<p>Patients with systemic lupus erythematosus (SLE) have elevated levels of interferon (IFN)-α in blood and IFN-α-producing cells in tissues. In the present thesis, we investigate the mechanisms behind the upregulated IFN-α-production in SLE and also show that the IFN-α system is activated in primary Sjögren’s syndrome (pSS), with IFN-α-producing cells in the major affected organ, the salivary glands. The IFN-α is a type I IFN, a family of cytokines counteracting especially viral infections, by acting directly on infected cells, and via many immunomodulatory effects. The latter may also contribute to autoimmune processes.</p><p>The type I IFNs are usually produced upon recognition of microbial structures. In SLE, however, DNA-containing immune complexes (ICs) that induce IFN-α production are found. Many autoantibodies in SLE and pSS are directed to nucleic acids or to DNA/RNA-binding proteins. We show that also RNA in complex with autoantibodies from SLE or pSS patients (RNA-IC) induces IFN-α-production. The RNA could be either in the form of RNA-containing material released from apoptotic or necrotic cells or as a pure RNA-containing autoantigen, the U1 small nuclear ribonucleoprotein particle. </p><p>The IFN-α-production induced by RNA-IC occurred in plasmacytoid dendritic cells (PDCs), also termed natural IFN-producing cells (NIPCs), via binding to Fcγ-receptor IIa, endocytosis and triggering of Toll-like receptors (TLRs), probably TLR7 and TLR9. The RNA-IC may also have other effects, and we found that they induce prostaglandin E2 (PGE2) production in monocytes and tumor necrosis factor (TNF)-α in both monocytes and NIPC/PDC. The PGE2 downregulated the IFN-α induction in NIPC/PDC, and the IFN-α induction was increased in monocyte-depleted cell cultures. </p><p>The findings presented in this thesis aids in the understanding of the mechanisms behind the activated IFN-α system in SLE and other autoimmune diseases, and shows that also pSS is one of these diseases.</p>

Patients with systemic lupus erythematosus (SLE) have elevated levels of interferon (IFN)-α in blood and IFN-α-producing cells in tissues. In the present thesis, we investigate the mechanisms behind the upregulated IFN-α-production in SLE and also show that the IFN-α system is activated in primary Sjögren’s syndrome (pSS), with IFN-α-producing cells in the major affected organ, the salivary glands. The IFN-α is a type I IFN, a family of cytokines counteracting especially viral infections, by acting directly on infected cells, and via many immunomodulatory effects. The latter may also contribute to autoimmune processes. The type I IFNs are usually produced upon recognition of microbial structures. In SLE, however, DNA-containing immune complexes (ICs) that induce IFN-α production are found. Many autoantibodies in SLE and pSS are directed to nucleic acids or to DNA/RNA-binding proteins. We show that also RNA in complex with autoantibodies from SLE or pSS patients (RNA-IC) induces IFN-α-production. The RNA could be either in the form of RNA-containing material released from apoptotic or necrotic cells or as a pure RNA-containing autoantigen, the U1 small nuclear ribonucleoprotein particle. The IFN-α-production induced by RNA-IC occurred in plasmacytoid dendritic cells (PDCs), also termed natural IFN-producing cells (NIPCs), via binding to Fcγ-receptor IIa, endocytosis and triggering of Toll-like receptors (TLRs), probably TLR7 and TLR9. The RNA-IC may also have other effects, and we found that they induce prostaglandin E2 (PGE2) production in monocytes and tumor necrosis factor (TNF)-α in both monocytes and NIPC/PDC. The PGE2 downregulated the IFN-α induction in NIPC/PDC, and the IFN-α induction was increased in monocyte-depleted cell cultures. The findings presented in this thesis aids in the understanding of the mechanisms behind the activated IFN-α system in SLE and other autoimmune diseases, and shows that also pSS is one of these diseases.

