Bacillus anthracis is one of the most feared agents of biological warfare and causes the deadly disease called anthrax. SVA (statens veterinärmedicinska anstalt) is working on a project together with SLV (statens livsmedelsverk) where the target is to find rapid and effective detection methods for Bacillus anthracis in animal feed and food samples. Bacillus subtilis, which is harmless, was used in this study as a model organism to Bacillus anthracis. A known concentration of vegetative Bacillus subtilis was spiked in animal feed and food samples. The genomic work flow was based on automated DNA isolation and real time PCR. The aim of the study was to screen for inhibitory components in the animal feed and food samples using two different DNA isolation robots; Magnatrix 8000 and Biorobot EZ1. The results showed that DNA of high quality was extracted from the samples with both robots. However, the CT-value generated by the real time PCR showed considerable variation depending on the sample matrix. Some samples, for instance egg and liver, were problematic and gave low concentrations and high CT-values probably due to inhibitory components in the samples. Further studies will be needed to solve these problems and optimize the methods that were used in this study.

Sarcoptes scabiei is a disease-causing parasitic mite of humans and animals that is prevalent worldwide. The parasite lives in burrows in the epidermis of its host. These burrows are formed by a combination of mechanical destruction by the mite and secretion of various factors. The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. In this project, the enzyme was expressed in transformed Escherichia coli cells. The SOD cDNA from S. scabiei was ligated into two different expression vectors: pPU16 and pET-14b. The S. scabiei SOD open reading frame reported here is 696 nucleotides long and yields a protein with a molecular weight of  69.5 kDa. Only one of the constructs was successfully created, using pPU16. The construct was designated pPU110 and has a sequence coding for a hexahistidine tag downstream of the SOD cDNA and has a sequence coding for the maltose binding protein (MBP) upstream. The expression plasmid pPU110 was verified by DNA-sequencing and the tested in different expression experiments. Analysis using SDS-PAGE showed that recombinant fusion SOD could be readily expressed in E.coli.

Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism. In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phase Bacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase. In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.

Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP. PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP. Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

Background: Mastitis in dairy cattle is a common ailment worldwide. A cause of mastitis can be bacteria such as Escherichia coli. Mastitis is not a deadly ailment and sometimes the dairy cows show no symptoms but if certain virulence genes are present in the bacteria that cause the mastitis, the bacteria can be transmitted to humans and cause severe diseases. The potential presence of enterohemorrhagic Escherichia coli (EHEC) in particular would be a major concern for human health. Aim: The aim for this study was to analyze the presence of virulence genes known to be present in E.coli strains isolated from dairy cows with mastitis in Sweden. Method: A Qiagen BIO ROBOT EZ1 was used to purify DNA from 90 bacterial cultures. A panel of virulence genes were amplified and biotinylated from the purified DNA by PCR and an E.coli based DNA microarray was used to detect presumed virulence genes in E.coli. Result: There were no samples that had all the genes traditionally used to classify E.coli as EHEC or potential EHEC. 63 samples were analyzed without any problems but 27 samples were not fully analyzed. Conclusion: The DNA based microarray proved to be a reliable method to detect genes from pathogenic bacteria but it needed high concentration of purified DNA which was not always easy to obtain. There were some samples in this study that contained virulence genes.

<p>Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases.</p><p>The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of <em>Lactobacillus spp</em>. in fecal samples retrieved from a study of the probiotic strain<em> L. reuteri</em> ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained <em>L. reuteri</em> belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.</p>

