Högskolan i Skövde

his.sePublications
Change search
Refine search result
12 1 - 50 of 73
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • apa-cv
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Abdullahi, Fardosa
    University of Skövde, School of Bioscience.
    Investigating possible differential expression level of hsa-miR-708-5p in Neuroblastoma2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Neuroblastoma (NB) is one of the most common extracranial cancers found in children under the age of five. The cause of NB is not well understood, about 2% of the cases have been linked to rare germline mutations in the anaplastic lymphoma kinase (ALK) gene. However, NB is thought to be mainly caused by genetic mutation at the early stages of development. Clinically, NB can be grouped into three risk groups: low, intermediate and high-risk disease. The survival rate of patients with high-risk NB is less than 50% of the diagnosed cases. Survival rates emphasizes the necessity for future NB diagnostic therapy. One potential study area is miRNA, studies have demonstrated both prognostic and predictive usefulness to therapies. MiRNA is a single-stranded RNA that is 18-24 nucleotides long. Its function is to regulate numerous cellular activities, and to act as tumor suppressors or oncogenes. Genetic anomalies such as MYCN amplification and 11q deletion cause NB by disrupting the expression patterns of certain miRNAs. In this experiment the miRNA, hsa-miR-708-5p, was examined in three genetically diverse NB cell lines; NB69 without MYCN amplification and 11q deletion, SKNBE with MYCN amplification, and Kelly with a chromosome 11q deletion, the cell lines were used to see if the expression levels of hsa-miR-708-5p differed. The expression level of hsa-miR-708-5p, was assessed using qPCR; variation in gene expression was identified between the cell lines. Therefore, miR-708-5p could be a viable option when looking at gene expression of hsa-miR-708-5p for future diagnostic or prognostic in NB.

  • 2.
    Abela, Sohunda
    University of Skövde, School of Bioscience.
    Molecular detection of Sclerotinia sclerotiorum from petals of oilseed rape by Nanopore sequencing using MinIon2023Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 3.
    Adindu Uzowuru, Cosmas
    University of Skövde, School of Bioscience.
    Inflammasome: Investigating the effect of NEK7 in the activation of the NLRP3 Inflammasome2020Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Inflammation is a biological defence mechanism applied by living organisms against foreign invaders. In the response to DAMPs and PAMPs, organisms use inflammatory multi-protein complexes to fight the attackers. The most studied inflammasome proteins are NLRP3, ASC and Caspase-1. This study is aimed at understanding the role of NEK7 protein in the NLRP3 inflammasome’s activation, using CRISPR/Cas9 system. To determine the effect of CRISPR/Cas9 and transfection, mRNA expression was analyzed. The results obtained suggest that neither the transfection nor the NEK7 protein knockout have sufficiently worked. This study could not experimentally establish that NEK7 triggers NLRP3 inflammasome activation because ELISA was not conducted to verify the levels of cytokines emitted, due to there being no statistical differences between the samples. Above all, the research question in this thesis project was not answered because the instability of the ACTB reference gene negatively influenced the results. However, previous related studies conclude that NEK7 plays a crucial role in the activation of the NLRP3 inflammasome.

    Download full text (pdf)
    fulltext
  • 4.
    Ajaj, Asil
    University of Skövde, School of Bioscience.
    The ecotoxicity effect of metronidazole on Raphidocelis subcapitata2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 5.
    Alghazali, Raghad
    University of Skövde, School of Bioscience.
    GSK-3 post-transcriptionally regulates TNF-α biosynthesis in THP-1 macrophages2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Few things are more fascinating than finding new interactions between previously unrelated pathways. Glycogen synthase kinase-3 (GSK-3), a ubiquitous kinase initially known for its role in regulating glycogen metabolism, has recently been found to be an indispensable regulator of the TLR4-mediated inflammatory response. GSK-3 inhibition exhibits potent anti-inflammatory effects by acting on both arms of the inflammatory response, reducing the secretion of pro-inflammatory cytokines, and promoting the production of anti-inflammatory cytokines. Tumor Necrosis Factor-α (TNF-α) is among the most important inflammatory cytokines. Aberrant TNF-α expression is associated with various inflammatory conditions, including sepsis and cancer. Thus, understanding the mechanisms regulating TNF-α production could reveal potential therapeutic strategies for TNF-α-associated diseases. Consequently, this study aimed to examine the effect of GSK-3 inhibition on TLR4-induced TNF-a production by THP-1 macrophages. THP-1 macrophages were stimulated with LPS and nigericin in the presence and absence of GSK-3 inhibitor, and TNF-α protein and mRNA levels were evaluated by ELISA and Real-time PCR, respectively. GSK-3 inhibition significantly attenuated TNF-α protein levels in a dose-dependent manner, whereas TNF-α mRNA levels remained unaffected, reflecting a possible post-transcriptional modulation of TNF-α biosynthesis by GSK-3. However, more comprehensive research is needed to elucidate the precise contribution of GSK-3 to TNF-α biosynthesis and to identify novel therapeutic mechanisms to alleviate inflammatory diseases associated with abnormal TNF-α production.

