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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No description available.

The requirement of DNA synthesis for the release of Ɣ-radiation-induced mitotic inhibition in mammalian cells has been studied. Mammalian cells in which DNA synthesis had been inhibited by treatment with fluorodeoxyuridine (FUdR) were not released from radiation-induced mitotic inhibition until the FUdR block was removed. After removal of the block, mitotic figures reappeared, but only after a time equivalent to the usual mitotic delay caused by the particular radiation dose employed. This suggests that repair of the mitotic inhibition lesion can not proceed unless the pathway for DNA synthesis is intact. Further evidence for the requirement of DNA synthesis in the release of mitotic inhibition came from the observation of radiation-induced synthesis of DNA during G₂, a stage in the cell cycle normally not associated with such synthesis. / Thesis / Master of Science (MSc)

Chloramphenicol has been used to study mitochondrial biogenesis in mammalian cells by examining its effect on: the incorporation of radioactive amino acids into protein by isolated mitochondria, the growth of L cells, the level of representative enzymes and cytochromes in the mitochondria and cytoplasm and the structure of mitochondria and L cells. A reversible inhibition of synthesis of cytochrome c oxidase was obtained by treating cells with D-threo-chloramphenicol for 90 hr. Recovery of cytochrome c oxidase activity was inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis. Cycloheximide also reversibly inhibited cytochrome c oxidase formation in cells which were not treated with D-chloramphenicol. It is suggested that the mitochondria and the nucleus have a joint control in the formation of a functionally active cytochrome c oxidase enzyme. / Thesis / Doctor of Philosophy (PhD)

No description available.

One of the main difficulties of addiction treatment is the high risk of relapse even after a longabstinence and fully detoxification. Therefore, discovering the underlying molecular principlesof relapse is essential. The metabotropic glutamate receptor, mGluR5, is considered to beinvolved in this aspect. One of the brain structures expressing mGluR5 is the striatum, an areawith well-established role in addiction which is largely composed of medium-sized spinyneurons (MSNs). These neurons are basically divided into two major subpopulationscharacterized based on their projections and protein properties. It is known that the mGluR5receptor is expressed on both subpopulations of MSNs. Consequently, it can be used to establishthe proportional contribution of each of MSNs subpopulations in relapse to addiction. In ourconstellation, we have generated a mouse line designed to have a selective mGluR5 knock-downin one of these subpopulations – the dopamine D1 receptor (D1R) expressing neurons. It hashowever been unclear if the expression of the transgene is indeed limited to only D1R-expressingneurons. By immunofluorescence technique, I here show that the construct is expressed only inMSNs and is restricted to the D1R-expressing cell population in the striatum. Thus the transgenicmouse line is a good tool for the study of mGluR5 selectively in D1R expressing neurons.

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

People are, in their daily lives, exposed to a number of airborne foreign compounds that do not normally affect the body. However, depending on the nature of these compounds, dose and duration of exposure, various airway symptoms may arise. Early symptoms are often manifested as upper airway mucosal inflammation which generates changes in protein composition in the airway lining fluid. This thesis aims at identifying, understanding mechanisms and characterizing protein alterations in the upper airway mucosa that can be used as potential new biomarkers for inflammation in the mucosa. The protein composition in the mucosa was studied by sampling of nasal lavage fluid that was further analyzed with a proteomic approach using twodimensional gel electrophoresis and mass spectrometry. Additionally, by studying factors on site through environmental examination, health questionnaires and biological analyses, we have tried to understand the background to these protein alterations and their impact on health. Respiratory symptoms from the upper airways are common among people who are exposed to irritative and microbial agents. This thesis have focused on personnel in swimming pool facilities exposed to trichloramine, metal industry workers exposed to metalworking fluids, employees working in damp and moldy buildings and infants diagnosed with respiratory syncytial virus infection. The common denominator in these four studies is that the subjects experience upper airway mucosal inflammation, which is manifested as cough, rhinitis, phlegm etc. In the three occupational studies, the symptoms were work related. Notably, a high prevalence of perceived mucosal symptoms was shown despite the relatively low levels of airborne irritants revealed by the environmental examination. Protein profiling verified an ongoing inflammatory response by identification of several proteins that displayed altered levels. Interestingly, innate immune proteins dominated and four protein alterations occurred in most of the studies; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin. Similarly, these proteins were also found in nasal fluid from children with virus infection and in addition a truncated form of SPLUNC1 and two other S100 proteins (S100A7-like 2 and S100A16), not previously found in nasal secretion, were identified. Altogether, the results indicate the potential use of a proteomic approach for identifying new biomarkers for the upper respiratory tract at an early stage in the disease process after exposure to irritant and microbial agents. The results indicate an effect on the innate immunity system and the proteins; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin are especially promising new biomarkers. Moreover, further studies of these proteins may help us to understand the molecular mechanisms involved in irritant-induced airway inflammation.