Högskolan i Skövde

Extracellular vesicles are emitted by almost all cell types. They play an important role in cell-to-cell communication by passing biomolecules such as mRNAs to other cells by endocytosis. It is crucial to isolate and purify them from complex body fluids for studying exosomes. Various techniques, including ultracentrifugation, ultrafiltration, precipitation kits, and immunoprecipitation, are used to isolate exosomes (Bu et al., 2019). Each of these techniques has a significant impact on the properties and purity of the EVs obtained. This project aims to understand the effects of different isolation methods on exosome content by comparing the methods of UC and SEC, with three objectives. The first objective was to compare UC and SEC samples and identify DEGs from native EVs. The second objective was to analyze DEG functional annotation between isolation methods to understand their impact on EV content in biological processes and cellular components. The last objective was to investigate the protein-protien interactions (PPI) between the differentially expressed genes. To investigate the effects of EVs isolation methods at the transcriptional level, RNA-seq data were analyzed from a dataset of three different cell lines, including human lung epithelial cells (HTB-177), umbilical vein endothelial cells (HUVEC), and cardiac progenitor cells (CPC). RNA-seq analysis used an available transcriptomic dataset of EV samples isolated by UC and SEC methods. It identified 10, 15509, and 8995 DEGs from HTB, HUVEC, and CPC, respectively, and mapped them to pathways using EnrichR software. The study found that isolation methods and cell line sources affect analysis results. EnrichR analysis revealed the isolation method's impact on exosomal RNA content and regulation of biological processes.

Low rates of allergy are observed in children growing up on a farm, but the factors that contribute to this protective effect are unclear. This study aimed to investigate if living on a farm alters the infant gut microbiota and thereby reduces the risk of developing allergic diseases in later childhood. This study was based on the prospective Farmflora cohort, which included 28 children living on a farm and 35 control children living in a rural area, who were followed from birth to 8 years of age. The gut microbiota was analyzed from 122 fecal samples collected from 48 children during the first 6 months after birth, using quantitative microbiome profiling. This novel method integrates 16S rRNA gene sequencing data with total bacterial load to obtain absolute counts of bacterial abundance. A significant increase in microbial diversity was seen in the gut microbiota of all the infants in the cohort over the first 6 months after birth. Similar alpha and beta diversity levels were observed in the farm and the control children. However, Sutterella, Megasphaera, and Dorea were more abundant in the gut microbiota of farm children. It has previously been observed that the farm environment was associated with low rates of allergy in children at 3 years. Taxa Akkermansia was more abundant in the gut microbiota of infants who were evaluated with allergy at 3 years. In addition, children who were healthy at 8 years had a higher abundance of Bifidobacterium in their gut microbiota at 6 months of age. However, the abundance of Bifidobacterium could not be linked to farm residence in this study. The findings were consistent with previous studies which link the higher abundance of Sutterella, Dorea, and Megasphaera with protection against allergic diseases. In conclusion, the study observed differences in the gut microbiota of children growing up on a farm, who have low rates of allergy, and showed Bifidobacterium may be protective against allergy development.

Sepsis is a dangerous and potentially fatal condition that has a mysterious origin, underscoring the significance of prompt and accurate diagnosis and treatment. Bacterial whole-genome sequencing, which is widely used in clinical microbiology, stands at the forefront of sequencing technologies, particularly to combat sepsis. The aim of this thesis is to improve sepsis treatment by examining the genetic characteristics and drug resistance patterns of the common sepsis-causing bacteria Pseudomonas and Proteus spp., by analyzing the whole-genome sequencing data of bacterial isolates using an in-house-developed pipeline. The result was compared with a commercial cloud-based platform from 1928 Diagnostic (Gothenburg, Sweden), as well as the results from a clinical laboratory. Using Illumina HiSeq X next-generation sequencing technology, whole-genome data from 88 isolates of Pseudomonas and Proteus spp. was obtained. The isolates were obtained during a prospective observational study of community-onset severe sepsis and septic shock in adults at Skaraborg Hospital in Sweden's western region. The collected isolates were characterized using approved laboratory techniques, such as phenotypic antibiotic susceptibility testing (AST) in accordance with EUCAST guidelines and species identification by MALDI-TOF MS analysis. The species identification result matched the phenotypic method, with the exception of two isolates from Pseudomonas samples and four isolates from Proteus samples. When benchmarking the in-house pipeline and 1928 platform for Pseudomonas spp., predicted 97% of the isolates were resistant to at least one class of the tested antibiotics, of which 94% shows multi-drug resistance. In phenotypes, 88% of the isolates had at least one antibiotic resistance future, of which 68% shows multi-drug resistance. The most prevalent sequence types (STs) identified were ST 3285 and ST111 (9.3%) and ST564 and ST17 (6.98%) each, and both pipelines accurately predicted the number of multilocus types. The in-house pipeline reported 9820 Pseudomonas virulence genes, with PhzB1, a metabolic factor, being the most common gene. It was discovered that there was a significant correlation between the virulence factor gene count and the multilocus sequence typing (MLST) (p = 0.00001). With a Simpson's Diversity Index of 0.98, the urine culture specimens showed the greatest ST diversity. Plasmids were detected in twelve samples (20.93%) in total. In general, this study provided a detailed description of the bacterial future for Pseudomonas and Proteus organisms using WGS data. This research shows the applicability of the in-house and 1928 pipelines in the identification of sepsis-causing organisms with accuracy. It also showed the need for an organized and easy-to-use international pipeline to implement and analyze WGS bacterial data and to compare it with laboratory results as needed.

Pancreatic Ductal Adenocarcinomas initiates in the exocrine part of the pancreatic tissue and represents over 90% of all the pancreatic cancers. Pancreatic Ductal Adenocarcinomas are extremely aggressive and are one of the most lethal malignant neoplasms. The five-year relative survival is currently less than 8% of the patients. The main reason behind such a low survival rate is that most of the cases are diagnosed at a very late stage. Although substantial advancement in pancreatic cancer research has been done, there has not been any remarkable significance in the mortality to incidence ratio. This is mainly a result of the scarce of early diagnostic characteristic symptoms and reliable biomarkers besides the unresponsiveness to the treatments. In this study, transcriptomics and proteomics data were used for the construction of a genome-scale metabolic model that was used in the detection of altered metabolic pathways, genes and metabolites using gene set analysis and reporter metabolites analysis. As a result, altered metabolic pathways in PDAC tumours were detected, including the lipid metabolism-related pathways as well as carbohydrate metabolism, in addition to nucleotide metabolism, which are considered as potential candidates for diagnostic biomarkers. Moreover, classification of the filtered DIRAC tightly regulated network genes, based on their prognostic values from the pathology atlas, detected two groups of PDAC patients that have significantly different survival outcome. The differential expression analysis of the two groups showed that six of the eight genes used in clustering were showing significantly altered expression, which suggests their importance in PDAC patient stratification. As a conclusion, this study shows the valuable outcome of the GEM reconstructions and other systems-level analyses for elucidating the underlying altered metabolic mechanisms of PDAC. Such analyses results should provide more insights into the biomarker discovery and developing of potential treatments.

Abstract

Sepsis is a serious clinical condition that is characterized by a systemic inflammatory response syndrome resulting from a known or suspected infection. The major clinical symptoms involve an abnormal WBC count, elevated body temperature, respiration and pulse rate. Reported cases with high mortality rate range between 13 - 20 million. In order to treat Sepsis, the detection of bacteria in blood culture is extremely crucial. Treating patients with broad spectrum antibiotics is usually related to adverse effects, drug resistance, increased mortality, and high cost. In the past decades, researches had detected that E. coli and S. aureus are the major role players that cause sepsis. These microbes are molecularly tested by methods like MALDI TOF, FISH and Microarrays.  

