Examensarbeten för masterexamen
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- PostImpact of stabilizing methods on the in vitro protein digestibility of Ulva fenestrata(2024) Helgesson, Klara; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Undeland, Ingrid; Vall-Llosera Juanola, Mar; Larsson, KarinThe seaweed Ulva fenestrata has gained interest from a dietary perspective due to its promising protein content, well-balanced amino acid composition and high levels of vitamin B12. It is however highly perishable and needs to be stabilized quickly after harvesting, which can be done by e.g. drying or sous vide cooking. Fresh water rinsing is often employed before stabilizing. Existing studies have reported a low or moderate digestibility for crude seaweed proteins, but there is a lack of studies exploring how the digestibility U. fenestrata proteins is affected by stabilizing methodssuch as drying and sous vide cooking; with or without pre-rinsing. Time and temperature are two critical factors decisive for whether the digestibility is improved or impaired during processing. In this thesis, the effect of drying at 40 and 60 °C, rinsing with fresh water prior to drying at 60 °C, and sous vide cooking at 60 and 80 °C on the in vitro protein digestibility of U. fenestrata was studied. A modified INFOGEST 2.0 static in vitro digestibility protocol was used, including casein as a reference. The protein digestibility was assessed based on the degree of hydrolysis (DH), and the chemical composition of the biomass was analyzed. Results showed no significant difference in DH between the different treatments, partly due to high variances. It was thus concluded that the chosen stabilizing methods did not alter the protein digestibility, and can be used without compromising the nutritional quality of the seaweed proteins. Future prospects and method improvements were discussed.
- PostExploring Liquid Chromatography Method Development Strategies: Enhancing Analytical Performance in Pharmaceutical Applications(2024) Habibollahi, Porya; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Savolainen, Otto; Wu Ludvigsson , JufangFrom discovery to the final phase of drug development, Liquid Chromatography (LC) is considered one of the core separation techniques. Its broad capabilities in identifying and quantifying complex compound mixtures find application in quality control, formulation studies, pharmacokinetics, environmental monitoring, and purity determination [1]. This 60-credit thesis aims to develop and optimize LC methods through exploration of three individual studies, each detailed in separate chapters.Chapter one is focused on evaluating separation performance under various conditions using a selected compound mixture as reference. This study examined factors such as buffer selection, mobile phase composition, and instrument compatibility based on the separation of these reference compounds. Despite high separation capability of trifluoroacetic acid (TFA) due to its strong ion pairing strength, its significant drawbacks include signal suppression in mass spectrometry (MS) and environmental impacts. The obtained results suggest that difluoroacetic acid (DFA) and ammonium acetate (AA) by showing acceptable performance, can be potential replacements for TFA. In chapter two, the LC method development process for separating impurities of metoprolol was examined. Fusion QbD software was employed in this section to assist in the design of experiments (DoE), analysis of screening experiments, and optimization of the separation methods. Ultimately, the investigation revealed a method using a 10 mM potassium phosphate buffer with a pH of 8.1 and a gradient from 15 to 42% acetonitrile over 7 minutes at 50°C yielded the most optimal separation of the target compound from its impurities. Furthermore, the study compared the performance of an MS-friendly buffer, ammonium acetate, with potassium buffers based on the parameters of the optimized method.In the final chapter, an optimized experimental procedure for assessing the stability of Inclisiran, a commercial siRNA used for reducing low-density lipoprotein cholesterol (LDL C), was studied. This exploration focused on measuring the melting point (Tm) and involved testing various parameters such as the number of thermal cycles, the effectiveness of silicone oil, and the choice of temperature ramp. In the end, it was demonstrated that setting only one cycle, adding 10 µL of silicone oil, and using a temperature ramp of 0.5°C/min were the optimal parameters for obtaining reliable data. On the other hand, experiments assessing Inclisiran stability in the TEA/HFIP system revealed that TEA concentration did not significantly impact stability at 5 and 15 mM, but adverse effects were observed at 100 mM TEA, resulting in a lowering of the melting point by 11.6°C. LC separation experiments provided additional insights into Inclisiran's behaviour, showing complete denaturation at 65°C but non denaturation conditions at lower temperatures.