    Download full text (pdf)
    fulltext
  • 6.
    Alsayed Ahmad, Alaa
    University of Skövde, School of Bioscience.
    Azithromycin effects on R. subcapitata on molecular levels: Ecotoxicological study on the effects of a pollutant on chlorophyll contents, pcna and cyt P450 genes expression2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 7.
    Babena, Omar
    University of Skövde, School of Bioscience.
    Expression of the chloride channel CLCC1 is downregulated after 24 hours in LPS-primed THP-1 monocyte-like cell line2021Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Inflammation is the body's response to infection or injury and is mediated by the innate immune system. The NLRP3 inflammasome is a multi-protein complex that is a major contributor to many inflammatory disorders. Emerging evidence suggests the involvement of the Endoplasmic reticulum stress with the NLRP3 inflammasome. The endoplasmic reticulum stress is a series of stress signals that can activate the unfolded protein response and usually accompanies inflammation and eventually causes cell death. Recently, a localized endoplasmic reticulum micro-protein called the chloride clic like-1 channel was found to be involved in the endoplasmic reticulum homeostasis. Recent evidence suggests the involvement of endoplasmic reticulum stress in the inflammation pathways of the NLRP3 inflammasome. The relationship between the ER and the NLRP3 inflammasome has not been clearly described. This study aimed at investigating the expression levels of the microprotein CLCC1 to shed a light on the relationship between the endoplasmic reticulum stress and the NLRP3 inflammasome. The expression levels of CLCC1 were analyzed by qPCR in cultured monocytes under different time points of Lipopolysachaaride immuno-stimulation. The stability of expression in candidate reference genes was investigated for normalization purposes. This study reported the regulation of CLCC1 as a novel finding under prolonged LPS exposure of monocytes and stable reference genes such as GUSB and ACTB were identified. The relationship between CLCC1 and NLRP3 inflammasome priming by LPS indicated that CLCC1 is regulated and may be involved in the inflammatory mechanisms of endoplasmic reticulum stress and NLRP3 inflammasome inflammatory diseases, contributing to a potential therapeutic target in the endoplasmic reticulum and inflammasome related diseases.

    Download full text (pdf)
    fulltext
  • 8.
    Backlund, Kristina
    University of Skövde, School of Bioscience.
    microRNA-200 Family Expression Level Changes in Stimulated THP-1 Cells Following NLRP3 Inflammasome Activation2020Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

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

    Download full text (pdf)
    fulltext
  • 9.
    Bansal, Divya
    University of Skövde, School of Bioscience.
    Adipocytes from SERCA2 knockout mice exhibit a dysregulation in the secretion of adiponectin and resistin2020Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Obesity leading to Type-2-diabetes is a major health issue all over the world. Obesity characterized by expansion of adipose tissue, in particular white adipose tissue (WAT) which controls the metabolic physiology in the body by secreting proteins like adiponectin and resistin. Adiponectin has a protective role against diabetes development whereas resistin causes insulin resistance. Protein folding, maturation and translocation is performed by the Endoplasmic reticulum using calcium ions. The calcium homeostasis is maintained by calcium pumps and channels, chief of this is sarco-/endoplasmic reticulum (SR/ER) Ca2+ ATPase pump (SERCA) which restores calcium back to the ER. To study the effect of SERCA2 on adiponectin and resistin secretion in different adipose tissue depots using an adipocyte specific tamoxifen-inducible SERCA2 Knock-out mice, short term secretion experiments were performed. Chemical inhibition of SERCA2 and ER stress was performed in in-vitro experiments using adipocyte like 3T3-L1 cell line. The experiments revealed that SERCA2 dysfunction led to decrease in adiponectin and resistin secretion in normal and stimulant conditions in both male and female mice. In-vitro experiments revealed that ER stress led to misfolded protein accumulation affecting exocytotic events of adiponectin containing vesicles. Therapeutic agents can be formulated to tackle the SERCA2 dysfunction and to maintain calcium homeostasis by identifying these key mechanisms for diabetes and related metabolic disorders.

    Download full text (pdf)
    fulltext
  • 10.
    Bansal, Vanisha
    University of Skövde, School of Bioscience.
    Blood interactions with bioactive peptidefunctionalized nanocellulose: An evaluation of the activation of the coagulation and complement system2022Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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. 

    Download full text (pdf)
    fulltext
  • 11.
    Belekar, Prajakta Ashok
    University of Skövde, School of Bioscience.
    Quantifying extracellular vesicle secretion from single neuro-endocrine cells to understand how they affect hormonal secretion2020Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Extracellular vesicles (EV’s) are small lipid bilayer vesicles that are generated by almost all kind of cells in a body. EV’s are considered as one of the key intercellular messengers regulating cell signalling mechanisms. Earlier studies have shown that, in metabolic diseases like diabetes and obesity, as well as during hypertension and neurodegenerative disease there is increased production and secretion of EV’s. Secretion mechanism of EV’s is yet unknown. The aim of this study was to investigate the EV production and secretion mechanism in type-2-diabetes and in parallel to EV studies, measurement of SST secretion, to elucidate how it is influenced by EV’s. Tetraspanin was used to label EV’s and the efficiency was evaluated by using TIRFM and considering how many exosomes they label per cell and how well they express. Further, the EV marker were exploited in studying trafficking events of EV’s at the plasma membrane. This included EV approach to PM through docking/visiting, and EV loss from PM through undocking. EV labelling showed that CD63 and CD151 were two efficient markers for live-cell imaging by TIRF microscopy (TIRFM). Trafficking analysis of EV’s showed that number of visiting events were significantly higher compared to docking and undocking events. To know how many of total EV’s in a cell are ready to fuse with plasma membrane, rate of displacement of EV’s was monitored. This showed, small fraction of EV’s were slow-moving, probably docked at the PM while rest EV’s were fast-moving, either visiting or undocking EV’s. Docked EV’s fuse with plasma membrane. SST secretion from δ-cells was studied using pancreatic islets. There are no currently reliable means to measure δ-cells SST secretion. Commercially available antibodies against SST were evaluated compared to antibodies developed in the lab. Efficiency of the antibodies was studied by analyzing number of δ-cells and their distribution in an islet. The results showed that the antibodies against SST that were developed in the lab have a higher efficiency compared to the commercially available antibodies in δ-cells in an islets and tissue. These antibodies were used for staining δ-cells in type-2 diabetic vs healthy islets. Decrease in number of δ-cells in diabetic islets was observed. Therefore, these developed antibodies can be used for future hormone secretion studies.

