Inflammasomes are multiprotein complexes that play a role in the innate immune system. One inflammasome is the NLRP3 inflammasome, which can be activated and primed by different stimuli that bind to pattern recognition receptors (PRRs). There are many theories of how the NLRP3 inflammasome can be regulated, one of which is deubiquitination by deubiquitinating enzymes (DUBs). The NLRP3 inflammasome is also involved in many diseases, for example, diabetes, cancer and neurodegenerative diseases. The main aim of this study is to increase knowledge of the protein-protein interactions with and within the NLRP3 inflammasome. Thus, this study will give further insight into NLRP3 inflammasome pathways and can lead to novel treatment targets for different NLRP3-associated diseases in the future. The NLRP3 inflammasome and its regulation were described in this study and protein-protein interaction (PPI) networks of the individual NLRP3 inflammasome key components (NLRP3, PYCARD, caspase-1) were obtained from STRING for human, mouse and macaque orthologs. The obtained PPI networks were then compared. The types of PPI in all PPI networks, either functional or physical, were verified by KEGG or research literature, respectively. Mass spectrometry data of unstimulated and stimulated THP-1 cells were also analyzed. During this study the BRISC complex and its members, a DUB, was also further explored. All in all, the study increased the knowledge about the protein-protein interactions with and within the NLRP3 inflammasome. Further research can aid in the discovery of novel treatment targets of diseases related to inflammasomes.

Freshwater mussels are members of phylum Mollusca, which live in freshwater habitats such as lakes and rivers. Freshwater mussels are essential ecologically in the aquatic ecosystems, they have a high capacity for water purification and play a significant role in calcium recycling. The genomic DNA of many freshwater mussels' species has not yet been sequenced. Knowledge of such a sequence can be useful in the development of a multi-biomarker panel to identify water pollution, and it also helps to develop a method to identify freshwater mussels' species according to their genomic DNA. This study aims to use nanopore sequencing technology to sequence the genomic DNA of Anodonta anatina, a species of freshwater mussel common in Europe. The DNA used in this experiment was extracted from the foot tissues, and two tissue homogenization methods were tested in this experiment to determine the best approach. The genomic DNA was sequenced by using Oxford nanopore MinION device, and the reads were assembled and polished using multiple software tools. The reads obtained from sequencing the DNA cover 3.5x of the estimated genome size of Anodonta anatina. 20x coverage is required for a complete genome assembly, and due to the low coverage, only a partial sequence of the genomic DNA was obtained during this experiment. This indicates that nanopore sequencing could be used to sequence the genome of freshwater mussels, but further sequencing runs are required to get enough coverage to assemble the whole genomic DNA.

Inflammation is the body’s innate immune system responding to harmful stimuli. The body reacts to this stimuli by forming and activating inflammasomes. Inflammasome protein activation plays major roles seen in metabolic and autoimmune disorders, showing the importance of comprehending the processes involved. The NLR protein family is involved in regulating innate immune responses. These types of proteins can sense pathogen-associated molecular patterns as well as damage-associated molecular patterns. Several miRNA families have been known to be regulators of the NLRP3 inflammasome, indicating that an improved understanding of how miRNAs work together to balance the inflammatory response is an area to be focused on. As well as increasing understanding of the miRNA networks and how those can be used to optimize the response of the inflammasome. The aim of this study was to determine the expression levels of the miR-34 family in relation to the NLRP3 inflammasome activation. Using THP-1 cells, mirRNA was isolated from cells taken at different time points after stimulation of the cells with LPS and ATP, followed by performing a two-step RT-qPCR. The Livak method with the RNU48 reference gene was used and indicated potential downregulation of the miR-34 family during NLRP3 inflammasome activation. Further studies should be carried out to confirm these findings.

Sepsis, defined as a life-threatening organ dysfunction, is a condition triggered by an adverse immune reaction often leading to a considerable cost in human lives. A fast and early detection is the cornerstone for treating sepsis, however, current therapeutic standard relies on blood culturing, a slow and non-specific indicator. Modern research has heightened an interest in a new set of biomarkers collectively named, microRNA, to fight against sepsis induced mortality. MicroRNAs are highly stable in biofluids and attractive candidates as biomarkers due to being detectable by non-invasive means, however, methods for their detection remains unclear. The study at hand aimed to optimize microRNA extraction from 100 μL initial blood plasma and subsequentially quantify a target microRNA-223 with the newly developed two-tailed RT-qPCR priming technology (TATAA Biocenter AB). Blood plasma was taken from self-assessed healthy donors and microRNA extraction was conducted using the miRNeasy Serum/Plasma advanced kit (QIAGEN) and QIAcube® (QIAGEN). Each extraction was analysed in a Qubit 3.0 (Thermo Fisher Scientific) and DS-11+spectrophotometer (DeNovix). Absolute quantification was used to quantify microRNA, two-tailed RT-qPCR to detect and obtain a Cq-value in a 7300 Real-Time PCR System (Applied Biosystems). Using this system, a standard curve was optimized to achieve a 103% efficiency and correlation coefficient R2=0.99 to secure technical excellence. The two-tailed RT-qPCR platform returned quantifiable microRNA-223 data which allowed for a theoretical profiling of microRNA-223 by absolute quantification. The study demonstrated a promising setting of using two-tailed RT-qPCR to detect and characterize microRNAs extracted from human plasma for future biomarker research.