In this analysis, DNA fluorescence in situ hybridization (FISH) assessment for the identification of S. aureus, one of the most frequent blood pathogens, was optimized in the labs of Högskolan i Skövde. As a result, the growth of S. aureus was observed very carefully, optimizing the FISH procedure for gram positive bacteria was done and the sensitivity, stability and specificity of the DNA probe were examined under variant conditions like the continuous decrease in the bacteria cells number and utilizing a mixture of different types of bacteria cells. 

Sepsis is a life-threatening organ dysfunction that is caused by a dysregulated host response to infection. Sepsis is a substantial health care and economic burden worldwide and is one of the most common reasons for admission to the hospital and intensive care unit. Early diagnosis and targeted treatment of sepsis are the bases to reduce the mortality and morbidity. Conventional blood culturing is the gold standard method for sepsis diagnostics. However, blood culturing is a time consuming method, requiring at least 48 to 72 hours to get the first results with very low sensitivity and specificity. The aim of this study was to determine and assess the direct sepsis-related costs for PCR-based diagnostic strategies (SeptiFast and POC/LAB). A mathematical model was constructed to compare PCR-based diagnostic strategies with the conventional blood culturing. Three case scenarios were investigated based on data from the United Kingdom, Spain and the Czech Republic. It was found that, POC/LAB was the most cost effective strategy in all countries if it could reduce the hospitalization length of stay with at least 3 days in the normal hospital ward and 1 day in the intensive care unit. Reducing the hospitalization length of stay had the greatest impact on the economic outcomes. While, reducing the costs of the diagnostic strategies did not show a remarkable effect on the economic results. In conclusion, the findings suggest that PCR-rapid diagnostic methods could be cost-effective for the diagnosis of patients with sepsis if they could reduce the hospitalization length of stay.

The use of mice as a model organism in biomedical research is widespread due to their similarities with humans in anatomy and physiology. However, the genetic isolation of inbred strains from natural populations raises concerns about the reliability of these models. Studies have shown differences in immune responses between wild and laboratory mice, and the use of exploratory tools for large datasets can help to analyze and interpret these differences. The aim statistical analysis of dataset can provide insights into the differences between variables and individuals and guide further investigations. The results showed that LDA provided clear separation between the different groups, and successfully differentiated between stimulation types and mouse strains, with distinct clustering of data points. The KNN algorithm performed best for smaller values of K. However, the selected gender characteristic did not possess strong discriminatory power in separation and further investigation into alternative features or methodologies may be necessary. In conclusion, the aim of providing comparative immunological analysis of wild and laboratory mice types is achieved. This study underscores the importance of careful statistical analysis, acknowledges the limitations of imputation methods, and highlights the potential of LDA and KNN algorithms in analyzing immune response data. As well, highlighting the need for improved models that are able to capture the complexities of immune responses and their relevance to human immunology.

Common variable immunodeficiency (CVID) is a primary immune deficiency present in about 1 in 25,000 people, characterized by recurrent infections, low serum immunoglobulin (Ig) levels (IgG, IgA, and sometimes IgM), and reduced vaccine responses. It is genetically a heterogeneous illness that often affects adults or teenagers and requires lifetime clinical care. CVID patients experience recurrent or chronic sinopulmonary tract infections, gastrointestinal disorders, and malignancies. Ig reconstitution administered intravenously or subcutaneously is the main treatment. Although the fundamental causes of CVID are still undefined, studies suggest that a variety of variables, including impaired somatic hypermutation (SHM), B cell maturation, primary B cell dysfunctions, abnormalities in T cells, and antigen-presenting cells are implicated. Understanding the molecular mechanisms driving this disease's genome regulation requires a deep understanding of the gene expression. It is today possible to study both coding and non-coding sections of RNA using next-generation RNA-seq, which allows detecting differentially expressed genes in massive amounts of data, particularly in multifaceted illnesses like CVID. The aim of this study was to identify the differentially expressed genes between unstimulated (ex vivo) and stimulated (in vitro) naïve B cells of CVID patients and healthy donors (HD), and also to identify the underlying biological processes by gene enrichment analysis. The results of this study showed that both in CVID and HD, the stimulated and unstimulated cells were well separated. In the gene set analysis, it was discovered that significantly enriched CVID pathways were mostly involved in immune system-related processes such as adaptive immune response, cytoplasmic translation, granulocyte activation, lymphocyte activation, and lymphocyte differentiation. Therefore, the transcriptomic analysis of this study concluded that the majority of the genes that regulate the immune cell activation process function may have a greater impact on CVID patients than on HD which helps to understand the immunological defects in CVID patients. 

The multifunctional purpose of hepatocytes, the functional liver cells within the metabolic, endocrine and secretory functions highlights key importance in emphasizing the research and treatment methods that utilize these cells. Forming 80% of the liver's cells, hepatocytes are involved in many of the primary functions of the liver including the delivery of immune response against pathogens and aiding in the detoxification of drugs. As a result, it provides a valuable basis for medical research. Through the findings of Ghosheh et al. (2017), a method of generating mature hepatocytes was achieved through the human pluripotent stem cells (HPSC), but the generation of hepatocytes in which all the genes are expressed at the right amount through this method proves to be a difficult endeavor. The primary goal of this project is to utilize the established findings to enhance and improve the efficacy of the process that goes behind the generation of mature hepatocytes. The approach towards the current project was initiated with culturing and differentiating three human embryonic stem cell lines and three human-induced pluripotent stem cell lines into mature hepatocytes. In the study mentioned, k-means clustering along with Pearson correlation as the distant measure was run in R to subdivide the top 2000 genes with the highest differential expression into 10 clusters. The cluster data from this paper was used to do the current study, in which the up-regulated and down-regulated gene were first identified for clusters 2, 4 & 6 and 9. The interactions of up-regulated genes in these clusters were further analyzed using Enrichr to identify the different miRNAs for various genes from the clusters. Within cluster 2, a total of 8 genes showed the possibility of being regulated using 4 miRNAs. Transcription factors were also identified for cluster 2 and a combination of HNF1A, EP300, AHR, NFKB1 and HIF1A could repress 8 genes that were not repressed by miRNAs. In cluster 4 & 6, most of the up-regulated genes showcased tumorigenicity and all 20 genes identified could be regulated with the combination of 7 miRNAs. In cluster 9, a combination of 11 miRNAscould be used to regulate 26 out of 27 genes that were analyzed. Ensuring that stem cell products do not turn cancerous is a priority in the medical field. Conducting the analyses of the other clusters aside from 2, 4 & 6 and 9 will prove highly beneficial in reducing the risks pertaining to stem cell mutation due to overexpression of genes.

Globally, the cases of cancer have been on the rise. This has led to increasing research to find a lasting solution to carcinogenesis. The increase in cancer cases can be due to a change in people’s lifestyles, such as diet and exercise routines which have changed for the worse. In most farms, chemicals as pesticides are added to plants to fasten their growth. These chemicals are carcinogenic, contributing to the increased number of cancer cases worldwide.

The objective of this research was to observe the cell response of brain, pancreatic, and breast cancer cells to different cortisol levels/concentrations. The three cell lines of interest in this research and present in the mentioned types of cancer are T-47D, PANC-1, and T98G. Their dynamics and roles are identified in this study. Cortisol excretion concentrations during stress and cancer growth are also monitored and compared. Additionally, the study solves the unpredictability of the impacts of stress on the three cancer types.

The three experimental setups in this study were as follows: 1. Breast cancer cells, obtained from a 54 year old woman with metastatic carcinoma. 2. Brain cancer cells, obtained from a 61 year old female with neuroblastoma. 3. Pancreatic cancer cells, obtained from a 56 year old male with metastatic carcinoma. The cells in the above setups were treated with cortisol in order to see what effect this induced stress has on cell growth.

The findings of the study concluded higher cortisol concentration increased cancer growth and spread within the body. This was that all three cancer cell types saw an increased effect of cortisol at either 24 hours or 48 hours at a specific concentration, which highlights that cortisol has an important role to play in cancer cells viability.

