Browsing by study line "Genetik och genomik"

Bacteraemia, the presence of bacteria in the bloodstream, may lead to severe and costly health issues. Sepsis, a serious complication of bacteraemia, is one of the top causes of mortality globally. Early and specific diagnostics as well as fast acting are essential in successful treatment. However, current diagnosis relies mainly on time-consuming blood culturing and clinical symptoms, which are unspecific for the causative agent. With the advanced technology and decreasing cost, state-of-art sequencing-based (Next generation sequencing) methods provide a new way to investigate the bacteria present. Metagenomics, which means sequencing and studying all DNA extracted from a microbial community sample, is widely used, but it only describes the genetic potential of a community and does not differentiate live from dead microbes. Metatranscriptomics, in which essentially all RNA from a sample is sequenced, provides information about expression and activity together with identification of viable bacteria, However, the high amounts of host cells and host RNA complicate the detection of bacterial transcripts from complex host-microbe samples. In this thesis, I investigated solutions for the efficient isolation and enrichment of bacterial RNA from whole blood to be used in sequencing and metatranscriptomics analysis. Firstly, I tested the capability of bacterial cell lysis of two commercial blood sampling tubes with Escherichia coli and Staphylococcus epidermidis suspensions. Both tubes, Tempus and RNAgard, were able to lyse gram-negative E. coli cells and good-quality RNA was extracted in measurable quantities with their respective RNA extraction methods. With Tempus tubes the RNA yield was clearly higher. With gram-positive S. epidermidis, RNA quantities from both extractions were below the measurement limits indicating insufficient lysis and need for further optimization. Secondly, I investigated the depletion of polyadenylated (poly-A) transcripts in order to reduce the host transcripts and thus to enrich the bacterial transcripts prior to costly sequencing step. I evaluated the performance of a previously designed in-house protocol, based on the capture of poly-A -transcripts with oligo-dT -beads, and tested different parameters to see whether the depletion efficiency could be enhanced. Most significantly, the amount of oligo-dT -bead suspension was reduced to half from the original protocol. In-house protocols were also compared to a commercial solution, which they clearly outperformed. Depletion performances were tested with a RT-qPCR and dot blot assay, which I designed along this thesis work. Finally, to make the poly-A depletion better suited for blood samples infested with globin transcripts (representing up to 80% of all poly-A transcripts extracted from whole blood), I tested and successfully pipelined the leading commercial method for depleting globin transcripts with the in-house poly-A depletion protocol. The optimized sample preparation protocol provides a platform for further bloodstream infection and sepsis studies. Next steps of the process, such as sequencing and testing with clinical samples, are already ongoing with promising preliminary results. In the future, the metatranscriptomics approach can be utilized in fast and specific identification of the pathogens and their antibiotic susceptibilities. In addition, infection mechanisms and host-pathogen interactions may be studied possibly providing novel insights for sepsis diagnostics and treatment.

Diabetic ovarian cancer patients who take metformin as part of their anti-diabetic medication generally respond better to DNA-damaging cancer treatment. The molecular mechanisms of the anti-cancer effects of metformin are currently being investigated, but they remain poorly elucidated. Not much is understood about the metformin effect on DNA damage in ovarian cancer cells, where it is of particular importance. When chemotherapy-induced double-stranded DNA breaks are unrepaired, cells reach a point when they cannot tolerate the accumulated DNA damage and die. However, some ovarian cancer cells efficiently employ DNA repair mechanisms, the most prominent being homologous recombination (HR), to overcome DNA damage. Efficient HR causes chemoresistance. An important question is whether metformin has the ability to induce the HR-deficient state in cancer cells, thereby sensitizing them to treatment. This study did not examine HR directly, but it assessed HR indirectly by observing the effect of metformin on recovery from DNA damage in two ovarian cancer cell lines: OVCAR4 (HR-proficient) and Kuramochi (HR-deficient). Additionally, this study evaluated the metformin effect on cell proliferation and apoptosis. OVCAR4 and Kuramochi cells were exposed to varying metformin concentrations (0,5 mM, 5 mM, 10 mM, 15 mM, 20 mM and 25 mM) and for varying durations (24 hours and 48 hours). This study also tested how metformin pretreatment affected the cells’ ability to repair externally (ionizing irradiation) induced DNA damage. The cells were imaged with a high-content imaging system, and percentages of nuclei that were positive for markers for different cellular processes (i.e., DNA damage, proliferation, and apoptosis) were calculated. The study found that only high metformin concentrations, such as 20 mM were able to increase DNA damage and reduce cell proliferation in HR-proficient OVCAR4 cells, both non-irradiated and irradiated. The HR-deficient Kuramochi cell line was generally more sensitive to metformin, particularly with regards to DNA damage, which increased using metformin concentrations < 20 mM. However, 20 mM concentration resulted in the most significant effects. Similarly, only high metformin concentration (25 mM) increased apoptosis, although data were obtained only for a limited number of Kuramochi cells. More experiments on apoptosis would be beneficial. Also, more extensive experiments for the irradiation part are needed to validate these preliminary findings, as well as examining whether high metformin concentrations (> 20 mM) affect specifically the HR-mediated DNA repair pathway.