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- PostA Step Toward Personalized Cancer Treatment Simultaneous Detection of Multiple Types of Chemotherapyinduced DNA Damage Using Single Molecule Imaging(2022)Chemotherapy is commonly used to treat cancer today, either alone or more commonly as part of combination therapy. Response to a certain chemotherapeutic agent is highly individual, both in terms of treatment efficacy and the extent to which healthy cells are affected. For several drugs, induced DNA damage provides the main cytotoxic effect, and a method for evaluating this damage could therefore prove a powerful tool in treatment planning. In this thesis, a single molecule imaging approach is used to assess chemotherapy-induced DNA damage, allowing visualisation of damage sites on individual DNA strands. While previous studies have focused on one damage type, or collective damage without distinction between types, a novel modification to pre-existing techniques that allows for this distinction has recently been demonstrated. In this thesis, the alkylating agent temozolomide was used to illustrate how different damage types can be distinguished with a single molecule imaging approach. This is done using repair enzymes associated with different DNA repair pathways. The repair enzymes sequentially incorporate spectrally distinct fluorescent nucleotides at the damage site which are then visualized as fluorescent spots of two different colours on individual DNA molecules. This distinction could be shown with high repeatability in terms of colour ratio. While both enzymes used separately clearly repaired the treated DNA, there appeared to be an overlap when applying them sequentially. This could suggest a problem with enzyme specificity. Further exploration of this issue is needed to verify the feasibility of single molecule imaging for the purpose of simultaneous detection of chemotherapy-induced DNA damage types.
- PostEvaluating longitudinal aspects of cerebrospinal fluid biomarkers for cognitive disease(2022)The most common neurodegenerative disease that millions are affect by today are Alzheimer’s disease (AD) and Parkinson’s. Diseases and disease progression can be diagnosed and evaluated using cerebrospinal biomarkers. The aim of this project is to evaluate these biomarkers (Aβ 1-42, T-tau, P-tau, albumin ratio, soluble APPα/β) to certain variables and how they affect cognition. Patient data was obtained from the participants of in the Gothenburg Mild Cognitve Impairment Study, and contained baseline data and date from a check up 2 years after baseline. The group included 862 patients and controls. Statistical analysis was performed to calculate normality plots, distribution between groups, medians, interquartile ranges Bonferroni correction and linear regression analysis. Results showed that there were significant differences in baseline for the variables between the groups. For year 2, there were significant differences between all the group for the variables expect for sAPPα. The delta values showed significant differences between the groups in biomarkers P-tau and sAPPβ and all the cognition test. Linear regression analysis showed that all biomarkers went toward the state that is considered sick with increasing age, but biomarkers sAPPα/β were considered stable. Results showed that some biomarkers are better for diagnosing certain diseases and the new biomarkers could potentially introduce a more accurate way of predicting cognitive disease.
- PostImproved mRNA drug efficacy in hepatic cells through mannose functionalization of lipid nanoparticles(2022)The recent development and worldwide use of messenger RNA (mRNA) vaccines against Coronavirus Disease 2019 (COVID-19) have shed light on the huge potential of mRNA therapeutics. Apart from viral vaccines, mRNA therapeutics can be utilized as treatment for abnormal gene expression, insufficient protein production and as targeted cancer drugs, addressing increasingly important medical needs in a world with an aging population. mRNA vaccines have a great potential of becoming the leading vaccine type due to their high safety and easily scalable production, but the current problem of low efficacy of cellular uptake and functional delivery (estimated to be 1.5-3.5 %) must first be tackled. In the COVID-19 mRNA vaccines, the mRNA is delivered through encapsulation in Lipid Nanoparticles (LNPs), which transport the mRNA into the cell and deliver it to the cytosol where it is translated into protein. In this Thesis, I have optimized LNP designs by introducing receptor-targeting ligands on their surface, in the form of the sugar moiety mannose, conjugated to a poly ethyleneglycol (PEG) lipid. The mannose conjugates were used to improve the efficacy of mRNA-LNPs, to ultimately decrease the proportion of mRNA-LNPs that are degraded and thus increase the fraction of functional delivery and release of mRNA into the cytosol per given dose. To achieve this, mRNA-encapsulating LNPs with varying ratios of incorporated mannose-PEG lipids were formulated and biophysically characterized, including controls for lipid and sugar specificity. Subsequently, live-cell confocal laser scanning microscopy, automated image analysis and flow cytometry techniques were used to study the mRNA uptake and functional delivery in human hepatic Huh7 cells and human neuronal SH-SY5Y cells. Interestingly, substitution of DMPE-PEG lipid in a standard LNP formulation for a DSPE-PEG-mannose lipid increased the mRNA uptake and functional delivery, presumably through targeting of mannose-receptors which could have subsequent redirection effects of the endocytic route. The mannosylated LNPs had an enhancing effect in Huh7 cells, but not in SH-SY5Y cells indicating that the sugar functionalization confers cell specificity. The optimal mannose-PEG lipid substitution for increased mRNA uptake and functional delivery was found to be 0.5 mol%. Improving mRNA uptake and functional delivery, as explored here, is essential to lower the dose of the mRNA therapeutic administered to patients. This is desirable since lower mRNA doses decrease the risk of potential drug side effects but also decrease expenses for both healthcare and patients. The findings in this Thesis, that mannose functionalization improves the mRNA delivery efficacy mediated by LNPs, represent a step forward in this respect, and may thereby serve as useful inspiration for future developments in LNP design. This work was performed at the Division of Chemical Biology in the Department of Biology and Biological Engineering at Chalmers, Gothenburg, and at the Advanced Drug Delivery department at AstraZeneca, Mölndal, within the Swedish industrial research centre for functional RNA delivery; FoRmulaEx.
