<p>The reversible phosphorylation of proteins is central to regulating most aspects of cell function. Malfunction in this critical cellular process have been implicated to cause diseases such as diabetes, cancer, and Alzheimer’s. Recent advances in mass spectrometry have made it possible to study this important post translational modification on a proteome-wide scale. However, to be able to do so, enrichment of phosphorylated peptides is required. Pairwise comparison of individual steps in an enrichment procedure and simultaneous improvement of data analysis resulted in a protocol which allowed high confidence identification of 2,131 unique phosphorylated peptides from 1,026 proteins. Thereby not only establishing a working protocol for phosphopeptide enrichment in the Griffin Lab, but also generating the largest list of proteins phosphorylated under normal conditions in yeast to date.</p>

The reversible phosphorylation of proteins is central to regulating most aspects of cell function. Malfunction in this critical cellular process have been implicated to cause diseases such as diabetes, cancer, and Alzheimer’s. Recent advances in mass spectrometry have made it possible to study this important post translational modification on a proteome-wide scale. However, to be able to do so, enrichment of phosphorylated peptides is required. Pairwise comparison of individual steps in an enrichment procedure and simultaneous improvement of data analysis resulted in a protocol which allowed high confidence identification of 2,131 unique phosphorylated peptides from 1,026 proteins. Thereby not only establishing a working protocol for phosphopeptide enrichment in the Griffin Lab, but also generating the largest list of proteins phosphorylated under normal conditions in yeast to date.

Biofuels that are renewable and environmentally benign constitute an important area of research, as the supply of fossil fuels decreases and the amount of green house gases in the atmosphere increases. Biohydrogen is not as well explored as other biofuels, but its properties render it a promising complement, as it is clean and can be used directly in fuel cells to generate electricity, the only waste products being water and heat. Hydrogenproducing microorganisms have the potential to be used to recycle industrial waste, such as carbohydrates from food manufacturing. Hence the cost of waste disposal could be reduced whilst biofuel is being produced through microbial processes. Escherichia coli is a well-known microorganism that produces hydrogen under fermentative conditions, through the conversion of formate to hydrogen gas and carbon dioxide, via an enzyme complex called formate hydrogenlyase (FHL). The complex is anchored to the inner cell membrane and consists of seven subunits: a formate dehydrogenase, a [Ni-Fe] hydrogenase, three electron carrier proteins, which together make up a large ‘hydrophilic domain’, and two integral membrane proteins (the ‘membrane domain’). Even though the entire bacterial genome is known, the FHL complex remains little understood and has proven difficult to isolate and characterise. During this project, a genetically modified strain producing only the hydrophilic domain of FHL was constructed, and the resultant sub-complex was purified. It was hoped that, if a stable and homogenous core complex could be isolated, it might be subjected to further analysis, such as elucidating the subunit stoichiometry and solving the structure. Furthermore, FHL is notoriously oxygen labile, which hampers its study and technological development. However, oxygen tolerance is a natural feature found in some other [Ni-Fe] hydrogenases, and recent research shows that this property is likely dependent on the presence of extra cysteine residues near an important metal cluster in the enzyme. These cysteines are not present in FHL and a complex that could be active in both aerobic and anaerobic conditions may be a useful tool in optimising microbial biohydrogen processes. Thus, three strains that each expressed a modified FHL variant carrying single Cysteine-for-Glycine substitutions were constructed. The modified FHL complexes proved to remain active in vivo, and can serve as the basis of genetically engineering oxygen tolerance into this important enzyme.

<p>Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus <em>Phytophthora </em>are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus <em>Saprolegnia</em>, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen <em>Phytophthora infestans</em> and the fish pathogen <em>Saprolegnia monoica</em>.</p><p> </p><p>A new Oomycete <em>CesA</em> gene family was identified. It contains four homologues designated as <em>CesA1, CesA2, CesA3</em> and <em>CesA4</em>. The gene products of <em>CesA1, 2</em> and <em>4 </em>contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete <em>CesA </em>genes. <em>CesA3</em> is the dominantly expressed <em>CesA </em>homologue in the mycelium of both <em>S. monoica</em> and <em>P. infestans</em>, while <em>CesA1</em> and<em> CesA2</em> are up-regulated in virulent life stages of <em>P. infestans</em>. <em>CesA4</em> was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole <em>CesA</em> gene family in <em>P. infestans</em> lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on <em>SmCesA</em> gene expression, which was accompanied by an increased b-glucan synthase activity <em>in vitro</em>. In addition, these inhibitors slowed down the growth of the mycelium from <em>S. monoica</em>. Zoospores from <em>P. infestans</em> treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the <em>CesA</em> gene family.</p><p>Altogether these results show that at least some of the <em>CesA1-4</em> genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by <em>P. infestans</em>.</p><p> </p>

