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In recent decades, the study of the gut microbiota has gained attention as a crucial element in human health and the onset of diseases. Technological advancements, such as high-throughput sequencing, have enabled a deeper study of intestinal microbiota composition and functions. Among the environmental factors modulating the gut microbial configuration, the impact of nutrients is of particular significance. Nonetheless, research on dietary lipids, particularly omega-3 fatty acids, remains underexplored. This study aimed to contribute to the metagenomic analysis of the 16 subunit of the ribosomal RNA gene in faecal samples. A nutritional intervention was carried out focused on fish consumption rich in omega-3. Successful DNA extraction from faecal samples yielded notable, with defined genomic DNA bands in the gel electrophoresis and an adequate concentration. Polymerase chain reactions, essential for library preparation, revealed distinct products in the gel electrophoresis, confirming the successful amplification of the V3-V4 16 ribosomal RNA region with proper adapter addition for DNA sequencing. Comprehensive quality control assessment of sequenced samples confirmed the effectiveness of the refined laboratory procedures. The steps undertaken in this project were critical for subsequent bioinformatics and biostatistics analyses, providing insights into the impact of omega-3-rich fish consumption on the relative abundance and diversity of studied microbial taxa. Integrating metagenomics analysis with other omics and non-omics disciplines, alongside standardising methods across different laboratories, will pave the way for the future application and democratisation of precision nutrition to enhance overall wellbeing.

Background Thymidine kinase 1 (TK1) plays a pivotal role in DNA synthesis and cellular proliferation. TK1 has been studied as a prognostic marker and as an early indicator of treatment response in human epidermal growth factor 2 (HER2)-negative early and metastatic breast cancer (BC). However, the prognostic and predictive value of serial TK1 activity in HER2-positive BC remains unknown. Methods In the PREDIX HER2 trial, 197 HER2-positive BC patients were randomized to neoadjuvant trastuzumab, pertuzumab, and docetaxel (DPH) or trastuzumab emtansine (T-DM1), followed by surgery and adjuvant epirubicin and cyclophosphamide. Serum samples were prospectively collected from all participants at multiple timepoints: at baseline, after cycle 1, 2, 4, and 6, at end of adjuvant therapy, annually for a total period of 5 years and/or at the time of recurrence. The associations of sTK1 activity with baseline characteristics, pathologic complete response (pCR), event-free survival (EFS), and disease-free survival (DFS) were evaluated. Results No association was detected between baseline sTK1 levels and all the baseline clinicopathologic characteristics. An increase of TK1 activity from baseline to cycle 2 was seen in all cases. sTK1 level at baseline, after 2 and 4 cycles was not associated with pCR status. After a median follow-up of 58 months, 23 patients had EFS events. There was no significant effect between baseline or cycle 2 sTK1 activity and time to event. A non-significant trend was noted among patents with residual disease (non-pCR) and high sTK1 activity at the end of treatment visit, indicating a potentially worse long-term prognosis. Conclusions TK1 activity increased following neoadjuvant therapy for HER2-positive BC but was not associated with patient outcomes or treatment benefit. However, the post-surgery prognostic value in patients that have not attained pCR warrants further investigation. Trial registration ClinicalTrials.gov, NCT02568839. Registered on 6 Oct

Introduction : Pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor beta (TGF-beta), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-beta s are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods : Source of TGF-beta s was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP((R)) technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results : All three TGF-beta isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-beta isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-beta isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion : Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-beta isoforms and in their safe transport from the male to the female reproductive tract., Funding Agencies|Spanish Ministry of Science and Innovation - MCIN/AEI , Madrid, Spain [PID2020-113493RB-I00]; European Union [891382]; FORMAS, Stockholm [2019-00288]

Hepatitis C viral infection is a globally widespread blood-borne disease affecting the liver, causing cirrhosis and hepatocellular carcinoma. This type of liver cancer is mainly caused by chronic Hepatitis C and Hepatitis B. Specific detection with the following correct treatment is crucial to reduce the overall burden of the disease. This work focused on investigating whether the padlock probes and rolling circle amplification can detect Hepatitis C, determining the limit of detection, and if any blood components would inhibit the reactions. All oligonucleotides were tested for ligation functionality in 10% TBE-Ureal gel electrophoresis and used with rolling circle amplification and phi29 polymerase to determine if eye read-out was possible. The lowest concentration of detection was found to be 10 pM. To avoid inhibition in blood plasma, samples were pre-treated at 95 ˚C for five minutes. Eye read-out was possible after amplification, with 30% plasma at the highest and 5% plasma at the lowest in samples. In conclusion, this novel assay using padlock probes, a detection oligonucleotide, and rolling circle amplification holds promise in developing a simplified new detection technique for the diagnostics of Hepatitis C.

