Benign prostatic hyperplasia (BPH) and chronic prostatitis (CP) are common diseases in aging men. Though medications are available to alleviate these conditions, problems of possible side-effects of first-line synthetic drugs for prostatic conditions have allowed patients to switch to a safer plant-based medication. CernitinTM, a pollen extract, is used to alleviate these conditions. A recent in vitro study showed that CernitinTM inhibits cell proliferation and induce a regulatory effect on inflammatory parameters. To validate those results, the inter-batch variability of CernitinTM was assessed using the active ingredients CernitinTM T60 and CernitinTM GBX on the human prostatic cell lines BPH‐1 and WPMY‐ 1 and on human peripheral blood mononuclear cells (hPBMCs) in vitro. Cell proliferation assay was performed in prostatic cell lines, while inflammatory parameters were analyzed in hPBMCs. Results revealed that both CernitinTM active ingredients, regardless of batch production, significantly inhibited the proliferation of both prostatic cell lines after 48 and 72 hours, respectively (p < 0.05 to p < 0.001). Among the batches, there were no significant differences observed. Notably, the GBX batches 14164, 14548 and 14160 had a more pronounced effect on cell proliferation right after 48 hours on both cell lines. Whilst, T60 batches 11539 and 14144 had a pronounced effect right after 48 hours on BPH cells. In hPBMC, the production of the anti-inflammatory cytokine interleukin (IL)- 10 and its receptor IL-10 receptor subunit beta (RB), as well as pro-inflammatory cytokine IL-6 was significantly increased after treatment with the T60 formulation regardless of the batch, but not after treatment with the GBX batch. Moreover, IL-10 receptor subunit alpha (RA) and tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) expression increased after the use of both formulations (p < 0.05 to p < 0.001). The pro-inflammatory cytokine IL-8 and chemokine CXCL-10 was significantly decreased using both batches of T60 (p < 0.05 to p < 0.001). Collectively, these results support the claim of the role of CernitinTM as an anti-proliferative agent and as a cytokine regulator.

Giardia intestinalis is a two-staged flagellated protozoan parasite which infects the lumen of the small intestine causing the gastrointestinal disease called giardiasis. This disease infects millions of people per year, commonly caused by inadequate water treatment and proximity to livestock. The main line of treatment against this disease has been metronidazole, a member of the nitroimidazole family of prodrugs, which when activated cause non-specific damage to the proteins and genetic material within anaerobic cells. Until recently, G. intestinalis has shown little resistance to metronidazole, but as resistance climbs it has become clear that genetic regulation is key. This study attempts to investigate a select number of key genes through the use of modified transfected plasmids for overexpression and with knockdown using morpholino oligos. Wild type (WB) G. intestinalis cells, transfected with plasmids modified to both overexpress specific genes and incorporate the human influenza hemagglutinin tag (HA-tag) were seen to change in survival rate when exposed to metronidazole. Localization of a select group of these genes was also found using the HA-tag. Two of these genes (quinone oxidoreductase and nitroreductase 1) had protein folding simulations run in order to be visualizee and compare their structures to closely matching equivalents. Finally, morpholino oligo nucleotides were used to knockdown expression in these two genes, leading to a significant decrease in survival for those targeted against quinone oxidoreductase when exposed to metronidazole.

Macrophages are an important key effector cell in the immune system which can practically be found in every tissue. Macrophages have for a long time been considered a population of cells only responsible for pro-inflammatory responses and anti-microbial activities. But over the past decade, many have come to realize the amazing plasticity of macrophages in response to different stimulations. The anti-microbial and pro-inflammatory macrophage is known as classically activated macrophages but newly discovered phenotypes have been revealed named wound-healing macrophages and regulatory macrophages. Through systematic screening we have identified an inducible macrophage activation state which has both wound-healing and regulatory capabilities activated by the novel cytokine combination IL-4/IL-10 with or without TGF-β.