Homologous genetic recombination, because of its fundamental roles in the maintenance of genome stability and evolution, is an essential cellular function common to all organisms. This process also plays important roles in the repair of damaged DNA molecules, generation of genetic diversity and proper segregation of chromosomes. The genetic exchange is a highly orchestrated process that entails a plethora of control mechanisms and a large number of proteins, of which RecA and SSB are two proteins that have been chosen for further investigation(s) in the present study. In addition, we have also investigated the interaction between SSB and UvrD1, which plays an important role in DNA repair pathways, especially nucleotide excision repair (NER) and mismatch repair as well as DNA replication and recombination. Chapter 1 reviews the literature regarding various aspects of homologous recombination, with an emphasis on the biochemical and the biophysical aspects of RecA and SSB proteins. In addition, it provides an overview of the study of DNA repair and recombination in mycobacteria. RecA protein is ubiquitous and well conserved among bacterial species. Many archaeal species possess two RecA homologues (RadA and RadB) and eukarya possess multiple homologues of RecA including, Rad51, Rad51B, Rad51C, Rad51D, DMC1, XRCC2, or XRCC3. RecA or its homologues function as polymers, consisting of hundreds of monomers that cooperatively polymerize on single-stranded DNA to form a nucleoprotein filament. E. coli RecA protein participates in Trans Lesion Synthesis (TLS) of DNA and forms the minimal mutasome in association with DNA polymerase V (UmuD’2C). The fundamental mechanism underlying HR, i.e. DNA strand exchange, is one of the most fascinating examples of molecular recognition and exchange between biological macromolecules. Since the isolation of E. coli recA gene and the subsequent purification of its gene product and also from other organisms, RecA protein has been studied extensively for more than three decades. E. coli RecA protein has pivotal roles in DNA recombination and repair, and binding to DNA in the presence of ATP, is a fundamental property of RecA protein resulting in the formation of a nucleoprotein filament. This is the slow step of the HR process, and is considerably faster on ssDNA than on duplex DNA. Binding of RecA to dsDNA is slower at physiological pH, is accelerated at acidic pH, and the lag in binding at the higher pH values is due to slow nucleation. The ATP and the DNA binding functions of RecA display allosteric interaction such that ATP- binding leads to an increase in affinity to ssDNA-binding and vice-versa. X-ray structures of E. coli RecA complexed with nucleotide cofactors have implicated a highly conserved Gln196 in Mycobacterium smegmatis RecA in the coupling of ATP and the DNA binding domains. The carboxyamide group of Gln196 makes an H-bond with the γ-phosphate group of ATP and the side chain of this residue is observed to move by approximately 2Å towards the ATP, relative to the other residues involved in ATP binding. In addition, a highly conserved Arg198 has also been postulated to interact with the γ-phosphate group of bound ATP and position it for a nucleophilic attack by a conserved residue-Glu96 leading to ATP hydrolyses. To elucidate the role of Gln196 and Arg198 in the allosteric modulation of RecA functions, we generated MsRecA variant proteins, where in Gln196 was substituted with alanine, asparagine or glutamate; Arg198 was mutated to a lysine. The biochemical characterization of MsRecA and its variant proteins with the objective of defining the allosteric interaction between the ATP- and the DNA-binding sites has been described with in Chapter 2. We observed that while the mutant MsRecA proteins were proficient in ATP-binding they were deficient in ATP hydrolyses. We assayed for the ability of these proteins to bind ssDNA using either nitrocellulose filter binding or Surface Plasmon Resonance (SPR). While we did not detect any ssDNA-binding by the mutant MsRecA proteins in the filter binding assay, we observed only ten-fold reduction in the affinity for ssDNA as compared to wild type MsRecA protein in MsRecAQ196A, Q196N and R198K in the SPR assay. MsRecA Q196E did not show any binding to ssDNA, in both nitrocellulose filter-binding as well as SPR assays. We assayed for the ability of the mutant RecA proteins for their ability to promote DNA-pairing as well as DNA strand exchange. While we observed limited pairing promoted by the mutant proteins relative to the wild-type MsRecA, we observed a complete abrogation of strand exchange in the case of mutant proteins. In addition, we assayed for the co-protease function of MsRecA, by monitoring the cleavage of MtLexA. We observed that only the wild-type MsRecA protein was able to cleave MtLexA, while none of the mutant RecA proteins were able to do so. In order to understand the differences observed between the wild -type and the mutant MsRecA proteins, we analyzed the conformational state of MsRecA and its variant proteins by circular dichroism spectroscopy upon ATP-binding. We observed that while MsRecA and MsRecAQ196N displayed a reduction in the absorbance at 220 nm upon ATP binding, we did not observe any such structural transitions in the other mutant MsRecA proteins that we tested. Based on our observations and the crystal structure of E. coli RecA bound to ssDNA, in Chapter 2, we propose a dual role for the Gln196 and Arg198 in modulating RecA activities. In the presynaptic filament Gln196 and Arg198 sense the presence of the nucleotide in the nucleotide binding pocket and initiate a series of conformation changes that culminate in the transition to an active RecA nucleoprotein filament. In the active RecA nucleoprotein filament these residues are repositioned such that they now form a part of the protomer-protomer interface. As such they perform two vital functions; they stabilize the protomer-protomer interface by participating in the formation of hydrogen bonds that span the interface as well transmit the wave of ATP hydrolysis across the interface leading to a coordinated hydrolyses