Sepsis is a very dangerous and life-threatening disease that develops when the body’s reaction to infection causes damage to the body’s tissues and organs. It is difficult to diagnose it and it develops fast leading to a high mortality rate. Current methods rely on blood culturing and multiple biomarkers, such as C-reactive protein and procalcitonin, that take too long to produce results. A possible solution to this problem lies in specific biomarkers such as microRNAs, which are small non-coding single stranded RNA molecules that contain around 22 nucleotides and have a big role in regulating gene expression. Being specific biomarkers for particular disease makes microRNAs promising biomarkers for sepsis. The aim of the project was to optimize the process from extraction to quantification of microRNAs using the miRNeasy Serum/Plasma Advanced Kit-Qiagen Kit (manual) and to see if the Two-tailed RT-qPCR (TATAA Biocenter) technique could quantify the samples. Blood plasma from healthy donors was used for microRNA extractions and was separated into two categories- spiked-in samples and non-spiked samples. Spiked-in samples were spiked with a synthetic microRNA- miR-223 and served as a positive control. All samples were quantified using the absolute quantification and the Two-tailed RT-qPCR method (TATAA Biocenter). Quantification was successful for all samples showing that the method was optimized, parameters for optimization were within the wanted range, and quantifiable. More research is needed, however, the method has potential in becoming a simple and quick novel tool in diagnosing sepsis in the early stages and thus saving lives.

Sepsis is a life-threatening syndrome that occurs due to dysregulated body response to pathogenic infections. More than 30 million cases are recorded annually worldwide, with a high mortality rate of up to 40% of the recorded cases. Early diagnosis of sepsis will help clinicians to start proper therapy as early as possible and save lives. Circulating miRNAs in biofluids were found previously as potential biomarkers that can be used in a multi-marker panel to develop a rapid, friendly user diagnostic kit for the early sepsis diagnosis. This study assessed miRNAs from healthy donors’ human plasma by two extraction methods, manually and robotically via QIAcube. In addition to optimizing two-tailed RT-qPCR (TATAA Biocenter) technology for miRNAs detection and quantification. The extraction of miRNAs was using miRNeasy® Serum/Plasma Advanced kit (Qiagen) for the two methods. Plasma was spiked in with synthetic miRNA-210 to ensure miRNA detection and was used as a positive control for the study. The concentration and the purity of the RNA eluates were measured and statistically analyzed to identify which method could be better in conventional laboratory practice. QIAcube results showed its ability to compete with manual RNA extraction protocols. However, more studies are required for RNA extractions with different kits using QIAcube. The two-tailed RT-qPCR technology successfully detected many miRNAs, but more samples are required to be tested for accurate conclusions. The results emphasize the ability of two-tailed RT-qPCR to detect and quantify miRNAs from human plasma as potential biomarkers in a multi-marker panel for early sepsis diagnosis.

