The elucidation of regulatory mechanisms in higher organisms represents a front line problem in biochemical genetics. In Man the only material available for experimental studies of regulatory mechanisms is cells cultured in vitro. Enzymes which are differentiated into isozymes may have a complexgenetic background involving the action of more than one gene locus. The study of isozyme systems in cultured cells has developed into a valuable tool of increasing importance for the understanding of the genetic regulatorymechanisms in normal cells as well as in cancer cells. The purposes of this investigation were: 1. to elucidate the isozyme differentiation of amino acid naphthylamidasein cultured human cancer cells and normal cells. 2. to study the regulatory effects of steroid hormones especially hydrocortisoneon the levels of the different isozymes. / digitalisering@umu.se

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.

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.

The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using the two technologies combined in pump probe experiments. Three main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding on the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; and the development of theory, with sample applications, for infrared X-ray pump probe spectroscopy - the main theme of the thesis. Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found that the X-ray photoelectron spectrum of the water dimer differs strongly from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. Our simulations of infrared X-ray pump-probe spectra were carried out using wave packet propagation techniques. The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-ray pump probe spectroscopy - are examined in detail in two sample applications - on the NO molecule and on the dynamics of proton transfer in core ionized water dimer. It is found that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential. This results in a phase dependence of the X-ray pump probe spectra. A proper choice of the delay time of the X-ray pulse allows to directly observe the X-ray transition in the proton transfered well of the core excited potential. / QC 20101125

Cardiac hypertrophy is when heart muscles thicken as an adaptive response to several stimuli. Prolonged pathological cardiac hypertrophy can lead to heart failure and severe cardiovascular diseases. Scientists have faced challenges in studying cardiac hypertrophy due to the lack of human cardiomyocytes available. Recently, hypertrophic model using human induced pluripotent stem cell-derived cardiomyocytes was introduced. In this study, expression profiles of in vitroendothelin-1 induced cardiac hypertrophy model were investigated at different time points. The study aimed to examine molecular pathways associated with cardiac hypertrophy, identify biomarker candidates for cardiac hypertrophy, and investigate if there were known pharmaceuticals that putatively are targeting the suggested candidate biomarkers. Using the Ingenuity pathway analysis (IPA) software, GRM1, NPPA, and STC1 gene were identified as biomarker candidates for cardiac hypertrophy model across all time points. More biomarker candidates unique to the cardiac hypertrophy-stages were also identified using IPA. In vivomicroarray data of hypertrophied heart profiles were also used to compare to the in vitro data and preliminarily validate the gene candidates identified by IPA. Four genes were identified by IPA and were presented in the in vivo data. IPA also revealed the in activation of specific pathways of the early-stage cardiac hypertrophy model. The result suggested that the molecular mechanisms of the in vitro cardiac hypertrophy model did not fully represent the actual hypertrophic condition of the heart. More research and validation are required to understand the underlying mechanism fully and potentially, in the future, utilize the identified genes as cardiac hypertrophy biomarkers.