Type 1 diabetes is a disease resulting from the progressive immune-mediated destruction of insulin producing β-cells. In order to understand more about diabetes we need to understand the mechanisms governing β-cell death. The leukemia drug Gleevec is a tyrosine kinase inhibitor that targets c-Abl. Surprisingly, Gleevec also counteracts Type 2 diabetes and acts as a cell death inhibiting agent, via inhibition c-Abl. Since both Type 1 and Type 2 diabetes are characterized by an increased β-cell death, and the role of c-Abl is unknown in β-cells, we wanted to investigate the following: 1.Does Gleevec act via inhibition of c-Abl in β-cells? 2.Can Gleevec treatment prevent beta-cell death and diabetes? 3.Which downstream signaling pathways are affected by Gleevec? In paper I, in order to determine whether Gleevec acts by inhibiting c-Abl, we used RNA-interference. Interestingly, siRNA against c-Abl produced by recombinant Dicer mediate almost complete and non-toxic silencing of c-Abl mRNA in dispersed islet cells and conferred protection from streptozotocin and cytokines. In paper II we show that Gleevec protects β-cells from nitric oxide, pro-inflammatory cytokines and streptozotocin in vitro and that Gleevec can prevent diabetes development in the NOD mouse and the streptozotocin-injected mouse. We also present the hypothesis that Gleevec induces a state resembling ischemic preconditioning. Paper III presents an additional mechanism by which Gleevec might improve β-cell survival, i.e. via the inhibition of the downstream stress-activated protein kinase c-Jun N-terminal kinase (JNK), the activity of which has been implicated in β-cell death signaling pathways. In paper IV we explore the interactions between the adaptor protein Shb and c-Abl. We presently show an association between Shb-c-Abl and that Shb is a substrate for the c-Abl kinase that might regulate stress-induced c-Abl activity.

There is a substantial intracellular degradation of insulin in pancreatic islets. This may be a physiological process, which, in correspondence with biosynthesis and secretion of insulin, would optimize the secretory granule content of the pancreatic β-cell. Insulin degradation may be effected by crinophagy, a process where secretory granules fuse with lysosomes. The general aim of this thesis was to investigate possible control mechanisms for intracellular degradation of insulin and crinophagy in isolated pancreatic islets. In islets incubated at low glucose concentrations there was an insulin degradation and this correlated well with the ultrastructural findings, where a lot of secondary lysosomes containing secretory granules were found. In islets incubated at a high glucose concentration there was no insulin degradation and the ultrastructure revealed only a few insulin granules and mostly primary lysosomes, indicating that there was no crinophagic activity. With interleukin-1β the islet insulin degradation, nitric oxide production and prostaglandin E2 production were increased. The effects were abolished either by inhibition of inducible nitric oxide synthetase by aminoguanidine, or by the specific cyclooxygenase-2 inhibitor rofecoxib. These findings indicate that there is a connection between the intracellular degradation of insulin, production of nitric oxide and cyclooxygenase-2 activation. The nitric oxide donor DETA/NO enhanced the intracellular degradation of insulin and cyclooxygenase-2 activation with subsequent production of prostaglandin E2, suggesting that the link between nitric oxide and insulin degradation may be a cyclooxygenase-2 activation and subsequent prostaglandin E2 production. With corticosterone added to islet incubations the insulin degradation decreased, which paralleled with a diminished crinophagy and formation of prostaglandin E2. With progesterone there was instead an increase in insulin degradation and crinophagy and an increased formation of prostaglandin E2. These effects were abolished by mifepristone, an inhibitor of intracellular corticosterone and progesterone receptors. This suggests that the effects from these steroids are exerted via a change in islet gene expression and cyclooxygenase-2 activation. It was also concluded that phospholipase A2 is involved in insulin degradation and that the isoform secretory phospholipase A2 may be involved in triggering this process. This suggests that cyclooxygenase-2 activation with a subsequent production of prostaglandin E2 may provide a control mechanism for intracellular degradation of insulin and crinophagy in pancreatic islets.

