http://hdl.handle.net/10037/199 2012-02-12T06:24:40Z http://hdl.handle.net/10037/3726 Paunas, Teodora Ioana<br /> Coronary heart disease is the leading cause of death worldwide. Infarct size can be limited by interventions used after the ischemic event like the use of thrombolytic therapy or primary percutaneous coronary intervention. Paradoxically, however, the return of blood flow can also result in additional cardiac damage and complications, referred to as reperfusion injury. It has been shown that reperfusion injuries can be decreased by postconditioning- rapid intermittent interruptions of blood flow in the early phase of reperfusion, or post-treatment using various drug therapies which applied during reperfusion can reduce infarct size. H2S, a gas that is synthesized in mammalian tissue, has been reported to be cardioprotective during ischemia-reperfusion injury. The means by which H2S is cardioprotective during I/R are believed to be: the opening of the sarcolemmal KATP channel, the generation of antiapoptotic effects inside the cells as well as a direct antioxidant effect. Low levels of reactive oxygen species (ROS) are constantly produce within cells and play important roles in cell signaling, cellular homeostasis, differentiation and apoptosis. However an excessive increase in the level of ROS can be harmful and has been proposed to play crucial roles or contribute in the development of various diseases. The aim of our study was to investigate the effects of H2S in an acute ischemia-reperfusion model and to determine whether exogenous administration of H2S in both healthy rats and rats exposed to experimental models of cardiac disease influenced the production of ROS. In order to do this we established a method trough which we were able to measure the presence of ROS in heart tissue samples harvested from normal rats and rats with heart hypertrophy and ischemic heart disease.<br /> 2011-08-31T22:00:00Z Paunas, Teodora Ioana http://hdl.handle.net/10037/3568 Adou, Koman Mireille Sophie Chinan<br /> Human polyomavirus BK (BKV) is a non enveloped virus with a double-stranded, circular DNA genome. BKV infects >70% of the human population world-wide. Infection occurs predominantly during childhood and the virus remains in a latent state throughout life in the immune competent individuals. In the context of immunosuppression, however, reactivation occurs and can lead to renal stenosis and interstitial nephritis in kidney transplant patients, and hemorrhagic cystitis in bone narrow transplant patients. Moreover, BKV has been associated with several human cancers, but its causal role remains disputed. One of BKV’s protein known as agnoprotein may play a role in these pathogenic processes. To develop antiviral therapy it is required to elucidate the exact biological function of this protein. One way to examine the function of agnoprotein is by identifying possible cellular interaction partners. Another way is to understand agnoprotein’s role in the viral life cycle. Thereto, we examined the interaction of agnoprotein with another viral protein, large T-antigen (LT-ag) and the functional implication of this interaction. First, we investigated the effect of agnoprotein on the transcriptional activity of LT-ag on the BKV early promoter by transient transfection studies in HEK293. Our results revealed that LT-ag affects BKV early promoter in a concentration-dependent manner with low concentrations of LT-ag inhibiting, while high concentrations stimulated BKV early promoter activity. Co-expression of agnoprotein repressed LT-ag-induced activation of the BKV early promoter, suggesting that agnoprotein may exert a negative regulatory effect on transactivation by LT-ag. To test whether agnoprotein mediates its effect through direct interaction with LT-ag, we studied a possible association between these proteins. GST pulldown, co-immunoprecipitation (in vivo and in vitro), and mammalian two hybrid studies confirmed an interaction between LT-ag and agnoprotein.<br /> 2011-07-31T22:00:00Z Adou, Koman Mireille Sophie Chinan http://hdl.handle.net/10037/3567 Bohra, Pallavi<br /> Starting with LacZ of Escherichia coli, coding for β-galactosidase, the aim of the thesis project is to apply in vitro directed evolution techniques to help create other glycosidic bond hydrolysis activities. This was done using the main β-galactosidase backbone with limited amino acid sequence change. Any altered glycosyl hydrolase activity would lead to changed substrate specificity. Moreover, genetic changes leading to improved beta-galactosidase activity was also investigated. Error-prone PCR was applied to the LacZ gene (β-galactosidase) to achieve the desired aims. The technique used to introduce random mutagenesis was based on modifications of method developed by Xu et al., 1999.Optimization was performed with DNA polymerase selection, PCR conditions and various Mn and dITP concentrations to obtain best amplified PCR product for random mutagenesis library construction. Plasmid pTZ1 containing the entire coding sequence of LacZ was used a whole plasmid random Mutagenesis library construction strategy. The complete pTZ1 plasmid sequence had to be done