The testis is a complex organ responsible for sperm production and hormone synthesis. Spermatogenesis, the process of sperm formation, occurs in the seminiferous tubules and involves the coordinated development of germ cells with the support of Sertoli cells. These provide structural and nutritional support for germ cells, regulate spermatogenesis, and maintain the blood-testis barrier. Despite extensive research on Sertoli cells, their protein expression patterns and spatial localization in relation to other testicular cell types remain poorly characterized. This project aimed to investigate the expression patterns and spatial localization of proteins in Sertoli cells. Single-cell RNA sequencing data and immunohistochemistry stainings from the Human Protein Atlas were used to choose candidate proteins to be visualized in a multiplex Immunofluorescence panel. After staining, the slides were scanned and analyzed manually as well as through automated image analysis via QuPath. 101 proteins were successfully stained and localized within testis tissue and subcellular locations in Sertoli cells were annotated. Additionally, a list of uncharacterized proteins in Sertoli cells were identified. The employed workflow demonstrated its potential for identifying Sertoli cell-specific proteins and can be applied to future research. Additional validation is required to verify the presence and identify function of these proteins in Sertoli cells. For this purpose, techniques such as mass spectrometry-based proteomics and mouse knock-out models could be used.

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., Utbytesstudent

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.

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.

Chronic kidney disease greatly raises cardiovascular disease risk. Heart disease and death risk grow proportionately with renal disease progression. Investigate the link between cardiovascular disease prevalence and chronic renal disease severity and mortality using meta-analysis. In this study, 155 publications were found after searching several databases (including PubMed and Google Scholar). 48 studies that matched the inclusion criteria were included in the literature review, however, only 20 were included in the meta-analysis. 17101 people had CKD, while 8883 had CVD or non-CVD. Using the R programming language, a meta-analysis was performed to get a pooled impact of the influence of CVD on the severity of CKD (odds ratio OR), and a funnel plot was also generated to check for publication bias. The outcomes of the meta-analysis indicate that cardiovascular disease has a moderate impact on the severity of chronic kidney disease (OR=2.28, 95% CI, 1.90-2.73). All data will give essential insights into the epidemiology of the cardiovascular disease in chronic kidney disease (CKD), disclose the influence of individual risk variables on bad outcomes, and serve as the platform for future interventional research. Further investigation of the particular (non-traditional) risk factors associated with the renal illness that contribute to accelerated atherosclerosis in this population is necessary to improve the efficacy of cardiovascular treatments for patients with CKD. The purpose of this research is to determine whether and how these variables affect the development of CKD., Utbytesstudent.

ZC3H11A (zinc finger CCCH domain-containing protein 11A) is a stress-induced mRNA-binding protein required for efficient growth of nuclear-replicating viruses. The cellular functions of ZC3H11A during embryonic development are unknown. Here, we report the generation and phenotypic characterization ofZc3h11a knockout (KO) mice. Heterozygous null Zc3h11a mice were born at the expected frequency without distinguishable phenotypic differences compared with wild-type mice. In contrast, homozygous null Zc3h11a mice were missing, indicating that Zc3h11a is crucial for embryonic viability and survival. Zc3h11a -/- embryos were detected at the expected Mendelian ratios up to late preimplantation stage (E4.5). However, phenotypic characterization at E6.5 revealed degeneration of Zc3h11a( -/-) embryos, indicating developmental defects around the time of implantation. Transcriptomic analyses documented a dysregulation of glycolysis and fatty acid metabolic pathways in Zc3h11a(-/-) embryos at E4.5. Proteomic analysis indicated a tight interaction between ZC3H11A and mRNA-export proteins in embryonic stem cells. CLIP-seq analysis demonstrated that ZC3H11A binds a subset of mRNA transcripts that are critical for metabolic regulation of embryonic cells. Furthermore, embryonic stem cells with an induced deletion of Zc3h11a display an impaired differentiation toward epiblast-like cells and impaired mitochondrial membrane potential. Altogether, the results show that ZC3H11A is participating in export and posttranscriptional regulation of selected mRNA transcripts required to maintain metabolic processes in embryonic cells. While ZC3H11A is essential for the viability of the early mouse embryo, inactivation of Zc3h11a expression in adult tissues using a conditional KO did not lead to obvious phenotypic defects.

