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. 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. 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. 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. 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.

This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of Neisseria gonorrhoeae, N. meningitidis and Streptococcus pyogenes on host epithelial cells and in vivo. N. gonorrhoeae cause the sexually transmitted disease gonorrhoea and N. meningitidis 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, N. meningitidis generally induced an earlier response compared to N. gonorrhoeae, probably as a result of the meningococcal capsule. The role of N. meningitidis 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. S. pyogenes 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. S. pyogenes 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 S. pyogenes 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 S. pyogenes.

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 Staphylococcu aureus. It also addresses genetic mechanisms by which S. aureus develops resistance to fusidic acid. Pre-clinical microbiological tests were made on two structurally different groups of fusidic acid analogues developed by Leo Pharma. These drugs were tested against S. aureus and Streptococcus pyogenes strains, measuring MIC, in vitro concentration-dependent bacteriocidal or bacteriostatic effects, and in vivo 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 S. aureus and S. pyogenes. Some new compounds giving promising results will be further tested and developed by Leo Pharma. 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 (fusA), or, in some strains, the presence of a gene (fusB, fusC or fusD) coding for a protein that protects EF-G from fusidic acid. We discovered two novel classes of spontaneous FusR mutants in S. aureus 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 fusA mutations map overwhelmingly in domain III). The remaining FusR SCV’s are unmapped but their additive effect on MIC together with the fusB plasmid suggests the possibility that their mechanism of resistance is also associated with the translation machinery.

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. For this thesis, a comprehensive study of the cell cycle in the model archaeon Sulfolobus acidocaldarius 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. Sulfolobus 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. 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 Methanothermobacter thermautotrophicus 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.

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). 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. 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. Drankvatten kan rötas under termofila förhållanden. För att temperaturförändring vid biogasanläggningen i Norrköping ska var ekonomiskt försvarbart måste processen klara högre belastning.

Only two out of more than 200 V. cholerae serogroups, classified on the basis of LPS structure, are associated with epidemic or pandemic cholera. These toxigenic serogroups carry phage-derived pathogenicity islands coding for the main virulence factors for establishment of cholera disease – cholera toxin (CTX) and toxin-coregulated pilus (TCP). The latter also serves as a bacterial surface receptor for the CTXΦ – the filamentous phage which carries the cholera toxin genes into otherwise harmless to human, environmental bacterium V. cholerae. In its natural aquatic habitat V. cholerae is subject to predator grazing, bacteriophage killing, temperature and pH changes, seasonality of plankton blooms and other environmental factors. Therefore understanding V. cholerae pathogenic and virulence potential requires the knowledge of its interaction not only with human host but also members of aquatic environment and environmental factors. V. cholerae is capable of killing the nematode Caenorhabditis elegans. Using a reverse genetics approach, we demonstrated that the quorum sensing regulated protease PrtV is essential for this killing. Other proteases did not seem to contribute to virulence in this model. The data from this study suggest that the PrtV could be important to V. cholerae in its natural niche for its resistance to the grazing predators. The PrtV protease belongs to an M6 family of metallopeptidases which is represented by an Immune Inhibitor A protease from the insect killing bacterium Bacillus thuringiensis. To characterize the protease in more detail, the PrtV was cloned, overexpressed in V. cholerae and purified from the culture supernatant. The enzyme was calcium stabilized and inhibited by metal ion chelators. In tests with in vitro cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell detachment and death. Using human blood plasma as a source of potential substrates, and by tests with purified candidate substrate proteins, we have identified fibrinogen (all α, β and γ chains), fibronectin and plasminogen to be degraded by the protease. Additionally, PrtV was found to alter the stability of V. cholerae cytolysin implicating its role in modulation of the reactogenicity of V. cholerae secreted factors. Pigmentation has been considered to be important in microbial pathogenesis because it has been associated with virulence in many microorganisms. Using transposon mutagenesis we identified the mutated locus of a pigment producing V. cholerae strain to encode a gene of a tyrosine catabolic pathway. The mutation in a putative homogentisate 1,2-dioxigenase gene lead to accumulation of homogentisic acid, its spontaneous oxidation and formation of a dark pigment. The pigment producing strain was altered in its ability to survive UV exposure and H2O2 stress, and was more efficient in colonizing the suckling mouse intestine compared to the wild type strain. Under the in vitro growth conditions the major virulence factor TcpA and CT expression was found to be somewhat enhanced too.