in order to help establish a framework for primer design and establish a complete restriction map of the plasmid including the lacZ gene. The sequence analysis of the plasmid revealed that it has 5,502bp. Screening of random mutagenesis libraries was based on the colour development resulting from the glycosidic hydrolysis of chromogenic substrate to identify any glycosidic activity towards particular glycosyl hydrolase on LB plates or M9 plates. We have screened random mutagenesis libraries for any possible activity for β-glucosidase, β-xylosidase or for an improved β-galactosidase activity. Colonies that showed colour development on substrate even after retransformation of plasmid DNA for β-xylosidase activity were selected and its mutated plasmid DNA was sequenced. Two of the variants in which one has mutation at K552E position and another at N959Y were isolated, from two different clear blue colonies on β-xylosidase substrate. However, to the issue of change in substrate specificity (colour development on plates) was not clear. The direct evolution method applied here is seems simpler and promising in creating random mutagenesis libraries in order to select variants with useful novel properties.<br /> 2011-05-15T22:00:00Z Bohra, Pallavi http://hdl.handle.net/10037/3484 Svenning, Steingrim<br /> It is now well established that p62 and NBR1 are selectively degraded by autophagy and can act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates. Research on autophagy in plants is also well under way, but the mechanism by which target substrates are sequestered for autophagic degradation has not been elucidated. The uncharacterized plant protein Q9SB64 shares several important functional properties with p62 and NBR1, which indicates that it could act as a cargo receptor for the autophagic degradation of ubiquitinated substrates in plants. Results from this study show that Q9SB64 polymerize via an N-terminal PB1 domain, binds ubiquitin through a C-terminal UBA domain and interacts with the Arabidopsis family of ATG8 proteins. Based on sequence similarity Q9SB64 can be viewed as the Arabidopsis orthologue of vertebrate NBR1 and named AtNBR1. Plants do not seem to have a p62 orthologue. However, with regard to the functional properties studied here AtNBR1 behaves more similar to mammalian p62 than to NBR1.<br /> 2009-05-14T22:00:00Z Svenning, Steingrim http://hdl.handle.net/10037/3427 Lægreid, Kari Jenssen<br /> The mitogen activated protein kinases (MAPK) are a large and diverse family of protein kinases, contributing to the cells ability to respond to external stimuli by relaying messages in a well orchestrated way until they reach their final destinations. This is achieved through successive phosphorylation events. One member of this large family is mitogen activated protein kinase activated protein kinase 5 (MAPKAPK5/MK5), which is activated by the upstream atypical MAPKs extracellular signal-regulated kinases 3 and 4 (ERK3 and ERK4), and possibly also the conventional MAPK p38MAPK. MK5 has been shown to be implicated in F-actin rearrangement through phosphorylation of heat shock protein 27 (HSP27), tumor suppression through at least two different pathways and cell cycle arrest in response to energy depletion. There has been done relatively little research on this protein, and few bona fide substrates for MK5 are known, although knowledge is emerging. One possible interaction partner for MK5 is DNAJB1, which has been shown to be phosphorylated by MK5 in vitro. DNAJB1 is a member of the heat shock protein 40 (HSP40) family/DNAJ family, which is a subunit of the much larger heat shock protein superfamily. Heat shock proteins mostly function as chaperones and co-chaperones in the cell, assisting in the maintenance of protein homeostasis, by refolding or degrading misfolded proteins. Conditions of stress in the cell can lead to increased levels of misfolded proteins, which in turn are thought to initiate the increased transcription of heat shock proteins observed in stressed cells. DNAJB1 mainly serves as a co-chaperone for heat shock protein 70, and has been implicated to play a role in various types of cancer and neurodegenerative disorders. In this study we demonstrated that MK5 phosphorylates DNAJB1 in vitro, and that Tyrosine residue 6 and Serine residues 149, 151 and 171 in DNAJB1 are in vitro phosphorylation sites for MK5. Our results also indicate that other phosphorylation sites may be present. Further experiments are needed to elucidate the in vivo potential of these phosphoacceptor sites. We also found that MK5 and DNAJB1 exist in complexes. This interaction proved hard to reproduce, indicating that it might be of a transient nature, or perhaps a product of non-physiological conditions. We also showed that both proteins localize mainly to the nucleus in resting cells, when ectopically expressed, and that DNAJB1 seems to downregulate the level or the transcriptional activity of MK5 when both proteins are ectopically expressed in the cell.