  • 12.
    Beniamin, Armanos
    University of Skövde, School of Life Sciences.
    Establishment of an Expression and Purification System for Plasmodium falciparum Multi Drug Resistance (pfmdr) Transporter2007Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Malaria is a life threatening parasite disease caused and transmitted by infected female anopheles mosquito. However, the parasite, Plasmodium falciparum, has become resistant to most anti malarial drugs, such as chloroquine, which contributes to fever and anaemia because of its ability to digest the haemoglobin in the red blood cells. The aims of this project were to establish whether “Bac to Bac” Baculoviral Expression System is suitable for expression of pfmdr 1 gene and for purification of the pgh 1 protein. The pfmdr 1 gene encodes an ABC transporter protein, pgh 1, fixed in the cell membrane of the Plasmodium falciparuum gut, which assist in elimination of drug compounds. Furthermore, “Bac to Bac” Baculoviral Expression System uses vectors with histidine tags to clone the pfmdr 1 gene and subsequently transform these into DH10Bac cells to produce the recombinant bacmid DNA. Since pfmdr 1 gene is an AT-rich sequence, PCR was optimized, by lowering the annealing and extension temperature to 47Co and 66Co respectively. The results show that “Bac to Bac” Baculoviral Expression System can be used to express the pfmdr 1 gene, though further experiments has to be performed.

    Download full text (pdf)
    FULLTEXT01
  • 13.
    Callado Prat, Elia
    University of Skövde, School of Bioscience.
    Comparison of manual and semi-automatic RNA extraction methods using two-tailed RT-qPCR for absolute quantification as part of the sepsis diagnosis research2023Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 14.
    Camargo Romera, Paula
    University of Skövde, School of Bioscience.
    Isoform 2 of DLG2 gene as a possible candidate tumour suppressor of neuroblastoma2021Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 15.
    Chrifi, Wail
    University of Skövde, School of Bioscience.
    The effect of temperature on the innate immune response in the lungs against RSV2020Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    A constant flow of various pathogens enters the respiratory system on daily basis through the involuntary mechanism of breathing. Respiratory viral infections are common yet can be fatal in vulnerable populations. Respiratory syncytial virus (RSV) is one of the first and most common viruses that the human population acquire in the first two years of life. Despite the ability of most infants to recover from a RSV infection, many require hospitalization and, in few cases, die from such an infection. The pattern of seasonality of respiratory viruses also applies to RSV. In this work the temperature dependence of infectivity was studied in Hep-2 cells infected with RSV that had been incubated with bronchoalveolar lavage (BAL) fluid. The results indicate a temperature dependence of infectivity. Inhibition of the viral infectivity was observed at three different temperatures 37 ̊C, 40 ̊C and 42 ̊C. The inhibition appears to be linked to the appearance of large agglutinates that appear to reduce the infectivity of RSV. Such a study found that viral neutralization is dependent on a temperature-dependent agglutination reaction. The causality of agglutination formation requires further investigation in order to conclusively confirm the immunological component(s) of this reaction, and how temperature is contributing to this reaction.

    Download full text (pdf)
    fulltext
  • 16.
    de Jong, Anton
    University of Skövde, School of Bioscience.
    Drosophila Melanogaster as a model for studies on fertility associated biomarkers2015Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Many of the processes that regulate male fertility are intricate and subfertility strikes hard against both couples trying to concieve and cattle farmers where the fertility of males used for artificial insemination is the single most important factor viewed in relative economic terms. A recently published study by Fagerlind and collegues showed that seven microRNA sequences differed significantly in expression between bulls with moderate and high fertility.

    In order to study the effect these might have on fertility, a model organism is needed. The present study aim to assess if the fruit fly Drosophila Melanogaster could be used as such. It have served science for over a century, is cheap to grow and has a short generation time. A secondary objective of the present study was to elucidate if any of the observed microRNAs was expressed at a higher concentration at a specific life stage of the fly. Samples from eggs, the three larval stages and adult males and females were collected. Subsequently, after conversion into cDNA with primers for miR-34, miR-1249, miR-148b and miR-15b, the microRNA concentrations were evaluated  with Quantitative Real-Time PCR. Three out of four microRNA sequenses showed expression in the fly and for one of them, miR-34, a marked difference in expression between the developmental stages could be observed, but not confirmed statistically due to the low number of samples. This result enables further studies on these sequences and their role in male fertility.