Profilin is a key regulator of the microfilament system. It binds to actin monomers in a 1:1 complex, forming the profilin:actin complex, which is the major precursor of actin for filament formation in vivo. The distribution of profilin has been studied in a variety of cells. It is present not only in the cytoplasm but also in the nucleus. In the cytoplasm, it is evenly distributed in a dotted pattern, which is concentrated at the edge of advancing lamellipodia and in the perinuclear region. In the nucleus, it is localized to Speckles and Cajal bodies. However, the distribution of the profilin:actin has not been possible to establish due to the lack of specific reagents. In this thesis I present the localization of the profilin:actin complex and demonstrate the importance of profilin during cell migration. The distribution of the profilin:actin complex was studied using affinity purified antibodies generated against a covalently coupled variant of profilin:actin in colocalization experiments with VASP and the Arp2/3 complex. In both cases, close co-distribution with profilin:actin was found. In order to study the role of profilin in vivo in migratory cells, I used the siRNA-technique to deplete profilin from motile mouse melanoma B16 cells. The particular cell line employed expressed actin fused to green fluorescent protein, which enabled imaging of live cells. Upon profilin-deficiency severe effects on cell behavior were observed, e.g. the cells lacked the ability to form characteristic broad lamellipodia at advancing edges, instead small protruding structures were generated and extended with a significantly reduced rate compared to control cells. Observations were also made suggesting that profilin regulates the expression of actin in mammalian cells. A new experimental system for studies of myoblast fusion and subsequent myotube formation in vitro was also established during these studies. This will facilitate systematic studies of molecular processes connected to muscle development.

The genetic information of a human diploid cell is contained within approximately 2 metres of linear DNA. The DNA molecules are compacted and organized in various ways to fit inside the cell nucleus. Various kinds of histones are involved in this compaction. One of these histones, histone H1 is the topic of the present thesis. In addition to its structural role, H1 histones have been implicated in various processes, for example gene regulation and inhibition of chromatin replication. H1 histones, also termed linker histones, are relatively conserved proteins, and the various subtypes seem to have different and important functions even though redundancy between the subtypes has been demonstrated. Despite the sequence conservation of H1 subtypes, two sequence variations were detected within the H1.2 and H1.4 subtypes using hydrophilic interaction liquid chromatographic separation of H1 proteins from K562 and Raji cell lines in Paper I in the present thesis. The variations were confirmed by genetic analysis, and the H1.2 sequence variation was also found in genomic DNA of normal blood donors, in an allele frequency of 6.8%. The H1.4 sequence variation was concluded to be Raji specific. The significance of H1 microsequence variants is unclear, since the physiological function of H1 histones remains to be established. H1 histones can be phosphorylated at multiple sites. Changes in H1 phosphorylation has been detected in apoptosis, the cell cycle, gene regulation, mitotic chromatin condensation and malignant transformation. Contradictory data have been obtained on H1 phosphorylation in apoptosis, and many results indicate that H1 dephosphorylation occurs during apoptosis. We and others hypothesized that cell cycle effects by the apoptosis inducers may have affected previous studies. In Paper II, the H1 phosphorylation pattern was investigated in early apoptosis in Jurkat cells, taking cell cycle effects into account. In receptor-mediated apoptosis, apoptosis occurs with a mainly preserved phosphorylation pattern, while Camptothecin induced apoptosis results in rapid dephosphorylation of H1 subtypes, demonstrating that H1 dephosphorylation is not a general event in apoptosis, but may occur upon apoptosis induction via the mitochondrial pathway. The dephosphorylation may also be a result of early cell cycle effects or signalling.Therefore, the H1 phosphorylation pattern in the cell cycle of normal activated T cells was investigated in Paper IV in this thesis. Some studies, which have been made using cancer cell lines from various species and cell synchronization, have indicated a sequential addition of phosphate groupsacross the cell cycle. Normal T cells and cell sorting by flow cytometry were used to circumvent side-effects from cell synchronization. The data demonstrate that a pattern with phosphorylated serines is established in late G1/early S phase, with some additional phosphorylation occurring during S, and further up-phosphorylation seems to occur during mitosis. Malignant transformation may lead to an altered G1 H1 phosphorylation pattern, as was demonstrated using sorted Jurkat T lymphoblastoid cells. During mitosis, certain H1 subtypes may be relocated to the cytoplasm. In Paper III, the location of histones H1.2, H1.3 and H1.5 during mitosis was investigated. Histone H1.3 was detected in cell nuclei in all mitotic stages, while H1.2 was detected in the nucleus during prophase and telophase, and primarily in the cytoplasm during metaphase and early anaphase. H1.5 was located mostly to chromatin during prophase and telophase, and to both chromatin and cytoplasm during metaphase and anaphase. Phosphorylated H1 was located in chromatin in prophase, and in both chromatin and cytoplasm during metaphase, anaphase and telophase, indicating that the mechanism for a possible H1 subtype relocation to the cytoplasm is phosphorylation. In conclusion, data obtained during this thesis work suggest that H1 histones and their phosphorylation may participate in the regulation of events in the cell cycle, such as S-phase progression and mitosis, possibly through altered interactions with chromatin, and/or by partial or complete removal of subtypes or phosphorylated variants from chromatin.

