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.

Cell stress, caused by misfolded or aggregated proteins or exposure to certain environmental stressors, activates stress detectors and selected analysis processes within the cell, resulting intranscriptional regulation and synthesis of factors that influence appropriate folding or removal of misaligned proteins to recover proteostasis. Yeasts are given a sophisticated and interconnected system to restore from the disruption of proteostasis, which involves molecular chaperones and protein degradation pathways as significant participants. Hsp104 is a stress response protein that, through an unknown mechanism, stimulates the reactivation of heatdamaged proteins in yeast. The aim of this thesis study is to learn more about Hsp104 function and location in young and old yeast cells. On the one hand, we aim to see how Hsp104 wildtype vs. mutant forms are distributed in young vs. elderly yeast cells. As a negative control, we employed the mutant variant of this protein. We also wanted to stress Hsp104 wildtype and mutant versions to see whether there are any variations in behaviour or protein levels. All research was carried out in yeast, with biochemical tests and high-throughput technologies like flow cytometry. Hsp104GFP signal were increased in the nucleus of Hsp104 wildtype cells with the increasing of cellular age. As well as, after heatshock treatment the Hsp104GFP aggregation was raised in Hsp104 wildtype and mutant forms. The results data demonstrated that Hsp104 protein levels were increased with cellular age and heatshock treatment enhanced the Hsp104 aggregation in Hsp104 wildtype and mutant forms.

SARS-CoV-1 is a highly contagious virus. The fatality rate is 9.7%. Measures to decrease the transmission include distancing and protective gears. Reviews prior to this one have primarily focused on distancing, while this review is the first review that focuses on facemasks as part of the protective gears to healthcare personnel. The aim of the meta-analysis was to determine whether usage of surgical facemasks mitigates transmission of SARS-CoV-1 among the healthcare personnel. The research question was the following: To what extent does the usage of facemasks mitigate the transmission of SARS-CoV-1 among the healthcare personnel? A meta-analysis was performed through searches in databases. According to the findings, the forest plot indicated that an effect was present in mitigating SARS-CoV-1. The absence of publication bias was inconclusive, as the funnel plot had too few data-points. In conclusion, the usage of facemasks among the healthcare personnel likely does lead to a mitigation of the transmission of SARS-CoV-1. Further research may focus on whether fabric masks have a greater effectiveness in mitigating transmission than a N95 mask while taking into account daily use in a non-healthcare setting or whether protective gear are effective for individuals of a certain age-group / <p>Programmet: Studenten var inte antagen till ett magisterprogram. Läste kursen som fristående. </p>

Sepsis is a life-threatening organ disfunction, which is caused by a dysfunctional immuneresponse and develops when an infection overwhelms the body’s defense mechanism and causesand uncontrolled inflammatory response. Biomarkers have a great impact on helping diagnosisand treatments of sepsis. The biomarkers, like miRNA, are needed for both more accurate andquicker diagnosis of sepsis in patients. The future diagnostics are looking at other types ofbiomarkers, e.g. miRNA, but low amounts of miRNA are present in biofluids and make itchallenging to quantify. A new methodology is needed which is both accurate and does notrequire a lot of fluid. The aim of this project was to identify which kit of two kits and which oftwo volumes of plasma would lead to the highest concentration of miRNA and highest quality ofmiRNA extracted. This was quantified by using two different volumes, 100 μl and 200 μl, andextracting the two volumes with both exoRNeasy Serum/Plasma midi kit (Qiagen) and TotalRNA Purification kit (Norgen). There was no statistical difference between median miRNAconcentrations between the two volumes within the Qiagen kit. However, the mean miRNAconcentration (0.833 ng/μl) obtained from the Norgen kit (100 μl plasma starting volume) wasstatistically higher than the mean miRNA concentration (0.570 ng/μl) obtained from the samekit with 200 μl, p = 0.033. The optimal kit and volume of this study is the Norgen kit with 100 μl.Further studies are needed to verify these results.