The development of diabetes mellitus depends on the balance between beta-cell proliferation and death. As mitogen-activated protein kinases (MAPK) may control this balance, the aim of this study was to investigate the events leading to MAPK activation in beta-cells and the consequences of these events. Overexpression of the SH2-domain containing adaptor protein Shb resulted in the assembly and activation of multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpression cells. This was paralleled by an attenuated activation of the MAP kinases ERK1/2. Thus, Shb-induced alterations in the IRS-1/PI3K/Akt/ERK pathway might explain the increased proliferation and apoptosis of beta-cells overexpressing Shb. The importance of the MAP kinase p38 in nitric oxide- and cytokine-induced beta-cell death was also investigated. Knock-down of p38 expression resulted in a lowered cell death rate in response to a nitric oxide donor. In transient transfections MKK3 over-expression resulted in increased p38 phosphorylation in RIN-5AH cells. In addition, a short-term MKK3 expression resulted in increased cytokine-induced cell death. A nitric oxide synthase inhibitor abolished the MKK3-potentiating effect on cytokine-induced cell death and inhibitors of phosphatases enhanced MKK3-stimulated p38 phosphorylation. Finally, as the dominant negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In further support for an MKK3/6-indepedent mechanism, the adaptor protein TAB1 significantly increased the cytokine- and nitric oxide-stimulated phosphorylation of p38. The TAB1-mediated activation of p38 was paralleled by a compensatory inhibition of ERK and JNK. In summary, p38 MAPK, activated mainly by TAB1, promotes, at least in part, beta-cell death in response to cytokines or nitric oxide.

Innate immunity relies on a repertoire of germline-encoded non-rearranging pattern recognition receptors that bind to invariant microbial surface molecules. This event initiates a number of signal transduction cascades that lead to humoral and cellular defense responses like synthesis of antimicrobial peptides, phagocytosis and coagulation – mechanisms that efficiently fight infectious microorganisms and have been evolutionary conserved to exist in parallel with the antibody-based adaptive immunity found in vertebrates. The fruit fly, Drosophila melanogaster represents a widely used animal model for studies of a pristine innate immune system. Its immune responsive intracellular signalling pathways display a high degree of similarity with the NF-κB /Rel-signalling pathways that mediate the inflammatory response in mammals. Insects are also vectors for medically important parasitic diseases which can trigger immune responses in the vector so basal knowledge about the regulation and function of insect immune systems can contribute to our understanding of inflammation and microbial disease in higher animals and open new strategies for biological vector control. Drosophila hemocytes play a key role in executing and coordinating local and systemic defenses in response to infection. This thesis describes in vitro studies of Drosophila gene expression in response to bacterial infection using the larval hemocyte-like cell line – mbn-2. Our results show that immune challenge with bacterial cell wall components and intact live bacteria induces differential gene expression that gives clues to how cellular immune responses could be activated and regulated.

