Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

Several Fusarium species can cause Fusarium head blight (FHB) and Fusarium crown rot (FCR) diseases in wheat and these are of economic importance in wheat production systems globally. Fusarium graminearum represents a model pathogen species to study these diseases because it has a sequenced genome, commercially available gene expression arrays and an growing collection of mutants impaired in pathogenicity and virulence, at least for FHB. FCR occurs at the stem base of the wheat plant, causing major reductions in grain yield. FCR has been much less intensively researched than FHB and the infection process of F. graminearum during crown rot disease in wheat has not been studied previously at the molecular level. Fungal biomass estimations by real-time quantitative PCR analysis of DNA from inoculated plants identified three distinct phases of infection during FCR, an initial increase in fungal mass in phase 1 up to 2 days post inoculation (dpi), then a reduction during phase 2 until 14 dpi followed by a large increase thereon in phase 3 that corresponded to symptom development. Histological characterisation of F. graminearum colonisation during these three phases of infection showed that initially the spores germinated on the stem surface at the point of inoculation forming a superficial hyphal mat. This occurred within the first two days of infection. The second phase was characterised by a period of low amounts of fungal tissue present in the infected plants and 14 days following infection hyphae were only observed below the point of inoculation at the stem base of the wheat seedling and had penetrated and colonised the adaxial epidermis of the outer leaf sheath. Following this, the third phase was characterised by a major colonisation of the internal tissues of the crown which corresponded to visible symptom development around 35 days after inoculation. Fungal gene expression during all three phases of infection were examined using the Affymetrix GeneChip system comprised of 22,000 F. graminearum gene probe sets. This analysis showed 1,839 genes were significantly up regulated in planta compared to axenic vegetative mycelia, including some known FHB virulence genes (e.g. those involved in the biosynthesis of trichothecene toxins). Fungal genes differentially regulated between the phases were identified indicating that FCR disease development requires a coordinated process involving distinct fungal gene expression programs. A bioinformatic comparison of global F. graminearum gene expression during FCR of wheat with published data for FHB of barley indicated similarities at very early stages of infection but divergence thereafter. It was decided to functionally test whether F. graminearum utilises the same virulence genes in FCR and FHB diseases. Because no virulence genes have been previously identified from FCR studies a small group of genes were initially selected from the FCR gene expression studies for further functional analysis using gene knock-out technology. Only two of these genes showed a changed phenotype during Fusarium infection of wheat plants and they encoded a probable ABC transporter (FgABC1) and a probable superoxide dismutase (FgSOD1). It was interesting to note that even though both FgABC1 and FgSOD1 exhibited similar transcription profiles during both FCR of wheat and FHB of barley it was found that FgABC1 was specifically required for full FCR disease development on the wheat cultivar Kennedy whereas FgSOD1 was specifically required for FHB disease on the same cultivar. This indicated that F. graminearum virulence genes can show specificity to the infection of different plant tissues and that these types of genes cannot be predicted based only on their transcription profiles. It is suggested that F. graminearum induces a global set of virulence factors but only some of these may be effective in particular tissues. To test further whether there was tissue specialisation for specific tissues and FCR & FHB diseases, a group of F. graminearum genes that were known virulence factors during FHB were tested to see if they were also virulence factors for FCR. This analysis showed that two genes displayed specificity only for FHB and five were virulence factors for both FHB and FCR. One of the genes that was a virulence factor for both diseases was the Tri5 gene that is necessary for the biosynthesis of trichothecene mycotoxins. This gene and these toxins did not appear to be necessary for symptom development and the induction of host defence responses but were necessary for fungal colonisation of the crown and stem in later stages of infection. Interestingly there were parallels in the role played by the Tri5 gene in FCR and that reported for FHB where it is necessary for colonisation for the spike. This study is the first molecular analysis of any Fusarium species during crown rot of wheat. Importantly, it shows that there may be specialisation towards host tissues for some virulence genes but also suggests that some factors may be non-specifically required for infection and it is these factors that will represent attractive targets for future control measures of both diseases.

Universiẗat, Diss., 2006--Frankfurt (Main).

University, Diss., 2006--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

University, Diss., 2006--Frankfurt (Main). / Erscheinungsjahr an der Haupttitelstelle: 2005.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Frankfurt (Main), Univ., Diss., 2009 / Erscheinungsjahr an der Haupttitelstelle: 2008

Diss. (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 4 uppsatser.