    Download full text (pdf)
    fulltext
  • 17.
    Diaz Rivera, Alicia
    University of Skövde, School of Bioscience.
    Future diagnosis of sepsis: Evaluating the mNGS approach by using the MinION device2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 18.
    Elawad, Hazzim
    University of Skövde, School of Bioscience.
    Sepsis and circulating miRNA: The road towards absolute quantification of unknown miRNA levels in plasma utilizing two-tailed RT-qPCR, while testing two extraction methods, striving to create multi-marker panel for sepsis diagnosis2021Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [sv]

    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.

    Download full text (pdf)
    fulltext
  • 19.
    Erixon, Rebecka
    University of Skövde, School of Bioscience.
    Cytochrome P450 expression and growth inhibition in R. subcapitata in response to pharmaceutical cocktail exposure: An ecotoxicological study of metoprolol, omeprazole, paracetamol and diclofenac2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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. 

    Download full text (pdf)
    fulltext
  • 20.
    Farrokgi, Moloud
    University of Skövde, School of Bioscience.
    Association of RABL6 withAP3 in trafficking membrane2022Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 21.
    Flos Berga, Mario
    University of Skövde, School of Bioscience.
    Effect of Ibuprofen on the growth of Pseudokirchneriella subcapitata2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Pharmaceuticals are an important class of pollutants in aquatic ecosystems. Detected concentration are typically in the range 1 ng/L – 1 μg/L. Traditional wastewater treatment does not provide a complete removal of these contaminants; hence, they may have a negative impact on the environment. In addition, microalgae are an ecologically-meaningful target group of species for bioindication purposes as well as primary production and oxygen supply. The present work aimed to investigate the effect of Ibuprofen on the green alga Pseudokirchneriella subcapitata. Algal cultures were exposed to five different concentrations of the drug (5, 15, 45, 135, 405 mg/L) for four days. Absorbance measured at 680 nm was determined every day and obtained data were transformed into cell concentration (cells/mL) by a previously prepared calibration curve. Specific growth rate, generation time, percent inhibition and effective concentration were calculated. Moreover, one way ANOVA with Tukey’s test were applied to observe differences between groups and time periods. Based on this study, all the cultures treated with Ibuprofen had a growth inhibition as well as presenting a lag phase. Increasing the Non-Steroidal Anti-Inflammatory drug (NSAID) concentration reduced the growth rate and consequently, increased the percent inhibition in a concentration-dependent manner. According to this report, new research should be focused on the development of hybrid systems for degradation and removal of pharmaceuticals. NSAID pollution may lead to a reduction in the diversity and number of functional groups of eukaryotic algae. Finally, more research should be devoted to the toxicity of drugs in a variety of test organisms and development of reliable methods for toxicity test at low and chronic exposures to achieve more realistic conclusions.

    Download full text (pdf)
    fulltext
  • 22.
    Fransson, Cristian
    University of Skövde, School of Bioscience.
    The initial steps in the pursuit to diagnose trimethylaminuria with liquid chromatography-tandem mass spectrometry and UniSpray ionization at CMMS2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 23.
    Geetha, Bandhumithra
    University of Skövde, School of Bioscience.
    The severity of human papillomavirus- 16/18 infection and its prevention to cervical cancer: A systematic review and meta-analysis2022Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 24.
    Ghiasvand, Mohammad
    University of Skövde, School of Bioscience.
    Bacterial cell detection limits using Oxford Nanopore’s MinION2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 25.
    Ghobadi, Bita
    University of Skövde, School of Bioscience.
    Suggestions for optimal biomarker miRNA extraction from plasma of sepsis patients2020Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 26.
    Groenewald, Lourens
    University of Skövde, School of Bioscience.
    Manual and robotic RNA extraction from human plasma with absolute quantification of miRNA through two-tailed RT-qPCR as part of research into early diagnosis of sepsis2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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). 

    Download full text (pdf)
    fulltext
  • 27.
    Halan Söderberg, Jessica
    University of Skövde, School of Bioscience.
    The GSK-3 inhibitor has no effect on production of IL-1β in LPS- and Nigericin-stimulated THP-1 macrophages2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Inflammation is the body's natural defense reaction and is known since ancient times. The inflammation is divided into two main phases, acute and chronic inflammation dependent on the process and cellular mechanisms of the inflammation. Inflammation has become to be an important field in research by biomedical research where it is included in many cellular processes thus being phagocytosis, chemotaxis, mitosis, and cell differentiation. Inflammasomes are pro-inflammatory intracellular multimeric protein complexes that introduce the activation of pro-inflammatory cytokines, such as interleukin-1β and interleukin-18, upon trigger by PAMPs and DAMPs signals. The most studied inflammasome is the NLRP3 inflammasome that is activated by various trigger signals, like DAMPs, ATP, uric acid crystals and amyloid-β fibrils. GSK-3β is a kinase that controls various cellular processes, such as inflammation by regulating the activity of abundant transcription factors that are valuable for cytokine production. The aim of this thesis project was to investigate if GSK-3 Inhibitor IV, SB-216763, in a concentration-dependent manner had an effect on production of IL-1β in LPS- and Nigericin-stimulated THP-1 ASC-GFP-macrophages. In addition to the gene expression analysis of IL-1β, the amount of secreted IL-1β, and the possible correlation between treated THP-1 cells with and without GSK-3 inhibitor evaluated. The gene expression analysis was performed by using qPCR and the amount of secreted IL-1β was done using sandwich enzyme-linked immunosorbent assay. The results from this study showed no significant difference in gene expression and amount secreted of IL-1β in THP-1 cells when treated with the GSK-3 Inhibitor IV, SB-216763. 

