Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Zona pellucida glycoproteins (ZP) are important proteins for maturation of the oocytes in eukaryotes, these proteins are encoded by cluster of zp genes. zp2.3 and zp3.5 genes are expressed during the developing and maturation of the oocytes in zebrafish ovaries. Both of the gens have a CCAAT box in their promoter regions, playing a big role in the expression of the both genes in zebrafish oocytes. The transcription of the genes in the eukaryotes requires transcription factors to initiate and promote the transcription, the transcription factors can bind to the promoter region and initiate the transcription process. The nuclear factor y (NFY) regulates the genes by binding to the CCAAT boxes in their promoter regions, it consist from many subunit such as NF-YA and NF-YB. Here in this study we characterize the expression pattern of NF-YA and NF-YB by screening these genes expression in several organs and tissues, also to determine its roles in the expression of the zp2.3 and zp3.5 genes in the adultzebrafish ovary.

The aim of this examination project work was to develop one new sports drink for consumption during prolonged exercise. Most existing sports drinks contain carbohydrates and electrolytes but the drink developed during this project work aimed to contain protein/amino acids and other substances that potentially may help the athlete to perform better compared with sports dinks containing only carbohydrates and electrolytes. Since it is unclear if whole proteins, oligopeptides or free amino acids are preferred, three different sports drink where developed. All three sports drinks contain 25 mmol Na+/l, 5.5 mmol K+/l, 240 mg caffeine (per serving), high molecular weight glucose polymer (7%), aromas, beta-carotene and other substances supposed to improve the taste of the drinks. The three drinks contain different sources of amino acids: 0.47% branched-chain amino acids (BCAA) in the BCAA-sports drink, 2% whey protein in the Whey protein-sports drink and 0.8% hydrolyzed casein (oligopeptides) in the Peptopro®-sports drink. The serving size of each sports drink is 0.75 l. As the drinks should taste well and be easy to drink during exercise blinded tasting tests were performed. During these tests prototypes and the final versions of all three sports drinks were compared to a placebo drink containing no amino acids or caffeine. The results from the taste-tests show that none of the test drinks or the placebo drink exhibited significantly better taste (p>0.05). However, the BCAA-sports drink, the Whey protein-sports drink and the placebo drink was shown to taste better than the Peptopro®-sports drink. To investigate the effects of the sports drinks on performance, two elite trained cyclists were supplemented with the test drinks and the placebo drink during interval-trials on a cycle ergometer equipped to measure power output during blinded tests. The power output was compared to the heart rate of each test participant. The placebo drinks contained 8% carbohydrates but no caffeine or amino acids. The results indicate that all three sports drinks either were considered equal, or improved the performance, as compared to the placebo drink. All drinks were considered to taste well during exercise. These findings indicate that the three sports drinks developed during this project work, improved performance and tasted well. However, it is desired to improve the taste of both the Peptopro®-sports drink and the Whey protein-sports drink. Alternative drinks containing no caffeine should be developed in order for the athlete to intake large quantities of the developed sports drinks during prolonged exercise / Sportdrycker är i regel kolhydratbaserade i syfte att skapa goda förutsättningar för idrottslig prestation. De sportdrycker som vid detta arbete utvecklats innehåller substanser utöver kolhydrater som möjliggör en förbättrad prestation vid långtidsuthållighetsidrott relativt kolhydratsbaserade sportdrycker. Sportdryckerna skulle vara välsmakande och lätta att konsumera i stora volymer under arbete. Syftet var att utveckla en sportdryck, innehållande en protein-/aminosyra-källa, men då det mot bakgrund av aktuell forskning var oklart om helprotein, oligopeptider eller fria aminosyror var att föredra, utvecklades tre olika sportdrycker. Detta resulterade i följande tre sportdrycker: BCAA-sportdrycken, som innehåller 0.47 % grenade aminosyror (BCAA), Vassleprotein-sportdrycken, som innehåller 2 % vassleprotein och Peptopro®-sportdrycken som innehåller 0,8 % hydrolyserat kasein (i form av oligopeptider). Alla tre har en serveringsstorlek på 0,75 l. Övrigt innehåll i sportdryckerna är 240 mg koffein (per servering), 25 mmol/l Na+, 5,5 mmol/l K+, 7 % glukospolymer av hög molekylvikt (Vitargo®) samt smakämnen och betakaroten. De tre sportdryckerna jämfördes vid enkelblindade tester relativt en etablerad sportdryck innehållande 8 % kolhydrater (Vitargo®), både genom smaktester utförda på tränad smakpanel och genom prestationstester utförda på unga elitcyklister. Även osmolalitet och pH mättes på färdiga drycker. Resultaten från smaktesterna visade att både BCAA-sportdrycken och Vassleprotein-sportdrycken var välsmakande, men att Peptopro®-sportdrycken ej var färdig smakmässigt. Ingen signifikant skillnad i smak uppmättes mellan sportdryckerna och kontrolldrycken (p>0,05). Resultaten från prestationstesterna indikerade att samtliga testdrycker var likvärdiga med eller förbättrade prestationen under tidsintervallen relativt kontrolldryck med annorlunda elektrolytsammansättning samt utan koffein och aminosyror. Samtliga drycker uppfattades här som välsmakande och lätta att konsumera under arbete. Sammanfattningsvis bedöms samtliga produkter vara såväl prestationsmässigt som smakmässigt bra alternativ till etablerade sportdrycker för uthållighetsidrottare. Ytterligare smakförbättringar är önskvärda för såväl Vassleprotein-sportdrycken som Peptopro®-sportdrycken. 2008:L1

