The ribosome is a macromolecular machine that produces proteins in all kingdoms of life. The proteins, in turn, control the biochemical processes within the cell. It is thus of extreme importance that the machine that makes the proteins works with high precision. By using three dimensional structures of the ribosome and homology modelling, we have applied molecular dynamics simulations and free-energy calculations to study the codon specificity of protein synthesis in initiation and termination on an atomistic level. In addition, we have examined the binding of small molecules to riboswitches, which can change the expression of an mRNA. The relative affinities on the ribosome between the eukaryotic initiator tRNA to the AUG start codon and six near-cognate codons were determined. The free-energy calculations show that the initiator tRNA has a strong preference for the start codon, but requires assistance from initiation factors 1 and 1A to uphold discrimination against near-cognate codons. When instead a stop codon (UAA, UGA or UAG) is positioned in the ribosomal A-site, a release factor binds and terminates protein synthesis by hydrolyzing the nascent peptide chain. However, vertebrate mitochondria have been thought to have four stop codons, namely AGA and AGG in addition to the standard UAA and UAG codons. Furthermore, two release factors have been identified, mtRF1 and mtRF1a. Free-energy calculations were used to determine if any of these two factors could bind to the two non-standard stop codons, and thereby terminate protein synthesis. Our calculations showed that the mtRF’s have similar stop codon specificity as bacterial RF1 and that it is highly unlikely that the mtRF’s are responsible for terminating at the AGA and AGG stop codons. The eukaryotic release factor 1, eRF1, on the other hand, can read all three stop codons singlehandedly. We show that eRF1 exerts a high discrimination against near-cognate codons, while having little preference for the different cognate stop codons. We also found an energetic mechanism for avoiding misreading of the UGG codon and could identify a conserved cluster of hydrophobic amino acids which prevents excessive solvent molecules to enter the codon binding site. The linear interaction energy method was used to examine binding of small molecules to the purine riboswitch and the FEP method was employed to explicitly calculate the LIE b-parameters. We show that the purine riboswitches have a remarkably high degree of electrostatic preorganization for their cognate ligands which is fundamental for discriminating against different purine analogs.

Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic. Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction. The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

The F1 part of F0F1-ATP synthase in the proteobacterium Rhodobacter blasticus contains five different proteins, but when the DNA was sequenced a sixth gene was found in the operon. The protein that corresponds to the sixth gene has been named Majastridin. When an amino acid BLAST search is performed with the Majastridin sequence, protein sequences have been found that are similar to Majastridin in other bacterial strains, and one of them is Streptococcus pneumonia. The hypothetical protein from Streptococcus pneumonia contains 242 amino acids and has a molecular weight around 30 kDa.   In this work the Majastridin-like protein from Streptococcus pneumonia was expressed in E. coli cells and purified with nickel affinity chromatography and size exclusion chromatography. The result was verified with SDS-PAGE and western blot. The purified protein was then crystallized with the hanging drop method, where crystals were formed and optimization was made. The protein was also used to produce antibodies.

digitalisering@umu.se

Some steroids in the brain and peripheral nervous system have been shown to have neuroprotective effects but the knowledge is limited. The present study examines the effects of steroids including oxysterols, vitamin D and vitamin D analogs on cell viability/growth and steroidogenesis in the nervous system. Both 24- and 27-hydroxycholesterol reduced staurosporine-induced toxicity in human neuroblastoma SH-SY5Y cells. In addition, 27-hydroxycholesterol decreased the staurosporine-mediated induction of caspases, known to be important in apoptotic events. From the findings it may be concluded that effects of oxysterols on cellular viability are dependent on the concentration and on the type of oxysterol. 24-Hydroxycholesterol was also found to attenuate oxidative stress both in SH-SY5Y cells and astrocytes. The results indicate that during some conditions, oxysterols may have neuroprotective effects. The vitamin D analogs tacalcitol and calcipotriol strongly reduced proliferation, cell viability and migration of human glioblastoma T98G cells, similarly as 1,25(OH)2D3 , the physiological form of vitamin D. Glioblastoma is the most lethal type of primary tumors in the CNS. These findings suggest that vitamin D analogs are potential candidates in treatment of brain tumors, most likely in combination with other therapies. Astrocytes were found to be a major site for expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) whereas expression of CYP17A1 was found in both astrocytes and neurons. 3β-HSD and CYP17A1 are important steroidogenic enzymes. Vitamin D inhibited both CYP17A1- and 3β-HSD -mediated activity and mRNA levels, with a stronger effect on mRNA expression than on enzyme activity. This indicates that 1,25(OH)2D3 could affect the production of sex hormones in the brain. In summary, results from this thesis contribute to the knowledge on the effects of oxysterols on cell viability and oxidative stress in cells from the CNS. Also the results provide data on the effects of vitamin D in the brain and suggest that vitamin D analogs may be promising candidates for treatment of certain brain tumors.