The pancreatic islets are collections of endocrine cells, dispersed throughout the pancreas. In adult islets, endocrine cells are closely associated with capillary endothelial cells and receive a high blood perfusion. Transplanted pancreatic islets, on the other hand, have a vascular disturbance, manifested as decreased blood vessel density. Besides impaired islet blood perfusion and oxygenation, this means that the normal close proximity between endothelial cells and β-cell in adult islets is interrupted. The aim of the thesis was to investigate if, and to what extent, β-cells and islet endothelial cells can interact with one another. This hypothesis was investigated during physiological growth of pancreatic islets, following transplantation and in vitro. We observed that islet endothelial and endocrine cell replication coincided immediately after birth, as well as during pregnancy. In pregnant animals, β-cell proliferation colocalized to islets with increased endothelial cell replication, indicating that the two processes were interconnected. The pregnancy hormone prolactin favored endothelial cell replication, and these activated cells could then augment β-cell proliferation. We found that prolactin pretreatment increased blood vessel density and oxygen tension in islets after transplantation. Furthermore, prolactin pretreatment improved endocrine function in a minimal islet transplant model. Partial pancreatectomy performed in association with islet transplantation improved revascularization, oxygen tension and glucose stimulated insulin release from the graft. In conclusion, the findings suggest that endocrine and endothelial cells interact with one another to regulate growth and function in pancreatic islets. This may form the basis for interventions aiming to improve revascularization and function of transplanted islets.

It is commonly stated that the cell is the smallest functional unit of life. By analogy, then, the immune cell is the smallest functional unit of the immune system. Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. Many phenotypically distinct subpopulations of NK cells have been discovered, usually by dividing cells on the basis of cell-surface markers. These subpopulations are typically described as related to activation or developmental status of the cells. However, how these distinct phenotypes correlate with behavior in e.g. NK–target interactions is less widely understood. There is therefore a need to study NK cell behavior down at the single-cell level. The aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells. Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they can be imaged over extended periods of time. We have performed experiments on both resting and IL-2-activated NK cells and quantified their cytotoxic behavior. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, which we termed “serial killers”, displayed faster and more effective cytotoxicity. Also, we identified differences between resting and activated NK cells in regard to their migration and contact dynamics. Activated NK cells were found to more readily adhere to targets cells than did NK cells freshly isolated from peripheral blood. Apart form migration and contact dynamics, we have also quantified killing behavior, where NK cells can be seen to exhibit a behavior we term multiple lytic hits on the basis of analyzing target cell fluorescence profiles. We have quantified these heterogeneities and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These methods, and automated single-cell analysis methods, will likely play a more important role in the study of immune responses in the future. / <p>QC 20140611</p>

No description available.

No description available.

The brain is arguably the most complex organ of the body: Controlling muscles, maintaining homeostasis, processing information. It has the longest developmental period of any organ, and hormones are essential during its development. Endocrine disruptive chemicals can disturb hormones in any part of their signaling pathway and can act as agonists or antagonists on their receptors. Should the balance of stem cell or progenitor cell proliferation be skewed in any direction during early brain development, it may lead to cognitive disfunctions or diseases. In this study, v-myc induced mouse-derived neural progenitor cells (NPCs) were exposed to retinoid and glucocorticoid receptor agonists and antagonists to investigate the involvement of these pathways on NPC proliferation. NPCs were exposed for 24 hours, after which they were read in a Tecan. Statistical analyses revealed that no treatment yielded statistically significant responses, however in the case for mifepristone (glucocorticoid receptor antagonist), dexamethasone (glucocorticoid receptor agonist) and Agn (retinoid receptor antagonist), a small negative response trend could be observed. Cell viability analyses also revealed that the concentrations of chemicals did not induce cytotoxicity. The unexpectedly high and variable data suggest errors were made during the seeding of the 96-well microplates. In conclusion, these findings suggest that with further development, this in vitro method could be used in conjunction with other in vitro models for the assessment of neurodevelopmental toxicity and for identification of EDCs in the future. / ENDpoiNTs

No description available.