Antimicrobial resistance (AMR) is concerning because it limits antimicrobial drug treatment options. AMR occurs by the overuse and misuse of antimicrobial drugs. In environmental settings, AMR can disseminate from places of high use, which leads to increased exposure to humans and animals. A previous study from our laboratory group showed extended-spectrum cephalosporinase-producing Escherichia coli/Klebsiella pneumoniae were isolated from fecal samples obtained in rural Cambodian community settings. Based on these isolates, this study has two aims. The first aim was characterization of selected Cambodian isolates with random amplification polymorphic DNA (RAPD) and antibiotic susceptibility test. From RAPD, the selected six isolates are diverse, except for C61 and C66 bacteria isolates with potential clonality. Additionally, the selected isolates are multidrug resistant (MDR) with reduced susceptibility to beta-lactams and fluoroquinolones. The second aim was to assess two developed methodologies, multiplex automated genome engineering (MAGE) and One-Step Curing Plasmid, by validation in bacteria laboratory strain and development for six Cambodian isolates. To modify AMR genetic elements, MAGE uses pMA7-SacB for homologous recombination with oligos for chromosomal gene disruption. Meanwhile, One-Step Curing Plasmid uses pFREE with the CRISPR/Cas9 system for plasmid and self-curing. Validation showed that MAGE can modify 8% of E. coli MG1655 with lacZ control screening oligos and almost 90% are cured from pFREE. Selected Cambodian isolates have antibiotic-resistance plasmids of IncR or IncFII replicon. For usage in Cambodian isolates, pFREE was modified to be pCAM-FREE by cloning IncR and IncFII plasmid as gRNA1 and gRNA5, respectively. Sequencing results showed pCAM-FREE have gRNA5. In conclusion, our study managed to characterize selected Cambodian isolates as MDR and diverse. In a laboratory strain, MAGE and One-Step Curing Plasmid are functional methods. Furthermore, pCAM-FREE was constructed to target IncFII and in the future, MAGE and pCAM-FREE could be tested in Cambodian isolates.

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Background Cancer immunotherapy has yielded many successes. Yet to some hard-to-treat brain tumors, such as glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), it still lacks substantial improvement. Neoantigens resulting from mutations in malignant cells are the key targets for employing adoptive cell therapies. A novel therapeutical strategy may be developed based on the identification of T cell receptors (TCRs) targeting specific neoantigens. Methods Previous work had been done to provide essential materials, including candidate neoantigen peptides, human leukocyte antigen (HLA) genotypes, and peripheral blood mononuclear cell (PBMCs) from patients and healthy donors (HDs). Autologous antigen-presenting cells (APCs) and T cells were isolated from PBMCs for in vitro assays. The activation of T cells against peptides was evaluated by the upregulation of 41BB utilizing flow cytometry (FACS). The cell populations with positive signals were sorted through FACS for TCR sequencing directly or after rapid cell expansion. Results T cells and APCs from 12 HDs were isolated. T cells from 10 HDs were analyzed after in vitro stimulation. T cells from HD30 showed reactions to several public neoantigens; while T cells from HD49 and HD53 showed reactions also to private neoantigens restricted in GBM patient C6. Conclusion The upregulation of 41BB indicated the activation of T cells and the existence of reactive TCRs against either public or private neoantigens in some HDs. Those reactive TCRs and their encoding sequences were the fundamentals of future works. Due to practical reasons, TCR sequencing cannot be done within this project. In future works, wildtype peptides will be included to further validate the results, ensuring identified TCRs recognize neoantigens specifically. Furthermore, the identified TCRs will be cloned and transferred to freshly isolated T cells to confirm their functionality. Keywords Cancer immunotherapy, brain cancer, neoantigen, MHC/HLA, TCR