Sepsis is defined as a syndrome of life-threatening organ dysfunction caused by a dysregulated host response to an infection. Early detection of sepsis and immediate treatment with antibiotics is critical for patient outcomes. Haemophilus influenzae (H. influenzae) is a gram-negative bacteria known to be a human-adapted pathogen that may cause a variety of communityacquired infections such as sepsis. A rapid increase in beta-lactam resistance in H. influenzae has been noticed and has become a major problem in clinical care. By implementing bacterial whole genome sequencing (WGS) in the clinical laboratory, it can provide a great amount of information such as species identification, serotype identification, antimicrobial resistance prediction, typing for epidemiologic purposes and tracking infectious disease outbreaks. The aim of this study was to analyze WGS data for clinical H. influenzae isolates using an in-house developed bioinformatic pipeline and an automated 1928 Diagnostics platform to evaluate and compare the predicted results in terms of species identification, prediction of resistance and virulence genes. Furthermore, the predicted genotypic antibiotic resistance genes were compared to the phenotypic antimicrobial susceptibility testing obtained from the clinical laboratory. For the in-house developed pipeline, the analysis of H. influenzae WGS data started with quality control and preprocessing (trimming) of FASTQ files. Following, de novo assembly and quality assessment of assembled contigs and lastly gene annotation tools were performed. For 1928 Diagnostics, the untrimmed FASTQ files were uploaded to the 1928 platform. Species identification resulted in a high agreement of predicted H. influenzae for both phenotypic and genotypic methods except for one sample that may have been contaminated. The analysis of antibiotic resistance genes resulted in both in-house developed pipeline and 1928 Diagnostics having a high agreement regarding the prediction of broad-spectrum beta-lactamase in six clinical isolates, all of which predicted bla TEM-1B. The four most common sequence types found in the MLST analysis from the in-house pipeline were ST159, ST388, ST14 and ST12. The analysis of virulence genes yielded a large number of different virulence genes and each of the identified virulence genes codes a specific function that is crucial to the pathogenesis of H. influenzae. In conclusion, the obtained results provide valuable insights into using WGS-based analysis as a reliable tool for determining the pathogen characteristics in clinical bacterial isolates.

Barley loose smut caused by the fungal pathogen Ustilago nuda is a global concern with detrimental effects on barley production. Early detection of this infection is vital for effective disease supervision. However, current seed health testing protocols suffer from limitations in terms of time and efficiency. The present research work aimed to produce a method using qPCR for simultaneous screening of barley and U. nuda. A set of primers, 1F and 1R, was employed for the detection of rRNA operon internal transcribed spacer 1 sequence from U. nuda and a part of the COX1 gene, present in barley seeds, was selected as an internal control for comparison with U. nuda. A specific 79 bp target amplicon from a part of the COX1 gene was successfully amplified using COX1 F and COX1 R primers, and cloned into a vector for standard curve generation. However, attempts to replicate the previously published qPCR method by bachelor researcher for U. nuda internal transcribed spacer 1 sequence detection using 1F and 1R primers were unsuccessful. Several efforts were made to reproduce the results, but amplification was not observed. Further optimization, including literature review, primers and probe optimization is required to improve this method. The successful amplification of a part of the COX1 gene in both normal and infected samples underscore its potential as a reliable internal control. However, further research is necessary to refine the detection of U. nuda. This study underscores the need for continuous advancements in disease screening methodologies to meet global market demands.

Co-occurrence of abiotic stresses, especially drought and salinity, is a natural phenomenon in field conditions and is worse for crop production than any single stress. Nowadays, rigorous methods of meta-analysis and systems biology have made it possible to perform cross-study comparisons of single stress experiments, which can uncover main overlapping mechanisms underlying tolerance to combined stress. In this study, a meta-analysis of RNA-Seq data was conducted to obtain the overlapping gene network of drought and salinity stresses in barley (Hordeum vulgare L.), which identified Rubisco activase A (RcaA) as a hub gene in the dual-stress response. Thereafter, a greenhouse experiment was carried out using two barley genotypes with different abiotic stress tolerance and evaluated several physiochemical properties as well as the expression profile and protein activity of RcaA. Finally, machine learning analysis was applied to uncover relationships among combined stress tolerance and evaluated properties. We identified 441 genes which were differentially expressed under both drought and salinity stress. Results revealed that the photosynthesis pathway and, in particular, the RcaA gene are major components of the dual-stress responsive transcriptome. Comparative physiochemical and molecular evaluations further confirmed that enhanced photosynthesis capability, mainly through regulation of RcaA expression and activity as well as accumulation of proline content, have a significant association with combined drought and salinity stress tolerance in barley. Overall, our results clarify the importance of RcaA in combined stress tolerance and may provide new insights for future investigations. 

It has long been held that concrete material has a processing advantage over abstract material, as predicted by Dual Coding Theory (Paivio,1991), although this has been challenged. For example, based on evidence for behavioural and neuroscientific studies, Kousta,, Vigliocco, Vinson, & Del Campo, (2011) proposed that emotional valance had a greater influence in the processing of abstract words, and that under some circumstances there may be no concreteness effect and might even be an abstractness effect. This would not be predicted by DCT. In addition, Isen and Daubman (1984) have claimed that emotional valence, and particularly positive emotion can influence cognitive processing. Specifically, they demonstrated that positive emotion was associated with more inclusive categorization of ambiguous category members. This current study was a 2 x 2 between group design to investigate the effect of positive and negative valence on recognition memory for concrete and abstract words and on categorization. Contrary to what was predicted by Dual Coding Theory, abstract words were generally better recognized than concrete, with there being an additional interaction with valence. A significant interaction between word type and valence on categorization was also found. Results partially support Kousta et al. (2011).

Neoehrlichia mikurensis is a bacterium transmitted to humans by tick bites, usually from Ixodes ricinus species, causing an infection known as neoehrlichiosis. The importance of B cells in host defence against this infection has previously been demonstrated in patients undergoing anti-B cell therapy for lymphoma. As a causal relationship between N. mikurensis and malignant B-cell lymphomas has not yet been established, the identification of a genetic signature caused by the infection would be a step forward in the development of therapeutics. Findings have been published on the heavy chain repertoire of the major B-cell clonotypes of N. mikurensis-positive lymphoma patients, and it is essential to gain a deeper understanding of the broader heavy chain repertoire. That can be achieved by analysing other aspects such as VDJ gene frequency, somatic hypermutations in complementarity determining regions (CDRs) and framework regions (FWRs) of the variable domain, and the physicochemical properties of the H-CDR3 repertoire, the latter being the most hypervariable part of the heavy chain and a major determinant of its specificity. Specifically, eight infected lymphoma patients were analysed for these variables. In short, the analysis of the results separates the data into at least two distinct groups of patients studied. It was also possible to group the patients by integrating the data into heatmap representations that clustered the patients in an unsupervised manner based on the use of IGHV, IGHD and IGHJ gene frequencies and the physicochemical properties of H-CDR3. Interestingly, this correlated closely with the lymphoma classifications of the patients infected with N. mikurensis. Although the sample size is not very large, and work is currently being to recruit more patients and controls, these results are promising as not only may there be an underlying signature related to the infection, but they may also help to separate the heavy chain repertoire data into groups, which in turn may help to diagnose lymphoma patients more accurately and perhaps more quickly and find the right treatment from the outset. 

