Inflammation is a part of the innate immune system. It protects the body against foreign invaders such as bacteria and viruses. Inflammation helps to restore the body by removing harmful stimuli and starting the healing process. Inflammation is produced in response to damage-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPS). Glycogen synthase kinase 3 (GSK-3) is a key regulator of a variety of pathways, making it a promising therapeutic target. Therefore, this experiment aims to see how inhibiting GSK-3 affects the generation of IL-8 in THP-1 ASC GFP macrophage cells. For this study qPCR was used to measure IL-8 expression, while ELISA was used for protein secretion. An ANOVA test was utilized for the statistical analysis. Obtained results from this study showed that there is a significant difference between stimulated cells with LPS and nigericin against unstimulated samples both in protein and mRNA levels. When it comes to the stimulated cells against inhibited cells, the ANOVA test showed there is no significant difference between the samples both in protein and mRNA levels. This might suggest that GSK-3 does not influence the development of inflammasomes in THP-1 macrophage cells. Another possible reason is that other pathways such as the MAPK and JAK-STAT may mask potential inhibitory effects on the NLRP3 inflammasome pathway by producing even more IL-8, which interfered with qPCR and ELISA results. In conclusion, additional research is needed to confirm the involvement of GSK-3 in NLRP3 inflammation.

The testis is a complex organ responsible for sperm production and hormone synthesis. Spermatogenesis, the process of sperm formation, occurs in the seminiferous tubules and involves the coordinated development of germ cells with the support of Sertoli cells. These provide structural and nutritional support for germ cells, regulate spermatogenesis, and maintain the blood-testis barrier. Despite extensive research on Sertoli cells, their protein expression patterns and spatial localization in relation to other testicular cell types remain poorly characterized. This project aimed to investigate the expression patterns and spatial localization of proteins in Sertoli cells. Single-cell RNA sequencing data and immunohistochemistry stainings from the Human Protein Atlas were used to choose candidate proteins to be visualized in a multiplex Immunofluorescence panel. After staining, the slides were scanned and analyzed manually as well as through automated image analysis via QuPath. 101 proteins were successfully stained and localized within testis tissue and subcellular locations in Sertoli cells were annotated. Additionally, a list of uncharacterized proteins in Sertoli cells were identified. The employed workflow demonstrated its potential for identifying Sertoli cell-specific proteins and can be applied to future research. Additional validation is required to verify the presence and identify function of these proteins in Sertoli cells. For this purpose, techniques such as mass spectrometry-based proteomics and mouse knock-out models could be used.

Hepatitis C viral infection is a globally widespread blood-borne disease affecting the liver, causing cirrhosis and hepatocellular carcinoma. This type of liver cancer is mainly caused by chronic Hepatitis C and Hepatitis B. Specific detection with the following correct treatment is crucial to reduce the overall burden of the disease. This work focused on investigating whether the padlock probes and rolling circle amplification can detect Hepatitis C, determining the limit of detection, and if any blood components would inhibit the reactions. All oligonucleotides were tested for ligation functionality in 10% TBE-Ureal gel electrophoresis and used with rolling circle amplification and phi29 polymerase to determine if eye read-out was possible. The lowest concentration of detection was found to be 10 pM. To avoid inhibition in blood plasma, samples were pre-treated at 95 ˚C for five minutes. Eye read-out was possible after amplification, with 30% plasma at the highest and 5% plasma at the lowest in samples. In conclusion, this novel assay using padlock probes, a detection oligonucleotide, and rolling circle amplification holds promise in developing a simplified new detection technique for the diagnostics of Hepatitis C.

Trimethylaminuria is an autosomal recessive disorder characterized by a decreased oxidation capacity of trimethylamine to trimethylamine-N-oxide in the liver. The condition is diagnosed by estimating concentrations of trimethylamine and trimethylamine-N-oxide in human urine and then evaluating their respective creatinine ratios and the oxidation efficiency percentage. Values previously retrieved with liquid chromatography-tandem mass spectrometry with electrospray ionization but not the novel ionization interface UniSpray. Thus, this project aims to initiate the development of a liquid chromatography-tandem mass spectrometry diagnostic method for trimethylaminuria with UniSpray ionization. The analytes were extracted from urine with a liquid-liquid extraction method and separated with hydrophilic interaction liquid chromatography using an isocratic profile with 5 mM of ammonium formate in water and methanol and multiple reaction monitoring. Overall, the mean coefficient of variation and recovery percentage from trimethylamine spiked urine samples were lower than expected, whereas the intra-precision for trimethylamine-N-oxide was acceptable. Three urine samples had estimated oxidation percentages, but only one had derived comparable creatinine ratios to an external laboratory. Due to the inadequacy of comparative data and the precision and recovery percentage deviations, the results presented in this report need cautious interpretation. Future development of the method could include manual tuning, reconsidering the calibration curve and reference values, and comparisons to the extraction method. Although there are apparent discrepancies in the precision and reliability of the derived trimethylamine and trimethylamineN-oxide concentrations, the initial steps in the pursuit of a liquid chromatography-tandem mass spectrometry diagnostic method for trimethylaminuria provide a practical foundation to continue the development.

