Examensarbeten för masterexamen


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  • Post
    Catalytic Activity of α-Synuclein Amyloid Fibers
    (2024) Rehnberg, Nikita; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wittung-Stafshede, Pernilla; Horvath, Istvan
    Amyloid fibers play a significant role in neurodegenerative diseases such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). They have for long been considered nonreactive dead-end products of protein aggregation. Recent research show that amyloid fibers, but not monomers, exhibit enzymatic catalytic activity in vitro. This project investigates the catalytic activity of α-Synuclein (aS) amyloid fibers. First, homogeneous aS amyloid fibers were made in a two-step seeding process and amyloids confirmed through ThT-fluorescence, far UV circular dichroism (CD) and atomic force microscopy (AFM). Then, an already established esterase activity assay, in vitro, was used to study ester bond cleavage of WT and A53T (disease-causing mutant) aS amyloid fibers. Another in vitro enzymatic assay was developed to study ATPase activity of WT, A53T and H50A (model of disease-causing mutants with altered residue 50) aS amyloid fibers. Results indicate that there is catalytic activity when aS amyloid fibers are incubated with both ester-bond and ATP substrates. However, the results are only qualitatively interpreted due to large variability in obtained kinetic parameters. To draw firm conclusions regarding the difference between the investigated aS mutants, additional experiments are needed.
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    Development of analytical and sensitive methods to detect and quantify therapeutic oligonucleotides
    (2023) Domagala, Izabela; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wenzel, Michaela; Ståhlberg, Anders
    Therapeutic antisense oligonucleotides (ASOs) had a breakthrough in 1998 when fomivirsen, an inhibitor of human cytomegalovirus, was approved for medical use. Scientists discovered the medical potential of therapeutic oligonucleotides in treatments of various conditions and diseases, leading to the research field gaining more popularity. A challenge within this research field is the detection and evaluation of different oligonucleotide sequences, as no established quantitative and sensitive methodologies are present for these. The aim of this thesis is to develop analytical and sensitive methods for detection and quantification of therapeutic oligonucleotides. Particularly antisense oligonucleotides (ASOs), for application in in vitro experiments associated with fields such as cancer research. The ASOs included in this project are EBER1-5 and EBER1-6. The two-tailed RT-qPCR method, used for detection of miRNA, was adapted and modified in this project. The method was changed so that instead of a reverse transciption, a PCR was performed to anneal and elongate the two-tailed primer with the ASO. The PCR was then followed by a qPCR analysis. Different deviations to the initial protocol as well as to the designs of the primers and oligonucleotides, were performed during the project. The results showed that the method is promising as detection was possible to approximately 105 molecules, which corresponds to a concentration of 83 fM. Still, improvements need to be done for the binding and the elongation in the two-tailed part of the method to be able to reliably detect lower amount of molecules.
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    Optical DNA Mapping as a Tool for Determination of EHEC Serotypes
    (2024) Mårtensson , Hanna; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Westerlund , Fredrik; Nambannor Kunnath, Radhika
    Enterohemorrhagic Escerichia coli (EHEC) is a pathogenic subgroup of E. coli, responsible for causing severe diarrheal disease though food-borne outbreaks. Infection can further progress to Hemolytic Uremic Syndrome (HUS), a critical condition associated with renal failure and potential fatality. In epidemiological surveillance of EHEC infection, it is of interest to determine the serotype of the EHEC strain, as the risk of severe disease is linked to a few serotypes. Currently, this process involves extensive hands-on involvement while also being time-consuming, suggesting the need to explore alternative methods to identify EHEC serotypes. In this project, Optical DNA Mapping was employed for bacterial typing. DNA from seven different EHEC samples was labeled through competitive binding and then stretched in nanochannels. Using fluorescence microscopy, the DNA molecules were imaged which due to the labeling resulted in unique fluorescence intensity profiles along the molecules corresponding to the DNA sequence. These intensity profiles were then matched trough reference-based alignment to a reference database in order to identify the bacterial strain associated with the DNA. The method demonstrated a 100% true positive rate identification at the species level. At the strain level, a majority of matches corresponded to the correct serotype. The results also indicated the potential to assign the matches to strain groups with high taxonomic resolution. In conclusion, the findings provide a positive indication of the possibility in using ODM for determining of EHEC serotypes.
