This study investigates upper secondary school students’ opinions on working with a computer simulation in microbiology. During one lesson, the students got to work with the simulation and answer questions about what was happening in the simulation. Collection of data was made through observation, surveys and interviews. The result shows an overall positive attitude among the students towards working with simulations in biology class. Despite this, using simulations as a learning tool can not be recommended without reservation. It is of importance that the simulation to be used is carefully selected according to learning goals and that students are supported in their interpretation of the simulation. The most important support is the teacher explaining what the simulation is showing.

During osteoclast differentiation and bone resorption the redox status in the cell display a decrease in reduction and a shift to an oxidized state. Structure, metabolism and function are some of the extensive changes that cells undergo during differentiation which alters both the extra- and intracellular redox environment. Osteoclasts express enzymes such as TRAP and NADPH oxidase which generates reactive oxygen species (ROS). ROS are molecules formed by oxygen reduction which gives these radicals at least one unpaired electron and makes them very reactive and chemically unstable. These are factors which stimulates differentiation of osteoclasts and bone resorption. RAW 264.7 cells will differentiate to osteoclasts when stimulated with RANKL and to activated macrophages when stimulated with LPS. The aim of this project was to analyze if the redox environment is affected during differentiation of RAW 264.7 cells to osteoclasts and macrophages. The reason for this was that we aimed to se if RAW 264.7 cells could be used as an in vitro system to study the effects of redox changes in osteoclasts and macrophages and their activation. Results from Western blot showed that protein expression of the Cysteine/Glutamate transporter xCT was up regulated with LPS and downregulated with RANKL. Results from the GSH/Cys assay show that the treatments with redox modulators did not affect the levels of GSH and Cys to a measurable extent. However the levels increased for both intracellular and extracellular GSH and Cys forms at day 4 in the control and stimulated cells. Addition of the disulfide reductant DTT affected differentiation to osteoclasts, leading to smaller osteoclasts probably due to interference with fusion of mononuclear pre-osteoclasts. Thus, down regulation of the xCT transporter could be an important mechanism to maintain a low level of free thiols shown to interfere with the differentiation to osteoclasts.

The aim of this work was to determine if the protein Majastridin found in the proteobacterium Rhodobacter blasticus has a functional relative in the hypothetical protein Q1U8S3/ B3XNV1 found in Lactobacillus reuteri. To be able to study the protein, it was overexpressed in  E. coli-cells and purified. As a starting material, the L. reuteri Q1U8S3 gene previously cloned into a pET SUMO vector from Invitrogen was used. The produced protein will be a fusion protein containing a His6-tag, a SUMO-protein and the protein of interest. A nickel column in combination with a gel filtration column was used to purify the protein and after purification, crystallization experiments were set up using standardized kits.

Mechanisms involved in the clearance of chylomicrons and aspects of the interactions at the vascular endothelium were studied in the rat. The poly-anion heparin, known to release lipoprotein lipase (LPL) from the vascular endothelium, enhanced the clearance of chylomicrons. Five minutes after heparin injection, the clearance of chylomicron triglycerides and retinyl esters was markedly accelerated. The rapid initial clearance was followed by a slower clearance of heavily lipolyzed chylomicrons. In contrast, one hour after heparin the clearance of both triglycerides and retinyl esters was retarded. This decreased removal of chylomicrons coincided with a decrease in the heparin releasable LPL activity, indicating that the previous release to plasma by heparin had resulted in net loss of functional LPL in the tissues. The poly-cation protamine released hepatic lipase and some LPL from their binding sites to plasma. One hour after protamine, plasma triglyceride levels were increased, indicating that chylomicron removal was impeded. It has been speculated that protamine inactivates LPL in vivo, but this was not the case. Ten minutes after injection of protamine normal amounts of LPL could be released by heparin. Thus, the accumulation of plasma triglycerides was not due to a rapid inactivation of LPL by protamine. LPL has specificity for sn-1,3-ester bonds. To investigate if this specificity is important in vivo, a lipid emulsion containing medium-chain fatty acids (MCFA) in the sn-1,3-position and long-chain fatty acids (LCFA) in the sn-2-position was synthesized, as well as an emulsion containing MCFA-TG mixed with LCFA-TGs (MMM/LLL). In vitro experiments showed large differences in the hydrolysis of the emulsions, but in vivo there were only small differences in the metabolism. To further study if lipid emulsions are cleared by the same mechanisms as chylomicrons, an emulsion was made by the same formulation as Intralipid® with addition of 3H-triolein and ,4C-cholesteryl ester. As measured by the removal of cholesteryl esters, the emulsion was cleared at the same rate as was chylomicrons. The triglyceride label was, however, removed more slowly from the emulsion droplets than from chylomicrons. Together with the lower recirculation of labeled free fatty acids (FFA) in plasma, this suggests that there was less lipolysis of the emulsion. The current view that removal of lipid emulsions in vivo is mainly dependent on LPL-mediated hydrolysis might thus not be correct. To further analyze the metabolism of chylomicrons, a compartmental model was developed. In this process, the distribution volume for chylomicrons was shown to be larger than the blood volume, a model for the metabolism of FFA in the rat was validated, and the full tissue distribution of injected chylomicrons was determined. According to the model, about half of the triglyceride label was removed from the circulation together with the core label while for the emulsion this number was about 80 %. In fasted rats all labeled fatty acids appeared to mix with the plasma FFA pool, while in fed rats about one-fifth of the fatty acids did not mix with the FFA but was apparently channeled directly to tissue metabolism. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p> / digitalisering@umu.se