    Download full text (pdf)
    fulltext
  • 28.
    Hassano, Ragad
    University of Skövde, School of Bioscience.
    GSK-3 and ROS (reactive oxygen species) inhibition modulate Vimentin expression2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.  

    Download full text (pdf)
    fulltext
  • 29.
    Hassen, Umaimah
    University of Skövde, School of Bioscience.
    The effect of GSK-3 inhibitor SB216763 on the expression and secretion of IL-8 in THP-1 ASC GFP macrophages cells2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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. 

    Download full text (pdf)
    fulltext
  • 30.
    Herrera Hernandez, Ana Guadalupe
    University of Skövde, School of Bioscience.
    Detection of Sclerotinia sclerotiorum in oilseed rape using Oxford Nanopore sequencing and qPCR2023Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 31.
    Hil Kafi, Abdulla
    University of Skövde, School of Bioscience.
    Effect of cardiovascular diseases on the severity of patients with renal failure2023Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 32.
    Hirche, Elin
    University of Skövde, School of Bioscience.
    AM I FUNNY NOW?: The Neurological Basis of Humor Styles2019Independent thesis Basic level (degree of Bachelor), 15 credits / 22,5 HE creditsStudent thesis
    Abstract [en]

    The present thesis will provide an overview of how the four humor styles, affiliative, self-enhancing, aggressive, and self-defeating humor, are connected to different brain areas. The thesis will also include an overview of how humor in general, and especially three factors of humor including, processing, appreciation, and comprehension is connected to different brain areas. The present study found a connection between these three factors of humor and activation in the prefrontal cortex (PFC) and inferior frontal gyrus (IFG). The four humor styles were all connected to activity in the midbrain and nucleus accumbens (NAc), though they were found to differ in other parts of the brain. Affiliative humor and self-enhancing humor are humor styles found to share activation of similar brain areas, whereas self-enhancing and aggressive humor was found to the least extent share activation of the same brain areas. No neural differences in relation to the four humor styles have been found between men and woman, or between cultures.

    Download full text (pdf)
    fulltext
  • 33.
    Janardanan, Sruthy
    University of Skövde, School of Bioscience.
    Explorative bioinformatic analysis of cardiomyocytes in 2D &3D in vitro culture system2021Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The in vitro cell culture models of human pluripotent stem cells (hPSC)-derived cardiomyocytes (CMs) have gained a predominant value in the field of drug discovery and is considered an attractive tool for cardiovascular disease modellings. However, despite several reports of different protocols for the hPSC-differentiation into CMs, the development of an efficient, controlled and reproducible 3D differentiation remains challenging. The main aim of this research study was to understand the changes in the gene expression as an impact of spatial orientation ofhPSC-derived CMs in 2D(two-dimensional) and 3D(three-dimensional) culture conditions and to identify the topologically important Hub and Hub-Bottleneck proteins using centrality measures to gain new knowledge for standardizing the pre-clinical models for the regeneration of CMs. The above-mentioned aim was achieved through an extensive bioinformatic analysis on the list of differentially expressed genes (DEGs) identified from RNA-sequencing (RNA-Seq). Functional annotation analysis of the DEGs from both 2D and 3D was performed using Cytoscape plug-in ClueGO. Followed by the topological analysis of the protein-protein interaction network (PPIN) using two centrality parameters; Degree and Betweeness in Cytoscape plug-in CenTiScaPe. The results obtained revealed that compared to 2D, DEGs in 3D are primarily associated with cell signalling suggesting the interaction between cells as an impact of the 3D microenvironment and topological analysis revealed 32 and 39 proteins as Hub and Hub-Bottleneck proteins, respectively in 3D indicating the possibility of utilizing those identified genes and their corresponding proteins as cardiac disease biomarkers in future by further research.