Changes in the milk microbiota during the course of mastitis are due to the nature of a sporadic occurring disease difficult to study. In this study we experimentally induced mastitis by infusion of Escherichia coli endotoxins in one udder quarter each of nine healthy lactating dairy cows and assessed the bacteriological dynamics and the milk microbiota at four time points before and eight time points after infusion. As control, saline was infused in one udder quarter each of additionally nine healthy cows that followed the same sampling protocol. The milk microbiota was assessed by sequencing of the 16 S rRNA gene and a range of positive and negative controls were included for methodological evaluation. Two different data filtration models were used to identify and cure data from contaminating taxa. Endotoxin infused quarters responded with transient clinical signs of inflammation and increased SCC while no response was observed in the control cows. In the milk microbiota data no response to inflammation was identified. The data analysis of the milk microbiota was largely hampered by laboratory and reagent contamination. Application of the filtration models caused a marked reduction in data but did not reveal any associations with the inflammatory reaction. Our results indicate that the microbiota in milk from healthy cows is unaffected by inflammation.

Iron plays a crucial role in various essential functions within the human body, participating in processes vital for overall health and well-being. To address iron deficiency, a wide array of iron supplements are commonly employed. However, it is important to recognize that certain types of iron supplements can have adverse effects on the body, including the induction of oxidative stress and inflammation. Therefore, extensive research is imperative to investigate the inflammatory potential of different sources of iron supplements in order to ensure their safety and effectiveness. In the pursuit of evaluating the inflammatory effects of various iron supplements, researchers frequently employ the Caco2 cell model. In this study, the focus was placed on examining the pro-inflammatory potential of different iron supplements by measuring the levels of a specific inflammatory biomarker, the cytokine IL-6, in the Caco2 cells. To mimic the physiological conditions, the supplements were subjected to a simulated gastrointestinal digestion protocol, ensuring that the Caco2 cells were exposed to digested forms of the supplement after which the levels of IL-6 were determined using ELISA. Surprisingly, the results of the study unveiled intriguing findings. Specifically, the two iron supplements derived from bovine sources exhibited no significant effect on IL-6 levels, indicating a lack of pro-inflammatory activity. However, it was the iron supplement derived from Spirulina, a marine-originated source that captured attention. This particular supplement showcased the ability to decrease the levels of IL-6, suggesting a potentially anti-inflammatory effect on intestinal cells., Utbytesstudent

Introduction: Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis. Methods: Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10(-9) M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard. Results: Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays. Discussion: These results demonstrate that th

Sclerotinia sclerotiorum is a plant pathogenic fungus that causes Sclerotinia stem rot in oilseed rape. In Sweden, the disease causes severe crop loss that varies by year. Previous studies have shown a relationship between the proportion of infected petals and disease incidence in infected fields in places with high humidity levels before and during flowering. In this study, the aim was to develop a technique to detect S. sclerotiorum and other fungi pathogens in the petals of oilseed rape from naturally infected fields by using nanopore sequencing from Oxford Nanopore Technologies. DNA was extracted from the petals of oilseed rape and subsequently amplified by performing PCR after optimizing the optimal annealing temperature. Using the forward primer ITS1catta and the reverse primer ITS4ngsUni, these primers targeted the ITS region, which is used as a marker for the identification of fungi. The resulting Amplicon concentrations varied. Five amplicon PCR samples were selected for MinION sequencing. These samples were selected since they had the best purity levels. Finally, bioinformatic analysis was done with Kraken2 and the Pavian tool and compared with UNITE databases. The result showed hundreds of thousands of reads were recovered from the Ascomycota and Basidiomycota fungi divisions; S. sclerotiorum was observed in one field sample; other Sclerotiniaceae species like Dumontinia tuberosa, Botrytis cinerea, and Sclerotinia bulborum were detected in two fields; and many other fungal pathogen species affecting rapeseed crops in Sweden were successfully detected. MinION was successful in identifying S. sclerotiorum and other plant pathogens.

Equine asthma (EA) is a heterogenous, complex disease, with a significant negative impact on horse welfare and performance. EA and human asthma share fundamental similarities, making EA a useful model for studying the disease. One relevant sample type for investigating chronic lung inflammation is bronchoalveolar lavage fluid (BALF), which provides a snapshot of the immune cells present in the alveolar space. To investigate the immune cell landscape of the respiratory tract in horses with mild-to-moderate equine asthma (mEA) and healthy controls, single-cell RNA sequencing was conducted on equine BALF cells. We characterized the major immune cell populations present in equine BALF, as well as subtypes thereof. Interestingly, the most significantly upregulated gene discovered in cases of mEA was FKBP5, a chaperone protein involved in regulating the activity of the glucocorticoid receptor.