Induced pluripotent stem cells (iPSCs) can self-renew and differentiate into many other cell types. IPSCs are derived from somatic cells, and upon reprogramming, they share an expression profile similar to embryonic stem cells (ESCs). Among their many applications, iPSCs are an advantageous tool for disease modelling, offering an accurate system to study human molecular networks associated with specific phenotypes. Moreover, progress in genome editing technologies improved the possibilities for investigation of genotype-phenotype relationship for diseases characterized by defined genetic variants. Indeed, CRISPR/Cas9 edited iPSCs lines from healthy donors offer the possibility to investigate molecular networks with comparison to an isogenic control line. Furthermore, the ability of iPSCs to differentiate into neural cells, makes them a good model for studying neurodevelopmental diseases (NDDs). NDDs are characterized by heterogenous genetics and phenotypes. Heterozygous gene variants in the alpha 1 subunit of the sodium-voltage gated channel 1.1 (SCN1A) and in Neurochondrin (NCDN) have been associated with epilepsy. While many variants defining NDDs are associated with genes of transcriptional networks, e.g. the zinc-finger E-box binding homeobox 2 transcription factor (ZEB2) or the RPB1 subunit of RNA polymerase II complex (POLR2A). Although published animal model systems are available, there is a lack of human derived systems to investigate the gene function in disrupted molecular networks in NDDs. In this project, IPSCs deficient for SCN1A, NCDN, ZEB2 and POLR2A were generated using CRISPR/Cas9. To further evaluate the quality of the cell lines as iPSCs model, a POLR2A knock down (K.D.) line carrying a 4 bp insertion and a ZEB2 knock out (K.O.) line carrying a 790 deletion were characterized. Pluripotency and differentiation potential were confirmed by flow cytometry analysis, immunostaining, and qPCR. Both lines maintained genome integrity and editing in the top predicted off targets was excluded with PCR and Sanger sequencing screening. Furthermore, ZEB2 is involved in induction of neural crest cells (NCC); ZEB2 deficient line and the control behave similarly after a week of NCC differentiation. In contrast, POLR2A variants suggest slowing of transcription compared to the wild-type, therefore rate of transcription was measured performing an activity assay. No relevant differences between POLR2A K.D. and control line were observed in transcription rate of early pre-mRNA.

Apis mellifera, otherwise known as the common honey bee, is an incredibly important social animal. Their important role in the world makes studying them of great importance. Their stomachs can be divided into three parts, the foregut, the midgut and the hindgut. The mouth and crop are located in the foregut, the midgut encompasses the ventriculus and the hindgut is made up of the ileum and rectum. Each part of a honey bee intestine hosts a different community of bacteria that vary in proportion with age, caste and season. These microbiota are essential for a honey bees mood, development and overall health. No two authors agree completely as to what the a honey bee’s gut microbiota is. In this thesis study the intestinal tract microbiome of four bee colonies, two of which belonging to the Apis mellifera carnica subspecies and two to the Apis mellifera buckfast subspecies, were sequenced. All four are from the same region in Sweden, Uddevalla. Many issues were found during this project, including one sick colony, but through them a more thorough and guaranteed method to sequence these honey bee intestinal bacteria was developed. The results of the sequencing showed that there is indeed a major difference in these intestinal communities even in bees from the same region or from the same subspecies. A possible culprit for the diseased colony was found.

Amyloidosis is a group of diseases where misfolded proteins aggregate in the body. These aggregates are called amyloid and today there are 37 different known amyloid proteins. Diagnosis of amyloidosis is done by Congo Red staining to find amyloid, and typing with immunohistochemistry together with mass spectrometry. An earlier study found an unknown amyloid fibril protein in the Ligamentum flavum of patients suffering from lumbar spinal stenosis (LSS), and another study found specifically amyloid of the precursor protein ApoA-I in the same tissue of patients with LSS. The aim of this project is to type an unknown amyloid fibril protein through immunohistochemistry and mass spectrometry as well as isolating the ApoA-I protein to be able to do further tests on the protein. The unknown amyloid protein was not characterized in this study, but it gave indications on what it is not and how to continue the search in future studies.

