Sepsis is a severe condition caused by a dysregulated host response to an infection that may leadto organ failure and death. Early diagnostic to be able to provide correct treatment is crucial forsurvival. Performing a blood culture is the most common way in the diagnostic of sepsis, a timeconsumingprocess with many false negative results. MicroRNA has been suggested as apotential biomarker for sepsis due to the stability of the microRNA and the possibility todiagnose more complex diseases. Blood was donated from self-assessed healthy individuals andthe plasma was used for extractions of microRNA. Two different isolation kits were used inextractions with a starting plasma volume of 100 μl and 200 μl to determine which of the kitsthat provide the highest amount of miRNA concentration with the highest quality, the kits werethen compared to each other. The two kits used were miRNeasy Serum/Plasma Advanced kit(Qiagen) and Total RNA Purification kit (Norgen). The result from the extractions were analyzedin SPSS and showed a statistically significant difference of microRNA concentration. ThemiRNeasy kit had a higher miRNA concentration in the extractions than the Norgen kit while thequality of the extractions did not show any significant difference between the two kits. ThemiRNeasy kit show a possibility of providing extractions with a high microRNA concentrationusing both a starting plasma volume of 100 μl and 200 μl and could be a possible kit to use infurther extractions in the research of sepsis.

Inflammation is a biological response to harmful stimuli which assists the overall immune response in defending the organism against potential threats like bacteria and other pathogens. This response is largely regulated by multiprotein complexes known as inflammasomes, which use one or more multi-stage networks to create a coordinated response across cell groups. The NLRP3 inflammasome in particular is thought to have many interacting partners in a complex inflammation and cell-death management network. RIPK3 is one protein that has been implicated in regulation of inflammation and cell death pathways in connection with the NLRP3 inflammasome. In this study, we attempt to create a CRISPR/CAS9 construct to knockout the RIPK3 gene in THP-1-ASC-GFP monocytes with ViafectTM Transfection in order to examine the apparent effects at various stages of activation. Transfected cells were then quantitatively examined using qPCR. While the Guide-itTM CRISPR/CAS9 Systems kit produced high quantity, high quality DNA plasmids, this study found that the ViafectTM system resulted in reduced RNA isolation among cells differentiated with PMA as compared to non-differentiated counterparts. qPCR performed on cDNA generated from the RNA extractions resulted in highly erratic Cq values with standard deviations well above the acceptable limitations among technical replicates in both experimental and control gene samples. Additionally, this study found that the reference gene used, ACTB, did not maintain stability across samples. Due to mistakes and time constraints, the experiment failed to provide any substantial evidence of activity; however, an architecture is developed for optimization of future studies.

Innate immunity is the immune systems rapid responses to infection after being attacked by a pathogen. Inflammatory responses are activated by the detection of pathogen-associated molecular patterns and danger-associated molecular patterns through pattern recognition receptors on inflammatory cells. NLRs are activated by intracellular PAMPs which warn cells of damage and have a major role in initiating the innate inflammatory responses as well as the development of infectious and inflammatory diseases. NLRP3 is a very large multiprotein complex and is the most studied inflammasome. The NLRP3 Inflammasome follows a two-signal model for activation, signal one forms the NLRP3 complex and signal two activates the inflammasome. NLRP3 initiates an inflammatory form of cell death called pyroptosis and triggers the release of pro-inflammatory cytokines IL-1β and IL-18. The miR-200 family has five members, miR-200a, miR-200b and miR-429 located on chromosome 1 and miR-200c and miR-141 located on chromosome 12. In this study, THP-1 cells were differentiated with PMA then stimulated with LPS and ATP. Various time samples were collected and isolated to obtain miRNA. Two-step RT-qPCR was then performed to quantitively monitor the changes in miRNA-200 family expression levels. The purpose of this study was to observe how miRNA-200 family expression levels change in stimulated THP-1 cells as the NLRP3 inflammasome is activated. This became a pilot study as all biological replicates could not be analyzed, miR-200 family is showing a potential response to the activation of the NLRP3 inflammasome and they should be investigated further.

Sclerotinia sclerotiorum is a notorious phytopathogenic fungus and is the causal agent of the disease Sclerotinia stem rot (SSR) of rapeseed (Brassica napus). SSR is one of the main diseases affecting the yield and oil quality of rapeseed crops worldwide. This disease is very hard to predict and control due to all the different factors that are involved in the development of the disease. Successful disease management depends on accurate identification and early detection of plant pathogens. qPCR is a fast, specific, reproducible, and reliable technique for plant pathogen diagnostics. However, one limitation of qPCR is that it is unsuitable to identify and study unknown species, other than those intended, making the detection of unknown pathogens very difficult. An alternative solution is to apply single molecule sequencing, which can provide information at species and strain level. In this study, a total sample of 15 rapeseed leaves coming from three different fields in Sweden with known incidence of SSR disease were analyzed using qPCR and other 15 leaves, coming from the same fields, were analyzed using Oxford Nanopore sequencing to attempt to identify pathogens, S. sclerotiorum being the main target. S. sclerotiorum was not identified with none of the previous mentioned techniques in any of the samples. Perhaps, S. sclerotiorum was not present on the samples at the time of the collection, due to the unfavorable weather conditions for the release of the spores. However, some issues were present during the development of the qPCR assays that also could have affected the results. Regarding Oxford Nanopore sequencing, other fungal species were identified instead.

