Plant hormones are responsible for plant growth and adaptation to the environment. Among them the most important are cytokinins. Plants undergo gene silencing processes called homology-dependent gene silencing processes. In Arabidopsis there are two homology-dependent gene silencing genes that were chosen for further study, namely AtHOG1 and AtHOG2. Transgenic plants were generated previously with ten different constructs containing AtHOG1 or AtHOG2 genes and were used in this study. Some of the constructs had GFP attached so that the protein expressed could be visualised in a confocal microscope. Transgenic plants generated were T1 and T2 generations. Their DNA was extracted from leaves. By means of PCR transgenic plants were identified. There were 147 samples. Among them there were 39 positiveswith BAR primers and 32 positives with construct specific primers. The localisation of the HOG2 protein was observed in a confocal microscope. Seeds used were T3 generation and were obtained from the lab. HOG2 protein was found to be localised in cell membrane, root tip and chloroplasts.

The primary function of adipose tissue is to store energy in the form of triacylglycerol, which is hydrolyzed to fatty acids to supply other tissues with energy. While insulin promotes the storage of triacylglycerol, catecholamines stimulate its hydrolysis. The development of type II diabetes is strongly associated with obesity, indicating a role of triacylglycerol metabolism in the pathogenesis of diabetes. Caveolae are plasma membrane invaginations found in most cells but are highly abundant in adipocytes. Insulin receptors are localized in caveolae and their function depends on intact caveolae structures. In the present thesis work, mass spectrometry-based methodology allowed identification of a number of new proteins and their posttranslational modifications in caveolae of human adipocytes. Variable N-terminal acetylation and phosphorylation of caveolin-1α and caveolin-1β were identified, which might regulate the function of caveolae. The transcription regulator protein PTRF was identified as the major caveolae associated protein. Specific proteolytic modifications of PTRF at the cytosolic surface of caveolae and phosphorylation on nine serine and one threonine residues were identified. Moreover, insulin induced translocation of PTRF from the plasma membrane to the nucleus. PTRF was previously shown to regulate the activity of both RNA polymerase I and polymerase II, thus a role of PTRF in mediating the anabolic action of insulin on protein synthesis and gene transcription is proposed. PTRF was also involved in an extranuclear function in the hormonal regulation of triacylglycerol metabolism in caveolae. PTRF was colocalized with the triacylglycerol regulator proteins perilipin and hormone-sensitive lipase (HSL) in the triacylglycerol-synthesizing caveolae subclass. We showed that, while perilipin was translocated to the plasma membrane, both PTRF and HSL were translocated from the plasma membrane to the cytosol as a complex in response to insulin. The perilipin recruited to the plasma membrane was highly threonine phosphorylated. By mass spectrometry, three phosphorylated threonine residues were identified and were located in an acidic domain in the lipid droplet targeting domain of perilipin. The insulin-induced recruitment of perilipin to the plasma membrane might, therefore be phosphorylation-dependent. Isoproterenol, which stimulates hydrolysis of triacylglycerol, induced a complete depletion of perilipin B from the plasma membrane, suggesting a function of perilipin B to protect newly synthesized triacylglycerol in caveolae from being hydrolyzed by HSL. The location of PTRF and HSL was not affected by isoproterenol, indicating that insulin is acting against a default presence of PTRF and HSL in caveolae. Taken together, this thesis expands our knowledge about caveolae and provided valuable information about their involvement in novel roles, particularly in the hormonal regulation of triacylglycerol metabolism.

