be separated. Consequently, the applicability of 62 as a possible dendrimer nucleus was investigated. Compound 62 was obtained by oxidisation of the diol 63 with sodium periodate. Attempts to synthesise the dioxime 69 from 62 failed and only the mono oxime 71 was obtained. Reduction of 71 with lithium aluminium hydride was unsuccessful. Molecular modelling revealed that the oxime carbon atom does not have a LUMO and that the carbonyl carbon atom would probably be unreactive towards nucleophiles. Several other attempts were made at reducing the oxime 71 to an amine. None of these attempts met with any success. The reason for the unreactive nature of 71 is less clear than in the case of 28. Failure to produce a cage amine from 1A or derivatives of 1A meant failure in functionalising the cage system with phosphine groups and coupling the Grubbs-I catalyst. It seems that cage compounds based on 1A are generally not suitable as starting materials for amine-functionalised dendrimer nuclei. / Thesis (M.Sc. (Chemistry))--North-West University, Potchefstroom Campus, 2011.

No description available.

Graft copolymers with a dendritic (arborescent) architecture and unimolecular micelle properties have been synthesized and examined for their solution properties as well as their ability to serve as templates for the preparation of metallic nanoparticles. The research focused on two types of arborescent copolymers, namely core-shell (CS) and core-shell-corona (CSC) architectures. Copolymer amphipolarity was provided by a hydrophobic polystyrene (PS) component and a polar poly(2-vinylpyridine), P2VP, phase. The CS copolymers were obtained by grafting P2VP onto linear or branched PS substrates to yield PS-g-P2VP unimolecular micelles. These copolymers exhibited solubility with limited aggregation in aqueous environments after protonation with HCl. Coordination of the coronal P2VP phase with HAuCl4 was achieved, and the resulting polymer-stabilized metallic nanoparticles had a spherical morphology. The tri-layered copolymers were synthesized in similar fashion, by grafting PS-b-P2VP block copolymers onto linear or branched PS substrates to afford a CSC morphology with a PS core, a P2VP inner shell, and a PS corona. While the grafting reaction proceeded with lower than expected grafting yields as compared to grafting reactions of homopolymer side-chains, significant P2VP content and molecular weight increases were achieved. It was determined that aggregation of the block copolymer side-chains hindered the coupling reaction of the reactive centers with the substrate. After purification of the PS-g-(P2VP-b-PS) arborescent copolymers, in part by developing a cloud-point centrifugation purification technique, the spherical copolymers were successfully loaded with various metallic compounds. The uptake of compounds such as HAuCl4 by the P2VP phase of the copolymers induced intramolecular phase segregation for copolymers of generations (G) 2 and above. Phase segregation produced some unique and intricate morphologies different from the ones observed previously for other unimolecular metal scaffolds. Intramolecular toroidal, nodular, and cylindrical morphologies were observed for the G2, G3, and G4 arborescent copolymers, respectively. Templates of generations 0 and 1 displayed a spherical morphology similar to their PS-b-P2VP intermolecular micelle analogues when loaded with metals. Phase segregation is believed to be mediated mainly by the characteristics of the PS core, the length of the P2VP segments having less influence on the morphologies obtained. Reduction of the metallic salts was performed using various agents and protocols. Stronger reducing agents yielded smaller and more narrowly distributed gold nanoparticles, while other reduction methods also removing the polymer scaffold resulted in larger nanoparticles.