<p>Cells experience numerous external signals which they must respond to. Such signals arriving at the cell surface are transduced via various signal transduction pathways and often ultimately result in regulation of transcription. NF-κB is a family of transcription factors involved in the regulation of genes important for processes such as immune and inflammatory responses, cell growth, development and cell survival. NF-κB proteins are normally kept inactive in the cytoplasm due to masking of their nuclear localisation signal (NLS) by inhibitory IκB proteins. A large number of stimuli lead to the activation of IκB-kinase (IKK). Active IKK phosphorylates IκB and thereby labels it for ubiquitination and, subsequently, degradation by the proteasome. Liberated NF-κB enters the nucleus, where it takes part in the regulation of its target genes. </p><p>Calmodulin (CaM) is a ubiquitous Ca2+-binding protein which is considered to be the predominant intracellular Ca2+ sensor. CaM plays a major role in the Ca2+-dependent regulation of a wide variety of cellular processes, including transcription. CaM regulates transcription both indirectly through CaM-dependent kinases and phosphatases and directly through interaction with transcription factors.</p><p>CaM was found to bind directly and in a Ca2+-dependent fashion to the two NF-κB family members c-Rel and RelA. The CaM-NF-κB interactions were strongly enhanced by NF-κB activating stimuli and this enhancement was blocked by the addition of IκB, suggesting that c-Rel and RelA can bind CaM after their signal-induced release from IκB. Compared to wild-type c-Rel, CaM binding-deficient mutants were shown to exhibit an increased nuclear accumulation and transcriptional activity on Ca2+-regulated cytokine promoters. The results suggest that CaM can inhibit transport of c-Rel, but not of RelA, to the nucleus and thereby differentially regulate the activation of NF-κB proteins following cell stimulation. CaM was also found to affect NF-κB activity indirectly through the action of a CaM-dependent kinase (CaMK). Studies of the events leading to IκBα phosphorylation revealed that CaM and CaMKII inhibitors blocked phorbol ester induced activation of IKK. Furthermore, CaM and CaMKII inhibitors also blocked T cell receptor/CD3 induced IκBα degradation, and expression of an inhibitor-resistant derivative of the γ isoform of CaMKII caused the inhibitors lose their effect on phorbol ester induced IκBα degradation. Finally, expression of a constitutively active CaMKII resulted in the activation of NF-κB. These results identify CaMKII as a mediator of IKK activation, specifically in response to T cell receptor/CD3 and phorbol ester stimulation.</p><p>In conclusion, this thesis describes the identification of CaM as a dual regulator of NF-κB proteins, acting both directly and indirectly to affect the activity of this family of transcription factors.</p>

<p>Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and visual problems due to a progressive and selective loss of neurons within the cerebellum, brainstem and retina. The disease is caused by the expansion of a CAG repeat in the first coding exon of the SCA7 gene, resulting in an expanded polyglutamine domain in the N-terminal part of ataxin-7, a protein of unknown function.</p><p>To expand our knowledge of the ataxin-7 protein and the mechanism by which mutant ataxin-7 causes disease, we have studied the expression and function of both the normal and the mutated ataxin-7 protein. </p><p>Ataxin-7 expression was examination in brain and non-CNS tissues from SCA7 patients and age-matched controls. Expression was predominantly nuclear in neurons throughout the brain of both healthy and SCA7 individuals. We also observed aggregation of mutant ataxin-7 in the nuclei of neurons. No obvious difference in the expression level of ataxin-7 or the formation of aggregates could be observed between affected and non-affected brain regions in SCA7 patients. Based on these findings, we could conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression levels or to the formation of ataxin-7 aggregates.</p><p>To widen our studies on ataxin-7 expression, we isolated and characterized the mouse SCA7 gene homolog. Cloning of the mouse SCA7 gene revealed two SCA7 mRNA isoforms that were highly homologous to their human counterparts. Immunohistochemical analysis also revealed a conserved expression pattern of ataxin-7 in adult mouse brain. In addition, ataxin-7 expression was observed during embryonic development in brain as well as in several non-neuronal tissues such as heart, liver and lung. </p><p>Besides SCA7, eight neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1-3, 6 and 17, DRPLA, SBMA and Huntington’s disease. The polyglutamine disorders have many features in common and a common pathological disease mechanism involving transcriptional dysregulation has been proposed. To investigate the possible involvement of transcriptional dysregulation in SCA7 pathology, we analyzed the effects of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP, the Purkinje cell-expressed nuclear receptor RORα1 or a basic TATA promoter. As previously shown for other polyglutamine disease proteins, expansion of the polyglutamine domain in ataxin-7 leads to reduced transcription. Surprisingly, strong repression of CBP-mediated, RORα1-mediated and basal transcription was also observed with wild-type ataxin-7, suggesting that the normal ataxin-7 protein may have a role in transcriptional regulation. </p>

<p>The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment. </p><p>We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002. </p><p>In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes. </p><p>The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.</p>