Inflammation is the body's natural defense reaction and is known since ancient times. The inflammation is divided into two main phases, acute and chronic inflammation dependent on the process and cellular mechanisms of the inflammation. Inflammation has become to be an important field in research by biomedical research where it is included in many cellular processes thus being phagocytosis, chemotaxis, mitosis, and cell differentiation. Inflammasomes are pro-inflammatory intracellular multimeric protein complexes that introduce the activation of pro-inflammatory cytokines, such as interleukin-1β and interleukin-18, upon trigger by PAMPs and DAMPs signals. The most studied inflammasome is the NLRP3 inflammasome that is activated by various trigger signals, like DAMPs, ATP, uric acid crystals and amyloid-β fibrils. GSK-3β is a kinase that controls various cellular processes, such as inflammation by regulating the activity of abundant transcription factors that are valuable for cytokine production. The aim of this thesis project was to investigate if GSK-3 Inhibitor IV, SB-216763, in a concentration-dependent manner had an effect on production of IL-1β in LPS- and Nigericin-stimulated THP-1 ASC-GFP-macrophages. In addition to the gene expression analysis of IL-1β, the amount of secreted IL-1β, and the possible correlation between treated THP-1 cells with and without GSK-3 inhibitor evaluated. The gene expression analysis was performed by using qPCR and the amount of secreted IL-1β was done using sandwich enzyme-linked immunosorbent assay. The results from this study showed no significant difference in gene expression and amount secreted of IL-1β in THP-1 cells when treated with the GSK-3 Inhibitor IV, SB-216763.

Corona virus disease caused by severe acute respiratory virus 2 causes blockage of the blood vessel which leads to thrombosis. Thrombotic events in covid-19 patients results to hospitalizations and death. And incidence of thrombosis in covid-19 patients have been increasing in most regions of the world. Due to the huge inequalities between developed and developing countries, incidence rates remain highest in more developed regions, but mortality is relatively much higher in less developed countries due to a lack of early detection and access to treatment facilities. The aim of this study was to investigate the incidence of thrombosis in covid-19 by performing a systematic review on research articles talking about thrombotic events in covid-19 patients, carrying out a meta-analysis on the generated data to make an inference on the statistical result in order to create an information concerning complications covid-19 patients suffer from. Literatures with cases of covid-19 that reported D-dimer elevation and that followed the WHO standard for covid-19 diagnosis were included. Literatures excluded were studies with pregnant women, cancer patients and patients undergoing chemotherapy and with no approved ethical considerations. Information sources included only original literatures with initial search yielding 55 results from 4 databases. After reviewing titles and abstracts of all 55 literatures, 35 studies were further screened and 10 were included in the analysis representing 3359 patients. A forest plot using the R programming language was done, and an overall pooled estimate using the random effect model was 20 % (95 % confidence interval 12.0 % - 29.0 %) with heterogeneity of 96 %, and p <0.01. The incidence of thrombosis among moderate cases of c ovid-19 patients was 12 % with (95 % confidence interval 8.0 % - 18.0 %) with heterogeneity 93 %, and the incidence of thrombosis among severe Covid-19 patients was 22 % (95% confidence 10.0 %-37.0 %) with 97 % heterogeneity, and p <0.01.

Sclerotinia sclerotiorum is a plant pathogenic fungus that causes Sclerotinia stem rot in oilseed rape. In Sweden, the disease causes severe crop loss that varies by year. Previous studies have shown a relationship between the proportion of infected petals and disease incidence in infected fields in places with high humidity levels before and during flowering. In this study, the aim was to develop a technique to detect S. sclerotiorum and other fungi pathogens in the petals of oilseed rape from naturally infected fields by using nanopore sequencing from Oxford Nanopore Technologies. DNA was extracted from the petals of oilseed rape and subsequently amplified by performing PCR after optimizing the optimal annealing temperature. Using the forward primer ITS1catta and the reverse primer ITS4ngsUni, these primers targeted the ITS region, which is used as a marker for the identification of fungi. The resulting Amplicon concentrations varied. Five amplicon PCR samples were selected for MinION sequencing. These samples were selected since they had the best purity levels. Finally, bioinformatic analysis was done with Kraken2 and the Pavian tool and compared with UNITE databases. The result showed hundreds of thousands of reads were recovered from the Ascomycota and Basidiomycota fungi divisions; S. sclerotiorum was observed in one field sample; other Sclerotiniaceae species like Dumontinia tuberosa, Botrytis cinerea, and Sclerotinia bulborum were detected in two fields; and many other fungal pathogen species affecting rapeseed crops in Sweden were successfully detected. MinION was successful in identifying S. sclerotiorum and other plant pathogens.

