Pax7 is essential for the function of muscle satellite cells. It was previously determined that Pax7 methylation is essential for its transcriptional activity and function in satellite cells. We investigated whether Pax7 displays other post-translational modifications playing a role in its function. By mass spectrometry using immunoprecipitated FLAG-Pax7, we identified two lysine residues (K105 and K193) within the Pax7 protein that are acetylated. Pax7 transcriptional activity was monitored using a luciferase reporter under the control of Myf5, a Pax7 target gene. Treatment with Trichostatin A, a histone deacetylase inhibitor, increased significantly luciferase activity, but this activity was progressively loss when the created Pax7 mutants (K105R, K193R) were introduced. This suggests that acetylation plays a role in Pax7 transcriptional activity. To identify the acetyltransferase modulating Pax7 activity, we used a candidate approach. Myst1 is expressed in muscle satellite cells. Myst1 is known for its interaction with Wdr5 and MLL1/2, a known Pax7 partner. We detected an interaction between Pax7 and Myst1 by co-immunoprecipitation in fibroblasts and in primary myoblasts. Myst1 knockdown decreases Pax7 acetylation status suggesting Myst1 as Pax7’s acetylase. Myst1 siRNA knockdown negatively impacts many Pax7’s target genes as well as primary myoblast proliferation. Moreover, primary myoblasts treated with siMyst1 express higher levels of MyoD. In satellite cells Myst1 reduction through siRNA knockdown significantly reduces satellite cells progenitor expansion as well as increases MyoD expression. In all, Myst1 modulating Pax7 activity through acetylation represents a novel mechanism in muscle stem cell biology.

The rheological and structural characteristics of gastric (MUC5AC) and duodenal (MUC2) mucin solutions, the structural basis of the adherent mucus layer in the two organs, and their interactions with polyphenols, the phytochemicals which are linked with a number of health benefits, were investigated using particle tracking microrheology and scattering techniques. We used biochemically well characterised porcine mucins as models for human mucins to characterise their viscoelasticity, structure and dynamics as a function of concentration and pH. Additionally, the mesoscopic forces that mediate the integrity of the network were investigated using reducing (dithiothreitol) and chaotropic agents (guanidinium chloride and urea). Mucins in solution were found to be flexible and three distinct viscoelastic regimes were identify ed. At neutral pH, both types of mucin were found to form flexible self-assembled semi-dilute networks above a critical concentration (c*) where the viscosity scales as c 0.53+-0.08 and c 0.53 +-0.06 for MUC5AC and MUC2 respectively. Above a second critical concentration, the entanglement concentration (Ce), the peptide backbones reptate and entangle and there is a sharp increase in viscosity, c 3.92+- 0.38 for MUC5AC and c 5.1 0+-0.08 for MUC2. At low pH, both types of mucin solution undergo a sol-gel transition, forming pH-switchable gels. The addition of tea-derived polyphenols and tea extracts to the mucin solutions has revealed the strong interaction of galloylated phenolic molecules with mucins, which eventually leads to the gelation of the solution. Cross-linking of mucins by galloylated polyphenols is thus expected to have an impact on the physicochemical environment of the stomach and small intestine; the alteration of the organisation of the mucin polymer network is expected to modulate the barrier properties of the two adherent mucus layers which will affect nutrient absorption and the viscoelastic microenvironment of intestinal bacteria.

The circadian clock is an endogenous timekeeper that synchronises biological processes with daily external rhythms such as light and temperature cycles. It provides organisms with a competitive advantage by allowing anticipation of daily events. The circadian clock encompasses a network of transcription-translational feedback loops (TTFLs) that orchestrates rhythmic expression of a large part of the genome. This network is regulated at post-transcriptional and post-translational level. Post-translational regulation of clock proteins is essential to ensure stable rhythms and robust timekeeping. Unlike the genes in the TTFL network, modifiers of clock proteins at post-translational level are conserved across taxa. SUMO, a small ubiquitin-related post-translational modifier, regulates timekeeping in mammals through modification of the clock transcription factor BMAL. In this study, SUMO is shown to contribute to oscillator function in Arabidopsis plants. Methods have been developed to prove that mutant lines defective in SUMO machinery, including SUMO-ligase and -protease mutants, display long circadian rhythms. Additionally, sumoylation on the crucial plant clock transcription factor CCA1 is observed in vivo. A fraction of the protein is sumoylated across the expression window of CCA1, with the phase of peak sumoylation in advance of peak total CCA1. The effect of sumoylation of CCA1 was investigated with respect to localisation, stability and DNA binding affinity of the protein, as these are previously described possible effects of sumoylation. The subcellular location of CCA1-YFP fusions in protoplasts was not altered in mutant lines of the SUMO machinery. In vitro experiments show that sumoylation negatively affects the affinity of CCA1 to its cognate promotor element, suggesting that SUMO could act as a reversible attenuator of CCA1 activity. Furthermore, effects of SUMO machinery mutations appear to be differential across a range of physiologically relevant temperatures, implying that sumoylation could be involved in the response to or buffering against fluctuating ambient temperatures. There is an increasing amount of evidence to suggest that metabolic oscillations are not only driven by transcriptional outputs of the clock, but are to some extent self-sustained and can feed timing information back into the clock. Glutathione was investigated as a possible metabolic feedback signal. Expression of clock gene CCA1 was found to be abolished in a mutant of the rate-limiting enzyme for glutathione synthesis (pad2-1). Surprisingly however, the amount of glutathione was not found to oscillate. Combined, the results discussed in this thesis provide a substantial advance on our understanding of post-translational regulation and the integration of metabolic and environmental information into the plant circadian clock.