The control of growth is a poorly understood aspect of animal development. This thesis focuses on body size regulation in Caenorhabditis elegans, and in particular, how worms grow to a certain size. In C. elegans, a key regulator of size is the TGFβ homologue DBL-1. Mutations that deplete the worm of DBL-1 result in a small body size, whereas overexpression of the gene renders long animals. The small mutants have the same number of cells as wild type suggesting that some or all cells are smaller. DBL-1 activates a TGFβ receptor leading to the nuclear localization of three Smad proteins which then initiate a transcriptional program for size control whose targets are mainly unknown. In order to learn more about how body size in C. elegans is regulated, we set up EMS mutagenesis screens to identify new loci that caused a long phenotype. A subset of the genes we have identified might function in the TGFβ signaling pathway regulating growth while others likely function in parallel pathways. One gene that we found in this screen, lon-3, encodes a cuticle collagen that genetically lies downstream of the DBL-1 TGFβ signaling pathway. Interestingly, loss of function mutations in lon-3 result in a Lon phenotype, whereas increasing the amount of LON-3 protein cause the worms to be dumpy, i.e. shorter, but slightly fatter than wild type. LON-3 is expressed in the hypodermis, the tissue from which the cuticle is synthesized and in which TGFβ signaling, regulating body size, has its focus. This study and previous work have shown that DBL-1 may affect body volume via effects on hypodermal nuclear ploidy, however this is unaffected in lon-3 mutants. Consistent with this finding, the volume of lon-3 mutant worms is not different from wild type. Taken together, our results suggest that another mechanism, by which TGFβ signaling can regulate body length, is by altering the shape of the cuticle via its effect on lon-3 and possibly other cuticle collagens. Studies in worms, flies and mice show that body size and nutrient allocation are closely connected. p70 S6-kinase (S6K) is a known regulator of cell and body size that also plays a role in metabolism. In mice and flies S6K mutants are much smaller than wild type. Our work on the worm homolog, rsks-1, shows that in worms as well, this gene is important for growth regulation and cell size. However, this effect seems to be at least in part independent of DBL-1 TGFβ signaling. Furthermore, rsks-1mutants have a 50 % increase in the amount of stored fat. Fatty acid metabolism has been shown to play an important role in environmental adaptation, especially in regards to temperature changes. Consistent with this idea, rsks-1 mutants appear to have difficulties in adjusting to such changes, reflected in a much-decreased fecundity at 15 and 25 °C compared to their cultivation temperature (20 °C). Within the nervous system the gene is specifically expressed in a subset of the chemosensory neurons that, when nutrients are abundant, secrete signals that promote growth. Intriguingly, this expression seems to be negatively regulated by insulin- like signaling, in contrast to the positive regulation of S6K by insulin in Drosophila and mice. Taken together we show that rsks-1 is an important regulator of growth and fat metabolism in Caenorhabditis elegans.

The vertebrate nervous system is extremely complex and contains a wide diversity of cell types. The formation of a functional nervous system requires the differential specification of progenitor cells at the right time and place. The generation of many different types of neurons along the rostro-caudal axis of the CNS begins with the initial specification of a few progenitor domains. This initial coarse pattern is refined by so-called secondary organizers arising at boundaries between these domains. The Isthmic Organizer (IsO) is a secondary organizer located at the boundary between the midbrain and the hindbrain. Although the function and maintenance of the IsO are well understood, the processes underlying its initial specification have remained elusive. In the present work we provide evidence that convergent Wnt and FGF signals initiate the specification of the IsO during late gastrulation as part of the neural caudalization process. The initial step in the generation of the nervous system is the division of the embryonic ectoderm into three cell populations: neural cells giving rise to the CNS, neural plate border cells giving rise to the peripheral nervous system, and epidermal cells giving rise to the outer layer of the skin. While the choice between neural and epidermal fate has been well studied, the mechanism by which neural plate border cells are generated is less well understood. At rostral levels of the neuraxis, the neural plate border gives rise to the olfactory and lens placodes, thickenings of the surface ectoderm from which sensory organs are derived. More caudally, the neural plate border generates neural crest cells, a transient population that migrates extensively and contributes to neurons and glia of the peripheral nervous system. How the early patterning of the central and peripheral nervous systems are coordinated has remained poorly understood. Here we show that the generation of neural plate border cells is initiated at the late blastula stage and involves two phases. During the first phase, neural plate border cells are exposed to Wnt signals in the absence of BMP signals. Simultaneous exposure to Wnt and BMP signals at this early stage leads to epidermal induction. Wnt signals induce expression of Bmp4, thereby regulating the sequential exposure of cells to Wnt and BMP signals. During the second phase, at the late gastrula stage, BMP signals play an instructive role to specify neural plate border cells of either placodal or neural crest character depending on the status of Wnt signaling. At this stage, Wnt signals promote caudal character simultaneously in the neural plate border and in the neural ectoderm. Thus, the choice between epidermal and neural plate border specification is mediated by an interplay of Wnt and BMP signals that represents a novel mechanism involving temporal control of BMP activity by Wnt signals. Moreover, the early development of the central and peripheral nervous systems are coordinated by simultaneous caudalization by Wnt signals.