Antibiotic resistance in microorganisms poses a global hazard to human and animal health. To combat antibiotic resistance, a deeper understanding of the mechanisms behind the spread of antibiotic resistance genes (ARGs) in bacteria is crucial. Plasmids are major vehicles for the spreading of ARGs, so their conjugation-mediated transfer is examined in this thesis. Escherichia coli (E. coli) was isolated from a wastewater treatment plant (WWTP) and clean water in Stockholm, Sweden and the isolates were used as donors of mobile plasmids and a lab strain of E. coli (CV601) was used as recipient. Transfer efficiency was analysed by calculating conjugation frequencies (CF), after testing conjugation by fast conjugation and filter mating method. The resulting transconjugants were analysed for antibiotic resistance pattern by the disc diffusion method. Additionally, sequence analysis was used to analyse the transferred plasmid(s) and ARGs. The qPCR was performed to check the expression of transfer genes (TraN and TraJ) during conjugation of IncN plasmids. The results showed that several isolates from WWTP were multi-resistant and successfully transferred plasmids to CV601 and donor strain 18 with a mobile IncN plasmid had the highest CF that depended mainly on the type of pili. The ARGs qnrS1, dfra14, and blaCTX-M-15 were transferred to transconjugants mainly by IncN plasmids and qPCR findings indicated that the level of gene expression of transfer genes in donor strains affected CF. The findings of this study expand our understanding of antibiotic resistance dissemination and knowledge of plasmids found in WWTPs and clean water.

Sclerotinia sclerotiorum, a notorious phytopathogenic fungus, causes sclerotinia stem rot in oilseed rape (Brassica napus), a disease with global consequences for oilseed rape productivity and oil quality. The complexity of factors contributing to sclerotinia stem rot makes prediction and control exceedingly challenging. Early and accurate identification of plant pathogens is crucial for effective disease management. This study aimed to establish a method utilizing MinION nanopore sequencing for identifying S. sclerotiorum and other fungi causing diseases in oilseed rape. Naturally infected leaf, soil and air samples were collected from oilseed rape fields in Sweden, and DNA was extracted. Two primer pairs targeting the ITS region known marker for fungus identification, were amplified by PCR. For nanopore sequencing, six PCR amplicon samples, two from each source were selected based on their purity and stem rot incidences, with one sample spiked-in with gDNA S. sclerotiorum as positive control. Bioinformatic analysis was performed using the CCMetagen tool and EPI2ME. Ascomycota and Basidiomycota constituted 34% and 45%, respectively, of the identified species. S. sclerotiorum was only detected in air field samples, and several other fungal species harmful to oilseed rape production in Sweden, such as Botrytis cinerea, Pyrenopeziza brassicae and many more, were identified. In conclusion, successful identification of plant pathogens, including S. sclerotiorum, was achieved using MinION nanopore sequencer.

The study of viral capsid proteins, such as VP2 of CtenDNAV-II, is essential for understanding viral capsid assembly, infection mechanisms, and host manipulation. Capsid proteins, the protein shells encapsulating viral genetic material, play critical roles in protecting the virus and aiding in its attachment to host cells. By capturing high-resolution images of individual virus particles embedded in vitrified ice, cryo-EM facilitates the reconstruction of three-dimensional (3D) structures of viral capsids. The study of viral capsid structures using cryo-EM provides insights into viral life cycles, host-virus interactions, and evolutionary events. The aim of this research was to gain deeper insights into viral capsid architecture and dynamics by reconstructing the small capsid particle (T=1) and investigating its role in the capsid assembly of CtenDNAV-II. The capsid protein was expressed using Sf9 insect cells that were inoculated with a baculovirus containing the VP2 gene. The capsid protein expression in E. coli was performed by transforming the cells with a ubiquitin-expressing plasmid. An already existing cryo-EM dataset, which has been used to reconstruct the large capsid particle (T=3) was used to reconstruct the small capsid particle and determine whether or not it was hollow inside. The single particle reconstruction of the T=1 capsid particles, realized using cryoSPARC, achieved a fairly high resolution and indicated that the particles are most likely empty inside, containing no genome. It was noticed that many of the small particles were broken, which indicates they were most likely intermediate or incomplete VLP assembly states.