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    Phospholipids interaction with graphene and graphene oxide. At the interface of quantum calculations and practical experiment
    (2023) Lanai, Victor; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Mijakovic, Ivan; Rahimi, Shadi
    It has been shown that vertically grown graphene flakes are effective in killing bacteria, whilst keeping mammalian cells intact. It was also reported that the graphene oxide sheet can attach to certain drugs and thereby can be used as a therapeutic drug carrier for cancer treatment. However, there is lack of knowledge on the mechanism of interaction between different kinds of cells and graphene-based materials. Therefore, a deeper investigation into the interaction between graphene and the plasma membrane of different types of cells was undertaken. The cell membrane consists of two key components, phospholipids and proteins. Various types of phospholipids exist, and the different types are present in various organisms. Since the constituent of phospholipids seems to differ between bacteria, mammalian normal and cancerous cells, we choose phospholipids as the main target for this thesis. Six phospholipids were studied together with graphene and its derivative, graphene oxide. This thesis is divided into two parts, theoretical and practical parts. In the theoretical part, density functional theory (DFT) is utilized to enhance the understanding at a quantum level. Phospholipids are simulated as isolated single molecules, in pairs and together with graphene and graphene oxide. The theoretical calculations show that the most abundant phospholipids in mammalian cells have stronger bonding to each other, compared to bacterial phospholipids. Further, when the graphene/ graphene oxide sheet is approaching the phospholipid pairs, the bacterial pair exhibits less repulsive interactions, thereby a more stable system with the sheets was found. In the practical part, phospholipids were assembled into liposomes, mimicking a cell membrane and treated with fluorescein functionalized graphene oxide. With fluorescent microscope, we assessed the internalization of graphene oxide by liposomes. Furthermore, differential scanning calorimetry revealed that the constituent of phospholipids affects liposomes heat capacity, which is in line with the theoretical calculations. There would be some other interactions between the cells and approaching material, but we present phospholipids as the key player in this study.
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    Development of xylan- and xylose assimilating Saccharomyces cerevisiae using CRISPR-Cas9 technology
    (2023) Lönnqvist, Elin; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Geijer, Cecilia; Heitor Colombelli Manfrão Netto, João
    Biorefineries produce a vast array of products for industrial, pharmaceutical and food applications. To allow for an enviromentally and economically sustainable production, the input biomass should not compete with crops cultivated for other purposes such as food or feed. Instead, lignocellulosic biomass from e.g. fast-growing wood and grasses has emerged as an alternative. However, one of the most important industrial microorganisms, the yeast Saccharomyces cerevisiae, is unable to efficiently degrade and utilise the polysaccharides present in the plant cell wall, including hemicellulose, a major constituent of lignocellulosic biomass. This project was aimed at developing a yeast strain able to utilise xylan, one of the most abundant hemicelluloses. A CRISPR-Cas9 system was used to introduce the hemicellulases endo-xylanase and β-xylosidase for degradation of the xylan polymer, and the XR/XDH pathway for assimilation of the xylose monomers by S. cerevisiae. The integration of the XR/XDH pathway and the hemicellulases was successful and growth analysis of the resulting strains showed ability to grow on xylose based media as well as on xylooligosaccharides, indicating a functional XR/XDH pathway and β-xylosidase enzyme. β-xylosidase activity was also demonstrated through enzymatic assays. However, capability of degrading the xylan polymer could not be demonstrated via enzymatic assays, indicating insufficient endo-xylanase activity. While further studies are required to optimise the hemicellulase expression in the constructed strain, the xylose-degrading ability of the strain was comparable to that of other genetically modified strains expressing the XR/XDH pathway. Overall, the successful development of an S. cerevisiae strain capable of efficiently degrading xylan and utilising xylose would be of great industrial value and further research on this topic should be pursued.
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    A Step Toward Personalized Cancer Treatment Simultaneous Detection of Multiple Types of Chemotherapyinduced DNA Damage Using Single Molecule Imaging
    (2022) Foss, Ebba; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering; Westerlund, Fredrik; Akwasi Aning, Obed
    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.
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    Evaluating longitudinal aspects of cerebrospinal fluid biomarkers for cognitive disease
    (2022) Basic, Emir; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Westerlund, Fredrik; Kettunen, Petronella
    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.
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    Improved mRNA drug efficacy in hepatic cells through mannose functionalization of lipid nanoparticles
    (2022) Brännudd, Ingrid; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Esbjörner Winters, Elin; van Leeuwen, Daniël
    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.