<br /> 2011-05-01T22:00:00Z Lægreid, Kari Jenssen http://hdl.handle.net/10037/3389 Killie, Ida Løken<br /> Neonatal alloimmune thrombocytopenia (NAIT) is most commonly caused by destruction of foetal platelets by maternal antibodies reactive to human platelet antigen (HPA)-1a. The activation of antigen-primed B cells to differentiate to antibody-secreting plasma cells usually requires help from CD4 T cells. The strong association between anti-HPA-1a-production and the MHC allele HLA-DRB3*0101 supports that this notion is also valid in the context of NAIT, and suggests the activation of HPA-1a-specific T cells as the most critical event of immunization. In this study, an improved protocol for enrichment, identification and efficient isolation of HPA-1a-specific CD4 T cells is presented. By replacing foetal bovine serum with human serum, enrichment of antigen-specific CD4 T cells improved dramatically. Identification and isolation of HPA-1a-specific CD4 T cells greatly improved when combining the CFSE proliferation assay with a second stimulation with antigen and subsequent assay for surface detection of TNF production. HPA-1a-specific CD4 T cells could also be identified in the CFSE proliferation assay as proliferating T cells with down-regulated expression of CD4. HPA-1a-specific T cells isolated from immunized women may serve as useful tools for investigating the cellular immune response to HPA-1a, and for developing strategies to prevent immunization in HPA-incompatible pregnancies, e.g. through TCR epitope mapping and examinations of the immunogenicity of the HPA-1a antigen at the amino-acid level.<br /> 2010-11-14T23:00:00Z Killie, Ida Løken http://hdl.handle.net/10037/3115 Haldorsen, Bjørg Christina<br /> Aminoglycosides represent an important class of antimicrobial agents. The prevalence of aminoglycoside resistance among Gram-negative bacteria in Norway is low, but an increased prevalence among clinical isolates of Escherichia coli has been observed during the last years. The most prevalent resistance mechanism is aminoglycoside modifying enzymes. In addition, resistance may occur when bacteria produces 16S rRNA methylases, which causes high level and broad-spectrum aminoglycoside resistance. In this study, we analysed the susceptibility pattern of different aminoglycosides in different Norwegian strain collections of E. coli, Klebsiella spp. and Pseudomonas aeruginosa. Among E. coli and Klebsiella spp. isolates from the Norwegian surveillance programme for antimicrobial resistance (NORM) 2009 the prevalence of reduced susceptibility to tobramycin (4.1%) was slightly higher compared to gentamicin (3.8), in blood culture isolates of E. coli and 2% and 1.4% in in blood culture isolates of Klebsiella spp., respectively. The prevalence of reduced susceptibility to amikacin was low in both species; 0.6% in E. coli and 0.4% in Klebsiella spp. In a collection of ESBLA-positive Enterobacteriaceae isolates the prevalence of reduced susceptibility was much higher with 56% and 44% of the isolates showing reduced susceptibility to tobramycin and gentamicin, respectively. The prevalence of reduced susceptibility to amikacin (7%) was also lower than for tobramycin and gentamicin in the ESBLA-positive isolates. The same pattern was also observed in the collection of carbapenem non-susceptible P. aeruginosa isolates. In both Enterobacteriaceae collections the aminoglycoside modifying enzymes AAC(3)-II and AAC(6’)-Ib were the dominating enzymes causing aminoglycoside resistance. The 16S rRNA methylases rmtB and rmtD were detected in one E. coli and one P. aeruginosa isolate resistant to all aminoglycosides tested, respectively. Three different methods for detection of reduced susceptibility were used; Etest, EUCAST disk diffusion and VITEK2 AST. The results from the three methods were compared. Discrepancies were mainly observed when comparing Etest and EUCAST disk diffusion for detecting tobramycin resistance in Enterobacteriaceae, and comparing Etest and EUCAST disk diffusion for detecting gentamicin resistance in P. aeruginosa. In conclusion, aminoglycoside resistance in Norway is low, but increasing. Worryingly, aminoglycoside resistance is coupled with other resistance mechanisms such as ESBLA resulting in multidrug resistance limiting treatment options. Method comparison indicates a need for evaluation and frequent maintenance of breakpoints.<br /> 2011-04-05T22:00:00Z Haldorsen, Bjørg Christina http://hdl.handle.net/10037/2975 Berg, Vivian<br /> Common features of arthritis include destruction of extracellular matrices in cartilage and bone as a result of chronic inflammation. Cartilage deterioration is generally described as a result from the effect of immune cells and their inflammatory mediators. However, recent reports suggest a role of chondrocytes in the initiation of inflammation in joints, and that they play a pivotal role in the destruction of their own matrix. Chondrocytes express multiple immune receptors and can produce and bind several cytokines, thus rendering them possible targets for therapy. We have demonstrated that serially cultured human articular chondrocytes posses the Chemerin receptor, ChemR23, and that this receptor is also present on chondrocytes in native human cartilage. In cultured chondrocytes we detected mRNA for the Chemerin receptor, and observed that stimulation with Chemerin resulted in phosphorylation of p44/p42 MAPK and Akt. Moreover, the Chemerin stimulation resulted in a marked increase of the pro-inflammatory cytokines IL-6 and IL-8, a modest increase in TNF-α and IL-1β, and marked increase in MMP-2, MMP-3 and MMP-13 in the culture supernatants. These results show that ChemR23 conveys pro-inflammatory signalling and affects MMP production when binding its ligand Chemerin. These observations indicate that ChemR23 takes part in inflammation initiation and cartilage degradation in joint disease.