Sepsis is a life-threatening organ dysfunction caused by the spread of infectious diseases throughout the body, resulting in a dysfunctional immune system response. It includes a wide range of symptomsa ffecting multiple organs, making accurate diagnosis challenging. Early detection is crucial for reducing the severity and improving outcomes of sepsis. While bacterial infections are the primary cause, viral and fungal infections can also lead to sepsis and often go underdiagnosed despiteaccounting for a significant number of cases. Current diagnostic tests, like blood cultures, have long turnaround times, hindering early diagnosis. Thus an accurate early diagnostic test is needed for faster and more targeted sepsis treatment. MicroRNAs (miRNAs) have shown promise as biomarkers forsuch a test, and combining multiple miRNAs in a biomarker panel may enhance diagnostic accuracy. This study aimed to compare manual and robotic methods for miRNA extraction from plasma samples to assess the viability of incorporating robotic miRNA extractions into a diagnostic kit. Furthermore, the study aimed to assess the performance of two- tailed RT-qPCR in detecting and quantifying a candidate miRNA biomarker (mirSeps-4) from human plasma samples. The results demonstrate the capability of two-tailed RT-qPCR to detect and quantify the candidate miRNA. Additionally, absolute quantification of qPCR results showed that robotic extractions yielded a significantly greater quantity of mirSeps-4 in unspiked samples.

Sclerotinia sclerotiorum, a fungus with a broad host range, causes plant diseases, while its closely related counterpart, Sclerotinia subarctica, has a more limited host range and prefers colder environments. Sclerotinia stem rot is a disease caused by the fungus, leading to economic losses for farmers. In this study, the aim was to utilize PCR and Sanger sequencing to identify and differentiate various isolates of S. sclerotiorum and S. subarctica obtained from sclerotia collected in eight fields, while also employing nanopore sequencing to examine oilseed rape leaves from three distinct fields for the detection of fungal pathogens. To confirm the identification of S. sclerotiorum or S. subarctica isolates, the ITS regions of ribosomal DNA from the leaves were amplified using the primer pairs ITS1Catta and ITS4ngsUni for targeted amplification, and ITS2AF and ITS2AR for amplification of the rRNA ITS region from the sclerotia. Based on the Sanger sequencing results from sclerotia samples, the study determined that S. sclerotiorum was the identified fungi in all of the samples. Nanopore sequencing was performed on the PCR amplified fungal ITS region from leaves, and the resulting data was analyzed using Kraken2 and UNITE databases. The analysis using Kraken2 and UNITE databases revealed successful identification of fungal sequences, with S. sclerotiorum not detected but other plant-infecting fungi identified. Ascomycota and Saccharomycodes ludwigii were predominant using Kraken2, while Streptophyta and Brassica napus were abundant using UNITE. Molecular-based methods like fungal ITS sequencing are essential for accurate identification of plant-infecting fungi.

Nowadays, sepsis has become a major healthcare problem. Its variance of symptoms and the lack of time to act makes it greatly difficult to treat. An early diagnosis using biomarkers, particularly miRNA, could potentially increase the patient’s prognosis as well as reduce the use of antibiotics for the treatment. The lack of method optimization for miRNA extraction and quantification calls for investigation prior to the construction of a multi-biomarker panel for sepsis diagnosis. The aim of this project was to examine and compare manual and semi-automatic extraction methodologies through the small RNA quantity and RNA quality, as well as test the detection and quantification abilities of the novel technique, two-tailed RT-qPCR. 30 extractions have been performed, their extracted elutions have been subjected to quality and quantity control and detection and absolute quantification through the two-tailed RT-qPCR. The results show no significant differences between the quantity and quality of the RNA extracted using both methods. Time management, on the contrary, reported significant differences between the two methods. On the other hand, the two-tailed RT-qPCR successfully amplified the miRNA candidate from as little as 100 µL of healthy plasma. The absolute quantification showed the miRNA candidate’s low concentration in plasma. Moreover, the qPCR efficiency was irregular during the project which may alert of contamination or unspecific primers. However, the melt curve showed a single amplicon which suggests great specificity. The detection and quantification of the miRNA candidate have been successful, though further investigation is recommended. / <p>Utbytesstudent</p>

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.