<p>Silver was used for its antimicrobial effect by the ancient Greeks, long before the existence of microorganisms were first suspected. Nowadays a wide range of antimicrobial dressings containing silver, either incorporated within or applied on the dressings, are available for clinical use. This type of dressings is designed to provide the antimicrobial activity of silver in a more convenient application.</p><p>The aim with this master thesis was to evaluate if silver release and antimicrobial effect of nine silver containing dressings are dependent on the test medium and if there is any relation between silver release and antimicrobial effect.</p><p>Release of silver and antimicrobial effect was evaluated by using a 6-well co-culture system, with inoculated test medium in the wells and dressing pieces in the culture well inserts. Three different test media with increased complexity and nutrient value were inoculated with either</p><p>Results show that release of silver depends on the test fluid used; for phosphate buffered saline (PBS), the silver concentration was as most 1.2 ppm, but for a complex media containing calf serum (SWF), it varied from 9 ppm to 134 ppm. The viable counts in PBS were reduced by at least 3 log units for all dressings and bacteria, whereas in SWF there were no reduction and instead growth was observed. In general, a high release resulted in less bacterial growth. Results also indicated that kinetics of silver release affect the antimicrobial effect. It is likely to assume that it is important for a dressing to release silver quickly.</p><p>It has previously not been possible to correlate silver release of wound care dressings and antimicrobial effect, since the two factors have been measured in different test systems and in different media. Since both factors depend on test medium and method used, it is shown in the present study that it is important to use relevant test medium for in-vitro evaluation. When measuring silver release and antimicrobial effect in the same test system, a relation is found.</p>

<p>Biogasprocessen är en komplex anaerob nedbrytningskedja där olika mikroorganismer är inblandade. Vanligast är att biogas produceras i mesofil rötning (cirka 38 ^oC), men även termofil rötning används (> 50 ^oC).</p><p>Svensk Biogas i Norrköping använder återstoden av etanoldestillationen hos en närliggande etanolproducent (drankvatten) som substrat. Substratets höga temperatur vid leverans motiverar termofilt rötningsförsök av drankvatten.</p><p>Försöket genomfördes i 55 ^oC med två kontinuerligt omrörda tankreaktorer (CSTR) och en termofil ymp. Biogasproduktion av drankvatten undersöktes. Sätt att hantera och motverka höga ammoniumhalter, samt effekter av näringslösningstillsats undersöktes. Det tog cirka 30 dagar för ympen att acceptera det nya substratet och då hade tillsats av processhjälpmedel KMB1 samt järnklorid använts. Reaktorerna kunde belastas med 3 g VS / (L • dygn) (VS, volatile solids, glödförlust). Den specifika gasproduktionen var 0,6 – 0,7 L / g VS och metanhalten ungefär 45 %. Höga ammoniumhalter motverkades genom förkortning av uppehållstiden. Under en period tillsattes nickelklorid i en av reaktorerna och under denna period hade reaktorn med nickelkloridtillsats något bättre specifik gasproduktion jämfört med reaktorn där ingen nickelklorid tillsattes.</p><p>Drankvatten kan rötas under termofila förhållanden. För att temperaturförändring vid biogasanläggningen i Norrköping ska var ekonomiskt försvarbart måste processen klara högre belastning.</p>

