Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases. The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A. Finally, the structure of YegS, an Escherichia coli protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.

In recent years, the use of alcohol and psychoactive drugs in combination withdriving has recieved increased attention. The lack of in-vehicle devices capable ofdetecting recent drug consumption and the difficulties associated with the breathbasedalcolocks in use today makes it interesting to investigate methods that areable to non-invasivelly measure analytes directly in the blood. The assignment of this project, commissioned by Volvo Technology Corporationand Volvo Car Corporation, is to map substances that constitute a possible threatto traffic safety, identify suitable detection markers as a proof of administrationof these substances, and study possible non-invasive techniques to detect thesemarkers. The objective is to present for Volvo if and how to continue evaluatingand developing a non-invasive detection device. The project has been carried out by performing an extensive literature study and averification experiment. From the literature review, a number of substances affectingdriving performance could be identified, and a metabolic study was performedfor each drug to map suitable biomarkers. Furthermore, two potential techniquesfor non-invasive detection, near-infrared Raman spectroscopy and near-infraredspectroscopy, were found and evaluated. The experiment was conducted usingnear-infrared Raman spectroscopy, with the aim of investigating the sensitivityand linearity of the method for ethanol detection. Based on the theoretical evaluation, both near-infrared Raman spectroscopy andnear-infrared spectroscopy are expected to have potential for non-invasive detectionof ethanol. The experiment further proved the theoretical conclusionsmade for near-infrared Raman spectroscopy. However, neither of the techniquesis thought to have potential for drug detection.Altogether, we believe that non-invasive ethanol detection is possible, but suggestfurther experiments in order to determine which technique to be preferred.

In humans, noroviruses (NVs) cause acute epidemic and viral gastroenteritis. NVs do not only infect humans; viruseshave also been found in pigs, cows, sheep, mice and dogs. The focus in this project has been on the murine norovirus(MNV). MNV is a member of the viral family Caliciviridae and it consists of a single-stranded, positive sense RNAgenome. The genome includes three open reading frames (ORFs), ORF1 encodes for a polyprotein that consists of theprecursor to the 6-7 non-structural (NS) proteins. The polyprotein is cleaved by the NS6 protease. The NS6 isresponsible for all the cleaving in ORF1 and that makes it an attractive target for antiviral drugs. The NS6 proteinstructure has been determined at 1.66 Å resolution using X-ray diffraction techniques. Surprisingly, the electrondensity map revealed density for a peptide bound in the active site. The peptide had a length of 7 residues andoriginated from the C-terminus of another chain in an adjacent asymmetric unit. The active site triad was composed ofthe conserved residues; histidine 30, aspargine 54 and cysteine 139, however in the structure the cysteine 139 ismutated to an alanine to inactivate the protease. Activity assays were performed to probe the importance of the residuein position 109 in the β-ribbon located close to the active site. The three full-length constructs with the mutations;I109A, I109S and I109T were found to have less activity than the full-length wt (1-183). A truncated protease, lacking9 residues in the C-terminus, also had less activity. This indicates that the terminal residues are also important foractivity.

Encystation of unicellular organisms is of considerable medical relevance since cysts are encapsulated byresilient cell walls, rendering them resistant to biocides and immune clearance. This survival strategymakes it complicated to produce effective treatment of diseases caused by many protozoan pathogens,e.g. species of Acanthamoeba which causes fatal granulomatous amebic encephalitis (GAE) and keratitis.Due to genetic limitations in most protists, the machinery of encystation is so far little understood.However, the signalling pathways can be investigated in the non-pathogenic social amoebas, Dictyostelia.In this master’s project, five genes in Polysphondylium pallidum were investigated, which might beinvolved in encystation. Research has demonstrated a relationship between encystation and the cyclicadenosine monophosphate (cAMP) signalling pathways in Dictyostelia spore formation. This indicates thatcysts are ancestral to spores, and hence are the sporulation genes: pkaC, yakA, regA, cudA and srfAselected for this study. The genes were individually knocked-out by a standard homologous recombinationapproach and the genes’ contribution to encystation was determined. Five knock-out constructs werecompleted and a preliminary analysis of the role of the intracellular cAMP phosphodiesterase RegA inPolysphondylium pallidum encystation process was performed.

The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO2-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.