- PostChromatin binding patterns of SWI/SNF chromatin remodeling complex components and interaction partners in FET sarcoma(2024) Grönqvist, Kajsa; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Westerlund, Fredrik; Lindén, Malin; Ståhlberg, AndersSarcoma is a group of cancers originating in supportive tissues in the body, such as muscle, bone, and fat. FET sarcoma, often found in young patients, is a subgroup of sarcoma. Treatment plans for certain types of FET sarcomas, can leave patients at a high risk of developing a secondary cancer later in life. FET sarcomas are characterized by the presence of fusion genes consisting of a member from the FET family partnered with a transcription factor for example DDIT3, and these fusion genes encode FET fusion oncoproteins, also called FET-FOPs. The FET fusion oncogenes are in several cases the only mutation present in FET sarcomas, indicating that the mutation can induce oncogenesis. FET-FOPs have been found to interact with the SWI/SNF chromatin remodeling complex, which is a complex responsible for remodeling nucleosomes to enable access to DNA for processes such as transcription. This project aimed to determine the chromatin binding strength of SWI/SNF components and known interaction partners in the cell line HT1080 with stable expression of different FET fusion oncoproteins. This was done by using sequential salt extraction (SSE), a method used to solubilize nuclear proteins iteratively with increasing salinity. The salt concentration at which the proteins were extracted reflects their binding strength. The protocol for SSE was optimized to use on HT1080 as the nuclei in the cells were prone to rupturing, resulting in gel pellets that did not allow collection of the nuclear fractions. The change in the protocol that gave desirable results was to resuspend the cell pellet in a buffer with low salt concentration before adding a higher salt concentration buffer as this minimized the occurrence of local high salt concentration, which was believed to be the cause of the ruptured nuclei. Three antibodies targeting the SWI/SNF components BCL7A/B/C were also optimized to be used on Western blot to ensure that they targeted the correct proteins. The final extracts from SSE were then evaluated on Western blot with antibodies targeting SWI/SNF components and known interaction partners to assess if the expression of FET-FOPs alter the chromatin binding strength. SWI/SNF components displayed a unique salt extraction profile, different from most interaction partners but no patterns in regard to functional module or SWI/SNF subtype was observed. The results indicated that the expression of FET-FOPs could have an influence on the binding strength of SWI/SNF components and interaction partners as different expression profiles were detected for some proteins. However, more experiments are needed to confirm this conclusion. By discovering more about the cellular mechanisms between FET-FOPs and SWI/SNF, new targeted treatments for FET sarcomas may be explored.
- PostAggregation of α-synuclein in primary neuron models of Parkinson’s disease(2024) Allgén, Elin; Borg, Olivia; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Esbjörner Winters, Elin; Ghaeidamini, Marziyeh; Agholme, Lotta; Esbjörner Winters, ElinParkinson’s disease (PD) is the second most prevalent neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons, leading to impaired motor function. In PD pathology, substantial evidence suggest that the neuronal loss is caused by the protein α-synuclein (α-syn) which aggregates into insoluble amyloid fibrils Mutations in the SNCA gene, encoding α-syn, can accelerate disease progression by enhancing the protein’s propensity to aggregate. Synthetic amyloid fibrils called pre-formed fibrils (PPFs) have recently been developed as a valuable tool for studying disease progression in vitro, by effectively induce endogenous aggregation in cells. In this project, five α-synuclein variants were studied: wild-type (WT) and pathological mutants A30P, E46K, H50Q, and A53T. The study aimed to characterize the biophysical properties of PFFs, identify differences among the variants, and assess their ability to induce endogenous α-syn aggregation and potential neurotoxicity. PFFs were generated and characterized using atomic force microscopy (AFM), circular dichroism (CD), and Thioflavin T fluorescence spectroscopy (ThT Assay). Cellular assays with primary cortical cultures from rat and mouse were then employed to evaluate cellular health, viability, and α-syn aggregation. This thesis demonstrated that within a given experiment PFFs could be generated and sonicated in a reproducible manner. Biophysical characterization revealed differences among the α-syn variants, including distinct morphologies, variations in monomer conversion to PFFs, and differences in fluorescence intensity and molar ellipticity. Furthermore, all α-syn variants induced endogenous aggregation in both cell models, with distinct aggregation patterns observed across variants and between models. E46K PFFs caused the highest level of aggregation in the mouse model without affecting cellular health, whereas A53T PFFs led to the highest aggregation in the rat model and impaired dendritic networks. H50Q induced minimal aggregation in both models, but negatively affected dendritic networks, suggesting potential neurotoxicity. Notably, aggregates induced by E46K displayed a distinct morphology compared to other variants. These findings highlight the complexity of α-syn pathology and suggest that different α-syn variants contribute uniquely to PD progression
- PostDNA Microscopy - Establishing an immuno protocol(2024) Andersson, Anna; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Westerlund, Fredrik; Högberg, BjörnAs we continue to explore and understand the world, the demands on our methods and techniques evolve too. DNA microscopy is a developing method that offers an alternative to conventional light microscopy. Instead of relying on light and a convex lens, it utilizes nature’s building blocks, DNA, to create spatial images and describe different proteins near one another using sequencing. Björn Högberg’s lab has previously shown the method to work in an artificial system and the project is now ready to start exploring one of the options for a cellular setup. One possibility is to create a multi-omic system that utilizes antibodies to deliver target strands to specific cells, mimicking the original artificial setup. This requires the conjugation of designed DNA strands, targets, to the antibody. An appropriate protocol was developed and established in this thesis. The four cell lines used were A549, HeLa, MCF-7, and MDA-MB-231. In situ immunocytochemistry (ICC) was used to visually confirm the relative specificity of the four cell types to the nine different antibodies. Both SiteClick and oYo-Link were trialed for the conjugation of targets, using both ProFIRE and Amicon to purify the conjugates. The result showed that all four cell lines had general specificity to antibodies CD44 and BASP1, but only HeLa and A549 to SLC38A1. A549 was the only cell line that showed specificity for ALDH3A1 and AKR1B10. For the conjugation protocol SiteClick in combination with ProFIRE and Amicon gave the best result for conjugate concentration and purity. The results of the thesis provide the beginning of a library with appropriate cell lines and antibodies to use in a cellular setup. It also establishes a protocol for the conjugation of target strands to antibodies. To fully realize a cellular version of the artificial system more study needs to be performed on appropriate cell lines and antibodies for a more complex setup. To further evaluate the conjugates’ effect on the DNA reactions the reaction requires sequencing and re-construction.