Diabetes mellitus is a bihormonal disorder with hyperglycemia due to deficiency of insulin and hypersecretion of glucagon. To improve diabetes treatment it is important to clarify the signal transduction of glucagon secretion. The cytoplasmic Ca2+ concentration ([Ca2+]i), an important determinant of hormone secretion, and the membrane potential were recorded in individual mouse α-cells. Glucagon and insulin secretion were measured from mouse islets and glucagon secretion from hamster glucagonoma cells. Glucose inhibited glucagon secretion from islets and glucagonoma cells with maximal effect at 7 mM, indicating a direct action on the α-cells. High concentrations of glucose paradoxically stimulated glucagon secretion. Whereas glucose inhibition of glucagon release was associated with lowering of [Ca2+]i, stimulation of secretion at high glucose concentrations was Ca2+-independent. Adrenaline, which is a potent stimulator of glucagon secretion, increased [Ca2+]i by α1- and β-adrenergic mechanisms involving mobilization of intracellular Ca2+ from the endoplasmic reticulum (ER) and influx of the ion across the plasma membrane. Ca2+ mobilization could be attributed to generation of inositol 1,4,5-trisphosphate and cAMP, and influx occurred through voltage-dependent L-type channels activated by a depolarizing store-operated current. Glucose hyperpolarized the α-cells and inhibited adrenaline-induced [Ca2+]i signalling. At 3 mM, glucose had a pronounced stimulatory effect on Ca2+ sequestration in the ER, shutting off store-operated Ca2+ influx. The α-cells express ATP-regulated K+ channels but pharmacological blockade of these channels neither interfered with the hyperpolarizing and [Ca2+]i lowering effects of glucose nor with the inhibition of glucagon secretion. In contrast, activation of the depolarizing store-operated mechanism prevented glucose-induced, hyperpolarization, lowering of [Ca2+]i and inhibition of glucagon secretion. It is proposed that adrenaline stimulation and glucose inhibition of glucagon release involve modulation of a store-operated depolarizing current. The U-shaped dose response relationship for glucose-regulated glucagon secretion may explain the hyperglucagonemia in diabetes.

The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear. Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell. The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects. The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane. In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

Specification of the many unique neuronal subtypes found in the nervous system depends on spatiotemporal cues and terminal selector cascades, mostly acting in sequential combinatorial codes of transcription factors (TFs) to dictate cell fate. Out of 10,000 cells in the Drosophila embryonic ventral nerve cord (VNC), only 28 cells selectively express Nplp1. The Nplp1 neurons in the Drosophila VNC can be subdivided into the thoracic ventro-lateral Tv1 and the dorsal-medial dAp neurons. Nplp1 expression in both cell subtypes is activated by the same terminal selector cascade: col > ap/eya > dimm > Nplp1. However Tv1 and dAp neurons are generated by different neuronal progenitors (neuroblasts, NB), and depend on different upstream cues to activate the cell specification cascade. The Tv1 cells are generated by NB5-6T, and in these cells the Nplp1 terminal selector cascade is triggered by spatio-temporal input provided by Antp/hth/exd/lbe/cas. Our studies identified that NB4-3 gives rise to the dAp cells and that the Nplp1 terminal selector cascade in dAp cells is activated by Kr/pdm>grn. I demonstrated how two different spatio-temporal combinations can funnel on a shared downstream terminal selector cascade to determine a highly related cell fate, in different regions of the VNC. I tested this scenario at the molecular level, by identification of cisregulatory modules (CRMs) for the main factors involved in the Nplp1 terminal selector cascade. Intriguingly, I found that col is under control of two separate CRMs, which are controlled by either Antp/hth/exd/lbe/cas in the NB5-6T lineage, and Kr/pdm/grn in the NB4-3 lineage. In addition, CRISPR deletion of the endogenous col CRMs did not result in loss of Col and Nplp1, indicating that col might be under control of more, yet unidentified CRMs. Nplp1 is expressed in one out of four cells in the thoracic Apterous cluster (Ap cluster); the Tv1 cell. The allocation of the right cell fate to each of the four Ap cluster cells, is regulated by the sub-temporal cascade including the factors Sqz/Nab/Svp, acting downstream of the temporal factor Cas. The sub-temporal factors have a repressive action on Col and Dimm, and thus on the terminal selector cascade regulating Nplp1 expression in the Tv1  cell. We demonstrated that the late and Tv1 specific expression of the early temporal factor Kr suppresses Svp in the Tv1 cell and allows for the progression of the Nplp1 cell fate specification cascade. Hence, early temporal factors involved in temporal progression of neuronal progenitors, can be re-utilized late and postmitotically to specify cell fate. It is tempting to speculate that similar mechanisms act to generate similar cell fate in different regions of the CNS, as well as the issue of sub-temporal multitasking, are common features both in Drosophila and higher organisms.