Uropathogenic Escherichia coli is a leading cause of urinary tract infections and bacterial prostatitis, the most common UTI complication in men. The initial stages of a successful infection involve bacterial adhesion to host cells through specialized adhesins. FimH, a protein located at the tip of type 1 pili, plays a crucial role as the main mediator for UPEC binding to bladder cells. While the host partners of FimH in the bladder are well-established, the interactions between FimH and prostate cells remain elusive. Consequently, the overarching goal is to enhance comprehension of the initial steps in prostate infection by investigating the interaction of FimH with prostate proteins. To achieve this, a recombinant FimH was constructed and expressed in an inducible expression vector, and an immunofluorescence staining assay was performed which demonstrated distinctive binding patterns in prostate cells compared to the bladder cell line. A Far Western overlay assay, revealed six distinct protein bands in human prostate cells and two in mouse prostate cells, indicating different potential protein partners. These interactions were examined under native conditions by establishing and optimizing a co-immunoprecipitation assay with cell proteins derived from both human and mouse prostates, with the 5637 cell line serving as a positive control. In summary, this study reveals striking differences between FimH binding to prostate and bladder cells, emphasizing the importance of FimH in adhesion and the need for further exploration of FimH interaction with prostate cells.

Antibiotic resistance in microorganisms poses a global hazard to human and animal health. To combat antibiotic resistance, a deeper understanding of the mechanisms behind the spread of antibiotic resistance genes (ARGs) in bacteria is crucial. Plasmids are major vehicles for the spreading of ARGs, so their conjugation-mediated transfer is examined in this thesis. Escherichia coli (E. coli) was isolated from a wastewater treatment plant (WWTP) and clean water in Stockholm, Sweden and the isolates were used as donors of mobile plasmids and a lab strain of E. coli (CV601) was used as recipient. Transfer efficiency was analysed by calculating conjugation frequencies (CF), after testing conjugation by fast conjugation and filter mating method. The resulting transconjugants were analysed for antibiotic resistance pattern by the disc diffusion method. Additionally, sequence analysis was used to analyse the transferred plasmid(s) and ARGs. The qPCR was performed to check the expression of transfer genes (TraN and TraJ) during conjugation of IncN plasmids. The results showed that several isolates from WWTP were multi-resistant and successfully transferred plasmids to CV601 and donor strain 18 with a mobile IncN plasmid had the highest CF that depended mainly on the type of pili. The ARGs qnrS1, dfra14, and blaCTX-M-15 were transferred to transconjugants mainly by IncN plasmids and qPCR findings indicated that the level of gene expression of transfer genes in donor strains affected CF. The findings of this study expand our understanding of antibiotic resistance dissemination and knowledge of plasmids found in WWTPs and clean water.

Background: Milk vendors play an important role in India's dairy value chain; however, their food safety practices are poorly understood. From the standpoint of milk safety, vendor behavior is significant because it has the potential to affect both consumer and producer behavior. This study investigates vendors' hygienic knowledge and practices toward milk safety, and antimicrobial resistance (AMR) Methods: A cross-sectional study was conducted in the states of Assam and Haryana (India). In selected villages, all the milk vendors identified at the time of the visit were interviewed. A questionnaire was used to assess the knowledge and practices on antibiotics, milk safety, and hygiene. The milk samples were tested for the presence of antibiotic-resistant bacteria using antibiotic susceptibility testing. Results: A total of 2444 milk vendors were interviewed during the survey. Out of these 156 (59.8%) of the vendors traded in raw milk while 40.2% traded in pasteurizes milk. Vendors were categorized depending on whom they supplied milk. Five categories were identified: (a) those who sold at grocery shops; (b) those who sold on the roadside; (c) those who sold from door to door; (d) those who sold to sweet makers/tea stalls; (e) those who sold from own home/other entity. The level of training among vendors on milk hygiene was non-existent and the knowledge related to antibiotics was low. Most of them (86.07%) agreed that boiled milk is always safer than raw milk but almost half (48.77%) of them admitted that sometimes they drink milk without boiling it. Most vendors believed that they could identify whether milk is safe or not for consumption just by its appearance and smell. Out of 124 milk samples collected from surveyed milk vendors and tested for the presence of antibiotic-resistant bacteria, 80 (64.52%) tested positive. Conclusion: This study highlights the low levels of knowledge regarding food safety among milk vendors. It shows the predominance of informal mil

