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

<p>Compaction of chromosomes in the eukaryotic cell is due to interactions between DNA and proteins and interactions between proteins. These two types of interaction form a dynamic structure, known as "chromatin". The condensation of chromatin must be carefully regulated, since the structure is an obstacle for factors that need access to the DNA. An extensive range of components, one group of which is the ATP-dependent chromatin remodel-ling complexes, controls the accessibility of DNA. These complexes have been studied in a variety of eukaryotic systems, and their functions in major events in the cell, such as replication, DNA-repair and transcription have been established, as have their roles in the assembly and maintenance of chromatin. All of the complexes contain a highly conserved ATPase, which belongs to the SWI2/SNF2 family of proteins, one group of which is known as the ISWI proteins. There are two forms of ISWI in human, known as "SNF2h" and "SNF2l".</p><p>We have identified a human SNF2h-assembly, B-WICH, that consists of SNF2h, William’s syndrome transcription factor (WSTF), nuclear myosin (NM1), and a number of additional nuclear proteins including the Myb-binding protein 1a (Myb bp1a), SF3b155/SAP155, the RNA helicase II/Guα, the proto-oncogene Dek, and the Cockayne Syndrome protein B (CSB). The 45S rRNA, 5S rRNA and 7SL RNA are all parts of the B-WICH assembly. The formation of B-WICH depends on active transcription, and is implicated in the regulation of both RNA transcription by both pol I and pol III. The B-WICH provides a link between RNA and the chromatin structure.</p>

<p>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.</p><p>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:</p><p>1.Does Gleevec act via inhibition of c-Abl in β-cells?</p><p>2.Can Gleevec treatment prevent beta-cell death and diabetes? </p><p>3.Which downstream signaling pathways are affected by Gleevec?</p><p>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.</p><p>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.</p><p>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. </p><p>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.</p>

<p>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.</p>

Compaction of chromosomes in the eukaryotic cell is due to interactions between DNA and proteins and interactions between proteins. These two types of interaction form a dynamic structure, known as "chromatin". The condensation of chromatin must be carefully regulated, since the structure is an obstacle for factors that need access to the DNA. An extensive range of components, one group of which is the ATP-dependent chromatin remodel-ling complexes, controls the accessibility of DNA. These complexes have been studied in a variety of eukaryotic systems, and their functions in major events in the cell, such as replication, DNA-repair and transcription have been established, as have their roles in the assembly and maintenance of chromatin. All of the complexes contain a highly conserved ATPase, which belongs to the SWI2/SNF2 family of proteins, one group of which is known as the ISWI proteins. There are two forms of ISWI in human, known as "SNF2h" and "SNF2l". We have identified a human SNF2h-assembly, B-WICH, that consists of SNF2h, William’s syndrome transcription factor (WSTF), nuclear myosin (NM1), and a number of additional nuclear proteins including the Myb-binding protein 1a (Myb bp1a), SF3b155/SAP155, the RNA helicase II/Guα, the proto-oncogene Dek, and the Cockayne Syndrome protein B (CSB). The 45S rRNA, 5S rRNA and 7SL RNA are all parts of the B-WICH assembly. The formation of B-WICH depends on active transcription, and is implicated in the regulation of both RNA transcription by both pol I and pol III. The B-WICH provides a link between RNA and the chromatin structure.

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.