of ATP essential for the heteroduplex extension phase of strand exchange reaction. The members of the super family of single stranded DNA binding proteins (SSB) play an important role in all aspects of DNA metabolism including DNA replication, repair, transcription and recombination. Prokaryotic SSBs bind ssDNA with high affinity and generally with positive cooperativity. Several lines of evidence suggest that prokaryotic SSBs are modularly organized into three distinct domains: the N-terminal DNA binding domain and acidic C-terminal domain are linked by a flexible spacer. Studies from our laboratory have revealed that M. smegmatis SSB plays a concerted role in recombination-like activities promoted by the cognate RecA. The C- terminal of SSB is known to be involved in its ability to interact with other proteins. We have previously reported that the C-terminal domain of M. smegmatis SSB, which is not essential for interaction with DNA, is the site for the binding of cognate RecA. The data in Chapter 3 describes the characterization of the SSB C-terminus with the objective of delineating the elements responsible for mediating protein-protein interaction, as well as to define the mechanism by which SSB is able to modulate the activities of RecA. To map the RecA interaction domain of SSB we created deletion mutants in MsSSB lacking 5, 10, 15 or 20 residues from the C-terminal. The truncated SSB proteins were expressed with a His- tag at the N- terminus and purified to homogeneity using a Ni-NTA affinity matrix. We observed unlike MsSSB, MsSSB∆C5 and MsSSB∆C10, MsSSB∆C15 and MsSSB∆C20 were unable to support three-strand exchange catalyzed by MsRecA. Based on the observation that interaction with SSB is essential for MsRecA to catalyze the strand Exchange reaction, we postulate that the RecA interacting domain of SSB is situated between the 15th and the 20th residue from the C-terminal. Further, the C-terminal of MsSSB modulates the transitions between DNA binding modes. Unlike the case with EcSSB where deletion of the last 8 residues from the C-terminal stabilizes the (SSB)35 mode of ssDNA binding, we observe that in case of MsSSB the deletion of C-terminal seems to destabilize the (SSB)35. In addition, the transition from the low density binding mode to a high density mode involves the formation of several intermediates when the C-terminal residues are deleted. With the objective of understanding the functions to the C-terminal of SSB independent of its DNA-binding domain in modulating RecA functions, we employed a peptide corresponding to the 35 residues from the C-terminal of the MsSSB. We observed that the C-terminal region alone is capable of interacting with RecA. In addition we also observed that the C-terminal domain of SSB stimulates RecA functions independent of its DNA binding domain. To address the question, whether the stimulatory effect of the C-terminal domain of SSB in the absence of its DNA-binding domain is restricted to RecA or is a generalized phenomenon associated with all SSB interacting proteins; we tested the effect of C-terminal domain of SSB on UvrD which is known to interact with SSB. UvrD participates in several pathways of DNA metabolism, which include the nucleotide excision repair (NER) and mismatch repair pathway, replication and recombination. Genetic evidence suggests that UvrD and SSB interact in vivo. We tested the effect of mycobacterial SSB on M. tuberculosis UvrD1 (MtUvrD1) functions in vitro. We observe that MtUvrd1 physically interacts with SSB. Further, presence of SSB has an inhibitory effect on the helicase activity of MtUvrD1 and that this effect is dependent on the C-terminal region as the deletion of residues from the C-terminal of SSB abrogates the inhibitory effect of SSB. However, unlike RecA, the C-terminal region of SSB alone had no effect on the helicase activity of UvrD1. We also observed that MsSSB has opposing effects on the ATPase activity of MtUvrD1. In the presence of low concentrations of SSB the ATPase activity is enhanced, while we observed an inhibition when the concentration of MsSSB is high. The precise mechanistic details of how SSB is able to act as an accessory protein to RecA, in context of homologous recombination and stimulates its biochemical activities have been a subject of debate. Whereas research from some groups has shown that the stimulatory effect SSB is mediated through its ability to melt DNA secondary structure, thereby allowing RecA to overcome the kinetic barrier imposed by the presence of secondary structure in ssDNA, others postulate that SSB plays a direct role in the stabilization of RecA nucleoprotein filament and prevents its dissociation. Chapter 3 discusses the experimental evidence in favor of the aforesaid models and based on the results of our experiments; we propose that the accessory functions of SSB may be mediated by a mechanism that involves elements of both models. While interaction with SSB can bring about a conformational change in RecA that is reflected in the enhanced levels of strand exchange and co-protease activity, the helix destabilizing function of SSB is essential during heteroduplex extension and to sequester the displaced strand such that it does not participate in any further pairing reactions. The novel finding that we present in Chapter 3 is that the interaction of SSB C-terminal alone has a stimulatory effect upon RecA activities. Furthermore, we observed that M. tuberculosis UvrD1 is a weak interaction partner of SSB. The physical and functional interactions between MsSSB with RecA on the one hand, and MsSSB and UvrD1 on the other highlight different types of cross-talk between the components of HR and DNA repair pathways. In contrast to the results of earlier studies, our results indicate that protein-protein interactions alone between SSB and RecA may modulate the RecA mediated processes of presynapsis, homologous pairing and strand exchange between homologous DNA molecules as well as modulate its co-protease activity. In addition, our studies indicate that a direct protein-protein interaction is responsible for the modulation of UvrD1 activities by SSB.