<p>Gustav Kährs AB är ett företag som funderar på att byta transportsätt från lastbil till järnväg. Kährs är beläget i Nybro, tillverkar golv och lagerhåller sina produkter i ett centrallager i Kalmar. I dagsläget sköts all transport från centrallagret med lastbil, varorna transporteras sedan vidare till Helsingborgs hamn där de lastas och skeppas vidare till Bremen. Det Kährs funderar över är att göra ett skifte till järnväg på denna transportsträcka.</p><p>Följande problemformulering har legat till grund för denna rapport:</p><p>”Är det värt att ställa om sina godstransporter från lastbil till järnväg med avseende på följande aspekter: miljö, ledtid, kostnad och investeringar?”</p><p>För att få en djupare insikt i vad ett skifte från järnväg till lastbil skulle kunna få för konsekvenser i fråga om ledtider, kostnader, investeringar och miljön har vi valt att göra en fallstudie. Fallstudiens syfte är inte att komma fram till ett definitivt svar utan vara mera öppen för tolkning, vilket även är fallet för vår problemformulering, då frågan om det är värt ett skifte är subjektiv.</p><p>Vi har hela tiden i rapporten arbetat ifrån en modell uppbyggd av de aspekter som nämns i vår problemformulering. Utifrån denna modell har vi sedan skapat en beräkningsmodell för att komma fram till om det är värt ett skifte från lastbil till järnväg. Det har då tagits fram olika scenarion där nuläget med lastbil ställs mot andra alternativ med järnväg som transportsätt. De olika scenariona innefattar olika hamnar och investeringar i form av kranar, truckar och förlängning av räls.</p><p>Beräkningsmodellen fokuserar på de kostnader som de olika scenarierna ger upphov till. Resultatet av analysen blev att lastbilen fick fler fördelar än järnvägen, men vilket alternativ som slutligen väljs beror mer på vilka egenskaper som Kährs prioriterar. Om exempelvis miljön är en viktig faktor, det vill säga om företaget har en stark önskan att profilera sig som miljövänligt, då kanske järnvägen är extra intressant. Frågan blir bara om företaget är beredda att ta de extra kostnader som blir följden. Kanske företaget inte anser att de miljöbesparingarna som järnvägen ger är en tillräcklig anledning för att ta på sig de extra kostnader som transportsättet är förknippat med.</p><p>Svaret på problemformuleringen blev slutligen att det inte var värt ett skifte från lastbil till järnväg. Men i slutändan handlar det om vilka egenskaper som Kährs värderar högst.</p> / <p>Gustav Kährs AB is a company that is considering a change of transportation from trucks to railroad. Kährs headquarter is situated in Nybro. The company makes wooden floors and stores them in their central warehouse in Kalmar. Today all transports from the warehouse are carried out by trucks; the goods are then transported to the harbor in Helsingborg and then shipped to Bremen. Kährs is considering switching to railroad transportation between Kalmar and Helsingborg.</p><p>The problem has been formulated as follows:</p><p>“Is it worth to switch transportation method from tuck to railroad considering the following aspects: the environment, lead times, costs and investments?”</p><p>To gain a deeper insight of the consequences that a switch would have on lead times, costs, investments and the environment we have chosen to conduct a case study. The purpose of this case study is not to reach a definitive answer to the problem but rather to give an open discussion on the subject. This fits our formulated problem fine, because the question if it is worth or not is a subjective matter.</p><p>This thesis has been built around a model that consists of all the different aspects that have been mentioned within the formulated problem. From this model we have derived a calculation model to calculate if it is worth to make a switch from tucks to railroad from a cost perspective. From the calculation model we have further derived different scenarios were today’s situation is compared to alternative means of transportation involving railroad. The different scenarios include different harbors and investments like cranes, forklifts and extension of railroad.</p><p>The calculation model focuses on the costs being generated by each scenario. The result of the analysis is that today’s truck solution had the most advantages compared to the other scenarios. But what final solution is chosen depends on what characteristics Kährs values. If the environment is an important factor, if Kährs feels that it is important to position itself as an environmental friendly company then the railroad would be an interesting alternative. The question is if Kährs is prepared to deal with the increased costs that this alternative generates. Perhaps the company doesn’t feel that the savings on the environmental side are reason enough to stand the extra costs.</p><p>The answer to our formulated problem is that we don’t consider it worth shifting from tuck to railroad. But in the end it is all about what characteristics Kährs consider to be important.</p>