<p>To further develop cancer therapy, targeted delivery of cell killing agents directly to tumor cells is an interesting approach. This thesis describes the development of PEG-stabilized liposome conjugates targeting either epidermal growth factor receptor (EGFR) using its natural ligand EGF, or human epidermal growth factor receptor 2 (HER-2) using the antibody trastuzumab. Both receptors are known to be overexpressed on a variety of tumors. The liposomes were loaded with the boronated compounds water soluble boronated acridine (WSA) or water soluble boronated phenantridine (WSP), compounds primarily developed for boron neutron capture therapy, BNCT. </p><p>The liposome conjugates bound specifically to their receptors in cell culture. Because the WSA conjugates exhibited the most favorable boron uptake this compound was chosen for further study. The WSA-loaded liposome conjugates was internalized, an important characteristic for BNCT, and had a long retention inside the cells. The cellular localization of WSA, studied using fluorescence was found to be mainly cytoplasmic. </p><p>To increase the boron uptake studies comparing different incubation methods was performed. It was shown for both EGF and trastuzumab targeted liposomes the uptake could be increased over 10 times by changing from incubation in monolayer culture to incubation in cell suspension in roller flasks. With this treatment the boron concentrations reached after 24 h incubation time was 90 ppm for EGF-liposomes and 132 ppm for trastuzumab-liposomes, levels that are clinically interesting. </p><p>To study the cell-killing efficacy of the liposome-conjugates an experimental BNCT study was performed using EGF-liposome-WSA on cultured glioma cells. About half the number of thermal neutron was needed to inactivate 90% of the cells if the cells had been incubated with EGF-liposome-WSA compared to control cells. When comparing the survival to dose it was shown that to inactivate 90% of the cells 2.9 Gy was needed for EGF-liposome-WSA and neutrons compared to 5.6 Gy with ¹³⁷Cs gamma. </p><p>The biodistribution of EGF-liposomes was also studied in mice. It was compared to EGF and it was found that the addition of a PEG-stabilized liposome to EGF significantly reduced EGF uptake in liver and kidneys, the circulation time in blood was prolonged as well. The reduced liver uptake might be due to inability of the 100 nm liposomes to pass the sinusoidal fenestrations of the liver and bind to the EGFR-rich hepatocytes. The reduced liver uptake potentates the use of EGF-liposome conjugates for systemic injection.</p>

<p>Escherichia coli is a natural inhabitant of the intestines of both humans and animals, but there are also several pathogenic types of E. coli which cause disease in humans.</p><p>Strains of enterohemorrhagic E. coli (EHEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of shigatoxin 1 and 2 or combination of these toxins. Other major virulence factors include EHEC hemolysin and intimin, the product of the eae gene that is involved in attaching and effacing adherence phenotype. EHEC has also been associated with uncomplicated diarrhea.</p><p>The capacity to control EHEC disease and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection.</p><p>The principal reservoirs of EHEC are cattle and food products, which presumably have come into contact with domestic animal manure and/or are inadequately pasteurised, these are important vehicles of infection.</p><p>In the present study, the PCR technique with primers detecting the verocytotoxin genes was shown to be a possible method to screen for and identify EHEC.</p><p>In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.</p>

<p>Unwanted fungal growth is one of the most common causes of food spoilage throughout the world, and is causing health risks for both humans and animals and economical losses for the food- and agricultural industries. In Europe the mycotoxin producing Fusarium species F. sporotrichioides, F. culmorum, F. poae and F. graminearum is of greatest importance, due to their production of the trichothecene deoxynivalenol (DON) among other mycotoxins. Today’s conventional determination methods of these Fusarium species is time-consuming and quicker screening methods directly on cereals is therefore of interest to develop. In this project a species-specific PCR-protocol targeting the trichodiene synthase (tri5) gene in F. sporotrichioides, F. culmorum, F. poae and F. graminearum was used to evaluate two different DNA-extraction methods for cereals. The PCR-protocol was first verified with pure fungal cultures and optimized with a PCR-gradient before it was applied on cereals. The PCR-gradient resulted in a background reduction in the PCR-analysis of F. sporotrichioides infected cereals.</p><p>The two methods, called the Hammer-method (cereals was crushed with a hammer) and the Nitrogen-method (cereals were crushed in a mortar together with liquid nitrogen), is both combinations of a published DNA-extraction method (CTAB-based) for cereals and a DNA-purifying kit (chaotropic agent-based). Within these two methods some modifications were made and a comparison of the results showed that the Nitrogen-method indicated to be more stable than the Hammer-method. Too few analyses have though been made for a definite conclusion. A verification of the Nitrogen-method showed that the PCR-protocol can be considered as stable and reliable also on cereals but the DNA-extraction method for cereals is still to be optimized and stabilized. Sonification of the cereals is under consideration for further tests and studies.</p> / <p>Mögelsvampsinfektioner av spannmålsprodukter är ett av de vanligaste problemen inom mat- och jordbruksindustrierna runt om i världen. Enligt FNs Food and Agriculture Organization beräknas att cirka 25 procent av världens spannmålsgrödor är infekterade med mykotoxinbildande mögelsvampar vilket kan leda till stora hälsorisker för konsumenterna och ekonomiska förluster för mat- och jord-bruksindustrierna. Mykotoxiner är sekundära produkter från svampens ämnesomsättning som troligen har betydelse för svampens överlevnad, men kan ge toxiska effekter hos människa och djur. I Europa är mögelsvampsläktet Fusarium den vanligaste och viktigaste av mykotoxinbildande svampar och producerar de för jordbruksnäringen två viktigaste mykotoxingrupperna trichotechener och fumonisi-ner.</p><p>På grund av den breda förekomsten av dessa Fusarium-svampar finns det idag ett behov av att utveckla snabba och pålitliga metoder för att detektera och identifiera mögelsvamparna redan direkt på de färska spannmålen. Under hösten 2002 startades projektet Bestämning av potentiella mykotoxin-producerande Fusariumsvampar med PCR-metodik vid Mikrobiologiska enheten på Livsmedelsver-ket i Uppsala. Projektet syftar till att utveckla en molekylärbiologisk bestämningsmetod där identifie-ringen av svamparna sker med hjälp av dess DNA istället för via mikroskopiska undersökningar. Det-ta examensarbete har varit en del av det projektet och har i huvudsak inriktats på att utveckla en stabil metod för själva utvinningen av svamp-DNA från de färska spannmålen. De slutsatser som nåtts är att de utvinningsmetoder som examensarbetet omfattade inte kan anses stabila i nuläget utan behöver utvecklas och stabiliseras ytterligare. Vad gäller de molekylärbiologiska metoderna har de kunnat visas stabila även på färskt spannmål.</p>