<p> </p><p>In orthopaedic practice, fractures are usually stabilised with metal screws or rods. This is done in order to keep the fracture parts in place during the rather slow healing process. The healing time can potentially be reduced by local- or systemic treatment with different bone promoting drugs. In later years, lithium, otherwise used to treat bipolar disease, has shown promise to be such a drug.</p><p> </p><p>The aim of this master thesis was to find a way to coat metal bone screws with lithium and to characterise the coating. The coating was to be designed in such a way that it could release lithium to the surrounding bone tissue.</p><p> </p><p>Lithium chloride was incorporated into a titanate sol-gel and attached to silicon wafers and stainless steel screws by dip coating. Wafers were used for initial <em>in vitro</em> studies of how lithium changed coating characteristics. This was studied using ellipsometry, AFM and SEM. Lithium is most probably physisorbed and not incorporated into the network building up the sol-gel. Coating structure is changed as more lithium is incorporated. For large amounts of lithium, the nanoparticles normally formed when curing the sol-gel are inhibited. One effect of this is reduced bioactivity, seen as a reduced ability for calcium phosphate crystals to nucleate on the coating when immersed in simulated body fluid.</p><p>Lithium release was investigated using AAS. Lithium is released from the coating, showing a burst effect. By changing the number of coating layers used, the release profile can be partly altered. The coating was also applied to screws, showing good attachment, and the lithium release profile was similar to the one seen from wafers.</p><p>Finally, a screw model was used in rats to assess the effect of local lithium treatment from screws and systemic lithium treatment on fracture healing. In the model, a screw was inserted in tibia, mimicking a fracture. When the bone around the screw was healed, a pullout test was performed, giving information about the strength of the bone surrounding the screw. No significant difference could be found for either local- or systemic lithium treatment compared to control. However, when evaluating the strength of intact bone in a similar way, a positive effect of systemic lithium treatment could be seen. Therefore, it is still likely that lithium has a positive effect on bone and further studies are needed to fully evaluate its role in fracture healing.</p><p> </p> / <p><p>Vid behandling av benbrott stabiliseras vanligtvis frakturen internt med metallskruvar och</p><p>metallstavar. Detta görs för att hålla brottbitarna på plats under den relativt långsamma läkprocessen. Det är möjligt att minska tiden för frakturläkning genom att lokalt eller systemiskt behandla med olika läkemedel som främjar bentillväxt. På senare år har det presenterats bevis för att litium, som annars används som psykofarmaka, fungerar som ett sådant läkemedel.</p><p> </p><p>Syftet med detta examensarbete var att hitta en metod för att fästa litium på benimplantat. Litium skulle fästas på ett sådant sätt att frisläppning till omgivande vävnad blev möjlig.</p><p> </p><p>Litiumklorid inkorporerades i en titanat-solgel och lager av detta lades på kiselytor och rostfria skruvar genom s.k. ”dip-coating”. Kiselytorna användes för initiala <em>in vitro</em>-studier av hur litium ändrade beläggningens egenskaper. Litium sitter antagligen fast på ytan av det tredimensionella nätverk som utgör solgelen, istället för att sitta inbundet i nätverket. Lagerstrukturen ändras ju mer litium som inkorporeras och vid stora mängder skapas inte de nanopartiklar som vanligtvis finns i en solgel-baserad beläggning. En följd av detta är reducerad bioaktivitet för beläggningen, dvs. en minskad förmåga för kalciumfosfatkristaller att bildas på ytan. Litium frisläpps från beläggningen, dock sker denna frisläppning snabbt. Genom att belägga ytan med flera lager av solgel kan frisläppningskinetiken delvis ändras. Solgelen kunde också med god vidhäftning appliceras på skruvar och frisläppningskinetiken från en skruv är liknande den från en kiselyta.</p>Slutligen användes en skruvmodell i råtta för att undersöka vilken effekt lokal respektive systemisk litiumbehandling har på frakturläkning. I modellen efterliknas ett benbrott genom att en skruv sätts in i skenbenet.  När benvävnaden runt skruven har läkt görs ett utdragstest på skruven vilket ger information om benets styrka. Ingen signifikant skillnad i skruvens utdragskraft kunde ses mellan de båda försöksgrupperna och kontrollgruppen. Däremot hade gruppen som fick systemisk litiumbehandling fått starkare ben totalt, vilket indikerar att litium har effekt på <em>intakt</em> ben. På grund av dessa resultat finns det fortfarande skäl att tro att litium har en positiv påverkan på ben, varför dess effekt på frakturläkning bör undersökas ytterligare. </p>

