Examensarbeten för masterexamen
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- 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.
- PostIntegration of Stem Cell-Derived Neurons with Multi-Electrode Array(2024) Simonsson, Isabella; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Asplund, Maria; Bengtsson, NiklasThe prevalence of serious mental illnesses (SMI) in the US adult population, in- cluding bipolar disorder (BP), major depressive disorder (MDD), and schizophrenia (SCZ), is estimated at 4-6%. While the creation of in vitro models from human induced pluripotent stem cells (hiPSCs) has transformed disease modeling in such illnesses, further research is needed to optimize these models. This project utilizes the human embryonic stem cell (hESC) line H1 to investigate the electrophysiological activity of stem cell-derived cortical neurons using Multi- Electrode Arrays (MEA). By integrating micro-electrodes into cell well-plates, neu- ral activity can be non-destructively measured over several weeks with MEA. The objective is to optimize the integration of stem cell-derived neurons with MEA for monitoring electrophysiological activity, evaluating different cell densities, coatings, and culturing mediums. Comprising a pilot study followed by a more extensive investigation, this project compares metrics such as activity and electrode coverage across various culturing conditions to identify optimal parameters. The most promising condition entails a cell density of 50k cells per well, coated with both Poly-L-Ornithine (PLO) and Bi- olaminin 521 (LN521), and cultured in either Neural Maintenance Medium (NMM) or BrainPhys (BP).
- PostVessel-on-a-Chip Development to Understand Systemically Administered NanoparticlesVessel-on-a-Chip Development to Understand Systemically Administered Nanoparticles(2024) Jia, Fan; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Stubelius, Alexandra; Svensson, ElinNanomedicine holds great promise for treating complex diseases, particularly due to its unique ability to sustain and target drug delivery. However, the validation method has been a great challenge to the development of nano-based therapeutics. The main reason is the unpredictable delivery dosage and lack of adequate preclinical trial methods that replicate the human biological environment for testing nanomedicine. This project introduced organ-on-a-chip (OOC) method to mimic the human vascular environment in vitro for studying the transportation of nanoparticles in human vessels. The goal was to use commercially available resources to increase the generalization and ease of adaptability for other labs working within the field of nanomedicine. The build-up of this vessel-on-a-chip (VoC) was successfully achieved using a microfluidic chip from Ibidi along with a microfluidic set-up to stimulate the human umbilical vein endothelial cells (HUVEC) seeded in the chip. The cells formed an oriented endothelial cell (EC) monolayer mimicking the endothelium of a capillary under a 24-hour flow of 10dyn/cm2. This monolayer acted as a barrier preventing NPs from extravasating into the tissue, which is an important aspect of nanoparticle delivery. In addition, a computational fluidic dynamic (CFD) simulation approach was conducted to further enhance the predictability of NP transportation. The microfluidic simulation helped to explain unexpected phenomena during the flow experiment.Although the blood flow simulation had some limitations, it could provide useful insights when evaluating a potential uptake amount of NPs when administered systemically. This integrated study of combining VOC with CFD simulation enhances our understanding of NP behavior in human vessels but requires further optimization to be fully operational.
- PostPurification of Adeno-Associated Viruses Using an Electrochemically Controllable Polyelectrolyte Brush - Separation of Genome-Filled Adeno Associated Viruses from Non-Genome-Corporated Virus Capsids(2024) Tranchell, Hanna; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Dahlin, Andreas; Ferrand-Drake del Castillo, GustavAdeno-associated Viruses (AAVs) are a promising gene therapy vector due to their low toxicity, long-term gene expression, versatility in transducing various cell types, and site-specific integration into the chromosome. However, several challenges remain in AAV production, such as low yield and difficulties in gene incorporation, which necessitate a thorough purification process that is both labor-intensive and expensive. Conventional techniques, that rely on chromatography column resins, face severe limitations, especially with diverse AAV serotypes. This thesis explores a novel purification approach using an electrochemical controllable polyelectrolyte brush (PE) brush. The PE brush serves as an adhesive surface for biomolecules, with high protein-binding capacity, and electrochemical signals induce controlled release. The electrochemical method avoids the drawbacks of conventional pH changes and hence, offers the potential to resolve impurity challenges. The objective was to validate the electrochemical PE brush system for AAV purification. Three milestones were established: confirming immobilization and elution, evaluating product purity and quantity, and optimizing the system. Results demonstrate successful immobilization and electrochemical elution using both PAA and PDEA brushes. The system shows the potential to up-concentrate capsids and enhance the percentage of filled capsids relative to the total amount of capsids. Recirculation further improves binding efficiency. Even though this study shows great potential to improve purification, the need for further purity enhancement needs to be acknowledged to unlock the advancement of the method in the aspect of AAV purification. Further refinements can put this innovative approach in a competitive position with conventional purification methods for AAVs.
- PostThe Role of LigD in Non- Homologous End-Joining in Mycobacterium tuberculosis(2024) Fornander, Selma; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Westerlund, Fredrik; Morati, Florian; Pavlova, EvgeniyaRepairing damaged DNA is necessary for the survival and accurate reproduction of organisms. To this end, numerous DNA damage repair pathways have evolved. Non-homologous end-joining is a repair pathway that repairs double-stranded breaks in DNA, without using a homologous sequence as a template. In bacteria, the two proteins necessary to facilitate non-homologous end joining are Ku and LigD, a DNA ligase. This project investigated how six different point mutations on LigD affected the DNA ligation activity. Both bulk and single molecule methods were utilized. For bulk assays, electrophoretic mobility assays, pulldown assays, and ligation assays were used to study how well Ku and LigD bind to and ligate DNA, and how the mutations affect this. The ligation activity was also investigated using nanofluidics, visualizing the DNA on a single molecule level. From these assays, it was shown that three of the point mutations (R198E, D162R, V194D) resulted in lowered ligation activity compared to wild type LigD. One of the mutations (K579E) increased the ligation activity. The mutation L580E caused DNA fragmentation, and for LigD D522R, the results from the assays were contradictory.