<p>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.</p><p>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.</p><p>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.</p>

Denna avhandling belyser hur elevdiskussioner om begreppskartor kan bidra till en ökad förståelse för innebörden av de utvalda biologiska begreppen hos de elever som deltar. Som bakgrund till avhandlingen redovisas forskningsresultat vilka visar ett antal svårigheter elever kan ha inom det cellbiologiska ämnesområdet. Två exempel på detta är de många ämnesspecifika begreppen och att det i cellbiologin finns flera organisationsnivåer. En viktig orsak till dessa svårigheter är naturvetenskapens karaktär, där konkreta fenomen förklaras med abstrakta modeller och teorier. I bakgrunden finns också tidigare forskning som visat att elevers diskussioner med varandra har positiv inverkan på lärandet. Syftet med studierna är att undersöka vilken typ av samtal som uppkommer i en elevgrupp vid konstruktionen av en begreppskarta och vad deltagarna tyckte om uppgiften.Resultat från två delstudier redovisas och diskuteras i avhandlingen. Den första av dessa studier redovisas i Artikel I. Insamlingen av data gjordes då eleverna genomförde en uppgift där de, under diskussioner i små grupper, konstruerade begreppskartor inom ämnet cellandning. Eleverna gick i en avgångsklass på det naturvetenskapliga programmet. Ytterligare data samlades in vid enskilda intervjuer med elever efter genomförandet. Analysen inriktades på i vilken utsträckning uttalanden i diskussionerna var biologiskt korrekta, vilken typ av samtal grupperna genomförde och hur deltagarna upplevde uppgiften. Resultaten visar att många uttalanden är helt eller delvis korrekta. Samtalen innehöll delar där eleverna enbart samtyckte till vad någon annan sagt, men också delar där deltagarna argumenterade för sitt synsätt. Eleverna uttryckte en positiv upplevelse av uppgiften i de efterföljande intervjuerna.Den andra studien redovisas i Artikel II. Studien gjordes i en klass som studerade genetik. Även här diskuterade elevgrupper hur en begreppskarta skulle konstrueras. Data insamlades under diskussioner och vid efterföljande, enskildavintervjuer. Analysen inriktades på tidigare kända svårigheter i genetik, det naturvetenskapliga innehållet i samtalen, samtalens karaktär och hur eleverna upplevde uppgiften. Resultaten visar att elevernas diskussioner utvecklas mot det naturvetenskapliga synsättet genom samtal där de flesta eleverna deltar aktivt och också uttrycker egna synpunkter. I intervjuerna uttrycker eleverna sig positivt om arbetsuppgiften och flera anser att de fått en bättre förståelse av ämnet.Sammanfattningsvis visar resultaten i avhandlingen att samtal om begreppskartor kan bidra till en förbättrad förståelse i cellbiologi.