A majority of SARS-CoV-2 recoverees develop only mild-to-moderate symptoms, while some remain completely asymptomatic. Although viruses, including SARS-CoV-2, may evade host immune responses by epigenetic mechanisms including DNA methylation, little is known about whether these modifications are important in defence against and healthy recovery from COVID-19 in the host. To this end, epigenome-wide DNA methylation patterns from COVID-19 convalescents were compared to uninfected controls from before and after the pandemic. Peripheral blood mononuclear cell (PBMC) DNA was extracted from uninfected controls, COVID-19 convalescents, and symptom-free individuals with SARS-CoV-2-specific T cell-responses, as well as from PBMCs stimulated in vitro with SARS-CoV-2. Subsequently, the Illumina MethylationEPIC 850K array was performed, and statistical/bioinformatic analyses comprised differential DNA methylation, pathway over-representation, and module identification analyses. Differential DNA methylation patterns distinguished COVID-19 convalescents from uninfected controls, with similar results in an experimental SARS-CoV-2 infection model. A SARS-CoV-2-induced module was identified in vivo, comprising 66 genes of which six (TP53, INS, HSPA4, SP1, ESR1, and FAS) were present in corresponding in vitro analyses. Over-representation analyses revealed involvement in Wnt, muscarinic acetylcholine receptor signalling, and gonadotropin-releasing hormone receptor pathways. Furthermore, numerous differentially methylated and network genes from both settings interacted with the SARS-CoV-2 interactome. Altered DNA methylation patterns of COVID-19 convalescents suggest recovery from mild-to-moderate SARS-CoV-2 infection leaves longstanding epigenetic traces. Both in vitro and in vivo exposure caused epigenetic modulation of pathways thataffect odour perception. Future studies should determine whether this reflects host-induced protective antiviral defense or targeted viral hijacking to, Funding Agencies: Swedish Heart and Lung Foundation [20200319, 20200067, 20210067]; Swedish Research Council [Covid-19/biobank 210202]; Open Medicine foundation [OMF190626]; Hjärt-Lungfonden; Open Medicine Foundation; Vetenskapsrådet

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).

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.

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.

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<0.001). Further research is needed to investigate optimum conditions for inhibition.

Sepsis is an irregular systemic response to an infection, in which a pathogen or some of its component(s)reaches the bloodstream of the host or sterile tissue, triggering a disproportionate immune reaction. The first three hours are critical in the diagnosis of sepsis, in order to ensure an effective treatment with less impact on the patient. Culture-dependent diagnosis is the present standard procedure which can take up to several days. Metagenomics Next Generation Sequencing (mNGS) is a culture independent diagnostics method which could be used to identify the presence of pathogens from DNA extracted from human whole blood enabling a more effective treatment procedure of infected patients. The aim of this research was to utilize the sequencing data obtained with the MinION Nanopore sequencing device, in order to systematize its use as a tool for the early detection of sepsis; furthermore, determine if this technology is effective to use on DNA extracted from whole blood. The main research question of this thesis focused on whether the MinION Nanopore sequencing is a reliable tool for the early detection of sepsis. Whole blood samples from healthy donors was spiked with bacteria and DNA was extracted and sequenced with MinION device. The sequencing results were interpreted with the MinKNOW v2.0 software, through the application What’s In My Pot (WIMP). Also, the web tool PATRIC 3.6.12. and KRAKEN2 algorithm. The reads from the taxonomic family where the bacteria belong to was analyzed, presuming the bacterial DNA was present in the DNA extracted but the genus was not detected. According to the KRAKEN2 and WIMP analysis, the bacteria used to spike the whole blood samples was detected up to the taxonomic family level. Thus, confirming the presence of the spiked bacteria in the purified DNA samples.