One of the main components of atherosclerotic plaque is the production and accumulation of cholesterol crystals (CCs), which could serve as a biomarker of atherosclerosis. Atherosclerosis is a chronic inflammatory artery disease that is the root cause of myocardial infarction and stroke. Endothelial dysfunction is one of the main contributors to the development of atherosclerosis. The aim of the study was to investigate whether Human Umbilical Vein Endothelial Cells (HUVECs) can uptake CCs and to examine CCs-induced inflammatory response in HUVECs. Using molecular and functional techniques, the distinctive characteristic of CC-mediated immune response was discovered in HUVECs. CCs were mostly taken up by HUVECs by macropinocytosis and phagocytosis. CCs were found to induce Signal Transducer of Activators of Transcription (STAT) 3 phosphorylation and Interleukin (IL)-6 release in HUVEC. In addition, Caspase activation and recruitment domain 8 (CARD8) knockdown drastically reduced CCs uptake and CCs induced IL-6 expression in HUVECs. Moreover, the stem cell growth factor (SCGF)-b protein release was downregulated in response to CCs. IL-1A and Colony Stimulating Factor (CSF) 2 were identified as the topmost hub nodes interacting with all other differentially expressed proteins. A significant increase in neutrophil adhesion on HUVECS was found in response to CCs and conditioned medium from CCs-treated HUVECs. In conclusion, the study findings suggest that the CCs induceSTAT3-mediated IL-6 release and neutrophil adhesion, thereby promoting inflammation in HUVECs.

Placebo treatments are inert but are known to alleviate symptoms across numerous clinical conditions. One of the most studied placebo effects is placebo analgesia, which is a placebo effect limited to pain relief. This thesis aims to introduce the current state of research regarding the neuroscience of placebo analgesia and specifically to present research findings regarding the neurotransmission. Studies have demonstrated that placebo analgesia can be elicited through two separate processes interacting with each other; manipulation of expectations and through conditioning. These processes seem to affect neurotransmission in different ways. Many brain areas have been found to be correlated to placebo analgesia. Besides the pain-processing brain areas, studies point to that the prefrontal cortex can have a vital role in the placebo analgesic effect. Known neurotransmitters that have shown to be involved in placebo analgesia are endogenous opioids, cholecystokinin (CCK), and endocannabinoids. Studies point to that endogenous opioids are involved in the placebo analgesic effect when elicited by expectation or conditioned by an opioid drug. CCK act on placebo analgesia by affecting the release of endogenous opioids and endocannabinoids seem to be involved in placebo analgesia while it occurs due to conditioning with non-opioid drugs. Getting a better understanding of placebo analgesia and find ways to apply this knowledge in the clinical context could powerfully develop the whole medical society.

Type 2 diabetes (T2D) is a widespread metabolic disorder characterized by chronic hyperglycemia and insulin resistance (IR), impacting over 90% of diabetes cases globally. Despite extensive research, the precise molecular mechanisms of T2D remain unclear, highlighting the need for further investigation into its pathophysiology. This study aims to elucidate the relationship between pancreatic islet cell electrophysiological activity and gene expression profiles to enhance our understanding of T2D’s cellular mechanisms.

Pancreatic islets from non-diabetic mice and humans were isolated, dissociated into single cells, and analyzed using high-density microelectrode arrays (HD-MEA) for electrophysiological across various glucose concentrations. The electrophysiological was monitored over 3-5 days, with validation through calcium fluorescence imaging. Single-cell RNA sequencing (scRNA-seq) was employed to analyze gene expression changes for alpha and beta cell types.

Results revealed distinct electrophysiological patterns in response to glucose, with variations inspike frequency. Differential gene expression analysis identified gene expression changes in alpha and beta cells. The TRIB3 and FFAR4 genes were found to be downregulated in T2D compared tonon-diabetic non-obese (NN) individuals, and both are involved in glucose transmembrane transport. Additionally, FFAR4 showed reduced expression in T2D relative to non-diabetic obese(NO) individuals, and is associated with peptide hormone secretion. Conversely, PDE4B was upregulated in T2D compared to NO individuals, impacting calcium channel voltage gating and cAMP signaling pathways. FXYD2, related to sodium channel regulation, was also downregulated in T2D compared to NO individuals. In alpha cells, the genes TMEM236, TMEM45B, and SYTL5 were upregulated in T2D compared to both NN and NO individuals, suggesting alterations in cellular membrane dynamics and signaling pathways.

The integration of HD-MEA with scRNA-seq provided insights into the functional and transcriptomic profiles of human pancreatic islets under different metabolic states. Findings highlight significant changes in gene expression and electrophysiology, emphasizing the impactof T2D and obesity on islet function. While this study enhances our understanding of islet dysfunction in T2D, it also points to limitations such as small sample sizes and the focus on only alpha and beta cells, suggesting the need for broader studies to further elucidate T2D mechanisms.

Idag sker stora populationsminskningar för många arter på grund av avsaknaden av livsmiljöer. Utifrån detta är det av stor vikt att stärka grön infrastruktur i hela landskapet och speciellt i sådana områden där det idag finns brist på livsmiljöer för olika djur- och växtarter. Värdekärna är den primära indikatorn för att beskriva kvalitéer i landskapet och kan till exempel utgöras av en nyckelbiotop i skogen. Den tretåiga hackspetten (Picoides tridactylus) anses i allmänt vara en specialist på barrskog och även svartmesen (Periparus ater) betraktas som en utpräglad barrskogsart. Syftet med studien är att undersöka om det finns områden i Götene, Karlsborg och Skövde kommun som kan utöka/förbättra barrskogsområden med värdekärnor. Detta genom att utföra en landskapsanalys och jämföra levnadsförutsättningarna mellan de två barrskogsarterna, tretåig hackspett (Picoides tridactylus) och svartmes (Periparus ater). Målet är att på lång sikt försöka förbättra områden med värdekärnor i berörda kommuner för att öka arternas förekomst och sannolikheten att överleva. Data för värdekärnor, avverkningsanmälningar och medelhöjd av träd har analyserats i ArcMap för eventuella hot mot arternas livsmiljö och lokalisering av potentiella framtida livsmiljö områden. Karlsborg kommun har stört areal av värdekärnor och minst avverkningsanmälning. Skövde kommun har mindre areal av värdekärnor och mer avverkningsanmälningar jämfört med Karlsborg. Götene kommun har minst areal av värdekärnor och mest avverkningsanmälningar. Skövde kommun har störts areal av potentiella framtida områden med värdekärnor för båda arterna. Karlsborg kommun har något mindre än Skövde och Götene kommun har mints areal av potentiella framtida områden med värdekärnor för båda arterna. Resultaten framtagna i denna studie kan inte betraktas som mer än ett första steg i arbetet med att försöka lokalisera framtida områden med värdekärnor för den tretåiga hackspetten och svartmesen. För framtida arbeten skulle det vara av intresse att i fält inventera de ”potentiellt framtida områdena” och på så vis avgöra om denna arbetsmetod är något som går att tillämpa i fler kommuner och eventuellt andra fågelarter för att finna potentiella framtida områden med värdekärnor för arter. 

The increasing number of biological databases today requires that users are able to search more efficiently among as well as in individual databases. One of the most widespread problems is redundancy, i.e. the problem of duplicated information in sets of data. This thesis aims at implementing an algorithm that distinguishes from other related attempts by using the genomic positions of sequences, instead of similarity based sequence comparisons, when making a sequence data set non-redundant. In an automatic updating procedure the algorithm drastically increases the possibility to update and to maintain the topicality of a non-redundant database. The procedure creates a biologically sound non-redundant data set with accuracy comparable to other algorithms focusing on making data sets non-redundant

Temporal stratification in lakes due to high air temperatures and dynamic wind conditions changes the environment for organisms in the depth gradient. For fish that have narrow requirements for temperature and oxygen conditions, stratification can lead to a significant reduction in habitat volume during the summer when surface temperatures are high. In Sweden, temporal stratification occurs between July and October. The vendace (Coregonus albula) is a cold-water fish that is found in several waters around Sweden. In Lake Mälaren, there is targeted commercial fishing for the species, but the total annual catch has drastically decreased in recent decades. Shrinking habitats and longer periods of temporal stratification during the summer could be a partial explanation. Understanding and predicting changes of the vendace populationin Lake Mälaren requires mapping of available habitat during the summer temporal stratification. This study has investigated the worst habitat conditions in terms of habitat thickness for vendace during the years 2017-2022 in Lake Mälaren based on the habitat requirements temperature ≤18 °C and DO concentration ≥2 mg/L. The spatial distribution in two basins where analysed. The results showed that the vendace had limited available habitat in three of seven water bodies investigated (habitat thickness ≤2.5 m). The spatial analysis of the two basins showed that habitat volume represented 41 % and 11 % of the total volume of water in each basin. The available habitat of the vendace during the summer temporal stratification in Lake Mälaren is thus limited in parts of the lake.