Neuroblastoma (NB) is the most frequent extracranial solid tumour in childhood. The clinical diagnosis of NB is difficult due to the age of the patient and the vague appearance of the symptoms. Moreover, there are two groups of aggressive NBs, one with MYCN amplification and the other with an 11q deletion. Some genes could be a candidate suppressor for NB, e.g., the DLG2 gene that resides within the 11q-deleted region. The DLG2 gene has a large number of exons and multiple isoforms depending on the alternative splicing process. Moreover, these isoforms can include the L27 domain or not. This study aimed to analyse, by applying bioinformatic tools, if isoform 2, which does not have L27 domain, could be a candidate suppressor for this disease. RNA-seq samples from different human cell lines were collected from NCBI and a quality analysis was performed. The filtered samples were run in R and Python programs to do a visualization of the exon expression level and the prediction of Rsubread for exon-exon junctions. The results showed that isoform 2 of DLG2 gene was not expressed in the samples of NB, which is a promising result for being a candidate suppressor of NB. Furthermore, the prediction of exon-exon junctions by Rsubread was confirmed to be very accurate. In conclusion, this study shows that isoform 2 of DLG2 gene could be a candidate tumour suppressor in NB that could, in the future, be used as a target to help to detect earlier the presence of NB and increase the life expectancy of children who suffer from this disease. / <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 a preventable yet life threatening condition, resulting from body response to infection. Time is crucial in sepsis diagnosis since deterioration in patients’ health can occur rapidly. Blood culturing is the gold standard for diagnosis, along with clinical assessment. The discovery of miRNA in biofluids as a biomarker, founded the way for extensive research on its capabilities. MiRNA showed promises in diagnosing, assessing outcome and reporting sepsis progression. Since being delicate to handle while present in biofluid, the need was uttermost to find an effective way for miRNA isolation and detection, to facilitate developing multi-marker panel that help diagnosing sepsis, more efficiently than blood culturing. The current study aimed at using manual and robotic (QIAcube) methods, with MiRNeasy Serum/Plasma Advanced (Qiagen) as kit and protocol, to extract miRNA from human plasma samples. Plasma was either spiked with synthetic miR-223 to act as a positive control, or non-spiked. Once extraction was done, quality-quantity assessment was conducted using Qubit and Nanodrop. Two-tailed RT-qPCR (TATAA Biocenter) was used for miRNA quantification. QIAcube showed better results in quantity, hands-on and turn-around time compared to manual extraction, while better purity was scored for the manual method. While amplification appeared in all spiked samples, absolute quantification detected miRNA in some of the non-spiked samples. The study verified using the extraction kit with 100 μl of plasma is effective for miRNA extraction. Although faced with difficulties, absolute quantification using two-tailed RT-qPCR demonstrates its success in detecting lowly expressed miRNA. Future studies are needed for more optimized verification.

FoxF2 is a transcriptional factor that initiates gene expression. Foxf2 is particularly expressed in central nervous system pericytes. Recent studies have displayed that an inactivation of Foxf2 in mice had an impact on the blood-brain barrier integrity with evidence of breakdown. During angiogenesis, the protein pdgf-B is synthesized by endothelial cells and is detected by pdgfr-𝛽 expressed on pericytes. Fibrinogen is a glycoprotein that participates in coagulation. It is undetectable in a healthy brain due to an intact barrier, making it an important biomarker in neurological diseases. Pericytes are a part of the microvascular unit and are wrapped around endothelial cells, which make up the blood-brain barrier. Pericytes play an important role in bloodvessel formation, development of vessel stability, regulation of blood flow, and the integrity and formation of vascular barrier. The aim of this study was to induce a pericyte specific knockout mutation of Foxf2 and investigate possible fibrinogen extravasation in the brain and pdgfr-𝛽 expression. Using Cre/loxP recombination mouse knockouts were created in vivo by Tamoxifen injections and the . Immunohistochemistry was performed to investigate fibrinogen and pdgfr-𝛽 expression. Pdgfr-𝛽 expression was further validated with RT-qPCR on an in vitro induced cell culture. Immunohistochemistry slides suggested that the knock-out mutation of Foxf2 caused a disrupted blood-brain barrier resulting in extravasated fibrinogen. However, immunohistochemistry images confirmed with RT-qPCR on pdgfr-𝛽 expression displayed no distinct difference. Due to a small sample size, RT-qPCR needs more validation and since immunohistochemistry only allows for interpretation, further study is required.

Parkinson’s disease is one of the leading neurodegenerative diseases that affect elderly people around the globe. In recent years, Parkinson’s disease has been connected to gut microbiome and associations have been made among several novel species of bacteria and the development and severity of Parkinson’s disease. The aim of this study was to identify general characteristics of the gut microbiome among incident Parkinson’s disease cases and controls and by this broaden our current understanding of the topic. Statistical analyses were performed in a FINRISK 2002 population-based cohort of over 7000 people, out of which 105 incident Parkinson’s disease cases were identified and further analysed. 𝛼-diversities among the case/control groups did not differ significantly, but there seemed to be gender-based differences in the 𝛽-dissimilarity matrix between Parkinson’s disease cases and healthy controls. Additionally, a total of ten bacterial species were associated with Parkinson’s disease by the generalized linear model with nominal p-values &lt;0.05, including for example E. hallii, C. comes, A. muciniphila, E. eligens and P. bivia. In conclusion, the gender-based variations in 𝛽-diversities and results from the regression analysis suggests that the gut microbiome may in fact be associated with the development of Parkinson’s disease.