Den tilltagande höga nivån av urbanisering och vägbyggen är en bidragande faktor till spridning av invasiva arter. Transporter och frekventa vegetationsstörningar resulterar i homogena, öppna och soliga landskap med minskad konkurrens där snabbt växande ruderala arter med hög ihållande fröproduktion etablerar sig, varav många är invasiva. Syftet med projektet är att undersöka spridningsfaktorer av invasiva växtarter inom c-verksamheter som återvunnit jordmassor mellan 2020–2021 inom Göteborg stad, Kungälv- och Härryda kommun. Dyker invasiva arter upp i samband med flytt av jordmassor? Utgör verksamhetsstrukturer med sina öppna ytor en spridningskorridor för redan befintliga arter i området? Vilken av verksamheterna bedöms utgöra störst risk för spridning? Invasiva arter (SE) inventerades inom sju kartlagda C-verksamheter (bullervallar, markhöjning på jordbruksmark, banarbete på motocrossbana och golfbana) med en buffert på 250 meter. Artförekomst noterades med värdet 1 för närvaro och värdet 0 för frånvaro. Små plantor av invasiva arter kunde även indikera på etablering efter masshantering. I QGIS 3.24 beräknades avståndet mellan förklaringsvariablerna vägar, stigar, järnvägar, vattenförekomst (NMD) och övriga marktäckedata (NMD). Alla bearbetade data exporterades till Rstudio för att utveckla en logistisk regressionsmodell (GLM) i syfte att förutspå invasiv artförekomst utifrån förklaringsvariablerna. Detta var nödvändigt då en logistisk GLMM inte visade någon skillnad mellan verksamheterna. Resultatet från regressionsanalysen (GLM) visade att det fanns ett samband mellan artförekomst och variablerna Vägar, Stigar och hantering av jordmassor. Detta beror delvis på att vägar och stigar öppnar upp för mänskliga aktiviteter som gynnar fröspridning. Verksamheter som bullervallar och motocrossbanor är starkt anknutna till vägstrukturer och mänskliga aktiviteter. Framtida studier bör inkludera förklaringsvariabler som befolkningstäthet inom verksamhetsområdet, nettoprimä, The project aimed to investigate dispersal factors of invasive plant species within C-operations, which recycled soil masses between 2020–2021 in the municipalities of Gothenburg, Kungälv and Härryda. Do invasive species appear in connection with the movement of soil masses? Do operationstructures constitute a dispersal corridor for already existing species in the area? Which of the activities is considered to pose the greatest risk of spread? A field inventory of invasive plant species (SE) were conducted within seven mapped C-operations (noise barriers, land elevation on agricultural land, course work (motocross course and golf course) with a buffer of 250 meters. Species occurrence was noted along a transect with the value 1 for presence and the value 0 for absence. Newly sprouted plants could also indicate establishment after mass handling of soil. In QGIS the proximity between the explanatory variables roads, paths, railways, water body (NMD) and other ground cover data (NMD) was calculated in order to develop a logistic generalized linear model (GLM) to predict invasive species occurrence based on the explanatory variables. The results from the regression analysis (GLM) showed that there was a connection between species occurrence and roads, paths, noice barriers and course work that handled soil masses. This is partly because roads and paths open up for human activities that promote seed dispersal. Activities such as noise barriers and motocross tracks are strongly linked to road structures and human activities. A GLMM- analysis did not show any difference between the operations. Therefore, in future studies a higher number of relevant operations of several types can be implemented, including landfills, sand and gravel pits. Also, explanatory variables such as population density in the area, net primary production, slopes, soil type and its seed content, as these variables have shown statistically significant predictions of species occurrence.

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.

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.

Trimethylaminuria is an autosomal recessive disorder characterized by a decreased oxidation capacity of trimethylamine to trimethylamine-N-oxide in the liver. The condition is diagnosed by estimating concentrations of trimethylamine and trimethylamine-N-oxide in human urine and then evaluating their respective creatinine ratios and the oxidation efficiency percentage. Values previously retrieved with liquid chromatography-tandem mass spectrometry with electrospray ionization but not the novel ionization interface UniSpray. Thus, this project aims to initiate the development of a liquid chromatography-tandem mass spectrometry diagnostic method for trimethylaminuria with UniSpray ionization. The analytes were extracted from urine with a liquid-liquid extraction method and separated with hydrophilic interaction liquid chromatography using an isocratic profile with 5 mM of ammonium formate in water and methanol and multiple reaction monitoring. Overall, the mean coefficient of variation and recovery percentage from trimethylamine spiked urine samples were lower than expected, whereas the intra-precision for trimethylamine-N-oxide was acceptable. Three urine samples had estimated oxidation percentages, but only one had derived comparable creatinine ratios to an external laboratory. Due to the inadequacy of comparative data and the precision and recovery percentage deviations, the results presented in this report need cautious interpretation. Future development of the method could include manual tuning, reconsidering the calibration curve and reference values, and comparisons to the extraction method. Although there are apparent discrepancies in the precision and reliability of the derived trimethylamine and trimethylamineN-oxide concentrations, the initial steps in the pursuit of a liquid chromatography-tandem mass spectrometry diagnostic method for trimethylaminuria provide a practical foundation to continue the development.