The stress system is essential for humans and other organisms to survive. However, when stress is prolonged it can have pathological effects on the brain. To experience long-term unemployment is often stressful, for it has been shown to correlate with depression, low self- esteem, learned helplessness and self-destructive behavior. Long-term unemployment also seems to have physiological consequences, for it has been shown to correlate with cortisol dysregulation. The hippocampus is a highly adaptable part of the brain located in the temporal lobe and is long known for its sensitivity to cortisol dysregulation due to stress. The aim of this thesis is to study how long-term unemployment affects physical and psychological well- being, focusing in particular upon finding out whether it affects the hippocampus. The results suggest that that the kind of stress caused by long-term unemployment is similar to the stress affecting the hippocampus. It thus seems to be a reasonably hypothesis that long-term unemployment has a negative influence upon the brain, and the hippocampus in particular.However, there is an additional issue that one needs to take into account. For some studies have shown that people with poor mental health are more likely to be unemployed. If poor mental health is associated with physiological disorders (including a damaged hippocampus), this implies that not only can long-term unemployment (via stress) affect the hippocampus, but a damaged hippocampus (along with other physiological factors) can increase the probability to become unemployed. This means that the relationship between long-term unemployment and a damaged hippocampus need not be a one-way causal relationship.

Consumption of antioxidant supplements is widely popular worldwide mainly because of their supposed anticancer benefits. Though these benefits against cancer are highly advertised by supplement industry and a well stablished perception in general population, there are not enough studies to support those claims. In fact, several studies have pinpointed antioxidants’ role in promoting tumor proliferation and migration in multiple types of cancer. Nevertheless, the mechanisms behind how antioxidants are actually mediating these events is largely unknown. This study aims to investigate how antioxidants are affecting lung cancer cells migration and to decipher the involvement of the transcription factor BACH1 in it. In this study, effect of antioxidant supplementation on three human Non-small-cell lung carcinoma (NSCLC) cell lines were investigated. Antioxidant administration was found to increase cell migration and BACH1 expression in all of them. Effect of inactivation and over activation of BACH1 confirmed significant relation between antioxidant induced migration and BACH1 expression. Along with increased migration, antioxidants increased glycolysis in the cells in a Bach1 dependent manner. Activation of NRF2, the master regulator of endogenous antioxidant expression had also been found to increase migration through up regulation of BACH1. Overall findings of this study confirm Bach1’s role in antioxidant-induced migration in lung cancer from different directions and provides a broader understanding how redox status of cancer cells influence their metastatic ability.

Organisms relevant for nature conservation dont follow administrative borders. Because of this there is a need for a landscape perspective within conservation and planning, and a need for the species of interest to have legal protection. Network analysis adapted for ecological purposes has grown to become a powerful tool for studying and communicating the relationships between species dispersion and access to habitat. In this study the following question is posed: How is the Osmoderma eremita and the Pernis apivorus dispersal possibilities in the small scale cultivated landscape of Borås affected by exploitation in respect to a) dispersal ability, b) habitat quality, c) position of habitat patches in a network? The analysis were based on municipal and regional nature conservation data, which in due to confidentiality is not accounted for in the report by maps, coordinates, etc. Several networks were established for both species to indicate how habitat patches are distributed today and how the species dispersal changes depending on which patches are excluded – this was done to imitate how exploitation can affect the species future survival and dispersion. The results showed that the O.e. is mainly inhibited by its poor dispersal abilities, followed by patch position, while the P.a. is the most affected by degrading habitat quality. The most important conclusions of the study were that the O.e. natural dispersal may be restricted but can be improved by linking small network components together and by maintaining the largest components. As for the P.a. it was concluded that a different type of analysis, focusing on its behaviour and need for different patches for different purposes, would generate more interesting results.

Enterotoxigenic Escherichia coli (ETEC) is a water-borne pathogen that causes diarrhoea. ETEC expresses virulence factors such as heat labile toxin (LT), and/or heat stable toxin (ST) that cause diarrhoea. ETEC also express colonization factors that enable attachment to the host epithelium. However, many of the virulence genes that are activated when the bacteria are exposed to stressful conditions are still unknown. In this study, the main focus was to analyse the survival of ETEC strain E2265 in an inefficient nutrient and stressful condition (freshwater) for a prolonged period in order to identify genes that are involved in mechanisms for survival. To thoroughly understand these mechanisms and particular gene activation in freshwater, transcriptomic analysis of ETEC strain E2265 was performed using Illumina paired-end RNA sequencing and differential expressed genes were analyzed using the programming language R. The results showed that genes responsible for metabolic mechanisms, cell repair and biofilm formation were activated after long period incubation in order to adapt and survive in a stressful environment (freshwater). It also indicate that the ETEC strain E2265 can survive in freshwater for long periods and remain culturable. During the more extended period the plasmid borne toxins (LT and ST) remained stable in the genome but not activated by the environmental conditions apart from a short burst of upregulated expression after 2 hours of incubation in freshwater. Also, biofilm formation of E2265 was identified, which is a thin protective layer formed by group of bacteria and genes involved in biofilm formation were activated. The results suggest that ETEC strain E2265 are able to survive for months in freshwater without losing plasmid virulence genes and that genes expression are actively modulated during different phases in early stages of freshwater incubation.

Sepsis is a life-threatening condition in which a dysregulated immune response causes damage to the patient’s organs. The condition moves fast leaving little time for diagnosis and starting treatment. Early diagnosis would help save lives and reduce the amount of antibiotics used. The aim of this study was to compare manual and robotic extractions for miRNA extraction and to quantify miR-seps4, a possible biomarker for sepsis, from human plasma. During the first part of the study manual extractions done by hand and robotic extractions done using QIAcube (Qiagen) of miRNA using the miRNeasy Serum/Plasma Advanced Kit (Qiagen) were compared using the quantity and purity of RNA they produced. Then two-tailed RT-qPCR was optimised for human plasma spiked with synthetic miR-seps4. The final objective was to use these methods to test whether two-tailed RT-qPCR could be used to identify and quantify miR-seps4 from non-spiked human plasma using the absolute quantification method. The QIAcube (Qiagen) extractions gave a higher concentration of RNA while manual RNA extractions had a higher purity A successfully optimised standard curve was made using synthetic miR-seps4 though the melt curve showed contamination and possible non-specific products. Non-spiked RNA extractions showed successful amplification of miR-seps4 that could be quantified through the absolute quantification method. This makes miR-seps4 a good target for further research and a possible addition to a multi-marker panel for early diagnosis of sepsis. 

Diffuse large B-cell lymphoma is a common non-Hodgkin lymphoma and covers about one third of global non-Hodgkin lymphoma cases each year. The lymphoma is characterised by B-cells with large nuclei which makes the cells unable to function normally as a part of the immune response. MicroRNAs, which are small noncoding RNA, can be used as biomarkers for the diagnosis, prognosis and treatment in many different diseases. However, the pathways regulated by microRNAs in connection to diffuse large B-cell lymphoma are not well understood. The aim of this study was to find hub microRNAs and their pathways in diffuse large B-cell lymphoma. This was done by identifying differentially expressed microRNAs, finding their target genes and building networks of the microRNAs and their targets. Gene ontology terms and KEGG pathways were identified for each network. This study found three microRNA hubs hsa-let-7f-1-3p, hsa-miR-200c-3p, and hsa-miR-507. Each of them has been shown to regulate tumours in other cancer types though they have not been connected to diffuse large B-cell lymphoma before. The target genes have also been found to play central roles in general tumour formation. This study found that known tumour suppressor genes were down-regulated while genes that aided in tumour formation were up-regulated. The gene ontology analysis showed that many of the target genes take part in the normal development of the human body, but the KEGG pathways were concentrated around cancer formation. The microRNAs show promise as prognostic markers and targets for developing treatments.

