<p>3’ end cleavage and polyadenylation are important steps in the maturation of eukaryotic mRNAs. Poly(A) polymerase (PAP), the enzyme catalysing the addition of adenosine residues, exists in multiple isoforms. In this study the functional significance of multiple poly(A) polymerases have been investigated. It is concluded (i) that at least three mechanisms generate the multiple isoforms i.e. gene duplication, post-translational modification and alternative mRNA processing and (ii) that the different isoforms of poly(A) polymerases have different catalytic properties. The study highlights regulation of poly(A) polymerase activity through modulation of its affinity for the substrate as visualised by the K_M parameter. We suggest that trans-acting factors modulating the K_M of poly(A) polymerase will play important roles in regulating its activity.</p><p>A new human poly(A) polymerase (PAPγ) encoded by the PAPOLG gene was identified. PAPγ is 65% homologous to the previously identified PAP. In human cells three isoforms of poly(A) polymerases being 90, 100 and 106 kDa in sizes are present. These native isoforms were purified. The PAPOLA gene encoded the 100 and 106 kDa isoforms while the 90 kDa isoform was encoded by the PAPOLG gene. Native PAPγ was found to be more active than 100 kDa PAP while the hyperphosphorylated 106 kDa PAP isoform was comparably inactive due to a 500-fold decrease in affinity for the RNA substrate. </p><p>The PAPOLG gene was shown to encode one unique mRNA while the PAPOLA gene generated five different PAP mRNAs by alternative splicing of the last three exons. The PAPOLA encoded mRNAs were divided into two classes based on the composition of the last three exons. Poly(A) polymerases from the two classes were shown to differ in polyadenylation activities. These differences revealed two novel regulatory motifs in the extreme C-terminal end of PAP, one being inactivating and the other activating for polyadenylation activity.</p>

3’ end cleavage and polyadenylation are important steps in the maturation of eukaryotic mRNAs. Poly(A) polymerase (PAP), the enzyme catalysing the addition of adenosine residues, exists in multiple isoforms. In this study the functional significance of multiple poly(A) polymerases have been investigated. It is concluded (i) that at least three mechanisms generate the multiple isoforms i.e. gene duplication, post-translational modification and alternative mRNA processing and (ii) that the different isoforms of poly(A) polymerases have different catalytic properties. The study highlights regulation of poly(A) polymerase activity through modulation of its affinity for the substrate as visualised by the KM parameter. We suggest that trans-acting factors modulating the KM of poly(A) polymerase will play important roles in regulating its activity. A new human poly(A) polymerase (PAPγ) encoded by the PAPOLG gene was identified. PAPγ is 65% homologous to the previously identified PAP. In human cells three isoforms of poly(A) polymerases being 90, 100 and 106 kDa in sizes are present. These native isoforms were purified. The PAPOLA gene encoded the 100 and 106 kDa isoforms while the 90 kDa isoform was encoded by the PAPOLG gene. Native PAPγ was found to be more active than 100 kDa PAP while the hyperphosphorylated 106 kDa PAP isoform was comparably inactive due to a 500-fold decrease in affinity for the RNA substrate. The PAPOLG gene was shown to encode one unique mRNA while the PAPOLA gene generated five different PAP mRNAs by alternative splicing of the last three exons. The PAPOLA encoded mRNAs were divided into two classes based on the composition of the last three exons. Poly(A) polymerases from the two classes were shown to differ in polyadenylation activities. These differences revealed two novel regulatory motifs in the extreme C-terminal end of PAP, one being inactivating and the other activating for polyadenylation activity.

Detta projekt behandlar metodutveckling för att modellera en trevägskatalysator som skulle kunna användas vidare för att uppskatta utsläppen från en Ottomotor. Modellen är byggd i AVL Cruise M för att bestämma omvandlingarna för de tre lagstiftade föroreningarna kolmonoxid, kväveoxider (NOx) och metan baserat på reaktioner som sker i trevägskatalysatorer. Projektet tar också upp olika experimentella metoder och specifika kalibreringsmetoder som används för att samla in data och bygga modellen. Projektet diskuterar två olika kalibreringsprocesser baserade på insamlade experimentella data och antalet reaktioner kalibrerade i varje steg. Dessutom diskuteras programvaran som används och ändringarna som gjorts i den fördefinierade modellen av programvaran. Resultatet efter kalibreringen visade att den andra kalibreringsprocessen gav bättre resultat, men vissa avvikelser observerades i den byggda modellen. Avvikelserna antas bero på följande tre anledningar. För det första, försummar ytreaktionsmodellen föroreningarnas diffusionshastigheter. Dessutom kan komplexiteten hos objektfunktionen som matas in i optimeraren samt de stationära data som används för kalibreringsändamål också ge avvikelser. Dessa tre argument testas i projektet och slutsatserna som dras för att kunna förbättra modellen är följande.  Objektfunktionen behöver förenklas så mycket som möjligt, diffusion av föroreningar genom washcoaten kan inkluderas i olika komplicerade steg och syrelagringsreaktioner blir viktiga när man tar hänsyn till transienta förhållanden. / This project discusses about development of a method to model the three-way catalyst which could be used further to estimate the emissions from an Otto engine. The model is built in the commercial software called AVL Cruise M to determine the conversions of three legislative pollutants namely, carbon monoxide, nitrogen oxide and methane based on reactions occurring within the three-way catalyst. The project also discusses about different experimental and calibration methods used for collecting the data and building the model. The project discusses two different calibration process based on the experimental data used and number of reactions calibrated on each step. Furthermore, the software used, and the modifications made in the predefined model of the software are also discussed. The result after the calibration showed that the second calibration process gave better results, but some deviations were observed in the model built. The deviations are assumed to be because of three arguments present. Firstly, considering the surface reaction model which neglects the diffusion rates of the species. Secondly, the complexity of the objective function fed into the optimiser. The optimiser is also a software by AVL named design explorer which helps to optimise the parameters. And thirdly, the use of steady state data only and not including transient conditions for the calibration purposes. These arguments are tested in the project, and it is concluded to improve the model, the objective function needs to be simplified as much as possible, diffusion of species through washcoat could be considered at advance stages and oxygen storage reactions become important when transient conditions are taken into consideration.