Neuroblastomas are the most common extra-cranial abnormal growths in immature nerve tissue within the sympathetic nervous system, in children ages 0-5. It is a heterogeneous malignancy, often emerging in and around adrenal glands, caused by rapid growth and division of nerve cells. From a clinical perspective it is difficult to treat as it has spontaneous regression, rapid progression and therapy resistance. MicroRNAs (miRNA) are small, single stranded RNA, 18-25 nucleotides long and act as human gene expression regulators which are deregulated in Neuroblastoma. The expression of miRNAs can be analysed to determine the progressive stage at which the neuroblastoma is, and the best course of treatment can be decided. The aim of this study was to analyse the expression of miRNA hsa-miR-34c-5p, in three different cell lines each having different genetic characteristics, NB69 without MYCN amplification and 11 q deletion, SN-BE with MYCN amplification and Kelly with 11q deletion. As hsa-miR-34c-5p has been found to have tumour suppressive capabilities in Neuroblastoma cell lines. The cell lines were cultured, the total RNA was isolated, Reverse Transcription was done to convert small RNA to cDNA and qPCR was run and Cq values were obtained. The miRNA expression was determined using qPCR and Livak´s method, however, no statistical difference was found in the expression of hsa-miR-34c-5p between the Neuroblastoma cell lines. These results are only a steppingstone in future research of miRNA and Neuroblastoma.

The corona virus disease of 2019 (Covid-19) is a deadly viral infection rampaging the world since 2019. Health practitioners have identified co-morbidities as one of the factors contributing to the severity of the disease among patients, with diabetes being one of the leading co-morbid. A systematic search was performed on PubMed, Google Scholar and Science hub databases to obtain articles that have addressed the link between diabetes and severity in Covid-19 infection. A meta-analysis to obtain a pooled effect of the effect of diabetes on the severity of Covid-19 (odd ratio OR) was calculated using R programming language and a funnel plot to check for publication bias was also plotted. Twelve studies with 3,180,125 diabetic patients with confirmed cases of Covid-19 (out of 61,820,553 confirmed Covid-19 participants) were included for the meta-analysis. The obtained pooled effect of diabetes on the severity of Covid-19 infection was (OR=1.47; 95% CI 1.33-1.63). From the meta-analysis results; Age, diabetic complications and drugs for the treatment of diabetes were identified as possible co-factors to the diabetic effect on Covid-19 infection, as diabetes was seen to be significantly related to its severity but not mortality. It is therefore very important for diabetic patients to adhere strictly to every laid down regulation regarding Covid-19. More clinical research on alternative diabetic therapy is needed as this will reduce the negative effect of insulin usage.

Corona virus disease caused by severe acute respiratory virus 2 causes blockage of the blood vessel which leads to thrombosis. Thrombotic events in covid-19 patients results to hospitalizations and death. And incidence of thrombosis in covid-19 patients have been increasing in most regions of the world. Due to the huge inequalities between developed and developing countries, incidence rates remain highest in more developed regions, but mortality is relatively much higher in less developed countries due to a lack of early detection and access to treatment facilities. The aim of this study was to investigate the incidence of thrombosis in covid-19 by performing a systematic review on research articles talking about thrombotic events in covid-19 patients, carrying out a meta-analysis on the generated data to make an inference on the statistical result in order to create an information concerning complications covid-19 patients suffer from. Literatures with cases of covid-19 that reported D-dimer elevation and that followed the WHO standard for covid-19 diagnosis were included. Literatures excluded were studies with pregnant women, cancer patients and patients undergoing chemotherapy and with no approved ethical considerations. Information sources included only original literatures with initial search yielding 55 results from 4 databases. After reviewing titles and abstracts of all 55 literatures, 35 studies were further screened and 10 were included in the analysis representing 3359 patients. A forest plot using the R programming language was done, and an overall pooled estimate using the random effect model was 20 % (95 % confidence interval 12.0 % - 29.0 %) with heterogeneity of 96 %, and p <0.01. The incidence of thrombosis among moderate cases of c ovid-19 patients was 12 % with (95 % confidence interval 8.0 % - 18.0 %) with heterogeneity 93 %, and the incidence of thrombosis among severe Covid-19 patients was 22 % (95% confidence 10.0 %-37.0 %) with 97 % hetero

Inflammation is a part of the innate immune system. It protects the body against foreign invaders such as bacteria and viruses. Inflammation helps to restore the body by removing harmful stimuli and starting the healing process. Inflammation is produced in response to damage-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPS). Glycogen synthase kinase 3 (GSK-3) is a key regulator of a variety of pathways, making it a promising therapeutic target. Therefore, this experiment aims to see how inhibiting GSK-3 affects the generation of IL-8 in THP-1 ASC GFP macrophage cells. For this study qPCR was used to measure IL-8 expression, while ELISA was used for protein secretion. An ANOVA test was utilized for the statistical analysis. Obtained results from this study showed that there is a significant difference between stimulated cells with LPS and nigericin against unstimulated samples both in protein and mRNA levels. When it comes to the stimulated cells against inhibited cells, the ANOVA test showed there is no significant difference between the samples both in protein and mRNA levels. This might suggest that GSK-3 does not influence the development of inflammasomes in THP-1 macrophage cells. Another possible reason is that other pathways such as the MAPK and JAK-STAT may mask potential inhibitory effects on the NLRP3 inflammasome pathway by producing even more IL-8, which interfered with qPCR and ELISA results. In conclusion, additional research is needed to confirm the involvement of GSK-3 in NLRP3 inflammation.