The main causative agent of human urinary tract infections is the uropathogenic Escherichia coli (UPEC) pathotype. It may cause disease due to its ability to express a number of bacterial virulence factors. Fimbrial adhesins are particularly important for the initial establishment of infection in the urinary tract. The fimbriae are hair-like structures protruding from the bacterial cell and by attaching to specific receptors in the urinary tract they mediate adherence to different cell types, allowing the bacteria to resist the shear forces from urine flow. The UPEC strains generally carry multiple determinants for fimbrial adhesins. Previous studies have indicated that there is a co-regulation between different fimbrial genes and one factor that has been implicated in this is the PapB protein, acting as a transcriptional regulator of P-fimbrial expression. The PapB protein can be regarded as the prototype of a family of fimbrial regulators that show high homology between different fimbrial operons. One homolog is FocB, regulator of F1C fimbriae. In this study, the role of the FocB protein in the regulation of F1C fimbriae as well as in the co-regulation with other fimbrial genes was investigated. It was observed that FocB binds to DNA, similarly to PapB, in an oligomeric fashion and that PapB and FocB can form hetero-oligomeric complexes, which appear to have a repressive role in the regulation of the F1C fimbriae. In addition, the FocB protein also had a repressive effect on transcription of the fim operon, which encodes theType 1 fimbriae. For further analysis of FocB in vitro, we developed efficient procedures for purification of the protein and established conditions for its crystal formation with the aim to conduct X-ray diffraction studies. By the hanging-drop vapour-diffusion method, we obtained crystals that in the X-ray analysis diffracted sufficiently well to allow modelling of a high resolution structure of FocB. The structural model was considered in relation to the DNA binding properties of the protein. The FocB analysis represents the first structural model of this family of transcriptional factors. This model should aid in further understanding of the roles and functions of these proteins in the regulation of the UPEC fimbrial operons. The complexity of the system, with multiple factors involved in the regulation of fimbrial operons, was revealed in earlier studies of the PapI protein showing that PapI activates transcription of the pap operon as a part of a complex with the global regulator Lrp. However, PapI itself did not appear to bind to DNA and its mode of action has remained unclear. By genetic analyses and in vitro studies we show that PapI may interact also with the α subunit of the RNA polymerase. This finding indicates that PapI might directly interact with the transcriptional apparatus and thus aid in the activation of pap expression. Bacteria are frequently releasing outer membrane vesicles (OMVs) from their surface. We studied the release of the haemolysin toxin from E. coli in connection with formation of OMVs and found that the toxin was tightly associated with the vesicles in an active form. By overproduction of the PapB or PapI regulators in order to maximise the population of bacteria expressing fimbriae, we could detect P fimbriae proteins associated with OMVs that displayed specific adhesion to receptor-coated beads. This suggests a possible scenario in which the vesicles canfunction as directed vehicles of bacterial virulence factors.

The study evaluated the Mutant Prevention Concentration (MPC) of Pradofloxacin on three Escherichia coli (E.coli) strains, 2 wildtypes and one first-step gyrA resistant mutant. We also measured the value of AUC (Under the Concentration)/MPC that prevents growth of resistant mutants. It is of importance to reach a concentration above MPC that prevent E.coli from developing resistance against the antibiotic. We used an in vitro kinetic model where we added bacteria? and antibiotic. The culture flask was attached to a pump with an adjustable pump-speed. This made it possible to dilute the antibiotics in a satisfying elimination half-life (t1/2= 7 hours) pace. Samples were removed with a syringe at different times in the study. The samples where then cultured on agar- plates to enable counting of the viable colonies after incubation. The optimal concentration to completely eradicate both E.coli wildtypes Nu14 and MG1655 with Pradofloxacin was Cmax ≥8 times MPC and AUC/MPC then became73. Additional experiments needs to be done on the resistant mutant LM378 before we can determine the optimal concentration. But results so far indicate that the concentration of Cmax would be about 8-12 timesMPC to completely eradicate that mutant.