Stress has been always associated with a deviating than normal feeding behavior. Both over-eating and under-eating accompanied by altered food choice towards palatable food have been reported in response to stress. The neuronal pathways that link stress with eating are still unclear. Locus Coeruleus (LC) is the main endogenous norepinephrine (NE) secreting nucleus. It lies in the center of the stress response mediating arousal state. LC-NE nucleus with its widespread innervations throughout the brain can modulate brain mechanisms linked with motivation towards food. In this study, the aim was to study the activity of NE neurons in the LC in relation to stress and food intake. The hypothesis was that NE neurons are activated by mild stressors and that this activity drives food intake. Because the association between LC activity and food intake is observational by nature, it is not expected to demonstrate a causal link but to show findings consistent with this hypothesis. Another aim was to standardize the photometry measurements and an analysis paradigm. In response to a stressor, animals showed freezing behavior, with photometry recordings displaying a significant reduction in Ca+2 signals right after the distressing stimulus. When a stressor preceded food intake, LC-NE activity significantly decreased right after the first meal the effect that did not last to the second meal with no difference between chow and palatable food. These results highlight the involvement of LC-NE in modulating feeding behavior by integrating environmental cues and internal needs. Future investigations of distinct, projection-defined, LC-NE sub-populations may reveal more specific food and stress interactions.

Inflammatory Bowel Disease (IBD) is a complicated and multifaceted illness marked by chronic inflammation of the gastrointestinal system. Understanding the underlying molecular causes of IBD is critical for creating tailored therapeutics and better patient outcomes. This thesis compares Over-Representation Analysis (ORA) and Gene Set Enrichment Analysis (GSEA) methods on two datasets, GSE66407 and GSE186582. Three ORA tools—ClusterProfiler, WebGestalt, and Enrichr—were tested to determine their sensitivity, specificity, and robustness in discovering enriched pathways. The results showed that the techniques were quite consistent, especially in the GSE186582 dataset, where there was perfect overlap in pathway discovery. However, small differences were seen in the GSE66407 dataset, with ClusterProfiler and Enrichr detecting distinct pathways. Furthermore, GSEA was conducted using clusterProfiler, WebGestalt, and fgsea, which provided a dynamic analysis of the whole ranked list of genes. GSEA results from both datasets consistently revealed immune-related pathways involved in IBD pathogenesis, including IL-17 signaling, NF-kappaB signaling, and TNF signaling. Particularly, ClusterProfiler, WebGestalt, and fgsea both identified many distinct pathways, demonstrating differences in pathway recognition depending on the approach utilized. While ORA approaches are strong and accurate for quickly identifying pathways, GSEA may give a more detailed knowledge of the biological processes by capturing minor gene expression variations.

The whole project consisted of two different parts. The first subproject were running optical density measurements to assist in finding optimal growth conditions and experimental setup for use of E.coli BL21 (DE3) bacteria with the PYCARD gene transformed into them. The transformed bacteria will be used for generating data for modeling. Previous laboratory attempts had problems with timing of the exponential phase when several tests were performed simultaneously. The optimizations of a method for displaying growth include trying out using different medium but also different inoculation ratios between culture and medium to see the effects on growth rate. Results have shown that lysogeny broth together with an inoculation ratio of 1:25 results in rapid absorbance increase in the bacterial growth curves.The second part of the thesis project was to study the kinetics of fading of phenolphthalein in sodium hydroxide solution in order to provide a basis for experiments illustrating this in laboratory environment. Phenolphthalein is used in many different applications such as an active ingredient in some laxatives but the perhaps most common use is as an acid-base indicator of pH. The experiments were performed by mixing alkaline solutions of varying sodium hydroxide concentration together with sodium chloride, then after addition of phenolphthalein to study absorbance variations over time to illustrate the rate law which correlates the rate of color fade to sodium hydroxide concentration. The results have proven that they correspond directly so that increasing the concentration of sodium hydroxide raised the color fading rate just as much in percent.

Corticotropin-releasing factor (CRF) is central in the stress response but also modulates several behaviors including anxiety-related behaviors and aggression. In this study, juvenile rainbow trout (Oncorhynchus mykiss) were tested for competitive ability, determined during dyadic fights for dominance, after intracerebroventricular (i.c.v.) administration of CRF, urotensin I (UI), the non-specific CRF antagonist alpha-helical RF(9-41) (ahCRF) or the CRF receptor subtype 1-specific antagonist antalarmin, when paired with a mass-matched con-specific injected with saline. In addition, isolated fish received the same substances. Plasma cortisol and brain monoamines were monitored in all fish. Most fish receiving CRF showed a conspicuous behavior consisting of flaring the opercula, opening the mouth and violent shaking of the head from side to side. When this occurred, the fish immediately forfeited the fight. Similar behavior was observed in most fish receiving UI but no effect on outcome of dyadic fights was noted. This behavior seems similar to non-ambulatory motor activity seen in rats and could be anxiety related. Furthermore, fish receiving CRF at a dose of 1000. ng became subordinate, whereas all other treatments had no effects on the outcome of dyadic fights. In addition, isolated fish receiving ahCRF had lower brain stem concentrations of 5-hydroxyindoleacetic acid, serotonin, 3,4-dihydroxyphenylacetic acid and dopamine. In conclusion, CRF seems to attenuate competitive ability, and both CRF and UI seem to induce anxiety-like behavior.

Background: There exist few, if any, practical guidelines for predictive and falsifiable multi-omic data integration that systematically integrate existing knowledge. Disease modules are popular concepts for interpreting genome-wide studies in medicine but have so far not been systematically evaluated and may lead to corroborating multi-omic modules. Result: We assessed eight module identification methods in 57 previously published expression and methylation studies of 19 diseases using GWAS enrichment analysis. Next, we applied the same strategy for multi-omic integration of 20 datasets of multiple sclerosis (MS), and further validated the resulting module using both GWAS and risk-factor-associated genes from several independent cohorts. Our benchmark of modules showed that in immune-associated diseases modules inferred from clique-based methods were the most enriched for GWAS genes. The multi-omic case study using MS data revealed the robust identification of a module of 220 genes. Strikingly, most genes of the module were differentially methylated upon the action of one or several environmental risk factors in MS (n = 217, P = 10− 47) and were also independently validated for association with five different risk factors of MS, which further stressed the high genetic and epigenetic relevance of the module for MS. Conclusions: We believe our analysis provides a workflow for selecting modules and our benchmark study may help further improvement of disease module methods. Moreover, we also stress that our methodology is generally applicable for combining and assessing the performance of multi-omic approaches for complex diseases. 