In recent time, several reports have been published about a more and more unhealthy population world wide, with increasing Body Mass Index (BMI) in welfare countries, such as Sweden. Diseases, such as obesity and diabetes, which is strongly connected to a high BMI, have increased and together with them also the medical expenses for society/state. Several initiatives have been started, in different countries, to tackle these problems and some have introduced a “sugar tax” on unhealthy products, like candy and soda, which has become a well- debated subject also in Sweden today. In this MBA master thesis, a literature study has been conducted with the goal of evaluating if an excise tax should be introduced in an efficient way on unhealthy sugar-sweetened beverages in Sweden. This case study is built on secondary data where reports and official statistics, from governments and health authorities/organizations, have been studied both for Sweden as well as from other countries. There has been a particular focus on Sweden's neighbouring countries Denmark and Finland, who has both experiences in the implementation of a “sugar tax”. Our theory is that introducing an excise tax on sugar-sweetened beverages will reduce the demand and consumption of these products, which will reduce welfare disease such as obesity and diabetes and yield a tax income for the state. However it is important to have in mind that the reduced consumption also will result in less tax income from the no longer sold goods, fewer personnel employed in the producing industries etc. The results showed that the overall sugar consumption actually has decreased in Sweden, as well as the overall consumption of sugar-sweetened beverages. However during the same time period the average calorie consumption and BMI has continued to increase resulting in a more unhealthy population that results in increased medical expenses. In conclusion an excise tax on sugar-sweetened beverages will not solve the welfare disease problems but may positively influence health. However it comes with a price also for the state from both gains and loss in tax incomes and increased administrations costs for managing the new tax. Finally it should be noted that since sugar-sweetened beverages are unhealthy products, which do not contribute to any positive health effects, sugar taxation might still be considered.

