Genomic imprinting is an epigenetic phenomenon that causes a subset of mammalian genes to be expressed from only one allele in a parent-of-origin manner. The defects in the imprinting regulation result in disorders that affect development, growth and metabolism. We have used the Kcnq1 imprinted cluster as a model to understand the mechanism of imprinted gene regulation. The imprinting at the Kcnq1 locus is regulated by a long noncoding RNA, Kcnq1ot1, whose transcription on the paternal chromosome is associated with the silencing of at least eight neighboring genes. By destabilizing Kcnq1ot1 in an episomal system, we have conclusively shown that it is the RNA and not the process of transcription that is required for the gene silencing in cis. Kcnq1ot1 RNA interacts with the chromatin modifying enzymes such as G9a and Ezh2 and recruits them to imprinted genes to establish repressive chromatin compartment and gene silencing. Using the episomal system, we have identified an 890 bp silencing domain (SD) at the 5’ end of Kcnq1ot1 RNA, which is required for silencing of neighboring reporter genes. The deletion of the SD in the mouse resulted in the relaxation of imprinting of ubiquitously imprinted genes (Cdkn1c, Kcnq1, Slc22a18, and Phlda2) as well as reduced DNA methylation over the somatic DMRs associated with the ubiquitously imprinted genes. Moreover, Kcnq1ot1 RNA interacts with Dnmt1 and recruits to the somatic DMRs and this recruitment was significantly affected in the SD mutant mice. By using a transgenic mouse, we have conditionally deleted Kcnq1ot1 promoter at different developmental stages and demonstrated that Kcnq1ot1 maintains imprinting of the ubiquitously imprinted genes by regulating DNA methylation over the somatic DMRs. Kcnq1ot1 is dispensable for the maintenance of repressive histone marks and the imprinting of placental-specific imprinted genes (Tssc4 and Osbpl5). In conclusion, we have described the mechanisms by which Kcnq1ot1 RNA establishes and maintains expression of multiple imprinted genes in cis.

Since the first signs of life on planet earth, organisms have had to adapt to the daily changes between light and dark, and high and low temperatures. This has led to the evolution of an endogenous time keeper, known as the circadian clock. This biological timing system helps the organism to synchronize developmental and metabolic events to the most favorable time of the day. Such a mechanism is of considerable value to plants, since they in contrast to animals cannot change location when the environment becomes unfavorable. Thus is the ability to predict coming events of central importance in a plants life. This thesis is a study of the molecular machinery behind the clockwork in the small weed plant Arabidopsis thaliana as well as its close relative perennial; the woody species Populus. We have characterized a novel component of the circadian clock, EARLY BIRD (EBI). EBI is involved in transcriptional and translational regulation, via interaction with the known post-translational clock regulator ZEITLUPE (ZTL). In Populus, we describe the role of the circadian clock and its components with respect to entry and exit of dormancy and show that gene expression of the Populus LATE ELONATED HYPOCOTYL (LHY) genes are crucial importance for freezing tolerance and thereby survival at high latitudes. Furthermore, the input to the Populus clock is mediated via the phytochrome A (phyA) photoreceptor. / Liv på jorden har alltid behövt anpassa sig till de dagliga växlingarna mellan främst ljus och mörker. Detta har lett till evolutionen av en intern, biologisk klocka, känd som den circadianska klockan, efter latinets ”circa diem”, som betyder ”ungefär en dag”. Denna inre klocka hjälper organismer att styra biologiska processer till den tid på dygnet som är mest gynnsam för deras utveckling och överlevnad. Denna mekanism är av stort värde för växter, eftersom de inte kan söka skydd på mera lämpliga platser om de blir utsatta för olika former av stress. Det gör att förmågan att förutse kommande händelser är av yttersta vikt för växter. Denna avhandling är en studie av det molekylära nätverk som styr denna biologiska klocka i den lilla örtplantan Arabidopsis thaliana (backtrav), och den besläktade träd-arten Populus (hybrid-asp). Vi har karaktäriserat en ny komponent i den circadianska klockan i Arabidopsis, EARLY BIRD (EBI). EBI är involverad i transkriptionell och translationell reglering av klockan, via interaktion med den kända post-translationella klock-regulatorn ZEITLUPE (ZTL). I Populus har vi beskrivit den interna klockan och dess roll i processer som invintring, vinterdvala och återstart av tillväxt. LATE ELONATED HYPOCOTYL (LHY) generna i Populus är avgörande för förvärv av köld-tolerans och således överlevnad på högre latituder. Dessutom har vi visat att signaler till den circadianska klockan i Populus är medierade via fotoreceptorn phytochrome A (phyA).

Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles. We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A0-A2 sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage. Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes. We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus. We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation. We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP. We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.

The Gram-negative bacterium Yersinia pseudotuberculosis employs a type 3 secretion system (T3SS) to establish infections. The T3SS translocates a diverse set of effector proteins directly into the host cells. The coordinate action of the translocated effectors blocks the innate immune system of the host and ensures extracellular proliferation of the bacterium. YopE is an essential effector that disrupts the actin cytoskeleton of infected host cells. This cytotoxicity is caused by the inactivation of RhoGTPases by the GTPase Activating Protein (GAP) activity of YopE. YopE was demonstrated to inactivate the RhoGTPases Rac1 and RhoA in vivo. However, Rac1 and RhoA inactivation was not a prerequisite for cytotoxicity or virulence. Thus, YopE must have additional targets during infection. Surprisingly, avirulent yopE mutants had lost the control of Yop expression in the presence of target cells and they all overtranslocated effectors. It appeared as if translocated YopE was able to control Yop expression and effector translocation via a feedback inhibition mechanism. This feedback inhibition was dependent on functional GAP activity. Translocation control could also be mediated by exogenous GAP activity, suggesting that effector translocation control might be a general property of all bacterial GAP proteins. Besides YopE, the regulatory protein YopK was also found to be involved in the effector translocation control process. Clearly, as demonstrated in virulence, the roles for YopE and YopK are intimately related.                       Further, YopE possesses a membrane localization domain (MLD) required for proper localization. A yopE∆MLD mutant had lost the feedback inhibition of YopE expression and was avirulent. Hence, the effector translocation control of YopE requires both proper localization as well as functional GAP activity.                                           In addition, fish keratocytes were established as a novel model system for Y. pseudotuberculosis infections. YopE was found to be the sole effector responsible for cytotoxicity towards the keratocytes. Further, induction of cytotoxicity required fully native YopE protein which indicated that the keratocytes would be useful as a sensitive model system for further studies of YopE mediated phenotypes. In summary, this thesis work has sought to unravel the multiple functions of translocated YopE. A novel role was elucidated where Yersinia utilizes translocated YopE to control the process of effector translocation into host cells. This regulatory control was connected to virulence in the mouse model of disease. Thus, perhaps YopE should be considered also as a regulatory protein besides being a classical effector.

The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

The use of fossil fuels presents a particularly interesting challenge - our society strongly depends on coal and oil, but we are aware that their use is damaging the environment. Currently, this awareness is gaining momentum, and pressure to evolve towards an energetically cleaner planet is very strong. Molecular hydrogen (H2) is an environmentally suitable energy carrier that could initially supplement or even substitute fossil fuels. Ideally, the primary energy source to produce hydrogen gas should be renewable, and the process of conversion back to energy without polluting emissions, making this cycle environmentally clean. Photoconversion of water to hydrogen can be achieved using the following strategies: 1) the use of photochemical fuel cells, 2) by applying photovoltaics, or 3) by promoting production of hydrogen by photosynthetic microorganisms, either phototrophic anoxygenic bacteria and cyanobacteria or eukaryotic green algae. For photobiological H2 production cyanobacteria are among the ideal candidates since they: a) are capable of H2 evolution, and b) have simple nutritional requirements - they can grow in air (N2 and CO2), water and mineral salts, with light as the only energy source. As this project started, a vision and a set of overall goals were established. These postulated that improved H2 production over a long period demanded: 1) selection of strains taking in consideration their specific hydrogen metabolism, 2) genetic modification in order to improve the H2 evolution, and 3) cultivation conditions in bioreactors should be exmined and improved. Within these goals, three main research objectives were set: 1) update and document the use of cyanobacteria for hydrogen production, 2) create tools to improve molecular biology work at the transcription analysis level, and 3) study cell division in cyanobacteria. This work resulted in: 1) the publication of a review on hydrogen evolution by cyanobacteria, 2) the development of tools to assist understanding of transcription, and 3) the start of a new fundamental research approach to ultimately improve the yield of H2 evolution by cyanobacteria.

Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations. Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes BRAF and NRAS, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with BRAF mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene MGMT was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15. These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment. / QC 20110121

Glucose is a monosaccharide and fuel for body, it cannot pass through membrane by simple diffusion so, integral transmembrane proteins named glucose transporters (Gluts) are involved in the regulation of the movement of glucose between the extracellular and intracellular spaces within the body. GLUT1 and GLUT3 have previously been shown by cold detergent extraction methods to reside in distinct plasma membrane domains in non-polarized mammalian cells, with GLUT1, but not GLUT3, residing  in detergent-resistant membrane (DRM) domains. To confirm this observation under less invasive conditions, molecular fusion tags are inserted in the first external loop in Glut1 with biotin ligase acceptor peptide (BLAP) between Ser-55 and Ile-56 and in Glut3 with Acyl carrier peptide (ACP) in between Val-57 and Leu-58 respectively. These Glut fusion proteins will be used in order to confirm these observations by fluorescence recovery after photobleaching (FRAP) and single molecule fluorescence microscopy in live cells. hGLUT1-EGFP, hGLUT1 (AgeI)-EGFP recombinants were constructed and transfected human embryonic kidney cells (HEK-293) quantum dot images supports the fact that EGFP transfected cells uniformly and is distributed in the cell cytoplasm, hGLUT1-EGFP transfected cells and is localized to the cell membrane and hGLUT1 (AgeI)-EGFP transfected cells and located to the plasma membrane with high intensity.

Biofilms are communities of sessile microorganisms attached to a surface and imbeddedin a matrix of extracellular polysaccharide substances. These communities can be foundin diverse aquatic environments, such as in industrial pipes and in humans. By formingmicrocolony structures, which are highly resistant to adverse physical conditions as wellas antimicrobial agents, biofilms are very problematic when associated with e.g.persistent infections. In order to find new ways of controlling biofilm growth, theprocesses involved in biofilm development must be investigated further. The maininterest of this study is the occurrence of void formation inside biofilms. Thisphenomenon has been observed in several studies and has been correlated to cell deathinside the microcolonies. The occurrence of cell death has recently been associated withthe presence of nitric oxide in the biofilm. In this study, the implications of nitric oxideaccumulation on biofilm development were investigated using an individual-basedmodel. Specifically, the role of nitric oxide in void formation was considered. A largenumber of simulations were run using different parameter settings in order to determine ifnitric oxide could account for the occurrence of void formation observed experimentally.The general predictions made by the model system showed agreement to someexperimental data, but not to others. Sloughing, the detachment of chunks of cells fromthe biofilm, was observed in the majority of simulations. In some cases, the model alsopredicted the presence of live cells inside the voids, which has been observedexperimentally.

Mushrooms are believed to have a great potential for production of bioactive metabolites e. g. terpenes, a group of interesting compounds with diverse chemical properties such as antitumour and antibacterial activity. Cop6 is a terpene cyclase isolated from the mushroom Coprinus cinereus that catalyzes the cyclization of farnesyl diphosphate (FPP) to mainly α-cuprenene. In this study gas chromatography combined with mass spectroscopy (GC-MS) is used to analyze the product profile of Cop6 mutants created by PCR based site directed mutagenesis. The goal is to produce trichodiene, the parent hydrocarbon in the biosynthesis of trichothecene antibiotics and mycotoxins. Valine instead of tyrosine in amino acid position 195 resulted in cyclisation of (E)-β-Farnesene and (3Z,6E)-α-Farnesene besides the products of the wild type enzyme. Another mutant with aspartic acid instead of asparagine in position 224 resulted in the synthesis of β-Bisabolene except for α-cuprenene and methionine in position 74 instead of isoleucine killed the activity of the cyclase. Furthermore, an attempt to saturation of position 98 was made, resulting in four mutants. Two of them essentially killed the activity of the cyclase whereas two had minor effect of the product profile compared to the wild type.