Background: The human gut microbiota is the complex microbial community that lives in our gut. The gut microbiota has a key role in health and disease and its disruption has been linked to several chronic diseases such as cardiovascular diseases. As antibiotics are well known disruptors of gut microbiota, the aim of this thesis work was to identify associations between previous antibiotic consumption and the abundance of seven gut microbiota species previously linked to subclinical coronary atherosclerosis in the large population-based Swedish CArdioPulmonary bioImage Study (SCAPIS) cohort. Materials and Methods: Faecal samples of 9,794 individuals from the SCAPIS Uppsala and Malmö cohorts were analysed by deep shotgun metagenomics sequencing in a cross-sectional study. Previous antibiotic use was retrieved using the Swedish Drug Prescribed Register and divided into three periods: one year, between one and five years, and between five and nine years before faecal sampling. Associations between antibiotic consumption and the gut microbiota species were evaluated using linear regression adjusted for covariates and corrected for multiple testing. Results: Our results showed that antibiotic consumption was associated with an increased abundance of Ligilactobacillus salivarius, Bifidobacterium dentium, Rothia mucilaginosa, Streptococcus parasanguinis and Streptococcus oralis subsp. oralis. Often these positive associations were present for antibiotic consumed between one and five years before sampling.  The strongest associations were for broad-spectrum antibiotics and lincosamides with L. salivarius, B. dentium, R. mucilaginosa and S. parasanguinis; and for nitrofurantoin with S. oralis subsp. oralis.   Conclusions: This study provides insights on how antibiotic consumption is associated with enrichment and higher abundance of species previously linked with subclinical coronary atherosclerosis in the gut. Hence, this study provides insights on unintended effects of using antibiotics for managing infections, which underscores antibiotic use as not only a concern for development of antibiotic resistance but also for disrupting the gut microbiota, which may contribute to disease development. Knowledge about effect of antibiotics in gut microbiota may help to adequate this therapy according to comorbidities of individual profiles and to design better diagnostic tools for the risk population with the goal of preventing cardiovascular events in the general population.

Abstract  Aggregatibacter aphrophilus, a Gram negative bacterium, found in the oral cavity, causing cerebral abscesses and infective endocarditis, has been shown to be serum resistant in previous studies. Bacterial secretion systems are important for bacteria as they transfer virulence factors into other bacteria or host cells as an attack. A. aphrophilus encodes a type VI secretion system, which is a spike-like membrane protein, mainly consisting of a hemolysin co-regulated protein (Hcp). In this work, it was tested if Hcp would contribute to serum resistance of A. aphrophilus. Firstly, to assess Hcp contribution to serum resistance, a bacterial serum killing assay-method was used and data was collected from three independent experiments. Two strains of A. aphrophilus were used in the experiments: the laboratory strain HK83 and a HK83 hcp mutant strain. The results showed that Hcp provided no significant effect on serum resistance of A. aphrophilus. Secondly, optical density measurements were made for growth curve analysis, to determine if the HK83 hcp mutant strain had an impact in growth compared to HK83. The results indicated that the HK83 hcp mutant strain had a somewhat reduced growth compared to its parental strain.

Evolution in bacteria occurs through the combined effects of spontaneous mutations and horizontal gene transfer (HGT). Several mechanisms can lead to HGT: (i) transformation, the uptake of DNA from the environment; (ii) transduction, the transfer of DNA carried by a bacteriophage into another bacterium during infection; and (iii) conjugation, bacterial mating mediated by a conjugative plasmid like the F-factor. HGT through conjugation can lead to the transfer of resistance and virulence genes, which often reside on conjugative plasmids. Conjugation can occur both within a species or between different species. The F-factor plasmid may sometimes integrate into the chromosome by recombination if there is homology between IS elements on the plasmid and the chromosome. Cells with an integrated F-factor can transfer chromosomal DNA with high efficiency and are called Hfr-cells. There are two clinical pathogens (Klebsiella pneumoniae ST258 and Escherichia coli ST1193) that are highly successful (pathogenicity-wise) and thought to originate through the transfer of chromosomal DNA via conjugation creating unique strains with hybrid chromosomes.  Our question was how frequently the transfer of chromosomal DNA occurs when we use clinical isolates of E. coli with a plasmid and mate them with an E. coli recipient. We hypothesized that any conjugative plasmid might integrate into the chromosome, thus creatingHfr-cells with the potential for transfer of chromosomal DNA to create hybrid strains. Our prediction was that some clinical isolates should be able to transfer chromosomal DNA to another bacterial strain. By plating conjugation mixtures on selective medium where neither donor nor recipient could grow, we were able to isolate 15 possible transconjugants with hybridgenotypes occurring at a frequency of ~10-10.