The microtubule cytoskeleton, which consists of dynamic polymers of alpha/beta tubulin heterodimers, organizes the cytoplasm and is essential for chromosome segregation during mitosis. My thesis addresses the significance and potential interplay between four distinct microtubule-regulatory proteins. The experimental approach included the development of a replicating vector system directing either constitutive expression of short hairpin RNAs or inducible ectopic expression, which allows stable depletion and/or conditional exchange of gene-products. Based on the originally observed activities in frog egg extracts, MCAK and TOGp have been viewed as major antagonistic proteins that regulate microtubule-dynamics throughout the cell cycle. Surprisingly, while my thesis work confirmed an essential role of these proteins to ensure mitotic fidelity, tubulin subunits partitioning is not controlled by the endogenous levels of MCAK and TOGp in human somatic cells. Our major discovery in these studies is that the activities of both CaMKII and TOGp are essential for spindle bipolarity through a mechanism involving protection of spindle microtubules against MCAK activity at the centrosome. In our search for the major antagonistic activities that regulates microtubule-dynamics in interphase cells, we found that the microtubule-destabilizing activity of Op18 is counteracted by MAP4. These studies also established Op18 and MAP4 as the predominant regulators of tubulin subunit partitioning in all three human cell model systems studied. Moreover, consistent with phosphorylation-inactivation of these two proteins during mitosis, we found that the microtubule-regulatory activities of both MAP4 and Op18 were only evident in interphase cells. Importantly, by employing a system for inducible gene product replacement, we found that site-specific phosphorylation-inactivation of Op18 is the direct cause of the demonstrated hyper-polymerization in response to T-cell antigen receptor triggering. This provides the first formally proven example of a signal transduction pathway for regulation of interphase microtubules. Op18 is frequently upregulated in various types of human malignancies. In addition, a somatic mutation of Op18 has recently been identified in an adenocarcinoma. This thesis work revealed that the mutant Op18 protein exerts increased microtubule-destabilizing activity. The mutant Op18 protein was also shown to be partially resistant to phosphorylation-inactivation during mitosis, which was associated with increased chromosome segregation aberrancies. Interestingly, we also observed the same phenotype by overexpressing the wild type Op18 protein. Thus, either excessive levels of wild type Op18 or normal levels of mutated hyper-active Op18 seems likely to contribute to tumor progression by exacerbating chromosomal instability.

Iron has a dualistic character. On the one hand it is essential for the life of most organisms, on the other hand it is involved in the generation of reactive oxygen species that are implicated in diseases and aging. During evolution efficient mechanisms for uptake, handling and storage of iron in a safe way have developed to keep the balance between iron availability and minimizing the hazards. In eukaryotes, mitochondria are the central organelle for “metabolizing” iron and consequently play an important role in cellular iron homeostasis. Mitoferrins are mitochondrial carrier proteins, which are involved in iron transport into mitochondria. In vertebrates two mitoferrins exist, one (mitoferrin1) of which is essential for heme synthesis during erythropoiesis, while the function of the other (mitoferrin2) is not well defined. In the fruit fly we found only one mitoferrin gene (dmfrn), which codes most likely for a functional homologueof vertebrate mitoferrin2. In Drosophila cell culture, dmfrn overexpression resulted in an overestimation of cell sensed iron levels. The signal responsible for this, is most likely a yet unidentified compound of ISC synthesis. In the cell culture system we also showed that iron chelation blocks the progression of the cell cycle in a reversible and therefore most likely controlled way. Study of different dmfrn mutants indicates a role of dmfrn during spermatogenesis and development to adulthood. As dmfrn deletion mutants are not lethal, it is likely that other lower affinity iron transporters exist. A similar conclusion has been drawn by others from the study of yeast mitoferrin homologuemutants. Rim2p/Mrs12p has recently been implicated in mitochondrial iron transport, and might be an alternative metal carrier. We identified a putative homologuein the fruit fly and found a possible link between mutants in this gene and iron. Our results emphasize the importance of the mitochondrial iron metabolism in cellular iron homeostasis. We also show for the first time, a direct connection between the mitochondrial iron metabolism and spermatogenesis. Mutants characterized and developed by us will help to study these processes in further detail and reveal the underlying mechanisms.

Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins by integrins and the following protein-protein interactions. The part of the research that I did, focused on the activation of PI3K by integrins and the question whether Ras is included in that pathway. I also studied the conformation changes of the integrins and tried to identify factors which regulate these changes. Known is that Ras can activate PI3K. But we wanted to know if this is a step in the activation of PI3K by integrins. So if this would be a fact then Ras must be activated by integrins. To see if integrins could activate Ras I did a pull down assay. GTP loaded Ras was isolated through its affinity for Raf. Only when Ras is in its activated state then it is GTP loaded, otherwise it is GDP loaded. In the experiment we also compared the β1A and the β1B splice variants. As result we could see that both splice variants probably can activate Ras. By blotting with anti-PI3K antibody we looked if PI3K had bound to Ras but no clear result could be obtained. Integrins presented on blood cells are mostly in the inactive state while adherent cells have integrins which are mostly in the active state. PI3K has been shown, for blood cells, to be involved in the conformation regulation of integrins. Possibly, there is a positive circle that for blood cells just has to be switched on. It could be that the integrins in adherent cells are active because the cells are adhesive. By being adhesive, PI3K is activated. PI3K may then activate the integrins, through which the integrins stay in the active state. This circle could be broken at two points: we could inhibit PI3K or we could make the cells un-adhesive. I analysed this in cell attachment assay and by binding of conformation-specific integrin antibodies in FACScan. From the results we could not find any evidence that the whole idea around the positive circle is correct. Surprisingly we saw that the integrin value at the surface decrease if you add PI3K inhibitor. This could be due to distribute recirculation of integrins from the cytoplasm to the cell surface. β1- and β3-integrins are both widely spread, but no functional difference could be shown already. Previous results suggest that there is a difference between migrations of those two types. To ensure this suggestion I did a wound assay. Hereby I compared the migration of different cell types, with different integrins on their surface and on different ligands.

Salt stress is one of the major problems in the present world’s agriculture. Plants encounter drought stress even in the availability of water because of osmotic imbalance in the cell due to excess salts. Plants avoid water uptake, which in turn decreases the photosynthetic activity. In this work, we measured the effect of salt stress on three accessions of Arabidopsis thaliana (Columbia (Col-0), Landsberg erecta (Ler-0), Wassilewskija (Ws-4)) by subjecting the plants to stress with 0-150 mM NaCl followed by recovery. The impact of the stress was clearly observed in all three accessions during stress and recovery period. Chlorophyll content in leaves decreased with increasing salt concentration. Proline levels increased during salt stress conditions. Non-photochemical quenching and PSII activity slightly decreased under stress conditions. Salt treated plants showed slow acidification of lumen with delayed Non-photochemical quenching in recovery phase. Ler-0 was the most sensitive ecotype to salt stress followed by Ws-4 and Col-0.

Autoimmune Polyendocrine Syndrome Type 1(APS I) is a monogenic autosomal recessive autoimmune disorder which is the result of mutations in the autoimmune regulator (AIRE) gene. Symptoms of the disease include circulation of multiple organ specific autoantibodies, which leads to the breakdown of several tissues, including the adrenal cortex and the parathyroid glands. The patients also develop a number of non-endocrine disorders. This study has investigated the peripheral tolerance mechanisms controlled by the AIRE gene in Aire deficient mice, an animal model of the disease. The B cell Activating Factor (BAFF), which is a cytokine involved in B cell survival and growth, is elevated in Aire-/- mice, resulting in an increased release of autoantibodies and B cell proliferation. Therefore the BAFF level differences between TCR-/- and B6 mice was studied, and the results showed significantly higher levels of BAFF in TCR-/- mice. This is not in accordance with earlier studies. ICOS and ICOSL are involved in the activation of follicular T helper cells. The expression of ICOSL on different subpopulations of DC from mice was studied to evaluate the possible influence of AIRE expression on the T cells in the spleen. The results showed that ICOSL is significantly higher expressed in peripheral 33D1+ DCs in Aire-/- mice, showing that AIRE has a role in the over-activation of the follicular T helper cells, which can lead to autoantibody production and inflammation. These results show that AIRE is involved in peripheral tolerance.

Evidence has shown that hypoxic hearts have greater heart/fetus mass ratio. However, it is still unclear if either hyperplasia or hypertrophy causes the relatively increased heart mass. Furthermore, the genes that might be involved in the process have not yet been identified. In the present study, the cardiac transcriptome was analyzed to identify differentially expressed genes related to hypoxia. Eggs were incubated for 15 and 19 days in two different environments, normoxic and hypoxic. Normalized microarray results were analyzed to isolate differentially expressed probes using the Affymetrix chip. Total RNA was also isolated from another set of fetuses incubated in the same conditions and used to perform a qPCR in order to confirm the microarray results. In the four groups (15N, 15H, 19N, 19H), some probes were differentially expressed. From the eggs incubated for 15 days, the microarray revealed five probes that were differentially expressed according to the criteria (p<0.01 and absolute fold change FC>2) in the two programs (PLIER & RMA) used to normalize the data. From the eggs incubated up to 19 days, eight probes were differentially expressed in both programs. No further tests were performed on the 19 days fetuses since there was no significant difference in that group after incubation for the heart/fetus mass ratio. Apolipoprotein-A1, p22, similar to ENS-1 and b2 adrenergic receptor were further tested in qPCR (15 days sample). The differently expressed genes are linked to cell division and should be further studied to identify their function, especially the similar to ENS-1.