Alzheimer’s disease (AD) is an aging-related neurodegenerative disorder with large implications for society and individuals. AD is a multi-factor disorder, with these factors having a direct or indirect correlation with each other. Despite many studies with different aspects on molecular and cellular pathways, there is still no specific treatment for AD. Identification of potential pathogenic factors can be done by transcriptomic studies of differentially expressed genes (DEGs), but the outcomes have been contradictory. Using both bioinformatics and meta-analysis methods can be useful for removing such inconsistencies. A useful and common approach for a better understanding of neurodegenerative disease is to assess its molecular causes, by comparing the gene expression levels in healthy and disease tissues. Next-generation RNA-sequencing is a valuable method for analyzing both coding and non-coding regions of RNA, and it has made it possible to identify differentially expressed genes in large-scale data. The aim of the current study was to get a better understanding of the transcriptional changes in AD models, and identify differentially expressed genes between healthy and AD individuals from the adult mouse brain model as well as detecting AD pathways. In this study, the transcriptomes of purified neuron, astrocyte and microglia cells from mouse brains were analyzed using publicly available RNA-seq datasets. The DEGs were identified for all three mentioned cell types using DESeq2 and EdgeR packages. All statistical analyses were performed by R software and the DEGs detected by DESeq2 and edgeR, respectively, were compared using Venn diagrams. Additionally, analyzing the AD pathway was performed using GOrilla tool for visualizing the enriched gene ontology (GO) terms in the list of ranked genes. From this project, it was found that there were very few significantly DEGs between AD and healthy samples in neuron cells, while there were more DEGs in astrocyte and microglia cells. In conclusion, comparing DESeq2 and egeR packages using Venn diagrams showed a slight advantage of DESeq2 in detection accuracy, since it was able to identify more DEGs than edgeR. Moreover, analyzing AD pathway using GOrilla tool indicated that identified enriched GO terms by each cell type differed from each other. For astrocytes, more enriched GO terms were identified than for microglia cells, while no significant enriched GO terms were detected for neuron cells.

The application of mechanistic models to natural systems is of interest to ecological researchers. We use the mechanistic Allometric Trophic Network (ATN) model, whichis well-studied for controlled and theoretical systems, to describe the dynamics of the aphidRhopalosiphum padi in an agricultural field. We diagnose problems that arise in a first attemptat a least squares parameter estimation on this system, including formulation of the modelfor the inverse problem and information content present in the data. We seek to establishwhether the field data, as it is currently collected, can support parameter estimation for theATN model.

Spermatogenesis is a complex process that involves the production of sperm cells from germ cells in order to transmit genetic information to progeny. The PIWI-interacting RNA (piRNA) is a non-coding small silencing RNA that guides the PIWI protein to silence target transposon transcripts to regulate the production of germ cells. In mammals, distinct sets of piRNAs function in gonocytes, spermatogonia, and developing spermatocytes. Coinciding with puberty, pachytene piRNAs, expressed at pachytene stage of meiosis I, silence mRNAs to regulate spermatogenesis. At the onset of meiosis I, the transcription factors A-MYB and TCFL5 initiate the transcription of pachytene piRNA genes via binding to their transcription start sites. This study aims to detect which pachytene piRNA genes are bound by these two transcription factors in two sub-species of mice, Mus Musculus musculus and Mus Musculus castaneus, and if there is a variation in binding regions between these sub-species. Here, chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to map the binding regions of A-MYB and TCFL5 in the genomes of mouse sub-species. Three testis tissues from each sub-species were sonicated for ChIP-seq on A-MYB and three from each mouse for TCFL5. Sonication was optimized at high power for 40 cycles of 60 sec on, 30 sec off, for 1 h. After 20 cycles, lysates were transferred to a fresh tube, vortex, and sonicated for 20 more cycles. Library DNAs were average 333-360 bp, and qPCR showed enough DNA for Illumina sequencing. Analysis of sequencing data is ongoing. The transcription start sites of pachytene piRNAs could be identified after analysis.

The rapid and reliable detection of pathogens is of utmost importance in healthcare settings to ensure the appropriate treatment thereby reducing morbidity and mortality for the patient. Current culturing, PCR based and NGS species detection methods are time consuming (Opota et al., 2015), limited in their detection (Buckley et al., 2015), or require specialist skills and are expensive (Basho and Eterovic., 2015). Oxford Technologies Nanopore devices could provide detailed genomic sequencing at a fraction of the cost and without the need for technical bioinformatic skills. This study evaluates the MinION device and analysis tools to suggest best practice. Classification and genotyping of 12 Klebsiella isolates were performed using EPI2ME automated workflows and manual de novo assembly.  Automated workflows using raw MinION reads provided clinically relevant information identified in ~6hrs. Manual de novo assembly and analysis used hybrid, and single source data took >24hrs. The inclusion of MinION long reads overcome problems assembling short reads. Hybrid genomes provided the most contiguous and highly detailed contigs. MinION only read assemblies contained more errors but still identified similar genotypic findings. Automated workflows are rapid and require minimal bioinformatic know-how. There should be a dialogue between clinicians and bioinformaticians to develop bespoke analysis tools.  Although challenges remain around compatible kits and vulnerable flowcells long read sequencing can be an effective tool for species detection and pathogen typing. Furthermore, hybrid assemblies have the potential to advance our genome detailing and discovery.

Mantle cell lymphoma (MCL)is a subtype of B-cell lymphoma characterized by chromosomal translocation t(11;14)(q13;q32), enhancing the expression of cyclin D1, resulting in alteration in cell cycle progression. The translocation is the key event for initiation of mantle cell lymphoma however is not sufficient for the development of the disease. Changes in the expression of the G-protein coupled receptors in different cancer types due to mutations and other regulating mechanisms can lead to changes in expression of proteins, resulting in defects in signalling pathways,favouring tumor dissemination. Growing evidences suggests that the disease progression in various B cell malignancies is due to the interactions between neoplastic B cells and stromal cells in tissue microenvironment. ONC201 is an imipridone, which is an antagonist to a type of G-protein coupled receptor: dopamine receptor D2. ONC201 has the property for inhibiting proliferation and inducing apoptosis. The objective of the project was to investigate the migration of MCL cells towards fetal bovine serum and by this, study the impact of ONC201 on the migration of mantle cell lymphoma cells using chemotaxis assay. Theapoptotic effect of the drug was examined by detection of activecaspase-3 and cleaved PARP proteins using western blotting. The chemotaxis assay and western blot demonstrated that ONC201 inhibited migration of the mantle cell lymphoma cell lines at the concentration-dependent manner without inducing apoptosis in the cells at the investigated time point. The expression of cyclin D1 in the cells treated with ONC201 was also investigated after incubation period of 24 hours and it was observed that ONC201 plays a role in reducing cyclin D1 expression in mantle cell lymphoma cells.

Endometrial cancer is a disease with serious impact on the human population, but not much is known about genetic factors involved in this complex disease. Female BDII rats are genetically predisposed to spontaneous endometrial carcinoma, and the BDII inbred strain provides an experimental animal model for endometrial carcinoma development. In the present study, BDII females were crossed with males from two nonsusceptible inbred rat strains. Endometrial adenocarcinomas (EACs) developed in a proportion of the F1 and F2 progeny. We screened 18 EAC solid tumors and 9 EAC cell cultures for loss of heterozygosity (LOH) using fluorescent-PCR-based marker allelotyping methodology with 47 microsatellite markers covering the proximal part of rat chromosome 10 (RNO10). Conclusive evidence was obtained for LOH/deletion involving about 56 cM in the proximal part of RNO10 in DNA from six out of seven informative tumor cell cultures. Analysis of the solid tumors confirmed the presence of LOH in this part of RNO10 in 14 of 17 informative tumors. However, from the studies in the solid tumors it appeared that in fact three separate segments in the proximal part of RNO10 were affected. These three LOH/deletion regions were located approximately in cytogenetic bands 10q11-12, 10q22, and 10q24. © 2001 Wiley-Liss, Inc.

The rat provides valuable and sometimes unique models of human complex diseases. To fully exploit the rat models in biomedical research, it is important to have access to detailed knowledge of the rat genome organization as well as its relation to the human genome. Rat Chromosome 10 (RNO10) harbors several important cancer-related genes. Deletions in the proximal part of RNO10 were repeatedly found in a rat model for endometrial cancer. To identify functional and positional candidate genes in the affected region, we used radiation hybrid (RH) mapping and single- and dual-color fluorescence in situ hybridization (FISH) techniques to construct a detailed chromosomal map of the proximal part of RNO10. The regional localization of 14 genes, most of them cancer-related (Grin2a, Gspt1, Crebbp, Gfer, Tsc2, Tpsb1, Il9r, Il4, Irf1, Csf2, Sparc, Tp53, Thra1, Gh1), and of five microsatellite markers ( D10Mit10, D10Rat42, D10Rat50, D10Rat72, and D10Rat165) was determined on RNO10. For a fifteenth gene, Ppm1b, which had previously been assigned to RNO10, the map position was corrected to RNO6q12-q13.