    Download full text (pdf)
    fulltext
  • 34.
    Karlsson, Andréa
    University of Skövde, School of Bioscience.
    A novel assay for the detection of Hepatitis C virus in blood plasma, using padlock probes and rolling circle amplification2023Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 35.
    Kjellman, Simon
    University of Skövde, School of Bioscience.
    16S Nanopore sequencing of Lactobacillus spp. in Apis mellifera, and investigation of their bacteriocin activity2022Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 36.
    Marinkovic, Lucija
    University of Skövde, School of Bioscience.
    Extraction of miR-223 from human blood plasma and quantification using the two-tailed RT-qPCR and absolute quantification2021Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 37.
    Mark Benjamin, Kirabo
    University of Skövde, School of Bioscience.
    The time dependence of processes related to the activated NLRP3 inflammasome2020Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Inflammasomes are large multiprotein complexes that are part of the innate immune response and assemble in response to microbial microorganisms. The function of the innate immune response is dependent upon the detection of pathogen-associated molecular patterns and Damage associated-molecular patterns by germline-encoded pattern recognition receptors. The NLRP3 is the most characterized member of the NOD-like receptors that are also capable of inflammasome activation, the activation leads to the cleavage of caspase-1 into its active form. Dysregulation of inflammasome activation has been linked to several autoimmune diseases such as type1 and 2 diabetes, and the mechanisms regulating the activation of the NLRP3 activation are not fully understood yet. This study aims to study the time dependence of processes related to the activated NLRP3 inflammasome. THP-1 cells were cultured and collected at set time points and the first objective was to perform a western blot analysis and the second was to do an RT-qPCR. The obtained results from the qPCR indicated that they were multiple products in the samples and therefore unspecific amplification during the run. Troubleshooting tests such as melting curve analysis was used to determine the melt profile of the amplicon and gel electrophoresis was used to determine if they were multiple products in the samples. The results from the western blot analysis showed unexpected bands and due to time constraints, further optimization for the antibodies could not be performed.

  • 38.
    Mehra, Ankita
    University of Skövde, School of Bioscience.
    Functional analysis of FBXL12 in response to replication stress2021Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    FBXL12 (UniProt_ID: Q9NXK8) is a largely uncharacterized F-box protein and bioinformatic analysis has revealed that its expression levels correlate with those of Cyclin-E and together play an unfavourable role in patient prognosis in breast cancer. This marks FBXL12 as a potential target for cancer therapy. In an MS-based screen, FANCD2 (Fanconi anaemia complementation group D2) was discovered as an interacting partner of FBXL12. FANCD2 is a protein of the Fanconi anaemia complementation group (FANC), a disorder of the bone marrow that leads to anaemia and other developmental defects. FBXL12 poly-ubiquitylates FANCD2, a modification that causes FANCD2 dissociation from the DNA and subsequent degradation. Presumably, FBXL12 inhibition results in FANCD2 accumulation on DNA that hinders the recruitment of DNA repair factors. The current study aims to focus on the implication of FBXL12-FANCD2 in cancer development and progression, particularly in triple-negative breast cancer cell lines. The role of FBXL12 during replication stress was assessed using stable FBXL12-KO cells, doxycycline regulated shRNAFBXL12 and siRNA-FBXL12-mediated depletion of FBXL12 treated with replication stressinducing drugs, including WEE 1 inhibitor AZD1775. Characterization of a putative phosphorylated FANCD2 site was analysed by Dot-blot and immunoblotting. The data suggest that depletion of FBXL12 sensitize cells to AZD1775-induced replication stress, presumably leading to lethal DNA damage and loss of cellular recovery. It can be concluded that FBXL12-FANCD2 interaction can be exploited as a potential target for cancer therapy to treat aggressive cancer types like triple-negative breast cancers that have poor patient outcomes and limited treatment options available.

  • 39.
    Miah, Md Masum
    University of Skövde, School of Bioscience.
    Pan-antiviral host cell mechanisms of kinase inhibitors as basis for novel treatment of human rhinovirus infections2022Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Human rhinovirus (HRV) is one of the leading causes of upper respiratory tract infections in humans. This infection is usually mild and self-limiting in the immunocompetent host, but in some cases HRV infection can be associated with bronchiolitis in infants, pneumonia in immune suppressed, and the deterioration of pre-existing respiratory diseases such as asthma. There is currently no vaccine or drug for clinical prophylactic/therapeutic use against HRV infection, mostly because of the fact that this virus exists in over 150 serotypes which are sufficiently different and thereby difficult to target by a single vaccine or antiviral compound.

    Here, a HeLa cell-based assay was developed to be used for screening the cellular kinase inhibitor library (KIL) for anti-HRV activity. The KIL library comprise 1200 of kinase inhibitors, and the screening identified 22 hits that completely protected HeLa cells against the development of HRV-induced cytopathic effect (CPE) at concentrations of 1 μM and 10 μM. Thereafter, these hits were assessed in a dose-dependent manner to establish their concentration that inhibited the viral CPE in 50% (EC50). Of these hits, two kinase inhibitors, i.e., a phosphatidylinositol 3-kinase (PI3Ka) inhibitor and an epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor were, investigated in more detail. In particular, it was found that the PI3K and EGFR inhibitors blocked HRV infection in HeLa cell cultures at EC50 of 0.64 μM and 0.49 μM, respectively. Furthermore, these two compounds exhibited 50% cytotoxicity (CC50) for HeLa cells at >100 μM and 13 μM, respectively, which resulted in a selectivity index (SI; CC50/EC50) of >156 for the PI3K inhibitor and 27 for the EGFR-TK inhibitor. These data indicated that the anti-HRV activity of tested of the non-toxic PI3K, and EGFR inhibitors was virus selective and occurred at comparatively low concentrations. Attempts to identify the mode of antiviral activity showed that both inhibitors lacked a direct virus-inactivating (virucidal) activity. Instead, the time-of-addition assay revealed that the PI3Ka inhibitor exhibited antiviral activity even when added as late as 10 h after HRV infection of HeLa cells while the EGFR-TK inhibitor was active when added at 10-12 h post infection. This suggests that both these inhibitors affected the late stage of HRV life cycle, most likely by abrogating release of progeny HRV particles from infected cells.

