Each hour´s delay in administering antibiotics has been shown to result in a 9% increase in the odds of mortality in sepsis cases. It is thus evident that the development of a diagnostic method that ensures an early time to diagnosis of sepsis is essential. MiRNAs have shown promise with regards to diagnostic capabilities concerning sepsis, with differential expression of circulatory miRNAs seen during various diseased states. MiRNA can be quantified directly from a blood plasma sample, greatly decreasing the time to diagnosis, as the requirement for culturing is eliminated. Quantification of miRNA by means of qPCR has proven rather challenging, due to their short length. A solution might be two-tailed RT-qPCR, a method which utilizes a two-tailed RT primer. The aim of the project was to optimize the extraction and quantification of miRNAs from minimal amounts of human blood plasma samples, as to create a standardized and reproduceable method for measuring biomarker miRNAs within human blood plasma. In this study, a significant difference between manual and semi-automated extraction of miRNA from plasma with regards to A260/A280 ratios (p = 0.00) was observed. It was also found that a correlation exists between A260/A280 ratios and miR-seps6 quantified, using the two-tailed RT-qPCR method. This method has shown to be effective at amplifying circulating miR-seps6 arising from 100 µL of human blood plasma. A linear standard curve, constructed from synthetic miR-seps 6 produced optimal amplification efficiencies, and the melt curve indicated a single product, which correlates with good specificity. As successful detection and amplification of miR-seps 6 had been achieved during this study, the next phase of the project can be initiated, where it will be attempted to detect miR-seps 6 from plasma stored in a human biological material bank (biobank).

Methionine is a proteinogenic amino acid critical amino for the pharmaceutical, biotechnological and agricultural industries. The overall methionine biosynthetic pathway is as follows: 1. Acetylation of homoserine and its subsequent activation 2. Formation of homoserine through the replacement hydroxyl group by a thiol group, 3. methyl group transfer to the thiol forming methionine. In bacteria this process can occur in two ways: through trans-sulfurylation or direct sulfurylation. Direct sulfurylation is a more efficient process and has been proven to yield more methionine, however the enzymes catalysing this reaction are not extensively described. MetX and MetY are two of these enzymes. The aim of this experimental work is to improve the methionine production in E. coli bacteria through the genetic and structural manipulation of Met X and Met Y. Hence this laboratory work was divided into three major components: screening system calibration, enzymatic kinetic assays and mutagenesis. Screening system calibration aimed to geneticall modify E.coli in order to later test the most efficient metionine production strain in M9 minimal media. Enzymatic assays were performed to quantitavely characterise methionine binding to either Acetyl-CoA or Homoserine through a DTNB reaction at 420nm. Lastly, mutagenesis was perfomed through an array of computational biology techniques such as Pymol, AlphaFold2 and PROSS to produce a table of mutations that in the future will be implemented using error-prone PCR.

Pseudokirchneriella subcapitata is a sickle-shaped freshwater green microalga that is normally found in unicellular form. It is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. Metronidazole (MTZ) is a routinely used nitroimidazole antibiotic that has caused environmental issues owing to incorrect use. A toxicity test was performed in order to understand the relationship between the MTZ concentrations and response at a physiological level. The study found a growth percentage of (0, 4.8571, 4.5714, -15.1429, -37.1429 %) accordingly. The changes on the transcriptomic level were tested by performing a RT-qPCR. Using ∆∆Ct method to compare the treated samples with low and high MTZ concentration against the control sample. The study found that Exposure to MTZ at the low and high concentrations gave rise to 1.45 fold upregulated pcna gene expression that was differentially expressed in control R. subcapitata. The high group of samples in the high group were clearly distinguishable from those in the control and low treatment groups.

The regulatory process of inflammation is very important for the well-being of an organism. Disruption of this process can lead to very dire consequences. The innate immune system is responsible in activating the inflammatory response. As a result of an inflammatory response, inflammasome complexes such as NLRP3 can activate. The activation of such a complex can lead to cytokine upregulation and even cellular death via pyrapoptosis. The NLRP3 inflammasome can be primed by LPS stimulation. The NFkB protein family functions as transcription factors that can increase the expression levels of genes involved in inflammation, immunology and cell survival, including NLRP3. The aim of this thesis project was to evaluate the expression levels of the NFkB family members RELA and NFkB2 after LPS priming. In order to accomplish this, stable reference genes are necessary in order to perform data normalization. In order to find stable reference genes, a panel of commonly used reference genes were amplified using RT-qPCR. Once stable reference genes have been identified and selected, the expression levels of the genes of interest were analysed using the statistical software SPSS. This was performed in order to determine whether or not LPS priming upregulates the expression of the genes of interest. The analysis of NFkB2 and RELA expression levels after LPS priming indicates that there is a significant difference between the unprimed and the primed samples, but the conclusions that can be drawn are limited as protein interactions were not observed and the scope of the research was relatively narrow, encompassing only two out of the family of five NFkB proteins.