Numerical simulation of stochastic biochemical reaction networks has received much attention in the growing field of computational systems biology. Systems are frequently modeled as a continuous-time discrete space Markov chain, and the governing equation for the probability density of the system is the (chemical) master equation. The direct numerical solution of this equation suffers from an exponential growth in computational time and memory with the number of reacting species in the model. As a consequence, Monte Carlo simulation methods play an important role in the study of stochastic chemical networks. The stochastic simulation algorithm (SSA) due to Gillespie has been available for more than three decades, but due to the multi-scale property of the chemical systems and the slow convergence of Monte Carlo methods, much work is currently being done in order to devise more efficient approximate schemes. In this thesis we review recent work for the solution of the chemical master equation by direct methods, by exact Monte Carlo methods and by approximate and hybrid methods. We also describe two conceptually different numerical methods to reduce the computational time when studying models using the SSA. A hybrid method is proposed, which is based on the separation of species into two subsets based on the variance of the copy numbers. This method yields a significant speed-up when the system permits such a splitting of the state space. A different approach is taken in an algorithm that makes use of low-discrepancy sequences and the method of uniformization to reduce variance in the computed density function.

The chemical master equation (CME) describes the probability for the discrete molecular copy numbers that define the state of a chemical system. Each molecular species in the chemical model adds a dimension to the state space. The CME is a difference-differential equation which can be solved numerically if the state space is truncated at an upper limit of the copy number in each dimension. The size of the truncated CME suffers from an exponential growth for an increasing number of chemical species. In this thesis the chemical master equation is approximated by a continuous Fokker-Planck equation (FPE) which makes it possible to use sparser computational grids than for CME. FPE on conservative form is used to compute steady state solutions by computation of an extremal eigenvalue and the corresponding eigenvector as well as time-dependent solutions by an implicit time-stepping scheme. The performance of the numerical solution is compared to a standard Monte Carlo algorithm. The computational work for a solutions with the same estimated error is compared for the two methods. Depending on the problem, FPE or the Monte Carlo algorithm will be more efficient. FPE is well suited for problems in low dimensions, especially if high accuracy is desirable.