This project deals with possibilities of using the software defined radio conception for radio amateur use in a short wave band and its subsequent implementation into properly designed hardware. The aim of this work is to design a transceiver that would be capable of working in AM, FM, SSB, and CW modes. Within a theoretical part of the project the architectures of software defined radios and their components are discussed. This part was focused mainly on analog parts of the chain, such as amplifiers, filters and converters. Signal processing algorithms for both receiver and transmitter working in desired modes are studied subsequently and their computer models are built. Designed algorithms are implemented into FPGA structure (Virtex -5).

Master’s thesis deals with design and practical realisation of electronic circuits, which are needed for assembling of DRM signal transmitter for ham short waves bands. There is presented DRM standard as well as there are described the differencies between DRM for radio broadcast and for ham using. There is described design of input audio circuits, modulator, mixer, local generator, amplifier and filters. Principle of used SSB modulator is based on phase method, often called Tayloe modulator. This principle is analysed in detail including mathematical description, which was derived. It is possible to control the transmitter by program running at computer, communication takes place via USB. There is described establishment of communication in this work too.

The dissertation thesis deals with a study of an influence of a simultaneous incidence of an additive thermal noise and a multiplicative phase noise on the useful signal transmission in narrowband satellite communication systems. While the additive thermal noise affects the useful signal only on the receiver side of the communication system, the multiplicative phase noise is produced in all system oscillators. One investigates how the receiver filter bandwidth reduction takes effect on the influence of individual noise types. The thesis is divided into four units. The first one (chapters 4 and 5) solves the ways of modeling of both noise types. In the second part (chapter 6), on the simple example, the primary analysis of the phase noise influence is made. Basic modulation schemes used in the satellite communication are also discussed (chapter 7). Third part (chapter 8) is devoted to the modeling of a general digital system with a M PSK modulation made directly on the main carrier wave. The last part (chapter 9) describes the modeling of a digital system with a BPSK modulation on the auxiliary subcarrier wave followed by an SSB modulation on the main carrier wave. General conclusions are deduced from obtained simulation results.

The design and analysis of an automotive structure is an important stage of the vehicle design process. The structural characteristics have significant impact on the vehicle performance. During the design process it is necessary to have knowledge about the structural characteristics; however in the preliminary design stages detailed information about the structure is not available. During this period of the design process the structure is often simplified to a representative model that can be analyzed and used as the input for the detailed design process. A vehicle model is developed based on the space frame structures where the frame is the load carrying portion of the structure. Preliminary design analysis is conducted using a static load condition applied to the vehicle as pure bending and pure torsion. The deflections of the vehicle based on these loading conditions are determined using the finite element method which has been implemented in developed software. The structural response, measured as the bending and torsion stiffness, is used to evaluate the structural design. An optimization program is implemented to improve the structural design with the goal of reducing weight while increasing stiffness. Following optimization the model is completed by estimating suitable plate thicknesses using a method of substructure analysis. The output of this process will be an optimized structural model with low weight and high stiffness that is ready for detailed design. / UOIT

The research conducted in this dissertation studies the issues regarding the improvement of phase noise performance in a BiCMOS Silicon Germanium (SiGe) cross-coupled differential-pair voltage controlled oscillator (VCO) in a narrowband application as a result of a tail-current shaping technique. With this technique, low-frequency noise components are reduced by increasing the signal amplitude without consuming additional power, and its effect on overall phase noise performance is evaluated. The research investigates effects of the tail-current as a main contributor to phase noise, and also other effects that may influence the phase noise performance like inductor geometry and placement, transistor sizing, and the gain of the oscillator. The hypothesis is verified through design in a standard 0.35 μm BiCMOS process supplied by Austriamicrosystems (AMS). Several VCOs are fabricated on-chip to serve for a comparison and verify that the employment of tail-current shaping does improve phase noise performance. The results are then compared with mathematical models and simulated results, to confirm the hypothesis. Simulation results provided a 3.3 dBc/Hz improvement from -105.3 dBc/Hz to -108.6 dBc/Hz at a 1 MHz offset frequency from the 5 GHz carrier when employing tail-current shaping. The relatively small increase in VCO phase noise performance translates in higher modulation accuracy when used in a transceiver, therefore this increase can be regarded as significant. Parametric analysis provided an additional 1.8 dBc/Hz performance enhancement in phase noise that can be investigated in future works. The power consumption of the simulated VCO is around 6 mW and 4.1 mW for the measured prototype. The circuitry occupies 2.1 mm2 of die area. Copyright / Dissertation (MEng)--University of Pretoria, 2010. / Electrical, Electronic and Computer Engineering / unrestricted

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