This thesis reports on cross-sectional surveys aimed at detecting and characterizing haemoprotozoan parasites infecting wild free-ranging non human primates (NHPs) in Kenya, East Africa. Blood samples from olive baboons (Papio cynocephalus anubis), vervet monkeys or African green monkeys (AGMs, Chlorocebus aethiops) and Syke's monkeys (Cercopithecus mitis) from five provinces of Kenya were analyzed. The haemoprotozoan parasites survey was performed with microscopic evaluation of blood smears, serological techniques and molecular tools. Blood specimens and serum samples from 121 NHPs were tested for the presence of Trypanosoma brucei (Study I). Indirect antibody enzyme-linked immunosorbent assay (Ab-ELISA) detected titers of anti-T. brucei antibodies in 19% (23/121) of the sera sampled. Subsequent field-oriented latex agglutination test (LAT) detected presence of T. brucei antigens in 16% (19/121) of the sera. However, there were no active infections detected on fixed blood smears, or wet blood films. Of the 378 NHPs sera samples tested for Leishmania major exposure using Ab-ELISA, 66% had detectable anti-L. major antibodies (study II). Western blot (WB) assay detected anti-L. major antibodies in sera from 46% (175/378) of the NHPs samples. Specific proliferation of peripheral blood mononuclear cells to L. major antigen was demonstrated in 23% (17/57) of AGMs samples. Haemoprotozoan parasites, Entopolypoides macaci and Hepatocystis kochi were detected by microscopic evaluation of Giemsa-stained blood smears from 179 NHPs (study III). The prevalence rate of E. macaci was 43% in African green monkeys, 35% in Syke’s monkeys and 33% in baboons. H. kochi infection rate was 18% in African green monkeys, 23% in baboons and 25% in Syke’s monkeys. Subsequent indirect immunofluorescent antibody test (IFAT) supported the morphologic appearance of E. macaci observed by microscopy. Molecular tools were used to detect and identify haemoprotozoan parasites in wild free-ranging NHPs (study IV). Nested polymerase chain reaction (PCR) targeting Babesia β-tubulin gene detected a 22% (27/125) B. microti infections in free-ranging NHPs in Kenya. PCR also detected 22% mixed infections by Hepatocystis and Entopolypoides, 12% Hepatocystis and Babesia and 7% Entopolypoides and Babesia (study V). Phylogenetic analysis inferred from mitochondrial cytochrome b (Cyt-b) gene confirmed the presence of Hepatocystis kochi whereas analysis of 18SS rRNA gene confirmed presence of two piroplasms, Babesia sp. and Entopolypoides macaci. In conclusion, epidemiological results from sero-prevalence studies provide strong circumstantial evidence that some species of Kenyan NHPs are naturally exposed to L. major and T. brucei infections and could be potential reservoir hosts for these haemoparasites. Molecular diagnosis revealed the occurrence of mixed parasite infections and confirmed the circulation of Babesia and Entopolypoides species in the same populations of Kenyan NHPs.

Drosophila melanogaster has been widely used as a model organism to study the immune responses against bacteria, fungi, parasites and viruses. Here, I present a D. melanogaster virus as a model to study persistent virus infections. I have discovered and characterized the Nora virus, a small picorna-like RNA virus able to persistently infect D. melanogaster. The Nora virus genome encodes four open reading frames; a feature not present in other picorna-like viruses. The Nora virus is not closely related to any other virus family, but rather is the first virus in a new family of picorna-like viruses. The major replicative proteins of this virus are encoded in the second open reading frame and the capsid proteins are encoded in the fourth open reading frame. The sequence of the capsid proteins are not obviously related to any other previously described protein. By looking at expressed sequence tags (EST) projects, we identified an EST sequence from the parasitic wasp Nasonia which appears to encode proteins that have sequence similarity to the Nora virus capsid proteins. I have shown that the Nora virus persists in the fly intestine however I did not observe serious pathological effects in the infected flies. The virus is shed through feces and the transmission occurs horizontally via the ingestion of virus-contaminated food. Moreover, I observed variability in the viral titers among single flies of the same infected stock. Some flies are able to clear the Nora virus but not others and this phenomenon seems to be titer-dependent. Surprisingly, none of the known Drosophila antiviral responses play a role against the Nora virus. In conclusion, my work shows that studying the Nora virus interaction with the Drosophila immune system can lead to new findings on viral persistence mechanisms of RNA viruses and of Drosophila viral innate immunity.