The T55 rat radiation hybrid (RH) mapping panel has been reported to retain the entire rat genome at retention frequencies between 22% and 37%. However, we found that a small segment of rat chromosome 10 harboring at least four different genes, including Tp53, was completely absent from the panel (retention frequency = 0%). Two other markers located in the vicinity exhibited much reduced retention (2–6%). RH clones are generated by transferring highly fragmented DNA into a recipient cell. There might be a strong selection against the transfer and retention of chromosome segments harboring an intact Tp53, as the action of this gene might prevent proliferation and establishment of the RH clone. Our finding further suggests that unexpected low retention or absence of chromosome segments in an RH panel may represent indications that the segments harbor genes with important functions in cell proliferation control.

Using FISH and RH mapping a chromosomal map of rat chromosome 10 (RNO10) was constructed. Our mapping data were complemented by other published data and the final map was compared to maps of mouse and human chromosomes. RNO10 contained segments homologous to mouse chromosomes (MMU) 11, 16 and 17, with evolutionary breakpoints between the three segments situated in the proximal part of RNO10. Near one of these breakpoints (between MMU17 and 11) we found evidence for an inversion ancestral to the mouse that was not ancestral to the condition in the rat. Within each of the chromosome segments identified, the gene order appeared to be largely conserved. This conservation was particularly clear in the long MMU11-homologous segment. RNO10 also contained segments homologous to three human chromosomes (HSA5, 16, 17). However, within each segment of conserved synteny were signs of more extensive rearrangements. At least 13 different evolutionary breakpoints were indicated in the rat-human comparison. In contrast to what was found between rat and mouse, the rat-human evolutionary breaks were distributed along the entire length of RNO10.

Apis mellifera is an important pollinator for agriculture, with a large distribution across the globe, generalist feeding behaviour, and high efficiency as a pollinator. The recent decline of Apis mellifera is affected by deforestation, climate change, spillover of pathogens, and a high demand for honey. The gut microbiota of honey bees can be a key to improving the health of the colonies and stopping the decline. This project aims to determine whether the gut microbiota of honey bees changes over winter and analyze the similarities and differences in the gut microbiota of the Apis mellifera mellifera subspecies by comparing samples collected in early autumn and spring. The study will specifically focus on the impact of the honey bee's diet, which varies between honey and sugar-water intake. This was accomplished by sequencing the 16S rRNA gene using nanopore sequencing technology. As a result, it was determined that the gut microbiota changes during the winter season, and Bartonella may play a crucial role in honey bees’ well-being in winter. The honey-fed group contained the most diverse bacterial composition, including species that were not determined in the sugar-fed and reference groups. Thus, it is believed, the diet affected the gut microbiota composition. The honey-fed group also contained a novel species Novisyntrophococcus fermenticellae, which was not previously found in honey bees.

Megasatellites are polymorphic tandem repetitive sequences with repeat-units longer than or equal to 1000 base pairs. The novel algorithm Megasatfinder predicts megasatellites in the human genome. A structured method of analysing the algorithm is developed and conducted. The analysis method consists of six test scenarios. Scripts are created, which execute the algorithm using various parameter settings. Three nucleotide sequences are applied; a real sequence extracted from the human genome and two random sequences, generated using different base probabilities. Usability and accuracy are investigated, providing the user with confidence in the algorithm and its output. The results indicate that Megasatfinder is an excellent tool for the detection of megasatellites and that the generated results are highly reliable. The results of the complete analysis suggest alterations in the default parameter settings, presented as user guidelines, and state that artificially generated sequences are not applicable as models for real DNA in computational simulations.

Multiple sclerosis is a common neurological disorder, characterized by increasing disability over time for the affected patient. The disease is considered an autoimmune disorder in which the immune system causes damage to nerves in the central nervous system through demyelination and inflammation. It is currently not understood what causes the disease, but both genetic susceptibility as well as environmental- and lifestyle factors are thought to contribute to disease development.In this project, data of known disease-associated risk factors were used to characterize the processes through which they may alter the risk of disease development. Modules for each risk factor was derived from experimental data, using the MODifieR disease-module inference algorithms. Of the five different risk factors included, each module was analysed using the PASCAL tool and disease specific GWAS data to evaluate the relevance towards the disease.Based on the modules derived using the Clique Sum Permutation module inference method a consensus module comprising 126 genes was identified, which proved to be significantly enriched for disease associated SNPs (single nucleotide polymorphisms). Additionally, the risk-factor consensus module was compared to disease specific consensus genes previously obtained within the research group. The comparison showed a significant overlap, which indicates that the methodology may provide means of examine the impact of risk-factors in the context of complex disease.

In the field of neuroscience, being in love and feeling romantically attached to a partner is described as a dynamic process. Romantic love may be viewed as a motivational system, changing throughout time and place, fluctuating on the interest and motivation of the individual. Early memories and attachment towards a caregiver, lay the foundation for later attachment behavior, also known as attachment styles. In this thesis, an exploratory approach is present. The thesis aims to introduce and describe the neural correlates of romantic love and romantic attachment. Brain regions concerned with reward, emotion and thought processing, such as the reward circuitry network of the brain and the limbic system, are being investigated. So are other brain areas involved in romantic love and romantic attachment. Research findings suggest that brain areas responsible for affection, emotional control, learning, memory and social judgment are all involved in the complex processes of being in love and feeling romantically attached. These findings are represented by the involvement of the frontal lobe, cerebral cortex, limbic system, orbitofrontal cortex, and hippocampus, anterior cingulate cortex (ACC), ventral tegmental area (VTA), caudate tail, including the reward pathways of the brain. Distribution and regulation of neurotransmitters such as; vasopressin, oxytocin, dopamine, corticosterone and serotonin are all present in the state of romantic  attachment and romantic love. Overlapping evidence confirms the involvement of the reward circuitry network, together with the limbic system as crucial in the formation and maintenance of a romantic relationship.

Genforskningen har koncentrerat sig på att urskilja vilka gener som påverkar eller skapar vilka beteenden och tendenser. Det finns till exempel en enda gen SRY, som möjliggör förekomsten av manligt biologiskt kön (Mukherjee, 2018). Det finns en gen FOX P2 som har med språkförmågan att göra. Men det finns tusen andra gener som också har med manligt kön och/eller språkförmåga att göra, som tillåter eller förhindrar manligt kön att uppstå i en viss individ, som hindrar eller tillåter en viss individ i en viss familj med FOX P2 att kunna utveckla normalt tal. Forskningen kring relationer mellan gener och beteenden och fysiologiska egenskaper pågår globalt. Ovan nämnda exempel är två av tusentals.

Relationerna mellan gener är komplexa; en viss genuppsättning kan inte förutsäga visst beteende med precision. Ändå har till exempel den intensiva adhd-debatten präglats mycket av just behovet att konstatera vissa beteenden och tendenser som bestämda och givna ”av naturen”. I fallet adhd gäller dessutom att man hittills inte har intensivstuderat eller spårat avvikelser på gennivå.

Neurofysiologin rymmer numera mycket kunskaper om relationen mellan specifika gener och specifika egenskaper. Exempelvis visar autism och Aspberger specifika drag i nervcellerna. Hos personer med autism är enligt vissa studier de trådar som förbinder nervcellerna kortare än hos den fysiologiska normalindividen, och genen FOXP2 är frånvarande hos individer med vissa svårigheter i språklärande. Men hos adhd finner man inte neurofysiologiska särdrag.