A Positive Electron Tomography/Computed Tomography devise was installed in theX-ray section at US Linköping in May 2007. Positive Electron Tomography examinations with 18F-fluoro-deoxy-glucose are mainly used for tumor examinations. During 2007 occurred approximately 200 examinations and in 2008 600 are planned. Today there are two reconstruction methods commercially available, Filtered Back projection and Maximum Likelihood Expectation Maximiza tion, used in the faster version called Ordered Subsets Expectation Maximization. The image quality in Positive Electron Tomography depends on the choice of reconstruction method and the settings of its parameters. We have performed a physical phantom study with Positive Electron Tomography to determine optimal parameters for the iterative reconstruction algorithm Ordered Subsets Expectation Maximization. To find out whether or not the quality of the image can be improved, so that the patient received radiation dose and/or examination time can be lowered. The phantom used was a NEMA IEC Body PhantomTM, designed to mimic smallhot lesions typical in 18F, Fluorine-18 PET, and all calculations were done according to the NEMA NU2-2001 protocol. The main conclusion from this project is that a higher level of contrast can be reached, compared to the one clinically obtained today. Using more iterations then recommended from the manufacturer. / I maj 2007 installerades en Positiv Elektron Tomografi /DatoriseradTomografi-kamera, PET/CT, i Röntgenavdelningen Linköping US. PET med 18F-fluoro-deoxy-glucose används huvudsakligen för tumörundersökningar.2007 genomfördes ca 200 undersökningar, och för 2008 är ytterligare 600 planerade. Idag finns två olika bildrekonstruktionsmetoder kliniskt tillgängliga; Filtered Back projection och Maximum Likelihood Expectation Maximization, där den  vidareutvecklade versionen kallad Ordered Subsets Expectation Maximizationanvänds. Bildkvalitén från en Positive Electron Tomographykamera påverkas av valet av rekonstruktionsmetod och dess ingående parametrar. I detta projekt har en fantomstudie genomförts med syfte att bestämma de optimala parametrarna för den iterativa metoden Ordered Subsets Expectation Maximization. För att utreda huruvida stråldosen och/eller undersöknings tiden kan minskas. Det testfantom som användes var en NEMA IEC Body PhantomTM. Projektet följde metoden angiven i NEMA NU2-2001 protokollet. Det resultaten visar är att de rekommenderade inställningarna från tillverkaren inte är de optimala.

<p>Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.</p>

<p>A Positive Electron Tomography/Computed Tomography devise was installed in theX-ray section at US Linköping in May 2007. Positive Electron Tomography examinations with 18F-fluoro-deoxy-glucose are mainly used for tumor examinations. During 2007 occurred approximately 200 examinations and in 2008 600 are planned. Today there are two reconstruction methods commercially available, Filtered Back projection and Maximum Likelihood Expectation Maximiza tion, used in the faster version called Ordered Subsets Expectation Maximization. The image quality in Positive Electron Tomography depends on the choice of reconstruction method and the settings of its parameters. We have performed a physical phantom study with Positive Electron Tomography to determine optimal parameters for the iterative reconstruction algorithm Ordered Subsets Expectation Maximization. To find out whether or not the quality of the image can be improved, so that the patient received radiation dose and/or examination time can be lowered. The phantom used was a NEMA IEC Body PhantomTM, designed to mimic smallhot lesions typical in 18F, Fluorine-18 PET, and all calculations were done according to the NEMA NU2-2001 protocol.</p><p>The main conclusion from this project is that a higher level of contrast can be reached, compared to the one clinically obtained today. Using more iterations then recommended from the manufacturer.</p> / <p>I maj 2007 installerades en Positiv Elektron Tomografi /DatoriseradTomografi-kamera, PET/CT, i Röntgenavdelningen Linköping US. PET med 18F-fluoro-deoxy-glucose används huvudsakligen för tumörundersökningar.2007 genomfördes ca 200 undersökningar, och för 2008 är ytterligare 600 planerade. Idag finns två olika bildrekonstruktionsmetoder kliniskt tillgängliga; Filtered Back projection och Maximum Likelihood Expectation Maximization, där den  vidareutvecklade versionen kallad Ordered Subsets Expectation Maximizationanvänds. Bildkvalitén från en Positive Electron Tomographykamera påverkas av valet av rekonstruktionsmetod och dess ingående parametrar.</p><p>I detta projekt har en fantomstudie genomförts med syfte att bestämma de optimala parametrarna för den iterativa metoden Ordered Subsets Expectation Maximization. För att utreda huruvida stråldosen och/eller undersöknings tiden kan minskas. Det testfantom som användes var en NEMA IEC Body PhantomTM. Projektet följde metoden angiven i NEMA NU2-2001 protokollet. Det resultaten visar är att de rekommenderade inställningarna från tillverkaren inte är de optimala.</p>

Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight into chemical energy in the permanently changing natural environment. For successful survival and growth photosynthetic organisms have developed complex sensing and signaling acclimation mechanisms. The environmentally dependent protein phosphorylation in photosynthetic membranes is implied in the adaptive responses; however, the molecular mechanisms of this regulation are still largely unknown. We used a mass spectrometry-based approach to achieve a comprehensive mapping of the in vivo protein phosphorylation sites within photosynthetic membranes from the green alga Chlamydomonas reinhardtii subjected to distinct environmental conditions known to affect the photosynthetic machinery. The state transitions process regulating the energy distribution between two photosystems, involves the temporal functional coupling of phosphorylated light-harvesting complexes II (LHCII) to photosystem I (PSI). During state transitions several of the thylakoid proteins undergo redox-controlled phosphorylation-dephosphorylation cycles. This work provided evidences suggesting that redox-dependent phosphorylation-induced structural changes of the minor LHCII antenna protein CP29 determine the affinity of LHCII for either of the two photosystems. In state 1 the doubly phosphorylated CP29 acts as a linker between the photosystem II (PSII) core and the trimeric LHCII whereas in state 2 this quadruply phosphorylated CP29 would migrate to PSI on the PsaH side and provide the docking of LHCII trimers to the PSI complex. Moreover, this study revealed that exposure of Chlamydomonas cells to high light stress caused hyperphosphorylation of CP29 at seven distinct residues and suggested that high light-induced hyperphosphorylation of CP29 may uncouple this protein together with LHCII from both photosystems to minimize the damaging effects of excess light. Reversible phosphorylation of the PSII reaction center proteins was shown to be essential for the maintenance of active PSII under high light stress. Particularly dephosphorylation of the light-damaged D1 protein, a central functional subunit of the PSII reaction center, is required for its degradation and replacement. We found in the alga the reversible D1 protein phosphorylation, which until our work, has been considered as plant-specific. We also discovered specific induction of thylakoid protein phosphorylation during adaptation of alga to limiting environmental CO2. One of the phosphorylated proteins has five phosphorylation sites at both serine and treonine residues. The discovered specific low-CO2- and redox-dependent protein phosphorylation may be an early adaptive and signalling response of the green alga to limitation in inorganic carbon. This work provides the first comprehensive insight into the network of environmentally regulated protein phosphorylation in algal photosynthetic membranes.

Microbial respiration of oxochlorates is important for the biotreatment of effluents from industries where oxochlorates are produced or handled. Several bacterial species are capable to use perchlorate and/or chlorate as an alternative electron acceptor in absence of oxygen. The present study deals with the electron transport from the membrane-bound components to the periplasmic chlorate reductase, in the gram-negative bacterium Ideonella dechloratans. Both chlorate reductase and the terminal oxidase of I. dechloratans were found to utilize soluble c cytochromes as electron donors. For further investigation, two major heme-containing components were purified and characterized. The most abundant was a 9 kDa c-type cytochrome (class I), denoted cytochrome c-Id1. This protein was shown to serve as electron donor for both chlorate reductase, and for a terminal oxidase. The other major component was a 55 kDa homotetrameric cytochrome c', (class II). A function for this cytochrome could not be demonstrated but it does not appear to serve as electron donor to chlorate reductase. A gene predicted to encode a soluble c cytochrome was found in close proximity to the gene cluster for chlorate reduction. The predicted sequence did not match any of the cytochromes discussed above. The gene was cloned and expressed heterologously, and the resulting protein was investigated as a candidate electron donor for chlorate reductase. Electron transfer from this protein could not be demonstrated, suggesting that the gene product does not serve as immediate electron donor for chlorate reductase.