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This thesis presents how a solution to fetch and stream a video feed from the astrovisualization software OpenSpace to a web page can be designed. The streaming protocol that was used was WebRTC. Three different methods for fetching data and creating a video feed were investigated: WebRTC, Spout, and GStreamer. Through user tests, the GStreamer method was determined to be the best option for the streaming solution., Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet

This thesis presents how a solution to fetch and stream a video feed from the astrovisualization software OpenSpace to a web page can be designed. The streaming protocol that was used was WebRTC. Three different methods for fetching data and creating a video feed were investigated: WebRTC, Spout, and GStreamer. Through user tests, the GStreamer method was determined to be the best option for the streaming solution., Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet

tRNA molecules get heavily modified post-transcriptionally. The N-1 methylation of purines at position 9 of eukaryal and archaeal tRNA is catalyzed by the SPOUT methyltranferase Trm10. Remarkably, while certain Trm10 orthologs are specific for either guanosine or adenosine, others show a dual specificity. Structural and functional studies have been performed on guanosine- and adenosine-specific enzymes. Here we report the structure and biochemical analysis of the dual-specificity enzyme from Thermococcus kodakaraensis (TkTrm10). We report the first crystal structure of a construct of this enzyme, consisting of the N-terminal domain and the catalytic SPOUT domain. Moreover, crystal structures of the SPOUT domain, either in the apo form or bound to S-adenosyl-L-methionine or S-adenosyl-L-homocysteine reveal the conformational plasticity of two active site loops upon substrate binding. Kinetic analysis shows that TkTrm10 has a high affinity for its tRNA substrates, while the enzyme on its own has a very low methyltransferase activity. Mutation of either of two active site aspartate residues (Asp206 and Asp245) to Asn or Ala results in only modest effects on the N-1 methylation reaction, with a small shift toward a preference for m1G formation over m1A formation. Only a double D206A/D245A mutation severely impairs activity. These results are in line with the recent finding that the single active-site aspartate was dispensable for activity in the guanosine-specific Trm10 from yeast, and suggest that also dual-specificity Trm10 orthologs use a noncanonical tRNA methyltransferase mechanism without residues acting as general base catalysts., SCOPUS: ar.j, info:eu-repo/semantics/published

The Minimal Tied Trefoil from Thermotoga maritima (MTTTm) represents an ideal system for investigating knots in proteins. It is one of the smallest knotted proteins to contain a deep trefoil knot, and one of the smallest SPOUT methyltransferases. To date, however, little has been accomplished on this system probing into the knots purpose in the protein. The work presented here characterizes the role of the knot in MTTTm. There are two main facets for understanding the knot: its role in the native state, and its role in the denatured state. To probe these, the work following uses a myriad of biophysical techniques to further understand the knots role in both states. These experiments show that in the native state the knot helps to tether down what was expected to be a flexible loop, which if flexible would impact binding. As well, there exist a series of hydrophobic contacts beneath the knot that are critical for allostery, and would be disrupted if the knot were not there. In the denatured state, there appears to be a linkage between the knot and critical packing residues that would complicate refolding in vivo, but the knot helps to maintain those contacts to allow for easier refolding. The studies here give us more information on why the knot exists in MTTTm, and help us to further understand the role of knots in proteins as a whole.

The Minimal Tied Trefoil from Thermotoga maritima (MTTTm) represents an ideal system for investigating knots in proteins. It is one of the smallest knotted proteins to contain a deep trefoil knot, and one of the smallest SPOUT methyltransferases. To date, however, little has been accomplished on this system probing into the knots purpose in the protein. The work presented here characterizes the role of the knot in MTTTm. There are two main facets for understanding the knot: its role in the native state, and its role in the denatured state. To probe these, the work following uses a myriad of biophysical techniques to further understand the knots role in both states. These experiments show that in the native state the knot helps to tether down what was expected to be a flexible loop, which if flexible would impact binding. As well, there exist a series of hydrophobic contacts beneath the knot that are critical for allostery, and would be disrupted if the knot were not there. In the denatured state, there appears to be a linkage between the knot and critical packing residues that would complicate refolding in vivo, but the knot helps to maintain those contacts to allow for easier refolding. The studies here give us more information on why the knot exists in MTTTm, and help us to further understand the role of knots in proteins as a whole.

During translation, the transfer RNAs (tRNAs) play the crucial role of adaptors between the messenger RNA and the amino acids. The tRNAs are first transcribed as pre-tRNAs which are then maturated. During this maturation, several nucleosides are modified by tRNA modification enzymes. These modifications are important for the functions of the tRNAs and for their correct folding. Many of the modifications are methylations of the bases or the ribose. Four families of tRNA methyltransferases are known, among which the SPOUT superfamily. Proteins of this superfamily are characterised by a C-terminal topological knot where the methyl donor is bound. With the exception of the monomeric Trm10, all known SPOUT proteins are dimeric and have an active site composed of residues of both protomers. Interestingly, depending on the organism, the same modification can be catalysed by completely unrelated enzymes. On the other hand, homologous enzymes can have different specificities or/and activities. These differences are well illustrated for the TrmJ and Trm10 enzymes.In the first part of this work we have identified the TrmJ enzyme of Sulfolobus acidocaldarius (the model organism of hyperthermophilic Crenarchaeota) which 2’-O-methylates the nucleoside at position 32 of tRNAs. This protein belongs to the SPOUT superfamily and is homologous to TrmJ of the bacterium Escherichia coli. A comparative study shows that the two enzymes have different specificities for the nature of the nucleoside at position 32 as well as for their tRNA substrates. To try to understand these shifts of specificity at a molecular level we solved the crystal structure of the SPOUT domains of the two TrmJ proteins.In the second part of this work, we have determined the crystal structure of the Trm10 protein of S. acidocaldarius. This is the first structure of a 1-methyladenosine (m1A) specific Trm10 and also the first structure of a full length Trm10 protein. The Trm10 protein of S. acidocaldarius is di, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The 2'-O-methylation of the nucleoside at position 32 of tRNA is found in organisms belonging to the three domains of life. Unrelated enzymes catalyzing this modification in Bacteria (TrmJ) and Eukarya (Trm7) have already been identified, but until now, no information is available for the archaeal enzyme. In this work we have identified the methyltransferase of the archaeon Sulfolobus acidocaldarius responsible for the 2'-O-methylation at position 32. This enzyme is a homolog of the bacterial TrmJ. Remarkably, both enzymes have different specificities for the nature of the nucleoside at position 32. While the four canonical nucleosides are substrates of the Escherichia coli enzyme, the archaeal TrmJ can only methylate the ribose of a cytidine. Moreover, the two enzymes recognize their tRNA substrates in a different way. We have solved the crystal structure of the catalytic domain of both enzymes to gain better understanding of these differences at a molecular level., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

The 2'-O-methylation of the nucleoside at position 32 of tRNA is found in organisms belonging to the three domains of life. Unrelated enzymes catalyzing this modification in Bacteria (TrmJ) and Eukarya (Trm7) have already been identified, but until now, no information is available for the archaeal enzyme. In this work we have identified the methyltransferase of the archaeon Sulfolobus acidocaldarius responsible for the 2'-O-methylation at position 32. This enzyme is a homolog of the bacterial TrmJ. Remarkably, both enzymes have different specificities for the nature of the nucleoside at position 32. While the four canonical nucleosides are substrates of the Escherichia coli enzyme, the archaeal TrmJ can only methylate the ribose of a cytidine. Moreover, the two enzymes recognize their tRNA substrates in a different way. We have solved the crystal structure of the catalytic domain of both enzymes to gain better understanding of these differences at a molecular level., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published