In orthopaedic practice, fractures are usually stabilised with metal screws or rods. This is done in order to keep the fracture parts in place during the rather slow healing process. The healing time can potentially be reduced by local- or systemic treatment with different bone promoting drugs. In later years, lithium, otherwise used to treat bipolar disease, has shown promise to be such a drug.   The aim of this master thesis was to find a way to coat metal bone screws with lithium and to characterise the coating. The coating was to be designed in such a way that it could release lithium to the surrounding bone tissue.   Lithium chloride was incorporated into a titanate sol-gel and attached to silicon wafers and stainless steel screws by dip coating. Wafers were used for initial in vitro studies of how lithium changed coating characteristics. This was studied using ellipsometry, AFM and SEM. Lithium is most probably physisorbed and not incorporated into the network building up the sol-gel. Coating structure is changed as more lithium is incorporated. For large amounts of lithium, the nanoparticles normally formed when curing the sol-gel are inhibited. One effect of this is reduced bioactivity, seen as a reduced ability for calcium phosphate crystals to nucleate on the coating when immersed in simulated body fluid. Lithium release was investigated using AAS. Lithium is released from the coating, showing a burst effect. By changing the number of coating layers used, the release profile can be partly altered. The coating was also applied to screws, showing good attachment, and the lithium release profile was similar to the one seen from wafers. Finally, a screw model was used in rats to assess the effect of local lithium treatment from screws and systemic lithium treatment on fracture healing. In the model, a screw was inserted in tibia, mimicking a fracture. When the bone around the screw was healed, a pullout test was performed, giving information about the strength of the bone surrounding the screw. No significant difference could be found for either local- or systemic lithium treatment compared to control. However, when evaluating the strength of intact bone in a similar way, a positive effect of systemic lithium treatment could be seen. Therefore, it is still likely that lithium has a positive effect on bone and further studies are needed to fully evaluate its role in fracture healing. / Vid behandling av benbrott stabiliseras vanligtvis frakturen internt med metallskruvar och metallstavar. Detta görs för att hålla brottbitarna på plats under den relativt långsamma läkprocessen. Det är möjligt att minska tiden för frakturläkning genom att lokalt eller systemiskt behandla med olika läkemedel som främjar bentillväxt. På senare år har det presenterats bevis för att litium, som annars används som psykofarmaka, fungerar som ett sådant läkemedel.   Syftet med detta examensarbete var att hitta en metod för att fästa litium på benimplantat. Litium skulle fästas på ett sådant sätt att frisläppning till omgivande vävnad blev möjlig.   Litiumklorid inkorporerades i en titanat-solgel och lager av detta lades på kiselytor och rostfria skruvar genom s.k. ”dip-coating”. Kiselytorna användes för initiala in vitro-studier av hur litium ändrade beläggningens egenskaper. Litium sitter antagligen fast på ytan av det tredimensionella nätverk som utgör solgelen, istället för att sitta inbundet i nätverket. Lagerstrukturen ändras ju mer litium som inkorporeras och vid stora mängder skapas inte de nanopartiklar som vanligtvis finns i en solgel-baserad beläggning. En följd av detta är reducerad bioaktivitet för beläggningen, dvs. en minskad förmåga för kalciumfosfatkristaller att bildas på ytan. Litium frisläpps från beläggningen, dock sker denna frisläppning snabbt. Genom att belägga ytan med flera lager av solgel kan frisläppningskinetiken delvis ändras. Solgelen kunde också med god vidhäftning appliceras på skruvar och frisläppningskinetiken från en skruv är liknande den från en kiselyta. Slutligen användes en skruvmodell i råtta för att undersöka vilken effekt lokal respektive systemisk litiumbehandling har på frakturläkning. I modellen efterliknas ett benbrott genom att en skruv sätts in i skenbenet.  När benvävnaden runt skruven har läkt görs ett utdragstest på skruven vilket ger information om benets styrka. Ingen signifikant skillnad i skruvens utdragskraft kunde ses mellan de båda försöksgrupperna och kontrollgruppen. Däremot hade gruppen som fick systemisk litiumbehandling fått starkare ben totalt, vilket indikerar att litium har effekt på intakt ben. På grund av dessa resultat finns det fortfarande skäl att tro att litium har en positiv påverkan på ben, varför dess effekt på frakturläkning bör undersökas ytterligare.

The RNA polymerase T7 is utilized as a component of motor complexes in DNA nanotechnology due to its high promotor specificity, the lack of external transcription factors and its very high processivity, but there is no experience of its application on small double stranded DNA circles. Circular templates from 210 to 126 bp in circumference sharing a common promotor termination motif were synthesized and transcription was monitored at end point on gel and in real time with a 2’ O methyl RNA molecular beacon. The RNAP T7 was found to be able to utilize circular dsDNA templates down to 126 bp with moderate impact on transcription rate for saturated systems and rolling circle transcription products were evident with denaturizing PAGE gel electrophoresis for templates down to 167 bp.