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    Can AD be identified through metabolic models?
    (2024) Sandstig, Gaston; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Kerkhoven, Eduard; Polster, Annikka
    The most common form of dementia is Alzheimer’s Disease (AD), a progressive neurodegenerative disorder, but so far no methods of curing or preventing it are known. Amyloid-β proteins have been the focus of therapeutic development but clinical trials focused on reducing amyloid-β production or preventing its aggregation have failed, leading to increased interests in other areas of AD pathology. There is evidence of impaired metabolism in those with AD, and in this project gene expression data from the ROSMAP study was used to construct single-sample genome-scale metabolic models in an effort to see what differences can be discerned between models from those with AD and not. Although there was no observed separation between the groups when using PCA and t-SNE, the aggregate metabolic coverage between groups differed in several metabolic subsystems. The differences found had support in literature, but subsystems C5-branched dibasic acid metabolism, GPI anchor biosynthesis, heparan sulphate degradation, and lipoic acid metabolism had not been identified as differing in comparable in silico metabolic AD model studies
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    Characterization of the antibacterial properties of human amyloid proteins and antimicrobial peptides based on bacterial membrane-binding domains.
    (2023) Qvist, August; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Wenzel, Michaela; Wenzel, Michaela
    Antibiotic resistance is one of the largest current threats to public health and food security, being directly responsible for 1.27 million deaths worldwide in 2019 and is estimated to rise to 10 million deaths per year by 2050. Therefore, there is an urgent need to find new treatments against resistant infections. This can be done by finding new antibiotic compounds with new mechanisms of action or through optimizing and potentiating existing antibiotics. The antimicrobial mechanisms of action for the human proteins S100A12 and α-synuclein, and 18 peptides derived from peripheral membrane proteins of Mycobacterium tuberculosis were investigated. Antimicrobial activity of each protein and peptide was assayed by determining the minimal inhibitory concentration (MIC). The 18 peptides derived from M. tuberculosis were screened for outer membrane permeabilization effects on Gram-negative bacteria (Escherichia coli) and mycobacteria (Mycolicibacterium smegmatis and Mycobacterium marinum) using the fluorescence probe 1-N-phenylnaphtylamine (NPN). The NPN assay was adapted from the E. coli protocol and investigated for use in mycobacteria. The fluorescent probe 3,3′- dipropylthiadicarbocyanine (DiSC35) was used in a single cell fluorescence microscopy assay to investigate effects on membrane polarization, which can indicate activity toward the plasma membrane. To determine the resistance frequency of the peptides a disk diffusion assay was used. The disk diffusion plates were checked for colonies inside of the inhibition zone after 24 and 48 hours. For S100A12 and α-synuclein-treated Bacillus subtilis grown in a minimal medium, no MIC or significant change in growth could be observed at the tested concentrations. Addition of calcium ions or copper ions to the proteins did not change the growth or MIC. This suggests that these proteins do not have any antimicrobial effect on B. subtilis under the tested conditions. However, some older batches of the S100A12 protein showed a decrease in growth rate suggesting some antimicrobial activity, likely caused by a degradation product or toxic oligomer formation. The antimicrobial activity of the OMPPs ranged from no activity to relatively high activity. The NPN assay in E. coli showed that out of the eighteen peptides, six showed no outer membrane permeabilization, and six peptides showed outer membrane permeabilization of 20% or higher at 10 μg/ml. With the DiSC35 assay, treating E. coli with the peptides for 15 minutes before imaging, an increase in fluorescence emission could be seen for the peptides that also showed a higher outer membrane permeabilization. However, no significant change in fluorescence of the dye could be seen for most peptides. The increased fluorescence of DiSC35 is likely caused by an increase in uptake of the dye due to increased permeability of the outer membrane. The disk diffusion assay showed no colonies within the inhibition zone after 24 or 48 hours indicating low frequency of resistance development towards the peptides. The collective results of the screenings in E. coli showed that two peptides may be selective outer membrane permeabilizers and should be investigated further at different concentrations. The rest of the peptides showed other or unspecific modes of action that may be of interest as antimicrobials and should also be investigated further. In mycobacteria, using NPN to assess outer membrane permeabilization needs to be further developed and optimized to produce proper results, as no reliable positive control is known. Further assays should be used to evaluate the activity of these peptides in mycobacteria.