Higher plants cannot move to a more favorable place when the environmental conditions are changing. To adapt to changes in light, temperature and access to water the plants had to evolve special mechanisms at the molecular level. Post-translational modifications of proteins, like phosphorylation, often serve as “on-and-off” switches in regulation of cellular activity and may affect protein-protein interactions. Photosynthesis in higher plants is regulated by reversible protein phosphorylation events, in a unique light- and redox-controlled system. Several biochemical methods are effectively used for characterization of phosphorylated proteins in photosynthetic membranes. Nevertheless, mass spectrometry is the most effective technique when it comes to identification of exact phosphorylation site(s) in the protein sequence, which is the ultimate evidence of protein phosphorylation. The same tandem mass spectrometry analysis identifies other in vivo post-translational modifications as well, such as acetylation of the N-terminus of mature protein. To study membrane proteins is a challenging project. In the present work the “shaving” of surface-exposed part of the membrane proteins, where phosphorylation occur, is used. In combination with mass spectrometry, this technique does not require the use of radioactive labeling or antibodies. The present work in spinach and Arabidopsis thaliana has identified and characterized several known phosphoproteins, new phosphorylation sites in well-known photosynthetic proteins, as well as two phosphoproteins previously unknown to be present in the photosynthetic membrane. Several photosystem II (PSII) core proteins become phosphorylated in their N-termini (D1, D2, CP43, PsbH), process involved in the regulation of the repair cycle of photo-damaged PSII complexes. The protein-protein interactions between PSII and its light harvesting complex (LHCII) seem to be affected by phosphorylation events in the interface area. In higher plants, phosphorylation sites have been identified in LHCII polypeptides, in one of the proteins (CP29) present in the interface area, as well as in the peripheral TSP9 protein. The TSP9 protein is unique among photosynthetic phosphoproteins, since it is a plant-specific soluble protein that becomes triple-phosphorylated in the middle part of the protein. It is also shown that photosystem I (PSI) is subjected to protein phosphorylation. The extrinsic PSI subunit PsaD becomes phosphorylated in its N-terminus. In addition, the latest characterized subunit of PSI, PsaP, is identified as a phosphoprotein. PsaP is an intrinsic protein assembled on the same side of the PSI complex as LHCII attaches. Several kinases are involved in phosphorylation of photosynthetic proteins, some more specific to PSII core proteins whereas others recognize LHCII proteins better. The STN8 kinase does not phosphorylate LHCII proteins, but is involved in the phosphorylation of the PSII core proteins D1, D2, CP43 and PsbH. STN8 is light-activated and is also specific in phosphorylation of threonine-4 (Thr-4) in the PsbH protein, but only after another kinase has phosphorylated Thr-2 first. A common feature of all kinases in plant photosynthetic membranes is the specificity for Thr residues and that the phosphorylation reactions occur in the N-terminal sequence of the proteins, except for the TSP9 protein. Nowadays, research is on the way to solve the complex network of regulation of photosynthetic activity via protein phosphorylation, but far more efforts are needed to get a complete view of the importance of all phosphorylation events and enzymatic specificity.