- PostEstablishment and Characterisation of New Immunoreagents for Diagnosis of Neurodegenerative Disease(2022)Neurodegenerative diseases are a collection of disorders which can develop any time in adulthood, but with the majority occurring in the elderly population. A common denominator for these diseases is the gradual degradation of neurons in the central nervous system, often taking place long before the manifestation of clinical symptoms. One of the proteins taking part in this process is called neurofilament light, which upon degradation can be found both in the cerebrospinal fluid as well as the blood. In vivo diagnostic tools utilising antibodies have been developed for the detection of NF-L in the CSF, allowing for earlier diagnosis. However, for screening to be able to take place at local healthcare centers, a diagnostic test for NF-L in blood would be more beneficial. Since the concentration of the antigen is much lower in the blood compared to the CSF, antibodies with high specificity and sensitivity are necessary. The aim of this project was to establish high affinity monoclonal antibodies against the protein neurofilament light, specifically when present in plasma, using classic hybridoma technology. Mice were immunised with full length bovine NF-L as well as two shorter custom made peptides, originating from the Coil-2 region of NF-L, having shown to be a valuable epitope sight for clinically effective antibodies. Over 20 hybridomas producing relevant antibodies were established, detected through ELISA screens. All succesfull hybridomas originated from the mice immunised with peptides. The majority of the established antibodies had the desired isotypes and a few had a strong response towards full length bovine NF-L, indicating clinical relevance. To conclude, a number of antibodies with the potential of being used in a sandwich ELISA diagnostic test for the detection of NF-L in blood have been established. These antibodies were proven to recognise the native form of bovine NF-L, epitope mapped to the clinically relevant Coil 2 region and were not reactive to other proteins present in clinical samples of CSF or serum. This project was performed at Fujirebio AB located in Gothenburg, Sweden.
- PostExploring Genetic Tools for Metabolic Engineering of Yeast Yarrowia lipolytica(2022)Abstract The oleaginous yeast Yarrowia lipolytica has several properties that gives it a high potential as a microbial cell factory for production of oleochemicals, proteins, and natural products. Although genetic tools have been developed for the yeast, more are needed to expand microbial fermentation using Y. lipolytica. The purpose of the thesis is to explore two genetic tools for metabolic engineering of Y. lipolytica. First, the functionality of three enzymes repurposed as colorimetric malonyl-CoA biosensors was tested. Second, a set of 18 tunable promoters with differential expression in different nitrogen conditions was investigated. In the biosensor project, a series of shake flasks experiments were performed to explore the properties of polyketide synthases repurposed as a biosensor. The biosensor signal strength was measured through absorbance in the UV-visible spectra. The dose-dependency of the biosensor was tested by employing it in three strain backgrounds, each assumed to have a different malonyl-CoA availability. The biosensor signal varied depending on the strain background, but not always as expected. Overexpression of the biosensor enzyme gene showed that the strain with increased lipid accumulation that was expected to give the lowest signal, actually gave the highest. This could be explained by the strain oxidising lipids, thereby increasing the malonyl-CoA concentration. In the promoter project, a strain for each promoter was constructed. GFP was used to measure the expression strength. The strains were grown in different media in a microbioreactor system which measured the biomass and fluorescence intensity. Some strains were also investigated in a fluorescence microscope. None of the tested promoter strains showed GFP expression, meaning the promoters did not initiate GFP gene transcription. The troubleshooting steps taken could not explain the unexpected results. More research is needed to establish the tools tested in this thesis. Instead of acting as biosensors, the polyketide synthases could be used for polyketide production. The polyketide produced would then need to be identified, followed by metabolic engineering strategies for increased production. To increase the detection range for the tested promoters, a brighter fluorescent protein and a more sensitive instrument could be used.