<br /> 2008-12-16T23:00:00Z Berg, Vivian http://hdl.handle.net/10037/2790 Aljabri, M. Belal<br /> P53 is a key tumor suppressor and transcription factor protecting us from cancer. The wild type p53 protein functions as a regulatory protein, triggering a variety of cellular responses to different signals. Activation of p53 can lead to cell division arrest, DNA repair, or apoptosis. More than 60% of all human cancers contain p53 mutations. P53 is also reported in many studies to play a role in the control of other cellular important activities such as angiogenesis and glycolysis. In this study we aimed to to identify novel target genes of p53 by investigating the difference in down-stream gene expression of wt p53 in a the Saos-2 cell line which is devoid of p53 expression, in comparison to mutated form of p53 that has been reported to be associated with cancer and in relation to the lack of p53 expression. The aim was also to study protein-protein interactions between p53 and its protein partners in the different p53 variants. Two different p53 mutations (R249S and R273H), considered as hot mutations, were constructed by site-directed mutagenesis. The GeneSwitch system was used to make stable inducible p53 cell lines. This expression is controlled by mifepristone (inducer). Total proteins were isolated from the different cell lines and separated on 2D gels. The total protein expression in Saos-2 cells containing wild type p53, R249S or R273H mutants, in addition to cells with no p53 copy were compared. The expression patterns of the different samples were similar but not identical. Our results showed a different expression patterns in some vital proteins. Our results suggest a role of p53 in transcriptionally activating the β subunit of Prolyl 4-Hydroxylase which plays an important role in angiogenesis. Also our results show different patterns in expressing vimentin, which is the most abundant intermediate filament protein in various cell types, between the different cell lines. Also our results show a clear difference in the protein expression patterns of four proteins, which are essential in glycolysis (TIM, enolase 1, α-enolase and aldolase A.) suggesting a role of p53 in the metabolism of tumors particularly in glycolysis. Recent studies have implicated some of these proteins in cancer if not to p53 as well. Studies dealing with P53 and its partner proteins from the total protein using anti V5 antibody were attempted, but was not pursued further due to the short time. The way of doing this was proteomics.<br /> 2006-10-31T23:00:00Z Aljabri, M. Belal http://hdl.handle.net/10037/2613 Jansen, Marta Karoline<br /> The arctic is exposed to a wide range of pollutants which originate from industrialized areas located in temperate regions. Halogenated organic contaminants (HOCs) are of special concern due to their persistency and ability to bioaccumulate. Consequently, animals high in the food chain, such as marine seabirds, are prone to accumulate high levels of these compounds. The HOCs are known to increase the formation of free radicals through metabolism and thereby jeopardise the balance between pro and antioxidant forces, which could lead to oxidative stress and therefore sever biological cell dysfunctions. The antioxidant system of kidney tissue of arctic seabirds has previously not been investigated as an organ for measuring effects of HOCs. In this study, single antioxidant parameters were measured in kidney tissue of three species of seabirds to investigate their species specific differences in antioxidant defences. The antioxidant parameters, catalase and glutathione peroxidase, glutathione reductase and total glutathione levels, revealed different species-specific antioxidant strategies in common eider, kittiwake and northern fulmar which are related to abiotic and biotic factors. In addition an experimental study was performed on herring gull chicks to assess the cause-effect relationship of exposure to HOCs, following parental transfer during feeding ,and fasting on the total oxidative scavenging capacity (TOSC) of their kidney tissue The TOSC assay provides an antioxidant scavenging profile, showing that exposure to HOCs significantly decrease the TOSC towards hydroxyl radicals and that exposure and fasting significantly increase the TOSC towards peroxynitrite and peroxyl radicals. We can conclude that parental transfer of HOCs to bird chicks can lead to biological effects in kidneys and effects are different between non fasting and fasting treatment. Finally, TOSC can be used as a biomarker on effects of HOCs in birds.<br /> 2010-06-14T22:00:00Z Jansen, Marta Karoline