    In conclusion, these results indicate that the cellular kinases represent an important host-related target for anti-HRV intervention. A further identification and characterization of kinase inhibitors that block the virus infection of cells, and their responsible mechanisms of action, may help in the development of potential anti-HRV drugs.

  • 40.
    Miah, Sabia
    University of Skövde, School of Bioscience.
    Future Sepsis Diagnosis: Difference between manual and Semi-automatic extraction methods and quantifying unknown miRNA2023Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Sepsis is defined by the body's inflammatory response to infection. It is a rapidly progressive condition in which a systemic response to an infection mediated by endogenous mediators, can result in a broad inflammatory reaction in organs distant from the actual injury and end in endorgan dysfunction or even failure. As of a 2020 study, sepsis remains the leading cause of death inhospitals. Currently, a single test for the diagnosis of sepsis does not exist, therefore hospitals rely on testing a combination of vital parameters and observing common symptoms associated with sepsis. However, these methods are often time-consuming, making them fatal for the patients. The University of Skövde is looking into combining different biomarkers into one multimarker panel. Their current research focuses on miRNA as a potential biomarker for sepsis. Despite being a promising field, miRNA extraction from plasma has its difficulties. The study extracted miRNA using miRNeasy Serum/Plasma Advanced Kit (Qiagen) kit and QIAcube (Qiagen) to determine whether manual or semi-automatic extraction is better in terms of time, quality, and quantity. The second aim of the study was to quantify and identify miR-Seps 3 using the RT-qPCR method. The two methods of extraction quality, quantity, and time did not have a significant difference. TheRT-qPCR method was able to amplify miR-Seps 3, though further research is needed on whether the miRNA can be a possible candidate for multimarker panel.

  • 41.
    Mohamad, Hamno
    University of Skövde, School of Bioscience.
    Possible novel drug treatment for neuroblastoma2023Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Neuroblastoma, cancer mostly affecting children, consists of undifferentiated neural crest cells in the spinal cord and adrenal medulla that proliferate uncontrollably. Current treatment involves retinoic acid as maintenance therapy. Research is ongoing to see if retinoic acid can be used in combination with other drugs. Drug X, a myosin inhibitor and drug Y, a sigma-1 receptor agonist, have not been tested together with retinoic acid and this project’s aim was to test how these drugs affect neuroblastoma cells. Proliferation assay was performed on neuroblastoma cells lines treated with drug X, drug Y and retinoic acid. Reverse transcription with quantitative polymerase chain reaction was used to measure the expression of the genes tyrosine hydroxylase, neurofilament 200, novel calcium channel, sigma-1-receptor and novel long non-coding RNA. Live cell imaging of mitochondria and calcium ions were performed for cells treated with retinoic acid and drug Y. Proliferation of cells using different concentrations of drug Y showed no significant difference with control over a 72 h test period. High concentration of drug Y showed a significant decrease in proliferation compared with control after 72 h, while drug Y + drug X showed an even more significant decrease in proliferation. Retinoic acid +drug Y showed a significant increase in gene expression for novel calcium channel and tyrosine hydroxylase and higher calcium ion activity. The combination treatments seem to work synergistically and enhance the expression of some important genes involved in differentiation of neuroblastoma cell lines. However, more tests are needed to validate the results.

  • 42.
    Mohammed, Afzal
    University of Skövde, School of Bioscience.
    Biofilm formation and antibiotic resistance in klebsiella pneumoniae: a meta-analysis study2021Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The study explored the prevalence of biofilm formers and its association with multidrug resistance in Klebsiella Pneumonia, a gram-negative bacterium that has high propensity to form antibiotic resistant strains and forms biofilms. Biofilms are complex microbial community with attributes that vary from planktonic cells. Antibiotic resistance is a property that has shown evidence to be higher in biofilms as compared to planktonic cells. Multi-drug resistance, a higher form of antibiotic resistance, is defined as resistance to at least one agent in three or more antibiotic categories. A single-armed and a two-armed meta-analysis was done to assess prevalence of biofilm formers and to find association between biofilm formation capacity and multi drug resistance. The one-armed meta-analysis revealed 74% (95% CI: 64%-83%) prevalence of biofilm formers among clinical isolates of Klebsiella Pneumonia. The prevalence rate is comparable with that of prevalence rate attained by other bacterium by similar meta-analysis studies. This high prevalence of biofilm formers warrants for a paradigm shift in treatment strategies for treatment of infections. The two-armed meta-analysis showed that there was identical risk of multi drug resistance among the biofilm formers and non-biofilm formers. The result challenges the intrinsic capacity of planktonic cells to resist against antibiotics to achieve multi drug resistance. Further research to update the biofilm formation profiles and to understand the resistance mechanism in commonly occurring bacterial infections in of utmost importance.