While bacterioplankton indisputably control vital biogeochemical paths in the cycling of carbon and nutrients in the world’s oceans, our knowledge about the functional and genetic diversity of bacterioplankton communities is negligible. In this thesis, molecular and more traditional microbiological methods were used to study the specific function of N2-fixation and in a general sense diversity of marine bacterioplankton species. Most oceans are nitrogen limited and, therefore, adaptive to bacterioplankton capable of N2-fixation. Recent studies have found nifH genes (coding for the nitrogenase enzyme) related to diverse heterotrophic bacteria in oceanic seawater samples indicating that, along with cyanobacteria, also heterotrophic bacteria benefit from N2-fixation. Here, molecular and cultivation methods were used to examine diazotrophic bacterioplankton in the Baltic Sea. We successfully isolated heterotrophic N2-fixing bacteria belonging to the γ-proteobacterial class by means of low-nitrogen plates and semi-solid diazotrophic medium tubes. The isolates required low-O2 conditions for N2-fixation. Using Real-time PCR it was found that heterotrophic bacterioplankton carrying the nifH gene was abundant (3 x 104 nifH gene copies L seawater-1) at locations in the Southwest Baltic proper. With the aim to identify the main N2-fixing organisms in Baltic Proper surface waters, a clone library of nifH gene transcripts (RNA) was generated. Clone inserts were exclusively related to Aphanizomenon sp. and Nodularia sp. Using quantitative real-time PCR it was found that the nifH gene expression from Nodularia sp. was highly variable between stations in the Baltic Proper but was 10-fold higher during mid summer relative to early summer and fall. A diel study showed a 4-fold increase in Nodularia transcript concentrations at early to mid day relative to rest of the day. Real-time PCR was found to be a powerful and highly sensitive method for measuring gene expression. Since nucleic acids are a prerequisite for molecular analyses of bacterioplankton dynamics a protocol to extract DNA from seawater samples was developed with the aim to maximize the yield of high-quality DNA. Each step in the protocol was important for the efficiency of extraction. The obtained extraction efficiencies were up to 92% for seawater samples and up to 96% for isolates. The protocol provides a guideline for DNA extraction from seawater samples for other studies. In a global sampling campaign (9 locations from polar, tropical and temperate regions) we sampled DNA from surface water and constructed 16S rRNA gene libraries to investigate diversity and biogeography of bacterioplankton. Approx. 80% of the sequences found were similar to sequences already deposited in GenBank, indicating that a large fraction of the marine bacterioplankton already has been sampled, which in turn suggests a limited global bacterioplankton diversity. This thesis have improved our knowledge about the composition and nifH gene expression of the diazotrophic bacterioplankton community in the Baltic Sea and contribute significantly to the discussion on global marine bacterioplankton diversity and biogeography. / Östersjön är ett av världens största brackvattensystem. Den ekologiska balansen i detta hav är hotad på grund av övergödning. Mycket arbete har därför fokuserats på att reducera utsläppen av näringsämnen, speciellt kväve. Dessa ansträngningar kan dock motverkas av bakterier som har förmåga att omvandla luftens kväve till metaboliskt användbart ammonium (kvävefixering). På sommaren är Östersjöns primärproduktion begränsad av kväve, med följden att det årligen uppstår massiva blomningar av kvävefixerande bakterier, framför allt cyanobakterier. Dessa är främst Aphanizomenon och Nodularia, men inte endast de fototrofa cyanobakterierna har förutsättningar att fixera N2. NifH gener (genen som kodar för nitrogenas) bärs också av heterotrofa bakterioplankton, vilket har visats i studier i främst Atlanten och Stilla havet. Med hjälp av två olika odlingsmetoder lyckades vi isolera heterotrofa kvävefixerande bakterier tillhörande klassen γ-proteobakteria från Östersjön. Svårigheten med att finna dessa bakterier ligger i att de kräver en miljö med mycket låg syrehalt för att kunna fixera kväve. Resultaten från denna studie