Global illumination techniques are used to improve the realism of 3D scenes. Calculating accurate global illumination requires a method for solving the rendering equation. However, the integral form of this equation cannot be evaluated. This thesis presents research in non real-time illumination techniques which are evaluated with a finite number of light rays. This includes a new technique which improves realism of the scene over traditional techniques. All computer rendering requires distortion free texture mapping to appear plausible to the eye. Inverse texture mapping, however, can be numerically unstable and computationally expensive. Alternative techniques for texture mapping and texture coordinate generation were developed to simplify rendering. Real-time rendering is improved by pre-calculating non real-time reflections. The results of this research demonstrate that a polynomial approximation of reflected light can be more accurate than a constant approximation. The solution improves realism and makes use of new features in graphics hardware. / May 2007

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Genom webbaserade enkäter har data insamlats för att undersöka huruvida det finns en korrelation med de olika personlighetsdragen inom The Big Five och olika dimensioner av prosocialt beteende. Till detta utformades en enkät genom sammanslagning av Ten Item Personality Inventory (TIPI) och Prosocial Tendency Measure (PTM), där index togs ut för alla olika dimensioner. Respondenter rekryterats genom ett tillgänglighetsurval (N= 79, varav 24 stycken män, M= 39,6 år, SD= 13.6, 55 stycken kvinnor, M= 40.1 år, SD= 13.6 ). Totalt av urvalet var 13,8 procent uppvuxna i en storstad, 28,8 procent uppvuxna i småstad och 57,5 procent inom landsbygd. Inom urvalet hade 10 procent en grundskoleutbildning, 38,8 procent gymnasieutbildning, och 51,3 högskole- eller universitetsutbildning. Resultatet visar att det finns samvariationer mellan de olika personlighetsdragen och olika dimensioner av prosocialt beteende.

Global illumination techniques are used to improve the realism of 3D scenes. Calculating accurate global illumination requires a method for solving the rendering equation. However, the integral form of this equation cannot be evaluated. This thesis presents research in non real-time illumination techniques which are evaluated with a finite number of light rays. This includes a new technique which improves realism of the scene over traditional techniques. All computer rendering requires distortion free texture mapping to appear plausible to the eye. Inverse texture mapping, however, can be numerically unstable and computationally expensive. Alternative techniques for texture mapping and texture coordinate generation were developed to simplify rendering. Real-time rendering is improved by pre-calculating non real-time reflections. The results of this research demonstrate that a polynomial approximation of reflected light can be more accurate than a constant approximation. The solution improves realism and makes use of new features in graphics hardware.

Global illumination techniques are used to improve the realism of 3D scenes. Calculating accurate global illumination requires a method for solving the rendering equation. However, the integral form of this equation cannot be evaluated. This thesis presents research in non real-time illumination techniques which are evaluated with a finite number of light rays. This includes a new technique which improves realism of the scene over traditional techniques. All computer rendering requires distortion free texture mapping to appear plausible to the eye. Inverse texture mapping, however, can be numerically unstable and computationally expensive. Alternative techniques for texture mapping and texture coordinate generation were developed to simplify rendering. Real-time rendering is improved by pre-calculating non real-time reflections. The results of this research demonstrate that a polynomial approximation of reflected light can be more accurate than a constant approximation. The solution improves realism and makes use of new features in graphics hardware.

Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes, but also present a solid obstacle in the proteome analysis. Identification and mapping of PTMs in proteins have improved dramatically, but to comprehend complex mechanisms and biological functions, one must address also a very low abundant proteins. Here, in this thesis entitled 'Analysis of DELLA protein ubiquitination' we demonstrate the use of a recombinant protein standard for the determination of in vivo modified peptides of the DELLA family protein RGA. The candidate peptide sequences were targeted in an SRM-based analysis to detect the ubiquitination site and the results of this analysis and that of an MS/MS data processing indicate that the modification is localized in the conserved N-terminal region of RGA protein.

Extracellular signal-regulated kinases 1/2 (ERK1/2) are important kinases regulating cell proliferation and cell migration, and have been established as therapeutic targets for cancer treatment. Previously, we found that ERK1 phosphorylates histone deacetylase 6 (HDAC6) to regulate its enzymatic activity. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we have discovered that ERK1/2 are acetylated proteins and shown that HDAC6 manipulates ERK1’s kinase activity via deacetylation. We demonstrated that both ERK1 and ERK2 interact with HDAC6 physically. We showed that the acetylation level of GST-ERK1/2 increased in a dose- and time-dependent manner upon treatment with a pan-HDAC inhibitor, Trichostatin A. Furthermore, the treatment by HDAC6-specific inhibitor, ACY-1215, also increased the level of acetylated GST-ERK1/2. We also noted that ERK1/2 acetylation levels increased in HDAC6-knockout mouse embryonic fibroblasts and in HDAC6-knockdown A549 cell lines compared with controls. In addition, we determined that acetyltransferases CBP and p300 acetylate ERK1/2. We have identified novel acetylation sites located in ERK1 and ERK2 by mass-spectrometry analysis. Among these acetylation sites, ERK1 lysine 72 acetylation status is related to ERK1 phosphorylation. The acetylation-mimicking mutant exhibits a decreased kinase activity toward ELK1, while the deacetylation-mimicking mutant exhibits a similar level of kinase activity as the wild-type ERK1, suggesting that acetylation/deacetylation alters ERK1 enzymatic activities. Taken together, our results suggest that HDAC6 may regulate ERK1’s kinase activity via deacetylation of its lysine 72 residue.