<p>Leishmaniasis is a disease affecting more than 12 million people worldwide. It is caused by the protozoan parasite Leishmania, which is transmitted to humans and dog hosts through bites of infected sand flies belonging to genus Phlebotomine. Several studies have shown Polymerase Chain Reaction (PCR) to be effective for the diagnosis of VL in clinical samples compared to the classical methods. The aims of this study were first to compare four different sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and furthermore to find a method that is sensitive, rapid, cost benefit, simple and easy to perform. Two preparation methods were compared for the isolation of leukocytes (with Ficoll and Tris –EDTA buffer) and two DNA isolation methods (with Proteinase K and QIAgen kit). From the methods that were compared, lysis of erythrocytes with TE and the QIAgen kit seems to be the most suitable to use.</p>

<p>This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of <i>Neisseria gonorrhoeae, N. meningitidis</i> and <i>Streptococcus pyogenes</i> on host epithelial cells and <i>in vivo</i>. </p><p><i>N. gonorrhoeae</i> cause the sexually transmitted disease gonorrhoea and <i>N. meningitidis</i> is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, <i>N. meningitidis</i> generally induced an earlier response compared to <i>N. gonorrhoeae</i>, probably as a result of the meningococcal capsule. The role of <i>N. meningitidis</i> serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. </p><p><i>S. pyogenes</i> may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. <i>S. pyogenes</i> ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of <i>S. pyogenes</i> is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of <i>S. pyogenes</i>.</p>

<p>Controlling bacterial infections with antibiotics is central to modern health care. However, increasing bacterial resistance to antibiotics threatens effective therapy. This thesis concerns the use of the antibiotic fusidic acid, and novel analogues of fusidic acid, to treat topical infections caused by the bacterial pathogen <i>Staphylococcu aureus</i>. It also addresses genetic mechanisms by which <i>S. aureus</i> develops resistance to fusidic acid.</p><p>Pre-clinical microbiological tests were made on two structurally different groups of fusidic acid analogues developed by Leo Pharma. These drugs were tested against <i>S. aureus</i> and <i>Streptococcus pyogenes</i> strains, measuring MIC, <i>in vitro</i> concentration-dependent bacteriocidal or bacteriostatic effects, and <i>in vivo</i> efficacy in clearing topical infections. We developed a new superficial skin infection animal model (the ‘tape-stripping model’) designed for testing topical antibiotics, including the novel fusidic acid analogues, against <i>S. aureus</i> and <i>S. pyogenes</i>. Some new compounds giving promising results will be further tested and developed by Leo Pharma. </p><p>Fusidic acid inhibits protein synthesis by binding to elongation factor EF-G on the ribosome. Previously described resistance mechanisms are mutations in the gene coding for EF-G (<i>fusA</i>), or, in some strains, the presence of a gene (<i>fusB, fusC</i> or <i>fusD</i>) coding for a protein that protects EF-G from fusidic acid.</p><p>We discovered two novel classes of spontaneous FusR mutants in <i>S. aureus</i> with the small colony variant (SCV) phenotype which is associated with persistent infections. The FusR SCV’s are very frequent, slow growing, cross-resistant to aminoglycosides, and auxotrophic for hemin or menadione. Some of the FusR SCV mutations are in structural domain V of EF-G (classic <i>fusA</i> mutations map overwhelmingly in domain III). The remaining FusR SCV’s are unmapped but their additive effect on MIC together with the <i>fusB</i> plasmid suggests the possibility that their mechanism of resistance is also associated with the translation machinery. </p>