    Download full text (pdf)
    fulltext
  • 43.
    Mohite, Komal
    University of Skövde, School of Bioscience.
    Glucose uptake in neuroendocrine cells through glucose transporters2020Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Glucose being the primary source for cellular energy, maintains the glucose homeostasis. Glucose transporters (GLUTs) are carrier proteins for glucose uptake in cells. They are expressed in a tissue- and cell-specific way, and have unique kinetic and regulatory characteristics that illustrate their specific functional roles. This study focuses on uptake of glucose into the neuroendocrine cells by GLUTs and explores the distribution of the GLUTs and their trafficking. The use of PC12 cell line has provided a great deal of knowledge about the role of the proteins that underlie vesicle fusion. GLUT1 and GLUT2 were tagged with fluorescence proteins that is easily expressed in PC12 cells. Live cell imaging was done using total internal reflection microscope (TIRFM) after 24-48 hours of transfection and these data were analyzed using ImageJ. The distribution pattern highlights the prominent expression of GLUT1 compared to GLUT2. To understand the dynamics of these vesicles, the vesicle-plasma membrane interaction was explored. Three behavioral characteristics of the vesicles; docking, undocking and visits were observed based on the residence time of the vesicles. This experiment is the first to investigate the trafficking and fusion of GLUT1 and GLUT2 in PC12 cells, and to create a model system for studying neuronal glucose transporter regulation.

  • 44.
    Monte, Ralph
    University of Skövde, School of Bioscience.
    Protein-protein interactions with and within the NLRP3 inflammasome: Evidence from STRING and literature studies2020Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 45.
    Mukhopadhyay, Maitreyee Banibrata
    University of Skövde, School of Bioscience.
    MYC/VHL Interplay and it's Impact on the Cell Cycle2021Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The MYC oncogene/transcription factor is a well-known factor causing tumorigenesis in many tumor types and is deregulated in almost all types of cancers, correlating with advanced aggressive disease and poor survival. The crucial role of MYC-family oncoproteins in cancer development make them attractive targets for therapy in cancer. An interaction between the oncoprotein MYC and VHL has recently been found in the cell nucleus, involving non-proteolytic ubiquitylation of MYC. The possibility that MYC and VHL regulate each other’s function through direct interaction and thereby influence the fate of tumor cells is largely unexplored in the literature and therefore constitutes a new field of research. This project was focused to understand the impact of MYC/VHL interplay on the cell cycle. In order to elucidate the impact of MYC/VHL on the progression of the cell cycle and to detect the expression levels of major cell cycle proteins, flow cytometry and western blot experiments were conducted respectively, on 786-OccRCC and U2OS osteosarcoma MYCER cell lines with regulatable MYC activity and different VHL status. Here, we demonstrate that VHL does not degrade MYC, but rather promotes its expression in 786-O ccRCC as well as VHL stimulated the cells to overcome the checkpoint barriers and progress through the cell cycle. VHL loss or depletion negatively affected MYC’s ability to regulate critical cell cycle proteins and thereby stimulate progression through the cell cycle. The results from this project warrants further investigation on how pVHL affects MYC function. 

  • 46.
    Nilsson, Andreas
    University of Skövde, School of Bioscience.
    Future diagnostics of sepsis: Defining optimization methods in detection and quantification of circulating microRNA using the QIAcube and two-tailed RT-qPCR2021Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 47.
    Okeke, Ugonna
    University of Skövde, School of Bioscience.
    Thrombotic events in Covid-19 patients using Meta-Analysis2022Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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 % heterogeneity, and p <0.01.

    Download full text (pdf)
    fulltext
  • 48.
    Patel, Angana Heet
    University of Skövde, School of Bioscience.
    Unravelingthe molecular mechanism behind metabolic reprogramming caused by alterations of the enzyme PI3-kinase2019Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Oncogenes and tumor suppressor genes play a key role in cancer induction and progression. They directly or indirectly regulate critical metabolic pathways, phosphatidylinositol-3 kinase pathway being frequently activated pathway in cancer. The catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), p110α, is the most frequently mutated kinase in human cancer, E542K, E545K, and H1047R mutations being the most common. Expression of hepatic E545K and H1047R p110α mutants in vivo shows marked and rapid increase in hepatic lipid and glycogen accumulation in mice with developmental (chronic) liver-specific deletion of p110α, which was not seen in mice when wildtype p110α is overexpressed. To investigate the logical pathways that could explain the lipid accumulation in mutant expressing mice, RNA sequencing from wildtype, knockout and mutated mouse livers was performed. Read alignment and count quantification was done using the Rsubread package and the statistical analyses are performed using the DeSeq2 package. Differentially expressed genes were identified with adjusted p-value of 0.05. Gene ontology analysis was performed on the differentially expressed genes using clusterProfiler, an R package to identify several key pathways which were upregulated and downregulated among the different sample groups. Signaling pathways related to cell cycle processes were mainly upregulated in the mutated samples when compared with the wildtype as well as knockout samples while signaling pathways related to many metabolic processes seem to be downregulated in mutated samples, even though these mutants showed upregulated metabolism by accumulation of lipids and glycogen physiologically. To confirm the results of gene expression data the results have to be cross validated with the gold standard quantitative Real Time Polymerase Chain Reaction.

    Download full text (pdf)
    fulltext
  • 49.
    Phadnis, Anushka
    University of Skövde, School of Bioscience.
    The influence of different forms of iron, of marine and animal origin on the inflammatory IL-6 pathway2023Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
  • 50.
    Plontke, Tjorven
    University of Skövde, School of Bioscience.
    Evaluation of manual and robotic techniques for Micro-RNA extraction in early sepsis diagnosis2023Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    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.

    Download full text (pdf)
    fulltext
12 1 - 50 of 73
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • apa-cv
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf