- PostValidation of newly developed surveillance platform in operating rooms, to monitor microbial contamination in orthopedic implant surgery(2023) Hovland, Ludvig; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Siewers, Verena; Stålfelt, FransNo access to the Master's thesis.
- PostDevelopment and optimization of stable CHO cell lines for expression of Recombinant antibodies for use in the diagnosis of cancer(2023) Pernsved, Monica; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Molin, Mikael; Lidqvist, Maria; Wising, CatharinaThis master's thesis project aimed to investigate and evaluate the development of stable CHO cell lines for the expression of recombinant antibodies (rAbs), for use in the diagnosis of cancer and thereby increase the know-how of this technique at Fujirebio Diagnostics AB (FDAB). Three antibodies targeting cancer biomarkers; C192, Ov185, and SCC140 were selected for recombinant expression in an attempt to increase yield and to get more stable productivity, to meet the increased demand at the market. Biomarkers produced from cancer cells can be detected by antibodies in in vitro diagnostics. In this project, the suspension cell line CHO-S adapted to high density serum-free culture was used in combination with the pCHO1.0 vector, constructed for the expression of two-subunit proteins, such as IgG. An alternative signal sequence (B), originating from human albumin was evaluated in comparison with the original signal sequence to increase antibody secretion from the cells further. Transient expressions with concentrations of 0.8-1.5 μg/ml were achieved 48 h post-transfection for pCHO 1.0 construct containing C192 with alternative signal sequence (C192B), Ov185 and Ov185 with alternative signal sequence (Ov185B). After selection with Methotrexate (MTX) and Puromycin stable expression with concentrations of 45 and 39 μg/ml were achieved for C192B and Ov185, respectively. The data indicate that a transient expression with concentrations of at least 0.8 μg/ml is a prerequisite for success in the selection phase, and if this is not achieved, that the transfection should be repeated. To select high producing clones from the cell pool achieved, in the selection phase cloning by limiting dilution was performed on stably transfected pools. High producing C192B clones were obtained that produced up to 490 μg/ml in productivity assessment, corresponding to at least 10 times increase in expression as compared to the original hybridoma. Recombinant C192B was purified and characterised regarding immunoactivity, with high purity and with immunoactivity in comparison to hybridoma produced C192. In this project two antibodies were successfully expressed in recombinant form and the technique can now be considered established at FDAB. Next step will be to scale up for manufacturing of rAbs at FDAB.
- PostEffect of mutations in E. coli membrane components on bacterial conjugation(2023) Ahlstedt, Emelie; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Bengtsson-Palme, Johan; Farewell, Anne; Fransson, Alfred; Lundgren, AndersThe rise of antibiotic resistant bacteria is a major threat to global health care as antibiotics are an important tool in modern medicine. Antibiotic resistance genes can spread between bacteria in a process called conjugation. Conjugation is a form of horizontal gene transfer where a donor cell transfers genetic material to a recipient cell. A possible method to stop the spread of antibiotic resistance genes is to target conjugation. In this project seven different single gene deletions were introduced in the donor. The genes are related to membrane components in Escherichia coli and their effect on conjugation was investigated. The genes of interest were fabF, fabH, lpp, plsX, rfaD, rseA, and wzzE. fabF, fabH, plsX, rfaD, and wzzE were investigated through liquid mating assays while lpp and rseA were investigated by a microfluidics method. However, this microfluidic method is not suited for large scale experiments and was therefore further developed in this project to enable a microfluidics system with four parallel channels. The liquid mating assay with mutant strains showed that ΔfabH and ΔplsX have a small effect on the transfer efficiency of the donor. ΔfabF and ΔwzzE were shown to have a moderate effect on transfer frequency while ΔrfaD showed a large effect on transfer frequency. ΔfabF and ΔrfaD were investigated further by a solid mating assay as these two mutations showed the biggest effect on transfer frequency. The results from the solid mating assay were similar to the liquid mating assay. It is hypothesised that the effects seen for ΔfabF and ΔrfaD could be due to altered membrane fluidity. Thus, the effect of altered membrane fluidity was investigated by treating the donor cells with the membrane softener pentanol in liquid mating assays. Pentanol concentrations of 0 M to 20 mM were examined. The liquid mating assay performed with pentanol showed that increased membrane fluidity decreases the transfer frequency of conjugation. Lastly, the development of the microfluidics method tested different surface modifications of the channels for the best adhesion of cells. A four-channel system was developed but needs further development to be used for conjugation experiments. As the microfluidics method showed problems with, for example, pressure sensitivity and adherence of cells, and issues with fluorescent markers in the mutant strains Δlpp and ΔrseA, conjugation with the two mutants were ultimately not investigated.
- PostAttempted Isolations of Cholesterol-to-Coprostanol Reducing Bacteria in the Human Gut(2023) Antonsson, Selma; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Landberg, Rikard; Tremaroli, ValentinaAtherosclerotic cardiovascular disease (ASCVD) is the leading cause of death globally. In early development of atherosclerosis, retention of cholesterol in artery walls is a key step. Cholesterol both endogenously produced and absorbed from the diet ends up in the gut, where bacteria may reduce it to coprostanol. Unlike cholesterol, coprostanol cannot be reabsorbed into circulation from the intestines, leading to long-standing hypotheses that high cholesterol-to-coprostanol conversion may lower blood cholesterol and thus the risk of ASCVD. Metagenomic evidence for both conversion being health-associated and ASCVD being a microbiota-modulated disease is mounting, emphasising the potential importance of microbial cholesterol metabolism in the gut. This project thus aimed to isolate cholesterol-converting bacteria from the human gut to further characterise them. Conversion was initially studied in a pure Eubacterium coprostanoligenes culture as a positive control. However, the type strain was quickly outcompeted by a contaminant whereupon culturing of faecal samples from two healthy donors was initiated. Over four sample series, several media compositions and cultivation approaches were investigated. Coprostanol was not observed in any culture. Pathway intermediates could not be analysed and as such it is possible, although improbable, that partial conversion took place. There was also no way of detecting the potential presence coprostanoligenic but non-converting bacteria. Altogether, these results reiterate on the previously established fastidiousness of coprostanoligenic bacteria. Further attempts would benefit from more research determining the conditionssupporting cholesterol conversion, as a better understanding of those conditions is a first step in characterising the bacteria performing conversion. Eventually, this might enable research on supporting their growth using prebiotics or the feasibility of their application as next-generation probiotics for improved cardiovascular health.
- PostLipid oxidation and in vitro protein digestibility in new protein ingredients from herring (Clupea harengus) co products produced with lingonberry press cake(2023) Hong, Bovie; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Undeland, Ingrid