Parkinson’s disease (PD) is a progressive neurodegenerative disease and characterized by the loss of dopaminergic (DA) neurons in the substantia nigra in the midbrain. The causes of the disease are still unknown. The most commonly used treatment is administration of L-DOPA, however, another possible treatment strategy is to transplant DA neurons to the striatum of PD patients to substitute the loss of neurons. Clinical trials have demonstrated beneficial effects from transplantation, but one obstacle with the grafting trials has been the variable outcome, where limited graft reinnervation of the host brain is one important issue to solve. To improve and control the graft DA nerve fiber outgrowth organotypic tissue cultures can be utilized. Cultures of fetal ventral mesencephalon (VM) have been used to investigate astrocytic migration and dopamine nerve fiber formations at different time points and under varying conditions to study how to control nerve fiber formation. The early appearing DA nerve fibers as revealed by tyrosine hydroxylase (TH) –immunoreactivity, form their fibers in the absence of glial cell bodies, are not persistent over time, and is called non-glial-associated TH-positive nerve fiber outgrowth. A monolayer of astrocytes guides a second persistent subpopulation of nerve fibers, the glial-associated TH-positive nerve fiber formation. Investigations of the interactions between the astrocytic migration and nerve fiber formations were made. In embryonic (E) day 14 VM cultures the mitosis of the astrocytes was inhibited with the antimitotic agent β-D-arabinofuranoside. The results revealed decreased astrocytic migration, reduced glial-associated TH-positive outgrowth, and enhanced presence of the non-glial-associated TH-positive outgrowth in the cultures. Thus, astrocytes affect both the non-glial- and the glial-associated growths by either its absence or presence, respectively. The astrocytes synthesize proteoglycans. Therefore the nerve fiber formation was studied in VM or spinal cord cultures treated with the proteoglycan blockers chondroitinase ABC (ChABC), which degrades the proteoglycans, or methyl-umbelliferyl-β-D-xyloside (β-xyloside), which blocks the proteoglycan synthesis. β-xyloside inhibited the migration of the astrocytes and the outgrowth of the glial-associated TH-positive nerve fibers in both VM and spinal cord cultures, whereas ChABC treatment had no effect in E14 VM or spinal cord cultures. E18 VM and spinal cord cultures were evaluated to investigate how the different developmental stages influence astrocytes and the two nerve fiber formations after 14 DIV. No nerve fiber formation was found in E18 VM cultures, while the non-glial-associated nerve fiber outgrowth was obvious as long and robust fibers in E18 spinal cord cultures. The astrocytic migration was similar in VM and spinal cord cultures. β-xyloside and ChABC did not affect nerve fiber growth but astrocytic migration in E18 VM cultures, while no effects was found in the spinal cord cultures. However, the neuronal migration found in control cultures was abolished in both VM and spinal cord cultures after both ChABC and β-xyloside. Neuroinflammation plays a critical role in the development of PD. Increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) are observed in postmortem PD brains and the levels of TNFα receptors on circulating T-lymphocytes in cerebrospinal fluid of PD patients are increased. The effects of TNFα were studied on E14 VM cultures. The outgrowth of the non-glial-associated TH-positive nerve fibers was inhibited while it stimulated astrocytic migration and glial-associated TH-positive nerve fiber outgrowth at an early treatment time point. Furthermore, blocking the endogenous levels of TNFα resulted in cell death of the TH-positive neurons. Furthermore, cultures of E14 mice with gene deletion for the protein CD47 were investigated. CD47 is expressed in all tissues and serves as a ligand for the signal regulatory protein (SIRP) α, which promotes e.g migration and synaptogenesis. CD47-/- cultures displayed massive and long non-glial-associated TH-positive nerve fiber outgrowth despite a normal astrocytic migration and the presence of glial-associated TH-positive nerve fiber outgrowth. For the first time, it was observed that the non-glial-guided TH-positive nerve fiber outgrowth did not degenerate after 14 DIV. Taken together, there is an interaction between astrocytes and TH-positive nerve fiber formations. Both nerve fiber formations seem to have their task during the development of the DA system.

Cell motility is a fundamental process, enabling cells to migrate, for instance during embryogenesis, tissue repair and defense. Force is generated by two protein systems, which also participate in cell proliferation, control macromolecular and organelle distribution and determine the fine structure of the cell interior. The major components of these are actin and tubulin, respectively, and they are referred to as the microfilament and the microtubule systems. This thesis focuses on tropomyosin, one of many microfilament associated proteins coupled to actin dynamics and organization and expressed in several isoform variants. Altered distribution and isoform expression of tropomyosin are signatures of malignant cells and are dealt with in the current thesis. The presence of tropomyosin isoforms in protruding lamellipodia of migrating cells is demonstrated, and a method to fractionate tropomyosin depending on its organization in an easily extractable, and a more tightly bound cytoplasmic form is presented. Analysis of the loosely associated tropomyosin fraction by gel filtration chromatography revealed that most of the tropomyosins in this fraction exist in a multimeric form. It was also observed that the distribution of tropomyosin varied between non-transformed and transformed cells with most of the isoforms enriched in the loosely bound fraction in the latter category of cells. Possibly this reflects the extensive reorganization of the microfilament system observed in cancer cells and which, depending on the context, can be normalized by introduction of certain tropomyosin isoforms. Many anti-cancer drugs target the microtubule system, inhibit cell division and promote apoptosis. Here it is shown that picropodophyllin, which has promising anticancer properties has a destabilizing effect on microtubules and via the microfilament system causes cells to detach from their substratum. Furthermore, picropodophyllin interferes with stimulation of the insulin-like growth factor receptor, which is involved in growth stimulation, differentiation and survival and whose expression is up-regulated in cancer cells.