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<p>In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations.</p><p>Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species.</p><p>Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain.</p><p>In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing.</p><p>Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.</p>

<p>Transmission of bacterial strains between patients is a serious problem in hospitals and with the increasing rate of antibiotic resistance the problem has farther escalated. Enterobacteriaceae produced extended-spectrum beta-lactamses (ESBLs), especially Escherichia coli (E-coli), are increasingly important nosocomial pathogens (7, 8). These bacteria are often multiple resistant and are responsible for many intestinal infections and urinary tract infections (2, 5). With the more frequent use of invasive devices in hospital care, these types of nosocomial infections have increased, particularly in seriously ill patients.</p><p>In order to diminish transmission of bacterial strains between patients and to study the epidemiology of these bacteria, it is of great importance to develop rapid and specific methods to be able to subtype on strain-level, i.e. to create a fingerprint of the isolates. The method may be based on phenotypic or genotypic characteristics of the microorganism. Any typing method must have high reproducibility and discrimination power to differentiate unrelated strains and also to demonstrate relationship of organisms deriving from the same source. In the present project, a Pulsed-Field Gel Electrophoresis (PFGE) assay for genotyping clinical E. coli isolates was used. PFGE can be used as a genotyping tool and is widely used to type bacteria and trace nosocomial infection. However, the method is time-consuming and relatively expensive in compare with other methods like PCR. In this study, a total of 93 strains were collected. The study was aimed to investigate the genotypes of the collected isolates and to identify and potential the outbreak strains.</p><p>The isolates investigated were genotypically diverse shown by a variety of PFGE banding patterns. However, clusters of closely related isolates involved in outbreaks were also identified.</p><p>In conclusion, when analyzing a large number of strains, a combination of a rapid phenotyping or genotyping method and a powerful genotyping method like PFGE would be an appropriate strategy for studying clonal relationship among isolates e.g. for detecting cross-transmission of nosocomial pathogens.</p>

<p>Plant resistance hypothesis says that under a period of time when a plant is exposed to powerful herbivore attack it will prioritise defence as a major metabolic function. In theory, induced plant defence (resistance) will provide opportunities for this organism to “invest” in other functions, in example growth when attackers are absent.</p><p>One of the compounds taking part in plant defence is callose. This β-1,3-glucan is synthesised by callose synthase and broken down by β-1,3-glucanase. Deposition of callose occurs as a reaction to aphid attack an varies, depending on cultivars, and aphid species. In this experiment barley (Hordeum vulgare) cultivar Clipper is being infested with two types of aphids: Russian wheat aphid (RWA, Diuraphis noxia) and bird cherry-oat aphid (BCA, Rhopalosiphium padi) over a time period. Infestation by those two insects results in different callose formation and deposition level.</p><p>Six sequences encoding for putative callose synthase genes and nine sequences encoding for β-1,3-glucanase were examined by RT-PCR and Real – Time PCR methods for different expression patterns.</p><p>The results did not show any significant regulation of gene expression during RWA and BCA attack for any of these genes. Thus the pathway regulating aphid – induced callose deposition in barley reminds unresolved.</p>

Transmission of bacterial strains between patients is a serious problem in hospitals and with the increasing rate of antibiotic resistance the problem has farther escalated. Enterobacteriaceae produced extended-spectrum beta-lactamses (ESBLs), especially Escherichia coli (E-coli), are increasingly important nosocomial pathogens (7, 8). These bacteria are often multiple resistant and are responsible for many intestinal infections and urinary tract infections (2, 5). With the more frequent use of invasive devices in hospital care, these types of nosocomial infections have increased, particularly in seriously ill patients. In order to diminish transmission of bacterial strains between patients and to study the epidemiology of these bacteria, it is of great importance to develop rapid and specific methods to be able to subtype on strain-level, i.e. to create a fingerprint of the isolates. The method may be based on phenotypic or genotypic characteristics of the microorganism. Any typing method must have high reproducibility and discrimination power to differentiate unrelated strains and also to demonstrate relationship of organisms deriving from the same source. In the present project, a Pulsed-Field Gel Electrophoresis (PFGE) assay for genotyping clinical E. coli isolates was used. PFGE can be used as a genotyping tool and is widely used to type bacteria and trace nosocomial infection. However, the method is time-consuming and relatively expensive in compare with other methods like PCR. In this study, a total of 93 strains were collected. The study was aimed to investigate the genotypes of the collected isolates and to identify and potential the outbreak strains. The isolates investigated were genotypically diverse shown by a variety of PFGE banding patterns. However, clusters of closely related isolates involved in outbreaks were also identified. In conclusion, when analyzing a large number of strains, a combination of a rapid phenotyping or genotyping method and a powerful genotyping method like PFGE would be an appropriate strategy for studying clonal relationship among isolates e.g. for detecting cross-transmission of nosocomial pathogens.