As cyanobacteria can be found in a wide variety of environments and can cause great damage to ecosystems, human health, and economy, it is important to monitor and understand their behaviour. In this case, the study aims to understand the spatial variations of cyanobacteria communities in the Gulf of Bothnia, by analysing environmental factors that affect them. I hypothesize that the variations from north to south and coast to offshore of nutrients such as phosphorus and nitrogen promote the occurrence of different cyanobacteria orders, i.e. Chroococcales, Oscillatoriales, and Nostocales. The methods used were 16S amplicon DNA sequencing and the traditional microscopy method. Both showed similar results: Nostocales seem to predominate south-offshore zones and Oscillatoriales coast-north stations. Nevertheless, contrary to what was hypothesized on the beginning, Chroococcales were widely distributed between the stations. Both analysis methods used have limitations and strengths, thus, they should complement each other. Moreover, the DNA method must be implemented in cyanobacterial monitoring for the Baltic Sea. On the other hand, it can be said that environmental variables such as nutrients, jointly with other parameters, such as salinity, pH, humic substances and DOC (dissolved organic carbon), influence cyanobacteria communities’ composition. Overall, the environmental factors in the Baltic Sea give cyanobacteria the perfect conditions for living and develop. Therefore, it is important to implement water management and monitoring plans, as temperatures are predicted to rise globally with climate change, thus, cyanobacterial blooms are also predicted to be more common.

Campylobacter are probably the most important bacterial pathogen related to food-borne illnesses; specifically, gastroenteritis and diarrheal diseases. These bacteria can be isolated from various environments, but always originate from the intestine of warm blooded animals. Particularly, Campylobacter are found in the intestinal tract of poultry, and due to contamination of poultry meat and also further contamination of other food they can cause human infections. Sometimes this results in larger outbreaks, such as during 2016-2017 in Sweden where thousands of persons got infected by a single strain of Campylobacter jejuni sequence type 918 (ST-918). The same strain was also identified amongst a large number of poultry farms and suspicions were directed towards dirty transport cages for poultry as a main route for transmitting the strain between different farms. Similar scenarios with large outbreaks related to one or two single strains (ST-50 and ST-257) had also been observed in previous years and this raised questions about certain strains being especially adapted to survive outside the intestine. The aim here was to examine whether outbreak strains and other strains of C. jejuni have different potential to resist different stress conditions that may be encountered between the poultry farm and the human intestine.

The epidermis is composed of multiple cell strata where viable keratinocytes, in the basal layer (stratum basale (SB)), go through a range of steps with the final stage of being dead corneocytes in the outer most layer (stratum corneum (SC)). The differentiation, which can be thought of as programmed cell death, include several key processes that are essential for an intact skin barrier. The route from SB to SC is accompanied by changes, such as osmotic pressure and pH, that are believed to trigger some of these processes. In this project, HaCaT cells were incubated with, commonly used, skin care substances (urea, glycerol, transcutol and salicylic acid) to assess their impact on cell viability, by MTT-assay, and gene expression, by qPCR. Further, the relationship between osmotic pressure, viability and gene expression was studied. The excipients showed a dose-dependent decrease of keratinocyte viability which also was explained by elevated osmotic pressure when concentration was increased. Exceptions were however observed for transcutol, which showed protective features against osmotic stress. Upregulation of the genes were mainly observed when cells were treated with high concentrations. Involucrin was affected by the substances to a greater extent when compared to other markers. The upregulation of involucrin was however seen to be driven by the osmotic pressure rather than biological effects of the molecules. The project conclude that the viability and gene expression of the keratinocytes are highly related to the osmotic pressure and probably influences the differentiation to a greater extent than the molecules themselves.

Natural products and their derivatives have and are still used by humans for various health ailments due to their rich sources of drug discovery. New biologically active compounds from natural products play a key role in drug development. Marine sponges and their associated microbes contain a lot of bioactive compounds that are potential for drug development. These compounds produce chemical compounds with useful pharmaceutical properties such as antitumor, anti-infective, anti-inflammatory, and antibacterial properties. The main focus of this project was on the antibacterial activity of six different sponge specimens. The aim was to screen the antibacterial activity of the sponge specimen’s extracts. In order to do so, a Minimum Inhibitory Concentration assay was performed to screen the sponge's antibacterial activity against E. coli and S. aureus. Analytical HPLC was used for separation and Solid Phase Extraction (SPE) was used for determining the effect of salts towards the inhibition of anti-bacterial activity for two selected extracts. Ethanolic extract of Stylissa massa showed antibacterial activity against S. aureus. SPE would be a rapid purification step to remove the salts present in sponges at a high concentration but it has not shown a significant effect on the inhibition of antibacterial activity. However, further separation and purification need to be done to be able to completely screen for all the six different sponge specimens.

No description available.