The diversity of microbial communities in hypoxic environments has resulted in the establishment of syntrophic relationships. The recently described anaerobic amoeba Anaeramoeba flamelloides is found in the metamonad group. It contains mitochondria-related organelles, MROs, which produce hydrogen gas. The MROs, or hydrogenosomes, are closely positioned next to bacterial symbionts, which use the hydrogen waste from the hydrogenosomes to fuel their sulfate-reducing metabolism. This study aims to establish effective compounds and protocols for further experiments with these organisms by performing assays for antibodies, antibiotics, and metabolic inhibitors. A protocol for the upscaling of cultures was streamlined, and genomic extraction, sequencing, and analysis of novel bacterial symbionts were performed. The genomic analysis yielded a syntrophic model of two bacterial symbionts within the A. flamelloides strain BUSS. The effective concentrations and activities of several antibiotics were established, including ones that might be used in transfectant screenings in future research. Two effective protocols for the elimination of the Desulfobacter spp. symbiont bacteria from the amoebae were established, as well as a promising protocol for the establishment of monoclonal amoebae cultures.

Most fungi, as well as many environmental bacteria, have an ideal growth temperature rangeof about 12-30 ℃, and are unable to grow in 37 ℃. This means that our internal temperatureworks as a thermal barrier that protects us from infection by most of these organisms.However, with the advent of global warming, rising environmental temperatures could lead toan increased temperature tolerance in environmental fungi and bacteria. This, in turn, risksleading to novel pathogens emerging due to a newly acquired ability to cross the mammalianthermal barrier. In this project, a library of environmental psychrotrophs with a maximumgrowth temperature below 37 ℃ were isolated and identified, and their ability to adapt tohigher temperatures was investigated. Initial experiments showed that for mostpsychrotrophs, 37 ℃ not only inhibits growth but is lethal to over 99% of cells, andspontaneous adaptation to a drastic temperature increase appears to be infrequent. Forisolated yeasts, a transformation protocol was adapted for future experiments with horizontaltransfer of thermotolerance, and future experiments are discussed.

No description available.

No description available.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

No description available.

Honey is a natural product composed of numeral substances and microorganisms. Although honey presents physicochemical properties that confer antimicrobial characteristics, certain microorganisms are still able to survive and multiply in honey. Some of these microorganisms are important to human health, such as Lactobacillus bacteria, which can be used as probiotic to improve human gastrointestinal condition. However, there is still a lack of knowledge about the microorganisms found in honey, and further research is necessary to better identify and understand these microorganisms. The aim of this study was to detect and identify Lactobacillus species from honey by sequencing the 16S ribosomal RNA gene with the universal primers 27F and 1492R. This was partly obtained by searching for Lactobacillus spp. directly in pure honey and after growing the unknown viable Lactobacillus spp. from honey in selective media. The microorganisms were isolated by centrifugation, had their DNA extracted, and only the bacterial 16S rRNA gene was amplified using Polymerase Chain Reaction and sequenced using the Oxford MinION Nanopore sequencing technology. EPI2ME, Oxford Nanopore Technology data analysis platform, was used to identify the bacteria. Results showed the presence of numeral Lactobacillus species in honey such as L. melliventris, L. mellifer, L. mellis, and L. helsinbgborgen, but in very low amounts. The analysis of honey samples was quite difficult. Low PCR product yield during the entire study, microscopic visualization and microbial analysis using fungicides supported the presence of fungi. In conclusion, a diversity of Lactobacillus spp. can be found in honey but in low quantities.

Giardia intestinalis is a protozoan parasite that causes diarrhea, and due to its 200 million human cases each year it has become important to try and reduce the infected. Giardia intestinalis expresses so called variant surface proteins (VSPs) on its surface. VSPs are surface antigens that the immune system can detect and react to. In order to hide from the immune system and protect itself Giardia is able to perform antigenic variation, where it switches the expressed VSP to another one of the 200 VSPs in its genomic repertoire. Understanding the regulation and expression of the VSPs is an important steppingstone towards being able to create a vaccine or a better treatment to try to reduce the negative health effects created by this parasite. The main components that regulate VSPs that are being expressed on the surface are Dicer and Argonaute. They are both involved in the RNA interference (RNAi) machinery that has been suggested to silence the VSPs that are not being expressed post-transcriptionally. The aim of this study is to create mutants of Giardia that will no longer express Argonaute or Dicer and thereby be able to analyze the VSP expression and to see if a difference in expression can be detected. This can lead to an understanding of the regulatory mechanism of antigenic variation. We were able to create mutants that most likely did not express Argonaute or Dicer, however since PCR verification of successful knockout did not work, except for mutant Dicer nr. 12, genome sequencing is needed for verification of all the knockout mutants. When analyzing the VSP expression using immunostaining the only surviving Argonaute mutant and the two Dicer mutants that had gone through an initial limiting dilution showed that there was a significant difference in VSP expression for the Argonaute mutants and one of the Dicer mutants. There was a higher fluorescence detected in the nr. 8 Argonaute mutant and the 12#2 Dicer mutant, which indicate a higher VSP expression for those two strains compared to the Cas9 expressing wild type. Furthermore, fluorescence dots could be detected around the parasites especially for Dicer 12#2 which could indicate that this mutant has a higher turnover of VSPs and is releasing more of them into the surrounding area. Due to time limitations many follow up experiments were not able to be performed, however the results obtained give an important insight into the regulation of VSPs in Giardia intestinalis.

Wolbachia is an intracellular bacterial symbiont found in nearly half of all arthropod species. In some mosquito vector species, it is known to exhibit repressive effects on viral replication and transmission. Sindbis virus (SINV) is an arthritogenic alphavirus that causes yearly human infections in Northern Europe. Its transmission cycle involves passerine birds and two main mosquito vectors, Culex pipiens and Cx. torrentium. Cx. pipiens is almost always infected with Wolbachia, yet the impact on its vector competence for SINV has never been investigated. The purpose of this study was to establish in vivo (Aim 1) and in vitro (Aims 2 and 3) platforms for the investigation of the effect of Wolbachia on vector competence and viral replication in mosquitoes of the Cx. pipiens complex. For Aim 1, a colony of lab-reared Cx. pipiens molestus was cleared of its natural Wolbachia infection through tetracycline treatment. For Aim 2, a proliferating primary cell culture was initiated from the eggs of Cx. pipiens molestus. For Aim 3, attempts were made to infect Hsu cells with Wolbachia from Cx. pipiens molestus ovaries with varying success. Protocols for all three primary aims have been established. Preliminary experiments suggest that Hsu cells do not support SINV replication. Additionally, the vector competence of Wolbachia-infected Cx. pipiens molestus for SINV has been investigated, however with inconclusive results. Continued maintenance of the Wolbachia-free mosquito line and primary cell culture is projected to allow future studies of Wolbachia-mediated phenotypes and its effect on virus transmission by Cx. pipiens.