Plant resistance hypothesis says that under a period of time when a plant is exposed to powerful herbivore attack it will prioritise defence as a major metabolic function. In theory, induced plant defence (resistance) will provide opportunities for this organism to “invest” in other functions, in example growth when attackers are absent. One of the compounds taking part in plant defence is callose. This β-1,3-glucan is synthesised by callose synthase and broken down by β-1,3-glucanase. Deposition of callose occurs as a reaction to aphid attack an varies, depending on cultivars, and aphid species. In this experiment barley (Hordeum vulgare) cultivar Clipper is being infested with two types of aphids: Russian wheat aphid (RWA, Diuraphis noxia) and bird cherry-oat aphid (BCA, Rhopalosiphium padi) over a time period. Infestation by those two insects results in different callose formation and deposition level. Six sequences encoding for putative callose synthase genes and nine sequences encoding for β-1,3-glucanase were examined by RT-PCR and Real – Time PCR methods for different expression patterns. The results did not show any significant regulation of gene expression during RWA and BCA attack for any of these genes. Thus the pathway regulating aphid – induced callose deposition in barley reminds unresolved.

Biological processes depend upon the structural and functional quality of the molecules that comprise living organisms. The integrity of molecules such as DNA, RNA, proteins, carbohydrates and lipids is crucial and the precise three-dimensional shape and the detailed chemistry of these molecules orchestrate the biochemical processes vital for life. Within a cell, each protein must be present at a specific concentration during certain specific conditions. To maintain cellular homeostasis and the ability to respond to the environment the proteome is in a dynamic state of synthesis and degradation. In eukaryotic cells the ubiquitin-proteasome pathway is the principal mechanism for regulated protein turnover in both the cytoplasm and the nucleus. The 20S proteasome is a cylindrical multi-subunit protease. Proteasomes play an essential role in the targeted and timely ordered degradation of key regulatory proteins and their inhibitors. The 26S proteasome is a 2.500 kDa complex composed of the 20S proteasome sandwiched between two 19S regulators. This is the enzymatic complex responsible for ATP-dependent ubiquitin mediated protein degradation. A polyubiquitin chain attached to a protein serves as a general recognition signal for destruction via the 26S proteasome. It is known that the 19S regulator confers ubiquitin recognition and substrate unfolding to the 20S proteasome, however, the specific functions for many of the different subunits within the 19S complex are not known. We have used RNA interference to study the S13/Rpn11 and S5a/Rpn10 subunits of Drosophila melanogatser proteasomes. We have produced stable cell lines with the human S13 gene under inducible promoters that was used to rescue the knockdown phenotype after RNA interference. The rescue was successful in demonstrating that the human protein is a functional homologue to the Drosophila protein. We call the technique RNAi+c (RNA interference + complementation). This procedure enabled us to also test different mutants of the human S13 protein for their ability to function in the proteasome. Using RNA interference to a Drosophila proteasome subunit in combination with complementation with a corresponding human protein we have been able to study residues important for the deubiquitinating activity of this subunit (Paper I). Interestingly, upon a decrease of either S13 or S5a we see an induction in the levels of active 20S proteasomes. Increase in the levels of the non-targeted 19S subunit can be detected when RNAi treatment is carried out on either S13 or S5a. We have used RNA interference and proteasomal inhibition together with whole genome microarray analysis to reveal a co-regulated network of proteasome genes. This network likely contributes to an overall regulatory system that maintains proper proteasome levels in the cell. Initial studies of the mechanism of transcriptional co-regulation of proteins involved in the 26S proteasome pathway were also performed (Paper II). Finally, the biological function of the proteasome regulator PA28g/REGg is not known. We have studied this regulator in Drosophila using RNA interference and promoter mapping (Paper III).

In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations. Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species. Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain. In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing. Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.

<p>Different mutations within the <em>NALP3</em> gene are thought to be associated with development of several types of hereditary auto inflammatory disorders such as neonatal onset multisystem inflammatory disorder (NOMID) and muckle-wells syndrome (MWS). In this work two separate mutations E688K and G569R were supposed to be constructed by site-directed mutagenesis in the cloned wild type <em>NALP3</em> genes and further expressed in bacterial and mammalian host cells for functional studies in protein -protein interaction models.</p>

<p>Under det senaste årtiondet har molekylärbiologin genomgått stora förändringar. Genom utvecklingen av tekniker för DNA-sekvensiering har man kunnat utforska stora mängder information. Denna information lagras i biodatabaser som i många fall är länkade med varandra för att ge användaren ett bredare sökfält. SWISS-PROT är ett exempel på en sådan biodatabas som lagrar proteininformation.</p><p>Användare som har mindre kunskap om databasens uppbyggnad och unika identifierare kan dock stöta på problem i form av en mängd sökresultat, där endast en mindre del är intressanta för användaren. Missförstånd och tvetydigheter kan också uppstå i länkade biodatabaser, då databaserna har olika uppbyggnad och definitioner på olika "byggstenar" i databasen.</p><p>Arbetet i denna rapport ska därför fokusera på att skapa en databas för molekylärbiologidata som tar hand om dessa problem. För att kunna åstadkomma detta ska ett databashanteringssystem som har funktionaliteter som erbjuder lösningar till dessa problem användas. Den data, i form av proteininformation, som ska lagras hämtas från en SWISS-PROT-domän. I arbetet kommer databashanteringssystemet Lore att användas.</p>