ledde oss vidare till att undersöka vilka organismer som uttrycker nifH genen (och då troligen även fixerar kväve) i Östersjön. En av de bakterier som isolerats kunde påvisas med Realtids PCR i ett relativt stort antal (3 x 104 nifH genkopior per liter) vid en av de ursprungliga provtagningsstationerna. För att söka rätt på de olika organismtyper som uttrycker nifH skapades ett klonbibliotek baserat på mRNA extraherat från havsvatten. Det visade sig då att alla de närmare 100 kloner som sekvenserades tillhörde antingen Aphanizominon eller Nodularia. De heterotrofa bakteriernas nifH genuttryck var troligen i jämförelse med dessa cyanobakterier alltför lågt för att kunna detekteras. Realtids PCR mätningar av Nodularias nifH genuttryck visade på en stor variation mellan de olika provtagningsstationerna samt mellan de olika provtagningstillfällena. Vi fann dock en kraftig ökning under juli med en nedgång igen i augusti. En dygnscykelstudie visade att Nodularia nifH genuttrycket ökade under förmiddagen med en topp mitt på dagen för att sedan minska igen. Detta troligen med anledning av att den energikrävande kvävefixeringsprocessen sker under de ljusa timmarna då cellen får energi från fotosyntesen. I de molekylärbiologiska metoderna som används för att få information om identitet och aktivitet hos skilda organismer krävs att DNA och RNA kan extraheras från prover tagna i naturliga vattenmiljöer. Även om antalet bakterier tillsynes är högt, så är mängden DNA och RNA per liter havsvatten relativt låg, därför krävs ett väl fungerande protokoll för denna extraktion. I en inledande studie i denna avhandling optimerades en metod för att utvinna DNA. Ett antal sådana protokoll finns publicerade men dessa har ofta lågt utbyte. Det nya protokollet har hög effektivitet, vilket gör att små provvolymer kan användas (2 ml jämfört med tidigare flera liter) och därmed ökar hanterbarheten. Vi visar i denna studie att varje steg 7 i DNA-extraktionsprotokollet är viktigt för att ge en hög effektivitet. Detta protokoll kan med fördel användas som vägledning för många olika typer av studier. På grund av att många havsbakterier inte kan bilda kolonier och alltså inte växa på traditionella medier har det varit svårt att få en klar bild av artrikedomen. Molekylärbiologin har dock gjort det möjligt att identifiera bakterier med hjälp av 16S rRNA genen, en enorm mängd gensekvenser från världens alla hav har inkommit till den gemensamma databanken (GenBank). År 2002 gjordes en studie där man sammanställde informationen i denna databank, för att få en bild av artrikedomen i världshaven. Resultatet av denna studie var att det i världshaven fanns färre bakterietyper än vad många forskare har spekulerat i. I denna avhandlig har vi utfört en studie där vi gjorde en stor global provtagning för att se om denna undersökning överensstämde med den datainformativa. Provtagning från nio lokaliteter gjordes i de tempererade, tropiska och polarhaven. Ett genbibliotek från varje lokal gjordes och kloner sekvenserades. Resultatet visar i likhet med den datainformativa undersökningen på en begränsad artrikedom. 80% av gensekvenserna fanns redan i databanken, vilket tyder på att de flesta arter redan har blivit funna. Dessutom visade det sig att få av bakterierna återfanns på alla ställen och många återfanns endast på ett ställe. Utöver detta visade det sig att det fanns en ökad artrikedom ju närmare ekvatorn man kom, vilket tidigare har visats för större organismer. Studierna i denna avhandling har ökat förståelsen för hur sammansättningen av det kvävefixerande bakteriesamhället i Östersjön ser ut samt bidragit till diskussionen om den globala artrikedomen bland bakterioplakton och dess utbredning.

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. 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. 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. 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. 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. In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.

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. 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. / 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. 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.

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.