Infectious diseases caused by human papillomavirus (HPV) are among the most common sexually transmitted diseases in the world. Currently, all countries of the WHO Eastern Mediterranean Region (EMRO) except the United Arab Emirates and Libya do not have a national vaccination program including the HPV vaccine. Cervical cancer risk can be reduced through the use of prophylactic HPV vaccines. Hence, the aim of this study was to examine the severity of HPV-16/18 infection in cervical cancer through a systematic review and to evaluate the effectiveness of vaccines against HPV-16/18 variants to prevent cervical cancer via a meta-analysis. Both the systematic review and meta-analysis contain nine relevant studies with 66154 and 78308 cervical cancer participants respectively. Statistical analyses were performed using pooled odds ratios (OR) with 95% confidence intervals (95% CI). Publication bias was examined using the funnel plot graph. The findings stated that overall 70% of cervical cancer was attributed to either HPV 16 or HPV 18. Heterogeneity for this meta-analysis was found to be I2= 80% with a p-value<0.01 and overall OR (odds ratio) was 0.09 (95% CI= 0.04-0.20) for the random effect model. The lower odds ratio (less than 1) indicated fewer occurrences of cervical cancer in the HPV 16/18 vaccinated group than in the unvaccinated individuals. The overall vaccination efficiency was found to be 91% from the odds ratio ((1-0.09)x100=91). Thus, the present findings support that a prophylactic vaccine against HPV16/18 prevents the severity of HPV-associated cervical cancer.

Sepsis is a potentially fatal emergency medical condition that reflects the presence of the body’s systematic inflammation. Around 260 biomarkers have been determined to sepsis. The gold standard of blood culturing has remained the best technique for finding sepsis etiology up to this data. However, some vital drawbacks, such as laboriousness, have encouraged a global attempt to find new techniques. This study aimed to optimize a method from which earlier sepsis diagnosis compared to the gold standard could be resulted. DNA was extracted from both spiked and non-spiked whole blood samples. After quality control of the DNA elutions, library preparation and nanopore sequencing using the MinION device were carried out. Basecalling and demultiplexing were done using Guppy GPU and barcoded FASTQ-files were analyzed using What’s-In-My-Pot and Kraken2 taxonomy classification programs. Three different DNA extraction methods were compared from which the second and third methods opted as the optimized methods. Although spiked species were not found in the used taxonomy classification databases, their respective families were spotted. Kraken2 program indicated a relationship between the read percentage of the families and the spiking level of initial blood samples. On the other hand, What’s-In-My-Pot did not show such a trend and only the highest spiking concentration had indicated the families within the reads. A possible justification for not finding the species within the reads is the patchiness of the two databases. Despite the failure in determining the species within FASTQ-files, the whole experiment has gathered valuable experiences for future studies. / <p>Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.</p><p>There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.</p>

Sepsis is an often fatal, critical health condition that arises when the body’s reaction to an infection systemically damages its own tissues and organs. The reality of sepsis is that it can kill within four to twelve hours from the incidence of symptoms. As of 2017, sepsis accounts for one in five deaths worldwide. The current golden standard for diagnosis is blood culturing, which requires 72 hours to cultivate and identify the pathogen and may yield no results due to low microbial concentration in the blood stream. The future diagnostics of sepsis project aimed to reduce the time from admission to sepsis diagnosis by investigating new methods of microbial DNA extraction from spiked whole human blood. The extracted DNA was sequenced through the MinION device, a new technology developed by Oxford Nanopore Technologies. A metataxonomic analysis of the results was then conducted to identify microbes to a species accurate level. The newly investigated methods were successful in isolating microbial DNA from spiked whole human blood. The metataxonomic identification analysis was conducted through the EPI2ME platform using the Fastq What’s In My Pot directory. Although the barcoding system did not work as intended, the analysis resulted in two fully developed taxonomic trees in which the most identified taxa belonged to the Staphylococcus family. This study is a first that moves forward, towards a new day. A day in which hopefully we are able to provide early and precise care, and see more and more patients fully recover. / <p>Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.</p><p>There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.</p>