<p>Archaea is the third domain of life, discovered only thirty years ago. In a microscope archaea appear indistinguishable from bacteria, but they have been shown to be more closely related to eukaryotes than to bacteria. Especially central information processing is homologous to that of eukaryotes. The archaea, previously thought to be limited to extreme environments, constitute a large part of life on Earth to an extent that has only begun to be understood. Despite their abundance little is known about several central cell-cycle features, such as cell division and genome segregation.</p><p>For this thesis, a comprehensive study of the cell cycle in the model archaeon <i>Sulfolobus acidocaldarius</i> was performed, describing the majority of its cell-cycle regulated genes. Several known DNA replication genes, as well as genes previously not known to have a role in the cell cycle, displayed cyclic transcription. Several transcription factors, kinases and DNA sequence elements were identified as cell-cycle regulatory elements. Among the most important findings were putative cell division and genome segregation machineries.</p><p><i>Sulfolobus</i> species were discovered to have three origins of replication, constituting the first known prokaryotes with multiple origins. All origins initiate replication in a synchronous manner. Cdc6 proteins were shown to bind to origin recognition boxes conserved across the Archaea domain. Two Cdc6 proteins function as replication initiators, while a third paralog is implicated as a negative factor. Replication was shown to proceed at a rate similar to that of eukaryotes.</p><p>A particular type of cell cycle organization was found to be unusually conserved in the Crenachaeota phylum. All the studied species displayed a short prereplicative phase and a long postreplicative phase, and cycle between one and two genome copies. Genome sizes were determined for several species. The euryarchaeon <i>Methanothermobacter thermautotrophicus</i> was also studied, and it was shown to initiate genome segregation during, or just after, replication. In contrast to the crenarchaea it never displayed a single genome copy per cell.</p>

<p>ABSTRACT</p><p>Nontypable Haemophilus influenzae is a common cause of otitis, sinusitis and conjunctivitis. It is the most common bacterial pathogen associated with chronic obstructive pulmonary disease (COPD). Studies have shown that nonpathogenic Haemophilus haemolyticus are often mistaken for Haemophilus influenzae due to an absent hemolytic reaction on blood agar. Distinguishing H. haemolyticus from H. influenzae is important to prevent unnecessary antibiotic use, and to understand the role of H. influenzae in clinical infections. In this study, PCR-primers for amplifying 16S rDNA sequences were used to set up a method for distinguishing H. haemolyticus from H. influenzae. The aim was to use the method for analyzing apparent H. influenzae strains, to investigate if some strains were in fact H. haemolyticus. However, because of problems with unspecific primerannealing,no conclusions could be drawn regarding misclassification of H. haemolyticus.</p><p>Moraxella catarrhalis is the second most common bacterial pathogen associated with COPD. It also causes otitis and sinusitis. An important virulence factor of M. catarrhalis is the outer membrane protein Moraxella catarrhalis IgD-binding protein (MID). One part of the protein; MID764-913 , has been shown to function as an adhesin, and this part has been fragmented to further investigate its adhesive properties. The aim of this second, independent study, was to express some of these proteinfragments by cloning in E. coli. The time spent on this project was too short, and no proteins could be expressed duing this period.</p>