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., Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.

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., Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.

When exposed to pathogenic stress, cellular processes and survival are dependent on cytoskeletal proteins for structure and organisation of the cell to adapt and maintain homeostasis during inflammation. Vimentin is type III cytoskeletal protein, with an extensive cytoplasmic meshwork, across the cell and regulate the cell structure and cellular space and expressed strongly under tumorigenic events. GSK-3, a regulatory component of inflammation expressed in abundance of cell together with reactive oxygen species (ROS), a group of key complex signalling molecules that are oxygen metabolites which are partially reduced, with robust oxidising abilities, are believed to influence inflammasome formation and specifically vimentin expression upon inflammation. This project investigated the potential modulation vimentin mRNA expression utilising the two signal NLRP3 inflammasome activation theory, by inhibiting GSK-3 and ROS in signal I and or signal II in LPS and nigericin stimulated THP-1 cells, compared to non-inhibited LPS and nigericin THP-1 cells. Inhibition of GSK-3 in signal II downregulated vimentin expression, reflecting repressed phosphorylation of GSK-3 hence also the components required for vimentin; whilst upregulation of vimentin in signal I, reflects possible alternative pathways phosphorylating vimentin components. Overall upregulation of vimentin upon inhibiting ROS in both signal I and II, further proved that inflammasome activation is independent of ROS in the priming step. More research is required integrating vimentin activity and either GSK-3 or ROS, as the potential of these prominent inflammatory markers and their major regulatory presence across an abundance of cell may contribute to the future of drug development for inflammatory diseases.

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.

Via the membrane trafficking system, proteins and macromolecules dispense in different pathways into cells. Moreover, it transports proteins inside and outside of cells. AP3 and LAMP1 are crucial in the biogenesis of lysosomes and sorting the cargo proteins in the trafficking membrane. The result of immunoprecipitation and mass spectrometry proteomics that Paul Manna carried out showed that the RABL6 protein has a relation to AP3. RABL6 has been known as a proto-oncogene previously, and this is the first time that RABL6 may have a function in trafficking membrane. This study aimed to find the association of RABL6 with AP3 and the function of RABL6 via the distribution of LAMP1. HeLa cell line was transfected by a plasmid containing RABL6/GFP. The cells transfected by RABL6/GFP plasmids and wild-type cells were fixed and prepared for immunostaining. After immunostaining, confocal microscopy was used to show the interaction of AP3 and RABL6 and the distribution of LAMP1. In addition, HeLa cells were knockout for RABL6 to show the function of AP3 in RABL6 knockout cells. Optimizing immunostaining, the ratio of 1:100 for RABL6 antibody with PFA fixing shows the best result. The colocalization of RABL6 and AP3 was calculated in cells transfected by RABL6/GFP plasmid and wild-type cells and interpreted the association to each other. The knockout RABL6 cells were unsuccessful, although some differences were observed in the size of the cells. The distribution of LAMP1 in wild-type cells and RABL6 cells transfected by RABL6/GFP plasmid displayed statistical differences. In overexpressed cells, LAMP1 showed more intensity. In conclusion, the RABL6 is involved in trafficking membrane with AP3 and LAMP1.

A current trend utilizing the biomedical approach in the field of wound care is focused on the increased potential to develop wound healing materials designed to address specific types of wounds or underlying pathologies to achieve improved healing. The work presented in this thesis evaluates the blood response to wood-derived nanocellulose functionalized with a peptide, with the ultimate aim of characterizing the material as a potential wound dressing for chronic wound care. The material was evaluated based on the response toward the innate immune system. These interactions between the material and blood were studied using an in vitro whole blood loop model, and then, the coagulation and complement system activation markers were quantified using enzyme-linked immunosorbent assays. The platelet count and the levels of the thrombin-antithrombin complex reported for the material showed no activation of the coagulation cascade whereas there was an activation caused in the complement system showing higher levels of C3a and s-C5b9 components as compared to the controls. The observations obtained from this interdisciplinary project can be considered as a stepping stone toward the need for further analysis of the material in advanced wound care applications. This can be achieved by targeting the specific phases of the wound healing process in order to promote effective wound management.