Pharmaceuticals and their metabolites are present in the environment, with levels expected to rise in the future. The effects on off-target organisms are still mostly unknown and of increasing concern, as are combination cocktail effects. Microalgae are of importance to ecosystems due to being primary producers and responsible for approximately half the global photosynthetic activity. Additionally, in some algal species a detoxification ability has been uncovered, linked to the enzymatic metabolism of cytochrome P450. However, the molecular mechanisms involved in the xenobiotic metabolism of algae are poorly studied, especially regarding cocktail effects. The aim of this study was to investigate the cocktail effect on growth inhibition and gene expression in Raphidocelis subcapitata, from environmentally relevant levels of pharmaceuticals. This was achieved by performing a growth inhibition test, according to the OECD 201 guidelines. Four of the most prescribed pharmaceuticals in Sweden, paracetamol, metoprolol, omeprazole and diclofenac were tested as a cocktail and compared to metoprolol exposure. Test concentrations ranged from naturally occurring levels up to that of a tenfold. In addition to optimization of RNA extraction, a transcriptomic analysis was performed on cocktail treated groups to evaluate expression of cytochrome P450. For all concentrations, metoprolol exposure resulted in algal growth inhibition, while cocktail exposure surprisingly promoted growth. Here the lowest and medium concentrations tested yielded a downregulation of the gene, while the highest concentration elicited an upregulation. The prospect of predicting cocktail toxicity is discussed, likewise the possibility of drug-drug interactions in microalgae due to cocktail exposure.

Antibiotics are considered a type of antimicrobial that particularly has an impact on bacteria or fungi in humans and animals. The widespread use of common antibiotics, combined with the fact that the majority of active antibiotics and their metabolites are water-soluble, results in persistent pollution in aquatic environments, as well as a potential threat to ecosystems. Moreover, there are inadequate ecotoxicological data on many antibiotics, such as azithromycin, which has been quantified at elevated levels in the aquatic system. Raphidocelis subcapitata is a globally distributed green alga that is commonly used as a model species for evaluating chemical toxicity due to the availability of a sequenced genome and its rapid growth, which allows assessing chemical effects across many generations. the aim of this project is to provide an insight on genotoxicity for R. subcapitata and study the effects of azithromycin antibiotic on algae, on both growth rate and molecular levels by determining gene expression levels, specifically, its effect related to chlorophyll pigments,biosynthesis, and DNA replication levels. In order to do that, toxicity test according to OECD guidelines for 7 days, photosynthetic pigment extraction and qRT-PCR were utilized. In the present study, an EC50 of 24 µg/L was obtained, while low risk in the Swedish water streams was indicated, significant induction in Chlorophyll a and b at high concentrations while no effects on carotenoids were observed, no significant difference in pcna and cyt P450 at LOEC and lower concentrations was obtained. This might suggests testing higher concentrations in upcoming research.

Apis mellifera is the most common honeybee species in the world. In recent years, there have been several reports of declines in wild, and domesticated populations. Central to honeybee health are the mutualistic relationships they have with their intestinal microbiome. The Lactobacillus species living in their digestive tract assist with nutrient digestion and pathogen protection. The aim of this study was to investigate which Lactobacillus spp. were present in the intestines of subspecies A. mellifera mellifera, and A. mellifera ligustica, and if they were able to inhibit growth of the pathogen Melissococcus plutonius. The 16S rRNA gene was amplified from gDNA extracted from two complete intestines per sample, in a PCR reaction with barcoded primers. Fragments were then analyzed with nanopore sequencing. In vitro assays of catalase-treated cell-free supernatants from Lactobacillus cultures were set up against living cultures of M. plutonius on KBHI agar plates and liquid broth media, in two experiments. The same seven Lactobacillus species previously found in honeybees were confirmed to be present in the bees of this study. The ratio of species was different between individual samples, which supports earlier findings suggesting the variation is dependent on factors such as individual health, food source, and sampling season. Liquid broth in vitro assay resulted in no inhibition of early growth phase, while the last cell count measure at24 h, recorded statistically significant difference in mean values between A. apinorum and negative control (p&lt;0.001). Further research is needed to investigate optimum conditions for inhibition.