Ribosomes are macromolecular machines that are responsible for production of every protein in a living cell. Yet we do not know the details about how these machines are formed. The ribosome consists of four RNA strands and roughly 80 proteins that associate with each other in the nucleolus and form pre-ribosomal complexes. Eukaryotes, in contrast to prokaryotes, need more than 200 non-ribosomal factors to assemble ribosomes. These associate with pre-ribosomal complexes at different stages as they travel from the nucleolus to the cytoplasm and are required for pre-rRNA processing. We do however lack knowledge about the molecular function of most of these factors and what enables pre-rRNA processing. Especially, information is missing about how non-ribosomal factors influence folding of the pre-rRNA and to what extent the pre-ribosomal complexes are restructured during their maturation.  This thesis aims to obtain a better understanding of the earliest events of ribosome assembly, namely those that take place in the nucleolus. This has been achieved by studying the essential protein Mrd1 by mutational analysis in the yeast Saccharomyces cerevisiae as well as by obtaining structural information of nucleolar pre-ribosomal complexes. Mrd1 has a modular structure consisting of multiple RNA binding domains (RBDs) that we find is conserved throughout eukarya. We show that an evolutionary conserved linker region of Mrd1 is crucial for function of the protein and likely forms an essential module together with adjacent RBDs. By obtaining structural information of pre-ribosomal complexes at different stages, we elucidate what structuring events occur in the nucleolus.  We uncover a direct role of Mrd1 in structuring the pre-rRNA in early pre-ribosomal complexes, which provides an explanation for why pre-rRNA cannot be processed in Mrd1 mutants. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Proteins have to be folded to their native structures to be functionally expressed. Misfolded proteins are proteotoxic and negatively impact on cellular fitness. To maintain the proteome functional proteins are under the constant surveillance of dedicated molecular chaperones that perform protein quality control (PQC). Using the model organism yeast Saccharomyces cerevisiae this thesis investigates the molecular mechanisms that cells employ to maintain protein homeostasis (proteostasis). In Study I the role of the molecular chaperone Hsp110 in the disentanglement and reactivation of aggregated proteins was investigated. We found that Hsp110 is essential for cellular protein disaggregation driven by the molecular chaperones Hsp40, Hsp70 and Hsp104 and characterized its involvement via regulation of Hsp70 ATPase activity as a nucleotide exchange factor. In Study II we found out that Hsp110 undergoes translational frameshifting during its expression resulting in a nuclear targeting. Nuclear Hsp110 interacts with Hsp70 and reprograms the proteostasis system to better deal with stress and to confer longevity. Study III describes regulation of Hsp70 function in PQC by the nucleotide exchange factor Fes1. We found that rare alternative splicing regulates Fes1 subcellular localization in the cytosol and nucleus and that the cytosolic isoform has a key role in PQC. In Study IV we have revealed the molecular mechanism that Fes1 employ in PQC. We show that Fes1 carries a specialized release domain (RD) that ensures the efficient release of protein substrates from Hsp70, explaining how Fes1 maintains the Hsp70-chaperone system clear of persistent misfolded proteins. In Study V we report on the use of a novel bioluminescent reporter (Nanoluc) for use in yeast to measure the gene expression and protein levels. In summary, this thesis contributes to the molecular understanding of chaperone-dependent PQC mechanisms both at the level of individual components as well as how they interact to ensure proteostasis. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that affect millions of people worldwide. Aggregation of Amyloid-β (Aβ) monomers create toxic oligomers that can interact with cellular membranes and disturb cellular functions, resulting in cell death and neurological dysfunction. Increased levels of oxidative stress have been shown in the brains of AD patients, something that besides the obvious cell and tissue toxicity, also favors the amyloidogenic pathway and generates more Aβ monomers. Here we show that Edaravone, a free radical scavenger can affect the aggregation rate of different lengths of Aβ. We show that Aβ-40 that is more commonly found in vivo aggregates faster with addition of Edaravone, while Aβ-42 aggregates slower or not at all. These findings add up to previous findings where free radical scavengers and antioxidants such as Edaravone have been suggested as a potential treatment in Alzheimer’s disease.

AtPreP (Arabidopsis thaliana Presequence Protease) is a zink metallooligopeptidase that is dually targeted to both mitochondria and chloroplasts. In these organelles it functions as a peptidasome that degrades the N-terminal targeting peptides that are cleaved off from the mature protein after protein import, as well as other unstructured peptides. In A. thaliana there are two isoforms of PreP, AtPreP1 and AtPreP2.  We have performed characterization studies of single and double prep knockout plants. Immunoblot analysis revealed that both PreP isoforms are expressed in all tissues with highest expression levels in flowers and siliques. Furthermore, AtPreP1 was shown to be the most abundant isoform of the two. When comparing phenotype, the atprep2 mutant was similar to wild type, whereas the atprep1 mutant had a slight pale-green phenotype in the early developmental stages. The atprep1 atprep2 double knockout plants showed a chlorotic phenotype in true leaves, especially prominent during the early developmental stages. When analysing the first true leaves of double knockout plants, we found a significant decrease in chlorophyll a and b content. Mitochondrial respiratory rates measurements showed partially uncoupled mitochondria. Ultrastructure analysis using electron microscopy on double knockout plants showed aberrant chloroplasts with altered grana stacking and clearly fewer starch granules. Older plants showed less altered  phenotype, although there was a significant decrease in the accumulated biomass of about 40% compared to wild type. Peptidolytic activity studies showed no sign of compensatory mechanisms in the absence of AtPreP in mitochondria; in contrast we found a peptidolytic activity in the chloroplast membranes not related to AtPreP. In addition to zinc located in the catalytic site, crystallographic data revealed two Mg-binding sites in the AtPreP structure. To further investigate the role of these Mg-binding sites, we have made AtPreP variants that are unable to bind metal ions. Our data shows that one of these sites located close to the catalytic site is important for the activity of AtPreP. We also measured proteolytic activity of four human PreP-SNP variants and observed that the activity of all the hPreP-SNPs variants was lower; especially the hPreP-SNP (A525D) variant that displayed only 20-30 % of wild type activity. Interestingly, the activity was fully restored for all SNP-variants by addition of Mg2+.

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